key: cord-023100-0pqnsiid authors: nan title: abstract date: 2008-04-10 journal: j small anim pract doi: 10.1111/j.1748-5827.1991.tb00876.x sha: doc_id: 23100 cord_uid: 0pqnsiid nan five 10-week-old puppies were inoculated oronasally with 10"' tcid50 of ccv-c54, an isolate of canine coronavirus (ccv), and killed three, five, seven, 10 and 14 days later. the dogs had mild diarrhoea from three to 11 days after inoculation, approximately at the same time as the virus was excreted in the faeces. virus could be isolated from the tonsils on day 3, and then from small and large intestinal tissues up to 14 days after inoculation; it was also isolated from liver and lung tissue. no histological changes were observed in any of the tissues, but antigen to the virus was detected mainly in the epithelium overlying gut-associated lymphoid tissue. virus neutralising antibody was first detected on day 10. specific anti-ccv igm was first detected in plasma three days after inoculation and igg on days 4 to 7. small amounts of anti-ccv igg, igm and iga were detected in duodenal secretions, but none in bile. neuromuscular diseases. 7. neuropathies chronic relapsing (dysimmune) polyneuropathy: diagnosis and treatment remarkable recovery of a steroid-responsive recurrent neuropathy chronic relapsing polyradiculoneuritis in a cat peripheral neuropathy in cats with inherited primary hyperchylomicronaemia the authors wish to thank karen wadewell and denise wigney for expert technical assistance, and dr a. d. j. watson for assistance with the manuscript. key: cord-266183-uzuda3ir authors: renieris, georgios; katrini, konstantina; damoulari, christina; akinosoglou, karolina; psarrakis, christos; kyriakopoulou, magdalini; dimopoulos, george; lada, malvina; koufargyris, panagiotis; giamarellos-bourboulis, evangelos j. title: serum hydrogen sulfide and outcome association in pneumonia by the sars-cov-2 corona virus date: 2020-05-18 journal: shock doi: 10.1097/shk.0000000000001562 sha: doc_id: 266183 cord_uid: uzuda3ir background: the pneumonia of covid-19 illness has often a subtle initial presentation making mandatory the use of biomarkers for evaluation of severity and prediction of final patient disposition. we evaluated the use of hydrogen sulfide (h2s) for the outcome of covid-19 pneumonia. materials & methods: we studied 74 patients with covid-19. clinical data were collected, and survival predictors were calculated. blood was collected within 24 hours after admission (day 1) and on day 7. h2s was measured in sera by monobromobimane derivation (mbb) followed by high performance liquid chromatography and correlated to other markers like procalcitonin (pct) and creactive protein (crp). tumor necrosis factor alpha (tnfα) and interleukin (il)-6 were also measured in serum. results: survivors had significantly higher h2s levels on day 1 and 7 after admission. a cut-off point of 150.44 μm could discriminate survivors from non-survivors with 80% sensitivity, 73.4% specificity and negative predictive value 95.9%. mortality after 28 days was 32% with admission levels lower or equal to 150.44 μμ and 4.1% with levels above 150.44 μμ (p: 0.0008). mortality was significantly greater among patients with a decrease of h2s levels from day 1 to day 7 greater or equal to 36% (p: 0.0005). serum h2s on day 1 was negatively correlated with il6 and crp and positively correlated with the absolute lymphocyte count in peripheral blood. conclusion: it is concluded that h2s is a potential marker for severity and final outcome of pneumonia by the sars-cov-2 coronavirus. its correlation with il6 suggests anti-inflammatory properties. since december 2019, humanity is experiencing a novel pandemic by the novel sars-cov-2 coronavirus-19 that is causing the disease known as covid-19 (1) . as of april 17 th 2020, 2,265, 271 confirmed cases were reported worldwide, causing 154,900 deaths (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). the main reason for death is severe respiratory failure (srf) developing in the field of community-acquired pneumonia (cap). it seems that derangement of the lung endothelium is the hallmark of disease pathogenesis. this is hypothesized by published evidence showing elevated levels of d-dimers and vascular endothelial growth factor in these patients (2, 3) . hydrogen sulfide (h2s), long thought of solely as an environmental toxicant, is now known to be an endothelial product that drives angiogenesis, promotes vasorelaxation, reduces atherosclerosis and prevents ischemia-reperfusion injury (4, 5) . in spite of being considered having anti-inflammatory properties through inhibition of nuclear factor-kb (6) , a recent animal model showed complex interaction with angiotensin-2 that is the receptor sars-cov-2 is using to invade host epithelia (7) . in light of these observations suggesting a pivotal role of h2s in the pathogenesis of covid-19, we studied the serum levels of h2s and its association with final outcome in a cohort of patients with covid-19 pneumonia. due to the described anti-inflammatory properties, we hypothesize that elevated levels of h2s in serum are associated with a favourable outcome of covid-19 pneumonia. this study was conducted in eight departments participating in the hellenic sepsis study groupfrom beginning of march 2020. the study protocol was approved by the ethics committees of the participating hospitals. patients were included after written informed consent was provided by themselves or by first-degree relatives in case of patients unable to consent. we enrolled patients admitted with lower respiratory infection as diagnosed by the presence of infiltrates in chest x-ray or in computed tomography of the lung and who were tested positive upon admission by molecular testing of respiratory secretions for sars-cov-2. blood was sampled within the first 24 hours of hospital admission and repeated after seven days. exclusion criteria were: a) hiv-1 infection and b) neutropenia defined as less than 1000 neutrophils/mm 3 . srf was defined as any ratio of partial oxygen pressure to fraction of inspired oxygen below 200 necessitating mechanical ventilation. the clinical study flow chart is shown in figure 1 . copyright © 2020 by the shock society. unauthorized reproduction of this article is prohibited. the following variables were recorded: i) demographics; ii) vital signs; ii) admission acute physiology and chronic health evaluation (apache) ii score, charlson's comorbidity index (cci), sequential organ failure assessment (sofa) score (8) and pneumonia severity index (psi) (9); (iii) absolute blood cell counts and biochemistry on admission and follow-up; and 28-day survival. immediately after sampling, blood was collected into sterile and pyrogen-free tubes and transported ice-cold for centrifugation within less than 10 minutes. h2s was measured in patient serum by using monobromobimane (mbb) derivation followed by high performance liquid chromatography as it has previously been described (10, 11) . mbb, monosodium phosphate (nah2po4), disodium phosphate (na2hpo4) and 5-sulfosalicylic acid (ssa) were were carried out at excitation and emission wavelengths of 390 nm and 475 nm respectively. the retention time of the derivatization product was 12.7 minutes. serum concentrations of tumor necrosis factor alpha (tnfα) and interleukin (il)-6 were measured in duplicate by an enzyme immunoassay (r&d, minneapolis, usa). the lowest detections limits were 40 pg/ml tnfα 40 pg/ml and 10 pg/ml for il-6. procalcitonin (pct) was measured by a time-resolved amplified cryptate emission technology assay according to the manufacturer's instructions (kryptor, brahms, hennigsdorf, germany). the lower detection limit was 0.06 ng/ml. c-reactive protein (crp) was estimated in duplicate by a nephelometric assay (behring, berlin, germany). the lowest limit of detection was 0.2 mg/dl. the association between h2s levels on day 1 and 28-day survival was the primary study endpoint. the association between change in h2s levels between day 1 and 7 and 28day survival was the secondary study endpoint. categorical data were presented as frequencies and quantitative variables as mean ± se. comparisons between groups were done using the fisher exact test for categorical data, the two-sided student's t test or mann-whitney u test for quantitative data. correlations were performed using the spearman's rank of order. survival was compared by the log-rank test. odds ratios (or) and 95% confidence intervals (cis) for were calculated by the mantel and haenszel's statistics. receiver operating characteristic (roc) curves were analyzed for outcome prediction; the best cut-off was selected using the youden index. stepwise forwards cox regression analysis with hazard ratios (hrs) and confidence intervals (cis) was used to investigate independent variable associated with 28-day outcome. any p value below 0.05 was considered statistically significant. seventy-four patients were enrolled. their demographics in association to 28-day outcome are shown in table 1 and in supplementary table 1 http://links.lww.com/shk/b52. two patients died before day; five patients were discharged before day 7 and four patients denied blood sampling on day 7; therefore measurements on day 7 were run in 63 patients serum h2s of days 1 and 7 was significantly higher among 28-day survivors. il-6 and pct of days 1 and 7 were higher among non-survivors whereas crp of day 7 was higher among non-survivors. h2s was negatively associated with il-6, pctand crp (figure 1 ). copyright © 2020 by the shock society. unauthorized reproduction of this article is prohibited. non-survivors had higher absolute neutrophil counts and significantly lower lymphocyteswhich are consistent with already reported characteristics of covid-19 patients (12) . serum h2s was negatively correlated with the absolute neutrophil count; a positive correlation with the absolute lymphocyte count was found ( figure 2 ). the above-mentioned results led to further evaluation of admission h2s as a marker of survival. following roc curve analysis, it was found that serum levels of h2s on day 1 lower than 150.44μμ had the best trade-off for sensitivity and specificity for death (figure 3aand b ). in total, 49 patients had less or equal and 25 patients had more than 150.44 μm h2s in serum on day 1. mortality after 28 days was 32% and 4.1% respectively ( figure 3c ). the or for death was 11.11 (95% ci: 2.13-5.88, p: 0.001). roc curve analysis revealed the following baseline values to be associated with unfavourable outcome: cci greater or equal to 3, apache ii score greater or equal to 10, psi greater or equal to 113 and sofa score greater or equal to 4. forward stepwise cox regression analysis showed that serum h2s on day 1 above 150.44μm is an independent protective factor for unfavourable outcome of covid-19 even in the presence of severity scores (table 1) . we furtherinvestigated the association between over-time change of serum h2s and outcome. roc curve analysis showed a cutoff decrease of 36% of h2s by day 7 as the best discriminator for death (figures3d and e) . survival of these patients was prolonged ( figure 3f ). moreover, the change of serum h2s between day 1 and 7 was negatively associated with the duration of hospitalization ( figure 3g ). this study suggests the gasotransmitterh2s as a potentially predictive variable of the outcome of pneumonia by sars-cov-2 mainly due to the high negative predictive value. although one interpretation of the presented findings is for the use of serum h2s as biomarker, we do feel that the intrinsic value is for h2s as a reflection of the endothelial function of the body vasculature. the kinetics in the circulation reveal that 28-day survivors are those who consume less of this gas. disturbed bioavailability of h2shas been suggested as an indicator of enhanced pro-inflammatory responses and of endothelial dysfunction (13, 14) . both these conditions often accompany severe covid-19. the negative association between serum h2s and il-6is interesting. il-6 has been proposed as the principle pro-inflammatory cytokine involved in the cytokine storm that leads to severe lung injury, respiratory failure and death by covid-19 (15). this has even copyright © 2020 by the shock society. unauthorized reproduction of this article is prohibited. led to the evaluation of therapeutic strategies of modulation of il-6 production. tocilizumab, a blocker of il-6r, which can effectively block il-6 signal transduction pathway, is currently being evaluated in several clinical studies in covid-19 patients worldwide (16) . our results postulate that h2s is an endogenous down-regulator of il-6, the consumption of which is a driver to unfavorable outcome. it may also lead to considerations for exogenous h2s supplementation as treatment strategy. indeed, inhaled h2s has been shown to reduce proinflammatory cytokines, among which il-6, and increase the survival of mice after experimental endotoxemia (17) . additionally, serum h2s was positively correlated with the lymphocyte count. lymphopenia is a key characteristic of covid-19 patients (12) and is considered a predictor of mortality (15) . the negative association between endogenous h2s and the lymphocyte count may be consistent with in vitro data supporting a role of h2s as t cell activator (18). the significant role of h2s for evaluating covid-19 patients further relies on serial measurements. this study has shown that all patients who sustain elevated levels after 7 days had no risk of an unfavourable outcome. this finding could suggest that serial measurements of serum h2s could be utilized as an adjunctive criterion, together with other established biomarkers such as crp and pct, for decision making.however, these are data coming from a small cohort and mandate validation in larger cohorts of patients. a novel coronavirus from patients with pneumonia in china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china cii: pharmacological modulation of h2s levels: h2s donors and h2s biosynthesis inhibitors hydrogen sulfide signaling in mitochondria and disease sulfur compounds block mcp-1 production by mycoplasma fermentans-infected macrophages through nf-κb inhibition hydrogen sulfide attenuates atherosclerosis in a partially ligated carotid artery mouse model via regulating angiotensin converting enzyme 2 expression survival predictors in elderly patients with acute respiratory distress syndrome: a prospective observational cohort study pneumonia severity index in viral community acquired pneumonia in adults unauthorized reproduction of this article is prohibited analysis of endogenous h2s and h2sn in mouse brain by high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection measurement of plasma hydrogen sulfide in vivo and in vitro clinical characteristics of 138 hospitalized patients with 2019 novel coronavirusinfected pneumonia in wuhan the role of h2s bioavailability in endothelial dysfunction the assessment of endothelial function: from research into clinical practice clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality inhaled hydrogen sulfide prevents endotoxin-induced systemic inflammation and improves survival by altering sulfide metabolism in mice the study has received part funding from the european union's horizon 2020 research and innovation program under the marie skłodowska-curie grant agreement grant european sepsis academy (agreement no 676129) and in part funding by the hellenic institute for the copyright © 2020 by the shock society. unauthorized reproduction of this article is prohibited. key: cord-032240-xswtx940 authors: sauer, françois; dagrenat, charlotte; couppie, philippe; jochum, gaelle; leddet, pierre title: pericardial effusion in patients with covid-19: case series date: 2020-09-09 journal: eur heart j case rep doi: 10.1093/ehjcr/ytaa287 sha: doc_id: 32240 cord_uid: xswtx940 background: sars-coronavirus-2 [coronavirus disease 2019 (covid-19)] infection is a public health issue affecting millions of people. it started in wuhan in china in december 2019 spreading rapidly worldwide. case summary: three patients aged 51–84 developed a pericarditis related to covid-19, associated for two of them with a myocarditis. case 1 was a covid-19 cardiac tamponade without myocarditis, confirmed by a positive chest computed tomography (ct) scan. case 2 showed a covid-19 myopericarditis, confirmed by a positive chest ct scan and a sars-coronavirus-2 positive swab. case 3 was a cardiac tamponade due to covid-19 pericarditis, with a positive polymerase chain reaction on pericardial fluid. they were all treated by colchicine and their condition improved rapidly. discussion: presumably rare, we reported three cases of pericardial effusions (pes) occurring in a single cardiology centre. there is a higher incidence of covid-19-related cardiac diseases such as pericarditis that can manifest as a minimal pe to a cardiac tamponade, which should result in a higher awareness of cardiologists. a systematic measure of the high-sensitivity troponin kinetic in patients affected by covid-19 could be interesting in order to screen for potential myocarditis. any unexplained haemodynamic failure or increased cardiac biomarkers should make the medical team search for myopericarditis by a transthoracic echocardiography. since the outbreak of clusters of viral pneumonia due to the novel coronavirus (severe acute respiratory syndrome coronavirus 2 or sars-cov-2) in wuhan, china in december 2019, 1 coronavirus disease 2019 (covid-19) has spread worldwide infecting more than 5.4 million people and causing more than 349 095 deaths as of 27 may 2020. 2 coronavirus disease 2019 primarily infects the lungs, has demonstrated a wide spectrum of clinical manifestations and may even extend to other organs such as the cardiovascular system. • three cases of pericardial effusions (pes) comprising two tamponades occurred in a single cardiology centre, suggesting a higher cardiac risk than expected during the coronavirus disease 2019 (covid-19) pandemic. • a systematic screening for pe should be performed in covid-19 patients either via a chest computed tomography scan in case of respiratory symptoms, or via transthoracic echocardiography in case of chest pain, haemodynamic instability, or respiratory worsening. mounting evidence is now supporting that covid-19 affects the cardiovascular system with acute cardiac injury, high risk of thrombosis including stroke, pulmonary embolism, and acute coronary syndrome. conversely, very few attention has been paid to pericardial effusion (pe). only very few case reports described pe, revealed by chest pain or a deterioration of general condition. [3] [4] [5] [6] [7] we hereby report a case series of three patients with cardiac and pericardial manifestations of covid-19 at our institution. haguenau hospital is the secondary care centre for the north-east of france, with 482 covid-19 patients admitted to our institution to date. a 51-year-old man presented to our emergency department with chest pain suggestive of pericarditis (retrosternal, intensified when coughing and laying down, eased by sitting up position), dyspnoea on exertion, and deterioration of general condition. he had history of asthma and active smoking. haemodynamic parameters (heart rate and blood pressure) were normal without fever and there was no need for oxygen therapy at the time of admission. the physical examination was normal. c-reactive protein (crp) was markedly increased [peak value 223 mg/lreference range (rr) < 4 mg/l] with leucocytosis [neutrophils: 11.8 g/l (rr < 7 g/l)] and thrombocytosis [platelet count: 402 g/l (rr 140-400 g/l)]. other laboratory data showed an acute kidney injury [urea at 10.1 mmol/l, glomerular filtration rate (gfr) (ckd-epi) 39 ml/min/m 2 )], cholestasis characterized by levels of alkaline phosphatase gamma glutamyltransferase twice the upper limit of normal and arterial blood gas measurements on room air showed an isolated hypoxaemia (po2 of 62 kpa). the high-sensitivity cardiac troponin i (ctni) peak was 919 ng/l (rr < 54 ng/l). the baseline electrocardiogram (ecg) showed a diffuse and discrete elevation of the st segment with a low qrs voltage ( figure 1 ). chest computed tomography (ct) revealed typical findings of covid-19 with moderate peripheral ground-glass opacification and a voluminous pe ( figure 2) . transthoracic echocardiography (tte) confirmed the presence of a significant and circumferential pe (22 mm) with compression of the right heart chambers. following chest ct, the patient presented with acute respiratory failure, requiring oxygen therapy with a high concentration mask (flow rate: 10 l/min). an emergency pericardiocentesis allowed the extraction of 800 ml of a sero-hematic liquid. serologies (human immunodeficiency virus, heptitis b virus, hepatitis c virus, epstein barr virus, cytomegalovirus, adenovirus, picornavirus, parvovirus b19) and reverse transcription polymerase chain reaction (rt-pcr) testing on nasopharyngeal swab for sars-cov-2 were all negative. despite an inconclusive testing for covid-19, the pe was considered as covid-19 related due to (i) typical covid-19 findings at chest ct, (ii) clinical symptoms, and (iii) epidemiological criteria. the histological analysis showed an inflammatory exudate with few lymphocytes and no malignant cells. treatment with colchicine 0.5 mg twice a day was initiated on day 3. on day 7, cardiac magnetic figure 3) . clinical improvement was rapidly observed, and the patient was discharged home on day 7 with maintenance dose of colchicine 1 mg daily for 3 months. at 45-day follow-up, the patient remained asymptomatic. a 60-year-old man, with no medical history except active smoking, had severe ongoing asthenia for a week and acute anosmia. one month later, he had shivers for which an antibiotic therapy by amoxicillin-clavulanic acid was initiated by his general practitioner. two days later, a diffuse erythema appeared. on arrival at the emergency room (er), haemodynamic parameters were stable, the temperature was measured at 39 c and there was no need for oxygen therapy. the physical examination was normal. arterial blood gas measurements showed a moderate hypoxaemia at 75 kpa. ctni peak was 639 ng/l. a nasopharyngeal swab was positive for sars-cov-2 by rt-pcr. baseline ecg registered a normal sinus rhythm and flattened t waves in lateral leads (supplementary material online, figure s1 ). chest ct scan revealed common patterns and distribution of patients affected by covid-19 ( figure 4 ) and a moderate pe localized beside the left ventricle (lv) (<10 mm), later confirmed by tte. treatment with colchicine 0.5 mg twice a day was initiated on day 1. during hospitalization, termination of a new-onset atrial fibrillation was achieved with flecainide. no curative anticoagulation (chads-vasc 0/9) was introduced. the patient's condition rapidly improved with laboratory tests returning to normal. the patient was discharged home on day 5 with maintenance dose of colchicine 1 mg daily for 3 months. control tte on day 8 showed a pe growth with a 15 mm effusion measured around the lv, but no signs of cardiac tamponade and normal left ventricular ejection fraction. the same day, a cardiac mri confirmed a predominant effusion over the lateral wall of the lv ( figure 5 and video 1) and an inferolateral sub-epicardial delayed gadolinium-enhancement, consistent with myocarditis. the treatment remained unchanged as the patient's state was stable. control tte on day 15 showed a decrease in pe (6 mm). at 45day follow-up, the patient remained asymptomatic with no signs of recurrent pericarditis. an 84-year-old woman was referred to our institution for dyspnoea, fever, and severe asthenia. she had history of hypertension and dyslipidaemia. at the er, there was no fever on admission. there was leg swelling and attenuated basal breath sounds. laboratory testing demonstrated an inflammatory response with peak crp value 66 mg/l, leucocytosis (neutrophils: 11.1. g/l), lymphopenia (0.92 g/l), and thrombocytopenia (platelet count: 129 g/l). arterial blood gas measurements on room air showed an isolated hypoxaemia (pao2 62 kpa). chest ct revealed a large and bilateral pleural effusion, no lung findings for covid-19 but a pe ( figure 6 ). on tte, pe was circumferential and initially measured at 7 mm. a thoracentesis was achieved on day 2 and yielded 400 ml of a serous fluid. the histological analysis showed an inflammatory exudate with lymphocytes and a nasopharyngeal swab was negative for sars-cov-2 by rt-pcr. of note, covid-19 testing was not performed in pleural fluid. treatment with colchicine 0.5 mg once a day was initiated on day 1 and patient was discharged home on day 8. on day 15, control tte showed a large pe measured at 25 mm with right ventricular diastolic collapse (figure 7 and videos 2 and 3). thrombocytopenia (123 g/l) and lymphopenia (0.82 g/l) persisted without inflammatory syndrome. n-terminal prohormone of brain natriuretic peptide and ctni remained within normal laboratory range. a pericardiocentesis extracted 500 ml of serous fluid. rt-pcr testing on pericardial fluid for sars-cov-2 was positive. likewise, the anatomopathological technique revealed an inflammatory exudate. a chest-abdomen-pelvis scan was carried out on day 17 and showed no lung affection by covid-19 and no tumoural lesion. on day 37, a cardiac mri showed a mild pe without right heart chambers compression nor myocarditis (figure 8) . at day 45, the patient's condition subsequently improved. we report three cases of covid-19-related pe of which two of them were complicated by cardiac tamponade. differential diagnosis cannot be formally excluded (other viral infections, malignant disease, etc.). each case involved either a positive covid-19 rt-pcr and/or typical ct findings of covid-19. after careful literature review, we found five cases of cardiac tamponade requiring emergency pericardial drainage in italy, 3 in the usa, 4-6 and in the uk 7 . one of them was associated with a positive rt-pcr for covid-19 in the pericardial fluid. four cases of myopericarditis have been described. especially in italy, 8 a case of myopericarditis required dobutamine support for 2 days, antivirals and corticosteroids, but no pericardial drainage. pericardial involvement in covid-19 seems to be rare but remains unquantified to date. publications on radiological data highlight infrequent pes. 9 the pathogenesis of covid-19 myopericarditis is yet unresolved. two predominant mechanisms could be relevant. 10 first, the heart affinity of the virus could be explained by sars-cov-2 s protein direct binding to human angiotensin-converting enzyme 2 11 present in the human heart, which allows for a cellular infection. indirectly, myopericarditis could follow a viral replication and dissemination in the blood, from day 7 up to 1 month after symptoms beginning. this could lead to a cytokine storm syndrome and a direct myopericardial lesion by inflammatory cell infiltration, similarly to covid-19 direct pulmonary lesions. 12 so far, physicians treating patients with covid-19 are facing a lack of solid evidence and guidelines regarding the management of covid-19-related pericardial disease. non-steroidal anti-inflammatory drugs should not be introduced considering the risk of the respiratory worsening. various studies 13 have proposed a potential beneficial effect of corticosteroids as a treatment during cytokine storm syndrome. colchicine is a well-known and safe therapy for pericarditis 14 and seems to be an interesting treatment option in covid-19 affections, as suggested in ongoing prospective studies 15 (colcorona nct04322682), due to its action on nlrp3 inflammasomes and cytokine's release. no coronary circulation assessment was done in each case, because of the low probability of acute coronary syndrome. moreover, each patient had a cardiac mri showing no ischaemic injury. presumably rare, we reported three cases of pe occurring at our institution. there is a higher incidence of covid-19-related cardiac diseases such as pericarditis that can manifest from minimal pe to cardiac tamponade. cardiologists and emergency physicians should be aware and extensively look for pe at the time of the covid-19 outbreak. françois sauer is a third-year resident in cardiology, who aims to specialize himself in cardiac imaging and sports cardiology. he lives in strasbourg (france). supplementary material is available at european heart journal -case reports online. slide sets: a fully edited slide set detailing this case and suitable for local presentation is available online as supplementary data. the author/s confirm that written consent for submission and publication of this case report including image(s) and associated text has been obtained from the patient in line with cope guidance. conflict of interest: none declared. world heart organization. coronavirus disease (covid-2019) situation china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china sars-cov-2 detection in the pericardial fluid of a patient with cardiac tamponade cardiac tamponade secondary to covid-19 acute myopericarditis with pericardial effusion and cardiac tamponade in a patient with covid-19 acute pericarditis and cardiac tamponade in a patient with covid-19: a therapeutic challenge life-threatening cardiac tamponade complicating myo-pericarditis in covid-19 cardiac involvement in a patient with coronavirus disease 2019 (covid-19) coronavirus disease 2019 (covid-19): a systematic review of imaging findings in 919 patients end-stage heart failure with covid-19: strong evidence of myocardial injury by 2019-ncov virology, epidemiology, pathogenesis, and control of covid-19 pathological findings of covid-19 associated with acute respiratory distress syndrome covid-19 for the cardiologist: a current review of the virology, clinical epidemiology, cardiac and other clinical manifestations and potential therapeutic strategies icap investigators. a randomized trial of colchicine for acute pericarditis the greek study in the effects of colchicine in covid-19 complications prevention (grecco-19 study): rationale and study design key: cord-006344-de4dhv4b authors: seitsonen, e.; hynninen, m.; kolho, e.; kallio-kokko, h.; pettilä, v. title: corticosteroids combined with continuous veno-venous hemodiafiltration for treatment of hantavirus pulmonary syndrome caused by puumala virus infection date: 2006-03-21 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-006-0117-z sha: doc_id: 6344 cord_uid: de4dhv4b reported here are two cases of hantavirus pulmonary syndrome caused by puumala virus infection, which rapidly resolved after initiation of corticosteroid treatment combined with continuous veno-venous hemodiafiltration. these cases emphasize the role of the inflammatory response in the pathogenesis of hantavirus pulmonary syndrome. puumala virus (puu) is a hantavirus that is endemic in northwestern europe, the balkans, and western russia [1] . it is carried and spread by the bank vole. puumala virus infection is usually either asymptomatic or causes a mild form of hemorrhagic fever with renal syndrome. the incidence in finland is 19/100,000. the mortality rate is low (0.1-0.4%) [1] . the most common signs and symptoms are fever, headache, myalgia, back pain and gastrointestinal symptoms, often followed by an oliguric phase with proteinuria and hematuria, and later a polyuric phase. transient visual impairment (myopia) is a pathognomonic sign. hemorrhagic manifestations are present in about 10% of cases. however, mild mucosal bleeding is very common [1] . spontaneous recovery usually takes place in 2-3 weeks [2] . of the hospital-treated patients, about 5% require transient hemodialysis [1] , and over 20% have pulmonary abnormalities. most often these relate to both the inflammation-associated capillary permeability disorder and fluid retention caused by renal failure [3] . however, a few cases of acute non-cardiogenic pulmonary edema, similar to the hantavirus pulmonary syndrome (hps) encountered on the american continent, have been described in europe in association with puu and other hantavirus species [4] [5] [6] [7] . we describe two cases of puu-associated hps, in which administration of intravenous corticosteroids combined with continuous veno-venous hemodiafiltration (cvvhdf) was followed by rapid clinical improvement. both patients gave their consent to publication of the data. the first day of fever is defined as day 0 post-onset of symptoms (pos). a 72-year-old male patient was admitted to a district hospital in southern finland in february 2003 with a 3-day history of fever, upper abdominal pain and melena. an infectious infiltrate was suspected in the right lung, and an enlarged spleen was detected on abdominal ultrasound. creactive protein (crp) level was 94 mg/l, platelet count 84×10 9 /l, leukocyte count 7.1×10 9 /l, and hematocrit 37%. the qualitative urinalysis showed proteinuria. intravenous antimicrobial agents were started for suspected community-acquired pneumonia. urine output began to decrease, and serum creatinine increased from 89 to 295 μmol/l. two blood cultures on consecutive days were negative. the patient became hypotensive, and oxygenation deteriorated. the patient was transferred to a university hospital on day 7 pos. echocardiography showed normal left ventricular function. the patient needed continuous ventilation with a positive airway pressure mask and 70% inspiratory oxygen fraction, and he was transferred to the intensive care unit (icu). diffuse bilateral perihilar infiltrates were present on chest radiograph (fig. 1) . crp level was 77 mg/ l, hematocrit 50%, platelet count 130×10 9 /l, leukocyte count 46×10 9 /l, creatinine 463 μmol/l, blood urea nitrogen 26.9 mmol/l, potassium 5.5 mmol/l, and lactate 3.9 mmol/l. troponin-t and creatine-kinase mb mass were within normal limits. due to poor oxygenation and oliguria, the patient required endotracheal intubation and cvvhdf. initially, the ratio of arterial oxygen tension to inspired oxygen fraction (pao 2 /fio 2 ratio) was 100 mmhg, and intermittent prone positioning was used for 3 days to improve gas exchange. ceftriaxone (2 g/day) and fluconazole (200 mg/day) were given as antimicrobial treatment. the blood morphological examination showed both neutrophilia and lymphocytosis, including atypical lymphocytes, and a variable degree of maturation. on day 8 pos, puu-igm was positive by μ-capture enzyme-linked immunosorbent assay based on baculovirus-expressed rn-antigen [8, 9] . puu-igg was detected with fluorescein isothiocyanate-conjugated sheep antihuman igg, and demonstrated a granular fluorescence pattern associated with antibodies against the puu nucleocapsid protein, typical of a recent infection [10, 11] . on day 10 pos, the pao 2 /fio 2 ratio was still less than 200 mmhg with 14 mmhg pulmonary artery occlusion pressure, and intravenous methylprednisolone (120 mg/day in three doses) was started. on the following days, the pao 2 /fio 2 ratio rapidly stabilized at over 300 mmhg. the evolution of the pao 2 /fio 2 ratio and the lung injury score is shown in fig. 2a ,b. the patient required vasoactive support for 9 days. the minimum urine output was 65 ml on day 11 pos. cvvhdf was discontinued on day 16 pos. the calculated dialysis and total fluid balances are shown in table 1 . the lowest platelet count was 59×10 9 /l on day 14 pos, the highest leukocyte count 50.2×10 9 /l on day 10 pos, and the highest crp 97 mg/l on day 11 pos. the evolution of crp values and platelet counts is shown in table 2 . blood cultures taken on days 7 and 10 pos were negative. the patient was extubated on day 18 pos, and discharged from the icu on day 21 pos. methylprednisolone was tapered off and switched to oral prednisone. on day 22 pos, left laterobasal infiltration was still visible on chest radiograph. on that day, proteinuria was 0.6 g/24 h. the levels of c3 and c4 were normal, and there was no indication of c4 subtype insufficiency. the patient's hla genotype was b7 drb1*15. the patient confirmed there were signs of vole infestation in the yard and shed of his home. he had not recently traveled abroad. a 34-year-old male patient came to a university hospital in southern finland in august 2004 for treatment of upper abdominal pain. his symptoms had started 3 days previously with high fever, fatigue, and myalgia. he had vomited once and had diarrhea in which he detected traces of blood. on day 4 pos, his crp level was 245 mg/l, platelet count 46×10 9 /l, leukocyte count 9.8×10 9 /l, hematocrit 45%, sodium 126 mmol/l, and alanine aminotransferase 86 u/l. the chest radiograph showed bilateral perihilar infiltrates, and the patient was started on ceftriaxone (2 g/day) and levofloxacin (1 g/day). he received supplemental oxygen via nasal cannulae. abdominal radiograph and ultrasound examinations were normal. on day 5 pos the lung infiltrates had increased considerably (fig. 3a,b) , and the patient required continuous ventilation with a positive airway pressure mask. he became oliguric, proteinuria 1.7 g/24 h was detected, and his creatinine level increased from 87 to 255 μmol/l. the patient's platelet count decreased to 40×10 9 /l, and he experienced visual disturbances. he confirmed that 18 days before the onset of symptoms he had visited a summer cottage, where exposure to vole secretions was possible. he had not recently traveled abroad. the patient was transferred to the icu due to respiratory insufficiency and renal failure. oxygenation deteriorated rapidly, and he required endotracheal intubation and mechanical ventilation. the lowest pao 2 /fio 2 ratio was 63 mmhg on day 6 pos, and intermittent prone positioning was continued until day 11 pos. the patient required a low dose of vasopressor support for 7 days. on day 5 pos, puu-igg-and puu-igm-antibodies were positive by the same methods described for case 1, and igg-ifa showed the typical granular fluorescence pattern seen in the acute phase of immunity. metabolic acidosis was detected, the patient's creatinine level increased to 412 μmol/l and blood urea nitrogen to 23.8 mmol/l. cvvhdf was started on day 7 pos. as the patient was severely ill, and a concomitant bacterial infection could not be ruled out, antibiotics were continued until crp began to decrease on day 9 pos. the evolution of crp values and platelet counts is shown in table 2 . blood cultures taken on days 5 and 9 pos were negative. on day 9 pos the pao 2 /fio 2 ratio was still 100-150 mmhg with 16 mmhg pulmonary artery occlusion pressure, and intravenous methylprednisolone (120 mg/day in three doses) was started. the evolution of the pao 2 /fio 2 ratio and the lung injury score is shown in fig 2a,b . the minimum urine output was 80 ml on day 8 pos. cvvhdf was discontinued on day 11 pos. the calculated dialysis and total fluid balances are shown in table 1 . the patient was extubated on day 12 pos. he was discharged from the icu on day 15 pos. on that day, left mediobasal and right basal atelectasis and slight bilateral pleural effusion were visible on chest radiograph. the methylprednisolone dose was tapered, and an oral preparation was substituted on day 16 pos. the patient was discharged home on day 18 pos. the patient lacked one of the c4b-type genes. his hla genotype was b13,35 drb1*01,07. we describe two cases of puu-infected patients who presented with both renal and respiratory failure requiring renal replacement therapy and mechanical ventilation. radiologically, both patients had alveolar infiltrates separate from pulmonary vascular congestion, and no prominent cardiomegaly. nevertheless, fluid inevitably accumulated in the lung during the disease process. most likely, both the corticosteroid treatment and the negative fluid balance achieved during cvvhdf treatment and recovery of renal values in bold type indicate the day when corticosteroid treatment was started (days 10 and 9 pos for cases 1 and 2, respectively) function contributed to the subsequent improvement in gas exchange. in hps, a febrile prodrome is followed by a severe increase in pulmonary vascular permeability and myocardial depression. the syndrome progresses very rapidly from interstitial pulmonary edema to a clinical picture resembling adult respiratory distress syndrome (ards). the alveolar infiltrates are typically central or bibasilar, and pleural effusion is common [12] . the hantaviruses are not cytopathic, and the increased capillary permeability is likely caused by immunologic factors [13] . in pathological specimens of hps-afflicted lungs, intra-alveolar edema and interstitial lymphoid cell infiltration are observed, the pneumocytes are preserved, and neutrophils are notably scarce [14, 15] . the endothelial cells are also largely intact, and the alveolar walls are thickened due to extravasation [16] . activation of t-lymphocytes and cytokine production appear to be involved in both hps and hemorrhagic fever with renal syndrome [13, 17] . terajima et al. [18] demon-strated that at the onset of pulmonary edema, hps patients have abundant viremia which clears rapidly after the resolution of fever, while respiratory distress may persist. the increase in alveolocapillary permeability in acute lung injury results from the inflammatory response [19, 20] . cortisol acts in concert with catecholamines to maintain vascular tone and endothelial integrity, and it suppresses the inflammatory cascade by down-regulating the production of proinflammatory molecules [21] . animal studies on the efficacy of corticosteroids in acute lung injury have produced mixed results [22, 23] . the american-european consensus conference on ards stated that corticosteroids are not generally useful in the early management of sepsis and ards [19] . early or prophylactic use of high-dose corticosteroid therapy in septicemia and ards may even be harmful [24] . the consensus conference acknowledged, however, that corticosteroids may be of value in treating certain ards variants [19] . currently, there is no universally accepted drug treatment for the high permeability edema associated with acute lung injury/ards in humans [20] . an overaggressive inflammatory response may be an important factor in the outcome of ards patients [25] . late corticosteroid treatment in sustained lung injury has been shown to inhibit proinflammatory activity and to improve lung function and possibly outcome [26] . in the series of seven patients with puu-associated hps described by clement et al. [4] , only one patient required mechanical ventilation. the course of mechanical ventilation was prolonged (20 days), but the patient made a full recovery. the treatment of the european hps cases described by launay et al. [5] and klempa et al. [7] was also mainly supportive (i.e., no mechanical ventilation was required), and the patients recovered without sequelae. the patient with dobrava virus-associated hps reported by schütt et al. [6] received both cvvhdf and prolonged mechanical ventilation (25 days), and eventually recovered. in a series of 16 andes virus-associated hps cases in chile, five patients, all of whom were among the survivors, received corticosteroid treatment along with supportive treatment, but the efficacy of corticosteroid treatment could not be determined due to the low number of cases [27] . in the series of 14 hps cases described by hallin et al. [15] , antimicrobial and supportive treatment was given. the two table 2 platelet counts (pc, ×10 9 /l) and c-reactive protein values (crp, mg/l) in cases 1 and 2 on consecutive days pos during the period of icu treatment (days 7-21 pos and 5-15 pos for cases 1 and 2, respectively) survivors who required mechanical ventilation were extubated after 5 days and made a full recovery. renal histology in nephropathia epidemica is characterized by tubulointerstitial nephritis with mixed interstitial cellular infiltration and edema, tubular epithelial changes and occasional medullar hemorrhages [28] . in a recent retrospective survey of mainly drug-induced acute interstitial nephritis, no effect of corticosteroid treatment on renal recovery was found as compared with supportive therapy alone [29] . the haplotype hlab8dr3dq2 is associated with a severe form of nephropathia epidemica [30] . this haplotype is also associated with a high level of tnf-α production [31] and deletion of the c4a gene [30] . the hlab8dr3 haplotype is known to be associated with several chronic autoimmune diseases and immunologic abnormalities. neither of our patients had that hla haplotype, but one of them lacked one of the c4b genes, leading to lowered levels of c4b and possibly altered immune function. as far as we know, this report is the first to describe the effects of corticosteroid treatment for puu infection complicated by severe hypoxemia, consistent with the ards criteria. in our two patients, the implementation of intravenous corticosteroids combined with cvvhdf was associated with resolution of both respiratory failure and shock. we suggest that corticosteroid therapy is safe and may hasten recovery in severe cardiopulmonary forms of puu infection. hantavirus infections in europe pulmonary involvement in nephropathia epidemica: radiological findings and their clinical correlations hantavirus pulmonary syndrome in new england and europe pulmonary-renal syndrome due to hemorrhagic fever with renal syndrome: an unusual manifestation of puumala virus infection in france life-threatening dobrava hantavirus infection with unusually extended pulmonary involvement occurrence of renal and pulmonary syndrome in a region of northeast germany where tula hantavirus circulates antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells evaluation of puumala virus igg and igm enzyme immunoassays based on recombinant baculovirus-expressed nucleocapsid protein for early nephropathia epidemica diagnosis human b-cell epitopes of puumala virus nucleocapsid protein, the major antigen in early response human immune response to puumala virus glycoproteins and nucleocapsid protein expressed in mammalian cells viral pneumonias in adults: radiologic and pathologic findings spectrum of hantavirus infection: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. the hantavirus study group cardiopulmonary manifestations of hantavirus pulmonary syndrome hantavirus pulmonary syndrome: radiographic findings in 16 patients high levels of cytokineproducing cells in the lung tissues of patients with fatal hantavirus pulmonary syndrome high levels of viremia in patients with the hantavirus pulmonary syndrome ventilatory, pharmacologic, supportive therapy, study design strategies and issues related to recovery and remodeling vascular pharmacology of acute lung injury and acute respiratory distress syndrome anti-inflammatory therapy for acute lung injury. a review of animal and clinical studies dexamethasone and pentastarch produce additive attenuation of ischaemia/ reperfusion lung injury early methylprednisolone treatment for septic syndrome and the adult respiratory distress syndrome persistent elevation of inflammatory cytokines predicts a poor outcome in ards. plasma il-1β and il-6 levels are consistent and efficient predictors of outcome over time effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome. a randomized controlled trial hantavirus pulmonary syndrome due to andes virus in temuco, chile: clinical experience with 16 adults cytokines, adhesion molecules, and cellular infiltration in nephropathia epidemica kidneys: an immunohistochemical study acute interstitial nephritis: clinical features and response to corticosteroid therapy genetic susceptibility to severe course of nephropathia epidemica caused by puumala hantavirus highproducer allele of tumour necrosis factor-alpha is part of the susceptibility mhc haplotype in severe puumala virus-induced nephropathia epidemica acknowledgement this report was supported by a hus research grant. key: cord-000097-vueo83vk authors: beretta, chiara; leoni, veronica; rossi, mario renato; jankovic, momcilo; patroniti, nicolo; foti, giuseppe; biagi, ettore title: prolonged extracorporeal membrane oxygenation therapy for severe acute respiratory distress syndrome in a child affected by rituximab-resistant autoimmune hemolytic anemia: a case report date: 2009-04-01 journal: j med case reports doi: 10.1186/1752-1947-3-6443 sha: doc_id: 97 cord_uid: vueo83vk introduction: autoimmune hemolytic anemia in children younger than 2 years of age is usually characterized by a severe course, with a mortality rate of approximately 10%. the prolonged immunosuppression following specific treatment may be associated with a high risk of developing severe infections. recently, the use of monoclonal antibodies (rituximab) has allowed sustained remissions to be obtained in the majority of pediatric patients with refractory autoimmune hemolytic anemia. case presentation: we describe the case of an 8-month-old caucasian girl affected by a severe form of autoimmune hemolytic anemia, which required continuous steroid treatment for 16 months. thereafter, she received 4 weekly doses of rituximab (375 mg/m(2)/dose) associated with steroid therapy, which was then tapered over the subsequent 2 weeks. one month after the last dose of rrituximab, she presented with recurrence of severe hemolysis and received two more doses of rrituximab. the patient remained in clinical remission for 7 months, before presenting with a further relapse. an alternative heavy immunosuppressive therapy was administered combining cyclophosphamide 10 mg/kg/day for 10 days with methylprednisolone 40 mg/kg/day for 5 days, which was then tapered down over 3 weeks. while still on steroid therapy, the patient developed an interstitial pneumonia with acute respiratory distress syndrome, which required immediate admission to the intensive care unit where extracorporeal membrane oxygenation therapy was administered continuously for 37 days. at 16-month follow-up, the patient is alive and in good clinical condition, with no organ dysfunction, free from any immunosuppressive treatment and with a normal hb level. conclusions: this case shows that aggressive combined immunosuppressive therapy may lead to a sustained complete remission in children with refractory autoimmune hemolytic anemia. however, the severe life-threatening complication presented by our patient indicates that strict clinical monitoring must be vigilantly performed, that antimicrobial prophylaxis should always be considered and that experienced medical and nursing staff must be available, to deliver highly specialized supportive salvage therapies, if necessary, during intensive care monitoring. autoimmune hemolytic anemia (aiha) in children is usually characterized by a severe course with a mortality rate of approximately 10% [1] . the required prolonged immunosuppressive therapy often leads to steroid dependence [2] . the administration of non-steroidal immunosuppressive drugs such as cyclosporine a, cyclophosphamide and azathioprine, has been used in the past [1] [2] [3] [4] . nowadays, the use of monoclonal antibodies such as rituximab, has given promising results for pediatric refractory aiha [5] [6] [7] , with sustained remissions in the majority of patients. nevertheless, potentially life-threatening infections are known to occur with rituximab [7] . in the event of rituximab failure, there is no general consensus or guidelines available indicating precisely how to manage resistant forms of aiha. heavy immunosuppression consisting of the combined use of cyclophosphamide and high-dose steroids may be considered [8, 9] . we report the case of an 8-month-old caucasian girl referred to us for observation due to intense pallor, jaundice, lethargy and fever. serological evaluations revealed severe anemia (hb = 2.8g/dl) with a strongly positive direct antiglobulin test and high-titer warm igg autoantibody. aiha was diagnosed and steroid therapy with intravenous methylprednisolone at 2mg/kg/day was administered for 5 days (figure 1 ). an adequate hb increase was obtained and the child was discharged after 10 days with oral prednisone at 2mg/kg/day. during the subsequent months, several attempts were made to taper off the prednisone, but the patient had developed steroid dependence. considering this dependence on high steroid doses, a therapeutic course with four doses of rituximab was performed (375mg/m 2 /dose) at weekly intervals ( figure 1 ). before rituximab infusion, serum immunoglobulin levels were normal and subpopulation lymphocyte counts were within the normal range. the treatment with rituximab was well tolerated and the patient received intravenous substitutive therapy with commercially available immunoglobulin preparations (400mg/kg, every 3 weeks for 6 months). one month after the end of the first course of rituximab, while still receiving low-dose steroids, the patient presented with a clinical relapse of aiha, so prednisone was increased to 2mg/kg/day and two further rituximab infusions were performed ( figure 1 ). after these infusions, b lymphocytes became undetectable and the count returned to normal values 8 months after treatment. the patient remained in clinical remission and free from immunosuppressive drugs for 7 months, before presenting with a further relapse. a more intensive treatment was performed ( figure 1 ) with cyclophosphamide 10mg/kg/ day for 10 days and methylprednisolone 40mg/kg/day for 5 days, which was tapered over 20 days. hb level increased and the patient was discharged 10 days later in good clinical condition, without any antifungal or antiviral prophylaxis. two weeks later, the child was referred to the emergency room for respiratory failure, persistent fever and abdominal pain. laboratory examination showed an hb level of 12.8g/dl, total leukocyte count (wbc) of 710/µl, absolute neutrophil count (anc) of 90/µl, a platelet count (plt) of 339,000/µl, and low levels of immunoglobulin (igg = 360mg/dl, iga = 10mg/dl, igm = 33mg/ dl). chest x-ray and ct scan revealed an interstitial pneumonia ( figure 2 ). therapy with amikacin, ceftazidime, g-csf and voriconazole was started. within a few hours, her clinical condition deteriorated and the patient developed acute respiratory distress syndrome (ards), which required immediate admission to the intensive care unit (icu). acceptable gas exchange was initially maintained by non-invasive continuous positive airway pressure ( figure 3 ). serologic tests showed a level of aspergillus galactomannan antigen of 0.8. all tested virus and microbial antigens were negative. on day 4, concomitantly with an elevation of wbc from 400 to 10,400/µl (anc = 4900/µl), respiratory conditions precipitated and endotracheal intubation and mechanical ventilation were started ( figure 3) . a protective ventilatory strategy with tidal volume of 6ml/kg and positive end expiratory pressure (peep) of 10cmh 2 o was instituted. in the following days, gas exchange deteriorated and peep levels rose to 17cmh 2 o. recruitment manoeuvres, prone positioning, and high doses of inhaled nitric oxide (noi) were necessary to maintain viable gas exchange. endotracheal instillation of porcine surfactant and a trial with high frequency oscillation were ineffective. on day 10, owing to the refractory hypoxia, worsening hypercapnia, and chest x-ray evidence of a pneumomediastinum, the patient was placed on venous-venous extracorporeal membrane oxygenation (ecmo) (figure 3) . a double lumen 15 french catheter (maquet, jostra medizintechnik ag, hirrlingen, germany) was inserted into the right internal jugular vein. the ecmo circuit consisted of a polymethylpentene membrane oxygenator, permanent life support and a centrifugal rotaflow pump (maquet, jostra medizintechnik ag, hirrlingen, germany). ecmo was started with a blood flow of 0.8 to 0.9 l/minute and gas flow of 1 l/minute of 100% oxygen. following the institution of ecmo, respiratory rate decreased from 45 to 10 breaths/minute, and it was possible to stop noi. after commencing caspofungin with voriconazole, wbc, anc and c reactive protein (crp) slowly decreased, while pulmonary function slightly improved. on day 19, a multidrug-resistant pseudomonas aeruginosa was isolated from bronchoaspirate ( figure 3 ). in spite of antibiotic reinforcement with levofloxacin, pseudomonas aeruginosa antibiogram showed increased resistance to all antibiotics and to colimicine which was started on day 26. on day 28, a sudden increase in resistance on the return part of the circuit caused a massive thrombosis in the oxygenator. the entire circuit and the cannula were immediately changed and ecmo restarted within 2 hours. following the development of pulmonary embolism, the gas exchange rapidly worsened and noi had to be restarted. an echocardiographic assessment showed right ventricular dilatation, with paradoxical septal wall motion and pulmonary hypertension (systolic pressure 90mmhg). prostacyclin and sildenafil improved the right heart function and effectively attenuated pulmonary hypertension. in the following days, wbc, anc and crp slowly decreased, while pulmonary function improved. thirty days from icu admission, ecmo was stopped, the patient rapidly restored her spontaneous ventilatory functions and she was extubated 10 days later. she was finally discharged from the icu on day 44. at 16-month follow-up, the patient is alive and free from immunosuppressive drugs. at the time of last follow-up, hb level was 13.5g/dl, reticulocyte count was 11 × 10 9 /l, with total bilirubin, lactic dehydrogenase and haptoglobin all within the normal ranges. aiha in children younger than 2 years is in some cases characterized by a resistance to corticosteroids or dependence on high steroid doses and subsequent development of severe side effects [2] . splenectomy or immunomodulating agents have frequently been used, but there is no consistent demonstration of their efficacy in controlling hemolysis [3] . immunosuppressive drugs such as azathioprine, cyclosporine a, or cyclophosphamide, alone or in combination reduce steroid dependence and sometimes control hemolysis [1] [2] [3] [4] . clinical experience with monoclonal antibodies appears encouraging. in particular, rituximab is increasingly being used off-label, for difficult-to-treat auto-immune diseases and presents the advantage of inducing a selective b-cell depletion, sparing cellular immunity mediated by t cells and natural killer cells. even though prospective controlled studies are not currently available, the efficacy of rituximab has been shown in pediatric studies. quartier et al. [5] treated five pediatric refractory aiha patients, who achieved a complete remission within 15 to 22 months after rituximab therapy. these results were confirmed by zecca et al. [6] in a group of 15 children treated with rituximab. four other children were treated by motto et al. [7] , with the achievement of complete remission. nevertheless, the prolonged impairment of antibody production leads to an increased risk of viral and bacterial infections. for this reason, monthly intravenous immunoglobulin infusions are recommended for a minimum of 6 months following completion of therapy and prophylaxis for p. jirovecii pneumonia is also suggested [7] . the patient described in our report received four rituximab infusions in an off-label setting, followed by two additional doses over 6 months. clinical remission was achieved for 7 months after which it was possible to interrupt steroid treatment. the pattern of immune reconstitution after rituximab therapy revealed persistently low immunoglobulin levels, partially corrected by the substitutive therapy. immunoglobulin levels reached their normal range in 18 months and the lymphocyte subpopulations returned to normal range in 16 months. it is, nevertheless, difficult to quantify the real role of rituximab in this heavy immunosuppression, since a combined therapy with high doses of methylprednisolone and cyclophosphamide was subsequently started. even though our patient did not present any early side effects related to the rituximab infusions, a prolonged follow-up should be carried out to monitor and prevent long-term side effects of rituximab, which are still unknown. when our patient relapsed, an alternative treatment was required, since therapies with steroids, rituximab and intravenous immunoglobulins proved to be ineffective. the role of splenectomy in refractory aiha is still controversial [1] [2] [3] [4] . although effective vaccinations are available, this surgical treatment should be avoided in children younger than 6 years of age, due to the risk of developing severe bacterial infections. according to local policy, drug-based immunosuppressive therapy is to be preferred. we therefore decided to adopt a combined therapy approach, with high doses of methylprednisolone (40mg/kg/day for 5 consecutive days), which was then tapered down over 20 days, and cyclophosphamide (10mg/kg/day for 10 consecutive days). this approach appeared to be feasible and encouraging, since we had previously successfully treated two pediatric cases of refractory aiha with an identical approach [8, 9] . the administration of methylprednisolone and cyclophosphamide increased the already significant immunodepression which had resulted from prior therapies and further contributed to the severity of the infectious complication presented by our patient that required ecmo therapy. while in the icu, the patient underwent various ventilatory treatments, some of which are not considered conventional. modern ventilatory strategy in ards aims to provide viable gas exchange with high oxygen concentration and peep, while minimizing the injurious effects of mechanical ventilation by using low tidal volume ventilation (6ml/kg) [10] . although other techniques such as the prone position, noi and recruitment manoeuvres are effective in improving gas exchange, they did not prove effective in terms of survival [10] . nevertheless, before ecmo, the only means of providing minimal acceptable oxygenation was to use both noi and the prone position. despite using low tidal volumes, a respiratory rate of up to 45 breaths/minute was necessary to obtain acceptable co 2 levels, and the occurrence of pneumomediastinum demonstrated that we were unable to provide an effective protective ventilatory strategy. thus, ecmo was the only real means of providing such a strategy, while allowing adequate gas exchange. refractory aiha in pediatric patients is a challenging disease that forces us to weigh up the risks and benefits of heavy and prolonged immunosuppressive therapies that can reduce or even eradicate the hemolysis, despite the risk of infectious complications. for this reason, we feel that prolonged viral and fungal prophylaxis therapy should always be considered, during and after the immunosuppressive therapy. furthermore, strict clinical monitoring should be carried out, even when no evident symptoms are present. in our patient, we did not administer any prophylaxis and clinical monitoring was probably delayed for too long after discharge. resolution of the infectious complication was possible thanks to an advanced intensive care assistance, which consisted of ecmo and the management of its related complications. this case study shows that rituximab-resistant aiha in young children represents a significant challenge, requiring aggressive immunosuppressive therapy, which may potentially cause severe life-threatening complications. nowadays, it is not clear which is the best immunosuppressive agent to be administered in the event of rituximab failure. we found that the combination of methylprednisolone and cyclophosphamide could be a valid alternative, based on previous experience. nevertheless, a universal therapeutic flow-chart is still lacking and should be defined, which considers new therapeutic strategies such as alemtuzumab [11] or hematopoietic stem cell transplantation [12] . what is clear, however, in the case of heavy immunosuppressive therapy, is the importance of strict patient monitoring during and after immunosuppressive therapy and an antimicrobial prophylaxis, particularly for fungal agents and p. jirovecii. aiha, autoimmune hemolytic anemia; anc, absolute neutrophil count; ct, computed tomography; wbc, leukocyte count; plt, platelet count; ards, acute respiratory distress syndrome; icu, intensive care unit; peep, positive end expiratory pressure; noi, inhaled nitric oxide; ecmo, extracorporeal membrane oxygenation; crp, c reactive protein. autoimmune hemolytic anemia. in nathan and oski's hematology of infancy and childhood factors influencing prognosis in childhood autoimmune hemolytic anemia management of autoimmune hemolytic anemias highdose cyclophosphamide for refractory autoimmune hemolytic anemia treatment of childhood autoimmune haemolytic anaemia with rituximab rituximab for the treatment of refractory autoimmune hemolytic anemia in children rituximab for refractory childhood autoimmune hemolytic anemia a persistent severe autoimmune hemolytic anemia despite apparent direct antiglobulin test negativization successful use of high-dose cyclophosphamide in a child with severe autoimmune hemolytic anemia mechanical ventilation in ards: a state-of-the-art review alemtuzumab induced complete remission of autoimmune hemolytic anemia refractory to corticosteroids, splenectomy and rituximab two-step immunoablative treatment with autologous peripheral blood cd34(+) cell transplantation in an 8-year-old boy with autoimmune haemolytic anaemia written informed consent was obtained from the patient's parents for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. the authors declare that they have no competing interests. cb was the major contributor in collecting the patient's data and writing the manuscript. she gave final approval of the version to be published. vl made a substantial contribution in the data-analysis and interpretation, and has been involved in drafting the manuscript. she gave final approval of the version to be published. mrr was the major contributor in conception of the manuscript; he also revised the manuscript critically for important intellectual content. he gave final approval of the version to be published. mj made a substantial contribution in the manuscript drafting and in revising it critically for important intellectual content. he gave final approval of the version to be published. np was a contributor in acquisition of patient's data during the icu admission and has been involved in drafting the manuscript for the part concerning the icu admission. he gave final approval of the version to be published. gf made a substantial contribution in analysing and interpreting the patient's data during the icu admission and has been involved in drafting the manuscript for the part concerning the icu admission. he gave final approval of the version to be published. eb took direct medical care of the patient and was the major contributor in revising the manuscript critically for important intellectual content. he gave final approval of the version to be published key: cord-265891-jmpterrj authors: eilersen, andreas; sneppen, kim title: cost–benefit of limited isolation and testing in covid-19 mitigation date: 2020-10-29 journal: sci rep doi: 10.1038/s41598-020-75640-2 sha: doc_id: 265891 cord_uid: jmpterrj the international community has been put in an unprecedented situation by the covid-19 pandemic. creating models to describe and quantify alternative mitigation strategies becomes increasingly urgent. in this study, we propose an agent-based model of disease transmission in a society divided into closely connected families, workplaces, and social groups. this allows us to discuss mitigation strategies, including targeted quarantine measures. we find that workplace and more diffuse social contacts are roughly equally important to disease spread, and that an effective lockdown must target both. we examine the cost–benefit of replacing a lockdown with tracing and quarantining contacts of the infected. quarantine can contribute substantially to mitigation, even if it has short duration and is done within households. when reopening society, testing and quarantining is a strategy that is much cheaper in terms of lost workdays than a long lockdown. a targeted quarantine strategy is quite efficient with only 5 days of quarantine, and its effect increases when testing is more widespread. | (2020) 10:18543 | https://doi.org/10.1038/s41598-020-75640-2 www.nature.com/scientificreports/ within families, workplaces, and friend groups, everyone is assumed to know everyone. each agent is assigned one family and workplace, as well as two groups of friends. workplaces on average contain ten people, whereas each friend group on average contains five. in the simulation runs presented here, we use a population of n = 5000 agents. increasing the number of agents changes the outcome very little, except for minimising stochastic noise. we also do not allow migration in or out of the system. we use a discrete-time stochastic algorithm. at each time-step (0.5 days), each person has one interaction with some other person. a "die roll" decides whether the person will interact with family, friends, work, or the public. the respective odds are the above-mentioned percentages 40:30:15:15. if the public is chosen, an entirely random person is selected, otherwise a person is drawn from a predefined group (family etc.). for each interaction, an infectious person has a fixed probability of passing on the disease to the person they interact with. the family size distribution of is based on the distribution of danish households 12 . the average number of people per household is approximately 2, and large households of more than 4 people have been ignored, as they account for less than 10% of the population. we believe that in a country where family sizes are larger and there are fewer singles, the family would be more important to the spread of disease. we test the effect of larger families in the supplement (figs. s1-s3) and find that it does not change our overall conclusions. we simulate the progression of disease using an seir model with four exposed states, e = e 1 + e 2 + e 3 + e 4 , each lasting on average 1.25 days, corresponding to a mean incubation period of 5 days. the exposed states are presymptomatic, meaning that people will not get tested in the incubation period. we let stages e3;4 be as infectious as the i-stage, as data suggest that a substantial fraction of covid-19 transmission happens before the onset of symptoms 13 . multiple exposed states are included in order to get a naturalistic distribution of incubation periods. li et al. 10 report that the mean incubation period is approximately five days and the reported distribution is fitted well by the gamma distribution we obtain from our four e-stages. a further problem is the duration of the infectious period (i). viral shedding has been observed to last up to eight days in moderate illness 14 . on the other hand, according to linton et al. 15 , the median time from onset to hospitalisation is three days. a bedridden patient (even if not hospitalised) is likely to transmit the disease less. to fit the observed mean serial intervals of 4.6 days of nishiura et al. 13 we model the infectious period as a single state with an average duration of three days. in addition, the infectious presymptomatic period lasts on average 2.5 days. in comparison ref. 16 uses a serial interval distribution with mean of 6.5 days. other authors have suggested a longer serial interval 10 with presymptomatic infections. finally, the transmission rate of the disease is estimated from an observed rate of increase of 23% per day in fatalities in the usa. this also fits the observation of a growth rate of icu admissions of about 22.5% per day in italy 17 . with our parameters this is reproduced by a basic reproduction number r 0 ~ 3 (as we allow transmission figure 1 . a diagram of the model structure. each agent has a network consisting of a family, a workplace and two groups of friends. the family accounts for 40% of interactions. work accounts for 30% and socialisation with friends accounts for a further 15%. the members of each of these 3 groups are fixed throughout the simulation. finally, 15% of interactions happen "in public", which we implement as an interaction with a randomly chosen other agent. everyone in the work and friend sub-graphs are assumed to be connected to each other. below the graph, the underlying mechanisms of the disease are shown. we divide the exposed state into four in order to get a more naturalistic gamma distribution of incubation periods. the two last exposed states are infectious, but asymptomatic, meaning that individuals will not get tested. this is to include presymptomatic infection. in our simulation we set the family groups to an average of 2 people, and the work network to 10 completely interconnected people. the friend network consists of two groups with five in each. having calibrated the model in this way, we want to explore mitigation strategies for the corona epidemic. specifically, we will investigate the relative importance of the areas of social life, and the extent that reducing workplace size reduces disease spread. moreover, we will examine the possible gain and cost by simple contact tracing and light quarantine practices. to illustrate the relative importance of the workplace and public life, we consider the scenarios in fig. 2a . in the first scenario, nothing is done. in the second, contacts within the workplace are reduced by 75%, while in the third, contacts with friends and the public are reduced. finally, we compare these with similar scenarios, but where good hygiene or keeping a distance reduces the probability of infection from all types of encounters by half. in the figure, we see that the effects of reducing workplace and social contacts are roughly of the same magnitude. this reflects the assignment of 30% weight to each of these contact types. the slightly larger effect of social contacts reflects our assumption that these connections are less clustered than the workplace network. the two latter graphs show the scenarios where we both reduce infection probability within one group by 75% and overall infection probability by 50%. they show that an effective lockdown requires both restrictions of the time spent in the workplace and in the public sphere, and measures that reduce infection probability by increased hygiene and physical distancing. the above results provide one useful piece of information. if the effect of workplace and social contacts are of the same order, it is of little importance which one is restricted. ideally, both will be restricted for a period. however, when restrictions need to be lifted, authorities will primarily be able to control the workplace, whereas the social sphere relies on local social behavior. obviously, it is economically more sustainable to lift the one with the largest social consequences first, by allowing people to return to work while encouraging keeping social gatherings at a minimum. if restrictions are lifted before a substantial level of immunity is achieved, the epidemic will re-ignite. therefore, we now examine what can be done to minimise spread in the reopened workplaces. one possible strategy is to reduce the number of people allowed at any one time in each workplace. in fig. 2b , we compare an epidemic scenario where the average number of employees per workplace is 10 with an epidemic where this number is reduced to 5. we further assume that the number of contacts per coworker remains the same, meaning that the number of contacts per person drops when workplace size is reduced. it can be seen that fragmentation of physical spaces at workplaces could have a significant effect on the peak number of infected. in a situation with a risk of straining the healthcare system, this could be part of a mitigation strategy. once again, the strategy becomes relatively more effective if the infection probability per encounter is also reduced. compared to the cases with no workplace size reduction, making workplaces smaller leads to a greater relative reduction in peak size if infection probability is lower, completely eliminating the epidemic at an infection probability reduction of 50%. a more local strategy that can be employed when reopening society is widespread testing and contact tracing. as mentioned above, hellewell et al. 11 have suggested that this can be effective in containing covid-19 www.nature.com/scientificreports/ outbreaks provided high efficiency in detecting infected individuals. contact tracing has previously been modeled in relation to other epidemics 18 , and used successfully against smallpox 19 and sars 20 . one obstacle to the widespread implementation of this strategy is the difficulty of tracing contacts. therefore, we will here implement a crude form of contact tracing where we (1) close the workplaces of people who are tested positive for the disease, (2) isolate their regular social contacts for a limited period, and (3) keep symptomatic individuals in quarantine until they recover. we will see that such a 1 step tracing and quarantine strategy (1stq) can give a sizeable reduction in disease spread while costing fewer lost workdays than overall lockdown. our simulations include the limitations imposed by not being able to trace the estimated 15% of infections from random public transmissions. thus, the strategy does not require sophisticated contact tracing but could be implemented based on infected people being able to recollect their recent face-to-face encounters with friends. it should be noted that we here quarantine persons in their own households, thereby making our contact tracing strategy easier to implement in practice. in particular, family members of a quarantined person are still free to interact outside their home if they are not themselves tested positive. the drawback of such light quarantine practices is that infected persons in quarantine may still transmit the infection to their families. figure 3 examines how increased testing efficiency systematically improves our ability to reduce the peak disease burden. this would then be a more cost efficient way to mitigate the pandemic than a complete lockdown where each person would lose several man-months. even detecting as little as 5% of covid-19 infected per day (which with an average symptomatic disease duration of 3 days corresponds to finding approximately 15% of the infected) can potentially reduce the peak number of cases by 50%. if 10% efficiency is possible, corresponding to detecting about a third of infectious cases, then peak height could be reduced by a factor of almost three with to a 60% drop, if the probability of infected people being tested is only 10% per day of illness. however, the price of this is that each person is on average quarantined once during the epidemic. if testing is more widespread, the epidemic peak can be further reduced, until it finally becomes unstable at a testing probability of around 40% per day. (d) epidemic peak and time spent in quarantine as a function of quarantine length for a testing probability of 20% per day. the average time spent in quarantine increases linearly with the length of quarantine. on the contrary, the effect of quarantine on the peak height appears to stagnate at approximately 5 days. www.nature.com/scientificreports/ less than two weeks in quarantine per person during the entire epidemic. this is illustrated in fig. 3a where peak height is reduced from 0.13 to 0.04 at 10% testing efficiency. the main cost of the quarantine option is the quarantine time. figure 3d examines the efficiency versus cost of as a function of quarantine length. it can be seen that there is little gain in extending the quarantine period beyond the 5-day duration of the incubation period. for this reason we opted for 5 days in quarantine in panel (a, b). as a consequence, an average person will stay around 12 days in quarantine during the course of the epidemic with a testing probability of 10% per day. this time can be reduced if people can be convinced of smaller work environments and fewer face-to-face contacts per week. fragmentation of our networks into smaller groups will reduce both quarantine overhead and the direct transmission of the disease (fig. 2b, orange curve) . a prolonged lockdown will hugely disrupt society, and it is questionable whether a complete eradication of the virus is possible anyway. therefore, most governments have aimed at softening the epidemic curve, with varying degrees of success. the one step contact tracing with testing and quarantine is a means to this end and would work most effectively in combination with other efforts to reduce r 0 . finally, we investigate whether an aggressive testing and contact tracing strategy could work if implemented at a late stage in an epidemic. this could be relevant if for example the strategy is part of an effort to reopen society after a period of lockdown. in fig. 4 , we show two possible scenarios where testing and contact tracing is implemented after a 30-day lockdown with a 75% reduction of the work and social spheres. the lockdown is initiated when 1% of the population is infected. in (a) we subsequently test and quarantine the infected and their contacts for 5 days, while in (b) the required quarantine is set to 10 days. we assume a testing efficiency of 20% chance of detection for each day a person is symptomatic. the progression of the epidemic without testing is marked by a black graph for comparison. from the figure one sees that the strategy of even relatively short quarantines also works with a late onset. at a realistic detection probability, it prevents a resurgence of the epidemic. nonetheless, it is quite costly initially, with a very high peak in number of quarantined people. importantly, the effect does not increase with a longer quarantine period, but the cost is substantially larger. pandemics such as the one caused by covid-19 can pose an existential threat to our social and economic life. the disease itself is serious and leaves specific epidemic signatures and characteristics that make traditional contact tracing difficult. in particular it is highly infectious, can sometimes be transmitted already two days after exposure, and a large fraction of transmission happens before the onset of symptoms. as such it is difficult to contain without a system-wide lockdown of society. nonetheless, a successful containment in south korea used contact tracing. this motivated us to explore a one-step contact tracing/quarantine strategy (1stq). using reasonable covid-19 infection parameters we find that the 1stq strategy can contribute to epidemic mitigation, in the sense that it can reduce the peak number of infected individuals by about a factor of two even with a realistic testing rate of 10% per day of illness. this was illustrated systematically in fig. 3 . the main cost was people in self-quarantine and not contributing to the workforce. in comparison one has to consider that a society-wide lockdown with similar reduction in peak height would have to last for about 100 days (see fig. 2 ). thus, the lockdown would require of order 100 days of quarantine (or at least extensive social distancing) per person, whereas testing and isolation only requires on average around 15 days per person with a 5-day quarantine www.nature.com/scientificreports/ even at high testing probabilities. importantly these numbers can be reduced if people are able to lower their number of contacts. a noticeable objection to the 1stq strategy is the fraction of cases with so weak symptoms that people do not contact health authorities. the effect of such limitations is in our model parameterized through the detection probability. from fig. 3c one sees that when the detection probability goes below 3% (a rate of 1% per day) the peak reduction of the 1stq strategy becomes only of the order 1 percentage point. it should also be noted that, since we rely on symptoms to determine who stays in quarantine, and people in the infectious/symptomatic stage are assumed to always stay in quarantine, we implicitly assume that all infected persons develop at least some symptoms at some point. this may be a break from reality. the increasing availability of tests may also change the perspectives of the 1stq strategy. with widely available rapid tests, it will be possible to test everyone regularly, and to test all quarantined persons before they leave quarantine. supplementary figure s4 deals with the results of such a testing strategy and finds that it makes it possible to totally control the epidemic, or to mitigate it without quarantining any healthy individuals. to put this into perspective, the drawbacks of widespread, but slow testing is examined in the supplementary fig. s5 . here, we find that the 1stq strategy is most efficient with no test delay, and that delayed contact tracing is comparable to a primitive lockdown. one interesting point which we have not examined here, is that real-world social networks are heterogeneous, with a large variance in number of contacts. it may be expected, for example, that workers in customer-facing positions in shops will have a high risk of catching the disease and passing it on. the effects of this heterogeneity is examined more closely in ref. 21 here, it is concluded that heterogeneity in the number of contacts enhances the effect of contact tracing, since persons with many contacts are both more likely to pass on the disease and more likely to be quarantined. in ref. 11 , the authors suggest a 1stq strategy similar to the one we here model. the main points of the present analysis is the focus on mitigating instead of eradicating the epidemic, our suggestion of a shorter quarantine length, and the implementation of quarantine together with other members of the household instead of total isolation. our stochastic, agent-based approach also allows for local failures due to the limited duration of quarantine (people may not yet be symptomatic when exiting quarantine) and the non-traceable public contacts (set to 15%). finally, one noticeable finding is that contact tracing and reduction of contacts per person is still feasible even at a later stage of the epidemic. as can be seen in fig. 4 , a lockdown and subsequent reopening with testing and contact tracing is highly effective in controlling the epidemic. our study that lockdowns have an important role to play in epidemic mitigation, but that they can be replaced by a 1stq strategy once the epidemic is under control. the covid-19 pandemic has set both governments, health professionals, and epidemiologists in a situation that is more stressful and more rapidly evolving than anything in recent years. due to the uncertainties caused by a situation in flux, it is difficult to predict anything definite about what works and what does not. the empirical observation that lockdowns worked in both china, and in a milder form in denmark shows that our assumption of a 75% reduction in specific infection rates under lockdown is realistic. our main result is that some of these restrictions can be replaced by testing, one-step contact tracing and short periods of quarantine. this is far cheaper than total lockdowns. perhaps most importantly, these measures work best in combination. as is highly relevant to the current epidemic stage of covid-19, we pinpoint that 1stq can be successfully implemented also at a late stage of the epidemic where testing may become massively available. plots of alternative variants of our model (including alternative testing strategies and larger family sizes) can be found in the supplementary material. the code used to produce the plots shown in this article is available on figshare under the url https ://doi.org/10.6084/m9.figsh are.12206 735.v4. how will country-based mitigation measures influence the course of the covid-19 epidemic report 9: impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand social contacts and mixing patterns relevant to the spread of infectious diseases epidemic analysis of covid-19 in china by dynamical modeling the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study modelling transmission and control of the covid-19 pandemic in australia substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) contagion! the bbc four pandemic: the model behind the documentary contacts in context: large-scale setting-specific social mixing matrices from the bbc pandemic project early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia feasibility of controlling covid-19 outbreaks by isolation of cases and contacts fam44n: families 1. january by municipality, type of family, size of family and number of children serial interval of novel coronavirus (covid-19) infections ) pandemic: increased transmission in the eu/eea and the uk-seventh update epidemiological characteristics of novel coronavirus infection: a statistical analysis of publicly available case data report 13: estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in 11 european countries covid-19 and italy: what next? the effectiveness of contact tracing in emerging epidemics smallpox and its eradication epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong heterogeneity is essential for contact tracing we thank gorm gruner, bjarke frost nielsen, andreas roepstorff, and lone simonsen for enlightening discussions. this project has received funding from the european research council (erc) under the european union's horizon 2020 research and innovation program under grant agreement no. 740704. a.e. and k.s. both participated in devising the model. code was written and plots produced by a.e.. the functionality of the code was checked by comparison with an alternative algorithm written by k.s. a.e. and k.s. wrote and edited the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-75640 -2.correspondence and requests for materials should be addressed to a.e.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, 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 licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence 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. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-032806-o6p861ms authors: fenin, audrey; newman, jill c.; taylor, sarah n. title: very low birth weight infants receive full enteral nutrition within 2 postnatal weeks date: 2020-09-29 journal: j perinatol doi: 10.1038/s41372-020-00819-4 sha: doc_id: 32806 cord_uid: o6p861ms objective: identify whether an enteral nutrition goal of reaching full feeds by 7 postnatal days for infants 1–1.5 kg and by 14 postnatal days for infants <1 kg was feasible and its associated outcomes. study design: very low birth weight infant cohort admitted in the first postnatal day and categorized as either epoch 1 or epoch 2, 12 months before and after implementation of a revised feeding protocol were compared. result: in epoch 2, 83% infants born 1–1.5 kg and 77% infants born <1 kg reached full feeds by 7 and 14 days compared to 26% and 25%, respectively in epoch 1 (p < 0.0001). central line and parental nutrition days were significantly lower in epoch 2 compared to epoch 1 with sustained and potentially improved infant growth. conclusion: an evidence-based advancement feeding protocol was associated with achieving full feeds within the first 2 postnatal weeks for very low birth weight infants. debate persists in very low birth weight (vlbw) infant care about the initiation and advancement of enteral nutrition [1] [2] [3] . historically, early introduction to enteral feeding and rapid advancement of feeds were related to higher incidence of necrotizing enterocolitis (nec) [4] [5] [6] . however, current evidence demonstrates no benefit to slower enteral feed advancement (15-20 ml/kg/day compared to 30-40 ml/kg/day) and potential harm including a delay in time to achieve full feeds, longer time to regain birth weight, and an increased chance of invasive infection risk with no reduction in the risk of nec [7, 8] . on the other hand, no difference in nec or late-onset sepsis was observed in a recently published randomized, controlled trial [9] . despite the lack of difference in 2-year outcomes including survival without moderate or severe neurodevelopmental disability in this trial [9] , neonatal care centers may still find benefit if more rapid advancement of enteral feeds is associated with two common quality indicators in neonatal care-early discontinuation of central venous lines (cvl) and shorter duration of parenteral nutrition (pn), while maintaining infant growth. based on published guidelines recommending advancement of enteral nutrition to achieve full feeds by 7 days in infants born 1-1.5 kg and by 14 days in infants born <1 kg [10] , a nutrition-focused research team, whose work to initiate enteral nutrition in the first postnatal day has been published previously [11] , aimed to investigate its ability to establish full enteral nutrition (120 kcal/kg/day) by 7 postnatal days for infants born 1-1.5 kg and by 14 postnatal days for infants born <1 kg after instituting practices that included discontinuation of routine gastric residual monitoring, decreased days of trophic feeding (minimal enteral nutrition), and faster feed volume advancement. despite evidence that early total enteral feeding of 50-80 ml/kg/day are tolerated on the first postnatal day in infants 1-1.5 kg [12] , concern existed that aggressive early feeding would lead paradoxically to feeding intolerance or fear of feeding intolerance and, therefore, would be associated with a greater delay to achieve full enteral nutrition. therefore, this cohort study, with a retrospective control, was performed with a primary aim to determine if infants were able to reach the full enteral nutrition goal and with secondary aims to determine whether this outcome was associated with changes in pn exposure, cvl days, or growth. after obtaining institutional review board exemption at the medical university of south carolina, this retrospective cohort study was performed at a single university-based tertiary care neonatal intensive care unit. data were retrospectively collected from a nutrition quality improvement database. subjects were selected if birth weight ≤1500 g and admitted within the first 24 postnatal hours to the neonatal intensive care unit. infants with congenital anomalies, cardiac defects, or metabolic defects that precluded feeding within the first 24 postnatal hours were excluded as were infants who died in the first 24 postnatal hours, or those discharged home or transferred before 28 postnatal days as their gv at 28 days would not be calculated. infants born from march 1, 2017 to february 28, 2018 and meeting inclusion criteria were in the cohort labeled epoch 1. these dates were chosen as march 1, 2018 was the day of implementation of the new protocol. infants born march 1, 2018 to february 28, 2019 were included in the epoch 2 cohort. data collected included birthweight, gestational age (ga), sex, race, and ethnicity. in addition, antenatal steroid exposure, small-for-gestational age (sga) status, respiratory support in the first 28 postnatal days, feeding type, and hour of first feed were collected. sga was defined as birth weight <10th percentile by fenton growth reference chart [13] . feeding type was differentiated as mother's milk (mm) only, mm and donor human milk (dhm) combined, and dhm only. respiratory support was collected as any oxygen, conventional ventilator, or nasal continuous positive airway pressure (cpap) in the first 28 postnatal days. occurrence of sepsis, bronchopulmonary dysplasia (bpd), and nec was collected through the hospitalization. sepsis was defined as any culture-positive blood infection during hospitalization. bpd was defined as oxygen support required at 36 weeks' ga or at hospital discharge if prior to 36 weeks' ga. nec was determined if modified bell's stage 2 or greater was diagnosed during hospitalization. spontaneous perforation was determined if occurred in first 28 postnatal days and without a diagnosis of nec. cvl and pn number of days and time to full feeds were calculated by finding the difference between the recorded times of initiation and discontinuation for each measure. growth trajectory was measured as 28-day gv, days to return to birth weight, and the change in weight z-score from birth to 28 days as determined by the fenton growth reference chart [13] . 28 day gv was chosen as the primary indicator of growth and was calculated by the 2-point model (change in weight in grams from birth to 28 days/the average weight between birth and 28 days/28 days) [14] . days to return to birth weight was calculated as the first postnatal day at which the infant's weight was higher than the birth weight. infants in epoch 1 had feeds initiated and advanced per a previous feeding protocol adopted in 2010. infants in epoch 2 had feed initiation and advancement per the revised protocol instituted on march 1, 2018. the revised feeding protocol was the result of the vlbw infant feeding evidence and expert recommendations published prior to october 31, 2017 as reviewed by an institutional multidisciplinary team comprising neonatologists, nurses, neonatal nurse practitioners, and neonatal registered dietitians. the march 2018 revised protocol included continuation of the practice of initiating feeds at 6-24 postnatal hours, discontinuation of the practice of routine gastric residual monitoring, a decrease in days of trophic feeding of 12 ml/ kg/day from 5 to 3 days in infants <1 kg and from 3 to 1 day in infants 1-1.5 kg. the revised protocol also included institution of the practice of faster advancement of enteral feeds with infants <1 kg advanced at 25 ml/kg/day divided into two daily steps (morning and evening) with fortification to 24 kcal/oz at~100 ml/kg/day and infants 1-1.5 kg advanced at 30 ml/kg/day also with two daily steps with fortification at~110 ml/kg/day. in epoch 1, infants <1 kg advanced at~18 ml/kg/day, also divided in two daily steps with a two-step fortification of 22 kcal/oz for 12 h followed by 24 kcal/oz at 100 ml/kg/day. for infants 1-1.5 kg, advancement was also done in two daily steps of 25 ml/kg/day, fortified at 100 ml/kg/day in two steps also. the practice of introduction of enteral feeds at 6-24 postnatal hours was continued since an earlier study had shown this practice was associated with decreased central line infections, decreased feeding intolerance and improved gv [11] . infants in both epoch 1 and 2 were either fed mm or dhm after obtaining parental assent if mm not available. to facilitate institution of the protocol, physicians, nursing, and dietary staff were educated prior to the protocol change by oral and visual presentations. see fig. 1 for a schematic of the new protocol change. no other changes in nutrition delivery, including pn, occurred during the study period. power analysis demonstrated to achieve 90% power to detect a difference between the group proportions of 0.2, 119 subjects were needed in each epoch to identify whether the proportion of infants achieving full feeds differ by 0.2 from the 0.46 proportion at baseline. a two-sided z test with pooled variance was used with a targeted significance level at 0.05. with an estimated 160 eligible subjects born each year, 1-year pre-(epoch 1) and 1-year post (epoch 2) were chosen as the ranges for subject inclusion. descriptive statistics for demographic and outcome characteristics were reported as frequencies and percentages, means and standard deviations or median and interquartile ranges. for the primary aim, a dichotomous (yes/no) variable to indicate whether the infant met the goal for full enteral nutrition was defined. infants were stratified by birth weight (<1 kg and 1-1.5 kg). for infants whose birth weight was <1 kg, the infant met goal if the number of days to full enteral nutrition was less than or equal to 14 postnatal days. if the number of days was >14 postnatal days, then the infant did not meet the goal for full enteral nutrition. the same definition applied to birth weight 1-1.5 kg with the cut-point for number of days being 7. chi-square tests or fisher's exact tests were used to test for associations between categorical measures. since the distributions for number of days cvl and pn were skewed, wilcoxon rank sum tests were used for unadjusted associations. student's t test was used to test for associations of normally-distributed continuous measures. multivariate linear and generalized mixed models were used to assess differences in epoch for gv, number of cvl days, and number of pn days, after controlling for birth ga, birth weight, race/ethnicity, sex, and antenatal steroid exposure. due to concern for collinearity, further comparisons were performed using sga status (yes or no) instead of birth weight in all models. weight z-score change from birth to 28-days also was assessed as the dependent variable in this second regression model. a bar chart was created to show the proportion of infants that met the goal for full enteral nutrition by epoch and weight group. histograms were used to show the distributions of continuous outcome measures by group. a p value <0.05 was considered statistically significant and all analyses were performed using sas version 9.4 (cary, nc). a total of 272 infants met inclusion criteria. in epoch 1, 34 subjects were excluded (20 due to discharge or transfer to another hospital, 13 due to death, and 1 with congenital anomaly affecting feeding). in epoch 2, 32 subjects were excluded (14 due to discharge or transfer to another hospital, 14 due to death, and 4 with congenital anomaly affecting feeding). the two groups were similar in baseline demographics, including sga status at birth, as shown in tables 1 and 2. in comparison of feeding exposures which were not affected by the revised feeding guideline, the proportion of infants receiving a combination of dhm feeds, as well as those receiving only dhm were not significantly different between epochs. in addition, both epochs demonstrated initiation of feeds at a median of 9 h. respiratory exposure in the first 28 postnatal days did not significantly differ between groups except a significantly higher proportion of infants born 1 to 1.5 kg received nasal cpap in epoch 2. in epoch 2, 83% of infants born 1-1.5 kg achieved full enteral feeds by 7 days and 77% of infants born <1 kg achieved full enteral feeds by 14 days and these proportions were significantly higher than for similar infants in epoch 1 (fig. 2) . cvl and pn days were also significantly lower in epoch 2 compared to epoch 1 (table 1 ) and the significant difference remained apparent when stratified by weight group (table 2) . only 103 infants out of the total 124 infants in epoch 1 had cvl and 112 out of 148 infants in epoch 2 had cvl. the distribution of days with pn and cvl between epochs and by weight group is shown in fig. 3 . in unadjusted analyses, no difference in the mean 28-day gv, weight z-score change from birth to 28 postnatal days, or average days to return to birth weight were observed between epoch, even when stratified by weight group (tables 1 and 2 ). multivariate analyses were performed for three secondary outcomes, cvl and pn days and 28-day gv. initially, regression models were performed with independent variables ga, birth weight, sex, race and ethnicity, and antenatal steroid exposure with significantly lower cvl and pn day in epoch 2 versus epoch 1 (p value < 0.001 for both comparisons). in addition, in an adjusted model, gv from birth to 28 days was significantly higher in epoch 2 versus epoch 1 (β estimate (β) = 0.95, standard error (se) = 0.35 and p-value= 0.008). due to concern for collinearity with birth ga and weight in the same model, a second model was developed with sga versus non-sga as a covariate instead of birth weight. the results of significantly higher gv in epoch 2 versus epoch 1 did not differ in the new multivariate model (β = 0.85, se = 0.39 and p value 0.0286). consistent with previous findings, the number of cvl days remained significantly lower in epoch very low birth weight infants receive full enteral nutrition within 2 postnatal weeks 2 (β = −0.46, se = 0.07 and p value < 0.0001). similarly, the analysis for the number of pn days was also significantly lower in epoch 2 (β = −0.45, se = 0.05 and p value < 0.0001). since growth velocity was significantly different between epochs in adjusted analysis, similar regression analysis including sga status to replace birth weight, was performed to identify whether weight z-score change from birth to 28 postnatal days differed significantly by epoch when controlling for the other factors. it did not (p = 0.12). in comparison of morbidities between groups, no infants in this study experienced spontaneous intestinal perforation. no difference was observed between epochs in bpd, nec, or late onset-sepsis, except the proportion of infants born < 1 kg who developed nec was significantly lower in epoch 2. this difference likely is not related to the revised feeding protocol and, instead, demonstrates the natural variation in nec incidence at a single institution over a 24-month period. after implementing an evidence based revised feeding guideline which included discontinuing the practice of gastric residual monitoring, decreasing number of days of trophic feeding and progressive enteral feeding advancement, 83% of infants 1-1.5 kg and 77% of infants <1 kg superscript identify the descriptive statistics which is "b" for mean (standard deviation). c superscript identify the descriptive statistics which is "c" for median [interquartile range]. reached full feeds by 7 and 14 postnatal days, respectively. this change was associated with statistically and clinically significantly less days of cvl and pn in both unadjusted and adjusted comparisons. in addition, in adjusted models, mean 28-day growth velocity was significantly higher for the epoch receiving the revised feeding protocol (epoch 2). however, weight z-score change from birth to 28-days was not significantly different between epochs in either univariate or multivariate analysis. this study focused specifically on how the revision of the feeding protocol, as compared by epochs, related to the outcomes of interest. investigation of how other parameters such as ga, sga status, and antenatal steroids was not performed in this study but may be of interest in future research of vlbw infant feeding. superscript identify the descriptive statistics which is "b" for mean (standard deviation). c superscript identify the descriptive statistics which is "c" for median [interquartile range]. the revised feeding protocol had multiple components based on evidence review. the revisions included a discontinuation of routine gastric residual monitoring which was based on recent evidence demonstrating no utility of this practice and the potential that it is associated with a delay in achievement of full enteral nutrition [15] . recently, results of a randomized, controlled trial verified the results of earlier observational studies and showed increased enteral nutrition delivery and improved weight gain when gastric residuals were not routinely monitored [16] . the revised guidelines in our study also included a shortened duration of trophic feeds to 1 day for infants 1-1.5 kg and to 3 days for infants <1 kg. a previous retrospective cohort study demonstrated 3 days of trophic feeds was associated with faster achievement of full enteral nutrition in extremely preterm infants [17] . in our study, the increased daily feed advancement volume to 25-30 ml/kg/day was based on published systematic review of the evidence [7] . more recently, the results of 2-year outcomes of a multicenter randomized trial comparing 30 ml/kg/day with 18 ml/kg/ day were published [9] . in this recent publication, a more rapid advancement of volume was associated with shorter duration to achieve full feeds, but no significant difference in 2-year outcomes was observed except for infants receiving formula-only feeds who demonstrated better survival without moderate or severe neurodevelopmental disability in the slower feed volume advancement group. of note, the infants in this multi-center randomized trial did not start feeds on the first postnatal day [9] . in this study with human milk feeds initiated on the first postnatal day, full feeds were achieved with lower cvl and pn days and higher growth velocity when adjusted for potential confounders. other retrospective cohort studies have demonstrated similar results but occurred in an older patient population [18] , included parenteral nutrition revisions [19], or compared a decrease in the number of days prior to feed progression. a decrease in the number of days prior to feed progression was associated with improved 28-day gv [20] . a similar significant increase in gv was found in our study when adjusted for potential confounders. in addition in our results, the change in z-score for both epochs were similar to the change observed by rochow et al., though they measured change in z-scores from birth to 21 postnatal days, instead of 28 days, and included infants 25-34 weeks' ga [21] . in our study, no difference in the number of days to return to birth weight was observed between epochs which may reflect the fact that pn practices did not differ between groups [22] . the strengths of this study include [1] the similarity of infants in both epochs in terms of sex, birthweight, and ga, and [2] the inclusion of infants who were sga in these standard feeding protocols. days of exposure to pn were decreased in this study hence potentially decreasing the risks and costs associated with pn. days with a cvl were decreased hence decreasing the days on which line complications could occur as well as the risk for central line-associated blood stream infection. limitations of this study include that not every potential complication of a revised feeding potential was studied. for example, the number of abdominal radiographs to evaluate potential feeding intolerance was not measured. in addition, although the study center is a tertiary regional center covering an eight-county region of south carolina, this was still a single-center cohort study. gv was only measured at 28 postnatal days. therefore, the effect on long-term growth is not known. despite clinical concern that "pushing" infants to feed earlier and more quickly would lead to more stops and starts of feeding and therefore longer time to full enteral feeds, this study shows that achieving full enteral nutrition within 1 postnatal week for infants born 1-1.5 kg and within 2 postnatal weeks for infants born <1 kg is feasible and can be applied to clinical practice. the magnitude of the decrease in central line and parenteral nutrition demonstrates the potential for decreased patient morbidity and hospital cost associated with this strategy. postnatal malnutrition and growth retardation: an inevitable consequence of current recommendations in preterm infants? growth in the neonatal intensive care unit influences neurodevelopmental and growth outcomes of extremely low birth weight infants growth failure in the preterm infant: can we catch up? enteral feeding regimens and necrotising enterocolitis in preterm infants: a multicentre case-control study role of delayed feeding and of feeding increments in necrotizing enterocolitis prolonging small feeding volumes early in life decreases the incidence of necrotizing enterocolitis in very low birth weight infants slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants slow advancement of enteral feed volumes to prevent necrotising enterocolitis in very low birth weight infants. cochrane datab system rev controlled trial of two incremental milk-feeding rates in preterm infants guidelines for feeding very low birth weight infants early enteral feeding in very low birth weight infants early total enteral feeding in stable preterm infants: a systematic review and metaanalysis a systematic review and meta-analysis to revise the fenton growth chart for preterm infants an attempt to standardize the calculation of growth velocity of preterm infants-evaluation of practical bedside methods the value of routine evaluation of gastric residuals in very low birth weight infants effect of gastric residual evaluation on enteral intake in extremely preterm infants: a randomized clinical trial short versus extended duration of trophic feeding to reduce time to achieve full enteral feeding in extremely preterm infants: an observational study reducing time to initiation and advancement of enteral feeding in an all-referral neonatal intensive care unit very low birth weight infants receive full enteral nutrition within 2 postnatal weeks 19. thoene mk, lyden e, anderson-berry a. improving nutrition outcomes for infants < 1500 grams with a progressive, evidenced-based enteral feeding protocol implementation of feeding guidelines hastens the time to initiation of enteral feeds and improves growth velocity in very low birthweight infants physiological adjustment to postnatal growth trajectories in healthy preterm infants effect of early parenteral nutrition discontinuation on time to regain birth weight in very low birth weight infants: a randomized controlled trial acknowledgements the authors acknowledge dr. julie ross for her leadership in quality improvement initiatives in the neonatal intensive care unit, the entire medical university of south carolina milky weigh nutrition quality improvement team, and the physicians, nurses, and dietitians who follow these evidence-based protocols in their care of vlbw infants. in addition, the authors acknowledge dr. martina mueller who assisted in the statistical methodology. the authors have no disclosures or sources of any support for this work such as grants and/or equipment and drugs.author contributions af and snt provided substantial contributions to the conception and design of the work. af performed data acquisition. all authors made substantial contributions in data analysis, data interpretation, and drafting the work with final approval of the version to be published. conflict of interest snt has received research funding from nih and the allen foundation. snt has served as a consultant for alcresta therapeutics and serves as a volunteer member of the mother's milk bank northeast medical advisory board. af and jcn have no potential conflict of interest to declare.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-288051-wp8v2mc5 authors: sánchez-gonzález, álvaro; lópez-fando lavalle, luis; esteban-fernández, alberto; ruiz, mercedes; hevia, vital; comeche, belén; sánchez conde, matilde; álvarez, sara; lorca álvaro, javier; fraile poblador, agustín; hevia palacios, manuel; domínguez gutiérrez, ana; artiles medina, alberto; sanz mayayo, enrique; duque, gemma; gómez dos santos, victoria; moreno-guillén, santiago; burgos revilla, javier title: what should be known by a urologist about the medical management of covid-19’s patients? date: 2020-09-01 journal: curr urol rep doi: 10.1007/s11934-020-00995-y sha: doc_id: 288051 cord_uid: wp8v2mc5 purpose of review: the alarming number of confirmed covid-19 cases put a strain on the healthcare systems, which had to reallocate human and technical resources to respond to the emergency. many urologists became integrated into multidisciplinary teams, dealing with this respiratory illness and its unknown management. it aims to summarize the epidemiological, clinical, diagnostical, and therapeutical characteristics of covid-19, from a practical perspective, to ease covid-19 management to non-physician staff. recent findings: we performed a narrative review of the literature regarding covid-19, updated to may 8th, 2020, at pubmed and covid resource platforms of the main scientific editorials. covid-19, characterized by fever, myalgias, dyspnea, and dry cough, varies widely from asymptomatic infection to death. arrhythmias and thrombotic events are prevalent. lymphopenia and inflammatory reactant elevation on laboratory, as well as bilateral and peripheral ground-glass opacities or consolidations on x-ray, are usually found in its assessment. little is known about sars-cov-2 immunology. to date, no therapy has demonstrated efficacy in covid-19. of-level or compassionate-use therapies are prescribed in the context of clinical trials. we should become familiar with specific adverse events and pharmacological interactions. summary: the covid-19 pandemic has paralyzed the urological activity, and its long-term consequences are unpredictable. despite not being used to deal with respiratory diseases, the urologists become easily qualified to manage covid-19 by following protocols and being integrated into multidisciplinary teams, helping to overcome the pandemic. since its first description in december 2019, coronavirus disease 19 , caused by the new severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has dramatically spread worldwide [1] . the alarming number of confirmed cases overcrowded hospitals, increasing their occupancy to 120% [2] . it was necessary to reallocate human and technical resources [3] . the infection and isolation of many physicians reduced its availability to struggle against covid-19, so it became essential to recruit surgical staff to assist covid-19 patients. in this way, some urologists have been integrated into covid-19 multidisciplinary teams [3] . the remaining urologists continued screening and treating patients, some of whom were asymptomatic covid-19's infected patients or whose symptoms were masked under the suspicion of urinary infection. finally, many patients got infected by sars-cov-2 during their hospitalization [4] . most of the scientific associations, like the european association of urology, have considered the importance of urologists' support in this challenging situation [5] . some guidelines were drafted concerning the prioritization of the urological activity, helping clinical decision-making [6] . this great effort may avoid a harmful impact on both patients and healthcare urological providers whenever it is possible. covid-19's outbreak and its medium and long-term course uncertainty force physicians to learn from real-life experience and changing emerging evidence [3] . this effort is remarkable in urologists and other surgical staffs, who do not use to deal with respiratory diseases and their medical management. this document aimed to synthesize the clinical, diagnostical, and therapeutical characteristics of covid-19. it is made from a practical perspective, adapted to non-physician staff. the manuscript is based on the limited scientific data regarding sars-cov-2, updated to may 8th, 2020, and the authors' personal-experience managing covid-19 at their institutions. the authors reviewed the literature concerning covid-19 at pudmed, but also at covid-19 resource platforms published by editorials like elsevier, jama network, springer nature, biomed central, the bmj, cochrane library, wiley. the results were clustered in epidemiology, clinical intercourse, assessment, and treatment of covid-19. the initial drafted version was discussed and agreed upon by all authors via a telematics conference. on may 9th, 2020, the final version was approved. this document reflects an updated and practical perspective of the current evidence adapted to the urology staff. with 3,759,967 confirmed cases on may 7th, including 259,474 deaths over 215 counties [7] , several systematic reviews have been published about sars-cov-2's epidemiology [8, 9, 10••] . sars-cov-2 has high transmission efficiency, with a basic reproduction range (r0) estimated between 2 and 3 in most studies [9, 10••] . the incubation period was estimated to be 4-6 days, ranging from 2 to 11 days after exposure in most cases [9] . in a practical sense, 14 days are considered its upper limit [10••] . the transmission was estimated to start 1-3 days before symptoms onset [8] (fig. 1) . the risk of transmission from patients with sars-cov-2 infection varies by the kind and duration of exposure, use of preventive measures, and individual factors [8, 9, 15] . infected symptomatic or asymptomatic patients are the source of infection, being respiratory droplets and direct contact with an infected person/surface the most frequent vehicles. it also occurs by long-time exposure to high-virus concentration respiratory aerosols [10••] . sars-cov-2 has been fig. 1 diagram of covid-1 clinical course spectrum and immunological response [11•, 12-14] isolated on stool samples, but the feco-oral transmission is not significant [8] . the sars-cov-2 yield in urine has not been demonstrated [16] . not vertical transmission has been demonstrated, although impaired effects have been described in newborns from infected pregnants [9] . exposure to higher virus concentrations and re-expositions are related to a worse prognosis. the duration of virus transmissibility is uncertain and seems to be related to the severity of illness. seven days after the clinical onset, the risk of transmission decreases in mildsymptomatic patients, but it may be extended over 24 days in severe cases [11•, 15] . the clinical spectrum of sars-cov-2 infection varies widely, including asymptomatic infection, mild upper respiratory tract illness, severe viral pneumonia with respiratory failure, and even death [9, 11•] (fig. 1) . fever, fatigue, dry cough, anorexia, myalgias, dyspnea, diarrhea, anosmia, and dysgeusia have been described commonly in covid-19 since the first chinese reports [8, 11•, 12] . hospitalization rates increased with age, ranging between 2.5 in those aged 18-49 years and 12.2 or 17.2 in those aged 65-74 or ≥ 85 years, respectively [17] . some patients may progress from mild-symptomatic to severe disease (oxygen saturation < 93%, shortness of breath, pao2/fio2 < 300 mmhg). dyspnea development was commonly reported after a median of 5 days since symptoms onset, and admission occurred after a median of 7 days [12] . acute respiratory distress syndrome (ards) is a significant complication in patients with severe disease and can manifest shortly after the onset of dyspnea, being age over 65 years, diabetes mellitus, and hypertension risk factors. septic shock, thrombotic events (te), and multiorganic failure are also common complications [11•, 12] . covid-19 should be considered in every patient with fever and typical symptoms. all of them should be screened about contact with positive or suspicious cases or residence/travel to civid-19's community transmission areas in the previous 14 days. sars-cov-2 assessment should be performed by nasopharyngeal (np) swap real-time reverse transcriptionpolymerase chain reaction (rtpcr). on admission, hemogram, coagulation profile (including d-dimer), hepatic and renal profiles, and acute reactants (including ldh, ferritin, c-reactive protein, creatine kinase, and procalcitonin) should be performed when possible. also, arterial gasometry, thoracic x-ray, ecg, and sofa score should be assessed [11•] . microbiological studies (hiv, hbv, and hcv serologies, blood culture, pneumococci, and legionella urinary antigens), myocardial enzymes and interleukin-6 determinations may be recommended. lymphopenia is consistently present at 40% of patients [11•, 18••] . an abnormal coagulation function is commonly found in cases of severe covid-19 pneumonia [11•, 19] . elevated inflammatory markers, such as d-dimer, c-reactive protein, ldh, fibrinogen, are commonly present. other common laboratory findings are shown in table 1 . critical/fatal illness should be suspected of older patients and high sofa score punctuations. exuberant inflammatory response findings, d-dimer over 1 μg/ml, and lymphopenia are mortality markers [11•, 12] . according to the literature, we monitored laboratory results every 48-72 h [12] . qtc calculation based on the admission ecg and qtc prolongation score should be performed before the start of covid-19's treatment (table 1) [20] [21] [22] 23 ••]. we should control qtc at least 2-3 times a week, while on treatment with qtc-prolongator drugs. covid-19 shows non-specific imaging features. bilateral lung involvement is found in 75% of x-rays. it is characterized by multiple peripheral ground-glass opacities with a subpleural distribution (table 1 ; image a) and subsegmental consolidations (table 1 ; image b) [12, 24] . in more severe 19] cases, extensive subpleural and peripheral multifocal areas of consolidation, predominantly in lower lobes (table 1 ; image c) [24] . lung involvement increases throughout the illness course, with a peak in severity at 10 to 12 days after symptom onset. no pleural effusions or cavitations have been reported. the american college of radiology recommends not using chest ct for its screening or diagnosis [8, 24] . the rt pcr is the preferred sars-cov-2's diagnostic test. viral load is demonstrated in the respiratory tract within 5-6 days of the onset of symptoms [13] (fig. 1) . the pick of viral load is correlated with covid-19 severity. an np swap is preferred over oropharyngeal one, due to sensitivity differences (63% vs. 32%) [13] . at the icu, bronchoalveolar lavage can be performed. little evidence exists about the serological course in covid-19's patients. preliminary evidence suggests that some of these antibodies protect, but this, as well as how long any protective effect lasts, must be definitively established [8] . uncertain correlation between their plasmatic level and clinical severity exists. after 10 days from symptom onset, most patients increase igg or igm level. seroconversion occurs after 7 days of clinic onset in 50% of them, and in 100% after 14 days [16] . plasmatic igm/igg determination will be useful to study immunological status in healthcare workers [14] . (fig. 1 ). to date, no therapy has demonstrated efficacy in covid-19. current management consists of supportive care and oxygen support if necessary [10••, 12, 25] . also, many patients have received off-level or compassionate-use therapies in the context of ethically approved clinical trials or the monitored emergency use of unregistered interventions framework (meuri) [23••] . two main aspects should be considered: we may be unfamiliar to specific adverse events that are potentiated with polimedication, and concomitant medication should be reviewed due to multiple pharmacological interactions (fig. 2) . table 2 describes the clinical features of the main drugs proposed in covid-19's management. evidence that supports their indication is exposed below. chloroquine is a well-known and economic antimalarial drug. an in vitro study demonstrated that chloroquine was highly effective in reducing viral replication [21] . results from 100 chloroquine-treated patients resulted in improved radiologic findings, enhanced viral clearance, and reduced disease progression. a study based on 36 hydroxychloroquine-treated patients reported 70% of virologic clearance at 6th day, measured by nasopharyngeal swabs [23••] . a harmful increase in cardiac conduction alterations is described in combination therapy with qtc-prolonger drugs. the current evidence suggests a limited role for lopinavir/ ritonavir in covid-19 [22] . a 199 patient-based study (99 receiving lopinavir/ritonavir) was published [20] . no differences were found against the standard of care group in terms of mortality, virus clearance, or time to hospital discharge. only in the intention to treat analysis, a 1-day difference in the median time to clinical improvement was found (15 days vs. 16 days). it should start at the early peak of viral replication (initial 7-10 days) to be effective [23••] . we should perform a careful review of concomitant medication due to frequent pharmacological interactions and potential aes (https://www.covid19-druginteractions.org/). azithromycin has in vitro activity against zika and ebola viruses and is commonly prescribed to prevent severe respiratory tract infections when administrated to patients suffering from viral infection [22] . a 100% 6-day virus clearance was described when hydroxychloroquine is combined with azithromycin, but a harmful increase in arrhythmias is described [23••] . low evidence exists about its indication [22] . in a recent series of 201 patients who developed ards, methylprednisolone was associated with a decreased mortality risk (46% vs. 62%) [1] . corticosteroids have an immunomodulatory effect, decreasing inflammatory response that may lead to ards and lung fibrosis. nevertheless, they may increase the risk of secondary infections and delay viral clearance [23••] . corticosteroids are recommended in the treatment of septic shock, exacerbation of chronic obstructive respiratory disease and these covid-19's patients with respiratory deterioration and quick radiological progression associated with sings of cytokine storm (cytopenia, maintained fever, an increase of inflammatory reactants: d-dimer > 1000 ng/ml, ferritin > 1000 ng/ml, fibrinogen > 100 ng/ml, il-6 > 40 pg/ml) [6, 23••] . in this context, they should be used at low-moderate doses (0.5-1 mg/kg/day up to 7 days) [6] . it is a humanized monoclonal antibody against the il-6 receptor. it decreases the damage caused by the cytokine storm and the amplified immune response in the lung and other organs [23••] . a first report based on 21 patients [17 (85.7%) received 1 dose] showed that the symptoms, hypoxemia, and ct opacity changes were improved immediately after the treatment with tocilizumab. of them, 75% lowered their oxygen intake (1 patient was taken off the ventilator on the first day after tocilizumab). a total of 90.5% have been discharged within 2 weeks after tocilizumab initiation. lymphocytes' percentage normalization in 52.6% and crp normalization in 84.5% of patients were observed on the fifth day. ct scans' lesions were absorbed in 19 patients (90.5%) and a little improvement in the others. no aes were reported [27] . it can be used in patients with extensive bilateral lung opacities or in severe or critical patients who have an elevated level of il-6 (> 40 pg/ml) [28] . it is a nucleoside analog prodrug that inhibits viral rna polymerases. previously reported at the ebola disease, it has demonstrated in vitro activity against sars-cov-2 [23••]. despite the fda approval for its urgent prescription in confirmed patients with oxygen saturations under 94%, clinical trial results are controversial. the results from 53 patients (30 receiving mechanical ventilation and 4 treated with ecmo) at 18-day median followup reported 68% (36 patients) of oxygen-support class improvements (including 17/30 (57%) extubations) and 47% (25/53) of patient discharges. seven (13%) patients died (6 in those receiving invasive ventilation) [25] . results from 237 patients, 158 assigned to remdesivir, showed no differences in time to clinical improvement, 28day mortality, oxygen support, hospitalization, or viral load. also, described 66% of aes and 12% of discontinuation [26] . several reports suggest a prothrombotic status with an increased trend of te [11•] . te, d-dimer's elevation, prothrombin time lengthened, and thrombocytopenia are predictors for 28-day mortality and worse prognosis. moreover, in patients with d-dimer > 6-fold of the standard upper limit or sic score ≥ 4, the treatment with heparin decreases the 28-day mortality rate [29] . heparin has anti-inflammatory properties and a protective effect over endothelium and microcirculation [30] . the covid-19's treatment interacts with antiplatelets and anticoagulants. the recommendations concerning the management of the coagulation emitted by the spanish no clinically significant interacɵon expected. interacɵon was described, but no specificaɵon about its severity was found. these drugs should not be coadministered. interacɵon expected: may require monitoring or drug dosage. qt enlargement fig. 2 interactions between urological drugs and hydroxychloroquine or lopinavir/ritonavir [26] curr urol rep (2020) 21:44 -not-recommended kidney or hepatic adjustment. -may be used in pregnancy if the benefit outweighs the risk. -cumulative effect: plasmatic half-life: 32 days. -ecg monitorization. ♥ -hepatopathy. -gastrointestinal symptoms. -not-recommended kidney adjustment. -ecg monitorization. -hepatic enzymes monitorization. tocilizumab -il-6 inhibition/reduction. -400 mg iv or 8 mg/kg in 1 or 2 doses sparsed 8-12 h. -pregnancy. -ast/alt > 5 times the upper limit. -neutropenia < 500 cells. -thrombocytopenia > 50,000 cells. -transplanted patients or anti-rejection drugs or immunoregulatory drugs -to be included in other c. -severe (23%): multiorganic failure, acute kidney injury, hepatic failure, sepsis. -discontinuation (12%) -not-recommended kidney or hepatic adjustment. -no probed in severe kidney/hepatic impairment. -compassivity uses if saturation < 94%. -hepatic enzymes and renal function monitorization. immunomodulation. -anti-inflammatory. -antifibrotic. -0.5-1 mg/kg/day up to 7 day. -250 mg/day up to 3 day. -concomitant infection. -gastrointestinal ulcer. severe: hyperglycemia, psychosis, and avascular necrosis. -dese de-escalation in chronic treatment. -the patient should meet ards criteria -very limited evidence. -glycemic control. association for anesthesiology, resuscitation, and pain therapy (sedar) are shown in fig. 3 [30] . pulmonary thromboembolism should be suspected in case of sudden respiratory deterioration associated with ards and low arterial pressure [11•] . -absence of fever for more than 3 days [11•]. -symptomatic improvement and more than 7-8 days from clinical onset [11•] . -radiologic response and rtpcr negativization before discharge are controversial. covid-19 is one of the most shocking worldwide events in the last centuries. no reliable prevision on its personal or communitarian impact can be done. the consequences of respiratory fibrosis and psychiatric and mobility harm after prolonged isolated hospitalization the urology department implemented urgent adaptive measures. scheduled inpatient clinics were screened and resolved by telephone. the surgical activity radically decreased, non-urgent, or non-priority surgeries were canceled (fig. 4) . elective admissions were minimized. it may be appropriate to consider whether the telematic model can be integrated as part of the daily practice. we highlight the collaborative and engaging character of urologists. three residents and nine consultants joined multidisciplinary teams (three of them got infected), led by internal medicine, infectologists and pneumologists. we have evaluated the patients' medical status and complimentary assessment, modified treatment prescriptions, and supported them emotionally; we have also informed their families and other bureaucratic tasks. the remaining ones attended the scheduled activity. despite not having received specific training, as urologists, we did not find difficulties in wearing the personal protective equipments or adapting to protocols. the tremendous collaboration into multidisciplinary teams allowed proper clinical management. it is essential to find emotional support and relief since covid-19's wards are stressful and crowed. moreover, feelings of fear of contagion and threat to loved ones are prevalent, and it can lead to emotional isolation that must be avoided. we are effectively improving how to properly manage covid, thanks to the experience shared by extraordinarily engaged and unselfish healthcare professionals. its collaborative work in multidisciplinary teams allows overcoming the covid-19 pandemic. conflict of interest dr. lópez-fando lavalle reports personal fees from astellas pharma sa, personal fees from neomedic, personal fees from boston scientific, personal fees from wellspect, personal fees from coloplast, outside the submitted work. dr. moreno-guillén reports grants and personal fees from gilead sciences, grants and personal fees from viiv healthcare, personal fees from janssen cilag, grants and personal fees from msd, outside the submitted work. the rest of the authors have nothing to disclose. human and animal rights and informed consent not applicable. this article does not contain any studies with human or animal subjects performed by any of the authors. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china pandemia covid-19: impacto y reacción rápida de la urología urology in the time of corona clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection eau guidelines office rapid reaction group: an organisation-wide collaborative effort to adapt the eau guidelines 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in mild and severe cases of covid-19 virological assessment of hospitalized patients with covid-2019 hospitalization rates and characteristics of patients hospitalized with cdc clinical, laboratory and imaging features of covid-19: a systematic review and meta-analysis abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 infectious diseases society of america guidelines on the treatment and management of patients with covid-19 pharmacologic treatments for coronavirus disease 2019 (covid-19) a review covid-19) outbreak: what the department of radiology should know compassionate use of remdesivir for patients with severe covid-19 remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial effective treatment of severe covid-19 patients with tocilizumab why tocilizumab could be an effective treatment for severe covid -19 ? anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy propuesta de recomendaciones de manejo de fármacos anticoagulantes y antiagregantes en los pacientes graves con infección por covid-19 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-274497-tqceazdp authors: n. nuñez, luis fabian; santander-parra, silvana h.; de la torre, david i.; de sá, lilian r. m.; buim, marcos r.; astolfi-ferreira, claudete s.; piantino ferreira, antonio j. title: molecular characterization and pathogenicity of chicken parvovirus (chpv) in specific pathogen-free chicks infected experimentally date: 2020-07-25 journal: pathogens doi: 10.3390/pathogens9080606 sha: doc_id: 274497 cord_uid: tqceazdp chicken parvovirus (chpv) is an agent frequently associated with runting stunting syndrome (rss). this syndrome has been reported in association with chpv in many countries, including brazil; however, studies characterizing the virus on a molecular level are scarce, and chpv pathogenicity in day-old chicks remains unclear. the aim of the present work was to establish the molecular characteristics of chpv, determine the pathogenicity of chpv in spf chicks and detect and quantify chpv by qpcr in several tissues and chicks of different ages. the experimental challenge was performed at one day of age, and daily and weekly observations were performed and five birds from each experimental group (mock and infected birds) were euthanized to perform the different analysis. chpv genome copies were detected and quantified by qpcr in gut, spleen, thymus, kidney, pancreas, proventriculus and bursa. clinically, the infected group presented with diarrhea 24 h post-infection, which persisted until 42 days of age. the small intestine was distended, and its contents were aqueous and foamy. enteritis and dilated crypts with cyst shapes were observed in intestinal segments. acute pancreatitis associated with lymphocytic nodules, infiltrating lymphocytes and plasma cells between the pancreatic acinus was observed. koch’s postulate was demonstrated and the genetic characterization of the vp1 gene showed that the brazilian chpv isolate belongs to the chpv ii group. enteric disorders are considered the most important concern to poultry gut health due to their compromising effect on poultry growth. enteric disorders have a multifactorial etiology and are associated with bacteria, fungi, protozoa and viruses [1] [2] [3] [4] [5] [6] . runting-stunting syndrome (rss) is commonly reported in poultry production and is associated with several viruses, such as astrovirus, rotavirus, reovirus, coronavirus and parvovirus, and bacteria, including coccidia [7] [8] [9] [10] [11] [12] . the clinical manifestation of rss is characterized by ruffled feathers, diarrhea, cloacal pasting, apathy, depression, the sequences obtained in this work from the vp1 gene of chpv generated a sequence of 2061 bp, accession number mk440128.1, which corresponds to the complete gene sequence of vp1. the obtained vp1 nucleotide sequence was aligned and compared with previously published sequences of chpv. the analysis of complete vp1 nucleotide sequences showed high similarity of nucleotides (nt) and amino acids (aa) between the analyzed sequences ( table 1 ). the sequence of the isolated chpv showed 95-95.1% nt and 98-98.2% aa similarity to sequences from korea; 91.6-95% nt and 94.9-98.2% aa similarity to sequences from the united states; 91.8% nt and 96.7% aa similarity to sequences from china and 88.2% nt and 93% aa similarity to reference sequences from hungary. compared to tupv sequences, the obtained sequence showed low nt (77.9-79.7%) and aa (72.9-73.6%) similarity, as shown in table 1 . phylogenetic analyses showed two well-defined groups, a chpv group (100% bootstrap) and a tupv group (100% bootstrap). the sequence of the chpv isolate used in the present work clustered with a usa isolate (km598414.1) (bootstrap 86%) belonging to chpv group ii; the chpv reference sequence was grouped in a separate branch (97% of bootstrap) with the usa sequence (km598415.1; figure 1 ). in the experimental infection, depression, lethargy, somnolence, ruffled feathers and diarrhea were observed 12 h post-inoculation. after 48 h of infection, the animals presented with cloacal pasting, dirty and wet feathers at the cloacal region caused by watery feces and ruffled feathers ( figure 2 ). somnolence and lethargy increased, resulting in birds that moved less and had more apathy, in addition to an increase in diarrhea. the clinical signs of enteric disease, mainly diarrhea, were observed throughout the experiment ( table 2 ). the infected animals also showed differing characteristics of the feathers on their wings, namely, the wings were found folded in the outer direction. ten days after infection, animals in the two groups were observed to have different sizes, with the infected animals appearing smaller and stunted compared with animals from the mock group. the mock group did not show any clinical signs and exhibited normal movement and healthy appearance, as described above. outer direction. ten days after infection, animals in the two groups were observed to have different sizes, with the infected animals appearing smaller and stunted compared with animals from the mock group. the mock group did not show any clinical signs and exhibited normal movement and healthy appearance, as described above. the birds of the mock group did not show any macroscopic lesions in the organs at any age examined. the postmortem examination of the challenged birds showed distended coelomic cavity intestinal loops filled with aqueous and foamy content. the intestinal loops exhibited segmentations along the small intestine; there were dilated stretches as well as narrow ones, and the contents were liquid, containing foamy and undigested feed along the length of the intestine. the birds in all age groups presented with intestinal volvulus, in which there was rotation of the duodenal loop segment in the mesenteric axis that resembled a corkscrew ("j" appearance). the mesentery presented opacification in this segment, and there was atrophy of the pancreas. the birds showed persistence of the yolk sac. the rest of the organs did not show any macroscopic alteration (table 2; figure 3 ). pathogens 2020, 9, x for peer review the birds of the mock group did not show any macroscopic lesions in the organs at any age examined. the postmortem examination of the challenged birds showed distended coelomic cavity intestinal loops filled with aqueous and foamy content. the intestinal loops exhibited segmentations along the small intestine; there were dilated stretches as well as narrow ones, and the contents were liquid, containing foamy and undigested feed along the length of the intestine. the birds in all age groups presented with intestinal volvulus, in which there was rotation of the duodenal loop segment in the mesenteric axis that resembled a corkscrew ("j" appearance). the mesentery presented opacification in this segment, and there was atrophy of the pancreas. the birds showed persistence of the yolk sac. the rest of the organs did not show any macroscopic alteration (table 2; figure 3 ). the birds infected with chpv showed preserved villous:crypt ratios and mild to moderate hyperplasia of crypts with the presence of crypt bifurcations, which were dilated, elongated and tortuous in different evaluation periods. from the 7th to 42nd day after inoculation, the crypts of lieberkühn were lined by a squamous epithelium containing cellular debris and degenerated inflammatory cells that formed structures with a cyst shape; these structures were observed in the table 2 . clinical signs and macroscopic findings at postmortem examination of chickens from chpv infected group. the birds infected with chpv showed preserved villous:crypt ratios and mild to moderate hyperplasia of crypts with the presence of crypt bifurcations, which were dilated, elongated and tortuous in different evaluation periods. from the 7th to 42nd day after inoculation, the crypts of lieberkühn were lined by a squamous epithelium containing cellular debris and degenerated inflammatory cells that formed structures with a cyst shape; these structures were observed in the duodenum, jejunum and ileum (figure 4) . table 3 shows the distribution of histopathological parameters of the digestive system in both groups. between the 7th day and 28th day post-infection, necrosis of crypt cells was observed in all segments of the small intestine (p < 0.05). furthermore, there was an increased number of mitotic cells in segments of the duodenum and an increased number of intraepithelial lymphocytes from the 7th to 42nd day (p < 0.05). the density of lymphocytes and plasma cells found in the lamina propria of the duodenum was significant (p = 0.05) from the 7th to the 21st day after inoculation, that of the jejunum was significant from the 14th to 21st day (p < 0.05) after infection, and that of the ileum was significant from the 21st to 35th day after infection (p < 0.05). the pancreas showed a loss of zymogene granules; lymphocytic nodules that contained an infiltrate of lymphocytes and plasma cells between the pancreatic acinus were also present, indicating acute pancreatitis. lymphoplasmacytic mesenteritis was observed in the mesentery of the duodenal loop and the peripancreatic area, which is associated with pancreatitis. the birds of the mock group showed segments of the duodenum, jejunum and ileum within the standard of normal histology (table 3; figure 4 ). the qpcr assay for the detection and quantification of chpv showed that all analyzed animals were positive for chpv in all collected organs from the first day post-infection. peak titers of chpv was observed in the intestines, for the ileum, followed by the jejunum and duodenum. however, it was very interesting to note that replication of the virus seemed to commence at 2-3 days post-infection for the ileum followed by the jejunum and then the duodenum. interestingly, in this study, replication in the pancreas and an elevated number of genome copies were not observed. the gastrointestinal tract (duodenum, jejunum and ileum) showed the highest level of genome copies of chpv at day fourteen post-infection, and the viral concentration decreased through day 42. the pancreas, thymus, liver, proventriculus and kidney showed basal virus genome titer from the first day until the end of the experiment at 42 days old. the spleen had the highest viral concentration at day 21, after which the concentration decreased (table 4; figure 5 ). figure 5 . the figure compares the viral distribution in the days after infection with the chpv strain according to each organ/tissue. note that peak titer of chpv is greatest in the intestines. however, it is interesting to note that replication of the virus seems to commence at 2-3 days post-infection for the ileum followed by the jejunum and then the duodenum. interestingly, multiplication in the pancreas was not observed. in the present work, the pathogenicity, viral tissue distribution and molecular characterization of chpv in chicks from a strain isolated in brazil were determined with a demonstration of koch's postulates according to our previous description [21] . there are few complete genomes of chpv available, and the complete genome refers to the chpv reference strain abu-p1, which was followed figure 5 . the figure compares the viral distribution in the days after infection with the chpv strain according to each organ/tissue. note that peak titer of chpv is greatest in the intestines. however, it is interesting to note that replication of the virus seems to commence at 2-3 days post-infection for the ileum followed by the jejunum and then the duodenum. interestingly, multiplication in the pancreas was not observed. in the present work, the pathogenicity, viral tissue distribution and molecular characterization of chpv in chicks from a strain isolated in brazil were determined with a demonstration of koch's postulates according to our previous description [21] . there are few complete genomes of chpv available, and the complete genome refers to the chpv reference strain abu-p1, which was followed by viral genomes from korea [24] , the united states [4] and china [23] . thus, these genomes possess the classical conformation of parvovirus and contain structural and nonstructural proteins; based on their vp1 gene sequences, three groups of chpv can be identified [23] . the lack of chpv vp1 gene sequences could likely be related to the difficulty in chpv dna isolation for whole vp1 gene sequencing; however, the majority of published works have included a molecular characterization of the ns gene sequence [15, 16] . nevertheless, genetic information based on ns gene sequences is not sufficient for discriminatory genotyping because there are few differences among strains [30] . hence, we report the first molecular characterization of chpv in brazil based on complete vp1 gene sequence analyses, confirming that the chpv used in the experimental infection belongs to the chpv ii group. around the world, chpv has been reported and detected in healthy and sick chickens, but it has mainly been identified in chickens showing enteric disorders. this virus has the particularity that it is detected relatively more in chicks and young animals [15] [16] [17] [18] . chpv causes severe enteric problems that are characterized by the presence of diarrhea, high morbidity and mortality [5, 31] . in the field, outbreaks that affected chickens showed many pathological alterations in the intestine upon postmortem examination, showing mainly distended intestines filled with aqueous feces and foamy and undigested feed. moreover, the challenge with outbreaks in the field is the association of chpv with other enteric viruses or bacteria, which could decrease the quality of the gut integrity [4, 16, 22, 23, 31] . experimental infections with isolated chpv (abu-p1) have demonstrated that the virus causes enteric diseases, resulting mainly in chickens with diarrhea, cloacal pasting, impaired growth, runting and stunting [32] . as reported in the present investigation, the principal clinical signs present in the infected animals were also diarrhea, cloacal pasting, somnolence, apathy, ruffled feathers, impaired growth, runting and stunting. however, the chpv strain abu-p1 does not cause any macroscopic lesions in enteric tissues [22, 32] . interestingly, this investigation revealed the presence of intestinal volvulus characterized by the rotation of the duodenal loop segment in the mesenteric axis; thus, the duodenal loop was rolled, resembling a corkscrew and pancreatic atrophy was also observed. lesions were previously described in commercial chicken flocks affected with rss and reported by our own group [21] ; the duodenal loop presented the same features, demonstrating koch's postulates in relation to chpv and experimentally infected chickens. the enteric diseases caused by bacteria or viruses (rss) have the particularity of presenting alterations at the ciliated columnar epithelium, lieberkühn crypts, and in intestinal villi, which show atrophy and fusion or expansion of the lamina propria as a result of inflammatory infiltrates [7, 26, 33] . nevertheless, experimental infections with chpv (abu-p1) have reproduced rss, but microscopic alterations were not found in the intestines or in any other organ [22] . in the present investigation, we demonstrated the presence of crypt alterations, which were characterized principally by dilated crypts lined by a squamous epithelium that contained cellular debris and degenerate inflammatory cells, a cyst shape and crypt cell necrosis, all of which are present in the segment of the rolled duodenal loop and in other segments of the intestine; thus, these findings microscopically characterize the chpv strain isolated in brazil. chpv was detected initially in the intestinal content or in the intestinal wall [6, 16, 20, 34] , because the intestine is considered the primary target for enteric virus infection; however, other studies showed the presence of the virus in the spleen and pancreas [35] , suggesting another tropism of enteric viruses. these results were supported by qpcr targeting the chpv strain usp-362-3 [6] . furthermore, the presence of chpv was quantified and detected in the three segments of the small intestine (duodenum, jejunum and ileum) and increased after third day post-infection (p.i.) for ileum; after the sixth day for jejunum and after the seventh day for duodenum with high concentrations of genome copies per mg of tissue, however, in the thymus, liver, kidneys, pancreas, proventriculus and bursa, an increase in virus in the tissue was not detected. nevertheless, the peak of the genomic titer was observed between the first-and fourth-weeks post-infection. the presence of chpv in many organs until day 42 showed that the virus could persist for a long time, explaining the continuous reinfections through the presence of chpv in poultry litters [15] . the total weight of the infected animals showed a significant decrease compared to the mock group, which agreed with previously reported data from experimental infection with abu-p1 [22] and showed that the chpv strain isolated in brazil is an important virus causing enteric diseases. other studies should be performed to understand the interaction of chpv with the lymphopoietic organs (bursa of fabricius, spleen and thymus), the exocrine function of digestive organs and certainly the interaction of chpv with chicken immune system cells. a strain of chpv was isolated in spf chicken embryos as described previously and was designated usp 362-3 [36] . an aliquot of a macerated embryo from which the virus was isolated was transferred to a 2 ml microtube containing 1 ml of 0.1 m pbs at ph 7.4. the suspension was subjected to three freeze thaw cycles consisting of freezing at −80 • c for 10 min and thawing at 56 • c for one min; each cycle was followed by homogenization. the sample was centrifuged at 12,000× g for 30 min at 4 • c. the viral dna was extracted from 250 µl of the supernatant of the viral suspension using trizol (thermo fisher scientific, carlsbad, ca, usa) reagent according to the manufacturer's instructions. for molecular characterization of chpv, pcr was carried out in triplicate as described previously [4] , with some modifications. the amplified products were purified using gfxpcr dna and gel band purification kit (ge healthcare, piscataway, nj, usa) following the manufacturer's instructions. the purified dna was then ligated into the pcr™ 2.1-topo cloning vector (thermo fisher scientific, carlsbad, ca, usa), and the vector was transformed into e. coli top 10 competent cells according to the manufacturer's instructions. the bacteria were cultured on luria bertani (lb) agar containing ampicillin 50 µg/ml. three colonies were cultured in 3 ml of lb broth and shaken at 230 rpm for 20 h. plasmid dna was extracted from the lb broth bacterial suspensions using the qiaprep spin miniprep kit (qiagen, hilden, germany). the plasmid dna was then sequenced in the forward and reverse directions using a bigdye ® terminator v3.1 cycle sequencing kit (thermo fisher scientific, carlsbad, ca, usa) with m13 primers. sequencing reactions were performed with an abi 3730 dna analyzer (thermo fisher scientific, carlsbad, ca, usa). the obtained electropherograms were analyzed using geneious 11.1.4 software using de novo assembly. the consensus sequences were aligned and compared with other sequences of chpv available in genbank using the clustal w method in clustalx 2.0.11 software (european bioinformatics institute, saffron walden, uk). the similarity of nucleotides (nt) and amino acids (aa) was determined using bioedit 7.1.3. the phylogenetic tree was built using the neighbor-joining statistical method and p-distance substitution model with 1000 bootstraps of replications in the mega 7 software package [37] . eighty (n = 80) day-old spf chicks, supplied by ceva (ceva animal health, campinas, brazil), were divided into two groups of forty (n = 40) birds: group i was infected with 2 × 10 5 genome copies (gc) of chpv in 200 µl of pbs, as previously determined through qpcr [6] , and group ii was mock infected with 200 µl of sterile 0.1 m pbs ph 7.4. all chicks were challenged by gavage. both groups were maintained for 42 days in separate isolators under positive air pressure (alesco, campinas, sp, brazil), with water and food supplied ad libitum. the birds were maintained and used in accordance with the guidelines and the approval of the committee on the care and use of laboratory animal resources of the school of veterinary medicine, university of são paulo, brazil, under protocol number #2569/2012. the birds were observed daily and scored for clinical signs and mortality. the experimental challenge was performed at one day of age, and daily and weekly observations were performed (day 0 pre-inoculation; daily 1, 2, 3, 4, 5, 6 and 7 days post-inoculation; and weekly 14, 21, 28, 35 and 42 days post-inoculation). five birds (5) at each experimental phase/step were euthanized and subjected to necropsy examination. hereafter, each organ sample was collected separately, and selected organs included the duodenum, jejunum, ileum, proventriculus, pancreas, liver, spleen, bursa, thymus and kidney. the samples for qpcr were initially stored in liquid nitrogen and then stored at −80 • c until processing. a fragment of each organ listed above from the 7th to 42nd day was fixed in neutral-buffered 10% formalin and embedded in paraffin. sections of 5 µm thicknesses were prepared and stained with hematoxylin-eosin (h&e). the slides were examined by light microscopy. the intestinal segments were examined by observing the villous:crypt ratio, lieberkühn crypt morphology, intensity of mononuclear and polymorphonuclear infiltrate in the lamina propria, presence and distribution of lymphoid follicles, number of intraepithelial lymphocytes present in one hundred enterocytes, and the number of cells in mitosis observed in the crypts in three fields at 40×. the microscopic examination of fragments of intestine was semiquantitative according to the parameters described in table 3 . the organs, including the proventriculus, pancreas, spleen, bursa, liver, thymus and kidney, were examined for the presence of inflammatory infiltrate, cell degeneration and other microscopic lesions. three randomly selected birds were necropsied from both the infected and mock groups, as described above, and dna was extracted from 30 mg of tissue from each collected organ. chpv was detected and quantified using qpcr as described by nuñez et al. [6] . each organ was tested in duplicate, and absolute quantification of the chpv genome copies was performed. the weight, number of cells in mitosis and number of intraepithelial lymphocytes were evaluated using the mann-whitney u test. semiquantitative evaluations of the variables, namely, the presence of polymorphonuclear and mononuclear cells in the lamina propria and changes in crypt morphology, were evaluated using a chi-square test. the genetic characterization of the vp1 gene showed that the brazilian chpv isolate belongs to the chpv ii group. koch's postulate was demonstrated because rss was reproduced through detection of the "j" appearance in the duodenum of the infected group, as observed in field conditions. molecular characterization and typing of chicken and turkey astroviruses circulating in the united states: implications for diagnostics partial genome sequence analysis of parvoviruses associated with enteric disease in poultry molecular characterization of avian astroviruses host specificity and phylogenetic relationships of chicken and turkey parvoviruses enteric virus diversity examined by molecular methods in brazilian poultry flocks development of a sensitive real-time fast-qpcr based on sybr ® green for detection and quantification of chicken parvovirus (chpv) detection of rotaviruses and intestinal lesions in broiler chicks from flocks with runting and stunting syndrome (rss) detection of chicken astrovirus by reverse transcriptase-polymerase chain reaction development and evaluation of real-time taqman ® rt-pcr assays for the detection of avian nephritis virus and chicken astrovirus in chickens emergence of enteric viruses in production chickens is a concern for avian health capsid protein sequence diversity of avian nephritis virus chicken astrovirus as an aetiological agent of runting-stunting syndrome in broiler chickens presence of parvoviruses in the intestine of chickens showing stunting syndrome experimental infection of chicken embryos and day-old chickens with parvovirus of chicken origin high prevalence of turkey parvovirus in turkey flocks from hungary experiencing enteric disease syndromes samorek-salamonowicz, e. occurrence of chicken parvovirus infection in poland parvovirus host range, cell tropism and evolution recent progress in the characterization of avian enteric viruses determination and analysis of the full-length chicken parvovirus genome identification and phylogenetic diversity of parvovirus circulating in commercial chicken and turkey flocks in croatia molecular detection of chicken parvovirus in broilers with enteric disorders presenting curving of duodenal loop, pancreatic atrophy, and mesenteritis chicken parvovirus-induced runting-stunting syndrome in young broilers genetic characterization of three novel chicken parvovirus strains based on analysis of their coding sequences development of an enzyme-linked immunosorbent assay to detect chicken parvovirus-specific antibodies development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses a novel tool for specific detection and quantification of chicken/turkey parvoviruses to trace poultry fecal contamination in the environment chicken parvovirus viral loads in cloacal swabs from malabsorption syndrome-affected and healthy broilers avian parvovirus: classification, phylogeny, pathogenesis and diagnosis genetic characterization of parvoviruses circulating in turkey and chicken flocks in poland naturally occurring parvoviral infection in hungarian broiler flocks investigation into the aetiology of runting and stunting syndrome in chickens periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on the farms phylogenetic diversity of avian nephritis virus in hungarian chicken flocks detection of enteric viruses in pancreas and spleen of broilers with runting-stunting syndrome (rss) isolation and molecular characterisation of chicken parvovirus from brazilian flocks with enteric disorders mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare that there is no conflict of interest in this manuscript. key: cord-266156-xmf4emln authors: miller, tyler e.; garcia beltran, wilfredo f.; bard, adam z.; gogakos, tasos; anahtar, melis n.; astudillo, michael gerino; yang, diane; thierauf, julia; fisch, adam s.; mahowald, grace k.; fitzpatrick, megan j.; nardi, valentina; feldman, jared; hauser, blake m.; caradonna, timothy m.; marble, hetal d.; ritterhouse, lauren l.; turbett, sara e.; batten, julie; georgantas, nicholas zeke; alter, galit; schmidt, aaron g.; harris, jason b.; gelfand, jeffrey a.; poznansky, mark c.; bernstein, bradley e.; louis, david n.; dighe, anand; charles, richelle c.; ryan, edward t.; branda, john a.; pierce, virginia m.; murali, mandakolathur r.; iafrate, a. john; rosenberg, eric s.; lennerz, jochen k. title: clinical sensitivity and interpretation of pcr and serological covid‐19 diagnostics for patients presenting to the hospital date: 2020-08-28 journal: faseb j doi: 10.1096/fj.202001700rr sha: doc_id: 266156 cord_uid: xmf4emln the diagnosis of covid‐19 requires integration of clinical and laboratory data. severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. our goal was to examine the clinical sensitivity of two most common sars‐cov‐2 diagnostic test modalities, polymerase chain reaction (pcr) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. we conducted a single‐center, retrospective study. to derive clinical sensitivity of pcr, we identified 209 pcr‐positive sars‐cov‐2 patients with multiple pcr test results (624 total pcr tests) and calculated daily sensitivity from date of symptom onset or first positive test. clinical sensitivity of pcr decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%‐71% from days 9 to 11, and 30% at day 21. to calculate daily clinical sensitivity by serology, we utilized 157 pcr‐positive patients with a total of 197 specimens tested by enzyme‐linked immunosorbent assay for igm, igg, and iga anti‐sars‐cov‐2 antibodies. in contrast to pcr, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after day 7, >80% after day 12, and 100% by day 21. taken together, pcr and serology are complimentary modalities that require time‐dependent interpretation. superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection. while many measures to mitigate the multifactorial impact of covid-19 are being implemented, one critical component of this strategy is the widespread testing and identification of individuals currently or previously infected by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the delivery of effective care and mitigation of infection depend on the performance of sars-cov-2 diagnostic testing and the clinical interpretation of results. the lack of a full understanding of the natural history and immunopathogenesis of covid-19 infection creates unique challenges in the implementation of diagnostic testing strategies. sars-cov-2 diagnostic assays have fixed technical performance metrics (eg, sensitivity and specificity). clinical sensitivity depends on more than technical performance and is also a function of pre-analytical variables and the disease state of the patient. interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. the goal of this study is to examine the clinical sensitivity and provide insights into the interpretation of the two most common sars-cov-2 diagnostic test modalities: polymerase chain reaction (pcr) and serology. laboratory-based diagnosis of active sars-cov-2 infection relies on the direct detection of virus-specific nucleic acids, most commonly obtained from the nasopharynx of infected patients. indirect markers of infection include the detection of sars-cov-2 specific antibodies, generated as part of the human immune response to the virus. serologic testing holds promise as a blood-based diagnostic aid, as a marker of viral exposure, and potentially as an indicator of protective immunity. understanding the presence of these biomarker in relationship to one another over the natural course of infection is required to effectively utilize these available diagnostic tests in clinical practice. [1] [2] [3] here, we share our experience of sars-cov-2 pcr sensitivity and separately obtained igm, iga, and igg sensitivity of an in-house enzyme-linked immunosorbent assay (elisa) during the natural course of disease in a cohort of patients presenting to the hospital. the project was conducted within the clinical laboratories of the massachusetts general hospital (mgh), a clinical laboratory improvement amendments-certified laboratory. the study was designed as a single-center, retrospective review of pcr results and serology data. pcr results were obtained between 3 march 2020 and 15 april 2020 and we superimposed serology data obtained from confirmed covid-19 positive patients as part of ongoing clinical validation studies of an elisa for regulatory approval. the study was conducted with approval from the mass general brigham institutional review board. we also used previously published data as a comparison dataset (wölfel et al 4 ). nucleic acid testing was performed as part of clinical care at mgh using three real-time pcr assays, each of which received eua by the fda. our laboratory-developed realtime pcr assay uses the centers for disease control and prevention (cdc) primers targeting regions of the n gene of sars-cov-2, the cobas sars-cov-2 test performed on the cobas 6800 (roche) targets regions of the orf1a and e genes, and the xpert xpress sars-cov-2 assay run on the genexpert infinity (cepheid) targets regions of the n and e genes. choice of which testing platform to use was determined by access to reagents available at the time of clinical testing provided for patient care. our laboratory-developed assay was validated to detect sars-cov-2 at or above 5 copies/µl with 100% technical sensitivity and specificity. for commercial assays, we internally validated the assays and found 100% technical sensitivity and specificity. within our validation cohort of known positive patients, we found 100% concordance between all three platforms. despite excellent (technical) performance characteristics, pre-analytical factors may decrease the performance of viral detection. these factors may include timing during the course of infection, improper sampling, specimen handling and others. an in-house elisa developed by massachusetts general hospital (boston, ma) and the ragon institute of mgh, mit, and harvard (cambridge, ma), was used to measure igg, iga, and igm antibodies that target the sars-cov-2 receptor binding domain (rbd) within the spike protein. the optical density (od) was read at 450 and 570 nm on a plate reader. od values were adjusted by subtracting the 570 nm od from the 450 nm od. to estimate antibody titers, we generated isotype-specific standard curves using anti-sars-cov-1/2 monoclonal igg, iga, and igm antibodies. we used this standard curve to calculate the concentration of anti-rbd igg, iga, and igm expressed in u/ ml. positive specimens were identified as those that had an u/ml three standard deviations above the mean of negative control specimens. (data not shown january 2020) when no detectable antibody responses would be expected. the date of symptom onset was determined by review of the electronic medical record by physician investigators. the onset date was determined in by one of two ways from the medical record: (a) as explicitly defined in the chart from an md-written note as "covid-19+ date of symptom onset" or (b) determined from md and non-md notes that stated date of symptom onset for any covid-19-related symptom that developed acutely and was new from baseline (fever, chills, loss of smell or taste, body aches, fatigue, runny nose, congestion, sore throat, cough, and shortness of breath). cases for which the date of symptom onset could not be determine were excluded from analysis (21/359, or 5.8%, of all pcr and serology cases). clinical laboratory test results are stored in a laboratory information management system connected to the electronic medical record. we performed two data queries with different end-dates: an initial pcr-query (3 march 2020 to 15 april 2020) and a second pcr-query (3 march 2020 to 4 may 2020). the initial pcr-query was performed to delineate clinical sensitivity over time, and analysis was restricted to patients with multiple pcr test results and at least one positive (ie, the most informative subset). these patients were considered confirmed covid-19 positive and taken as true positives. all pcr test results regardless of specimen type were used to confirm a patient as sars-cov-2 positive; however, only pcr test results from a np-swab specimen were used for sensitivity calculations. the resulting dataset consists of 624 pcr results from 209 unique subjects. in this subset, 83% were inpatients, 13% were patients from the ed, and 4% were outpatients. for each specimen, we manually mapped date of symptom onset and all test results on a daily scale and calculated: (a) the time (in days) from the date of symptom onset to the date of specimen collection, (b) the duration from the first positive pcr test result to any subsequent positive pcr test result, and (c) clinical detection rates (pcr-positive over total tests per day) at each day in relation to symptom onset or first pcr-positive, respectively. we modeled a linear daily regression trend after first positive pcr test, to estimate the time when pcr sensitivity reaches zero (foot-point analysis). the second pcr query was performed to capture hospital-wide testing metrics, and to assess whether the above subset analysis of sars-cov-2 patients with multiple pcr results is representative of the entire tested population in our setting. we extracted admission date, encounter, discharge date (when applicable), age, gender, and collection types and times, reporting dates and times along with results from all sars-cov-2 pcr tests. by clinical encounter, 55.5% of orders originated in the outpatient setting, 12.1% originated in the emergency department (ed), and 32.2% of orders originated from the inpatient setting. the overall pcr positivity rate was 27.0% (n = 3163/11 703) in unique individuals and 28.3% of all tests performed (n = 4320/15 251; table 1 ). all test results were used for calculations of test number over time, positivity rate and age as well as gender calculations ( figures s1 and s2) . to compare our data of mainly hospitalized patients to a population with mild disease, we used data derived from wölfel et al, 4 in which patients with a known exposure were instructed to present to the clinic at the first sign of symptoms. a positive pcr was necessary for inclusion into the study. we applied the validated limit of detection of our laboratory-developed assay (5 copies/µl) to the data derived from wölfel et al, 4 and used the same calculations for time-dependent clinical sensitivity for pcr. serologic analysis of igm, iga and igg status was performed in a subset of the above sars-cov-2 pcr-positive patients for which we had excess material in the mgh core laboratories for clinical validation studies. for each sample, we determined the days post symptom onset at the collection date and calculated daily sensitivity for each antibody isotype as well as detection rate of any isotype. although serology is currently considered a diagnostic aid (as opposed to a primary diagnostic test), we calculated sensitivity as the number of seropositive samples over the total number of tested samples from patients with the disease (ie, who tested positive by pcr). we plotted the sensitivity for both test modalities (pcr and serology) as percentages per overlapping 5-day leading intervals against the days since symptom onset. statistical analysis consisted of fisher's exact test (association of sars-cov-2 status with dichotomous factors), χ 2 with yates correction, or t test (comparison of means). sars-cov-2 viral rna levels decline over the course of infection. 4 this decline of rna levels clearly impacts the clinical sensitivity of pcr testing. it is not possible to determine the false negative rate of the pcr test from patients with a single pcr result. therefore, we identified patients | 5 with multiple pcr tests who had at least one positive test result (considered true positives). the resulting dataset is composed of 624 test results from 209 patients (6.6% of all pcr-positive patients, table 1 ). we compared this subset to all tested patients ( figure s1 , table s1 ) and contingency analysis of the multi-pcr vs. all single pcr-positive patient subset showed no significant differences in age, gender, and test type (table s1, figure s2 ). thus, we consider the multi-pcr subset demographically representative of our tested patient population. to derive clinical sensitivity over disease course, we needed to define an anchor point. we chose two different anchor points: symptom onset (subjective) and first positive test (objective). first, we examined the time course by anchoring test results to the day of symptom onset (figures 1 and s3) . clinical sensitivity remains above 90% for the first 5 days after symptom onset. in our cohort of patients presenting to the hospital, patients were admitted to the hospital a median of 8 days after symptom onset (interquartile range: 4-16 days; mean: 10 days), and between days 6 and 8, the clinical sensitivity of pcr ranged from 84% to 76%. on subsequent days the sensitivity decreases, and at day 18 the sensitivity decreases below 50%. we also compared sensitivity data from an earlier study of mildly symptomatic patients 4 and noted a steeper pcr sensitivity decline, consistent with viral levels dropping more quickly in this population ( figure s4 ). this data provide sensitivity estimates at the time of presentation (eg, a patient presents at day 10 after symptom onset). second, we also modeled how pcr sensitivity decreases over time after the first positive pcr test (figures s3 and s5 ). regression modeling and extension of pcr positivity decay (foot-point analysis) revealed that in our cohort, npswab specimens could stay pcr-positive beyond 20 and up to 40 days ( figure s5 ). seroconversion is also a dynamic response to the virus and assay sensitivity changes over time. to assess the sensitivity of our serology assay over time, we tested for igm, igg, and iga antibodies against the rbd of sars-cov-2 spike protein in 157 sars-cov-2 pcr-positive patients using an in-house elisa (table 1) . for some patients, we were able to assess serology at multiple time points (n = 591 total isotype tests on 197 total specimens). anchoring our serologic results to days after symptom onset shows that seroconversion starts as early as symptom onset, is detectable in 50% of subjects after day 7, and continues to increase with >80% of patients showing seropositivity after day 12 (tables 2 and s2 ; figures 1 and s4 ). in the subset of patients with multiple serological tests we saw isotype switching occur as short as 1-4 days, consistent with recent reports. 5 of note, we detected iga prior to igm or igg in a number of individuals, with two subjects having only detectable iga within 3 days of symptom onset. we also documented cases of igg positivity prior to igm or iga, highlighting the utility of measuring seroconversion using all three isotypes (table s3 , figure s6 ). the superimposition of serologic sensitivities with pcr sensitivities shows that, after day 7, seroconversion is a reliable diagnostic aid indicating recent or prior infection (figure 1 ). we present the dynamic clinical sensitivity of sars-cov-2 pcr and our serology platform in patients presenting to the hospital. we performed this single-center, retrospective analysis to share real-world evidence that can inform interpretation of pcr and serologic testing. we focused on assessing the clinical utility of both modalities in conjunction by direct superimposition of both sensitivity time courses. the direct superimposition shows that serology can function as a reliable diagnostic aid indicating recent or prior infection-in particular at times when pcr sensitivity is lower than 70%. our findings emphasize that understanding the specific sensitivity kinetics of both modalities is paramount for interpretation and effective utilization of sars-cov-2 diagnostics. there is considerable interest in moving sars-cov-2 diagnostics to evidence-based principles. [6] [7] [8] [9] while clinicians await formal guidance from large, prospective, multi-center studies-which will be challenging during the ongoing pandemic-there is considerable uncertainty surrounding sars-cov-2 diagnostics in clinical practice. 3,10-13 using available published data [3] [4] [5] [6] [7] 12, 14 and data presented here from our hospital, we offer the following five diagnostic principles for consideration: 1. in symptomatic patients, all interpretations are anchored on days post symptom onset. understanding performance characteristics of sars-cov-2 diagnostics over the course of the infection is key to interpretation of results. we provide two distinct approaches to anchor interpretation over the course of infection: subjective (date of symptom onset) and objective (first pcr-positive result). both approaches are valid and have limitations. for example, the quality of patient histories is variable and in many cases the day of symptom onset is unknown or cannot easily be reconstructed. as a practical suggestion, to make this data easily obtainable and searchable, we recommend placing the date of symptom onset (when available) in the front page of the (electronic) medical record of patients diagnosed with covid-19. 2. pcr is the diagnostic gold standard during acute infection. pcr testing using consensus primers has an estimated specificity of >99%. 15 based on early reports from wuhan 16 the overall clinical sensitivity is reported around 70%. we found a clinical sensitivity around 95% in the first 5 days after symptom onset and although pcr is an imperfect standard, concurrent igm/iga/igg antibody assessment in the first 5 days post symptom onset does not significantly aid in rendering a current diagnosis; at no point during active infection should serology replace pcr for diagnosis. 3. clinical sensitivity of pcr decreases with days post symptom onset. in clinical practice many symptomatic patients present to medical care after day 1 of symptom onset. our data show pcr sensitivity decreases with days post symptom onset ( figure 1 ) and with days post first pcr-positive test result ( figure s5) . notably, some patients may have an initial pcr-negative result at presentation ( figure s3 ). our data also indicate that severely ill patients (many patients in our cohort) remain pcrpositive for a longer period than mildly ill patients (patients in the wölfel et al 4 cohort figure s4 ). both time since symptom onset and disease severity may be key elements for the interpretation of pcr results. 4. serological assay sensitivity increases with days post symptom onset. by positivity alone, in our cohort, seropositivity surpasses pcr positivity after days 8-10 post symptom onset. remarkably, we find seroconversion does not follow the typical kinetics of igm antibodies followed by class-switched igg and iga antibodies. rather, all appear simultaneously at a cohort level, with igg or iga seropositivity preceding igm responses in some cases. supporting this are other studies that report overall low igm responses to sars-cov-2 that are often preceded by igg. 14, 17 these data highlight the benefit of measuring all three anti-sars-cov-2 antibody isotypes to maximize sensitivity. in the case of sars-cov-2, it is unknown for how long igm, iga, or igg antibodies remain detectable after infection. it is important to note that a positive serologic result for igm, iga, and/or igg does not conclusively indicate that a patient's presenting symptoms are due to a current sars-cov-2 infection. distinguishing a prior versus an acute infection by serology will require isotype-specific interpretation, serial serologic testing to demonstrate seroconversion, and integration with clinical data. 5. negative results do not completely preclude sars-cov-2 infection. ruling out sars-cov-2 infection remains challenging. supplementing pcr results with serologic assessment can increase sensitivity (serology as a diagnostic aid). however, our data clearly show that there is a window period (day 6-12 from symptom onset) when clinical sensitivity of pcr and serological assays are below 90%. in a symptomatic patient, if multiple pcr tests are negative and serological results after 8-12 days are also negative, we believe the likelihood of active sars-cov-2 infection to be low. clinical judgment is needed in this situation as there are rare scenarios where pcr negativity may be due to disease at a different anatomic site and/or serologic negativity may be due to an immunocompromised state. limitations in our study include relatively small numbers, a retrospective design, and selection bias due to the specific setting and testing practice. we evaluated symptomatic, mostly hospitalized patients and we cannot derive recommendations for asymptomatic or mildly symptomatic patients from our data. due to limited availability of tests and time constraints during an ongoing pandemic, we did not perform daily sampling. date of symptom onset is not consistently available, subjective, and affected by recall biases-yet, it represents a useful anchor point for disease time course in symptomatic patients. notably, the eclipse period ranges from 2 to 14 days 18-21 and some patients already mounted a serologic response at the time of presentation, which can be taken as an argument for the early y-axis deviation from zero in the serology curve ( figure 1 ) and a confirmation of date of symptom onset as an imperfect marker. we caution that the presented serology data are specific to our elisa, and we cannot extrapolate to anti-sars-cov-2 antibody responses in general. nonetheless, other publications indicate that the time courses are comparable. 14, 17, 22 our serological studies measured antibodies to the rbd of sars-cov-2. we chose this viral antigen because of its specificity to sars-cov-2, and because anti-rbd antibodies are typically neutralizing. plaque reduction neutralization tests are the gold standard for assessing neutralizing ability, [23] [24] [25] [26] and ongoing studies are in progress to confirm anti-rbd antibodies are neutralizing in sars-cov-2 infection. 27 some commercially available assays measure the more abundant nucleocapsid protein, which may increase sensitivity to detect a serologic response early in the course of infection, therefore, shifting the seroconversion curve to the left. however, antibodies to nucleocapsid protein are unlikely to provide protective immunity as nucleocapsid protein is inaccessible to antibodies in an intact virus. therefore, serology results also depend on the specific antigen employed in testing. finally, our multi-pcr cohort and serologic patient cohort are largely non-overlapping pcrpositive patients (n = 20/209). to enable additive sensitivity calculations from combined pcr and serology assays, prospective and systematically obtained repeated parallel pcr and quantitative serologic data will be necessary. our real-world data outline the strengths and weaknesses of two sars-cov-2 test modalities over the natural course of infection. we hope these data, in conjunction with the five diagnostic principles for consideration, will contribute to effective utilization and interpretation of covid-19-related laboratory data for patient care. diagnostic testing for severe acute respiratory syndrome-related coronavirus 2: a narrative review profiling early humoral response to diagnose novel coronavirus disease (covid-19) interpreting diagnostic tests for sars-cov-2 virological assessment of hospitalized patients with covid-2019 temporal dynamics in viral shedding and transmissibility of covid-19 evidence based management guideline for the covid-19 pandemic -review article covid-19 diagnosis and management: a comprehensive review laboratory diagnosis of covid-19: current issues and challenges covid-19 diagnostics in context sars-cov-2 serology: test, test, test, but interpret with caution covid-19 pandemic-a focused review for clinicians multitiered screening and diagnosis strategy for covid-19: a model for sustainable testing capacity in response to pandemic waiting for certainty on covid-19 antibody tests -at what cost? antibody responses to sars-cov-2 in patients with covid-19 us cdc real-time reverse transcription pcr panel for detection of severe acute respiratory syndrome coronavirus 2 detection of sars-cov-2 in different types of clinical specimens profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan 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 incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data antibody responses to sars-cov-2 in patients of novel coronavirus disease potent neutralizing antibodies from covid-19 patients define multiple targets of vulnerability identification of a critical neutralization determinant of severe acute respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model potent neutralization of sars-cov-2 by human antibody heavy-chain variable domains isolated from a large library with a new stable scaffold. mabs potent human neutralizing antibodies elicited by sars-cov-2 infection the authors thank all patients and laboratory staff for making this study possible. cr3022 reference sequences, cr3022-iga and cr3022-igm isotypes, and elisa protocols were kindly provided by stephanie fischinger, caroline atyeo, and matthew slein from the laboratory of dr galit alter (ragon institute). we thank yael heher md for excellent discussions. elisa protocols were developed and optimized with the laboratory of dr alejandro balazs (ragon institute). this work is also in part supported by nih (ro1 ca225655) to jkl, centers for disease control and prevention u01ck000490 (etr, rcc), nigms training grant t32 gm007753 (bmh, tmc), nih r01 ai146779 (ags). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institute of health or any other organization. dr gelfand reports personal fees from henry schein inc, outside the submitted work; dr turbett reports grants from centers for disease control and prevention, outside the submitted work; dr anahtar reports personal fees and other from day zero diagnostics, outside the submitted work; dr ryan reports grants from cdc, during the conduct of the study. dr. branda reports grants from zeus scientific, grants from biomerieux, grants from immunetics, personal fees from t2 biosystems, personal fees from diasorin, personal fees from roche diagnostics, grants from bay area lyme foundation, grants from lyme disease biobank foundation, outside the submitted work. key: cord-274860-7ec2jcoq authors: salazar, eric; christensen, paul a.; graviss, edward a.; nguyen, duc t.; castillo, brian; chen, jian; lopez, bevin valdez; eagar, todd n.; yi, xin; zhao, picheng; rogers, john; shehabeldin, ahmed; joseph, david; masud, faisal; leveque, christopher; olsen, randall j.; bernard, david w.; gollihar, jimmy; musser, james m. title: significantly decreased mortality in a large cohort of covid-19 patients transfused early with convalescent plasma containing high titer anti-sars-cov-2 spike protein igg date: 2020-11-04 journal: am j pathol doi: 10.1016/j.ajpath.2020.10.008 sha: doc_id: 274860 cord_uid: 7ec2jcoq coronavirus disease 2019 (covid-19) convalescent plasma has emerged as a promising therapy and has been granted emergency use authorization by the u.s. food and drug administration (fda) for hospitalized covid-19 patients. we recently reported results from interim analysis of a propensity-score matched study suggesting that early treatment of covid-19 patients with convalescent plasma containing high titer anti-spike protein receptor binding domain (rbd) igg significantly decreases mortality. we here present results from 60-day follow up of our cohort of 351 transfused hospitalized patients. prospective determination of elisa anti-rbd igg titer facilitated selection and transfusion of the highest titer units available. retrospective analysis by the ortho vitros igg assay revealed a median signal/cutoff (s/c) ratio of 24.0 for transfused units, a value far exceeding the recently fda-required cutoff of 12.0 for designation of high titer convalescent plasma. with respect to altering mortality, our analysis identified an optimal window of 44 hours post-hospitalization for transfusing covid-19 patients with high titer convalescent plasma. in the aggregate, the analysis confirms and extends our previous preliminary finding that transfusion of covid-19 patients soon after hospitalization with high titer anti-spike protein rbd igg present in convalescent plasma significantly reduces mortality. coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused massive societal disruption and death globally. as of september 27, 2020, there have been more than 33 million covid-19 cases causing in excess of 1,000,000 deaths worldwide. 1 the united states has many areas where rising case rates continue to threaten multiple populations. very few effective treatments exist (https://www.covid19treatmentguidelines.nih.gov/, both links last accessed september 24, 2020), although hundreds of registered clinical trials are ongoing, including several phase 3 vaccine trials (https://www.nytimes.com/interactive/2020/science/coronavirusvaccine-tracker.html, subscription required). we and others have published safety and efficacy outcomes in patients who were transfused with covid-19 convalescent plasma. [2] [3] [4] aggregated available evidence stimulated the u.s. food and drug administration (fda) in late august 2020 to grant emergency use authorization (eua) for covid-19 convalescent plasma therapy (https://www.fda.gov/media/141477/download, last accessed september 24, 2020). in our previous study, interim analysis revealed that, relative to matched controls, patients transfused with convalescent plasma containing high titer anti-spike protein receptor binding domain (rbd) igg within 72 hrs of hospital admission had significantly reduced mortality at 28 days post-transfusion. 3 to further investigate these observations, and to address limitations inherent in an interim analysis, we here present results from 60-day follow up of our entire cohort of 351 transfused patients. the data confirm our previous findings that transfusion of patients soon after hospital admission with high titer anti-spike protein rbd igg present in convalescent plasma significantly decreases mortality. we analyzed data from patients cared for in all eight houston methodist hospitals from march 28, 2020, through september 14, 2020 , with the approval of the houston methodist research institute ethics j o u r n a l p r e -p r o o f review board and with written informed consent of the patient or legally authorized representative. details of the study, including inclusion and exclusion criteria, and criteria for the transfusion of multiple units have been described. 3 detailed protocols for convalescent plasma collection and anti-spike protein titer assessment have been described. 3, 5, 6 covid-19 convalescent plasma units were selected for transfusion based on antispike ectodomain and rbd igg elisa titers available on donor units obtained from april 7, 2020 onward. we previously published that plasma with an anti-rbd igg titer of ≥1:1350 corresponds to an ~80% probability of a live virus in vitro neutralization titer of ≥1:160. 7 this titer is the value initially recommended by the fda for transfusing covid-19 patients. 8 to facilitate the need for increased donor unit assessment, we standardized our elisa to four plasma dilutions: 1:50, 1:150, 1:450, and 1:1350. to select the highest titer unit available, elisa results were ranked based on highest titer and, subsequently by highest optical density at dilution 1:50. patients were transfused with the abocompatible convalescent plasma unit with the highest titer and highest optical density at dilution 1:50 available. frozen serum samples were assessed retrospectively with the ortho vitros igg assay (raritan, nj) according to manufacturer's instructions. we analyzed patients who met a 60-day outcome defined as having outcome data available 60 days post-transfusion (cases) and 60 days post-hospitalization (controls). control patients enrolled in other clinical trials were excluded from the analysis. patients discharged before day 60 were presumed to be on room air after discharge unless otherwise noted in the electronic medical record. baseline characteristics for covid-19 patients who met the 60-day outcome definition are shown in supplemental table s1 . we conducted a one-to-many nearest neighbor propensity score matching analysis without replacement using an initial ratio of case:control=1:3 and caliper of ≤1 between patients having plasma j o u r n a l p r e -p r o o f transfusion (cases) versus patients who did not have plasma transfusion (controls). the primary matching criteria included age (categorical, <30, 30-39, 40-49, 50-59, 60-69, 70-79, ≥80) , sex, bmi (+/-30), diabetes, hypertension, chronic pulmonary disease, chronic kidney disease, hyperlipidemia, coronary disease, and baseline ventilation requirement within 48 hrs of admission, use of any steroid, azithromycin, hydroxychloroquine, remdesivir, ribavirin, and tocilizumab. a secondary propensity score matching was conducted based on the ventilation status at day 0, defined as the day of transfusion for cases and the corresponding day in the hospitalization course for controls, using a case:control ratio of either 1:2 or 1:1 and caliper ≤1. 9 propensity score matching adjusts for the influences of potential confounding factors and minimizes bias in estimating outcome effect. 10 the approach has particular advantage in lieu of randomized controlled trials. the primary outcome (mortality within 60 days post-day 0) was displayed by kaplan-meier curves. differences between groups were compared with the log-rank test. cox proportional hazards modeling (with clustered sandwich estimator option for the matched cluster in the propensity-matched cohorts) was performed to determine the characteristics associated with the overall mortality within 28 days and 60 days. variables for the multivariable models were selected based on potential clinical relevance and using stata's lasso technique with cross-validation. 11, 12 receiver operating characteristic (roc) curve analysis with youden index was used to identify the optimal time (in hours) from admission-to-transfusion of first unit that discriminates 60-day mortality in patients that received covid-19 convalescent plasma. 13 roc analysis with youden index allows for the determination of the optimal cut point for continuous variables, at which the combination of the sensitivity and specificity of the evaluated variable is maximized. generalized linear modeling (glm) and multinomial logistic regression with a cluster variance estimator were also used to evaluate several exploratory endpoints. the evaluated covariates included supplemental oxygen requirements (room air, low-flow oxygen delivery, high-flow oxygen delivery, noninvasive positive pressure ventilation, mechanical ventilation, extracorporeal membrane oxygenation (ecmo), or death) at day 7, day 14, day 28, and day 60 post-transfusion; clinical improvement relative to day 0; intensive care unit (icu) stay requirement; icu length of stay; mechanical ventilation j o u r n a l p r e -p r o o f requirement; length of mechanical ventilation requirement; length of supplemental oxygen requirement; and inflammatory marker levels (interleukin-6, c-reactive protein, ferritin, fibrinogen, d-dimer) at day 7. clinical improvement relative to day 0 was defined as a 1 point improvement in ordinal scale [1, discharged (alive) ; 2, hospitalized, not requiring supplemental oxygen but requiring ongoing medical care (for covid-19 or otherwise); 3, hospitalized, requiring low-flow supplemental oxygen; 4, hospitalized, on non-invasive ventilation or high-flow oxygen devices; 5, hospitalized and on invasive mechanical ventilation or ecmo; 6, death]. all analyses were performed with stata version 16.1 (statacorp llc, college station, tx, usa) or the r statistical computing environment (http://www.rproject.org/, last accessed september 24, 2020). a p-value of ≤0.05 was considered significant. we had 5,297 hospitalized covid-19 patients available for analysis, 353 of whom were transfused with covid-19 convalescent plasma. two of the 353 patients received plasma without a titer assessment prior to transfusion, and these patients were excluded from the overall analysis, resulting in a cohort of 351 transfused evaluable patients. relative to non-transfused patients, transfused patients were significantly younger, predominantly male, predominantly hispanic, had a higher bmi, lower rates of comorbidities (specifically, chronic pulmonary disease, chronic kidney disease, hyperlipidemia, and coronary disease, but not hypertension and diabetes), a higher requirement for supplemental oxygen, and higher inflammatory biomarker concentrations. d-dimer was significantly lower in the transfused cohort at baseline by 0.2 fibrinogen equivalent units. use of steroids, azithromycin, remdesivir, and tocilizumab was more common among the transfused cohort (supplemental table s1 ). among 351 transfused patients included in the study, only seven (2.0%) had adverse events deemed related to plasma transfusion. six events were classified as allergic transfusion reactions and five of these six were mild and included only a transient rash. one patient developed transient worsening of j o u r n a l p r e -p r o o f shortness of breath that resolved with diphenhydramine. one case of possible transfusion-associated circulatory overload occurred, with associated transient worsening of dyspnea that improved with furosemide. these two events were deemed to be significant adverse events. thus, among the 351 transfused study patients, only two (0.6%) significant adverse events were deemed related to plasma transfusion. univariate and multivariate cox proportional hazards modeling assessing factors associated with a higher risk of death within 60 days post-transfusion day 0 was performed for all covid-19 patients admitted to our eight hospitals during the study period for whom data were available (supplemental tables s2 and s3). factors associated with a higher risk of death in the multivariate analysis included age, male sex, diabetes, chronic kidney disease, worst ventilation status within 48 hrs of admission, and/or administration of any steroids or tocilizumab. neither abo blood type, race, nor ethnicity were associated with higher risk of death in the multivariate analysis. importantly, the covariates that had a significant association with risk of death were included in the propensity score matching algorithm. we did not include baseline inflammatory concentrations in the multivariate analysis and in the propensity score matching algorithm because of the high proportion of missing data. most transfused patients (278/351; 79%) received only one ~300 ml unit of covid-19 convalescent plasma. the great majority of patients received an initial or sole unit of convalescent plasma with anti-rbd igg titer of ≥1:1350 (321/351; 91%); 24 patients received an initial or sole unit of convalescent plasma with an anti-rbd igg titer >1:150 but <1:1350; six patients received an initial or sole unit of convalescent plasma with anti-rbd igg titer of <1:150. for patients who received a second unit of convalescent plasma, 71 (71/75; 95%) received a second unit with an anti-rbd igg titer ≥1:1350, and four (4/75; 5%) patients received a second unit with an anti-rbd igg titer >1:150 but <1:1350. the fda issued an eua for convalescent plasma transfusion of covid-19 patients on august 23, 2020. the agency's guidance is to use convalescent plasma units with a signal/cutoff (s/c) level of >12, as defined by the ortho vitros igg test (https://www.fda.gov/media/141477/download, last accessed september 24, 2020). for 278 of the 351 (79%) initial plasma units transfused, a sample was available for retrospective assessment of anti-sars-cov-2 igg titer by the ortho vitros igg test. the median igg s/c ratio was 24.0 (range=0.01-35) and only seven units (3%) had a corresponding s/c ratio of <12. in addition, we found a very strong positive correlation between the elisa anti-rbd igg optical density at dilution 1:50 and the ortho vitros igg test for 1,142 samples with parallel assessment (r=0.88; p<0.001). the distribution of ortho vitros igg s/c ratios and anti-rbd igg elisa optical density for transfused plasma units confirms that high anti-spike protein igg titer units were being given to the enrolled covid-19 patients (figure 1 ). propensity score matching yielded a study population of 341 transfused patients and 594 matched controls, which were balanced across all matching criteria (figure 2 and supplemental table s4 ). kaplan-meier curves showed significantly decreased mortality within 60 days post-day 0 in the transfused cohort relative to propensity score-matched controls (p=0.02) (data not shown). statistical significance increased to p=0.003 when the matching algorithm and analysis were restricted to patients transfused with plasma with an anti-rbd igg titer of ≥1:1350 (figure 3 ). mortality was not significantly different within 60 days post-day 0 between cases and controls in patients who were intubated at day 0 or in patients who were transfused more than 72 hrs after admission, even when the analysis was restricted to patients who received plasma with an anti-rbd igg titer of ≥1:1350. there was no significant difference in mortality between cases and controls when the analysis was restricted to patients who received plasma with an anti-rbd igg titer of <1:1350. in contrast, mortality was significantly decreased in patients who received plasma with an anti-rbd igg titer of ≥1:1350 within 72 hrs of admission (figure 4 ). point estimates of the outcomes when the analysis was restricted to transfusion of high titer plasma confirm these findings ( table 1) . consistent with these observations, the unadjusted hr and adjusted hr in the univariate and multivariate cox proportional hazards models for mortality within 60 days was significant when the analysis was restricted to patients who received plasma with an anti-rbd igg titer of ≥1:1350 ( table 2) . due to small sample sizes, multivariate analysis could not be performed for patients who received plasma with a titer ≥1:1350 and were intubated at day 0, or who were transfused more than 72 hrs after hospitalization. in these two cohorts, the unadjusted hr in univariate analyses for mortality within 60 days post-day 0 was not significant (hr=1.61 for controls; p=0.44 and hr=1.93 for controls; p=0. 16, respectively) . similarly, the unadjusted hr for mortality within 60 days in the analysis restricted to patients who received plasma with a titer <1:1350 was not significant (hr=1.57 for controls, p=0.36). however, the unadjusted hr for mortality within 60 days was significant (hr=1.93 for controls, p=0.02) when the analysis was restricted to patients who received plasma with a titer ≥1:1350 within 72 hrs of hospital admission. for this cohort, the adjusted hr for mortality within 60 days was significant when assessed for a 28-day outcome (ahr=2.09 for controls; p=0.047) and approached significance when assessed for a 60-day outcome (ahr=1.82 for controls; p=0.051). we sought to identify the optimal window after hospitalization within which transfusion of convalescent plasma was most useful with respect to altering mortality. roc curve analysis with youden index revealed an optimal cut point of transfusion within 44 hrs of hospital admission for discriminating mortality within 60 days post-transfusion in all patients transfused with covid-19 convalescent plasma ( figure 5a ). the analysis identified the same cut point when restricted to patients transfused with convalescent plasma with an anti-rbd igg titer ≥1:1350. therefore, we performed the propensity score-matched analysis using this cut point as a restrictor. cohorts were again balanced across all matching criteria (data not shown). the resulting kaplan-meier curves showed significantly decreased mortality within 60 days post-day 0 in the cohort transfused with convalescent plasma with an anti-rbd igg ≥1:1350 within 44 hrs of admission relative to propensity score-matched controls (p=0.004) ( figure 5b ). point estimates of the outcomes for the analysis restricted to transfusion of high titer convalescent plasma within 44 hrs confirm these findings ( table 3) . univariate cox regression in this cohort revealed a significant unadjusted hr for mortality within 60 days (hr=3.26 for controls, j o u r n a l p r e -p r o o f p=0.01). likewise, multivariate cox regression showed a significant adjusted hr for mortality within 28 days (ahr=2.63 for controls, p=0.04) and within 60 days post-day 0 (ahr = 2.90 for controls, p=0.02) ( table 4 ). transfusion of convalescent plasma has emerged in the last six months as a promising therapy for covid-19 patients and has been granted emergency use authorization for hospitalized patients by the fda. because of the logistical challenges of planning and executing a study during a rapidly changing pandemic involving very complex medical patients, the results of few completed controlled studies assessing convalescent plasma efficacy have been published. here, we provide an analysis of a propensity score-matched study from a large cohort of hospitalized covid-19 patients who were transfused in one healthcare system with high-titer convalescent plasma qualified in one laboratory. in the aggregate, the data confirm and extend findings from our interim analysis suggesting that transfusion of convalescent plasma with high titer anti-rbd igg is safe and significantly decreases covid-19 mortality. 3 transfusion later in hospitalization or later in the disease course (e.g., postintubation) had no significant benefit on mortality, regardless of plasma titer. several lines of evidence support our findings, including survival analyses of specific cohorts of transfused patients relative to matched controls, point estimates from the generalized linear model and multinomial logistic regression, and univariate and multivariate analyses. the current analysis addressed several limitations we identified in our interim analysis. 3 first, the patient sample size is almost three times as large as that included in our interim analysis. second, we included additional covariates in the propensity score matching algorithm, including relevant concomitant medications (any steroid, azithromycin, hydroxychloroquine, remdesivir, ribavirin, and tocilizumab). importantly, factors identified as having a significant adjusted hr for mortality for all hospitalized covid-19 patients were included in the propensity match. third, because a large proportion of deaths occurred after 28 days post-day 0, we assessed a 60-day outcome. fourth, control patients enrolled in other clinical trials involving alternative experimental therapies were j o u r n a l p r e -p r o o f excluded. fifth, when possible, we performed multivariate analyses assessing factors associated with mortality within 60 days. finally, we used roc analysis with youden index to identify the optimal cut point at which transfusion of convalescent plasma is most useful with respect to altering mortality. our results bear on other recent studies treating patients with convalescent plasma. 4, 9, [14] [15] [16] [17] for example, a recent fixed-effect meta-analysis model assessing 12 controlled studies of covid-19 convalescent plasma found that the aggregate mortality rate of transfused covid-19 patients was significantly lower than that of non-transfused patients. 4 results from three randomized controlled studies and one large observational study have recently been released. 2, [18] [19] [20] the placid trial found convalescent plasma was not associated with significantly reduced mortality or progression to severe disease. 18 however, resolution of shortness of breath, fatigue, and negative conversion of sars-cov-2 viral rna at day 7 was higher in the transfused study arm. the authors acknowledged several limitations of their study. for example, the proportion of patients with comorbidities, especially diabetes, was higher in the transfused study arm. importantly, most of the convalescent plasma donors were young with mild disease and their median titer of neutralizing antibody was 1:40, a value considerably lower than the fda-recommended neutralizing antibody titer of 1:160. in addition, neutralizing antibody titers were not determined before transfusion, which means the highest titer units were not used for transfusion. similar results were reported for a randomized controlled trial conducted in chile in which neutralizing antibody titers in donor plasma were not determined prior to transfusion. 20 in contrast, interim analysis of a randomized controlled trial from spain with 81 randomized patients, reported that no patients progressed to mechanical ventilation or death among the 38 patients receiving convalescent plasma (0%), whereas six of 43 patients (14%) in the control arm did. 19 mortality rates were 0% versus 9.3% at days 15 and 29 for the active and control groups, respectively. all transfused convalescent plasma units had neutralizing antibodies with a titer >1:80 with a median titer of 1:292. unfortunately, the trial was stopped after the first interim analysis due to decreased recruitment related to better control of the pandemic. in contrast to several of the studies cited above, we methodically selected units for transfusion based on the elisa data identifying the highest level of igg antibody directed against spike ectodomain and rbd. we transfused compatible donor units determined to have j o u r n a l p r e -p r o o f the highest antibody titer available, an approach confirmed by our retrospective assessment of anti-sars-cov-2 igg by the ortho vitros assay (figure 1) . thus, the vast majority of our patients were transfused with convalescent plasma units with very high titer anti-spike protein igg. we think it reasonable to speculate that this strategy contributed to differences in outcomes observed between our study and several others that did not transfuse patients with plasma units specifically chosen to have very high igg antibody levels against spike protein. overall, the results from various published studies highlight the difficulty in drawing definitive conclusions for convalescent plasma efficacy from multiple studies with variable design, a problem that can extend to and thereby hobble randomized controlled trials with different study designs. substantial efforts to collect, use, and study covid-19 convalescent plasma continue worldwide. our study has several implications for these efforts. the data presented here may inform the design and conduct of ongoing or future studies. for example, we conclude that transfusing plasma units with low or no antibody titer against spike protein is unlikely to be beneficial. our data support the concept that identification of units contain high antibody titer by either a viral neutralization assay or a surrogate thereof prior to transfusion is essential, regardless of the type of trial being conducted. covid-19 convalescent plasma is now being administered widely in the united states under an eua, with an ortho vitros igg cutoff of >12 required by the fda for the designation of a high titer unit. in our study, we prospectively selected units based on anti-rbd igg elisa data. assessment of transfused units by the ortho vitros assay was done retrospectively. the relative lack of data from an adequate number of patients we transfused with convalescent plasma units near or below the s/c cutoff of 12 precludes evaluation of this cutoff value with respect to outcome. however, we conclude that prospective selection and transfusion of very high titer units likely contributed to the significantly decreased mortality we observed. in addition, transfusing soon after hospitalization will be more beneficial than the alternative. similarly, transfusion in patients that have progressed to the need for mechanical ventilation or otherwise have further deteriorated clinically likely confers no clear benefit. these findings support the notion that virus neutralizing antibodies present in covid-19 convalescent plasma impart therapeutic benefit when patients are in the relatively early viral infection/replication j o u r n a l p r e -p r o o f phase of covid-19 disease, but not after patients have progressed to manifest disease mechanisms such as a pathogenic severe inflammatory host response. 21, 22 prioritizing studies targeted toward early disease patients is, therefore, important. our finding that a large proportion of deaths in covid-19 patients occurs after day 28 may also have implications for study design, as findings at day 28 may not apply over a longer follow-up period. importantly, our study has several limitations. first, it is a propensity score-matched study rather than a randomized controlled trial. although we made every effort to control for all important covariates, potentially relevant covariates may have been omitted unintentionally from the matching algorithm. second, the background standard of care for covid-19 has evolved as new data emerged. thus, we may not have completely addressed the potential for variations over time in background standard of care and period effect as sources of confounding in our dataset. third, there was heterogeneity in the transfusion of two units versus one based on inventory limitations early in the study and on patient enrollment in other trials that specifically excluded redosing of convalescent plasma. fourth, our analysis was based on patient data available in the electronic medical record. fifth, we note that the results reflect the experience of one system of eight hospitals in the houston metropolitan region that have a fairly uniform approach to covid-19 patient care. our findings may not apply to all hospitalized covid-19 patients because of inter-institutional and/or regional heterogeneity in medical care. sixth, baseline inflammatory marker measurements were not included in the matching algorithm due to the high proportion of missing data points. our study approach facilitated rapid assessment of safety and efficacy of high-titer anti-sars-cov-2 convalescent plasma transfusion during early phases of a rapidly evolving pandemic with uncertain trajectory. the data presented here may help to inform the science and logistics of ongoing and future studies that address the use of convalescent plasma for other emerging and rapidly disseminating infectious diseases. to summarize, this propensity score-matched analysis of a large patient cohort confirms and extends our previous findings and suggests that transfusion of convalescent plasma containing very high titer anti-rbd igg early in hospitalization reduces mortality in covid-19 patients. we thank terumo bct for continuously and rapidly supplying blood collection devices and supplies. we also thank shmuel shoham, md for graciously sharing a draft study protocol for adaptation early in the study planning phase. statement of ethical assurance: jmm is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. interleukin steroids and tocilizumab were treated as time-varying covariates in the multivariate model. covid-19 dashboard by the center for systems science and engineering casadevall a: effect of convalescent plasma on mortality among hospitalized patients with covid-19: initial three-month experience treatment of coronavirus disease 2019 patients with convalescent plasma reveals a signal of significantly decreased mortality casadevall a: evidence favouring the efficacy of convalescent plasma for covid-19 therapy relationship between anti-spike protein antibody titers and sars-cov-2 in vitro virus neutralization in convalescent plasma treatment of coronavirus disease 2019 (covid-19) patients with convalescent plasma convalescent plasma anti-sars-cov-2 spike protein ectodomain and receptor binding domain igg correlate with virus neutralization convalescent plasma to treat coronavirus disease 2019 (covid-19): considerations for clinical trial design convalescent plasma treatment of severe covid-19: a matched control study the central role of the propensity score in observational studies for causal effects statistical learning with sparsity: the lasso and generalizations lasso ss: stata reference manual: release 16 index for rating diagnostic tests evaluating the efficacy and safety of human anti-sars-cov-2 convalescent plasma in severely ill adults with covid-19: a structured summary of a study protocol for a randomized controlled trial convalescent plasma use of convalescent plasma in hospitalized patients with covid-19 -case series effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening covid-19: a randomized clinical trial convalescent plasma in the management of moderate covid-19 in india: an open-label parallel-arm phase ii multicentre randomized controlled trial convalescent plasma for covid-19: a multicenter early anti-sars-cov-2 convalescent plasma in patients admitted for covid-19: a randomized phase ii clinical trial mechanisms underlying disease severity and progression the convalescent sera option for containing covid-19 c-reactive protein delta (day 7-baseline), (mg/dl), median (iqr) (n=169) -10.9 ferritin at baseline, (ng/ml), median (iqr) (n=358) fibrinogen at baseline, (mg/dl), median (iqr) fibrinogen delta (day 7-baseline), (mg/dl), median (iqr) d-dimer at baseline, (µg/ml feu), median (iqr) key: cord-002659-566uoozj authors: fujimoto, yousuke; hasegawa, shunji; matsushige, takeshi; wakiguchi, hiroyuki; nakamura, tamaki; hasegawa, hideki; nakajima, noriko; ainai, akira; oga, atsunori; itoh, hiroshi; shirabe, komei; toda, shoichi; atsuta, ryo; morishima, tsuneo; ohga, shouichi title: pulmonary inflammation and cytokine dynamics of bronchoalveolar lavage fluid from a mouse model of bronchial asthma during a(h1n1)pdm09 influenza infection date: 2017-08-22 journal: sci rep doi: 10.1038/s41598-017-08030-w sha: doc_id: 2659 cord_uid: 566uoozj asthmatic patients present more rapid progression of respiratory distress after a(h1n1)pdm09 influenza infection than after seasonal infection. here, we sought to clarify the pathophysiology of early deterioration in asthmatic patients after a(h1n1)pdm09 infection. cytokine levels and virus titres in bronchoalveolar lavage fluid from mice with and without asthma after a(h1n1)pdm09 or seasonal h1n1 infection were examined. in asthma/a(h1n1)pdm09 mice, il-6 and tnf-α levels peaked at 3 days post-infection and were higher than those in all other groups. ifn-γ levels in asthma/a(h1n1)pdm09 mice at 3 days post-infection were higher than in all other mice at any time point, whereas at 7 days post-infection, the levels were lowest in asthma/a(h1n1)pdm09 mice. virus titres in asthma/a(h1n1)pdm09 mice were highest at 3 days post-infection, and decreased by 7 days post-infection, although the levels at this time point were still higher than that in any other group. histopathological examination showed more inflammatory cell infiltration and lung tissue destruction in the asthma/a(h1n1)pdm09 group than in any other group. the distinct cytokine profiles in a(h1n1)pdm09-infected asthmatic mice indicated excessive inflammation and virus replication within a few days after infection. thus, bronchial asthma could be a more exacerbating factor for pandemic influenza infection than for seasonal influenza infection. the incidence of a(h1n1)pdm09 viral infection was significantly higher in children with asthma than in children without asthma 7 . paediatric patients with a(h1n1)pdm09 infection showed milder symptoms than those with seasonal h1n1 infection. however, severe respiratory issues, including pneumonia and acute respiratory distress syndrome (ards), have been reported in children and young adults with a(h1n1)pdm09 infection 5, [8] [9] [10] . bronchial asthma increases the risk of hospital and intensive care admission in infants and children [3] [4] [5] [6] [10] [11] [12] [13] [14] . we previously reported that a(h1n1)pdm09 infection, but not seasonal h1n1 infection, induces severe pulmonary inflammation in a mouse model of asthma 7 days after infection 14, 15 . however, we observed that the duration of the latent period for a(h1n1)pdm09 infection is shorter than 7 days, and patients present earlier progression of pulmonary disease and systemic conditions after infection. since exacerbation was frequently observed in asthmatic patients after viral infection, we suspect that a cytokine storm, including inflammatory cytokines (interleukin [il]-6 and tumour necrosis factor [tnf]-α), anti-inflammatory cytokines (il-10), anti-viral cytokines (interferon [ifn]-γ), and helper t (th) 2 cytokines (il-4 and il-13), may occur in the lung following a(h1n1)pdm09 infection. however, little is known about the early host response against a(h1n1)pdm09 infection in patients with underlying bronchial asthma. in the present study, we investigated the sequential changes in intra-tracheal cytokine production, viral loads, and pulmonary inflammation in a mouse model of bronchial asthma during the first 7 days after a(h1n1)pdm09 or seasonal h1n1 influenza infection. inflammatory cytokine concentrations in bronchoalveolar lavage (bal) fluid. il-6, tnf-α, and il-1β concentrations in bal fluid obtained 2, 3, and 7 days post-infection are shown in fig. 1 . although the mean il-6 levels in asthmatic mice challenged with a(h1n1)pdm09 were low (118.9 pg/ml) at 2 days post-infection, the levels were markedly increased (to 1578.2 pg/ml) at 3 days post-infection, and the levels in all groups remained high at 7 days post-infection. in contrast, il-6 levels in a(h1n1)pdm09-challenged control mice were 198.6 pg/ml at 2 days post-infection, peaked at 463.5 pg/ml by 3 days post-infection, and remained at similar levels at 7 days post-infection. after challenge with influenza a/puerto rico, il-6 levels in asthmatic mice slowly increased to 161.7 pg/ml by 3 days post-infection, and then reached 654.7 pg/ml at 7 days post-infection. in contrast, il-6 levels in control mice increased to 433.2 pg/ml at 3 days post-infection, similar to the levels after a(h1n1)pdm09 infection. il-6 levels in control mice at 3 days post-infection exceeded those of asthmatic mice at this time point (p = 0.015), and peaked to 1696.8 pg/ml at 7 days post-infection. the tnf-α levels in asthmatic/a(h1n1)pdm09 mice increased to 188.5 pg/ml at 3 days post-infection, which was the highest for all groups, and levels remained high at 7 days post-infection (p = 0.33). in contrast, tnf-α levels in the control/a(h1n1)pdm09 group increased to 63.7 pg/ml at 3 days post-infection, and peaked to 110.7 pg/ml by 7 days post-infection (p = 0.12). after seasonal virus infection, tnf-α levels in asthmatic mice increased to only 92.2 pg/ml at 3 days post-infection, which was similar to the levels in control/a(h1n1)pdm09 mice (p = 0.06), and these levels were maintained until 7 days post-infection. in contrast, the levels in control mice increased to 161.4 pg/ml by 3 days post-seasonal virus infection, which were similar to those in asthma/a(h1n1)pdm09 mice (p = 1.00), and these levels were maintained until 7 days post-infection. elevations in il-6 and tnf-α levels in bal fluid were not observed in the two mock-infected groups. the bal il-1β levels in control (145.6 pg/ml) and asthmatic mice (100.7 pg/ml) after seasonal infection were significantly higher than the a(h1n1)pdm09-infected groups (control/a(h1n1)pdm09: 16.5 pg/ml, asthmatic/a(h1n1)pdm09: 45.0 pg/ml), respectively. the early increasing pattern of il-6 and tnf-α levels (but not il-1β) in asthmatic mice after a(h1n1)pdm09 infection (but not control mice), was in contrast to the dynamics observed in a/puerto rico-infected mice. other cytokines in bal fluid. ifn-γ levels in asthmatic/a(h1n1)pdm09 mice significantly increased to 240.4 pg/ml by 3 days post-infection, which was the highest among all mice at this time point (vs. control/a(h1n1)pdm09, p = 0.007; vs. asthma/seasonal, p = 0.002; vs. control/seasonal, p = 0.001), and then levels increased to 651.5 pg/ml at 7 days post-infection (p = 0.001). ifn-γ levels in control mice at 3 days after a(h1n1)pdm09 infection increased to 70.8 pg/ml, which was significantly lower than the levels in asthmatic/a(h1n1)pdm09 mice at 3 days post-infection (p = 0.007). however, ifn-γ levels in control/a(h1n1)pdm09 mice peaked at 1209.4 pg/ml at 7 days post-infection, which were the highest of all the groups. il-10 levels were undetectable in all mice until 3 days post-infection. at 7 days post-infection, the levels in asthmatic/a(h1n1)pdm09 mice increased to 322.1 pg/ml, which was the highest of all the groups (vs. control/a(h1n1)pdm09, p = 0.007; vs. asthma/seasonal, p = 0.0001; vs. control/seasonal, p = 0.005). il-10 levels in control/a(h1n1)pdm09 mice increased to 202.4 pg/ml at 7 days post-infection. these levels were similar to those in control/seasonal mice (185.3 pg/ml, p = 0.44) but higher than those in asthma/seasonal mice (122.9 pg/ml, p = 0.03). after seasonal virus infection, the ifn-γ levels in asthmatic mice increased to 72.8 pg/ml at 3 days post-infection, which were similar to the levels in a/puerto rico-challenged control and asthmatic mice at 3 days post-infection, but were lower than those in asthmatic/a(h1n1)pdm09 mice at 3 days post-infection (p = 0.002). the ifn-γ levels in asthmatic/seasonal mice then increased to 420.6 pg/ml by 7 days post-infection, which were lower than that in any other group at this time point (vs. asthmatic/a(h1n1)pdm09, p = 0.03; vs. control/a(h1n1)pdm09, p = 0.002; and vs. control/seasonal, p = 0.002). ifn-γ levels in control/seasonal mice only increased to 80.7 pg/ml at 3 days post-infection, but then increased to 945.0 pg/ml by 7 days post-infection (p = 0.0001). neither il-10 levels nor ifn-γ levels were elevated in non-infected groups. bal il-13 levels in all asthmatic groups were higher than those in all non-asthmatic control groups, but the differences were not statistically significant. additionally, the levels in the control/a(h1n1)pdm09 and asthma/a(h1n1)pdm09 groups increased from 3 to 7 days post-infection. no significant differences in the levels of il-2, il-4, or il-17a, were observed among the groups, and the levels were all below the detection limits. p < 0.05, † † p < 0.01; control/seasonal vs. asthma/seasonal: || p < 0.05, || || p < 0.01; control/a(h1n1)pdm09 vs. control/seasonal: p < 0.05; asthma/a(h1n1)pdm09 vs. asthma/seasonal: § p < 0.05, § § p < 0.01; asthma/a(h1n1) pdm09 vs. control/seasonal: $$ p < 0.01; and control/a(h1n1)pdm09 vs. asthma/seasonal: ‡ p < 0.05, ‡ ‡ p < 0.01; mock/control or mock/asthma vs. each group: *p < 0.05, **p < 0.01; day 2 vs. day 3, ¶ ¶ p < 0.01; day 3 vs. day 7: fig. 2 . the numbers of total cells, lymphocytes, cd4 + cells, and cd8 + cells in all infected groups were increased at 3 days post-infection, and then decreased at 7 days post-infection. the numbers of cd8 + cells in the asthma/a(h1n1)pdm09 and control/seasonal groups were maintained until 7 days post-infection. the number of lymphocytes in control/seasonal group were higher than that in control/a(h1n1) pdm09 at 3 days post-infection, however, there were no significant differences between control/seasonal and asthma/a(h1n1)pdm09, control/a(h1n1)pdm09 and asthma/a(h1n1)pdm09 groups, respectively. additionally, the numbers of cd4 + cells, neutrophils and eosinophils on the 3 days and cd8 + cells on the 7 days post-infection were higher in the asthmatic/a(h1n1)pdm09 group than other groups respectively, but not statistically. in contrast, the numbers in the control/mock and asthma/mock groups were lower than those in the infected groups. virus titres in bal fluid. the virus titres in bal fluid at 2, 3, and 7 days post-infection are shown in fig. 3 . the mean titres at 2 days post-infection in the a(h1n1)pdm09-infected groups (asthma/a(h1n1)pdm09: 1.9 × 10 5 pfu/ml and control/a(h1n1)pdm09: 1.1 × 10 5 pfu/ml) were higher than those in the seasonal-infected groups (asthma/seasonal: 1.4 × 10 4 pfu/ml, control/seasonal: not detected); however, these differences were not statistically significant. the mean titre in the asthmatic/a(h1n1)pdm09 group at 3 days post-infection (2.2 × 10 7 pfu/ml) was the highest of all groups, and the differences were significant (vs. control/a(h1n1)pdm09, p = 0.001; vs. asthma/seasonal, p = 0.001), with the exception of the control/seasonal group. the virus titre in the asthmatic/a(h1n1)pdm09 group at 7 days post-infection (2.6 × 10 6 pfu/ml) was higher than those in any other groups at this time point (vs. control/a(h1n1)pdm09, p = 0.03; vs. asthma/seasonal, p = 0.001; vs. control/seasonal, p = 0.03). after a(h1n1)pdm09 infection, the titre in asthmatic mice at 3 days post-infection was higher than the titre at 7 days post-infection (p = 0.0002). the virus titres in control mice were also higher at 3 days post-infection than at 7 days post-infection (p = 0.006) after challenge with seasonal h1n1. histopathological findings in the lungs. figure 4 shows the h&e staining of lung tissues from mice at 2 (a), 3 (b) and 7 (c) days post-infection. the degrees of inflammatory cell infiltration and abscess formation in the asthma/a(h1n1)pdm09 group were more remarkable than in the control/a(h1n1)pdm09, asthma/seasonal, and control/seasonal groups on 2, 3 and 7 days post-infection. in addition, on 7 days post-infection, they were most severe in asthma/a(h1n1)pdm09 mice, compared with other mice. nucleoprotein antigen (infa-np) in the lungs of mice after a(h1n1)pdm09 or seasonal infection were observed by immunohistochemistry (fig. 5) . infa-np was detected in the epithelial cells and suspected macrophages in the lungs of asthmatic/a(h1n1)pdm09 (a, day 3) and control/a(h1n1)pdm09 group mice (a, day 2) since the early phase to day 7 (b) after infection, but not in mice from seasonal infected groups. infiltration of various inflammatory cells was noted, mainly in the alveolar walls in all four infected groups and more around the bronchioles in the a(h1n1)pdm09-infected groups, than in the seasonal h1n1-infected groups. only the asthmatic/a(h1n1) pdm09 group showed abscess formation with severe inflammation. the notable findings in the present study were the early peak in both il-6 and tnf-α levels, the high inflammatory cell infiltration in bal fluids, and the severe pulmonary inflammation at 3 days post-infection in asthmatic/a(h1n1)pdm09 mice. the pulmonary cytokine storm at 3 days post-infection in asthma/a(h1n1)pdm09 mice may mirror the rapid exacerbation observed in asthmatic patients 13 . in contrast, the delayed peak in il-10 levels and insufficient surge of ifn-γ levels in a(h1n1)pdm09 mice at 7 days post-infection could lead to ineffective exclusion of the viruses. the early potent inflammation associated with high viral loads in the lungs of asthmatic/a(h1n1)pdm09 mice may corroborate the rapid progression of asthmatic patients during outbreaks of pandemic virus infection. because the dynamics of il-1β was different from those of il-6 and tnf-α levels, il-1β may play other roles after influenza virus infection. in addition, the il-13 levels were increased in only the asthma/a(h1n1)pdm09 group after the infection. il-13 may be involved in pathophysiology of a(h1n1)pdm09 infection in asthmatic children. although il-2, il-4, and il-17a may be involved in the pathogenesis of bronchial asthma and influenza infection, they were undetectable in the bal fluid from mice in this study. this may be explained by the cytokines' short half-lives and/or limited roles in this microenvironment. in asthma/a(h1n1)pdm09 group, the number of cd4 + cells at 3 days and cd8 + cells at 7 days post-infection were higher than those of other groups, respectively. these results show that cd8 + cells may act anti-viral function during influenza infection. we could not recognized whether these were th1 or th2 lymphocytes, however both cd4 + and cd8 + cells may play important roles in pathophysiology of a(h1n1)pdm09-infected asthmatic patients. the histopathological findings in the early phase of infection in asthmatic/a(h1n1)pdm09 mice were severe pneumonia with abscess formation, and were not observed in any other groups (fig. 4) . these results demonstrated that pulmonary inflammation in asthmatic mice is induced beginning in the early phase of a(h1n1)pdm09 infection, which mirrors the finding that a(h1n1)pdm09 infection in asthmatic children induces severe pulmonary complications, including pneumonia, atelectasis, etc., after a shorter incubation period than with seasonal virus infection. after a(h1n1)pdm09 infection, the viral loads in bal fluid from asthmatic mice were higher than those from control mice, which was not typically observed after seasonal infection (fig. 3) . immunostaining of the virus showed many infa-np-positive cells in the lungs of asthmatic/a(h1n1) pdm09 and control/a(h1n1)pdm09 mice since early phase after viral infection, but not in the lungs of mice from seasonal h1n1 groups, as shown in fig. 5 . previous reports demonstrated that a(h1n1)pdm09 viral proteins were detected in damaged type ii pneumocytes, epithelial cells, and infiltrated macrophages in the lung by immunohistochemistry [16] [17] [18] . in addition, at autopsy after a(h1n1)pdm09 infection, acute diffuse alveolar damage was observed 19 . avian influenza viruses preferentially bind to saα2-3 gal, which is expressed on distal bronchioles and type ii pneumocytes in the lower respiratory tract 19 . in contrast, seasonal h1n1 influenza viruses bind to saα2-6 gal, which is expressed on epithelial cells in the upper respiratory tract. a(h1n1)pdm09 virus binds to both saα2-6 gal and saα2-3gal 19, 20 . predominant replication of a(h1n1)pdm09 virus in the lower respiratory tract, compared with that of seasonal influenza virus, could explain the distinct viral loads shown in fig. 3 . these findings suggested that a(h1n1)pdm09 virus may induce severe pneumonia in asthmatic patients, which is much less likely in seasonal influenza-infected asthmatic patients or non-asthmatic patients. we control/seasonal, : asthma/seasonal, : control/mock, : asthma/mock. control/a(h1n1)pdm09 vs. asthma/ a(h1n1)pdm09: † p < 0.05, † † p < 0.01; control/seasonal vs. asthma/seasonal: || p < 0.05, || || p < 0.01; control/ a(h1n1)pdm09 vs. control/seasonal: *p < 0.05; asthma/a(h1n1)pdm09 vs. asthma/seasonal: § p < 0.05, § § p < 0.01; asthma/a(h1n1)pdm09 vs. control/seasonal: $$ p < 0.01; and control/a(h1n1)pdm09 vs. asthma/seasonal: ‡ p < 0.05, ‡ ‡ p < 0.01; mock group vs. each group: *p < 0.05, **p < 0.01; day 2 vs. day 3, ¶ ¶ p < 0.01; day 3 vs. day 7: ## p < 0.01. concluded that severe pulmonary complications are caused not only by the characteristics of the infecting viruses but also by factors in the host defence of asthmatic children during a(h1n1)pdm09 infection. in the present study, cytokine levels in bal fluid ( fig. 1 ) appeared not to be associated with the lung histopathology. the histopathological analyses showed that airway inflammation was augmented in asthmatic mice when compared to control mice infected with either a(h1n1)pdm09 or seasonal influenza (fig. 4) . however, bal fluid cytokine levels showed no paralleled alterations. in fact, inflammatory cytokine levels in the non-infected groups were equivocally low in mice with or without bronchial asthma, which suggested that these histopathological changes without detectable cytokine elevations, were independent of asthma. at 3 days post-infection, ifn-γ levels in bal fluid were significantly higher in the asthmatic/a(h1n1) pdm09 group than in the control/a(h1n1)pdm09 group, in contrast to the pattern at 7 days post-infection. a previous report also showed that ifn-γ levels in asthmatic mice were lower than those in control mice at 7 days after a(h1n1)pdm09 infection 21 . virus titres in the asthmatic/a(h1n1)pdm09 group at both 3 and 7 days post-infection were significantly higher than those in control mice, and the titres of asthmatic/a(h1n1)pdm09 mice at 3 days post-infection were the highest among all groups at both 3 and 7 days post-infection. in contrast, the virus titres of the control/seasonal group were significantly lower than those of the asthmatic/seasonal group at both 3 and 7 days post-infection. ifn-γ levels were reportedly reduced in bronchial asthmatic patients, indicating an alteration in the cytokine milieu, with excess production of th2 cytokines and decreased production of th1 cytokines 22, 23 , and it has been reported that bronchial asthma patients show suppressed innate immunity [24] [25] [26] . ifn-γ is produced by th1 cells, cd8 + t (cytotoxic t) cells, natural killer (nk) cells, and nkt cells 27, 28 , and in our study, ifn-γ levels were elevated against the high virus load in the asthmatic/a(h1n1)pdm09 group at 3 days post-infection, but were not sufficiently augmented at 7 days post-infection. pulmonary inflammation, through not only the ifn-γ pathway but also other inflammatory molecules, might be involved in the exacerbation observed in a(h1n1)pdm09-infected asthma patients. we have some limited reasons for the unexplainable reciprocal pattern of virus titres in a(h1n1)pdm09-and seasonal influenza-infected asthmatic mice. the viral titre may depend on both the specificity of these viruses and the distinctive host defences in asthmatic individuals. however, further investigations are needed to characterize the immune responses against a(h1n1)pdm09 infection in asthmatic patients. in this study, the lung tissues of asthmatic/a(h1n1)pdm09 and control/a(h1n1)pdm09 mice were positive for infa-np antigens, whereas the lung tissues of mice in seasonal groups were not, even though virus titres were detected in the bal fluid of all infected groups. the reasons for this discrepancy may be the lower affinity for the virus receptors present in the lower respiratory tract or some yet unknown properties of the polyclonal antibodies and/or viral strains used, although the reason remains unclear. further research should be directed toward immunohistochemical studies of the upper respiratory tract along with lung function and airway hyperresponsiveness. in conclusion, a(h1n1)pdm09 infection can induce more severe pulmonary inflammation in patients with bronchial asthma than seasonal h1n1 infection, based on the dynamics of early excessive production of inflammatory cytokines and the reciprocal depression of anti-viral cytokines, along with high viral loads in a mouse model of bronchial asthma. sensitization of mice and allergen challenge. balb/c mice (age: 6-8 weeks) were obtained from chiyoda kaihatsu co., ltd. (tokyo, japan) and were sensitized and challenged with grade ii ovalbumin (ova; sigma-aldrich., st. louis, mo, usa), as previously described 14, 15 . all animal procedures were approved by the institutional animal care and use committee of yamaguchi university (no. 29-s01), and all methods were control/seasonal, ▲: asthma/seasonal. data are the mean ± sd of three independent experiments. control/ a(h1n1)pdm09 vs. asthma/a(h1n1)pdm09: † p < 0.05, † † p < 0.01; control/seasonal vs. asthma/seasonal: ||p < 0.05, || ||p < 0.01; asthma/a(h1n1)pdm09 vs. asthma/seasonal: § p < 0.05, § § p < 0.01; day 2 vs. day 3, ¶ ¶ p < 0.01; day 3 vs. day 7: ## p < 0.01. conducted in accordance with the approved guidelines. this study was performed independently of our previous reports 14, 15 . viruses, infection of mice, and preparation of bal fluid. mouse-adapted a(h1n1)pdm09 (strain: a/narita/1/09) or seasonal h1n1 (strain: a/puerto rico) viruses were provided by the national institute of infectious diseases (tokyo, japan). on day 31, influenza virus (concentration: 1 × 10 5 pfu/20 μl) or vehicle (mock-infection) was administered intranasally to mice. then, mice were euthanized at 2, 3, or 7 days post-infection, and samples were collected. bal fluids were collected on day 33, 34, and 38 (2, 3, and 7 days post-infection) with three consecutive 1-ml instillations of phosphate-buffered saline (pbs) at room temperature. the collected bal fluid was centrifuged at 1,500 rpm for 5 min at 4 °c, and the supernatants were stored at −80 °c for estimation of cytokine levels and virus titres. pg/ml, respectively. measurement of cd4 + cells, cd8 + cells, eosinophils, and neutrophils in bal fluid. cell pellets were resuspended in pbs and stained with fluorescein isothiocyanate (fitc)-conjugated anti-cd4 (bd biosciences) and allophycocyanin (apc)-conjugated anti-cd8 (bd biosciences) antibodies; erythrocytes were lysed by the addition of facs lysing solution epidemiology of 2009 pandemic influenza a (h1n1) deaths in the united states factors associated with death or hospitalization due to pandemic 2009 influenza a (h1n1) infection in california fatal cases associated with pandemic influenza a (h1n1) reported in greece does glycosylation as a modifier of original antigenic sin explain the case age distribution and unusual toxicity in pandemic novel h1n1 influenza? hospitalized patients with 2009 h1n1 influenza in the united states risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis increased h1n1 infection rate in children with asthma pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico acute respiratory distress syndrome induced by a swine 2009 h1n1 variant in mice three children with plastic bronchitis associated with 2009 h1n1 influenza virus infection plastic bronchitis in three children associated with 2009 influenza a(h1n1) virus infection a retrospective cross-sectional study of risk factors and clinical spectrum of children admitted to hospital with pandemic h1n1 influenza as compared to influenza a characteristics of atopic children with pandemic h1n1 influenza viral infection: pandemic h1n1 influenza reveals 'occult' asthma of childhood analysis of bronchoalveolar lavage fluid in a mouse model of bronchial asthma and h1n1 2009 infection cytokine profile of bronchoalveolar lavage fluid from a mouse model of bronchial asthma during seasonal h1n1 infection sudden death of a patient with pandemic influenza (a/h1n1pdm) virus infection by acute respiratory distress syndrome influenza a viruses target type ii pneumocytes in the human lung increased severity of 2009 pandemic influenza a virus subtype h1n1 infection in alveolar type ii cells from patients with pulmonary fibrosis histopathological and immunohistochemical findings of 20 autopsy cases with 2009 h1n1 virus infection receptor-binding specificity of pandemic influenza a (h1n1) 2009 virus determined by carbohydrate microarray the immune profile associated with acute allergic asthma accelerates clearance of influenza virus interferon-gamma: a historical perspective rhinovirus-induced interferon-gamma and airway responsiveness in asthma il-33 is more potent than il-25 in provoking il-13-producing nuocytes (type 2 innate lymphoid cells) and airway contraction influenza enhances caspase-1 in bronchial epithelial cells from asthmatic volunteers and is associated with pathogenesis innate immunity to influenza in chronic airways diseases inhibition of nk cell activity by il-17 allows vaccinia virus to induce severe skin lesions in a mouse model of eczema vaccinatum nk cells and nkt cells in innate defense against viral infections a mutant h3n2 influenza virus uses an alternative activation mechanism in tmprss2 knockout mice by loss of an oligosaccharide in the hemagglutinin stalk region we thank dr. takashi plaque assay. plaque assays were performed as described previously 10, 11 . briefly, madin-darby canine kidney (mdck) cells (lonza, walkersville, md, usa) were maintained at 37 °c in a humidified 5% co 2 chamber under stationary conditions. each well of a 6-well plate was seeded with 1 × 10 6 cells and cultured in α-minimum essential medium (mem; gibco/invitrogen, carlsbad, ca, usa) containing 10% foetal bovine serum (fbs), 100 units/ml penicillin (gibco), and 100 μg/ml streptomycin (gibco). after two washes with serum-free dulbecco's modified eagle's medium (dmem; gibco/invitrogen), the cells were maintained in serum-free dmem at 37 °c for 1 h. then, each well was overlaid with 200 μl of diluted bal (10 −3 , 10 −4 , and 10 −5 dilutions) and incubated at 37 °c for 1 h. after one wash in serum-free dmem, the cells were overlaid with serum-free dmem containing 0.8% agarose (becton, dickinson and company, sparks, md, usa), 0.1% diethylaminoethyl-dextran (sigma-aldrich), and 7 μg/ml trypsin (sigma-aldrich). the cells were cultured at 37 °c for 72 h, fixed in 10% formaldehyde (wako pure chemical industries, ltd., osaka, japan), and then stained with 0.037% methylene blue (wako pure chemical industries, ltd.). each experiment was performed in duplicate.histological and immunohistochemical examination of the lungs. lung tissues were fixed in 10% buffered formalin for 24 h at room temperature and then embedded in paraffin. serial sections (3-µm thick) were cut and stained with hematoxylin and eosin (h&e; muto pure chemicals co., ltd., tokyo, japan). the distribution of viral antigens was examined by immunological staining with a rabbit polyclonal antibody against infa-np, which recognize not only a(h1n1)pdm09 and seasonal h1n1, but also h3n2 influenza 29 . specific antigen-antibody reactions were visualized by 3,3′-diaminobenzidine tetrahydrochloride staining with the envision rabbit/hrp system (dako cytomation). the stained sections were observed by light microscopy to evaluate the degree of pulmonary inflammation and localization of a(h1n1)pdm09-infected cells.statistical analysis. the differences between groups were analysed by the mann-whitney u test. p values less than 0.05 were considered statistically significant. all analyses and calculations were performed using spss version 11.0 (spss inc., chicago, il, usa). yousuke fujimoto, shunji hasegawa, and shouichi ohga were the principal investigators who take primary responsibility for the study. yousuke fujimoto, shunji hasegawa, takeshi matsushige, hiroyuki wakiguchi, and tamaki nakamura performed the mouse experiments. hideki hasegawa, noriko nakajima, akira ainai, atsunori oga, and hiroshi itoh performed the histopathological examinations. komei shirabe, shoichi toda, ryo atsuta, and tsuneo morishima supported this study with helpful discussions. yousuke fujimoto, shunji hasegwa, and shouichi ohga wrote the first draft of the manuscript. competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, 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 article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article'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. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-266571-qbskh1uu authors: de arriba, m.l; carvajal, a; pozo, j; rubio, p title: lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus date: 2002-06-04 journal: j virol methods doi: 10.1016/s0166-0934(02)00063-0 sha: doc_id: 266571 cord_uid: qbskh1uu lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (pedv) strain cv-777 and challenged 21 days later with the virulent isolate of the same virus. a lymphoproliferative assay was developed in which mononuclear cells isolated from lymphoid tissues at different postinoculation and postchallenge days underwent a secondary in vitro stimulation with semipurified antigen obtained from pedv-infected cell cultures. vigorous lymphocyte proliferative responses were detected in the pigs inoculated with the virulent pedv at postinoculation days 4–21, especially in the mesenteric lymph nodes and the blood; however, in the spleen this response was lower and less regular. the pigs inoculated with the attenuated virus showed a less intense response, the higher lymphocyte proliferation also corresponded to the mononuclear cells from mesenteric lymph nodes. lymphocyte proliferation responses showed high correlations with protection against homologous challenge with virulent pedv, and this correlation was higher in the gut associated lymphoid tissues (mesenteric lymph nodes). the cell proliferation response detected in blood mirrored that detected in the mesenteric lymph nodes, and showed also good correlation with protection. the results confirm that t-cell-helper function, assessed by lymphocyte proliferation responses, contributes to establishing a protective immune response against pedv infections. porcine epidemic diarrhoea is an enteric disease in pigs caused by a member of the coronaviridae family, porcine epidemic diarrhoea virus (pedv) (cavanagh et al., 1994; murphy et al., 1999) . because of the high prevalence and the severity of clinical symptoms, porcine epidemic diarrhoea represents the most important viral infection of swine intestinal tract in spain and in many other european and asean countries (carvajal et al., 1995b; pensaert, 1999; sueyoshi et al., 1995; van reeth and pensaert, 1994) , although not in the usa where the pedv is not present but a high prevalence of transmissible gastroenteritis virus infections is still present (saif, 1998; saif and wesley, 1999) . the disease is characterized by acute watery diarrhoea, depression and anorexia. although morbidity is high and most of the animals in the herd can be affected within a week, mortality is usually low in adult animals (3%), which can recover in 7-10 days. however, when piglets less than 4-5 weeks are infected, mortality is usually 50% and can reach 90% in severe outbreaks, as usually happens in transmissible gastroenteritis. besides the mortality, pedv infection causes important economic loses due to the diminishing of the productive indexes (pensaert, 1999) . as in many other viral infections of food animals, the lack of effective treatment makes immunity the main key for prevention and control of the disease. however, the development of candidate vaccines needs previous knowledge of immunological aspects related to the infection. due to the enteric nature of the disease and the special configuration of the mucosal immune system, protection depends mostly on the local immune response (corthesy and kraehenbuhl, 1999; kagnoff, 1996; saif et al., 1994; van cott et al., 1994) , and is the reason why these studies cannot be limited to blood but require organs containing gut associated lymphoid tissues, in which local immune response can be measured. in spite of the fact that the importance of humoral immune response in gastroenteric viral infections of porcine is well recognized (corthesy and kraehenbuhl, 1999; saif et al., 1994; tô et al., 1998; van cott et al., 1994; yuan et al., 1996) , little is known about cell-mediated immunity, particularly in pedv infections. however, it has been proposed that cellular immune response may play an important role in the protection and recovery from infection, besides the fact that production of antibodies is regulated by cytokines produced by activated t lymphocytes and other mononuclear cells (corthesy and kraehenbuhl, 1999; kraehenbuhl and neutra, 1992; saif, 1999; totterdell et al., 1988) . the aim of this study was to assess the cellular immune response following natural infection with pedv and also after inoculation of an attenuated virus, and the contribution to the establishment of a protective response. virus-specific lymphoproliferative responses of systemic tissues (spleen and blood) and mesenteric lymph nodes were studied in conventional piglets after primary inoculation with the virulent, wild type, strain cv-777 of pedv or its cell culture attenuated form and after challenge, 3 weeks later, with a high dose of the virulent virus. vero cells were grown with eagle's minimum essential medium (gibco, life technologies) buffered with bicarbonate and supplemented with 5% (v/v) fetal calf serum (gibco), 0.04% (w/v) yeast extract (difco, mi, usa), streptomycin (10 mg/l) and penicillin (10,000 ui/l) (penicillin-streptomycin, gibco). the cell culture adapted strain of pedv cv-777, attenuated by many passages, was propagated in vero cells as described by hofmann and wyler (1989) , infecting confluent monolayers of cells after removing the growth medium and adding the viral inoculum diluted in medium without fetal calf serum but containing 10 ml/ml of trypsin (difco). a pedv-infected cell lysate was used as attenuated pedv inoculum. the wild type isolated of the cv-777 strain of pedv, kindly provided by dr peansert (gent, belgium), was amplified by passages in conventional 1-week-old piglets without antibodies against pedv and prepared in pbs for use as virulent pedv inoculum. after oral inoculation, the animals were killed in the acute phase of diarrhoea, and the intestinal contents and the small intestine were collected at necropsy. the small intestine from each animal was macerated in pbs (1:2 (w/v)) and, like the intestinal contents, clarified by centrifugation at 5000× g for 20 min at 4°c. the richest fractions were pooled and stored at − 70°c. a total of 62 conventional 11-day-old piglets, seronegative to pedv and from a herd with no previous history of the disease were assigned to three different experimental groups which were maintained in isolation facilities to prevent virus circulation. group 1 (n = 29) was inoculated with a low dose of the virulent isolate of pedv strain cv-777 that was adjusted in a previous experiment to produce a high morbidity without causing severe disease in the animals. pigs from group 2 (n =20) were inoculated with 2.55×10 5 fluorescent focus-forming units per pig of the attenuated isolate of the same pedv strain. the third group (n = 13) was mock-inoculated and served as control. twenty-one days after inoculation, the three groups of pigs were challenged with the virulent pedv, using a two times higher dose than that used to inoculate the group 1. animals were observed daily for clinical symptoms and rectal swabs were taken for 11 days after inoculation and for 9 days after challenge. faecal scores were recorded as normal faeces, pasty faeces, semiliquid (moderate diarrhoea) and liquid (watery diarrhoea). at different postinoculation and postchallenge days (postinoculation days 4, 7 and 14, and postinoculation/postchallenge days 21/0, 25/4 and 33/12) subsets of each group of pigs (n= 2-5) were killed by injection of barbiturate overdose (eutalender, normon, madrid, spain). hundred millilitre of blood were collected from cardiac cavities in 25% (v/v) acid citrate glucose. spleen and mesenteric lymph nodes were also collected aseptically and placed in ice-cold wash medium (rpmi 1640 containing 10 mm hepes and 200 mg of gentamicin and 20 mg ampicillin per ml). three pedv-seronegative, unexposed pigs served as negative control and were killed to obtain the background values for the lymphoproliferative assay. mononuclear cells from blood and tissues were isolated as described previously (de arriba et al., 2001a) . briefly, peripheral blood lymphocytes were obtained by density gradient centrifugation in ficoll-paque (ficoll-paque research grade, pharmacia biotech, upsala, sweden). lymphocytes collected from the interface were washed twice in hanks' balanced salt solution and suspended in rpmi 1640 containing 8% fetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 0.1 mm nonessential aminoacids, 20 mm hepes and 20 mg of ampicillin and 100 mg of gentamicin per ml (enriched medium). mononuclear cells from spleen and mesenteric lymph nodes were obtained by pressing the tissues through stainless steel screens (80 mesh) of a cell collector (cellecter; e-c apparatus corp., fla, usa). cell suspensions were centrifuged, and the mononuclear cells were removed from the pellet by continuous and discontinuous gradient centrifugation in percoll (pharmacia biotech.), washed twice with wash medium and resuspended in enriched medium. viability of all mononuclear cells preparations was confirmed by the trypan blue exclusion test, in every case being \ 95%. the lymphoproliferative assay for detection of pedv-specific t cells was adapted from methods published previously (brim et al., , 1995 ward et al., 1996) . semipurified pedv antigen for in vitro stimulation of mononuclear cells cultures was obtained from lysates of pedv-infected cell cultures that were concentrated 50 times by ultracentrifugation at 100,000×g for 2 h at 4°c and then semipurified by ultracentrifugation through 20% sucrose under the same conditions. the most favourable concentration of antigen for optimal antigenic stimulation of the mononuclear cells was established by dose-response curves. a pedvcontrol antigen was obtained giving the same treatment to mock-infected cultures. the t-cell mitogen phytohaemagglutinin (gibco) was used as positive control at a final concentration of 10 ml/ml, following the manufacturer's instructions. optimal conditions of number of cells and duration of incubation were determined by preliminary studies. mononuclear cells at a concentration of 5×10 5 cells per 100 ml and per well were placed in 96-well culture plates and stimulated in triplicate with the pedv antigen or the control antigen or the phytohaemagglutinin. cells were incubated for 72 h at 37°c in 5% co 2 and 18 h prior to harvest each well was labelled by pulsing 1 mci of [ 3 h]thymidine (amersham pharmacia biotech). harvesting of cells was carried out on glass fiber filters (filtermat, skatron inc., va, usa) and [ 3 h]thymidine incorporation was determined by liquid scintillation spectrophotometry. the lymphocyte proliferative responses for each mononuclear cells sample assayed was expressed as the stimulation index (si), calculated as si= mean cpm of pedv stimulated wells/mean cpm of control antigen stimulated wells, being cpm counts per minute. viral antigen was detected in faecal samples by the double antibody sandwich elisa described by carvajal et al. (1995a) . this elisa is based on the use of two monoclonal antibodies (lelsytad cvi-66.31 and lelystad cvi-66.49) directed specifically against the s protein of the virus. a blocking step with rabbit-anti pedv hyperimmune serum was included to increase the specificity. twofold serial dilutions of the samples were assayed starting at 1:2 and titres were expressed as the inverse of the lowest positive dilution. for calculation of the geometric mean titre (gmt), negative samples were given a titre of 1. one-way analysis of variance followed by the paired student's t-test was used to determine the nature of differences observed in virus shedding and lymphocyte proliferation responses among inoculated groups, tissues and days. correlation between proliferation responses at challenge day and protection against the infection was established by spearman's correlation coefficient (z). significance was assessed at pb 0.05. for the analysis the systat for windows v.5.03 (sys-tat inc.) and the spreadsheet microsoft excel v.7.0 (microsoft comp.) were used. a summary of faecal virus shedding and clinical disease after inoculation and challenge is given in table 1 . after primary inoculation of pigs from group 1 with virulent pedv, moderate to severe diarrhoea was observed in 33% of the animals and virus shedding in 100%. the onset of diarrhoea was observed between postinoculation days 2 and 4 and the average duration was 1.7 days. virus shedding was detected in some of the pigs at postinoculation day 1 but most shed pedv in faeces from postinoculation day 2 to 6. the average duration of the shedding period was 5.4 days. the gmt of viral antigen in faecal samples was measured using elisa; it increased strongly from postinoculation day 2 and reached a peak at postinoculation day 5. conversely, pigs from group 2 inoculated with the attenuated pedv did not show typical signs of the disease and only one pig had moderate diarrhoea for 1 day and virus shedding was detected in only one sample at postinoculation day 5 and with a low titre. differences between the gmt of antigen in the faeces of the two groups were significant statistically. on the challenge day, at postinoculation day 21, pigs from group 1 were protected against infection and disease, none developed diarrhoea and not viral antigen was detected in the samples taken after challenge. diarrhoea was not observed in pigs from group 2 after challenge either, but viral detection in rectal swab samples revealed that protection against the infection was only partial (25%), and the antigen was detected in 75% of the challenged animals (9 out of 12). in the control group moderate diarrhoea was seen in 46% of the pigs starting between postchallenge days 3 and 4 and with an average duration of 1.5 days. viral antigen was detected in faeces of all of the challenged pigs from this group in which the average duration of viral shedding (4.6 days) was significantly higher than in group 2 (2.6 days). the gmt of viral antigen detected in the control group was also significantly higher than in group 2. in order to obtain the optimal secondary antigenic stimulation for the lymphoproliferative assay, two different doses of antigen were used, 25.5 ng of the semipurified antigen were added to each 5× 10 5 mononuclear cells from spleen and blood, whereas in the mesenteric lymph nodes a minor dose, 12.8 ng, yielded the optimal stimulation of the cells. mean cpm obtained after stimulation of each tissue with the different antigens and the mitogen are shown in table 2 . background cpm, obtained after stimulation of each mononuclear cells culture with the control antigen, were low in mesenteric lymph nodes and blood (usually b 4000) but not in spleen, where these counts sometimes reached values close to 22,000. after inoculation of group 1, a specific lymphocyte proliferation response was detected for the first time in the mesenteric lymph nodes at postinoculation day 4 (fig. 1) and was maintained until challenge day (postinoculation day 21), when the maximum value of si was found (18.82). si in this tissue was significantly higher from postinoculation day 4 to 21 than that observed in unexposed pigs. the lymphoproliferative responses in mesenteric lymph nodes of pigs from group 2 increased significantly at postinoculation day 14 compared with their responses in previous days ( fig. 1) and si reached its peak that day at 5.73. values of the si between postinoculation days 14 and 21 were significantly higher compared with responses for mononuclear cells from mesenteric lymph nodes in unexposed pigs. group 1 si was greater than group 2 at any pid, however statistical significance was only detected at postinoculation day 4. group 3 was mock inoculated and served as control at challenge. statistically significant differences among groups are denoted by the letters: 'a' when differences are between groups 1 and 2, 'b' when differences are between groups 1 and 3 and 'c' for differences between groups 2 and 3. level of significance is defined by *p50.5, **p50.01. a pid: postinoculation days. b pcd: postchallenge days. c gmt: geometric mean titre of antigen detected in faeces by elisa. mononuclear cells were stimulated with positive, negative antigen or the t-cell mitogen phytohaemagglutinin (pha). background values correspond to pigs with no previous contact to pedv. pid, postinoculation day; pcd, postchallenge day. fig. 2 . correlations between lymphocyte proliferative responses in mononuclear cells collected from mesenteric lymph nodes, blood and spleen from pigs inoculated with virulent or attenuated pedv or mock-inoculated and protection against challenge 21 days later with virulent pedv. correlations were assessed by spearman rank correlation test. lymphocyte proliferative responses were expressed as mean cpm of pedv stimulated wells versus mean cpm of control antigen stimulated wells, being cpm counts per minute. fig. 1 . course of the virus-specific lymphocyte proliferative responses represented by si for mononuclear cells from mesenteric lymph nodes, spleen and blood from pigs after inoculation with virulent (group 1) or attenuated (group 2) pedv or mock-inoculation (group 3) and after challenge with virulent pedv. the si are the mean cpm of virus-specific stimulated wells versus mean cpm of control antigen stimulated wells, being cpm counts per minute. the mean value of the si obtained from the group of unexposed pigs is represented by a line crossing the y-axis. statistically significant differences (p 50.05) with values obtained in nonexposed pigs are noted as *. differences between groups are nodded as: 'a' when differences are between groups 1 and 2, 'b' between groups 1 and 3 and 'c' for differences between groups 2 and 3. mononuclear cells purified from blood of group 1 showed a vigorous proliferative response after inoculation starting at postinoculation day 7 with significant increases over the following days. the si obtained for this group in blood were significantly higher than values in blood of unexposed pigs between postinoculation days 7 and 21 and as in the mesenteric lymph nodes, the peak value occurred at postinoculation day 21 (fig. 2) . virusspecific lymphoproliferative responses in blood from group 2 occurred at postinoculation day 14, the only day in which si value was significantly higher than that in unexposed animals (si= 9.58, pb0.001). likewise in mesenteric lymph nodes, the si of group 2 were minor than the indexes of group 1, although the difference was statistical significant only at postinoculation day 21. the magnitude of the virus-specific proliferation in the spleen of group 1 after inoculation was lower than in the other tissues, being also less regular. at postinoculation days 4 and 7, the response of this group was low and similar to the proliferation shown by unexposed pigs. in the following days there was an increase in the si that peaked at postinoculation day 14, however, the value of si was not significantly higher than the background values at any postinoculation time. group 2 did not show a virus-specific proliferative response in mononuclear cells from spleen after inoculation, with an si similar to that in unexposed pigs. after challenge at postinoculation day 21, the lymphoproliferative responses in mesenteric lymph nodes of group 1 underwent an important increase and even though at postchallenge day 4 (postinoculation day 25) the si was lower than on challenge day (although not significantly), this value again reached its peak at postchallenge day 7 (postinoculation day 28) with a value of 29.25 (fig. 1) . in group 2 lymphocyte proliferation responses after challenge were low and only at postchallenge day 7 (postinoculation day 28) was the si significantly higher than that in unexposed animals. responses in group 3, the mock-inoculated control group, after challenge were similar to responses described in mesenteric lymph nodes of pigs from group 1 after inoculation with virulent pedv, but showed a higher intensity. the si in this group was significantly higher than in unexposed animals from postchallenge day 7 (postinoculation day 28) and reached 5 days later (at postchallenge day 12, postinoculation day 33) the highest value detected in the mesenteric lymph nodes of all the groups. when the si of the three groups were compared, statistically significant differences were found at postchallenge day 4 (postinoculation day 25) between group 1 and groups 2 and 3 and at postchallenge day 12 (postinoculation day 33) the si of groups 1 and 3 were significantly higher than index in group 2. lymphocyte proliferative responses in blood after challenge in group 1 were significantly lower at postchallenge day 4 (postinoculation day 25) compared to the challenge day, however, from postchallenge day 7 (postinoculation day 28) there were significant increases, reaching maximum value at postchallenge day 12 (postinoculation day 33) (fig. 2) , the only day that this value could be demonstrated significantly higher than that in unexposed pigs. similarly to mesenteric lymph nodes, the response detected after challenge in blood from group 2 pigs was low, only the si at postchallenge day 12 (postinoculation day 33) was significantly higher than unexposed animals index. in the control group, lymphoproliferative responses were low up to postchallenge day 12 (postinoculation day 33) and there was not statistical significance in the differences observed with regard to the unexposed animals. comparisons between the si in the blood after challenge in the different groups showed a higher response in group 1, although statistical significance was only shown at postchallenge day 12 (postinoculation day 33). the si was lower in group 3 than in group 2, but not significantly. in the spleen, responses after challenge of group 1 increased significantly at postchallenge day 7 (postinoculation day 28) with regard to the previous day, similar to the mesenteric lymph nodes. group 2 response after challenge was maximum at postchallenge day 4 (postinoculation day 25), this being the only time in which the si of mononuclear cells of spleen from this group was significantly higher than the si of unexposed animals. the control group underwent for the first time a specific proliferation response at postchallenge day 12 (postinoculation day 33). the si obtained in each group at each point in time were compared and no statistically significant differences were found. correlations between lymphoproliferative responses detected in each tissue and group at the challenge day and protection against challenge, represented by the protection rate against infection, were established by the spearman rank correlation test and are shown in fig. 2 . the magnitude of the response in all tissues examined at postinoculation day 21 correlated positively with protection against challenge, although statistical significance were not attained. the highest correlation was detected in the mesenteric lymph nodes (z= 0.99, p= 0.08). protection in swine gastroenteric viral infections, as ped, has been related almost exclusively to the antibody immune responses. however, cell-mediated immunity must play an important role in protecting and recovery from infection, besides the control function of the b cell-humoral responses carried out by t cell populations (corthesy and kraehenbuhl, 1999; kraehenbuhl and neutra, 1992; mcghee et al., 1992; saif, 1999; totterdell et al., 1988) . thus, without any b cell population deficiency in humans the lack of antibody and specific t-cell responses, resulting in rotavirus persistent infection with viral excretion in faeces for 15 months (totterdell et al., 1988) . moreover, welch et al. (1988) , in pigs inoculated with transmissible gastroenteritis virus, related peaks of lymphoproliferative responses ending up with final virus shedding in faeces and the beginning of recovery from the disease. in this study, an in vitro virus-specific proliferation assay was carried out as a method to estimate the cell-mediated immune response since this antigen-induced proliferation has been recognised as a property of cd4 +(t helper) cells in studies undertaken on pigs, mice and humans with rotavirus and coronavirus offit et al., 1992; ward et al., 1996) . the specific proliferative response after inoculation of pigs with virulent pedv was detected immediately in the mesenteric lymph nodes, the organs directly associated with the mucosal immune system. the maximum values were found around postinoculation day 21, just when a strong response of virus-specific antibody-secreting cells was detected in this organ and also in the duodenum and ileum lamina propria (de arriba et al., 2001b) . in pigs inoculated with the attenuated strain of pedv this specific lymphoproliferative response was detected later, at postinoculation day 14 and it was lower that in pigs inoculated with the wild virus. this minor response of group 2 also corresponded to a low response of pedv-specific antibody-secreting cells (de arriba et al., 2001b) . the difference observed between the lymphoproliferative responses of the two inoculated groups has also been described by other researchers (brim et al., , 1995 ward et al., 1996) in other gastroenteric viruses of swine, describing that the lymphocyte proliferative responses induced by attenuated strains of transmissible gastroenteritis and rotavirus were significantly lower than that induced by the homologous virulent virus. these results suggest that a protective antibody response to the virulent pedv could be associated with previous development of a strong specific cell-mediated immune response. this consideration could be reinforced by the fact that antibody production by specialised b cells requires t cell help (corthesy and kraehenbuhl, 1999) . the virus-specific lymphocyte proliferative response in the systemic lymph tissues (blood and spleen) was observed later than in mesenteric lymph nodes, as well as being considerably lower in the spleen than in other tissues. this delay could be explained if it is considered that the pedv-specific t cells located in blood and spleen originate in the inductive sites from the gut-associated lymphoid tissues, like the mesenteric lymph nodes, and its presence in systemic tissues is due to the homing process necessary for its maturation (corthesy and kraehenbuhl, 1999; kagnoff, 1996; kantele et al., 1997; salmi and jalkanen, 1997) . the specific cell proliferation response in the blood was more similar to that observed in the mesenteric lymph nodes than in the spleen, especially in the group 1, in spite of both, blood and spleen, being linked to the systemic immune system. however, the blood, together with the lymphatic system, is the main vehicle for lymphocyte migration (salmi and jalkanen, 1997) , and its lymphoid population may reflect primed t cells migrating to the gut for some time after an infection. the lymphocyte proliferative responses at the challenge day showed high correlation with protection against challenge. pigs from group 1 inoculated initially with the virulent pedv, were 100% protected against infection 21 days later with a higher dose of the same virus whereas protection in group 2, inoculated with the attenuated pedv, was just partial and only 25% of pigs were protected against infection with the virulent virus. the highest correlation was observed in mesenteric lymph nodes. this result again suggests that t-cell response, especially in the gut associated lymphoid tissues, contributes in an important way to the development of a protective immune response in pedv infections. the highly attenuated pedv conferred partial protection against challenge with virulent virus in conventional pigs, this protection is related to the inoculated dose and increases when a higher dose is used (de arriba et al., 2001b) . kweon et al. (1999) also described the induction of protective immunity by a attenuated strain of pedv inoculated intramuscularly. after the challenge, there was an increase in the lymphocyte proliferative response in pigs from group 1, however this increase was not reflected either by an enhancement of the virus-specific antibody secreting cell response or the gmt of pedv-specific serum igg and iga (de arriba et al., 2001b) . thus, ward et al. (1996) also reported that pigs inoculated and challenged with virulent rotavirus strains showed after challenge lymphoproliferative responses similar or poorer than after inoculation. although there is no clear explanation in this, the possibility remains that this secondary response after challenge could be related to the proliferation of cell clones involved in immune regulatory functions different to providing help for antibody production, such as t suppressor populations. in this study the development of cell-mediated immunity occurred in systemic and lymphoid tissues after inoculation with virulent and attenuated strains of the pedv. the results suggest that cell-mediated immune responses contribute significantly to the instauration of protective immune status against homologous virulent virus challenge. the lymphoproliferative responses both in gut associated lymphoid tissues and systemic tissues had a higher magnitude when virulent pedv was used versus attenuated pedv to inoculate pigs. however, higher doses and administration methods have to be assayed in order to develop vaccines. lymphocyte proliferation responses of transmissible gastroenteritis virus or porcine respiratory coronavirus cellular immune responses of pigs after primary inoculation with porcine respiratory coronavirus or transmissible gastroenteritis virus and challenge with transmissible gastroenteritis virus evaluation of a bloking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies seroprevalence of porcine epidemic diarrhea virus infection among different types of breeding swine farms in spain revision of the taxonomy of the coronavirus, torovirus and arterivirus genera antibody-mediated protection of mucosal surfaces isotype-specific antibody-secreting cells in systemic and mucosal associated lymphoid tissues and antibody responses in serum of conventional pigs inoculated with pedv mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) mucosal immunology: new frontiers homing potentials of circulating lymphocytes in humans depend on the site of activation molecular and cellular basis of immune protection of mucosal surfaces derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate the mucosal immune system: from fundamental concepts to vaccine development viral taxonomy and nomenclature rotavirus-specific helper t cell responses in newborns, infants, children and adults porcine epidemic diarrhea enteric viral infections of swine enteric viral infections of pigs and strategies for induction of mucosal immunity immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine transmissible gastroenteritis and porcine respiratory coronavirus how do lymphocytes know where to go: current concepts and enigmas of lymphocyte homing an immunohistochemical investigation of porcine epidemic diarrhoea serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease systemic lymphoproliferative responses to rotavirus contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening herds development of mucosal and systemic lymphoproliferative responses and protective immunity to human group a rotavirus in a gnotobiotic pig model cell-mediated immune responses of suckling pigs inoculated with attenuated or virulent transmissible gastroenteritis virus systemic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease we wish to thank dr m.b. pensaert for providing the wild type isolated of the pedv strain cv-777 and dr l.j. saif and dr l.a. ward for laboratory training. we also wish to thank g.f. bayó n and b. escudero for their excellent techni-cal assistance. this work was funded by the comisió n interministerial de ciencia y tecnología (cicyt) project no. agf-960486. salaries were provided by the excelentisima diputació n provincial de leó n. key: cord-009772-pzxvicee authors: grünberg, k.; timmers, m. c.; smits, h. h.; de klerk, e. p. a.; dick, e. c.; spaan, w. j. m.; hiemstra, p. s.; sterk, p. j. title: effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin‐8 in nasal lavage in asthmatic subjects in vivo date: 2006-04-27 journal: clin exp allergy doi: 10.1111/j.1365-2222.1997.tb00670.x sha: doc_id: 9772 cord_uid: pzxvicee background asthma exacerbations are closely associated with respiratory virus infections. however, the pathophysiological consequences of such infections in asthma are largely unclear. objective to examine the effect of rhinovirus 16 (rv16) infection on airway hypersensitivity to histamine. and on interleukin‐8 (il‐8) in nasal lavage. objective twenty‐seven non‐smoking atopic, mildly asthmatic subjects participated in a placebo‐controlled, parallel study. a dose of 0.5–2.9 ± 10(4) tcid50 rv16 or placebo was nasally administered. cold symptoms were recorded by questionnaire throughout the study. histamine challenges were performed at entry, and on days 4 and 11 after inoculation. nasal lavages were obtained at entry, and on days 2 and 9. the response to histamine was measured by pc(20) (changes expressed as doubling doses: dd). il‐8 levels were obtained by elisa, and were expressed in ng/ml. results rv infection was confirmed by culture of nasal lavage and/or by antibody titre rise in each of the rv16‐treated subjects. among the 19 rv16‐treated subjects, eight developed severe cold symptoms. baseline fev(1) did not change significantly during the study in either treatment group (p= 0.99). however, in the rv16‐treated subjects there was a decrease in pc(20) at day 4, which was most pronounced in those with a severe cold (mean change ± sem: –1.14 ± 0.28 dd, p= 0.01). in addition. il‐8 levels increased in tbe rv16 group at days 2 and 9 (p < 0.001). the increase in nasal il‐8 at day 2 correlated significantly with the change in pc(20) at day 4 (r=–0.48, p= 0.04). conclusion we conclude that the severity of cold, as induced by experimental rv16 infection, is a determinant of the increase in airway hypersensitivity to histamine in patients with asthma. our results suggest that this may be mediated by an infiammatory mechanism, involving the release of chemokines such as il‐8. virus infections and/or to airborne allergens, potentially leading lo a flare-up of airway inliammation ii.2|. several clinical and epidemiological studies have described a close lemporal as.^ociation of respiratory virus infections with asthma exacerbations [3|. respiratory viruses can be identified in 10 to 44% of the asthma exacerbations in adults |4.5|. whilst in children identification rates vary from 26 to 83'/f [6] [7] [8] [9] . the use of sen.sitive techniques to detect rhinovirus and coronavirus in the two most recent studies have resulted in the highest identification rates so far [5. 9|. among the various respiratory viruses identilied, rhinovirus predominates in most of these studies 15.7-91. interestingly, rbinovirus shedding in the absence of cold symptoms does not seem to be associated with clinical worsening of asthma [7] . the effects of experimental rhinovirus infection on airway responsiveness to inhaled histamine are somewhat controversial. lemanske et al. demonstrated an induction of hypersensitivity to histamine after experimental rbinovirus 16 (rvi6) infection in non-asthmatic patients with atopic rhinitis [lo] . whereas others have not observed such an effect when using other rhinovirus scrotypes [11.12] . in asthmatic subjects. halperin et al. found increased hypersensitivity to histamine in only four out of 22 subjects after experimental rhinovirus (serotype 39 and hh strain) infection 113|. whilst in the most recent study by fraenke! et al. a rather small, but significant increase in sensitivity to histamine could be detected in six asthmatic subjects after infection with rvi6 [ 14|. since none of these studies was placebo-controlled, it seems mandatory to examine the effect of experimental rhinovirus infection on airway sensitivity to histamine in asthmatic subjects by using such a design. this has successfully been employed in our previous study, showing excessive airway narrowing to methacholine after rvl6 infection in atopic asthmatic subjects | ls]. rhinovirus infection has been shown to lead to infiltration of inflammator>' cells into nasal secretions and mucosa fl6-18| as well us into the bronchia! mucusa |i4] in normal and/or atopic subjects. in vitro, there is evidence that bronchial epithelial cell lines, fibroblasts and mononuclear cells produce pro-inflammatory cytokines in response to infection with rhinovirus 119-211. in vivo, the levels of chemokines such as interleukin-8. rantes and mip-lo; were found to be elevated in nasal secretions of asthmatic children during naturally acquired colds [22.23] . consequently, one can postulate that chemokines such as il-8 drive recruitment of inflammatory cells |24!. thus promoting airway inflammation, and thereby airway sensitivity to histamine. in the present study, we hypothesized that experimental rvi6 colds in atopic asthmatic patients increase airway sensitivity to histamine. particularly in those with severe cold symptoms. in addition, we postulated that this effect is associated with a rise of il-8 in nasal secretion. to tbat end. we measured dose-response curves to inhaled histamine and levels of il-8 in nusul washings before and after placebocontrolled nasal inhalation of wild type rv16 in atopic. mildly asthmatic patients. twenty-seven non-smoking, atopic asthmatic subjects participated in this study. the subjects had not used inhaled or oral corticosteroids for at least 3 months, nor had they used theophyllines. antihistamines. sodium cromoglycate, or nedocromyl sodium for at least 6 weeks preceding the study. symptoms of asthma were stable and controlled by on demand usage of inhaled salbutamol alone, that was withheld for at least 8 h before the measurements. there was no history of relevant exposure to allergens from 2 weeks before until the end of the study. the patients were not selected on basis of a history of virus-induced exacerbations. among the eight subjects who received placebo-inocuiatit^n. four did not have neutralizing antibodies in their undiluted sent against 20-25 tissue culture infective dose (< 1:1). and four had titres between 1:2 and 1:128 serum dilution. fourteen out of the 19 .subjects who received virus did not have neutralizing antibodies, and live had titres in the range of 1:2 to 1:16 serum dilution. the study was conducted from july to december 1994. the study was approved by the hospital's medical ethics committee, and informed con.sent was obtained from all participants. the subjects" characteristics are listed in table i . the study had a double-blind, placebo-controlled parallel design. prior to tbe study, each subject was screened for inclusion and exclusion criteria. three days before the experimental inoculation o\ virus or placebo, a histamine inhalation test was carried out. subsequently, virus or placebo (diluent) was administered on two successive days. histamine challenges were repeated at days 4 and 11 after the first inoculation of virus or placebo. nasal washing was performed and a blood sample was taken immediately before the first inoculation of virus or placebo, and then at days 2 and 9. four weeks after inoculation all subjects returned to the laborator>' for a final nasal washing and a blood sample to determine the convalescent antibody titre. tbe rvl6 virus strain and stock was the same as used in previous experiments in humans in vivo by others |ioi and k. griinherg et ai. by ourselves [i5[. the virus was cultured according to standards of good laboratory practice and the int)culum was tested to be safe for human m vivo usage 125|. nasal inoculation of ihe rhino\ims was performed foiuiuing a previously described method j l()[, that was slightly modified by adding nasal virus inhalation [ i5|. a total dose of 0.5-2.9xin^ tclds,, diluted in 3ml hanks' balanced salt solution (hbss) with o.y/c (w/v) gelatin was administered to each subject. this dose was divided over 2 days. on each day the same procedure for virus inoculation was followed. first, 0.5 ml of the inoculum was inhaled through the nose by using a nebuli/.er (devilbiss 646: median mass aerosol diameter (mmad) 2.4/xm) connected to a lace mask. .second. 0.5ml was sprayed by atomizer (devilbiss 286. powered by a compressor. mmad: > io^m)intothe nostrils. and finally, 0.5 ml was instilled into the nostrils by pipette. we considered a four-fold or greater increase in virusspecific neutralizing antibody in the serum and/or recovery of the virus from nasal washes as confinnatitm ^^\ rv16 infection [ 1(m5|. before and 28 days after virus or placebo inoculation, levels of neutralizing antibodies were determined by a neutralization assay using homologous virus [ 10.i5j. nasal unages were obtained before the first virus or placebo administration, and subsequently on days 2. 9 and 28. human embryonic lung fibroblast (hel) cultures were inoculated with these lavages and incubated at 32"c for 14 days. if the culture showed the characteristic rhinovirusinduced cytopathic effects. rvl6 was identified by a neutralization assay, using rvl6 specific guinea pig immune serum (1126as/gp-vr: atnerican type culture collection. rdckville. md). all na.sal washes were also inoculated into rhesus monkey kidney <llc-mk2) cells, hep-2 cell cultures and hel cells, and cultured at 37°c. in order to exclude any intercurrent respiratory virus infection. throughout the study period, symptoms of common cold and asthma were evaluated with a questionnaire tbat was completed by the participants three times daily. cold symptoms included sneezing, sore throat, nasal discharge, stuffy nose, headache, cough, malaise, chills or fever, which were graded: 0 -absent. 1 = mild. 2 -moderate, 3 = severe and added up to a total cold symptom score. in subjects in whom infection was confirmed, those who recorded a total cold symptom score ^ 11 at any time-point after inoculation were defined as having a severe cold, whereas subjects with scores < 11 were considered to have a mild cold 110. 15.25] . in the same manner, asthma symptoms including cough, breathlessness. wheeze, chest tightness, and nocturnal symptoms were recorded |15]. the daily consumption of inhaled salbutamol as rescue medication was also noted. standardized histamine challenge tests |26i were performed using histamine-di-phosphate in phosphate buffered saline. histamine was stored at 4^c. and nebulized at room temperature in serial doubling concentrations ranging from 0.03 to 8mg/ml. a devilbiss 646 nebuli/er (devilbiss co., somerset, pa) (output 0.13 ml/min) was used, in connection tt) the central chamber of an in-and expiratory valve box with an expiratory aerosol filter (pall ultipor bb50t). the aerosol was inhaled by tidal breathing for 2min at 5min intervals witb the nose clipped. the lung function respk)n,se was measured as fev|. obtained by a dry rolling spiromcter (morgan spiroflow. rainham, united kingdom). first, baseline fev i was determined as the mean of three reproducible values (within 5*^). subsequently, single measurements of fev| were made 90s after each histamine dose. the tests were discontinued if fev, decreased by more than 20% from baseline. afterwards the subjects inhaled 2(k)^g salbutamol to provide immediate bronchodilalion. a nuklified "nasal pool device" was used for nasal lavage |27[. this consists of a syringe, connected to a rubber tube, equipped with an inflatable balloon, serving as a nasal adapter. the balkxin was inserted into the vestibulum nasi. and gently inflated. while the patient was sitting in a writing position, 10 ml of pre-warmed hanks' balanced salt solution (hbss) (one nostril with and the other without 0,5% gelatin) was instilled by gently compressing the syringe. after 5niin the fluid was recovered by retracting the syringe. this procedure was carried out in each nostril. the lavage recovered from the first (with 0.5'' ' f gelatin) and second nostril (without gelatin) was used for confirmation of rv infection and to determine the il-8 level, respectively. immediately after recover\', nasal washings were centrifugcd at 250 x^' for lomin. supernatant was removed and stored at -1q>^c until further analysis. the il-8 levels were determined by elisa (clb. amsterdam. the netherlands), according to the manufacturer's direction,*!. the detection limit of this assay was 40pg/mi. before, and on days 2 and 9 iifter placebo or virus administration, absolute and differential leucocyte counts were assessed by automated blood count analysis (technicon hi, technicon, tarrytown, ny). the highest individual total cold symptom score (referred to as cold score), and the cumulative asthma score recorded from day 0 to 5 after inoculation minus the cumulative scores from 4 days to i day before inoculation (referred to as asthma score) were used for correlation testing [15] . the response of fev| to histamine was expressed as percentage fall from baseline value |26|, and was plotted against log nebulized concentration of histamine in mg/ml. the concentration-response curves were characterized by their position, expressed as the provocative concentration causing 20% fall in fevi from baseline value (pcao), which was calculated by log-linear interpolation between the last two adjacent data-points |26]. the logarithm of pc20 was used in the analysis, and changes in pc20 were expressed in doubling doses (dd). il-8 levels were expressed in ng/ml. peripheral blood leucocyte numbers were expressed in cells/l. changes in the variables were analysed by repeatedmeasure analysis of variance (manova). with placebo. rv16 treatment or severe cold and mitd eold a.s betweengroup factors and time as a within-group factor. significant manova effects were explored with student's /-tests. differences in pc2(i. il-f^ levels and leucocyte numbers within the groups between the study days were examined using two-tailed paired r-tests, and differences between the groups were analysed using unpaired /-tests, the summary statistics were expressed as means ± sem. for evaluation of associations between the variables. ihe pearson's correlation test was used. p values less than 0.05 were considered statistically significant. one of the rvl 6-treated subjects (subject 9) dropped out of the study at 7 days after the first inoculation because of a moderate exacerbation of asthma, requiring treatment with oral prednisone, to which she responded well. one nasal washing sample was excluded from the analysis (subject 7, day 9), because of a recent nose bleed. in the plaeebo group all cultures of nasal washes remained negative for rvl6 during each visit. in the virus-treated group rv 16 could not be detected in the nasal lavage before inoculation, whilst at day 2 rvl6 was detected in the nasal lavage of all but one subject (subject 18). at day 9, rvl6 was identified in 10 out of 19 subjects, whereas at day 28 all nasal washings were negative. no other respiratory viruses were identified in any of the nasal washings (table i) . in the placebo group none of the subjects showed an increase in rv16 neutralizing antibodies. in the rv16 group all subjects but two (subjects 16 and 24) showed at least a four-fold increase in neutralizing antibodies in the convalescent sera (range: 4-fold to 128-fold increase) ( table 1) . in the placebo group, there was no significant change in cold score or asthma score (manova, p > 0.52). in the rvl6 group, there was a significant increase in cold score (manova. p<0.00\). that peaked i day after the first inoculation, gradually returning to baseline within 5 days. the highest cold scores were significantly different between the groups (p < 0.001). eight of the rvi6-infected subjects had a severe cold as shown by a symptom score > 11 ( table 1 ). in the rvl 6-treated subjects there was a significant increase in asthma symptoms {manova, p < 0.001) that peaked on the second and third day after the first inoculation, and returned to baseline within 5 days. the asthma score in the subjects with a severe cold was significantly higher than the asthma score in those with a mild cold and the placebo-treated subjects (/»< 0.001) ( table 1) . cold score and asthma score were significantly correlated in the rvi6 group (r^o.92. /'< 0.001). the use of salbutamol did not change significantly within the groups at any time point (manova, p = 1.00). before rv 16 or placebo inoculation, fev i % predicted was slightly higher in the rvl6 group as compared to the placebo group (p = 0.04) ( table 1 ). during the course of the study, there were no significant effects on baseline fev| within either the placebo group or in the rvl 6-treated subjects with a mild or severe cold (manova, p -0.99) ( figure 1 ). the maximal change in fev, after infection did not correlate significantly with the asthma score (p = 0.98). before inoculation of rvl6 or placebo, the mean pc20 was not different between the two treatment groups (p ^ 0.93). in the placebo group, there was no significant change in pc20 during the study (manova, p = 0.67) (figure 2 ). in the rvl6 group, there was a significant decrease in pc20 at day 4 (mean difference ± sem: -0.65 ± 0.25 dd, p = 0.02), which was no longer significant at day 11 (mean difference ± sem: -0.40 ± 0.30 dd, p = 0.19). these changes were not significantly different from placebo (p = 0.10 and p = 0.27, respectively). however, in the subjects with a severe cold, this decrease was more pronounced: mean difference ± sem: -1.14 ± 0.28 dd, p -0.005 at day 4. with a trend towards a decrease at day ii (mean difference ± sem: -0.75 ± 0.34dd. p = 0.07) {figure 2). this change was significantly different from placebo at day four, but not at day \i (p = o.oi and p = 0.09. respectively). in the tnild cold group there was neither a change in pc2(, at day 4 (mean difference ± sem: -0.30 ± 0.35 dd. p = 0.42). nor at day ii (mean difference ± sem: -0.18 ± 0.44 dd, p = 0.68) (figure 2) . the changes in pc20 in the five subjects with pre-existing neutralizing antibodies against rv16 were not statistically different from the changes in those without such antibodies {manova. p ^ 0.33) {day 4: mean change ± sem: -0.53 ± 0.46 dd. p = 0.31. and -0.69 ± 0.31 dd. f = 0.04. respectively. day 11: -1.10 ±0.28dd. p = 0.02. and -0.13 ± 0.38 dd. p = 0.73, respectively). il-8 in the nasal washings did not change significantly in the placebo group (manova. p = 0.06) {figure 3). in the rvl6 group, il-8 increased both at days 2 and 9 {p<o.ooi) as compared to day -5. in subjects with a severe cold this increase was significant at both time-points {within group. p < 0.04), and also as compared to placebo {p<0.05). in those with a mild cold the increase in il-8 was only significant at day 9, both within the group {p = 0.008) and compared to placebo (p<0.01) {figure 3). the increase in il-8 at day 2 in the rv16 group correlated significantly with the cold score {r ^ 0.80. p< 0.001). a.sthma score (r = 0.68. p^ 0.001), and with the change in pc2() at day 4 (r = -0.48, p = 0.04) (figure 4a, 4b) . in the rvl6 treated subjects differential leucocyte counts showed a significant rise in the number of neutrophils {p<o.ool) and a concomitant decrease in lymphocyte number at day 2 after inoculation {p< 0.001) ( figure 5 ). only in subjects with a severe cold were the cell numbers at day 2 significantly different from placebo {p^o.ol). neutrophil and lymphocyte numbers had returned to baseline at day 9 {p > 0.17). the number of neutrophils at day 2 correlated significantly with cold score (r = 0.59. p = 0.008). asthma score {r^o.65, p = 0.002), the change in pc2,) at day 4 (r --0.49, p = 0.03), whilst there was a trend towards a significant correlation with the change in il-8 levels at day 2 (r-0.39. p-o.io) (figure 4c) . furthermore, the number of lymphocytes at day 2 was also significantly related to the cold scores histamine. particularly in those patients who develop a severe cold. in addition, we have demonstrated that the levels of the pro-infiammatory chemokine il-8 in nasal secretions rise after infection. this rise is ass(x:iated with cold score, change in airway hyperresponsiveness. and numbers of neutrophils and lymphwytes in peripheral blthxi after infection. these findings suggest that the severity of the cold is a major determinant of rhinovirus-indiiced airway hypenesponsiveness in asthma. our results fit in with the hypeithesis that this is mediated through an inflammatory mechanism inviil\ing locally produced chemokines. this is the first placebo-controlled study showing the development of airway hypersensitivity to histamine after experimental infection with wild type rhinovirus. the change in airway hypersensitivity in the asthmatic subjects with severe colds was about 1 doubling dose, which is similar to what is usually observed after allergen challenge |28|. rhinovinis-induced hypersensitivity of such magnitude has also been demonstrated in patients with atopic rhinitis by lemanske et ai |l()|. however, in the latter subjects the histamine hypersensitivity lasted up lo 4 weeks after infection, whereas in our study the effect was no longer significant at day 11. one could speculate that the prolonged effect in atopic rhinitis may have been due to the additional allergen challenges during that study. two previous studies on experimental rhinovirus infection in asthma [!3.14| showed small and variable changes in airway hypersensitivity. our findings suggest that this might be explained by the severity of the colds that were induced. after taking this into account, it appears that experimental rhinovirus infection in asthma does lead to substantial worsening of pcio to histamine. interestingly, this also occurred in the small number of subjects who had preexisting rvl6 neutralizing, but possibly cross-reactive circulating antibodies. the latter may not be surprising, since atopic .subjects with low titres of neutralizing antibodies, as opposed to normal subjects with such titres, have been shown to develop severe cold symptoms after experimental rv16 inoculation [29] . the design of the present study allowed us to differentiate the responses to rhinovirus inoculation from normal fluctuations in symptoms and airway physiology that are characteristic to asthma. the study was performed in the months july to december, but no attempt was made to exclude coinciding allergen exposure (pollen, house dust mite), since this would have been hard to accomplish. however, the present circumstances can be considered as those encountered during naturally occurring infections. despite the fact that the subjects were clinically stable as assessed by history, and by symptom control with p.r.n. /st adrenergic medication alone, a moderate exacerbation of asthma developed in the subject who had the lowest pc^o at entry into the study. this underlines the potential of exacerbations after rhinovirus infection in patients with asthma (3.9,301, despite the usually small accompanying changes in lung function [13] [14] [15] . in this study, we applied validated procedures for inoculation and measuring the responses to rhinovirus infection. first, by using a combination of three methods of virus admini.stration, including nasal inhalation, the natural ways of transmission were mimicked [31] . in this way, the virus may even have reached the intrapulmonary airways [32] . second, commonly used and well-standardized methods for lung function testing and histamine challenges were used [26] . third, we applied a validated method for nasal lavage [27] , which allowed ll-8 to diffuse into the lavage fluid during a 5-min exposure period of the nasal epithelium, resulting in il-8 levels well above the detection limit of the il-8 elisa. how can the present results be interpreted? the increase in airway hypersensitivity, in the absence of a significant decrease in lung function, during the acute phase of infection might be explained by physiological phenomena such as airway wall swelling, potentiating the airway narrowing effect of smooth muscle shortening [33] . such an explanation would be in keeping with the observations by cheung et al. [15] , who showed that experimental rv16 infection leads to excessive airway narrowing in response to inhaled methacholine in subjects with asthma. airway wall swelling in asthma is generally considered to be a consequence of inflammation [2] . indeed, fraenkel et al. [14] recently described the infiltration of inflammatory cells, particularly lymphocytes and eosinophils into the bronchial mucosa in patients with asthma after experimental rvi6 infection. the presently observed correlation between the numbers of © 1997 blackwell science lid, clinical and experimental allergy. 11, 36-45 neutrophils and lymphocytes in peripheral blood after infeetion and the change in airway hypersensitivity indirectly supports an active role of these cells in the virus-induced airway inllammation. we found a marked rise in ll-8 in nasal secretions after rvl6 infection. in general, this confirms the ability of rhinovirus to increase the release of a number of proinflammatory mediators and/or cytokines within the airways, such as kinins [16, 34] and interleukin-1 [35] in nasal secretions, and histamine in broncho-alveolar lavage fluid [36] . the present results obtained by experimental rhinovirus infection are in keeping with the preliminary data of teran et al. [22] , who showed that levels of il-8 in nasal secretions were elevated in nasal secretions during a naturally acquired cold in children with asthma. our results extend these previous observations by showing an association between the increase in il-8 in nasal washings and cold or asthma symptoms, as well as the degree of worsening of airway hyperrespon.sivene.ss. il-8 is a cxc-chemokine, produced by tissue cells {epithelial ceils, fibroblast and endothelial cells), leucocytes, macrophages and mast cells [24, 37] and displays various activities, such as chemotactic activity for neutrophils, lymphocytes and basophils [38] . in addition, il-8 may be involved in the recruitment of primed eosinophils, implicating its involvement in allergic inflammation |39]. since rhinovirus in vitro induces the production of il-8 in epithelium, fibroblasts and peripheral blood mononuclear cells 120,21,40], our findings support the hypothesis that the release of mediators, such as the chemokine il-8, can drive the airway inflammation, and thereby the hypersensitivity to histamine after rhinovirus infection in allergic asthma. what are the clinical implications of this study? first, a common cold aggravates airway hypersensitivity in patients with asthma, fitting in with the close epidemiological association between rhinovirus infections and exacerbations of asthma [5, 9] . and .second, it appears that atopie asthmatic patients with low titres of neutralizing antibodies may not be fully protected against experimental rvi6 infection, and its detrimental effects on their asthma. this ohservation in a small number of subjects in the present study first needs confirmation in larger series of experimental or naturally occurring rhinovirus infections in patients with allergic asthma. in conclusion, experimental rv16 infection can be employed as a useful laboratory model for the development of airway hypersensitivity during an asthma exacerbation. one of the potential mechanisms for this might be the rhinovirus-induced release of pro-inflammatory chemokines. this hypothesis needs further testing in models of rhinovirus infection in vitro and in vivo, focusing on the pathological mechanisms in the intrapulmonary airways in patients with asthma. international consensus report on diagnosis and treatment of asthma wilson jw ci at. mucosal inflammation in asthma viruses as precipitanis ot asthma synipioni.s. i. epidemiology viral respiratory tract infection and exacerbations of asthma in adult patients respiratory viruses and exacerbations of asthma in adults the association of viral and bacterial respiratory inlections with exacerbations of wheezing in young asthmatic children viruses as precipitants of asthmatic attacks in children respiratory viral infection and whee/y bronchitis in childhood a community study of the role of virus infections in exacerhaiions of asthma in 9-11 ye:ir old children rhinovirus upper respirator;' infection increases ainvay hyperreactivity and late asthmatic reactions ht)lgate st. bronchial reactivity to histamine and bradykinin is unchanged after rhinovirus infection in normal subjects lower airway responses to rhinovirus 39 in healthy allergic and nonallergic subjects exacerbations of asthma in adult.s during experimental rhinovirus infection lower airways intlammalion jurinj; rhinovirus colds in nomia! and in asthmatic subjects rhinovirus inhalation causes long-lasling excessive airway narrowing in response to methacholine in asthmatic subjects in vivo kinins are generated during experimental rhinovirus colds nasal-secretion leuktkyte populations deteniiined by flow cytometry during acute rhinovirus infection histopatht)logic examination and enumeration of pt)lymorphonuclear leukixytes in the nasal mucosa during experimental rhinovirus colds rhinovirus enters but does not replicate inside monocytes and airway macniphages infection of a human respiratory epithelial cell line wiih rhinovirus. induction of cytokine release and modulation of susceptibility to infection by cytokine exposure rhinoviruses induce prolonged interleukin-8 release, increases in nirna and promotor activation in pulmonary epithelial and peripheral blood mononuclear cells increased levels of interieukin-8 in the nasal aspirates of children with virus-associatetl asthma immunoreactive rantes and mlp-ia are increa.sed in the nasal aspirates of children with virus-associated asthma interleukin-8 and related chemotactic cytokines -cxc and cc chemokines updated recommendations for safety*testing of viral inocula used in volunteer experiments on rhinovirus colds airway responsiveness. standardized challenge testing with pharmacological, physical and .sensitizing stimuli in adults the 'nasal pool' device applies controlled concentralions of solutes on human nasal airway mucosa and samples its surface exudations/ secretions allergen-induced airway hyperresponsiveness amplified rhinovirus colds in atopic subjects sudden death of an infant with rhinovirus infection complicating bronchial asthma: case report textb(k»k of pediatric infectious disea.ses. philadelphia. wb saunders comparison of nebulised aerosol deposition in the kings of healthy adults following oral and nasal inhalation clinical ami experimaittii allergy. 21. 36-45 airway narrowing and hypertesponsiveness in viral respiratory tract infections kinins are generated in nasal secretions during natural rhinovirus colds increa.sed levels of interleukin-1 are detected in nasal secretions of volunteers during experimental rhinovirus colds bus.se ww. a common cold vini.s, rhinovirus 16. potentiates airway inflammation after ,segmental antigen bronchoprovocation in allergic subjects ll-8 is expressed by human peripheral blood eosinophils. evidence for increased secretion in asthma cc chemokines in allergic inflammation upregulation of formyl-peptide and interleukin-8-induced eosinophil chemotaxis in patients with allergic asthma rhinovirus stimulation of inter!eukin-6 in vivo and in vino. evidence for nuclear factor kb-dependent transcriptional activation this study was supported by a grant of the nelherlands asthma foundation (project no. 93.17). key: cord-000705-w52dc97h authors: ríos, fernando g; estenssoro, elisa; villarejo, fernando; valentini, ricardo; aguilar, liliana; pezzola, daniel; valdez, pascual; blasco, miguel; orlandi, cristina; alvarez, javier; saldarini, fernando; gómez, alejandro; gómez, pablo e; deheza, martin; zazu, alan; quinteros, mónica; chena, ariel; osatnik, javier; violi, damian; gonzalez, maria eugenia; chiappero, guillermo title: lung function and organ dysfunctions in 178 patients requiring mechanical ventilation during the 2009 influenza a (h1n1) pandemic date: 2011-08-17 journal: crit care doi: 10.1186/cc10369 sha: doc_id: 705 cord_uid: w52dc97h introduction: most cases of the 2009 influenza a (h1n1) infection are self-limited, but occasionally the disease evolves to a severe condition needing hospitalization. here we describe the evolution of the respiratory compromise, ventilatory management and laboratory variables of patients with diffuse viral pneumonitis caused by pandemic 2009 influenza a (h1n1) admitted to the icu. method: this was a multicenter, prospective inception cohort study including adult patients with acute respiratory failure requiring mechanical ventilation (mv) admitted to 20 icus in argentina between june and september of 2009 during the influenza a (h1n1) pandemic. in a standard case-report form, we collected epidemiological characteristics, results of real-time reverse-transcriptase--polymerase-chain-reaction viral diagnostic tests, oxygenation variables, acid-base status, respiratory mechanics, ventilation management and laboratory tests. variables were recorded on icu admission and at days 3, 7 and 10. results: during the study period 178 patients with diffuse viral pneumonitis requiring mv were admitted. they were 44 ± 15 years of age, with acute physiology and chronic health evaluation ii (apache ii) scores of 18 ± 7, and most frequent comorbidities were obesity (26%), previous respiratory disease (24%) and immunosuppression (16%). non-invasive ventilation (niv) was applied in 49 (28%) patients on admission, but 94% were later intubated. acute respiratory distress syndrome (ards) was present throughout the entire icu stay in the whole group (mean pao(2)/fio(2 )170 ± 25). tidal-volumes used were 7.8 to 8.1 ml/kg (ideal body weight), plateau pressures always remained < 30 cmh(2)o, without differences between survivors and non-survivors; and mean positive end-expiratory pressure (peep) levels used were between 8 to 12 cm h(2)o. rescue therapies, like recruitment maneuvers (8 to 35%), prone positioning (12 to 24%) and tracheal gas insufflation (3%) were frequently applied. at all time points, ph, platelet count, lactate dehydrogenase assay (ldh) and sequential organ failure assessment (sofa) differed significantly between survivors and non-survivors. lack of recovery of platelet count and persistence of leukocytosis were characteristic of non-survivors. mortality was high (46%); and length of mv was 10 (6 to 17) days. conclusions: these patients had severe, hypoxemic respiratory failure compatible with ards that persisted over time, frequently requiring rescue therapies to support oxygenation. niv use is not warranted, given its high failure rate. death and evolution to prolonged mechanical ventilation were common outcomes. persistence of thrombocytopenia, acidosis and leukocytosis, and high ldh levels found in non-survivors during the course of the disease might be novel prognostic findings. on april 2009, a novel influenza a (h1n1) virus emerged in mexico and spread rapidly across the world [1, 2] . as of 17 june 2010, more than 214 countries had reported confirmed cases of infection with pandemic 2009 influenza a (h1n1) virus, including at least 18,156 deaths [3] . unlike seasonal influenza, in which hospitalizations occur among patients younger than 2 and older than 65 years, or in those with underlying diseases [4] , this novel virus affected otherwise healthy young and middle-aged adults and obese individuals [2, 5] . patients with previous respiratory disease, immunocompromised hosts and pregnant women were affected as frequently as with seasonal influenza [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . although a mild form of the disease was prevalent, it soon became evident that the 2009 influenza a (h1n1) virus could also provoke severe, acute respiratory failure requiring admission to the intensive care unit (icu) for mechanical ventilation [16] , which was reflected in the severe pathological injury found at autopsy [17] . the argentinian population was greatly affected during the pandemic, with a total of 1,390,566 cases of influenza-like illness requiring 14,034 hospitalizations. of the 11,746 confirmed cases of patients infected with the new strain, 617 died [18] . this represents a death rate per infection of 4.3% in hospitalized cases; an intermediate figure compared to 3.6% in brazil, 1.2% in chile, and approximately 6% in uruguay, colombia and venezuela [19] . it should be noted that these numbers reflect great uncertainty, particularly with regard to case diagnosis. lack of testing of mild disease and difficulties due to laboratory overload have also been well described [15, 20] . these general problems have been acknowledged by experts [21] . the severity of disease was rapidly perceived by health authorities and scientific societies. hence, a committee of experts of the argentinian society of intensive care medicine decided to focus on the most acutely ill patients: those presenting with diffuse viral pneumonitis requiring mechanical ventilation. they designed an epidemiological study, recently-published, to determine risk factors and outcomes [15] ; this is one of many series up to the present that have described epidemiological and clinical aspects of the 2009 influenza a (h1n1) pandemic [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . there remains, however, a paucity of data published on physiological evolution during icu stay [22] . this present study, concurrently planned with the first by the same committee of experts, thus aims to provide such information. our objectives were: first, to characterize alterations of oxygenation, respiratory mechanics and the use of mechanical ventilation; second, to explore compliance with protective lung ventilation; and, finally, to assess the evolution of laboratory findings and organ dysfunctions throughout the course of the disease. this was a multicenter, inception cohort study that included patients aged > 15 years admitted to the icu with a previous history of influenza-like illness, evolving to acute respiratory failure that required mechanical ventilation during the 2009 winter in the southern hemisphere. these patients had confirmed or probable disease caused by the 2009 influenza a (h1n1) virus and were included in the registry of cases of the argentinian society of intensive care medicine (sati), created to characterize local aspects of the pandemic. on 27 june 2009, a form to collect online epidemiological data was posted on the official sati website. a detailed description and analysis of this information was recently published [14] . there was also an optional, more comprehensive casereport form to complete, developed by experts of the sati's respiratory committee for recording certain prespecified variables throughout icu stay, which included mechanical ventilation (mv), respiratory mechanics, oxygenation, blood chemistry and organ failure variables. this information was collected over 10 days and is analyzed in the present study. patients were characterized as confirmed, probable or possible cases of 2009 influenza a (h1n1) [20] according to the findings in the respiratory samples collected on admission. some specimens, however, were not analyzed because laboratories soon became overloaded, especially at the beginning of the pandemic. as of 25 september 2009, the weekly update of the ministry of health reported that in patients ≥5 years with influenzalike illness, the 2009 influenza a (h1n1) virus had displaced other respiratory viruses in 93.4% of the samples processed [23, 24] . as a result of this, probable and suspected cases were considered as caused by the novel virus and were so included in the study. we collected dates of hospital and icu admission, and of mv onset; demographics; risk factors for influenza a; actual weight; height; severity of illness (acute physiology and chronic health evaluation ii, apache ii), organ failures (sequential organ failure assessment, sofa); type of mv used, as noninvasive (niv) and invasive; and date of intubation. ideal body weight (ibw, ml/kg) and body mass index (bmi) were calculated; obesity was defined as a bmi > 30. at mv onset (day 0) and on days 3, 7 and 10, until death or discharge, whichever occurred first, we recorded: (1) mv-related variables. (2) mv modes: volume-controlled ventilation (vcv); pressure-controlled ventilation (pcv); bilevel mode; pressure support ventilation (psv); other. (3) tidal volume (vt, in ml/kg of ibw) (4) pressures: peak, plateau pressures, total positive end-expiratory pressure (peep) and driving pressure (plateau pressure -peep), in cmh2o. the main outcome measure was hospital mortality; secondary outcomes were length of mv, of icu (losicu) and of hospital (loshosp) stays. in case of missing observations, local study coordinators were contacted to provide the corresponding values. proportions were calculated as percentages of existing data. no assumptions for missing data were made. statistical analysis was performed with spss 17.0 (spss inc., chicago, il, usa). data were analyzed for the entire population; for the subgroups of survivors vs. non-survivors; and for patients receiving niv on admission vs. those who did not. descriptive statistics used were: mean ± standard deviations (sd) and median and 25-75% interquartile ranges (iqr) for continuous data of normal and non-normal distribution, respectively; and percentages for categorical data. differences between subgroups were analyzed with unpaired t test, mann-whitney u test, and chi-square tests, as appropriate. a p-value of <.05 was considered statistically significant. a kaplan-meier curve was constructed to evaluate survival over the follow-up period. over time, normally distributed data were analyzed with two-way repeated measures of anova. at the pre-specified time points, differences within the entire group and subgroups, and between subgroups, were tested using paired and unpaired t tests, respectively. in non-normally distributed data, differences over time within the entire group and the subgroups were analyzed with friedman's and wilcoxon tests. comparisons between subgroups at the pre-specified time points were tested with mann-whitney u test. the bonferroni correction was used to adjustments for multiple comparisons. the local institutional review boards waived the need for informed consent, given the general lack of knowledge on the clinical and outcome characteristics of the ongoing pandemic and to the non-interventional study design. general characteristics (table 1) between 6 june and 28 august 2009, the sati's online registry included 337 patients admitted to 35 icus with confirmed/probable/possible diffuse viral pneumonitis caused by influenza a (h1n1), with acute respiratory failure requiring mv (14) . of these, 178 consecutive patients admitted to 20 icus were followed over time, and are presented in this study. to address any potential concern that unconfirmed cases could belong to a different population of patients, we performed a sensitivity analysis of clinical and outcome characteristics data after exclusion of these patients. the results of this analysis did not differ from those of the primary assessments, so the 178 patients are considered for evaluation. briefly, patients were middle-aged, with no gender preponderance; they had a history of symptoms of nearly one-week duration and were ventilated at 1 [0 to -2] day after hospital admission. pre-existent respiratory diseases, obesity, and diseases causing immunosuppression were the most frequent comorbid conditions; and prevalence of pregnancy was higher than in the general population, as expected [25] . non-survivors were sicker on admission; duration of previous symptoms was longer; and organ failures were more severe. obesity and immunosuppression were significantly more frequent as predisposing conditions. ninety-three patients survived (52%) (see figure 1 ). (table 2) during the study period, the entire group had vt values between 7.8 to 8.1 ml/kg of ibw, with plateau pressures remaining always < 30 cmh 2 o. non-survivors displayed a trend towards lower vt and higher plateau pressures, which differed significantly from survivors only at day 7. intermediate peep levels were used, and decreased in survivors from day 3 onwards. driving pressures were similar over time in all patients; only at admission did non-survivors exhibit higher values. pao 2 /fio 2 increased significantly over time in all patients and in survivors. it remained, however, < 200 in the whole group throughout the entire icu stay due to non-survivor values. non-survivors displayed significantly lower pao 2 /fio 2 at all time points. lung infiltrates (in quadrants) peaked at day 3 (3.1 ± 1.0 vs. 2.9 ± 1 at day 0, p < 0.01) and then decreased during the study in the entire group, especially at day 10 (2.8 ± 1.1, p < 0.83 vs. day 0), which reflected the improvement in survivors (3.1 ± 1.0 at day 3 vs. 2.9 ± 1.0 at day 10, p < 0.01). in figure 2 , the utilization of ventilation modes and rescue therapies in the entire group are shown. briefly, pcv use equaled vcv at day 10, preceded by deterioration in oxygenation and respiratory mechanics: pao 2 / fio 2 78 ± 24 vs. 128 ± 33, (p = 0.03); paco 2 44 ± 4 vs. 35 ± 3 mmhg (p = 0.04); ph 7.29 ± 0.03 vs. 7.39 ± 0.05 (p = 0.05), and plateau pressures of 30 ± 2 vs. 25 ± 3 cmh 2 o (p = 0.03). recruitment maneuvers became significantly more common in non-survivors at day 3 (46%, vs. 29% in survivors; p = 0.03), as did prone positioning (24%, vs. 14%; p = 0.001). after that, only prone positioning remained significantly more used in nonsurvivors (at day 7: 38%; vs. 14%, p = 0.004; and at day 10: 25%; vs. 5%, p = 0.02). six patients received tracheal gas insufflation; only one survived. neuromuscular blockers were prescribed in 18% of patients on admission; and their use was subsequently more frequent in non-survivors (day 3: 14% vs. 8%, p = 0.02; and day 7: 14% vs. 8%, p = 0.04). the main causes of death were refractory hypoxemia (64%); followed by multiorgan dysfunction syndrome (15%) and shock (10%). prolonged mechanical ventilation and long icu and hospital stays were frequent (table 1) . tracheostomy was performed in 29 patients (16%) at day 14 [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . acid-base variables and fluid balance (table 3) arterial ph increased over time in the whole cohort and in both subgroups, perhaps secondary to general resuscitation measures. despite this, non-survivors displayed significantly lower ph at all time points, owing to changes in base excess on days 0 and 3, and to pco 2 elevations thereafter. respiratory rates remained unchanged, only increasing at day 10 in non-survivors; nevertheless, this corresponded to the highest pco 2 values, indicating the more severe respiratory compromise. bicarbonate paralleled ph behavior. changes in fluid balance did not show clear trends: only at day 10 they decreased significantly, expressing survivors' behavior. forty-nine patients (28%) underwent a trial of niv on admission; they were significantly less ill and had a lower incidence of immunosuppression. oxygenation and outcome variables were similar to those of patients not receiving niv. sixty-one percent of patients (n = 30) receiving niv survived; duration of niv was of 8 (2 to 18) hours. there were no differences between survivors and nonsurvivors in the duration of the procedure, or in the type of interface or respirator used. of note, most patients on niv (46 out of 49; 94%) had to be intubated and ventilated invasively for hypoxemic failure. characteristics associated to niv success/failure are shown in table 5 . niv was also used for treating post-extubation respiratory failure in 12 of 178 patients (7%), with success (reintubation not needed) in 8 cases (66%). the most consistent changes over time were found in platelet count, which increased significantly in the whole cohort (p < 0.000 for days 3, 7 and 10 vs. day 0), secondary to elevations in survivors. at all time points, platelets differed between survivors and non-survivors. conversely, white blood cell count showed a progressive creatine-kinase and markers of liver injury (alanine/ aspartate aminotransferases, serum bilirubin; not shown) were mildly elevated and displayed no substantial changes. on the contrary, lactate-dehydrogenase levels were significantly higher in non-survivors throughout the study. creatinine levels were stable over the period, but were significantly higher in non-survivors on days 0 and 3. finally, sofa score diminished over time in all patients (p < 0.000 for days 7 and 10 vs. day 0), as a result of the decrease in survivors. sofa was significantly lower in survivors throughout the study. in figure 3 , the differences between survivors and non-survivors are displayed. we report on a large, prospective cohort of 2009 influenza a (h1n1) patients that were mechanically ventilated for acute respiratory failure due to diffuse pneumonitis during the pandemic in argentina. though most were middle-aged, previously healthy adults, patients with preexistent lung disease, immunosuppression, obesity and pregnancy were also affected. mortality was high and evolution to chronic critical illness was common, as shown by prolonged mechanical ventilation, high needs of tracheostomy, and lengthened icu and hospital stays. patients had characteristically a history of protracted symptoms and displayed severe compromise of oxygenation compatible with ards throughout the study period, which only improved in survivors. at all time points, pao 2 /fio 2 differed significantly between survivors and non-survivors, requiring higher fio 2 and peep in this last subgroup. yet the levels of applied peep were only in the intermediate range, similar to mean values of 8.7 cmh 2 o of peep in an international study on mechanical ventilation [26] , which may explain the relatively high fio 2 used in our study. driving pressures were similar in both subgroups most of the time, suggesting an intention to limit alveolar excursion as part of a protective strategy. it is striking that, as has been described in similar studies on mechanical ventilation performed during the 2009 influenza a (h1n1) pandemic [6, 7] , tidal volumes used were between 7.5 and 8.3 ml/kg ibw, certainly higher than the 6 ml/kg demonstrated as being lungprotective [27] . indeed, barriers to implementing lowtidal volume have been identified and might explain physician behavior [28] . despite this, plateau pressures did remain below 30 cmh 2 o [29] , indicating that lung compliance might have been preserved. perhaps clinicians focused on plateau pressures rather than on tidal volumes [30] since it still remains unclear which should be limited to avoid ventilator-induced lung injury [31] . we, like others [6, 7, 32, 33] , could not find differences in utilized tidal volumes between survivors and non-survivors. even so, non-survivors tended to display lower values, probably reflecting physician efforts to intensify protective ventilation strategies in the most severely compromised. some researchers [34, 35] have suggested that allowing higher tidal volumes in a population of young and previously healthy patients with strong ventilatory drive might reveal an attempt to restrain heavy sedation and neuromuscular blocker use. notwithstanding this, we believe that these findings may also represent clinicians' inadequate prescription, as described in other scenarios [36] . not unexpectedly, vcv was the most common ventilator mode used. pcv use increased throughout the study period, peaking at day 10. this is in contrast with the recently identified trend towards decreased pcv utilization. transition to pcv mode was associated with preceding physiological worsening, so clinicians might have perceived pcv utilization as part of a global lungprotective strategy [37] . refractory hypoxemia was the main cause of death. as in other studies [6, 7, 11] , rescue therapies were frequently applied, with utilization highest 72 hours after admission. recruitment maneuvers and prone positioning were the primary adjuvants utilized; ecmo and hfov are currently not available in argentina. a table 3 oxygenation and acid-base variables, and fluid balance in all patients, and in survivors and non-survivors. prolonged mechanical ventilation course was frequent as reported elsewhere [6] . niv was the first ventilation approach in 28% of cases, with 94% later requiring invasive ventilation, as has been documented in other studies [6, 7, 11] . these common experiences should caution against delaying proper ventilatory support in this group, given that rapid deterioration is common. a recent meta-analysis suggests that niv does not decrease the need for intubation, so evidence to support its use in severe ards is questionable [38] . in our study, improved outcomes with niv could be due to milder disease, evidenced by apache ii. the small number of patients that were not intubated precludes a statistical analysis; however, they were younger, with less severe disease and better oxygenation. significant changes in fluid balance were late and reflected changes in survivors. negative fluid balances could never be obtained, perhaps suggesting a continuing need for hemodynamic support: 72% of patients presented with shock [14] . on the whole, fluid balances remained between those achieved by "liberal" and "conservative" strategies of the fluids and catheters treatment trial, depending on the day evaluated [39] . thus far, it is not clear whether the negative fluid balance has a causal role in improving outcome in ali/ards, or if it simply expresses the global recovery of patients. another important finding was that arterial ph consistently and significantly differed between survivors and non-survivors, as described elsewhere [40, 41] . during the first 72 hours acidosis had a major metabolic component, likely as a sign of hemodynamic impairment. after the first week, respiratory acidosis ensued, indicating either the effects of protective ventilation, or merely deterioration due to progressive shunt, profound ventilation/perfusion mismatch and increased deadspace. with respect to blood chemistry, the usual findings of thrombocytopenia, leukocytosis and mildly elevated creatine-kinase blood levels were present [21, 42] . regrettably, the lymphocyte count was not recorded. in viral infections, thrombocytopenia occurred frequently. although the mechanisms by which the 2009 influenza a (h1n1) virus causes thrombocytopenia are unknown, its lack of resolution is a marker of poor prognosis. both leukocytosis and leucopenia have been found in hospitalized patients with 2009 influenza a (h1n1) [2, 43] ; in our study, persistent leukocytosis was associated with increased mortality. ldh elevations have been previously described in fatal cases [2] , which corresponded to our finding of higher ldh levels in non-survivors at all time points. such elevations have also been reported in seasonal influenza [44] . in experimental studies, increased ldh is a marker of human fetal membrane cell apoptosis induced by influenza virus [45] . finally, multiorgan failure was frequent, and predictably more severe in non-survivors. this study has several strengths: first, the clinical characteristics and time course of pandemic 2009 influenza a (h1n1) are thoroughly described and analyzed. second, data were collected prospectively in consecutive patients and with a standardized casereporting form, representing a large, nationwide cohort. third, temporal patterns of mechanical ventilation use, acid-base and blood chemistry variables, as well as fluid balance and organ failures, are carefully analyzed. prognostic implications are highlighted. finally, we present the largest experience with niv use during the pandemic. study limitations include the focus on mechanically ventilated patients, excluding less severe cases also admitted to the icu. many cases could not be confirmed because laboratories were overwhelmed with clinical samples, which is also described elsewhere [7, 14] . data about transmission to healthcare workers were not recorded, especially regarding niv. currently, most information about its use during an epidemic relies upon expert opinion [46] . in 178 patients with diffuse viral pneumonitis caused by the 2009 influenza a (h1n1) virus admitted to the icu and followed over time, ards was the rule, requiring high ventilation support and frequent use of rescue therapies. death, organ failures, and evolution to prolonged mechanical ventilation were common. in most cases, noninvasive ventilation failed to prevent endotracheal intubation. finally, elevated ldh levels, lack of recovery of platelet count and persistent acidosis and leukocytosis in non-survivors behaved as prognostic findings. • in 2009 influenza a (h1n1) patients, hospital admission with prompt indication of mechanical ventilation -a marker of severe disease -was associated with a history of symptoms of nearly one-week duration. • an initial niv trial was not effective to avoid intubation in most patients; thus, this ventilation approach should likely be discarded in this setting. • mortality and morbidity were frequent: death was common and was mainly caused by persistent, refractory hypoxemia. prolonged mechanical ventilation and icu and hospital stays were typical. • ph, platelet count, ldh and sofa differed significantly between survivors and non-survivors over time. lack of recovery of platelet count and persistence of leukocytosis might be markers of poor prognosis. • every effort should be done to increase adherence to protective ventilation in the real world. abbreviations ali: acute lung injury; ards: acute respiratory distress syndrome; bmi: body mass index; cxr: plain chest x-ray film; ibw: ideal body weight; icu: intensive care unit; ldh: lactate dehydrogenase assay; los: length of stay; mv: mechanical ventilation; niv: non-invasive ventilation; pao2/fio2: relation between patient arterial po 2 and inspired oxygen fraction used; pcv: pressure-controlled ventilation; peep: positive end-expiratory pressure; psv: pressure support ventilation; rr: respiratory rate; rt-pcr: real-time reversetranscriptase-polymerase-chain-reaction; sati: argentinian society of intensive care; sofa: sequential organ failure assessment; vcv: volumecontrolled ventilation; vt: tidal volume. the registry of the argentinian society of intensive care department intensive care, hospital general de agudos velez sarsfield, calderón de la barca 1550, (c1407ahh) department critical care department intensive care, hospital lagomaggiore, gordillo s/n 16 department intensive care mar del plata, argentina. 19 intensive care unit, hospital universidad abierta interamericana, portela 2975, (c1069aab) group on influenza: pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico pandemic (h1n1) 2009 -update 104. weekly update influenza-associated hospitalizations in the united states intensive care patients with severe novel influenza a (h1n1) virus infection-michigan critically ill patients with 2009 influenza a (h1n1) infection in canada critically ill patients with 2009 influenza a (h1n1) in mexico critical care services and 2009 h1n1 influenza in australia and new zealand pandemic influenza a (h1n1) virus hospitalizations investigation team: hospitalized patients with 2009 h1n1 influenza in the united states h1n1) working group: factors associated with death or hospitalization due to pandemic 2009 influenza a(h1n1) infection in california intensive care adult patients with severe respiratory failure caused by influenza a (h1n1) in spain influenza a pandemics: clinical and organizational aspects: the experience in chile national influenza a pandemic (h1n1) 2009 clinical investigation group of china: clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china registry of the argentinian society of intensive care sati: pandemic 2009 influenza a (h1n1) in argentina: a study of 337 patients on mechanical ventilation novel influenza a (h1n1) pregnancy working group: h1n1 2009 influenza virus infection during pregnancy in the usa severe respiratory disease concurrent with the circulation of h1n1 influenza pathology in fatal novel human influenza a (h1n1) infection influenza pandémica (h1n1) 2009. república argentina worldwide statistics of the h1n1 influenza a pandemic practical lessons from the first outbreaks: clinical presentation, obstacles, and management strategies for severe pandemic (ph1n1) 2009 influenza pneumonitis writing committee of the who consultation on clinical aspects of pandemic (h1n1) ventilator management for hypoxemic respiratory failure attributable to h1n1 novel swine origin influenza virus guidance on case definitions to be used for investigations of novel influenza a (h1n1) cases influenza pandémica (h1n1) 2009. república argentina. influenza pandémica (h1n1) 2009. report of the 34 epidemiological week direccion de estadisticas e informacion de salud. sistema estadístico de salud. serie 5 -número 51 evolution of mechanical ventilation in response to clinical research the acute respiratory distress syndrome network: ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and acute respiratory distress syndrome barriers to providing lung-protective ventilation to patients with acute lung injury tidal volume reduction in patients with acute lung injury when plateau pressures are not high. ards clinical trials network express) study group: positive end-expiratory pressure setting in adults with acute lung injury and acute respiratory distress syndrome: a randomized controlled trial anzueto a: tidal volume in mechanical ventilation: the importance of considering predicted body weight pressure-and volume-limited ventilation strategy group: evaluation of a ventilation strategy to prevent barotrauma in patients at high risk for acute respiratory distress syndrome the multicenter trail group on tidal volume reduction in ards: tidal volume reduction for prevention of ventilator-induced lung injury in acute respiratory distress syndrome mechanical ventilation in critically ill patients with 2009 influenza a (h1n1) mechanical ventilation in critically ill patients with 2009 influenza a (h1n1) the finnali study on acute respiratory failure: not the final cut effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome is there a role for noninvasive ventilation in acute respiratory distress syndrome? a meta-analysis the national heart, lung, and blood institute acute respiratory distress syndrome (ards) clinical trials network: comparison of two fluidmanagement strategies in acute lung injury metabolic correlates of oxygen debt predict posttrauma early acute respiratory distress syndrome and the related cytokine response osatnik j: incidence, clinical course, and outcome in 217 patients with acute respiratory distress syndrome swine influenza (h1n1) pneumonia: clinical considerations influenza pneumonia: a descriptive study lactate dehydrogenase leakage as a marker for apoptotic cell degradation induced by influenza virus infection in human fetal membrane cells should noninvasive ventilation be considered a high-risk procedure during an epidemic? on the role of non-invasive (niv) to treat patients during the h1n1 influenza pandemic lung function and organ dysfunctions in 178 patients requiring mechanical ventilation during the influenza a (h1n1) pandemic the authors declare that they have no competing interests. key: cord-027259-f4sgobcz authors: metsker, oleg; igor, vozniuk; kopanitsa, georgy; morozova, elena; maria, prohorova title: stroke icu patient mortality day prediction date: 2020-05-23 journal: computational science iccs 2020 doi: 10.1007/978-3-030-50423-6_29 sha: doc_id: 27259 cord_uid: f4sgobcz this article presents a study on development of methods for analysis of data reflecting the process of treatment of stroke inpatients to predict clinical outcomes at the emergency care unit. the aim of this work is to develop models for the creation of validated risk scales for early intravenous stroke with minimum number of parameters with maximum prognostic accuracy and possibility to calculate the time of “expected intravenous stroke mortality”. the study of experience in the development and use of medical information systems allows us to state the insufficient ability of existing models for adequate data analysis, weak formalization and lack of system approach in the collection of diagnostic data, insufficient personalization of diagnostic data on the factors determining early intravenous stroke mortality. in our study we divided patients into 3 subgroups according to the time of death up to 1 day, 1 to 3 days, and 4 to 10 days. early mortality in each subgroup was associated with a number of demographic, clinical, and instrumental-laboratory characteristics based on the interpretation of the results of calculating the significance of predictors of binary classification models by machine learning methods from the scikit-learn library. the target classes in training were “mortality rate of 1 day”, “mortality rate of 1–3 days”, “mortality rate from 4 days”. auc roc of trained models reached 91% for the method of random forest. the results of interpretation of decision trees and calculation of significance of predictors of built-in methods of random forest coincide that can prove to correctness of calculations. stroke is the second most deadly cause of death worldwide. in russia, brain stroke is the second leading cause of death after myocardial infarction. every year around 450000 people suffer from stroke, in fact it is the population of a big city [1] . the mortality rate in russia is 4 times higher than in the usa and canada [2] . among european countries, the mortality rate from cerebrovascular diseases is the highest in russia. according to the all-russian center for preventive medicine, in our country 25% of men and 39% of women die from cerebrovascular diseases. in the largest cities of the country the situation with this type of pathology is extremely unfavorable. in st. petersburg, for example, the frequency of stroke is about 528 cases per 100,000 residents, while the mortality rate for ischemic stroke is 39%. it is necessary to emphasize the catastrophic consequences of ischemic stroke -up to 84-87% of patients die or remain disabled and only 16-13% of patients fully recover [2] . according to the findings of a large-scale study of recent years, some modern epidemiological trends have been identified [3] : in general, global statistics show a decline in stroke mortality over the past two decades due to the introduction of new treatments (thrombolysis, thrombectrosis), but the absolute number of people who have stroke is only increasing every year [4] . this nosology is still the strong second leading cause of death from cardiovascular disease (cvd), remaining the undisputed leader among all nosologies leading to severe disability. hospital mortality remains one the most important quality indicator, which can be used to identify problems associated with the optimization of prehospital and hospital treatment process. it can be used to assess the effectiveness of primary and secondary care, routing, and the degree of implementation of modern diagnostic and treatment algorithms, including the quality of interaction between different levels of care [5] . it is important to note that regional characteristics of the populations may significantly differ from the global ones, and the development of specialized care programs for patients with a stroke has its national and institutional characteristics. understanding the factors that contribute to the reduction of hospital mortality will allow us to develop a targeted strategy for the development of services providing care to patients with a stroke in russia and in the world. thus, development of personalized models and algorithms for planning of individual treatment tactics for the stroke patients can reduce mortality and increase the standard of life. the development of such models and algorithms will ensure better continuity and efficiency of medical care and help reducing the number of complications. the basis for such models can be the scales of calculation of patients' mortality risks in emergency units, which are also absent in russia at present. most statistics are accumulated in national stroke registries or national databases: china national stroke registry ii (cnsr ii) [6] , the nationwide hospital discharge database (nhdd), berlin stroke register (bsr), german stroke register, the registry of the canadian stroke network (rcsn), national acute stroke israeli (nasis) registry, fleni stroke data bank, australian stroke clinical registry (auscr), national stroke register of ireland, the austrian stroke registy. the analysis of available literature revealed rather heterogeneous values of the share of hospital mortality of patients with stroke in different countries. at the same time, direct indicators of the share of hospital mortality had significant differences from 1,4% in china [7] to 22,7% in ethiopia [8] . significant differences in data can be explained both by the quality of care and by the nature of statistical data collection. in particular, most of the reports took into account only the ischemic type of stroke [9] [10] [11] [12] [13] [14] , different exclusion criteria were applied in a number of observations -daily mortality and stay exceeding 180 days [9] , inhospital stroke [15] , patients in need of admission to the general intensive care unit [16] or a general department. it should also be noted that samples are heterogeneous in terms of the number of patients: from 110 [8] to 12 million patients [17] . hospital mortality rates vary considerably between facilities within the same country. for example, the average hospital mortality rate in germany in 2011 was 4.6% when 26 stroke units were evaluated. [13] , at the same time as in the german study of 2015 on this parameter was 8.2% [18] . in australia, hospital mortality also varies significantly (from 7% to 23%) depending on the level of the hospital. [15] , in germany, there is a dependence on the size of the hospital -from 0% to 25% in small hospitals and from 0.4% to 9.3% in large hospitals. [11] . only 9 studies out of 22 provide data that allow tracking the dynamics of changes in the indicator of intra-hospital mortality. the average rate of decline in this indicator was 0.36% per year. rapid changes in this parameter are more typical of the ischemic type of stroke, and mainly the faster rate of decline was associated with the introduction and expansion of the vascular center network for stroke (with mandatory stroke unit). the most significant example of canada -where vascular center system was introduced, which led to the rate of change in the provinces was 0.28% per year, while in the provinces without the introduction of the vascular center system, the rate changed only by 0.11% per year [19] . the availability of prognostic models and scales that are understandable to clinical staff and easy to operate, reduces hospital mortality and allows for a more targeted and individualized approach to therapy. such models should take into account locally established practices. models should be available that can predict a fatal scenario for the disease, considering all relevant factors. to date, the international medical community has made repeated attempts to create such a prognostic scale. in the 2002 review, c. counsell and m. dennis analyzed 83 models with a total of 150 prognostic factors, and the assessment resulted in only 4 models meeting quality criteria [8] . the databases have a huge number of parameters including various tests and indicators. in some cases, the use of a large number of features leads to lower rates of learning and forecasting, reduces the predictive accuracy of the model, and prevents the model from being interpreted, which is an important requirement for models used in medicine. thus, finding the best set of features in the context of our task is one of the key factors ensuring high quality of the predictive model. on the basis of the analysis of 12 modern prognostic models from 10 countries we can identify some of the most stable (main) predictors for the causes of intra-hospital mortality: age [16, [20] [21] [22] [23] [24] ; type of stroke [25] ; lesion location [25] ; level of consciousness [11, 20, 23, 25, 26] upon admission; nihss stroke severity [10, 21, 22, 24] ; comorbidity [22, 27] , charlson comorbidity index [23] , atrial fibrillation [11, 22] , case history transitor ischemic attack (tia) [31]; hospital complications (high intracranial pressure) [16] , pneumonia, seizures, anxiety/depression, infections, limb pains and constipation [22, 27] . among the predictors related to the organization of care, the time of admission to hospital can be noted -in a japanese study, the 7-day mortality rate increased if the patient was admitted on weekends or holidays. [23] , hospital delivery method had a predictive value as well [16] , both these parameters are included in the gwtg-stroke program [14] . in order to identify priority areas for improving the outcome of the disease it is necessary to divide the selected factors (predictors) into modifiable and unmodifiable, respectively. modifiable mortality predictors can be referred to: time and method of hospital delivery; qualifications of medical personnel; stroke care model; history of stroke or tia, atrial fibrillation, diabetes mellitus, comorbidity indexparameters to which primary prevention should be directed; intra-hospital complications (high intracranial pressure pneumonia, seizures, anxiety/ depression, infection, extremity pain and constipation). a special form of complications in the form of extracerebral pathology -polyorgan failure syndrome -is distinguished separately. special attention should be paid to the prevention of this syndrome. the unmodifiable factors of stroke mortality include: gender, age, type of stroke, localization of lesion. as for the assessment of the impact of comorbid diseases, it is important to consider not only the presence of individual pathologies, but also their combination. in particular, the following groups can be distinguished: arterial hypertension + atrial fibrillation, arterial hypertension + atrial fibrillation + coronary heart disease, atrial fibrillation + postinfarction cardiosclerosis, and, arterial hypertension + postinfarction cardiosclerosis + atrial fibrillation ma и + diabetes mellitus. only two studies presented clear prognostic scales containing a scoring system for rapid assessment of the risk (probability) of in hospital mortality [14, 21] . the premise scale is simple, quick to calculate at > 85% of strokes and uses only variables that are available shortly after the onset of ischemic stroke when admitted to the stroke unit. it should be noted that the practical application of any analyzed scale above in different countries requires corrections to take into account regional peculiarities -social, geographical and medical and economic factors [26] . the creation of such scales and models in russia would provide a tool to assess the efficiency of care. the goal of this work is to identify features for the creation of validated risk scales for early hospital mortality. the study includes data about 36450 episodes (17158 outpatient 5565-inpatient patients 200-lethal patients 5565 patients who has international criteria for diagnosis icd i60 to i69.8) and were treated in the almazov national research center from 2011 to 2019. among the causes of admission: ischemic stroke, hemorrhagic stroke, embolic stroke, transitor attacks. as the initial data describing the condition the patient's examination data at the intake and use of clinical scales (nihss, mrs), conclusion of magnetic resonance imaging (mri), conclusion of ultrasound investigation, data from laboratory tests, data on treatment events from the medical information system. a separate more detailed analysis of the group of only deceased patients from 100 people was carried out to identify differences and mortality factors in different time periods (1 day, 2-3 days, 4-15 days) on the basis of data from the the saint petersburg research institute of emergency medicine n.a. i.i. dzhanelidze 1 . the data of the medical information system of the operating specialized center of mri, ultrasound and other characteristics of the volume of cerebral and vascular stroke examination were compared with the data on the duration and outcomes and time of 1 http://www.emergency.spb.ru/. death. information about hospital mortality was included in the study, if they met the following criteria: the fact of clinically confirmed diagnosis of acute cerebral circulation disorder (ischemic or hemorrhagic), with the presence of focal, general cerebral neurological syndromes, which lasted more than 24 h from the beginning of the disease; hospitalization in connection with stroke in the first day of the disease; the entire period of hospitalization in connection with acute case of the patient spent in one institution; lethal outcome was associated with an acute period of stroke. information confirming lesions of the brain substance has been obtained from data from the ct scan and/or mri of the brain, which have been repeated if necessary. the extent of precerebral and cerebral artery lesions was assessed using ultrasound duplex scanning, ct scan, mri or cerebral angiography. to obtain the optimal set of features a combination of classical methods based on different correlation coefficients of features (pearson correlation coefficient and spearman correlation coefficient) were used. ensemble algorithms, including ensembles built on the basis of models with the use of decision trees, and random forest are used as prognostic models. a scikit-learn library was used to implement machine learning methods. in the process of definition of hyperparameters of machine learning models, cross-validation by k blocks was applied. precision and recall (accuracy and completeness), as well as their harmonic mean (f-score) were used as metrics at this stage. construction of the confusion matrix of multiclass classification allowed to analyze errors, improve data sampling used for model training and initialize the next iteration of model training. the data on treatment of real patients from the almazov center were used for validation of the final resulting models. the data of patients who did not participate in any stages of model training and adjustment of hyper-parameters were used. auc roc -the area under the error curve -was used as the result metrics. p-value was calculated using two methods. the essence of the first method is that for each sample of dead (<1 day, 1-3 days, 4-10 days) we have calculated p-value for every feature of the corresponding test. chi-square criterion was used for categorical features, kolmogorov-smirnov's test was used for continuous features. the essence of the second method of calculating p-value by one attribute (mortality period) for three groups of patients according to the severity and type of stroke (group 1: ich+pvh, is +ich; group 2: is+bilat atr, is-foc 16-25hu; group 3: is-1/3<16hu, sub tent icv). the analysis obtained a general model of mortality for all patients with stroke auc roc-93% demonstrated random forest learned on the dataset with more than 60 laboratory and personal patient observation features. three separate models have also been developed for patients with different lethality periods (up to 1 day, from 1 to 3 days, and from 4 to 15 days) using decision trees that showed an auc roc of 85 to 91%. for these models, the dataset consisted of more than 70 specialized features, including a score on neurologic scales, brain examination data, assessment of the patient's consciousness and somatic state. the importance of features for different duration of lethality was also compared. moreover, a clinical interpretation of the comparison results is given below. the models were trained on the dataset describing 5565 patients who were treated as a binary classification models by machine learning methods from the scikit-learn library. the following parameters were used as features: patients age, male, pressure, area of brain damage, the size of the hematoma. moreover, the following laboratory tests were used as features: mchc-red blood cell index, endothelin, interleukin-10, interleukin-8, interleukin-6, interleukin-4, interleukin-1-beta, inr, fibrogen, vitamin d, paratohormone, urine nitrites, urine bilirubin, urine, bld urine, leu urine, urine nit, urine ket, urine glucose, urine pro, urine ph, urine color, d-dimmer, albumin, lipids, triglyceride, total cholesterol, prothrombin index, fibrinogen by klaus, k+ (vienna), neutrophils, monocytes, lymphocytes, mpv average, platelet volume, pdw width of platelet distribution by volume, rdw width of red blood cell distribution by volume, mchc the average concentration of hemoglobin in eritr, mch is the average hemoglobin content in 1 erythrocyte average volume of red blood cells, reactive protein, erythrocyte sedimentation rate, troponin, alt, ast, hgb hemoglobin, wbc white blood cells, rbc red blood cells, plt platelets, creatinine, bilirubin, hct hematocrit, glucose level. random forest demonstrated the best auc roc-93%. the nine most importantly lethality features of the stroke patient further: systolic pressure (0.06), rbc red blood cells (0.05), interleukin-8 (0.05), hct hematocrit (0.04), diastolic pressure (0.04), age (0.03), mchc -red blood cell index(0.03), ventricular damage(0.03), hematoma volume (0.03). the following conclusions emerge from the general analysis of the overall data: 1. terms of mortality. all cases of death of patients, which were distributed within 14 days, were estimated, with the greatest number of lethal outcomes occurring within 5 days. 2. patients age 60 to 90 years (at least 65% out of 200 dead) were most frequently encountered in the group of the deceased, the maximum frequency (25%) falls on the age of 60 to 70 years, in the same age group there is the maximum morbidity of stroke with their share is almost 35% of the number of diseased. 3. among the deceased, men prevailed (by more than 25%). 4. the proportion of deaths in the hemorrhagic stroke cohort was twice as high compared with the proportion of deaths in ischemic stroke patients. from the general analysis, several interlinked signs are evident indicating the likelihood of lethal outcomes in patients with cerebrovascular disease at an early stage: hemorrhagic type of stroke is most likely to be lethal in patients with acute cerebrovascular disease; stroke incidence and mortality are highest in patients aged 60 to 70 years; stroke with lethal outcomes are more likely in men; regardless of the type of stroke, lethal outcomes are most likely in patients aged 60 to 90 years. all patients were divided into 3 subgroups according to the time of death -up to 1 day, 1 to 3 days, and 4 to 10 days. early mortality in each subgroup was associated with a number of demographic, clinical, and instrumental-laboratory characteristics based on the interpretation of the results of calculating the significance of predictors of binary classification models by machine learning methods from the scikit-learn library 2 . the target classes in training were "mortality rate of 1 day", "mortality rate of 1-3 days", "mortality rate from 4 days". auc roc of trained models reached 91% for the method of random forest. the results of interpretation of decision trees and calculation of significance of predictors of built-in methods of random forest coincide that can testify to correctness of calculations. as a result of the decision trees, the following conclusions were drawn regarding the time frame of death: 1. factors that cause patients to be lethal on the first day: patient's age over 67 years; male sex; significant volume of brain lesions (more than 1/3 of the middle cerebral artery pool) hemispheric ischemic (or hemorrhagic with impregnation of the ischemic focus) stroke or patients with intracerebral hematoma (both less than 50 ml and 50 to 100 ml) with a breakthrough into the ventricular system of the brain; more important was the combination of ischemic or hemorrhagic lesions with displacement of the medial structures due to perifocal edema; right hemispheric cerebral lesion; severe condition at entry (with severe neurological deficit, up to 23 nihss points); unstable systemic hemodynamics, expressed by fluctuations in blood pressure, appearance of tachycardia and tachyarrhythmia, i.e., in the ventricular system.h. with sharp rise (>200 mm hg) or sharp decrease (<65 mm hg).) systolic and diastolic blood pressure and heart rhythm disorders (tachycardia and tachyarrhythmia); manifestations of decompensated hypersympathicotonia accompanied by hyperthermia and polyuria (densephalic syndrome, irritation of the densephalic region of the brain) and hemoconcentration (hypercoagulation); high degree of comorbidity (presence of significant number of concomitant diseases at the decompensation stage, comorbidity index > 6.5). 2. mortality in the group from 1 to 3 days is caused by the following factors: age over 55 years old; male gender; consciousness impairment not lower than stun; presence of extensive hemispheric ischemic (more than 1/3 of the middle cerebral artery basin) or large intracerebral hematoma against the background of pronounced brain atrophy, in some cases with hemorrhagic saturation of the ischemic focus; the greatest importance was given to the combination of ischemic or hemorrhagic lesions with displacement of the medial structures due to perifocal edema; lesion of the right hemisphere; instability of system hemodynamics -with indicators of sharp decrease (<65 mm hg.st.) of systolic blood pressure, heart rate -with indicators of sharp decrease.st.) systolic blood pressure, heart rhythm disorders (bradiarrhythmia and tachycardia); or with a high degree of comorbidity (presence of a significant number of concomitant diseases at the decompensation stage, comorbidity index > 6.5); vivid manifestations of vegetative regulation decompensation (hypersympathicotonia), accompanied by hyperthermia and polyuria (diencephal syndrome, irritation of the diencephalic region of the brain) and hemoconcentration (hypercoagulation); phenomena of systemic inflammatory reaction and presence of signs of hemoconcentration in blood tests; 3. the largest contribution to the patients' mortality from 4 to 10 days was made by the following factors: age from 30 to 90 years (the largest group of patients aged 60-70 years); female gender; extensive hemispheric ischemic (more than 1/3 of the pool of the middle cerebral artery) in combination with severe hemispheric atrophy, or the presence of intracerebral hematoma (much more often less than 50 ml), a breakthrough into the ventricles of the brain, the most important was the presence of dislocation, a combination of ischemic or hemorrhagic lesions with the displacement of medial structures due to general edema; conscious disturbance (stun, coma) or condition that required sedation (to provide prosthetics for breathing function); unstable systemic hemodynamics -with sharp rise (>200 mm hg) or (<65 mm hg) of systolic blood pressure; phenomena of moderate hemoconcentration and moderate systemic inflammatory response in blood tests; high degree of comorbidity (presence of a significant number of concomitant diseases at the decompensation stage, comorbidity index > 5). at the same time, it should be noted that in contrast to patients with 1-3 day mortality, in this case the side of the brain lesion did not matter. three groups of patients were compared by the terms of mortality (mortality in the first day, mortality from 1 to 3, lethality from 4 to 10 days) among themselves by means of standard t-test (non-parametric criterion chi) with thirty one parameter. the value p < 0.0005 was considered significant. the results of the interpretation of the obtained test are presented in tables 1, 2 and 3. the following calculation results have been obtained p-value using method 2: for the groups 1 and 2 p-value = 0.0052; for the groups 2 and 3 p-value = 0.0042; from the groups 1 and 3 p-value = 0. on the basis of the analysis calculations it is possible to draw a conclusion about a significant difference between the 1st and the 3rd group, where group 1: ich (intracerebral hemorrhage) +pvh (periventricular hyperintensity), is (ischemic stroke) +ich (intracranial hemorrhage); group 2: is+bilat atr, is-foc 16-25 hu; group 3: is-1/3<16hu, sub tent icv (intracerebroventricular). feature <1 day p 1-3 days p 4-10 days p interpretation gender 0,0003 0,0004 0,0048 gender showed the significance of differences between all subgroups, with the groups with mortality of 4-10 days dominated by women, and between the subgroups of mortality up to 1 day and mortality of 1-3 days, with a general prevalence of incidence of men among deceased patients, the frequency of occurrence in the subgroups also significantly differed period of admission less than 4. the difference between subgroups of up to 1 day and 1-3 days is insignificant, i.e. the fact of later hospitalization did not affect earlier mortality. the differences in subgroups 1-3 and 4-10 are significant, for lighter patients (with lower comorbidity index or with severe atrophy) the difference between the subgroups of up to 1 day and 1-3 days is insignificant, i.e. the fact of edema affected earlier mortality. the differences in subgroups 1-3 and 4-10 are significant due to the fact that edema developed later as a factor affecting mortality or did not determine the mortality (e.g. in patients with severe atrophy, small foci) dislocation 0,0003 0,0003 1,0000 the difference between the subgroups of up to 1 day and 1-3 days is insignificant, i.e. the fact of edema influenced earlier mortality. the differences in subgroups 1-3 and 4-10 are significant due to the fact that the brain substance dislocation developed later as a factor influencing mortality or also did not determine the mortality (e.g. in patients with severe atrophy, small focus, cortical-subcortical focus, without affecting the central structures of the brain) (continued) the difference between subgroups of up to 1 day and 1-3 days is insignificant, i.e. in each case the fact of hemorrhagic impregnation of the zone of brain matter ischemia affected mortality. in subgroups of 1-3 days and 4-10 days this difference is significant due to availability of reserve spaces due to brain atrophy and less probability of dislocation of brain substance amount of ischemia > 1/3 of the middle cerebral artery (mca) 0,0002 0,0002 0,4306 the difference between subgroups of up to 1 day and 1-3 days is insignificant, i.e. in each case the fact of extensive ischemic lesion had an impact on mortality in earlier periods. in subgroups of 1-3 days and 4-10 days this difference is significant due to availability of reserve spaces in connection with brain atrophy and less probability of threatening dislocation (constriction) of brain substance even in presence of a large focal point of ischemia and consequently edema and tissue swelling expressed atrophic changes in brain matter 0,0003 0,0003 0,6586 the difference between subgroups of up to 1 day and 1-3 days is insignificant, in the subgroups of 1-3 days and 4-10 days this difference is significant as the availability of reserve spaces due to brain atrophy reduces the probability of dislocation of brain matter even in the presence of a large focal point of ischemia or hemorrhage (large hematoma) a detailed study of electronic medical records data and combinations of clinical and laboratory characteristics of patients made it possible to reveal dependencies and develop descriptive models between the degree of lesion and the time of intra-hospital lethality of patients. further, based on a large array of correlated data, models were developed to identify major favorable and unfavorable patterns of early mortality of patients for control and correction of the treatment plan. decision-making models for predicting the outcome and duration of treatment of stroke patients have been developed using systems analysis, statistical analysis, mathematical modeling and machine learning methods. as a result, clinical and morphological predictors of early hospital stroke mortality have been identified. similar models can also be used to validate existing scales, to study the causes of mortality at the emergency stages and to develop clinical guidelines, including for the prevention, diagnosis and treatment of stroke. as a result of this study, descriptive and prognostic models of mortality in stroke patients have been developed. the significance of predictors was ranked using statistical and machine learning methods. clinical interpretation of the obtained results was made in the form of clear conclusions that can be used in the organization of continuity care for acute stroke patients, as well as the calculation of personal risks. provided that all standards of specialized medical care for patients with stroke are complied with, first of all, monitoring and intra-hospital logistics, completeness of the diagnostic scope, it is possible to make a prognostic assessment to identify predictors of early hospital lethality. a number of clinical, pathomorphological and instrumental parameters may indicate a high probability of early lethality, namely: charlson comorbidity index with a value greater than 3.0; six subtypes of stroke; for 3 subtypes, combination with an extended intracellular cma clot, with an age greater than 64 years and ind. ch-4.5 b. for 1 and 2 subtypes the severity of lesion volume and presence of dislocation complications determine the high risk of mortality. for 4 subtypes the greatest risk is associated with the combination of an acute focus in the deep parts of the temporal lobe with moderate perifocal ischemic oedema, compression of medial structures, with the age over 64 years old and high and ind. ch-4.5 b. for subtype 6, a significant contribution is made by global (diffuse atrophy or the presence of a fresh acute focus in the deep regions of the temporal lobe on the side of the opposite marked atrophy (including post-stroke). global, regional, and national burden of stroke, 1990-2016: a systematic analysis for the global burden of disease study human mortality database global burden of stroke and risk factors in 188 countries, during 1990-2013: a systematic analysis for the global burden of disease study global and regional burden of stroke during 1990-2010: findings from the global burden of disease study impact of microalbuminuria on incident stroke: a meta-analysis the china national stroke registry for patients with acute cerebrovascular events: design, rationale, and baseline patient characteristics gwtg risk model for all stroke types predicts in-hospital and 3-month mortality in chinese patients with acute stroke burden, clinical outcomes and predictors of time to in hospital mortality among adult patients admitted to stroke unit of jimma university medical center: a prospective cohort study analysis on geographic variations in hospital deaths and endovascular therapy in ischaemic stroke patients: an observational cross-sectional study in china in-hospital mortality among ischemic stroke patients in gondar university hospital: a retrospective cohort study predictors of in-hospital mortality and attributable risks of death after ischemic stroke the german stroke registers study group factors influencing in-hospital mortality and morbidity in patients treated on a stroke unit explaining the decrease of in-hospital mortality from ischemic stroke risk score for in-hospital ischemic stroke mortality derived and validated within the get with the guidelines-stroke program risk-adjusted hospital mortality rates for stroke: evidence from the australian stroke clinical registry (auscr) the quality of acute stroke units on a nation-wide level: the austrian stroke registry for acute stroke units recent trends in inpatient mortality and resource utilization for patients with stroke in the united states impact of atrial fibrillation on in-hospital mortality of ischemic stroke patients and identification of promoting factors of atrial thrombi-results from the german integrated systems of stroke care and reduction in 30-day mortality: a retrospective analysis predicting outcome after acute and subacute stroke: development and validation of new prognostic models predicting early mortality of acute ischemic stroke: score-based approach prediction of in-hospital stroke mortality in critical care unit derivation and validation of in-hospital mortality prediction models in ischaemic stroke patients using administrative data age and national institutes of health stroke scale score within 6 hours after onset are accurate predictors of outcome after cerebral ischemia: development and external validation of prognostic models prediction of in-hospital mortality after first-ever stroke: the lausanne stroke registry a prognostic index for 30-day mortality after stroke trends in management and outcome of hospitalized patients with acute stroke and transient ischemic attack: the national acute stroke israeli (nasis) registry acknowledgements. this work was financially supported by the government of the russian federation through the itmo fellowship and professorship program. this work is financially supported by national center for cognitive research of itmo university. key: cord-280821-kc0ut4oy authors: venturini, elisabetta; montagnani, carlotta; garazzino, silvia; donà, daniele; pierantoni, luca; lo vecchio, andrea; nicolini, giangiacomo; bianchini, sonia; krzysztofiak, andrzej; galli, luisa; villani, alberto; castelli-gattinara, guido title: treatment of children with covid-19: position paper of the italian society of pediatric infectious disease date: 2020-09-24 journal: ital j pediatr doi: 10.1186/s13052-020-00900-w sha: doc_id: 280821 cord_uid: kc0ut4oy a statement of consensus was formulated after reviewing available literature on pediatric treatment strategies for covid-19 by the steering and scientific committee of the italian society of infectious pediatric diseases in connection with the italian society of paediatrics. since december 2019, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection has been reported in hubei province, china, and from there it spread worldwide. starting from the end of february 2020, the number of cases of coronavirus disease 2019 (covid19) outside china rapidly increased, urging the world health organization (who) to declare covid-19 as a pandemic on march 11 [1] . most children with sars-cov-2 infection develop none or mild symptoms, needing only supportive treatment [2, 3] . however, few cases of severe covid-19 in children have been reported [3] [4] [5] , including multisystem inflammatory syndromes [6, 7] . as no evidence of at least good quality is available regarding therapy of covid-19, treatment regimens of covid-19 are not standardized. at present, few clinical trials for covid-19 treatment involving children are ongoing [8] . moreover, evidences are rapidly evolving and the therapeutic indications can change very quickly and even this work has changed quite a bit in the course of its writing. to date, many national and international guidelines have been issued for the adult population [9] [10] [11] . however, there is a need of guidance on covid-19 management in children from scientific societies and expert groups. recently, an expert opinion has been released by a group of pediatric infectious disease specialists from north america, suggesting that supportive care alone is indicated for the overwhelming majority of cases [12] . the italian society of pediatric infectious diseases steering and scientific committee developed a position paper on treatment of children with covid-19, reviewing the current literature on this topic and providing indications based on the available literature data. since new evidences will be available in the next weeks/months, the italian society of pediatric infectious diseases will guarantee to maintain treatment indication updated on the website www.sitip.org (updated english and italian versions of the present document). the consensus statement was formulated by the steering and scientific committee of the italian society of pediatric infectious diseases in connection with the italian society of pediatrics. decision was made after reviewing available literature on pediatric treatment strategies for covid-19 at 16 june 2020 on pubmed with the following search strategies: (sars-cov-19 or covid-19 or coronavirus) and treatment. moreover, national and international recommendations of who and scientific societies available online were evaluated. ongoing clinical trials were searched on https://clinicaltrials.gov/ and https://www. aifa.gov.it/sperimentazioni-cliniche-covid-19. clinical syndromes associated with covid-19 in children were defined adapting the who classification as follows [13] [14] [15] . asymptomatic case: infection identified during screening or contact tracing without symptoms. mild case: fever and/or fatigue and/or upper airways symptoms without radiological/ultrasound findings (if performed). moderate case: fever and/or fatigue and/or upper airways symptoms (cough or mild respiratory distress) and/ or poor feeding and/or pneumonia identified with chest x-ray or ultrasound. severe case: [16] . sepsis-associated organ dysfunction and septic shock were defined according to surviving sepsis campaign definition [17] . regardless of the early stage of the disease, the following indicators should be assessed as related to an increased risk of rapid progression to the severe/critical stage. clinical early warning indicators: increased tachypnoea, despite 2 h of intravenous rehydration and low flow nasal cannula oxygen therapy impaired consciousness progressive increasing of lactate values bilateral lung infiltration or multiple lobes involvement, pleural effusion or rapid progression of the lesions in a short period of time age < 3 months underlying diseases (congenital heart disease, bronchopulmonary dysplasia, anomalies of respiratory tract, abnormal hemoglobin, anemia, severe malnutrition, congenital or acquired immunodeficiency) children and adolescents 0-19 years of age with fever > 3 days and two of the following: skin rash or bilateral non-purulent conjunctivitis or muco-cutaneous inflammation signs (oral, hands or feet) hypotension or shock signs of myocardial dysfunction, pericarditis, valvulitis, or coronary abnormalities (including echocardiography findings or elevated troponin/ nt-probnp) evidence of coagulopathy acute gastrointestinal problems (diarrhea, vomiting, or abdominal pain) elevated markers of inflammation such as erythrocyte sedimentation rate, c-reactive protein, or procalcitonin. and no other obvious microbial cause of inflammation, including bacterial sepsis, staphylococcal or streptococcal shock syndromes. and evidence of covid-19 (positive real-time polymerase chain reaction, antigen test or serology), or a likely contact with patients with covid-19. asymptomatic cases: no treatment. moderate and mild cases: only antipyretic therapy. remdesivir if not available hydroxychloroquine or lopinavir/ritonavir all these drugs should be preferably administered within a clinical trial. warning: hydroxychloroquine can cause qtc prolongation. every 2 days perform electrocardiogram (ecg) and qtc assessment for qtc prolongation increased risk. for hydroxychloroquine, carry out glucose 6 phosphate dehydrogenase (g6pdh) dosage in case of risk factors for deficiency. remdesivir should be administered in patients with normal renal function. liver function assessment should be performed in all patients prior to starting remdesivir and every other day while receiving remdesivir. duration of therapy: 5-7 days, extendable according to clinical course. immunomodulating therapy: this therapy must be considered in case of: -ards or progressive deterioration of respiratory function. -multisystem inflammatory syndrome. -marked alteration or increasing trend of il-6 and/or d-dimer and/or ferritin and/or c-reactive protein. -interval of at least 7 days from the beginning of the symptoms. methylprednisolone or dexamethasone or anakinra (or tocilizumab) the available dosages and formulations of the various drugs are indicated in the text. covid-19 management and treatment in children, according to disease severity, are reported in table 1 . antipyretic therapy: prefer paracetamol (10-15 mg/kg every 4-6 h) in case of fever > 38.5°c. avoid ibuprofen in case of dehydration, vomiting and diarrhea, as it is associated with an increased risk of kidney failure. some authors have suggested a correlation between the use of ibuprofen and an unfavorable course of sars-cov-2 infection [19] . however, these data are not currently confirmed and the european medicines agency does not contraindicate the use of non-steroidal antiinflammatory drugs [20] . inhalation therapy: if topic steroids and/or bronchodilators are needed (e.g. patient with recurrent wheezing undergoing exacerbation and suggestive symptoms or confirmed sars-cov-2 infection) the use of pressurized suspensions with spacer chamber is suggested. conversely, the use of nebulisers is not recommended in order to avoid particles aerosolization and increased contagiousness. ongoing steroid treatment should not be stopped [21] . venous thromboembolism prophylaxis: severe covid-19 seems to be associated in adults with an increased risk of disseminated intravascular coagulation and venous thromboembolism. a study on 449 adult patients with severe infection showed a lower mortality rate in those receiving anticoagulant therapy [22] . therefore, recently adults protocols suggest strategies for prevention and management of coagulative disorders secondary to covid19 infection with low molecularweight heparin, especially in case of ards [13, 23] . however, children have a much lower incidence of thrombotic complications than adults, even under higher risk conditions such as major surgery or polytrauma [24] . therefore, such prophylaxis is not routinely suggested in children. an exception can be made for neonatal age and adolescents, where constitutionally the incidence of thrombotic complications is higher [25] . preventive anticoagulant therapy can therefore be considered for these age groups, in cases where severe inflammatory conditions occur and therefore hyperactivation of the clotting process could lead to the appearance of important thrombotic complications. the suggested treatment is with subcutaneous enoxaparin 100-200 u/kg/day, that can be increased to 150-300 u/kg/day in neonates. lopinavir/ritonavir lopinavir/ritonavir is a boosted protease inhibitor, used, in association with other drugs, in the therapy of human immunodeficiency virus (hiv) infection, starting from the age of 14 days of live [26] . its security profile is well known, as it has been largely used in hiv infections in the pediatric age, it is available both in oral suspension and tablets. on the base of some clinical trials, the chinese guideline on sars-cov-2 pneumoniae recommend the early use of lopinavir/ritonavir [27] . on 18 march 2020, the results of a double blinded, randomized, open-label trial, which compared lopinavir/ ritonavir associated to standard of care and standard of care alone, on 199 sars-cov-2 pneumonia hospitalized patients, have been published on new england journal of medicine [28] . the study did not evidence any statistically significant differences in the clinical improvement. mortality at 28 days showed a 5.8% difference in favor of lopinavir/ritonavir use, although not statistically significant (95% confidence interval, ci95%: − 17.3-5.7). patients in which lopinavir/ritonavir therapy has been started before the 12 days of symptoms had a significantly reduction of symptoms duration, hazard ratio: 1.25 (95%ci, 1.77-2.05). data about mortality, stay in intensive care unit and duration of hospitalization were not stratified for the start of therapy before and after 12 days. the results of this study cannot be transferred to pediatric population, to patients with mildmoderate symptoms, and to patients who underwent an early therapy. currently, american guidelines on covid-19 treatment published in may 2020, recommend both in children and adults to use lopinavir/ritonavir only in the context of clinical trials, given the lack of effectiveness reported now in literature [9, 12] . the latest chinese guidelines on sars-cov-2 pneumoniae do not recommend the use of a specific antiviral for the treatment of covid-19, and nevertheless include lopinavir/ritonavir among the available therapeutic options for hospitalized patients [29] . less than 40 clinical trials are currently registered to evaluate the effectiveness of lopinavir/ritonavir against covid-19. lopinavir/ritonavir is not indicated in premature neonates before the 42 weeks of corrected age and in all cases before 14 days of live [30] . remdesivir remdesivir is a nucleotide analogue, which is incorporated in the viral rna chain, determining its premature termination. it has been developed from gilead in 2017 for ebola therapy. in vitro studies, its high spectrum efficacy against different coronavirus has been demonstrated [31, 32] . interestingly, remdesivir seems to have a high genetic barrier to viral resistance development (demonstrated in sars studies). it has a short plasmatic half-life, but it is rapidly converted into its active form (in 2 h from the infusion) and it has a 14 h intracellular half-life [33] . in may 2020, following an assessment of the emergency use authorization criteria and available scientific evidence, the fda issued an emergency use authorization allowing for the administration of remdesivir intravenously by health care providers for the treatment of covid-19 suspected or laboratoryconfirmed in adults and pediatric patients hospitalized with severe disease [34] . in a guidance approved by the american pediatric infectious diseases society for covid-19 treatment in children, if an antiviral is used, panel suggested remdesivir as the preferred agent [12] . antivirals should preferably be used as part of a clinical trial, if available. however, a warning has been issued on the coadministration of remdesivir and chloroquine phosphate or hydroxychloroquine sulfate as it may result in reduced antiviral activity of remdesivir [34] . as at 26 june 2020, there are 39 ongoing trials evaluating clinical efficacy of this drug in the therapy of moderate and severe sars-cov-2 infections [35] . four studies (gs-us-540-5774, gs-us-540-5773, gs-us-540-5821 and gs-us-540-5823) are currently ongoing in italy. all these studies are sponsored by gilead and only one study involve children from birth to 18 years whereas the other three enroll children older than 12 years and adults [36] . dosage: adults: 1st day 200 mg iv in 30 min, followed by 100 mg iv /day for other 9 days children (< 40 kg): 1st day 5 mg/kg iv (in 30 min), followed by 2.5 mg/kg iv (in 30 min)/day for other 9 days. at present, the dosage has not been established for the first 2 weeks of life and weight < 2.5 kg. hydroxychloroquine (and chloroquine) could have an antiviral activity [37] . the mechanism is currently not fully clarified. however, in vitro studies suggest that they could act by increasing the endosomal ph required for virus / host cell fusion and interfering with the glycosylation of the sars-cov-2 cell receptor [38] . in particular, hydroxychloroquine appears to have better in vitro activity towards sars-cov-2. the anti-inflammatory activity of these molecules, through the inhibition of the production of interleukin (il) -6 and tumor necrosis factor (tnf)-α, could contribute to their effectiveness. in addition, these molecules have been in use for decades, showing a good safety profile. compared to chloroquine, hydroxychloroquine is a drug more readily available and with a higher safety profile [39] . in february 2020, a panel of experts in china summarized the results of the use of chloroquine (500 mg every 12 h for 10 days) in adults with covid-19, suggesting that its use would be associated with an improvement in the clinical success rate, reducing the hospitalization and improving the outcome [40] . since then, hydroxychloroquine has been widely used in adult patients [41, 42] . to date, there are more than 200 registered clinical trials for pre-exposure or postexposure prophylaxis of sars-cov-2 infection, and treatment of patients with mild, moderate, and severe covid-19 [35] . however, it is not yet clear if the benefits outweigh the risks, especially in children. in fact, on 24 april 2020, the fda warned against the use of hydroxychloroquine or chloroquine for covid-19 outside hospital or clinical trial setting due to the risk of qt interval prolongation and ventricular tachycardia [43] . this risk could be also increased when combined with some antibiotics such as azithromycin. moreover, since the majority of children manifest only mild symptoms, a widespread use of hydroxychloroquine may confer only minimal benefit. furthermore, pharmacokinetic modeling and simulation used to identify pediatric-specific dosages for hydroxychloroquine have raised concerns on plasma exposures lower than those needed to mediate an antiviral effect [44] . however, these findings do not exclude a potential utility of hydroxychloroquine for covid-19 treatment based on different mechanisms of action, such as immunomodulation. conclusive data are needed from ongoing trial to understand the possible role of this drug in children with covid-19. available formulations: hydroxychloroquine tablets 200 mg. dosage: adults: 400 mg twice a day the first day, followed by 200 mg twice a day for overall 5-7 days. children: 6 mg/kg (maximum: 400 mg/dose) twice a day on day 1, followed by 3 mg/kg (maximum: 200 mg/ dose) twice a day for up to 5 days. *. * hydroxychloroquine solution preparation is recommended for children. precautions for use: perform ecg before administering the drug to rule out long qt. in case of risk factors, dose g6pdh before use. other antiviral therapy favipiravir is an antiviral drug authorized in japan for the treatment of flu. it works by inhibiting rna polymerase-rna-dependent. the medicine is not authorized in europe or in the usa. preliminary results on 80 patients with non-serious sars-cov-2 infection, published only in chinese, seems to show a better efficacy of this drug compared to lopinavir/ritonavir [37] . few clinical trials are currently undergoing to evaluate the efficacy of favipiravir in sars-cov-2 infection and the aifa scientific technical commission approved a clinical trial program for this drug [36, 45] . ivermectin, an fda-approved anti-parasitic, seems to have broad-spectrum anti-viral activity in vitro, is an inhibitor of the causative virus (sars-cov-2), 2 h post infection with sars-cov-2 able to effect 5000-fold reduction in viral rna at 48 h [46] . a single center, double-blind, randomized, placebo-controlled trial is ongoing on ivermectin efficacy in reducing sars-cov-2 viral load in adult patients with low risk, non-severe covid-19 [47] . steroids at present, there are no clear evidences to support the use of systemic steroids during sars-cov-2 infection unless specific needs (e.g. asthmatic patient who does not respond to 3 doses of bronchodilator, severe allergic reaction). in particular, in patients on chronic systemic or inhaled steroid therapy, treatment stopping is not necessary. some data suggest that methylprednisolone could have an immunomodulating activity in case of ards, decreasing the risk of death [48] . its use is also indicated in case of a worsening of pulmonary function after at least 7 days from the beginning of symptoms, in association with marked alteration or tendency to increasing of il-6 and/or d-dimer and/or ferritin and/or c-reactive protein. in these cases, can be used: -methylprednisolone 1-2 mg / kg (max 80 mg) once a day. a short course is indicated (2-5 days). in severe cases high doses (methylprednisolone 30 mg/ kg) can be considered. recently some good results were reported by the interim analysis of the recovery trial [49], a study where 2104 adult patients were randomised to receive dexamethasone 6 mg once per day (either orally or intravenously) for 10 days and 4321 patients were randomised to usual care alone. dexamethasone significantly reduced deaths by one-third in ventilated patients (relative risk, rr 0.65) and by one fifth in those receiving only oxygen (rr 0.80), but there was no benefit among patients who did not require respiratory support. based on these results, a treatment with dexamethasone (0.2-0.4 mg/kg, maximum 6 mg) in patients requiring oxygen should be considered. anakinra the multisystem inflammatory syndrome reported in patients with covid-19 shares considerable biochemical overlapping with the cytokine storm complicating macrophage activation syndrome associated with rheumatic disease. anakinra is licensed for treating people with rheumatoid arthritis, aged at least 8 months. this is a 17 kd recombinant, non-glycosylated human il-1 receptor antagonist who inhibits the proinflammatory cytokines interleukin (il)-1α and il-1β, with a short half-life of about 3-4 h and good safety profile. anakinra has also been used with some success to treat macrophage activation syndrome caused by various inflammatory conditions. several case series report that anakinra reduced both need for invasive mechanical ventilation in the intensive care unit and mortality among patients with severe forms of covid-19, without serious side-effects [50, 51] . currently, there are 10 ongoing clinical trials on anakinra use in covid-19, all in adults [52] . however, there are few pediatric reports of children treated with anakinra showing that such treatment in serious cases can be safe and beneficial [53, 54] . indeed, anakinra has been proposed as the best option if such treatment is considered necessary, because it has a relatively short half-life and may be discontinued rapidly in case of adverse effect or concern of worsening the infection [55] . anakinra can be administered intravenously (off label) or subcutaneously and has a wide therapeutic window; when anakinra is effective for cytokine storm syndromes, it works within 2 to 3 days [56] . therapeutic scheme: -anakinra vial 100 mg/0.67 ml intravenously: 8-10 mg/kg/day in 2 or 4 administrations depending on the total dose (up to a maximum of 100 mg 4 times a day) after 48-72 h, repeat the plasma dosage of il-6 and/ or d-dimer. tocilizumab tocilizumab is a recombinant humanized monoclonal antibody belonging to the g1 immunoglobulin subclass and directed against both soluble and membrane il-6 receptors [57] . this drug is indicated for the treatment of moderate and severe rheumatoid arthritis, systemic juvenile idiopathic arthritis (from the age of 1 year), juvenile idiopathic polyarthritis (from the age of 2 years) and severe release of cytokines induced by car-t lymphocytes (chimeric antigen receptor t cell) (from the age of 2 years). studies suggested that the alveolar damage in covid-19 is caused by a cytokine storm (including il-6) and that symptoms improve with the use of tocilizumab [58, 59] . based on these results, different clinical trials have been started. the italian multicenter study tocivid-19 has been promoted by the national cancer institute, irccs, g. pascale foundation, naples. the recruitment of the prospective phase has been completed, while the observational study continues. the protocol includes the enrollment of patients of any age. two other studies started at the end of march in italy, but not enrolling pediatric patients. one of them, aimed at assessing the efficacy of the early tocilizumab administered early in patients suffering from recently emerging covid-19 pneumonia and requiring hospital care but not invasive or semi-invasive ventilation, was concluded early because it was not effective [36] . therapeutic scheme: -tocilizumab vial 20 mg/ml -first infusion at a dosage of 10-12 mg/kg < 30 kg and 8 mg/kg > 30 kg, (maximum dosage 800 mg, duration of infusion at least 60 min) -second infusion 12 h after the first (at the discretion of the doctor, in case of no response) -a possible third infusion after 24 h may be considered. after 24 h from the last administration, repeat the plasma dosage of il-6 and / or d-dimer. when to use biologic drugs: • serious or critical cases. • end of the initial phase of high viral load of covid-19 (afebrile> 72 h and/or at least 7 days after the onset of symptoms). • high levels of il-6 (> 40 pg/ml); alternatively, high levels of d-dimer and/or pcr and/or ferritin and/or fibrinogen increasing progressively. when not to use biologic drugs: • ast / alt value above 5 times normal levels. • neutrophils value lower than 500 cells/ml. • platelets value lower than 50,000 cells /ml. • documented sepsis from other pathogens other than covid-19. • presence of comorbidities related, according to clinical judgment, to an unfavorable outcome. • complicated diverticulitis or intestinal perforation. • immunomodulating and anti-rejection therapy. • known hypersensitivity to the drug. it is also recommended to avoid administration of biologics if anti-mrpv vaccination has been carried out in the past 30 days. if possible, before starting therapy: -quantiferon test -hbv and hcv markers other immunomodulant therapies in view of the fact that the alveolar damage of severe forms of sars-cov-2 infection is caused by a cytokine storm [60] , two other clinical trials have been started in italy on the use of emapalumab (anti-interferon gamma monoclonal antibody) and associated anakinra (il-1 receptor antagonist) and the use of sarilumab (monoclonal antibody that binds to the il-6 receptor). a belgian study comparing tocilizumab, anakinra and siltuximab is going to start soon. information and forms relating to the studies are available on aifa website, but there is no prevision for enrollment of pediatric patients [36] . among the new therapies planned for the future is the infusion of hyperimmune plasma from cured patients. this approach, already used in china and previously for ebola and sars, seems to give good results at least in the most serious cases. in the usa 3 studies have been launched that will evaluate this approach [61] . the choice to add empirical antibiotic therapy should only be made if there is reasonable evidence of bacterial superinfection. an increased procalcitonin, c-reactive protein and the presence of neutrophilic leukocytosis represent laboratory parameters suggestive of bacterial infection. from a clinical point of view, the persistence of fever for more than 3 days can be suggestive of bacterial infection [62] . tests for atypical bacteria (i.e. mycoplasma) should be performed in case of clinical suspect, in order to start targeted therapy. the start of an empirical antibiotic therapy is also recommended in the presence of comorbidities, such as immunodeficiency, cystic fibrosis, other chronic diseases of the respiratory tract, severe neuromotor disability. in case of productive cough, the collection of a sputum sample for culture examination before the start of antibiotic therapy would be indicated. in patients without risk factors, we recommend: -amoxicillin 90 mg /kg/day in 3 doses, in case of possible oral intake -ceftriaxone 80-100 mg/kg/day, in case of impossibility of oral intake. this drug is also recommended for the possibility of administering once a day and therefore reducing the risks for healthcare professionals. azithromycin preliminary data have suggested a possible efficacy of azithromycin in combination with hydroxychloroquine as a background therapy for sars-cov-2 infection. the exact role of this drug in covid-19 is unknown, as no in vitro data are available at present. it has been supposed a dual role, antiviral and anti-inflammatory. the anti-inflammatory action of azithromycin has been already demonstrated in many conditions. regarding the theoretical antiviral activity, the first published study was on a small sample size (36 patients, of whom only 6 receiving the combination of azithromycin/hydroxychloroquine) [41] . in this study, by gautret and colleagues, 100% of the patients taking hydroxychloroquine and azithromycin had negative nasopharyngeal swab 6 days from the start of therapy, compared to 57.1% of the hydroxychloroquine alone and 12.5% of the control group. the same results have been replicated in a larger population [63] . however, a more recent study on virological and clinical results of 11 adult patients with a serious disease raises doubts about the antiviral efficacy of this combination, as it reports results in contrast with those of gautret et al. [64] . this combination have been also evaluated in a larger observational study including 1061 adults, resulting to be safe and associate with a very low fatality rate. however, its efficacy has been doubted in a study on a large adult population in new york among hospitalized patients with covid-19, where treatment with hydroxychloroquine, azithromycin, or both were not associated with significantly lower in-hospital mortality [65] . considering the lack of a strong rationale and the absence of evidence of certain effectiveness in the treatment of covid-19 patients, the use of azithromycin should be considered carefully and the qtc interval strongly monitored. in fact, on april 2020, european medicines agency (ema) warned about risk of serious side effects particularly if associated with chloroquine and hydroxychloroquine [66] . dosage: adults: 500 mg the first day, then 250 mg/day for other 4 days children: 15 mg/kg the first day, then 7.5 mg/kg once a day for other 4 days the drugs used for the therapy of sars-cov-2 infection, especially lopinavir/ritonavir and azithromycin/ hydroxychloroquine, can have interactions with other drugs. before administering those treatment, evaluate possible interactions on drugs [67] . for all drugs, consent for off-label use must be requested and the procedures provided by the reference healthcare company must be followed. as for remdesivir, since it is an experimental drug, the procedures for compassionate use must be followed. in conclusion this position paper summarizes the suggested treatments in covid-19 infected children based on a review of the current literature carried out by the scientific committee of the italian society of infectious pediatric diseases. since most sars-cov-2 infections in children have a benign course, pharmacological treatment, other than supportive therapy, should be reserved to those with more severe cases. as new evidences are expected to be available in the next weeks/months, the italian society of pediatric infectious diseases will guarantee to maintain treatment indication updated on the website www.sitip.org world health organization. who director-general's opening remarks at the media briefing on covid-19 -11 systematic review of covid-19 in children shows milder cases and a better prognosis than adults the italian sitip-sip pediatric infection study group, et al. multicentre italian study of sars-cov-2 infection in children and adolescents screening and severity of coronavirus disease 2019 (covid-19) in children in madrid clinical features of severe pediatric patients with coronavirus disease 2019 in wuhan: a single center's observational study kawasaki-like multisystem inflammatory syndrome in children during the covid-19 pandemic in paris, france: prospective observational study an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study covid-19) treatment guidelines società italiana di malattie infettive tropicali. sezione regione lazio. raccomandazioni per la gestione clinica e terapeutica della covid-19 maggio 2020 multicenter initial guidance on use of antivirals for children with covid-19/ sars-cov-2 clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance multisystem inflammatory syndrome in children and adolescents with covid-19 pediatric acute respiratory distress syndrome: consensus recommendations from the pediatric acute lung injury consensus conference surviving sepsis campaign international guidelines for the management of septic shock and sepsis-associated organ dysfunction in children multicentre validation of the bedside paediatric early warning system score: a severity of illness score to detect evolving critical illness in hospitalised children covid-19: ibuprofen should not be used for managing symptoms, say doctors and scientists ema gives advice on the use of non-steroidal anti-inflammatories for covid-19 recommendations for inhaled asthma controller medications anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy isth interim guidance on recognition and management of coagulopathy in covid-19 hospital-associated venous thromboembolism in pediatrics: a systematic review and meta-analysis of risk factors and risk-assessment models developmental hemostasis: clinical implications from the fetus to the adolescent paediatric european network for treatment of aids (penta) guidelines for treatment of paediatric hiv-1 infection 2015: optimizing health in preparation for adult life national health commission & state administration of traditional chinese medicine. diagnosis and treatment protocol for novel coronavirus pneumonia a trial of lopinavirritonavir in adults hospitalized with severe covid-19 national health commission & national administration of traditional chinese medicine. diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7) european public assessment report (epar) for kaletra arguments in favour of remdesivir for treating sars-cov-2 infections remdesivir as a possible therapeutic option for the covid-19 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro remdesivir by gilead sciences: fda warns of newly discovered potential drug interaction that may reduce effectiveness of treatment sperimentazioni cliniche -covid-19 discovering drugs to treat coronavirus disease 2019 (covid-19) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) za zhi for the multicenter collaboration group of department of science and technology of guangdong province and health commission of guangdong province for chloroquine in the treatment of novel coronavirus pneumonia. expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia (article in chinese hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial information for clinicians on therapeutic options for covid-19 patients fda cautions against use of hydroxychloroquine or chloroquine for covid-19 outside of the hospital setting or a clinical trial due to risk of heart rhythm problems best pharmaceuticals for children act-pediatric trials network steering committee, et al. simulated assessment of pharmacokinetically guided dosing for investigational treatments of pediatric patients with coronavirus disease favipiravir: aggiornamento della valutazione della cts the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro the sars-cov-2 ivermectin navarra-isglobal trial (saint) to evaluate the potential of ivermectin to reduce transmission in low risk, non-severe covid-19 patients in the first 48 hours after symptoms onset: a structured summary of a study protocol for a randomized control pilot trial risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china anakinra for severe forms of covid-19: a cohort study interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study anakinra in covid-19: important considerations for clinical trials the lancet reumatology clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 novel paediatric presentation of covid-19 with ards and cytokine storm syndrome without respiratory symptoms intravenous anakinra for macrophage activation syndrome may hold lessons for treatment of cytokine storm in the setting of coronavirus disease 2019 the rheumatologist's role in covid-19 european public assessment report (epar) for roactemra (tocilizumab ema gives advice on the use of non-steroidal anti-inflammatories for covid-19 effective treatment of severe covid-19 patients with tocilizumab covid-19: consider cytokine storm syndromes and immunosuppression how blood from coronavirus survivors might save lives diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxy-chloroquine and azithromycin in patients with severe covid-19 infection association of treatment with hydroxychloroquine or azithromycin with inhospital mortality in patients with covid-19 in new york state covid-19: reminder of risk of serious side effects with chloroquine and hydroxychloroquine covid-19 drug interactions publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. all authors contribute to prepare the manuscript read and approved the final version. key: cord-268341-103xf3dw authors: parra, beatriz; hinton, david r.; lin, mark t.; cua, daniel j.; stohlman, stephen a. title: kinetics of cytokine mrna expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis date: 1997-07-07 journal: virology doi: 10.1006/viro.1997.8613 sha: doc_id: 268341 cord_uid: 103xf3dw abstract the potential role(s) of cytokines in the reduction of infectious virus and persistent viral infection in the central nervous system was examined by determining the kinetics of cytokine mrna expression following infection with the neurotropic jhm strain of mouse hepatitis virus. mice were infected with an antibody escape variant which produces a nonlethal encephalomyelitis and compared to a clonal virus population which produces a fulminant fatal encephalomyelitis. infection with both viruses induced the accumulation of mrnas associated with th1and th2-type cytokines, including ifn-γ, il-4, and il-10. peak mrna accumulations were coincident with the clearance of virus and there was no obvious differences between lethally and nonlethally infected mice. tnf-α mrna was induced more rapidly in lethally infected mice compared to mice undergoing a nonfatal encephalomyelitis. rapid transient increases in the mrnas encoding il-12, inos, il-1α, il-1β, and il-6 occurred following infection. nonlethal infections were associated with increased il-12, il-1β, and earlier expression of il-6, while lethal infections were associated with increased inos and il-1α mrna. these data suggest a rapid but differential response within the central nervous system cells to infection by different jhmv variants. however, neither the accumulation nor kinetics of induction provide evidence to distinguish lethal infections from nonlethal infections leading to a persistent infection. accumulation of both th1 and th2 cytokines in the central nervous system of jhmv-infected mice is consistent with the participation of both cytokines and cell immune effectors during resolution of acute viral-induced encephalomyelitis. introduction (wesselingh et al., 1994) . variations between viral infections resulting in cns the goal of the immune response during viral infection inflammation prompted an examination of the temporal is to limit replication via induction of both nonspecific and induction of cns cytokines during fatal and nonfatal cns specific antiviral effectors. acute viral infections of the ceninfections by variants of the jhm strain (jhmv) of mouse tral nervous system (cns) result in vigorous, but in some hepatitis virus (mhv). in immunocompromised hosts instances limited, host immune responses (sedgwick and jhmv replicates unchecked in the cns demonstrating dorries, 1991) . in contrast to responses in the periphery the importance of immune effectors in limiting cns virus where limiting virus replication can generally be carried replication ; houtman and out with minimal regard to tissue damage, within the cns fleming, 1996b; . effector checks and balances minimize inflammatory-mediated mechanisms implicated in protection and clearance of damage while limiting viral-induced cytopathology. al-jhmv from the cns include cell-mediated immunity and though a wide range of immune effectors are often induced, both neutralizing and nonneutralizing antibodies. jhmv predominant anti-viral mechanisms appear related to the provides an interesting paradigm of acute viral encephapathogenesis strategy of the individual agent. for example, litis not only because of its associated demyelination infection of mice with lymphocytic choriomeningitis virus (weiner, 1973; lampert et al., 1973) but also because induces a predominant cd8 / cytotoxic t lymphocyte (ctl) some immune effector mechanisms prevent death via response (lehmann et al., 1988) . by contrast, resolution of directly reducing cns virus replication while other immeasles virus-encephalitis in mice is mediated by cd4 / t mune effectors prevent death without significantly altercells and correlates with the local production of ifn-g ing virus replication hout-(finke et al., 1995) . finally, resolution of sindbis virus-inman and fleming, 1996b; . a duced encephalitis is related to induction of neutralizing common theme appears to be prevention of neuronal infection by reducing viral load or preventing neuronal infection, most likely via cytokines. tion and clearance of jhmv from the cns are not yet and rarely neurons (2.2v-1). these viruses contrast to the predominantly neuronotropic oblv-60 variant previously clear. the antiviral effects of cd8 / t cells appear to be due to direct lysis of infected cells; however, cd8 / and examined (pearce et al., 1994; . cd4 / t cells may also exert antiviral activity indirectly material and methods via cytokine secretion (biron, 1994) . neither infected neurons nor oligodendrocytes appear susceptible to major mice and viruses histocompatibility (mhc) class i-mediated killing in vivo, c57bl/6 mice were purchased from the jackson laboconsistent with the inability of jhmv-specific ctl to clear ratory (bar harbor, me) at 6 weeks and maintained in virus from infected oligodendroglia (stohlman et al., the university of southern california vivarium. all mice 1995b). furthermore, clearance of jhmv from the cns were used at 7 weeks of age. to produce a lethal infecis inhibited, but not abolished, in mice genetically defition, mice were infected by intracerebral inoculation (i.c.) cient in perforin-mediated cytolysis (lin et al., 1997) . with 100 pfu of the plaque-purified dm isolate of jhmv these data suggest the possibility that cytokines contrib(stohlman et al., 1982) in a volume of 32 ml. this virus has ute to either clearance or protection from jhmv infection. the plaque size and pathogenesis similar to the parental during jhmv infection of the cns there is an abrupt suckling mouse brain pool of jhmv originally described increase in mrna encoding interleukin-1 (a and b), ilby weiner (1973) and produces a lethal encephalomyeli-6, tumor necrosis factor (tnf)-a, and interferon (ifn)-g, tis with minimal demyelination apparent at the time of at the time of maximal decrease in virus replication and death. to produce a sublethal infection, mice were inmononuclear cell infiltration (pearce et al., 1994) . no ifnfected with 25 pfu of the 2.2v-1 monoclonal antibodyg mrna was detected in immunodeficient mice, sugderived neutralization-resistant variant of jhmv (fleming gesting this cytokine may be important during viral clearet al., 1986) . this variant replicates predominantly in oliance (pearce et al., 1994) . consistent with this concept, godendroglia producing a flaccid paralysis. although vimice treated with anti-ifn-g are more susceptible to ral antigen is cleared from survivors by 30 days postinfec-jhmv, while administration of ifn-g provides protection tion (p.i.), viral rna persists for at least 12 months (adami (smith et al., 1991) . il-6, tnf-a, and type 2 nitric oxide et al., 1995) . groups of at least 3 mice were sacrificed synthase (inos) have also been detected in the cns at various times p.i. immunosuppression was induced during acute jhmv infection (sun et al., 1995; by lethal irradiation (850r) 24 hr prior to infection. shamet al., 1995a; while il-1b, il-6, tnf-a, infected mice were injected i.c. with 32 ml of sterile endoand inos were detected in the cns of chronically intoxin-free phosphate-buffered saline (pbs). fected mice (sun et al., 1995) . the complex interactions of multiple immune effector mechanisms during jhmv virus titration infection may reflect both the relative immune privilege virus titers were determined by plaque assay using of the cns (sedgwick and dorries, 1991) as well as the monolayers of dbt cells as previously described (stohlspecific tropism of the virus for cns cell types. neuroman et al., 1982) . one-half of the brain was homogenized tropic mhv isolates differ in tropism and include viruses using tenbrock tissue homogenizers in 2.0 ml of dulbecwith predominant tropisms for astrocytes, microglia, and co's pbs, ph 7.4. the remaining half was taken for histooligodendroglia as well as neurons (fleming et al., 1986; pathology or rna extraction (see below). following cenperlman and reis, 1987; kyuwa and stohlman, 1990; trifugation at 1500 g for 7 min at 4њ, supernatants were pearce et al., 1994; stohlman et al., 1995a) . the balance assayed immediately or frozen at 070њ. data presented between limiting viral replication and preserving cns are the average titer of groups of three or more mice. function occasionally results in incomplete viral clearance and a persistent cns infection which may or may antibody titration not involve the continued presence of infectious virus jhmv-specific igm, igg1, and igg2a antibodies were quantitated by elisa as previously described (lin et al., 1996b) . persistence of infectious virus correlates with the 1997) using rabbit anti-mouse igm, igg1, or igg2a antipresence of ctl escape variants (pewe et al., 1996) . bodies (cappel, costa mesa, ca). concentrations of se-to understand the complex interrelationships between rum antibodies were expressed as the highest dilution encephalitis, protection, and viral clearance leading to a with o.d. values three times above background level. persistent infection of the cns, the expression of pro-neutralizing antibodies were tested in serum as preand anti-inflammatory cytokine mrnas in the cns of viously described (lin et al., 1997) . mice undergoing either lethal or sublethal jhmv infection were compared. the two jhmv chosen for study infect histology either primarily microglia and astrocytes, less frequently oligodendroglia and neurons (dm) or primarily oligoden-histopathologic analysis was performed as previously described (stohlman et al., 1995a) . briefly, tissues were droglia, much less frequently microglia and astrocytes, fixed for 3 hr in clark's solution (75% ethanol, 25% glacial with 32 p-atp-labeled internal oligonucleotide probes. membranes were washed (three times; 21 ssc, 0.1% acidic acid) and embedded in paraffin. sections were stained with hematoxylin and eosin or luxol fast blue. distri-sds; room temperature), exposed to storage phosphor screens (molecular dynamics, sunnyvale, ca), and bution of jhmv antigen was examined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlin-scanned using a phosphorimaging scanner (molecular dynamics). game, ca) using the anti-jhmv mab j3.3 specific for the viral nucleocapsid protein (fleming et al., 1983) . radioactive signals of cytokine cdna were quantified and normalized to the house-keeping enzyme hypoxanthine phosphoriboxyltransferase (hprt) values to adjust cytokine mrna expression for differences in cdna as previously described (cua et al., 1995 (cua et al., , 1996 . the sample with the highest specific brains were processed individually to prevent contamination. rna was isolated from half brains by homogeni-activity was designated the 100% maximal response and values for the remainder were derived as percentage of zation at room temperature in guanidinium isothiocyanate using tenbrock tissue homogenizers as previously the highest value. data shown are mean values for 3-4 mice at each time point { 1 standard deviation. described (cua et al., 1995) . samples were sheared prior to centrifugation through 5.4 m cesium chloride at 100,000 g for 18 hr to isolate rna. the cdna were pre-results pared using avian myeloblastosis reverse transcriptase acute and subacute jhmv-induced encephalitis (promega, madison, wi) and oligo dt primers (promega) for 60 min at 42њ. expression of cytokine mrna was fatal encephalomyelitis induced by jhmv is associated with minimal demyelination (kyuwa and stohlman, determined by semiquantitative pcr analysis, following procedures previously described (cua et al., 1995 (cua et al., , 1996 (cua et al., ). 1990 houtman and fleming, 1996b) . this contrasts with infection by 2.2v-1 which produces an acute nonfatal en-pcr was performed using amplitaq dna polymerase (perkin-elmer, branchburg, nj) and specific cytokine cephalomyelitis with extensive demyelination (fleming et al., 1986; wang et al., 1990) . although both viruses primers for ifn-g, il-1a, il-1b, il-4, il-6, il-10, tnf-a (murphy et al., 1993; cua et al., 1996) , and il-12p40. the replicated rapidly to high titer in the cns (fig. 1a) , jhmvinfected mice succumbed within 8 days while 2.2v-1-sequences of the il-12p40 oligonucleotides primers and probe used are as follows: 5 primer, gac cct gcc infected mice underwent a subacute disease with little or no mortality (fig. 1b) . peak 2.2v-1 replication was at cat tga act ggc; 3 primer, caa cgt tgc atc cta gga tcg; oligoprobe, tgt ctg cgt gca agc tca day 3 while the peak of jhmv replication was delayed until day 5. 2.2v-1 clearance began at day 5 p.i. and by gga. amplification was carried out using 35 cycles of one denaturation step at 94њ (45 sec), primer annealing day 7 virus was nearly undetectable. by contrast, titers in jhmv-infected mice initially decreased at day 7 p.i. at 59њ (45 sec), extension step at 72њ (1.5 min), followed by a final extension step for 7 min. for il-4 and inos a and detectable virus was still present in the cns of moribound mice at day 8 p.i. (fig. 1a ). during lethal jhmv nested pcr was performed by using internal primers in a second round of pcr (25 cycles) under the conditions infection, virus replication within the cns is not reduced as rapidly as in mice which survive infection ( fig. 1a ) described above. the oligonucleotide primers used in the second pcr for il-4 were the corresponding se-consistent with the notion that rapid clearance correlates positively with protection. consistent with these findings, quences for the 5-primer and the probe described by cua et al. (1996) . the nucleotide sequence for the il-4 immunohistologic examination of the brains of jhmvinfected mice at day 7 showed abundant viral antigen oligonucleotide probe was ttg aag gag gtc aca gga gaa ggga (sideras et al., 1987) . the 5 and 3 outer in regions of encephalitis while only focal residual viral antigen was found in 2.2v-1-infected animals (fig. 2) . en-primer sequences for inos were gcc ttc cgc agc tgg gct gt and atg tgg tag cca cat ccc gag cephalitis was prominent in mice infected with either 2.2v-1 or jhmv and no differences in the amount or distri-cc, respectively (lyons et al., 1992) . internal 5 and 3 inos primers were agc tac tgg gtc aaa gac aag bution of mononuclear cell infiltrates were found at day 7 (fig. 2) . no serum neutralizing antibodies were de-agg ct and the 3 outer primer, respectively. the oligonucleotide probe consisted of the sequence ctc cct tected in either group by day 9 postinfection, even though the virus titer in the cns had declined over 3 log 10 (lin tcc gaa gtt tct ggc agc a. for quantification, pcr products were diluted in dena-et al., 1997; data not shown). in contrast to neutralizing antibodies, igm was first detected at day 5 post-2.2v-1 turing solution (0.4 n naoh, 25 mm edta), neutralized with tris-hci (1.0 m; ph 8.0), and transferred to 0.45 infection (data not shown) and both igg1 and igg2a were detected as early as 7 days p.i. (fig. 1c) . the igg1 and mm nytran membranes (schleicher & schuell, keene, nh) using a minifold i dot blot apparatus (schleicher & igg2a response suggest the absence of a shift toward either a th1-or th2-type response reported to be in-schuell). membranes were hybridized (60њ; overnight) volved in the response to sindbis virus-induced encepha-deficient mice, which showed no evidence of ifn-g mrna, suggests tnf-a may also contribute to inos litis (wesselingh et al., 1994) . mrna induction (colasanti et al., 1995; gazzinelli et al., 1993) . consistent with this notion, tnf-a mrna was first proinflammatory cytokines detected at day 3 in mice undergoing a lethal infection the mrna encoding ifn-g increased in both groups and at day 5 in mice sublethally infected (fig. 3c ). similar of mice through day 5 postinfection, consistent with the to the kinetics of ifn-g, tnf-a mrna increased until rapid accumulation of both nk and t cells in the cns of death of lethally infected mice. in mice undergoing a infected mice (williamson et al., 1991; williamson, 1992) sublethal infection, tnf-a mrna declined following the (fig. 3a) . no ifn-g mrna was detected in either shampeak of virus replication and approached baseline levels infected mice or in infected immunodeficient mice. durby 14 days p.i. ing the lethal jhmv infection ifn-g mrna did not in-similar to both tnf-a and inos, il-12 is secreted from crease between day 5 and day 7. however, in mice macrophages during the induction of cell-mediated imundergoing a sublethal infection the level of ifn-g mrna munity and protects from a number of viral infections continued to increase to day 7 and remained elevated, via a ifn-g-dependent mechanism (ozmen et al., 1995; suggesting the possibility that ifn-g is important followorange and biron, 1996) . no il-12 mrna was found foling infection with a jhmv variant tropic for oligodendroglowing sham infection; however, il-12 mrna increased lia. even though ifn-g mrna increased during the early rapidly and peaked at 3 days following both infections phase of infection, a sharp transient increase in inos (fig. 4a ). increased il-12 mrna also occurred in immu-mrna was detected at day 5 p.i. in mice with a lethal nodeficient mice at 3 days p.i., suggesting a direct reencephalomyelitis (fig. 3b) . only a slight increase was sponse to infection which may be related to the recently detected in mice undergoing subacute encephalomyelidescribed ifn-g-independent induction of il-12 (heinzel tis. interestingly, infection of immunodeficient mice with et al., 1996) . il-12 mrna levels decreased after day 3 jhmv induced the accumulation of inos mrna to apand nearly approached base line levels found in uninproximately 50% the level found in infected immunocomfected mice by 14 days p.i. petent mice, suggesting a direct response to viral infec-the il-1b mrna level found at day 1 p.i. declined by 3 days p.i., consistent with induction of an early transient tion. the increase in inos and tnf-a mrna in immunoincrease in il-1b mrna in sham-infected mice (fig. 4b ). subacute infection (fig. 4c ) and then declined but never returned to baseline. in lethally infected mice the peak il-1b mrna peaked at day 5 following sublethal infection and subsequently declined as virus was cleared of il-1a mrna was delayed (day 5 p.i.) and then declined as the animals succumbed to infection (fig. 4c ). il-6 from the cns. following a lethal infection, the quantity of il-1b mrna increased from day 3 p.i. until death. il-mrna peaked at day 5 postinfection in lethally infected mice and declined by day 7 as virus was cleared from 1a mrna peaked at day 3 p.i. in the mice undergoing a the cns (fig. 4d) . in contrast to the lethal infection, the of il-4 mrna expression following acute and subacute infections showed that the levels increased in parallel levels of il-6 mrna increased rapidly and peaked at day 3 p.i. following subacute infection. the level then through day 7 p.i. (fig. 5a ). in 2.2v-1-infected mice, the level of il-4 mrna continued to increase until day 9 p.i. declined rapidly by day 5 and had reached baseline levels by day 9 p.i. no il-6 mrna was detected in sham-and then declined slightly by day 14. infected mice, suggesting a rapid response to virus infection. very low levels of il-6 mrna were detected in im-discussion munodeficient mice infected with either virus. jhmv produces an acute cns infection associated with several immune effector mechanisms, including th2-related cytokines both cd4 / and cd8 / t cells houtman and fleming, 1996b) . kinetic analysis of cellular igg1 and igg2a virus-specific antibodies were detected in survivors of jhmv infection; however, there appeared cns infiltrations during jhmv infection of mice shows that nk cells accumulate prior to cd8 / t cells, which in to be little relationship between induction of antibody and control of jhmv infection within the cns. induction of both turn precede accumulation of cd4 / t cells and macrophages (williamson et al., 1991; williamson, 1992) . there isotypes suggest that th1 and th2 cytokines are induced by jhmv infection. the kinetics of il-10 mrna induction is no direct evidence for a role of nk cells in suppressing jhmv replication (houtman and fleming, 1996a) ; how-was of interest due to the association of il-10 with reduced th1 activity in vitro and with remission during exper-ever, cd8 / ctl appear to be critical immune effectors (williamson and stohlman, 1990; stohlman et al., 1995b) . imental allergic encephalomyelitis (kennedy et al., 1992) . il-10 mrna was first detected at day 3 p.i. in lethally recent analysis of jhmv pathogenesis in mice deficient in perforin suggests that in addition to cytolytic effectors infected mice, but not until day 5 postinfection in the cns of the mice undergoing a subacute encephalitis. however, other immune components also contribute to sterilizing immunity (lin et al., 1997) . similarly, the adoptive transfer at the time most lethally infected mice were about to succumb to infection (day 7), there was no difference in the of virus-specific cd4 / t cells to jhmv-infected mice demonstrates that some clones protect via reducing viral peak levels of il-10 mrna between the two groups. the kinetics of il-10 mrna accumulation differed between the replication (yamaguchi et al., 1991) , while others protect without reducing virus replication , groups; il-10 mrna accumulation in mice undergoing a sublethal infection was slower and remained at peak lev-suggesting that cytokines may play an important role in providing sterile immunity. els until day 9 p.i., prior to declining to near basal levels by day 14. no il10 mrna was detected in the cns of in general the kinetics of cytokine mrna expression correlated with the temporal presence of cns infiltrating sham-infected or infected immunodeficient mice. no il-4 mrna was detected following a single amplification dur-mononuclear cells. many cytokine transcripts, with the exceptions of il-12, il-1a, and il-6, were maximally ex-ing lethal or sublethal jhmv infections. however, after a second amplification, low abundant mrnas were de-pressed by 7 day p.i., near the peak inflammatory cell infiltration and during the elimination of virus from the tected (fig. 5a) . no il-4 mrna was detected in either sham-infected or infected immunosuppressed mice fol-cns (williamson et al., 1991; williamson, 1992) . previous data using the oblv-60 jhmv variant which has a selec-lowing two amplifications (data not shown). the kinetics tive tropism for neurons suggested a correlation between increasing time following subacute infection, consistent with the resolution of encephalitis. it is interesting that ifn-g induction, t cell accumulation, and reduction of virus replication (pearce et al., 1994) . the semiquantita-the cns of mice with active macrophage-mediated demyelination (day 14 p.i.) showed little evidence of tnf-tive kinetic analysis of ifn-g mrna in the cns of mice undergoing both lethal and sublethal jhmv infections a mrna, consistent with the inability of anti-tnf-a to prevent jhmv-mediated demyelination (stohlman et al., supports the positive correlation between ifn-g and viral clearance. however, the oblv-60 jhmv variant is 1995a). a surprising number of mrnas peaked relatively early cleared from the cns of ifn-g-deficient mice , consistent with ifn-g exhibiting poor in vitro following jhmv infection. the mrnas encoding inos, il-12, il-1a, il-1b, and il-6 peaked either prior to or anti-jhmv activity (zhang et al., 1997) and inability of rifn-g to inhibit cns virus replication (smith et al., 1991) . coincident with initiation of viral clearance. in most cases (except inos mrna) the levels were either higher or these data contrast with other viral-induced encephalopathies in which ifn-g plays a significant role (kundig et increased more rapidly in the mice undergoing subacute infections. accumulation of inos mrna was first de-al., 1993; finke et al., 1995) , including some (yu et al., 1996) , but not all (wesselingh et al., 1994) , neuronotropic tected in mice undergoing a lethal infection coincident with the initial detection of ifn-g mrna. however, the viruses. the kinetics of ifn-g mrna induction suggests that it may play a more prominent role in the pathogene-mrna levels declined as virus replication declined, suggesting a direct effect of virus on inos induction. in sis of jhmv variants with predominant tropisms for microglia, astrocytes (jhmv), or oligodendroglia (2.2v-1). contrast to lethal infections, inos mrna lagged detection of ifn-g in mice undergoing subacute infections and the isotype diversity of the anti-jhmv antibody response suggests that both th1 and th2 subsets of cd4 / increased to less than 50% the level detected in mice undergoing a lethal infection. similar to the recent data t cells are activated during infection. il-4 mrna accumulation in the cns corresponds to infiltration of th2 cells demonstrating low levels of inos in the cns of both nude mice and mice deficient in ifn-g , (cua et al., 1995) and kinetic analysis suggests that t cells expressing th2 cytokine profiles are recruited into inos mrna in immunodeficient mice was approximately 50% the levels detected in the cns of intact mice at day the cns with nearly equal kinetics in both lethally and sublethally infected mice (fig. 5a) . il-4 increases the 3 p.i. although jhmv is susceptible to inhibition by inos in vitro, inos is not associated with in vivo protection severity of encephalitis (ikemoto et al., 1995) and could potentially play a role in jhmv persistence via inhibition . il-12, predominantly produced by cells of the myelo-of viral clearance (moran et al., 1996) . in support of the recruitment of th2 cells, il-10 mrna also increased with monocytic lineage, is associated with the induction of th1 cd4 / t cells (brunda, 1994) . il-12 mrna peaked kinetics similar to those of ifn-g and il-4. whether this difference in detection of th2 cytokines is due to differ-early (day 3) in mice undergoing both lethal and sublethal jhmv infections. however, no significant differences ences in mouse strains or the selective tropism of the virus is not known. it is interesting that although il-10 is were found comparing mrna levels in immunodeficient mice to intact mice. this may suggest that jhmv infection secreted by activated microglia in vitro (lodge and sriram, 1996) , no il-10 mrna was detected in sham-induces transcription of il-12 mrna in cns cells. in addition, 2.2v-1 infects predominantly, but not exclu-infected or immunodeficient mice. this contrasts with other cytokine mrna detected in either sham-infected sively, oligodendroglia, while jhmv infects predominantly microglia and astrocytes. the relatively higher or virus-infected immunodeficient hosts (see below). tnf-a mrna is induced following jhmv infection level of il-12 mrna in 2.2v-1-infected mice suggests the possibility that oligodendroglia transcribe il-12 mrna in (pearce et al., 1994; stohlman et al., 1995a; sun et al., 1995) and tnf-a is present during both the acute and response to jhmv infection, similar to the induction of il-12 mrna following measles virus infection of oligo-persistent jhmv infections. tnf-a mrna is not translated in jhmv-infected cells (stohlman et al., 1995a ), al-dendroglia (yamabe et al., 1994 . during both the lethal and sublethal infections the il-though it may be secreted by adjacent but not infected cells. in addition, inhibition of tnf-a, which prevents 1a mrna peaks appear to coincide with replication and not clearance, suggesting that infection induces a rapid experimental autoimmune encephalitis (ruddle et al., 1990) , has no effect on either jhmv-induced encephalitis induction of il-1a mrna. these data contrast to the association of il-1a mrna and the clearance of the oblv-or demyelination (stohlman et al., 1995a) . as anticipated, based on the relative tropism of the two viruses analyzed, 60 variant of jhmv (pearce et al., 1994) , suggesting an additional difference in cytokine responses depending tnf-a mrna accumulated initially in the cns of mice infected with jhmv. however, by day 5 p.i. there was on the tropism of the virus analyzed. il-1b mrna, previously detected in the cns of jhmv-infected mice little difference in the levels of tnf-a mrna in the two groups. finally, the level of tnf-a mrna decreased with (pearce et al., 1994) , increased directly after infection at day 1 p.i. however, the level was approximately the same suggests it may play a positive role in reducing the extent of cns inflammation thereby inadvertently contributing as the level detected in sham-infected mice, suggesting it was induced by trauma. in all mice the levels subse-to persistent infection. some aspects of our data, i.e., the rapid induction of il-12 mrna in mice infected with 2.2v-quently dropped by day 3 p.i. the levels of il-1b mrna peaked at day 5 following 2.2v-1 infection and at day 7 1, suggest that infection of specific cell types may influence the induction of cytokine mrna (yamabe et al., following jhmv infection, suggesting il-1b mrna was also induced by infection. analysis of the levels in immu-1994) . this supports the notion that the cytokine mrna patterns more closely reflect diversity of the immune re-nodeficient mice were consistent with the notion that infection, and not immune infiltrates, contributed the ma-sponse to an individual agent, although differential secretion of cytokines following infection of unique cns cell jority of the il-1b mrna levels. il-6, another pleiotropic cytokine with numerous ef-types cannot be ruled out (benveniste, 1992; sun et al., 1995) . while the kinetics of ifn-g, il-4, and il-10 showed fects on immune responses (van snick, 1990) , was also detected early following both lethal and sublethal infec-little difference between the groups undergoing lethal or sublethal infections, mrnas encoding il-6 and il-1b tions. by contrast, il-6 mrna was also only detected at 6 days p.i. with the neuronotropic oblv-60 jhmv variant either appeared more rapidly (il-6) or accumulated to higher levels (il-1b) following infection with 2.2v-1 virus. (pearce et al., 1994) . kinetic analysis shows that the levels of il-6 mrna peaked at day 3 post-2.2v-1 infection by contrast the induction of inos and il-1a mrnas were increased in mice undergoing a lethal infection. these and at day 5 post-jhmv infection. interestingly, analysis of the mrna levels in the immunodeficient mice showed data suggest that an early induction of il-6, and possibly il-1b, are associated with sublethal infection or the dif-virtually no induction of il-6 mrna, suggesting that in contrast to il-1a and il-1b an intact immune response ferent tropisms exhibited by these two jhmv variants. however, during both infections the mrna levels de-was required for il-6 mrna induction. rapid induction of il-6 mrna following jhmv infection is consistent with creased as virus was cleared. similarly, there appears to be an inverse correlation between a rapid induction other models of viral-induced encephalitis in which it also precedes ifn-g (moskophidis et al., 1991) . although of inos mrna and sublethal disease, consistent with the recent demonstration that although inos is protective in both il-6 and il-10 are cofactors for ctl induction (chen and zlotnik, 1991; takai et al., 1988) , kinetic analysis is vitro, inhibition of inos activity in vivo appears to have no effect on jhmv pathogenesis . taken consistent with the notion that il-6, and not il-10, may be involved in the induction or recruitment of jhmv-specific together, kinetic analyses of the induction of cytokine mrna during the lethal and sublethal jhmv infections ctl. jhmv infection induces il-6 secretion from both brain endothelial cells and astrocytes following in vitro are consistent with the accumulation of both th1-and th2-associated cytokines and support the interaction of infection with jhmv (joseph et al., 1993) , consistent with data showing that it is produced by resident cns cells multiple cellular and soluble effector mechanisms whose balance may be critical in providing protection and steri-following infection with lymphocytic choriomeningitis virus (frei et al., 1989) . it is interesting that il-6 mrna lizing immunity. peaks first in mice infected with the 2.2 v-1 variant compared to jhmv, which infects a significantly larger num-acknowledgments ber of astrocytes. the rapid induction of il-6 and il-we thank wen-qiang for excellent technical assistance. this work 1b following infection with 2.2v-1 is consistent with the was supported by grant ns18146 from the national institutes of health. induction of these mrna in oligodendroglia infected by escherichia coli lipopolysaccharide and tumor necrosis factor alpha a paradigm for virus-induced demyelinating disease. trends microbiol. 5, 9-14. stimulation recovery from acute virus infection. role of cytotoxic t lymphocytes in the eliminatiation factor self-antigen-intion of lymphocytic choriomeningitis virus from spleens of mice duced th2 responses in experimental allergic encephalomyelitis (eae)-resistant mice exposure to t helper 2 cytokines in vivo before encounter with antigen selects for perforin-mediated cytolysis regulation of microglial activation t helper subsets via alterations in antigen-presenting cell function gamma interferon is a major mediator of antiviral defense in experiing and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv-4) rus-infected mice production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of in-weiner on the cellular source and function of interleukin 6 produced in the central nervous system in viral disease. ase chain reaction method in schistosoma mansoni infected mice an absolute and restricted requirement for il-12 in natural killer cell ifn-g production and antiviral bral toxoplasmosis is induced by in vivo neutralization of tnf-a and correlates with the down-regulated expression of inducible nitric defense the in vivo oxide synthase and other markers of macrophage activation ifng independent production of il-12 during murine endotoxemia cytokine induction during t-cell mediated clearance of mouse hepa-immunol dissociation of demyelination titis virus from neurons in vivo the astrocyte is a target cell in mice and viral clearance in congenitally immunodeficient mice infected with murine coronavirus jhm pathogenesis of mouse hepatitis virus-induced demyelination cytotoxic t cell-resistant variants are selected in a virus-induced small amounts of exogenous il-4 increase the severity of demyelinating disease grunencephalitis induced in mice by the intranasal infection of herpes simplex virus type 1 interleukin-6 induction in vitro in mouse brain endothelial cells and astrocytes allergic encephalomyelitis the immune system response by exposure to mouse hepatitis virus (mhv-4, jhm) igg1 induction factor: a single molecular entity with multiple of mice with experimental autoimmune encephalomyelitis reveals that il-10 mrna expression correlates with recovery 89-100. dependent ifn-g exerts an antiviral effect in the central nervous system but not in peripheral solid organs pathogenesis of a neurotropic of two plaque morphology variants of the jhm neurotropic strain murine coronavirus strain jhm in the central nervous system. seminar virol mechanisms of demyelination in jhm virus encephalomyelitis. electron microscopic cells prevent a lethal infection but do not inhibit virus replication tumor necrosis between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus mouse hepatitis virus-specific cytotoxic t lymphocytes protect from lethal infection without eliminating virus from the central williamson hepatitis virus strain jhm virus-specific t cells in the central nervous activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus b cell stimulatory factor-2 is involved in the differentiation of yamabe 8, gene expression in measles-infected adult human glial cells protection of mice from a lethal coronavirus infection in the central induced by murine hepatitis virus, jhm strain (mhv-4) is immunologically mediated pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) role of interferon-g in immunity to herpes simplex virus expression of gamma interferon by a coronavirus defectivealphavirus encephalitis suggests a predominant type 2 t cell response effective clearance of key: cord-256015-tt58n0jk authors: zhu, juanjuan; zhou, wei; zhou, mingyu; liu, yang; yang, jing; li, haiyang; zhao, xueke title: clinical characteristics and therapeutic procedure for a critical case of novel coronavirus pneumonia treated with glucocorticoids and non-invasive ventilator treatment date: 2020-06-01 journal: revista da sociedade brasileira de medicina tropical doi: 10.1590/0037-8682-0227-2020 sha: doc_id: 256015 cord_uid: tt58n0jk the novel coronavirus pneumonia (ncp) outbreak occurred in wuhan, china at the end of 2019. here, we report the clinical characteristics and therapeutic procedure for a case of severe ncp. the patient was started on glucocorticoids and non-invasive ventilator treatment. after treatment, the patient’s symptoms improved, and the status was confirmed as ncp negative. our results may provide clues for the treatment of ncp. at the end of december 2019, the novel coronavirus pneumonia (ncp) outbreak occurred in wuhan, china. the world health organization officially named this new virus as the 2019 novel coronavirus, simply referred to as 2019-ncov 1 . this virus belongs to the β coronavirus family, and its genetic characteristics are significantly different in severe acute respiratory syndrome related coronavirus (sarsr-cov) and middle east respiratory syndrome related coronavirus (mersr-cov), and it has more than 85% homology with the bat sars-like cov 2 . herein, we report the clinical characteristics and diagnosis of a patient with severe ncp who was treated with glucocorticoids and non-invasive ventilation. a 61-year-old woman from guizhou was admitted to the affiliated hospital of guizhou medical university on january 30, 2020 with one-day history of fever and two-day history of cough. her body temperature was 37.8 o c. she was in good health without hypertension, diabetes, and coronary heart disease. at admission, her heart rate (85 times/min), respiratory rate (19 times/min) and blood pressure (121/60 mmhg) were in the normal range. her c-reactive protein (17.38 mg/l) and il-6 (6.85 pg/ml) levels were elevated. laboratory examinations showed normal white blood cell count, liver function, kidney function, and procalcitonin level. ct lung imaging showed patchy exudate in the lower lobe of the right lung, slight fibrosis in the middle lobe of the right lung and upper lobe of the left lung, nodules in the upper lobe of the right lung and lower lobe of the left lung, and aortic sclerosis (figure 1) . additionally, the sputum swab test was performed, and the nucleic acid test indicated positivity for 2019-ncov. based on her laboratory report, she was treated with lopinavir and ritonavir tablets (two tablets twice a day), arbidol tablets (two tablets three times a day), α-interferon atomization inhalation, xuebijing injection (100 ml twice a day), live combined clostridium butyricum and enterococcus tablets (two tablets three times a day), vitamin c (two tablets three times a day) and 2 l/min oxygen (twice a day, two hours every time) were also administered. on the fourth day of admission, the oxygen saturation of the patient was 89. considering the severity of the disease, intravenous injection of methylprednisolone (60 mg/d) and subcutaneous injection of thymalfasin (1.6 mg) were administered once a day. on the sixth day of admission, chest ct images revealed obvious exudative lesions in bilateral lungs (figure 2 ). on the eighth day of admission, she developed dyspnea and chest distress. physical exam revealed body temperature of 36.3 o c, respiratory rate of 20 breaths/min, 3 l/min oxygen by nasal catheter, and oxygen saturation less than 90%. blood gas analysis showed increased ph (7.58), decreased po 2 (58 mmhg), and normal pco 2 (43 mmhg). the findings were suggestive of type i respiratory failure with metabolic alkalosis. subsequently, moxifloxacin and intravenous injection of methylprednisolone (40 mg) were administered, and required assisted respiration with non-invasive ventilator. the dosage of methylprednisolone was reduced every 2-3 days until withdrawal (table s1) . changes in c-reactive protein and il-6 levels are shown in table s2 . the results indicated that the levels of il-6 and c-reactive protein had decreased with the increase in the dosage and duration of methylprednisolone therapy and returned to normal on february 10. in addition, she was placed on non-invasive ventilation four times a day for 2 hours each (table s3 ). blood gas analysis indicated normal pco 2 and spo 2 . however, the value of po 2 was lower than normal on february 7 and 8 and returned to normal on february 12 (table s4) . on the 15 th day of admission, the patient was administered 2 l/min of oxygen. spo 2 was maintained at more than 93%, po 2 , between 86 and 116 mmhg, and pco 2 , between 37.5 and 40 mmhg. on the 16 th day of admission, chest ct showed absorption of bilateral lung effusion lesions (figure 3) . on the 18 th day of admission, she was treated with cefoperazone sodium and sulbactam sodium. on the 19 th day of admission, a nasopharyngeal swab was performed for the first time after the treatment and the test result was negative for 2019-ncov infection. on the 20 th day of admission, antiviral drugs and antibiotics were stopped, and she was only treated with thymalfasin and traditional chinese medicine preparations. chest ct revealed that the exudation in both lungs was absorbed, and there was a small amount of fibrosis in the middle lobe of the right lung and the upper lobe of the left lung ( figure s1 ). on the 21 th day of admission, a nasopharyngeal swab was performed for the second time after treatment and the test result was negative for 2019-ncov infection. after consultation with the ncp expert group at our hospital, it was decided that she had reached the relief isolation criteria. her condition had healed, and she was discharged on 24 february, 2020. it was suggested that she be placed under continued medical observation for 14 days after discharge and return to the hospital for follow-up, two weeks and four weeks after discharge. methylprednisolone is one of the glucocorticoids, which is frequently used in clinical settings, it has anti-inflammatory and anti-immune effects and improves airway function 3 . studies have found that when inflammation occurs in the body, the concentration of glucocorticoids increase in the blood 4 . in the current report, the levels of il-6 and c-reactive protein decreased with the increase in methylprednisolone dosage, consistent with the above mentioned findings. in addition, the levels of il-6 and c-reactive protein returned to normal on day 8 of methylprednisolone use. similarly, zhou et al. suggested the median time of glucocorticoid use in cured patients was 9.5 days through the observation of critically ill patients with ncp 5 . for patients with severe acute respiratory infection and respiratory distress, oxygen inhalation through mask or noninvasive ventilation is necessary 6 . non-invasive ventilator treatment in the early stage of critical disease can reduce the probability of respiratory failure. in this report, the patient developed type i respiratory failure on day 8 of admission. after nearly one week of intermittent non-invasive ventilator-assisted respiration, the patient's arterial partial pressure of oxygen, oxygenation index, and oxygen saturation had significantly improved (table s3 and table s4 ). it was found that the use of non-invasive ventilation was effective in improving the patient's respiratory function. in summary, we reported the history, diagnosis, and treatment of a patient with ncp. glucocorticoids and non-invasive ventilator treatment could significantly improve the clinical symptoms in critically ill patients with ncp. it may provide a good reference for the treatment of ncp. clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures effect of montelukast combined with methylprednisolone for the treatment of mycoplasma pneumonia therapeutic mechanisms of glucocorticoids potential benefits of precise corticosteroids therapy for severe 2019-ncov pneumonia 2019 novel coronavirus of pneumonia in wuhan, china: emerging attack and management strategies writing -review & editing. all authors read and approved the final manuscript. the authors declare that they have no conflict of interests. key: cord-002945-29nj4f05 authors: ambrose, rebecca k.; gravel, jennifer l.; commins, margaret a.; fowler, elizabeth v.; mahony, timothy j. title: in vivo characterisation of five strains of bovine viral diarrhoea virus 1 (subgenotype 1c) date: 2018-01-19 journal: pathogens doi: 10.3390/pathogens7010012 sha: doc_id: 2945 cord_uid: 29nj4f05 bovine viral diarrhoea virus 1 (bvdv-1) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. to date many subgenotypes have been reported for bvdv-1, currently ranging from subgenotype 1a to subgenotype 1u. while bvdv-1 has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. as an example, bvdv-1 subgenotypes 1a and 1b are frequently detected in north america and europe, while the subgenotype 1c is rarely detected. in contrast, bvdv-1 subgenotype 1c is by far the most commonly reported in australia. despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. the aim of this study was to characterise the in vivo properties of five strains of bvdv-1 subgenotype 1c in cattle infection studies. no overt respiratory signs were reported in any of the infected cattle regardless of strain. consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. the quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. further studies are required to fully understand the variability and importance of the bvdv-1 subgenotype 1c. bovine respiratory disease (brd) is the most important disease of intensively finished cattle. while multiple factors contribute to the likelihood of cattle developing brd, a generally accepted model for brd development is a primary viral infection predisposing cattle to more severe secondary bacterial infections. several viruses have been associated with an increased risk of brd, including bovine herpesvirus 1 (bohv-1), bovine viral diarrhoea virus 1 (bvdv-1), bovine respiratory syncytial virus and bovine parainfluenza 3 virus. of the key brd associated viruses, bvdv-1 is the most genetically diverse with 21 subgenotypes, bvdv-1a to bvdv-1u, having been reported [1] . yesilbag et al. [1] recently reviewed the geographical distribution of the bvdv-1 subgenotypes. this analysis highlighted several unusual trends in the distribution of the bvdv-1 subgenotypes. as an example, the vast majority of genotyped strains identified in australia have been classified within the subgenotype 1c [2, 3] . this is in contrast to the usa where the subgenotype 1b is dominant, but the subgenotype 1a is also frequently reported [4] . while in europe the most common subgenotypes are 1a and 1b, also common are subgenotypes 1d, 1e, 1f and 1h [1] . however, the distribution of subgenotypes can vary between countries within europe, for example subgenotype 1b and 1d were recently reported to be the most frequently identified subgenotypes in germany [5] . strains of the subgenotype 1c have been rarely reported in the usa or europe. the drivers of these distributions are unclear, but do not appear to be influenced by vaccine use [6] . several studies have suggested that the subgenotypes, at least in part, also reflect the antigen diversity between strains using cross-neutralisation assays [3, 7, 8] . clearly the degree of cross-protection afforded by the subgenotype(s) included in a bvdv-1 in a vaccine against the subgenotypes circulating in a cattle population is an important issue. the overall biological importance of the bvdv-1 subgenotypes remains to be fully elucidated since the majority of published studies have focused on the bvdv-1a and bvdv-1b genotypes. it is reasonable to accept that the subgenotypes of bvdv-1 share similar properties with respect to their capacity to infect susceptible ruminants, and their contribution to the development of more severe clinical disease outcomes such as brd and the birth of persistently infected calves following transplacental infection. all of these outcomes are reported in cattle populations regardless of what subgenotype is present. the evaluation of any variation of the in vivo properties of bvdv-1 subgenotypes has received little attention. to assist in developing a better understanding of the importance of the bvdv-1 subgenotypes, the aim of this study was to assess the in vivo properties of five bvdv-1 strains from the poorly studied subgenotype 1c. no overt clinical scores were recorded for any of the trial animals, challenged or unchallenged with the bvdv-1c strains used in this study (data not shown). for all groups, except the cattle infected with bvdv-1c strain ns155, there was a general trend for an increase in the average rectal temperature on day 0 of the experiment, prior to any viral exposure, compared to the temperatures from day 1 to day 5 (figure 1a -f). the reason(s) for this increase are unclear. the cattle had been inducted into the containment facility seven days prior to the commencement of the trial and were therefore considered to have been well adapted/acclimatised to the environment. however, on day 0 there were extra staff and equipment present while the cattle were being inoculated which included extra handling of the animals/cattle potentially contributing to this apparent increase. due to this anomaly, the day 1 temperatures were used as the reference point in the subsequent statistical comparisons within each treatment group. the daily rectal temperatures were monitored for the trial cattle for 14 days following experimental infection. overall the mean rectal temperature results were variable ( figure 1 ). animals (n = 6) infected with bvdv-1c strain pi506 exhibited a significant temperature elevation on day 8 post infection (p < 0.001, figure 1a ). for animals infected with bvdv-1c strain trangie (n = 4), the temperature increase was gradual from day 6 to day 9 post infection, although the increase was only statistically significant on day 9 (p < 0.05, figure 1b ). animals infected with bvdv-1c strain ao554 (n = 4) exhibited elevated mean temperatures on day 7 and day 8 post infection, with only the day 8 mean temperature being statistically significant (p < 0.05, figure 1d) . a similar temperature profile was observed for bvdv-1c strain ns155 (n = 4), with elevated temperatures on day 8 and day 9, with only day 8 being statistically significant (p < 0.05, figure 1d ). although there was a suggestion of an elevated mean temperature on day 9 post-infection for the cattle (n = 3) infected with vr1112 this was not statistically significant ( figure 1e ). the rectal temperatures of the contact animals (n = 2) did not exceed 39.0 • c during the experiment (figure 1f ). no statistical comparisons were undertaken for this group due the low animal numbers. animals infected with bvdv-1c strain trangie (n = 4); (c) animals infected with bvdv-1c strain ao554 (n = 4); (d) animals infected with bvdv-1c strain ns155 (n = 4); (e) animals infected with bvdv-1c strain vr1112 (n = 3); (f) uninfected animals (n = 2). the asterisks above selected days indicate significant differences compared to day 1 post-infection for that group of cattle. level of significance; * p < 0.05; ** p < 0.01; **** p < 0.0001. the course of the bvdv-1c nasal shedding by the trial cattle was assessed by testing extracts from nasal swabs collected from day 0 to day 14 and serum samples collected on day 7 and day 14 post-infection with a bvdv-1 specific quantitative real-time (qpcr). a summary of these results expressed as the threshold cycle (ct) is shown in table 1 . overall, the distribution of positive nasal swabs was variable between and within the groups of cattle infected with the different strains of bvdv-1c. the earliest that virus was detected in nasal swabs was day 2 post-infection for animals infected with pi506 (1 of 6 animals), trangie (1 of 4 animals) and ao554 (2 of 4 animals). one nasal swab from an animal infected with strain pi506 tested positive at day 14 post infection. the bvdv-1c strain ao554 was the virus most consistently detected in the nasal swabs with all infected animals reacting with the qpcr at day 6 and day 7 post infection, and three of the four animals also reacted in the qpcr on day 8 ( table 1 ). the serum samples collected from animals infected with ao554 at day 7 post-infection also reacted and were deemed to be positive for bvdv-1c (table 1 ). (b) animals infected with bvdv-1c strain trangie (n = 4); (c) animals infected with bvdv-1c strain ao554 (n = 4); (d) animals infected with bvdv-1c strain ns155 (n = 4); (e) animals infected with bvdv-1c strain vr1112 (n = 3); (f) uninfected animals (n = 2). the asterisks above selected days indicate significant differences compared to day 1 post-infection for that group of cattle. level of significance; * p < 0.05; **** p < 0.0001. the course of the bvdv-1c nasal shedding by the trial cattle was assessed by testing extracts from nasal swabs collected from day 0 to day 14 and serum samples collected on day 7 and day 14 post-infection with a bvdv-1 specific quantitative real-time (qpcr). a summary of these results expressed as the threshold cycle (ct) is shown in table 1 . overall, the distribution of positive nasal swabs was variable between and within the groups of cattle infected with the different strains of bvdv-1c. the earliest that virus was detected in nasal swabs was day 2 post-infection for animals infected with pi506 (1 of 6 animals), trangie (1 of 4 animals) and ao554 (2 of 4 animals). one nasal swab from an animal infected with strain pi506 tested positive at day 14 post infection. the bvdv-1c strain ao554 was the virus most consistently detected in the nasal swabs with all infected animals reacting with the qpcr at day 6 and day 7 post infection, and three of the four animals also reacted in the qpcr on day 8 ( table 1 ). the serum samples collected from animals infected with ao554 at day 7 post-infection also reacted and were deemed to be positive for bvdv-1c (table 1) . table 1 . detection of bovine viral diarrhoea virus 1 subgenotype 1c in extracts from cattle samples using quantitative real time pcr (qpcr). nasal swabs (n) and serum (s) samples were analysed using qpcr. reactive samples were deemed positive for bvdv-1c with the cycle threshold value shown (shaded cells). samples which did not react (>40) with the qpcr were deemed to be negative (−) for the virus. with respect to the other groups, bvdv-1c was consistently detected in nasal swabs collected on day 5, day 6 and day 7 post-infection from two of the six animals infected with bvdv-1c strain pi506 ( table 1) . one of the animals in this group tested positive (nasal swabs) from day 3 to day 7 post-infection and the swabs from day 13 and day 14 were also positive. for cattle infected with bvdv-1c strain trangie, one of the four animals tested positive on day 2 post-infection, while all the other samples were negative throughout the sampling period (table 1) . of the animals infected with bvdv-1c strain ns155, two animals had positive nasal swabs. one of these animals was positive on day 6. the strain ns155 was detected sporadically between day 7 and 13 in samples from animal 2680. the serum samples from day 7 and day 14 from this animal were both reactive with the qpcr assay (table 1) . two animals infected with bvdv-1c strain vr1112 tested positive for the virus in a sporadic manner. the remaining animals did not return any positive results ( table 1) . none of the sample extracts from the two contact animals reacted with the qpcr during the sampling period (table 1) . bvdv-1c was not detected via qpcr in the nasal swab or serum samples collected from all animals on day 21, day 28, day 42 and day 55 post-infection and were deemed to be negative (data not shown). attempts were made to isolate the bvdv-1c strains from the nasal swabs collected at day 7 post-infection from selected animals. no bvdv-1c was detected in any of the culture supernatants by qpcr after three passages of animal 2684 (pi506 infected and tested positive from day 2 to day 7), animal 2686 (trangie infected), animal 2666 (ao554 infected) or animal 2669 (ao554 infected). with respect to the other groups, bvdv-1c was consistently detected in nasal swabs collected on day 5, day 6 and day 7 post-infection from two of the six animals infected with bvdv-1c strain pi506 ( table 1) . one of the animals in this group tested positive (nasal swabs) from day 3 to day 7 post-infection and the swabs from day 13 and day 14 were also positive. for cattle infected with bvdv-1c strain trangie, one of the four animals tested positive on day 2 post-infection, while all the other samples were negative throughout the sampling period (table 1) . of the animals infected with bvdv-1c strain ns155, two animals had positive nasal swabs. one of these animals was positive on day 6. the strain ns155 was detected sporadically between day 7 and 13 in samples from animal 2680. the serum samples from day 7 and day 14 from this animal were both reactive with the qpcr assay (table 1) . two animals infected with bvdv-1c strain vr1112 tested positive for the virus in a sporadic manner. the remaining animals did not return any positive results ( table 1) . none of the sample extracts from the two contact animals reacted with the qpcr during the sampling period (table 1) . bvdv-1c was not detected via qpcr in the nasal swab or serum samples collected from all animals on day 21, day 28, day 42 and day 55 post-infection and were deemed to be negative (data not shown). attempts were made to isolate the bvdv-1c strains from the nasal swabs collected at day 7 postinfection from selected animals. no bvdv-1c was detected in any of the culture supernatants by qpcr after three passages of animal 2684 (pi506 infected and tested positive from day 2 to day 7), animal 2686 (trangie infected), animal 2666 (ao554 infected) or animal 2669 (ao554 infected). monocytes: seven days post-infection, animals infected with bvdv-1c strain pi506 had significantly reduced concentration of monocytes compared to the pre-infection sample (p < 0.01, figure 4a ). no significant changes in the numbers of monocytes were detected for any of the animals infected with the remaining bvdv-1c strains or the contact animals ( figure 4 ). (e) (f) (e) (f) (e) (f) platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv-1c strains on day 7 and/or day 14 post infection compared to day 0. the only significant reduction was for the animals infected with bvdv-1c strain ns155 on day 14 (p < 0.05, figure 7d) . a reduction in platelet numbers was also apparent on day 14 for animals infected with strain pi506, trangie and vr1112, but these differences were not statistically significant (figure 7a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv-1c strain ao554 appeared stable for the duration of the experiment, apart from a significant increase on day 21 postinfection (p < 0.05, figure 7c ). platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv-1c strains on day 7 and/or day 14 post infection compared to day 0. the only significant reduction was for the animals infected with bvdv-1c strain ns155 on day 14 (p < 0.05, figure 7d) . a reduction in platelet numbers was also apparent on day 14 for animals infected with strain pi506, trangie and vr1112, but these differences were not statistically significant (figure 7a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv-1c strain ao554 appeared stable for the duration of the experiment, apart from a significant increase on day 21 post-infection (p < 0.05, figure 7c ). platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv-1c strains on day 7 and/or day 14 post infection compared to day 0. the only significant reduction was for the animals infected with bvdv-1c strain ns155 on day 14 (p < 0.05, figure 7d) . a reduction in platelet numbers was also apparent on day 14 for animals infected with strain pi506, trangie and vr1112, but these differences were not statistically significant (figure 7a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv-1c strain ao554 appeared stable for the duration of the experiment, apart from a significant increase on day 21 postinfection (p < 0.05, figure 7c ). (a) (b) (c) (d) all trial animals were monitored for the development of bvdv-1 specific antibodies throughout the course of the experiment. virus specific antibody was first detected on day 14 post-infection with 10 of the 21 infected animals testing positive. however, none of the animals infected with strains trangie or ao554 had detectable antibodies by this time point. by day 21 post-infection, 18 of the 21 animals had detectable bvdv-1 specific antibody ( table 2 ). all the bvdv-1c challenged animals had detectable bvdv-1 antibodies by day 28 post-infection ( table 2 ). one of the animals infected with strain ao554 was positive on day 14 but subsequently tested negative on day 21 (table 2) . no bvdv-1 antibody was detected in the serum samples from either of the two contact animals at any of the sampling time points (table 2) . table 2 . serological responses (igg) of cattle challenged with one of five strains of bovine viral diarrhoea virus 1 genotype 1c. the level of virus specific igg were determined using a commercial elisa and assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++). all trial animals were monitored for the development of bvdv-1 specific antibodies throughout the course of the experiment. virus specific antibody was first detected on day 14 post-infection with 10 of the 21 infected animals testing positive. however, none of the animals infected with strains trangie or ao554 had detectable antibodies by this time point. by day 21 post-infection, 18 of the 21 animals had detectable bvdv-1 specific antibody ( table 2 ). all the bvdv-1c challenged animals had detectable bvdv-1 antibodies by day 28 post-infection ( table 2 ). one of the animals infected with strain ao554 was positive on day 14 but subsequently tested negative on day 21 (table 2) . no bvdv-1 antibody was detected in the serum samples from either of the two contact animals at any of the sampling time points (table 2) . table 2 . serological responses (igg) of cattle challenged with one of five strains of bovine viral diarrhoea virus 1 genotype 1c. the level of virus specific igg were determined using a commercial elisa and assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++). animal id infection 0 7 14 21 28 35 55 pi506 2661 − − − + ++ +++ ++ 2664 − − + +++ ++++ +++++ ++++ 2665 − − + +++ ++++ ++++ +++ 2676 − − − ++ +++ ++++ ++++ 2682 − − − ++ ++ ++++ ++++ 2684 − − − +++ +++ ++++ ++++ trangie 2675 − − − +++ ++++ ++++ +++ 2677 − − − ++ +++ +++ +++ 2685 − − − +++ +++ ++++ +++++ 2686 − − ++ +++ +++++ +++++ +++++ ao554 2666 − − ++ +++ ++++ +++++ ++++ 2668 − − +++ − +++++ +++++ +++++ 2669 − − +++ ++++ +++++ +++++ +++++ 2673 − − − ++ ++++ +++++ ++++− − + + +++++ +++++ ++++ 2674 − − − − ++++ ++++ ++++ 2680 − − + ++++ +++++ +++++ +++++ vr1112 2662 − − − − ++ ++ ++ 2678 − − +++ +++ +++++ +++++ +++++ 2683 − − − + + ++ ++ contact 2663 − − − − − − − 2681 − − − − − − − several studies have explored the potential links between the subgenotypes and antigenic variation through cross neutralisation studies [3, 7, 8] . clearly, knowledge of any such relationship is important as it would facilitate the selection of vaccine components to match the circulating bvdv-1 subgenotypes, while also enabling the ongoing monitoring of field strains to detect any change in the dominant subgenotype. the importance of the bvdv-1 subgenotypes with respect to the in vivo biology has received minimal attention and more research is required to define commonalities and divergences between each group such as virulence. several of the parameters measured in this study showed similar effects of the bvdv-1c strains on their bovine hosts. these commonalities were not unexpected as the strains used in this study were all bvdv-1 subgenotype 1c. currently, there are no specific criteria proposed to evaluate bvdv-1 virulence, however clinical signs (respiratory and/or digestive), biphasic pyrexia, biphasic leukopenia and thrombocytopenia have been reported for bvdv-1 subgenotypes 1a, 1b, 1d, 1e and 1k from various countries [9] [10] [11] [12] [13] . in the current study, no respiratory or digestive clinical signs were observed in the bvdv-1c inoculated cattle. while a significant pyrexia was identified in cattle infected with four of the five bvdv-1 strains, however, there was no evidence of a biphasic pyrexia (figure 1 ). four of the five bvdv-1 infected groups exhibited significant leukopenia at day 7 post-infection, three of which were biphasic (figure 3 ). only the group infected with vr1112 did not have detectable leukopenia. as vr1112 was the only cytopathic bvdv-1 strain included in the current study, further research is required to determine why this was the case. with respect to thrombocytopenia, only the cattle infected with strain ns155 had a significant loss of platelets that was detected 14 days after infection (figure 7d ). collectively these data suggest the bvdv-1 subgenotypes 1c evaluated in this study have low virulence in transiently infected animals under the experimental conditions utilised. one additional parameter which is commonly considered in the assessment of viral virulence is transmission capacity [14] . there is general agreement that there is either no or limited horizontal transmission of bvdv-1 between transiently infected cattle [15] [16] [17] . sarrazin et al. [18] concluded that the field strains of bvdv-1 subgenotype 1a and 1b evaluated in their study were unlikely to play an important role in transmission. the detection of bvdv-1c in the nasal swabs of infected cattle the current study was sporadic and where detected the results suggested low quantities of virus ( table 1 ). the range of ct values from nasal swabs and serum samples in the current study were 31.6 to 39.0 and 32.0 to 39.0 respectively (table 1) . previous studies have evaluated the use of qpcr to differentiate persistently and transiently infected animals. hanon et al. [19] estimated that a ct value below 28.89 from a blood sample would identify all persistently infected animals, although this value was likely to misclassify some transiently infected animals as being persistently infected. while hay et al. [20] estimated that a ct value below 33 from serum was indicative of an animal being persistently infected with bvdv-1. the results of the current study, suggest a ct value of 33 is too high for the differentiation of transiently and persistently infected animals based on a single sample. noting that both prior studies utilised field samples for these estimates, thus direct comparison to the current study requires caution. the use of these estimates, would also require sample preparation and analyses to be comparable, particularly volume of sample extracted and subsequently used in the qpcr assay. associated with these results, the two uninfected control animals included in the study did not test positive for bvdv-1 or seroconvert to bvdv-1 over the course of the experiment. unchallenged animals could only be included in one of the containment rooms of the study for logistical reasons. consequently, there were no uninfected animals penned with the animals infected with bvdv-1c strain ao554 or strain pi506, the viruses most consistently detected in nasal swabs and in the highest quantities (table 1) . collectively, these results suggest that minimal amounts of the challenge viruses were present in the nasal secretions of the infected animals and as a result the risk of virus transmission to other animals was very low for these bvdv-1c strains. evans et al. [21] recently reported the absence of horizontal transmission from sheep experimentally infected with an australian strain of bvdv-1 subgenotype 1c to sentinel sheep. the possibility of animals transiently infected with the bvdv-1c strains used in these studies producing and shedding sufficient quantities of virus to facilitate transmission if subjected to stressful conditions cannot be excluded. it has recently been demonstrated that the bvdv-1 strain h0916 (subgenotype 1a) was only transmitted to sentinel animals when the infected animals were immunosuppressed with dexamethasone [22] . if the low risk if transmission from transiently infected animals extends to all bvdv-1 subgenotype 1c it could have important implications in the implementation of effective bvdv-1 control plans with persistently infected animals as the sole source of virus [3, [23] [24] [25] . further research is required to determine if any bvdv-1 subgenotype 1c strains replicate sufficiently in the nasal epithelia at levels to facilitate transmission to susceptible sentinel animals. the bvdv-1c strains pi506 and ao554 would be excellent candidate viruses for these studies. while the detection of virus in nasal swab and serum samples was sporadic, the cattle were clearly infected as demonstrated by the serological analyses. infected animals started to seroconvert by day 14 post-infection with 10 of the 22 infected animals testing positive for bvdv-1 specific antibody. the number of positive animals increased by day 21, with all infected animals being antibody positive by day 28. these data are consistent with other bvdv-1 infection studies [17, 26] . there did not appear to be anything specific to the bvdv-1c strains used in this study in relation to the serological data with animals from all groups becoming seropositive in a fourteen day period and all animals being seropositive by day 28. the impacts of some bvdv-1c strains on cattle in the current study may have been under-estimated due to the smaller number of cattle in each group, particularly for strain vr1112 where one animal was withdrawn from the experiment immediately prior to commencement of the trial for ethical reasons (lameness). the number of cattle in this study was constrained by the capacity of the facility and need to evaluate the properties of several bvdv-1 subgenotype 1c strains. another potential limitation of the current study was the viral inoculums used were quantified using rt-qpcr of the final cell culture supernatants. while previous studies have demonstrated correlations between rt-qpcr results and measures of in vitro infectivity such as plaque forming units and/or 50% cell culture infectious dose (tcid50) for other viruses, such relationships have not been reported for bvdv-1 as yet [27] [28] [29] [30] . future studies which aim to directly compare the in vivo properties of the bvdv-1c strains used in the study would need to establish the relationship between rt-qpcr results and measures of in vitro infectivity to enable the standardisation of the challenge doses. future studies will be required to better understand the relationships between the bvdv-1 subgenotypes and virulence. strong et al. [22] also identified that the challenge dose can influence the clinical outcomes of cattle challenged with bvdv-1a. data was also reported which suggested an influence of calf age on clinical outcome. as a consequence, future studies aiming to characterise the interactions of bvdv-1 and its bovine host should aim to do so under a standardised challenge system, including route of infection, challenge dose (where possible multiple doses) and age of animals. it is imperative that the subgenotype of the bvdv-1 isolate(s) used also be included. while the current study has focused on the respiratory component of the bvdv-1c infection, it is well accepted the virus can have profound impacts on the reproductive capacity of individual animals and cattle herds overall. the bvdv-1c isolate trangie was used in several earlier studies that reported the capacity of this virus to significantly impair bovine reproductive function [31] [32] [33] [34] . the capacity of specific bvdv-1 strains and/or subgenotype groups to cause both respiratory and reproductive disease are yet to be investigated, and may be required to fully understand this important cattle pathogen. this study is the first to characterise the in vivo properties of bvdv-1 strains confirmed as belonging to the subgenotype 1c. interestingly, the overall impacts of the infection of the different strains on the infected cattle were in general similar to those reported for other bvdv-1 subgenotypes with transient pyrexia, leukopenia, and quantities of virus in nasal swabs which are unlikely to facilitate horizontal transmission [9] [10] [11] [12] [13] 22] . of the bvdv-1c strains examined in this study pi506 had the most consistent impact on the experimentally infected cattle and is a strong candidate for use in cattle studies to further define the in vivo properties of the subgenotype 1c, including direct comparisons to strains of other subgenotypes. all experimental procedures involving animals were reviewed and approved by the university of queensland animal ethics committee, approval number qaafi/399/13/mla. the bvdv-1c isolates used in the cattle trial are described in table 3 . viral inoculums were prepared by adding 100 µl of primary stock of each bvdv-1c strain to culture medium of subconfluent monolayers of mdbk cells in tissue culture flasks (25 cm 2 ) and incubated at 37 • c in a 5% co 2 atmosphere for seven days. the supernatants were clarified at 5000 g, aliquoted and stored at −80 • c until required. as the aims of this study did not include intergroup statistical comparisons, the viral supernatants were used as harvested to inoculate cattle at the maximum possible titre. cattle were sourced by veterinary health research ltd. pty (armidale, nsw, australia). the animals were black angus and 6 to 9 months of age. prior to enrolment into the study, cattle were tested multiple times and confirmed negative for serological evidence of prior bvdv-1 exposure/infection. elisas were performed using the bio k284 elisa, as described by the manufacturer (bio-x diagnostics, jemelle, belgium). seven days prior to commencement of the trial, cattle (n = 24) were moved into the large animal pc2 facility at the queensland animal science precinct (gatton, qld, australia). the cattle were randomly assigned to one of four pens (4 × 6) with two pens per room ( table 4 ). the rooms are operated independently, including separate air handling systems. to minimise the risk of virus transmission between rooms, separate teams of staff were used to maintain/care for and collect samples from the animals in each room daily. on day 0, the rectal temperature for each animal was recorded and two blood samples collected via the jugular vein. the cattle groups were inoculated with one of the bvdv-1 viral strains as shown in table 4 . briefly, the animal was restrained with the nose elevated and the viral inoculum (1 ml) was slowly dripped into each nostril. the nose was held in this position for 15 to 20 s and the animal then released. two animals in room 2 were not inoculated with virus. clinical assessments: cattle were monitored from day 1 to day 14 post-infection for clinical signs in respect to nasal discharge, coughing, behavior/demeanour and loss of appetite (feed residue). temperature: the rectal temperatures for each animal was recorded from day 1 to day 14. the expected rectal temperature of healthy cattle was 38.5 • c [36] . nasal swabs were collected from day 0 to day 14, day 21, day 28, day 42, day 56 and day 60. nasal swabs were immediately placed on ice for transport back to the laboratory for storage at 4 • c until required. nasal swabs were immersed in 500 µl of pbs containing 5 × antibiotic-antimycotic (thermofisher scientific, waltham, ma, usa) and gently agitated. the swab was subsequently removed and discarded. a 200 µl aliquot of this resuspension was used for total nucleic acid extraction using the dneasy blood & tissue kit (qiagen, hilden, germany) as described by the manufacturer, except for the exclusion of rnase a. total nucleic acid extracts were also prepared from aliquots (200 µl) of cattle serum samples, collected as described below, using the same methodology. sample extracts prepared from nasal swabs and sera were analysed by qpcr for the presence of bvdv-1 rna as previously described [37] . samples yielding a ct value ≥ 40 were deemed to be negative for bvdv-1. an aliquot of the resuspended nasal swab from day 7 post-infection from selected animals were utilised for virus isolation. aliquots, 10 µl and 100 µl of the nasal swab resuspension diluted 1:10 with pbs were added directly to culture medium of subconfluent monolayers of mdbk cells in 6 well plates and incubated at 37 • c in a 5% co 2 atmosphere for seven days. the monolayers were freeze/thawed once and a 100 µl aliquot of the culture supernatant added to new subconfluent monolayers of mdbk cells in 6 well plates and incubated at 37 • c in a 5% co 2 for seven days. this process was repeated three times. total nucleic acids were extracted from a 200 µl aliquot of each culture supernatant and tested using qpcr for the presence of bvdv-1 as described previously. blood sampling: blood for serum harvesting (6 ml bd vacutainers™, bd biosciences, franklin lakes, nj, usa) and blood cell count analyses (6 ml edta bd vacutainers™, bd scientific, franklin lakes, nj, usa) were collected on day 0, day 7, day 14, day 21, day 28, day 42, day 56 and day 60 post infection. serum samples were tested for the presence of bvdv-1 specific antibodies using the bio k284 elisa, as described by the manufacturer (bio-x diagnostics, jemelle, belgium). the level of virus specific igg in each serum sample was assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++) according to the manufacturer's instructions. whole blood samples were submitted to the veterinary science diagnostic services (school of veterinary science, university of queensland, gatton, qld, australia) for analyses of the cell populations using standard blood smearing and cell counting methodologies. data generated from the animal trial were analysed using a one-way analysis of variance (anova) with dunnett's multiple comparisons test and statistical significance attributed where p < 0.05 within graphpad prism™ (version 7.03, graphpad software, inc., la jolla, ca, usa). variability and global distribution of subgenotypes of bovine viral diarrhea virus genetic analysis of bovine viral diarrhoea viruses from australia prevalence and antigenic differences observed between bovine viral diarrhea virus subgenotypes isolated from cattle in australia and feedlots in the southwestern united states bovine viral diarrhea virus (bvdv) 1b: predominant bvdv subtype in calves with respiratory disease eradication of bovine viral diarrhea virus in germany-diversity of subtypes and detection of live-vaccine viruses bovine viral diarrhea virus: global 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dairy cattle early reproductive loss due to bovine pestivirus infection increased reproductive losses in cattle infected with bovine pestivirus around the time of insemination a field investigation of the effects of bovine viral diarrhea virus infection around the time of insemination on the reproductive performance of cattle a single amino acid is critical for the expression of b-cell epitopes on the helicase domain of the pestivirus ns3 protein a manual for the primary animal health care worker; food and agriculture organization (fao multiplex real-time rt-pcr detection of three viruses associated with the bovine respiratory disease complex the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results". key: cord-292970-32mql9nq authors: camdessanche, jean-philippe; morel, jérôme; pozzetto, bruno; paul, stéphane; tholance, yannick; botelho-nevers, elisabeth title: covid-19 may induce guillain-barré syndrome date: 2020-04-15 journal: rev neurol (paris) doi: 10.1016/j.neurol.2020.04.003 sha: doc_id: 292970 cord_uid: 32mql9nq nan a 64-year-old man without medical history was admitted to our hospital after he fell and hurt his left shoulder leading to a tear of the rotator cuff. he had fever and cough for two days. in the context of covid-19 pandemic, sars-cov-2 rt-pcr on nasopharyngeal swab was performed and positive. clinical presentation was moderate with high grade fever for three days requiring oxygen 2-3 l/min through nasal cannula for five days. he received paracetamol, preventing thromboembolism by low molecular weight heparin and lopinavir/ritonavir 400/100 mg twice a day for ten days. thoracic ct scan showed only 10-25% of ground glass opacities. eleven days after the symptom onset, while he did not need oxygen anymore having had no fever for five days, the patient complained of paresthesia in feet and hands. in three days, he covid-19 pandemic is a worldwide disaster. pulmonary disorder and respiratory insufficiency are the main problems linked to sars-cov-2 infection, which explains difficulties in icu to treat numerous patients [2] . recently, zhao et al. questioned the link between covid-19 and gbs [3] . our case is the first gbs with a chronology undoubtedly in favor of a complication of covid-19 infection. this must be known by clinicians as gbs may lead to icu admission and needs to be differentiated from a possible icu-acquired weakness after icu treatments. diagnosis of guillain-barré syndrome and validation of brighton criteria clinical characteristics of coronavirus disease 2019 in china guillain-barré syndrome associated with sars-cov-2 infection: causality or coincidence? key: cord-268081-ytx6sf3x authors: guionie, olivier; courtillon, céline; allee, chantal; maurel, stéphan; queguiner, marilyne; eterradossi, nicolas title: an experimental study of the survival of turkey coronavirus at room temperature and +4°c date: 2013-04-22 journal: avian pathol doi: 10.1080/03079457.2013.779364 sha: doc_id: 268081 cord_uid: ytx6sf3x turkey coronavirus (tcov) is a gammacoronavirus (coronaviridae, nidovirales) responsible for digestive disorders in young turkeys. tcov has been associated with poult enteritis complex, a syndrome that severely affects turkey production. no medical prophylaxis exists to control tcov, therefore sanitary measures such as cleaning and disinfection are essential. it is thus important to evaluate temperatures that allow persistence of tcov in the environment. two series of aliquots of a suspension of a french isolate of tcov (fr tcov) were stored at room temperature or +4°c for 0 to 40 days. as tcov does not grow in cell culture, the presence of residual infectious tcov in the stored samples was tested by inoculating embryonated specific pathogen free turkey eggs. as tcov does not induce lesions in the embryo, virus replication in the jejunum and ileum of the embryos was detected 4 days post inoculation, using rna extraction and a real-time reverse transcriptase-polymerase chain reaction based on the nucleocapsid gene. no surviving virus was detected after 10 days storage at +21.6±1.4°c or after 40 days storage at +4.1±1.6°c, these temperatures being representative of the mean summer and winter temperatures, respectively, in the major french poultry-producing region. the relatively short survival of the virus at room temperature should contribute to limited virus survival during summer months. however, infectious virus was still detected after 20 days storage at the cooler temperatures, a finding that suggests prolonged survival of fr tcov and easier transmission between poultry farms in a cool environment are possible. turkey coronavirus (tcov) belongs to the genus gammacoronavirus in the subfamily coronavirinae, part of the family coronaviridae in the order nidovirales (de groot et al., 2008) . tcov is an enveloped virus with a 28-kb-long single-stranded positive-sense rna genome (cao et al., 2008; gomaa et al., 2008; jackwood et al., 2010) . together with infectious bronchitis virus (ibv), tcov is one of the two most economically significant avian coronaviruses. unlike ibv, however, tcov has not been adapted to grow in cell cultures and can only be propagated in ovo or in vivo. tcov was shown in the 1970s to be one of the causative agents of an enteric disease known as bluecomb, transmissible enteritis or coronaviral enteritis of turkeys (guy, 2008) . more recently, tcov and several other agents (viruses, bacteria, protozoa, fungi) have been associated with poult enteritis complex (pec) (barnes & guy, 2003) . pec is a general term for a group of multifactorial, transmissible infectious diseases of young turkey poults up to 7 weeks of age, with signs including enteritis, retarded development, impaired feed utilization and frequent immune dysfunction (barnes et al., 2000) . pec severely affects turkey production (barnes et al., 2000) . in europe, tcov has been detected in the uk (cavanagh et al., 2001) , and more recently in france (maurel et al., 2009 (maurel et al., , 2010 (maurel et al., , 2011 and poland (domanska-blicharz et al., 2010) . a coronavirus was also detected in turkeys with enteritis in italy (moreno martin et al., 2002) . in france, since 2003, the number of turkey flocks exhibiting clinical signs compatible with pec has increased and pec has become an important concern (germain & rousseau, 2005) . a survey performed from 2007 to 2009 and based on a real-time reverse transcriptase-polymerase chain reaction (rt-pcr) assay specific for the nucleocapsid gene of ibv and tcov (maurel et al., 2009) revealed that 37% of turkey flocks suspected of pec were positive for tcov, whereas only 17% were positive among flocks without enteritis or with an enteritis not evocative of poult enteritis (pec)/ mortality syndrome (pems). the virus was found predominantly in the digestive contents (41% in the jejunum, 39% in the caecum) (maurel et al., 2011) . the genetic characterization of french strains of tcov (fr tcov) demonstrated that although they are related to their north american (na tcov) counterparts, they form an independent sublineage and might have been generated by recombination events involving different ibv strains than those involved in the emergence of na tcov (jackwood et al., 2010; maurel et al., 2011) . interestingly, the ibvs most related to fr tcov appeared to be 4/91 and qx (ita/90254/2005), respectively, which represent some of the more recently emerged ibv serotypes in europe (worthington et al., 2008) . to date, no medical prophylaxis exists to control tcov infection. it is therefore essential to control this virus by sanitary measures, such as cleaning and disinfection. in this respect, evaluating the survival of fr tcov at room temperature or lower is an important step. intra-amniotic route of inoculation for embryonated turkey eggs. eighteen-day-old to 21-day-old embryonated specific pathogen free (spf) turkey eggs were candled to ensure embryo viability. they were placed vertically with the air chamber uppermost. the top of the shell was disinfected with iodine and alcohol, and a small hole was drilled at the centre of the air cell. a 21-gauge (0.8 mm), 1.5-inch (40 mm) needle mounted onto a 1 ml syringe held vertically was slowly but completely inserted into the hole and 0.1 ml inoculum was injected. the needle was removed slowly along the same vertical axis and the hole was sealed with melted wax. the eggs were returned to the incubator at '378c, where they were kept vertically with the air chamber uppermost until 4 days post inoculation. virus stock. the 080385d virus used in this study was isolated in november 2008, from the duodenal contents of 42-day-old turkeys affected by pec. a search for astrovirus, reovirus, adenovirus, and serial passage in chicken hepatocyte cultures failed to detect any virus other than fr tcov. the virus was propagated serially five times in embryonated spf turkey eggs (anses, ploufragan, france), inoculated as described above. as fr tcov does not induce embryo lesions, the jejunum and ileum of the inoculated embryos were harvested 3 to 6 days post inoculation (depending on the age of the inoculated embryos). the collected material was diluted w/v in phosphate-buffered saline at ' 48c, then ground with a thurrax blender (ika, staufen, germany). the suspension was centrifuged at 3000 )g for 10 min and the supernatant centrifuged a second time at 3000 )g for 5 min. the collected supernatant was considered the virus stock. virus titration in specific pathogen free turkey eggs. the virus was serially diluted in a stabilizing solution (phosphate buffer 'sucrose 20 g/l) supplemented with 0.2% of a penicillin (100,000 u/ml)á streptomycin (100 mg/ml) antibiotic solution and 0.4% of fungizon preparation (0.5 mg/ml), from 10 (1 to 10 (5 or from 4 (1 to 4 (5 when high or low virus titres were expected, respectively. each dilution was inoculated in embryonated spf turkey eggs as described above (four to eight eggs per dilution). replication of the virus in each inoculated embryo was assessed by harvesting the jejunum and ileum followed by the molecular detection of the tcov genome (see below). titres were calculated using the reed and muench method for the determination of 50% endpoints (reed & muench, 1938) and were expressed as median infectious dose per millilitre of virus (eid 50 /ml). molecular detection of the tcov genome. three freezeáthaw cycles were performed to increase the lysis of the tissues and possibly maximize the extraction of tcov genetic material. then 140 ml phosphate-buffer saline solution was added to the tissues. seventy microlitres of this mixture were mixed with 330 ml rlt lysis buffer (qiagen, courtaboeuf, france) and incubated at room temperature for 15 min to inactivate the virus. rna was extracted from the resulting 400 ml using the magattract rna tissue mini m48 kit for automated extraction and the m48 biorobot (qiagen). the presence of tcov genome was assessed using a real-time rt-pcr based on a conserved part of the nucleocapsid gene of avian coronaviruses (primers and protocol according to maurel et al., 2011) . in this real-time rt-pcr, a cycle threshold (ct) higher than 35 was considered non-specific (maurel et al., 2011) . with such a protocol, embryos containing infectious fr tcov typically produced values ranging from 23 to 35 ct. virus incubation at room temperature and 48c. fr tcov was diluted to obtain an initial titre of 3.2 log 10 eid 50 /ml. to mimic the presence of organic material and possibly promote virus stability, the dilution medium (memh) was supplemented with 5% foetal calf serum. aliquots of the initial virus suspension (400 ml/tube) were placed in an isothermal box containing a thermometer at room temperature. duplicate tubes were removed from the box and stored at (708c after 0, 2, 5, 7, 10, 14, 21, 28, 35 and 42 days storage at room temperature. another series of aliquots was placed in a cold room at '48c, also containing a thermometer. duplicate tubes were removed from this second series and were frozen at (708c after 5, 10, 15, 20 and 40 days of exposure. the temperatures of both rooms were registered daily. assessing the stability of fr tcov rna at room temperature. the first duplicates, harvested after 0 and 42 days of storage at room temperature, and 40 days of storage at '48c, were directly submitted to real-time rt-pcr as described above, prior to egg inoculation. assessing fr tcov persistence in samples stored for different durations. the first duplicates harvested after different times of storage at the different temperatures were used to test for the presence of residual infectious virus in the sample by inoculating spf embryonated turkey eggs as described above. whenever the first duplicate was shown to still contain live fr tcov, the second duplicate was used to titrate the virus as described above. optimization of the intra-amniotic route of inoculation for embryonated turkey eggs. to achieve reliable virus delivery into the desired embryonic cavity, some efforts were made to optimize inoculation via the intra-amniotic route into embryonated spf turkey eggs. using this methodology, the proper targeting of the inoculum was checked by inoculating 1% methylene blue dye into 20day-old embryonated spf turkey eggs, then opening the eggs to check the location of the dye deposit. out of 40 eggs inoculated by four people, 38 were suitable for analysis of the inoculation point. inoculation proved to have been performed into the amniotic sac, yolk sac or allantoic fluid in 34 (89%), three (8%) and one (3%) of the inoculated eggs, respectively. the comparison of the results by the different manipulators showed that the percentage success ranged from 6/9 (66%) for one person to 10/10 (100%) for two others. only those who succeeded best in the intra-amniotic inoculation test inoculated eggs in the subsequent experiments. validation of the method for the detection of residual live virus after storage. the tcov rna signal from the initial virus suspension kept for 0 or 42 days at room temperature was 2794 ct. this demonstrated that the amount of viral rna in the inoculum was not affected by the duration of storage. however, when the same samples were inoculated into embryonated spf turkey eggs, viral rna was detected in the digestive tracts of the inoculated embryos after 0 days of storage (27.5 ct), but not after 42 or 40 days ( 40 ct) at room temperature or '48c, respectively. the results observed at 42 and 40 days strongly suggested that the starting inoculum was so diluted when inoculated into the embryonated eggs that the viral genome could not be detected, unless tcov replication occurred in the inoculated eggs, as was observed after 0 days of storage. consequently, in the subsequent experiments, a positive real-time rt-pcr signal from the digestive tract of inoculated embryos was interpreted as the presence of infectious virus, not as residual non-infectious rna derived from the inoculum. detection of residual infectious virus after different storage times. the mean room temperature was '21.68c (minimum: '19.78c; maximum: '25.38c; standard deviation: 1.48c). after 2 and 5 days of storage, all inoculated eggs were positive (ct 026.6). after 7 days, only one out of four amplified the virus (ct 0 29.1). from 10 days of storage onwards, none of the inoculated eggs allowed detection of any virus replication (table 1) . the mean refrigeration temperature was '4.18c (minimum: (0.28c; maximum: '9.98c; standard deviation: 1.68c). after 5 days of exposure, all inoculated eggs were positive (ct 029.0). after 10 or 15 days, three out of four had supported virus replication (ct 030.4). after 20 days, two out of three inoculated eggs were still positive (ct 030.8), whereas no virus was detected after 40 days of storage (table 1) . quantitative decrease in virus titre during storage. the virus titre measured after 0 days of storage was 2.8 log 10 eid 50 /ml, which was consistent with the initial titre of the virus stock (3.2 log 10 eid 50 /ml), considering the possible effect of one freeze (thaw cycle (titres were calculated after the aliquots had been stored at (708c). after storage at room temperature, the titres of the residual virus at days 2 and 5 were similar, and lower than or equal to 1.5 log 10 eid 50 /ml (decrease of at least 1.3 log 10 eid 50 /ml). the decrease continued at 7 days, with a titre lower than or equal to 0.7 eid 50 /ml (decrease of at least 2.1 log 10 eid 50 /ml). finally, no virus titre could be detected at 10 days (figure 1) . after refrigerated storage, the titre of the residual virus proved stable from 0 to 5 days (2.8 log 10 eid 50 /ml) and a virus titre of 2.3 log 10 eid 50 /ml (decrease of 0.5 log 10 eid 50 /ml) was still detected after 20 days of storage. no surviving virus was detected after 40 days of storage at '48c ( figure 1 ). the protocol used here was developed to try to simulate the survival of fr tcov in the environment at '208c and '48c. these temperatures were selected as representative of the mean summer ('16.6 to '19.28c) and winter ('5.9 to '6.28c) temperatures in brittany, the largest poultry-producing area in france, over the 1981 to 2010 period (http://www.meteo-bretagne.fr/normalesclimatiques-rennes). indeed, in this region and during this period, the mean number of days above '258c or below 08c did not exceed 39.6 and 33.5 days, respectively. in addition, the '48c temperature is likely to be comparable with temperature during refrigerated shipment of unfrozen biological samples. however, our study does not mimic the survival of virus in litter or dried faeces, and so forth, where relative humidity is different. the use of a virus suspension, instead of dried viruses on surfaces, decreased the risk of titre variation, simplified the study, and gave consistent data. this simplified protocol was all the more interesting as this virus does not grow in cell culture, requiring a multi-step procedure for titration of the virus, comprising embryonated spf turkey eggs, embryo dissection and real-time rt-pcr. another significant improvement would have been to start with a virus titre initially higher, in order to obtain values of decrease in virus titre on a larger scale. however, this was not possible because titres obtained when propagating fr tcov in turkey eggs currently did not exceed 4 log 10 eid 50 /ml, a titre that could not be increased either by inoculating the embryonated spf nd, not done. a cycle threshold determined using a real-time rt-pcr specific for avian coronaviruses (maurel et al., 2011) . turkey eggs at a younger age or by increasing the passage level of fr tcov in eggs (data not shown). our data suggest that fr tcov in suspension survives for less than 10 days at room temperature and for 20 to 40 days at '48c. the virus titre loss was only 0.5 log 10 eid 50 /ml after 20 days at '48c, which strongly suggests that the virus might survive much longer than 20 days. however, survival between 20 and 40 days at '48c was not evaluated. coronaviruses, being enveloped viruses, are expected to be less stable in the environment than non-enveloped viruses. data available for the severe acute respiratory syndrome (sars) coronavirus infecting humans are in the same range as reported here for fr tcov. sars-cov dried on plastic retained its infectivity for as long as 6 days (loss of 3 log 10 tcid 50 /ml) at room temperature ('21 to '258c), the total loss of the initial 7 log 10 tcid 50 /ml being obtained after 9 days (rabenau et al., 2004) . this stability is much higher than previously described for human coronavirus hcov-229e, which completely lost its infectivity within 72 h under the same conditions. another study using a suspension of sars-cov showed a loss of 3.7 log 10 tcid 50 /ml after 7 days and 5.7 log 10 tcid 50 /ml after 13 days, at '22/ '258c (chan et al., 2011) . the fr tcov titre reduction was less in the first days (2.1 log 10 eid 50 /ml after 7 days) than that previously described for a suspension of sars-cov (chan et al., 2011) but fr tcov could not be detected at 10 days. the effect of temperature was recently studied in two animal coronaviruses: transmissible gastroenteritis virus (tgev), an enteric pathogen of swine; and mouse hepatitis virus (mhv), a respiratory and enteric pathogen of laboratory mice (casanova et al., 2010) . this study investigated viral inocula placed on stainless steel surfaces, then in a controlled relative humidity environment (20% and 80% of relative humidity). our data, being obtained from viruses in suspension, most closely compare with data obtained under 80% relative humidity. at '208c, after 7 days, the titre decrease was 1.7 log 10 for tgev and 4 log 10 for mhv. after 10 days the titre decrease was 2 log 10 for tgev and at least 5 log 10 for mhv, indicating that infectious mhv was no longer detected. compared with our data, the titre decrease was higher with mhv than fr tcov, but similar between tgev and fr tcov, an interesting finding considering that both viruses are enteric pathogens. at '48c mhv and tgev persistence was longer, showing a titre decline after 28 days of 3.2 and 2.5 log 10 for tgev and mhv, respectively. the fr tcov titre in suspension at '48c was more stable (decrease of only 0.5 log 10 after 20 days). as far as avian viruses are concerned, it is difficult to compare the temperature sensitivity of fr tcov with that of ibv, because very limited published information is available regarding the latter. an early study of ibv survival in lyophilized preparations (hofstad & yoder, 1963) can hardly be compared with the present study due to the presentation of the samples being so different. lyophilized ibv strains 33 and 97 both exhibited a similar decay in titre (about 2.3 log 10 eid 50 /ml) when stored for 30 days at '378c, but no loss in virus titre was measured when the viruses were stored at '48c for the same time. this is consistent with a greater thermal stability of avian coronaviruses at lower temperatures. in another study dating back to the same period, the presence of salts in solution was shown to stabilize the titre of the ibv-42 (beaudette) strain at '258c. indeed, the virus titre strongly decreased by 3.5 log 10 eid 50 /ml when incubated for only 15 minutes at '258c, whereas it was stable in salt solution (hopkins, 1967) . clearly, more recent studies on the stability of ibv in the environment are required if comparisons are to be made between tcov and ibv. in general, the data available regarding animal coronaviruses are in the same range. virus survival ranges from 1 to 2 weeks at '208c, whereas survival is longer than 20 days at '48c. the longer survival of tcov at cooler temperatures could be a factor allowing an increased persistence or transmission of the virus during the cooler seasons of the year. indeed, the strain used in this study (080385d) was isolated from a diseased flock in november, usually a rather cold and humid period in western france. however, this may not reflect the seasonality of enteric disorders in france. in their survey of 81 french turkey flocks, performed during the period 2007 to 2009, maurel et al. (2009) detected 24 flocks positive for fr tcov. fifteen of these flocks (63%) were detected during warm periods (springásummer), as compared with only nine flocks (37%) in autumn and winter. although this survey was not designed to precisely assess seasonality of fr tcov infections, the annual distribution of pec reports to the french nationwide network for epidemiological observations in poultry (rnoea) (souillard et al., 2007) confirms that the proportion of pec cases declared from 2007 to 2011 was higher during the warmer periods (77% of the total declared pec cases) than during the colder ones (23% of the total reported cases) (toux & souillard, personal communication, 2011) . the period of highest pec incidence hence appears not to be the most favourable for fr tcov survival in the environment. however, it should be remembered that pec is a multifactorial syndrome, so that the reported pec cases may be caused by a variety of agents (fr tcov, astroviruses, reoviruses, adenoviruses, bacteria, etc.), some of which may account for additional summer cases. furthermore, the survival of fr tcov is only one possible factor that might contribute to the seasonality of the disease, and other possibly more significant interfering factors, such as possible vectors or seasonality of turkey production, might also play a role, so that fr tcov virus survival according to the outdoor temperature might not be the most important parameter for its transmission between turkey flocks. based on the data of the present study, it can be concluded that a fr tcov strain survives for less than 10 days in a liquid culture medium kept at room temperature, but for more than 20 days at '48c. interesting future work would be to assess virus survival: at the higher temperatures that are reached during steam cleaning on the surfaces of farm equipment; at '108c (the temperature for pre-licensing activity testing of agricultural disinfectants); and at (58c, the usual temperature during shipment of frozen samples to diagnostic laboratories. the results of such studies might help understanding of tcov epidemiology and ensure efficient shipment of samples for virus isolation. temperature effect on turkey coronavirus 251 poult enteritis-mortality syndrome poult enteritis complex. revue scientifique et technique (international office of epizootics) complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus effects of air temperature and relative humidity on coronavirus survival on surfaces detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens the effects of temperature and relative humidity on the viability of the sars coronavirus taxonomic proposal to the ictv executive committee: revision of the family coronaviridae experimental infection with turkey coronavirus isolate enteritis, cold sensitivity and growth depression syndrome in turkeys: a field case study in france in 2004 complete genomic sequence of turkey coronavirus turkey coronavirus enteritis inactivation rates of some lyophilized poultry viruses at 37 and 38c thermal stability of infectious bronchitis virus in the presence of salt solutions emergence of a group 3 coronavirus through recombination. virology first full-length sequences of the s gene of european isolates reveal further diversity among turkey coronaviruses molecular identification and characterization of a turkey coronavirus in france viruses associated with poult enteritis complex in france diagnosis of turkey viral enteritic diseases by electron microscopy and identification of coronavirus in a case of turkey enteritis stability and inactivation of sars coronavirus a simple method of estimating 50 per cent end points rnoea: french nationwide network for epidemiological observations in poultry a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from the authors acknowledge the financial support of conseil général des cô tes d'armor (cg22), conseil régional de bretagne, conseil régional des pays de loire, franceagrimer, comité interprofessionnel de la dinde française (cidef), and the help of pô le agronomique de l'ouest for project coordination. key: cord-280242-2w2kl0uf authors: kaya, yildiz; kara, simay; akinci, canan; kocaman, ayse sagduyu title: transient cortical blindness in covid-19 pneumonia; a pres-like syndrome: a case report date: 2020-04-28 journal: j neurol sci doi: 10.1016/j.jns.2020.116858 sha: doc_id: 280242 cord_uid: 2w2kl0uf nan the world health organization declared the outbreak of the 2019 novel coronavirus, in march 12th, 2020, a global pandemic after widely spreading of the epidemic covid-19 pneumonia cases 1. it has been reported that, in addition to the respiratory tract infection symptoms, patients can also have neurologic signs and symptoms; like acute cerebrovascular disease, polyneuritis, encephalitis and encephalopathy 2. in this report, we describe a patient who developed bilateral reversible cortical blindness, who presented by covid-19 related pneumonia. a 38 years old male patient, admitted to emergency department with a history of fever for 5 days. his body temperature was 38,5 °c, blood pressure was 130/80 mmhg and oxygen saturation 98% while he was breathing ambient air. breath sounds were normal with adventitious sounds on both sides. his chest ct scan showed multiple, multilobar, peripheral ground-glass opasifications in both lungs ( figure 1 ). laboratory tests results showed highly elevated crp and ferritin levels with marked lymphop enia. his nasopharyngeal swab reverse transcription-pcr (rt-pcr) was positive for sars-cov-2. after admission, he received hydroxychloroquine (400 mg for the first day, 200 mg/day for four days), azitromicin 500 mg/day, and osetalmivir 150 mg/day combined with nasal oxygen therapy. his oxygen saturation was declined to 88% on the second day and noninvasive mechanical ventilatory support was started at intensive care unit (icu). on the fifth day of icu, he suddenly developed acute confusional state with agitation and his blood pressure observed to be at high levels for a few hours. meanwhile, the patient complained about vision loss in both eyes. in his neurological examination he was awake, but apathic and hardly obeying commands. his pupils were 2 mm and equally reactive to light. fundus examination was normal. his visual acuity was severely impared on both eyes; he could only recognize waving hands and there was perception of light. his entire neurological examination was normal. brain magnetic resonance imaging (mri) showed bilateral, especially left occipital, frontal cortical white matter and splenium of corpus callosum t2/fluid-attenuated inversion recovery (flair) hyperintensities and diffusion restriction in diffusion weighted imaging (dwi) (figure 2 ) revealing vasogenic edema similar to posterior reversible leucoencephalopathy (pres). hydroxychloroquine treatment was stopped and the dexamethasone with a 24mg/day dose is started. on second dose of corticosteroid treatment, patient was able to obey commands and his visual impairment fully recovered. in his neurocognitive assessment we determined visual agnosia which lasted in a week. the corticosteroid therapy tapered and stopped in two weeks' time. his neurological examination and neurocognitive assessment were completely normal on the tenth day. the brain mri performed two weeks later, showed complete regression of the lesions (figure 3 ). we still don't know why focal neurological deficits may arise during sars-cov-2 infection. common suggestions for pathological mechanisms are direct virus infection invasion or inflamatory factors. recent autopsy reports have revealed that, like many viral infections sars-cov-2 can cause brain tissue edema and partial neuronal degeneration [3] . infectious toxic encephalopathy is a reversible brain dysfunction syndrome caused by systemic toxemia, metabolic disorders and hypoxia during the process of acute infection [4] . in this disease, main pathological change is brain edema without evidence of inflammation on cerebrospinal fluid analysis. hypoxia in the brain causes anaerobic metabolism in the mitochondria of neurons and this leads cerebral vasodilatation, swelling of neurons, interstitial edema and obstruction of cerebral blood flow [5] . pres is a result of a systemic inflammatory state causing endothelial dysfunction [6] . that hypothesis is supported by the observation that pres is usually associated with a systemic inflammatory process such as sepsis, eclampsia, transplantation, and autoimmune disease [7] . although we could not determine the exact ethiology in our case, regulating the blood pressure controlling the vasogenic edema by corticosteroid treatment and controlling the virus related pneumonia have helped fort he recovery of our patient. unfortunately, evidences are lacking to determine which of these features were due to infectious toxic encephalopathy, and which features were specific to sars-cov-2 infection. the authors declare that there are no competing interests associated with the manuscript nervous system involvement after infection with covi̇d-19 and other coronaviruses pathological findings of covi̇d-19 associated with acute respiratory distress syndrome encephalopathy of infection and systemic inflammation interaction brain-lungs posterior reversible encephalopathy syndrome, part 2: controversies surrounding pathophysiology of vasogenic edema posterior reversible encephalopathy syndrome key: cord-285096-g9y3au1a authors: mitchell, judy a.; brooks, harriet w.; szladovits, balázs; erles, kerstin; gibbons, rachel; shields, shelly; brownlie, joe title: tropism and pathological findings associated with canine respiratory coronavirus (crcov) date: 2013-03-23 journal: vet microbiol doi: 10.1016/j.vetmic.2012.11.025 sha: doc_id: 285096 cord_uid: g9y3au1a canine infectious respiratory disease (cird) occurs frequently in densely housed dog populations. one of the common pathogens involved is canine respiratory coronavirus (crcov), however little is known regarding its pathogenesis and the role it plays in the development of cird. the pathogenesis of five geographically unrelated canine respiratory coronavirus (crcov) isolates was investigated. following experimental infection in dogs, all five crcov isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. the presence of crcov was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. the successful re-isolation of crcov from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of crcov for respiratory tissues and fulfils the final requirement for koch's postulates. by study day 14 dogs had seroconverted to crcov and the antibodies raised were neutralising against both homologous and heterologous strains of crcov in vitro, thus demonstrating antigenic homogeneity among crcov strains from the two continents. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. here we have successfully developed a model for studying the pathogenicity and the role of crcov in cird. tropism and pathological findings associated with canine respiratory coronavirus (crcov) a b s t r a c t canine infectious respiratory disease (cird) occurs frequently in densely housed dog populations. one of the common pathogens involved is canine respiratory coronavirus (crcov), however little is known regarding its pathogenesis and the role it plays in the development of cird. the pathogenesis of five geographically unrelated canine respiratory coronavirus (crcov) isolates was investigated. following experimental infection in dogs, all five crcov isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. the presence of crcov was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. the successful re-isolation of crcov from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of crcov for respiratory tissues and fulfils the final requirement for koch's postulates. by study day 14 dogs had seroconverted to crcov and the antibodies raised were neutralising against both homologous and heterologous strains of crcov in vitro, thus demonstrating antigenic homogeneity among crcov strains from the two continents. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. here we have successfully developed a model for studying the pathogenicity and the role of crcov in cird. ß 2012 elsevier b.v. all rights reserved. since its initial discovery in 2003 (erles et al., 2003) ; canine respiratory coronavirus (crcov) is now considered to be a significant cird pathogen, most frequently detected in dogs with mild respiratory clinical signs during the early stages of cird onset (erles et al., 2003) . although crcov has been found worldwide (decaro et al., 2007; erles and brownlie, 2008; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 yachi and mochizuki, 2006; knesl et al., 2009) ; little is known regarding its pathogenesis, tissue tropism or virulence differences among global isolates in the canine host. it is postulated that crcov plays an important role during the early stages of cird by predisposing the dog to more severe clinical disease from secondary infections. through the use of an in vitro tracheal explant culture system, a moderate reduction in ciliary function and a down-regulation of pro-inflammatory cytokine mrna levels (tnf-a, il-6 and the chemokine il-8) was observed in response to crcov exposure (priestnall et al., 2009) . such alterations in the mucociliary and innate immune systems could be linked to increased susceptibility to secondary infection and is consistent with the proposed role for crcov in cird. however, the limitation of this in vitro model precludes the understanding of the clinical relevance and pathogenesis of a crcov infection in the dog. furthermore, given the global presence of this virus, insight into crcov pathogenesis among isolates originating from geographically distinct locations would be valuable to determine the need for a global vaccine. recently our group has collected findings from a preliminary in vivo challenge study of crcov. in that study we demonstrated that young dogs were susceptible to experimental infection with both crcov isolates, which gave rise to clinical signs of respiratory disease consistent with naturally occurring infection. crcov was detected in the oropharynx of infected dogs and spread rapidly to sentinel dogs which also displayed clinical signs of disease (mitchell et al., unpublished data) . here we extend this study to gain a better understanding of crcov pathogenesis in vivo. analyses specifically focused on the histopathological changes in the canine upper and lower respiratory tissues, virulence differences among crcov isolates derived from cird cases representing wide geographical locations; uk and usa [mo, ne, ut and mi] , and the demonstration of koch's postulates. the information obtained from this study vastly enhances our understanding of crcov pathogenicity and its involvement in the cird complex. five crcov isolates originating from geographically distinct regions of the uk and usa were used in this study (table 1) . crcov isolates uk 4182, np631, np787, and np742 were propagated in culture on human rectal tumour cells (hrt-18g; american type culture collection cell line, manassas, va, usa), and used at 10 6 tcid 50 /ml for intranasal challenge. crcov lu298 was not was not propagated or expanded in vitro, instead 0.2 mm filtered viral fluids obtained from the tissues of a crcov infected dog in the usa were used to challenge animals in to1. prior to challenge each of the crcov virus and control hrt-18g cell culture fluids were satisfactorily tested for sterility and canine extraneous agents (including canine distemper virus, measles, canine adenovirus-2, canine parainfluenza virus, canine rotavirus, rabies, canine parvovirus and canine enteric coronavirus). thirty-six, 12-16-week old specific pathogen free (spf) purpose bred beagle dogs were used in this study. all dogs were demonstrated as crcov negative and seronegative for crcov and b. bronchiseptica prior to the study. dogs were housed in temperature controlled isolation rooms with dedicated shower in and out procedures, disinfection and sterilisation of all items prior to entry and a pasteurised diet were used to maintain bio-security. colony dogs are screened quarterly to determine the spf status. throughout the study dogs from the same treatment group were housed in pairs to minimise stress. dogs were randomly divided into six treatment groups (t1-t6) (table 1) , each consisting of six dogs. dogs in groups t1-t5 inclusive were challenged with the crcov isolates as described in table 1 . dogs in t6 were mock challenged with uninfected hrt-18g cell culture supernatant to serve as negative controls. intranasal inoculations took place over two consecutive days (study day zero and one). on each challenge day dogs were sedated prior to intranasal administration with 1.0 ml of virus or control material (0.5 ml/nostril). dogs were monitored throughout the trial for clinical signs of disease. on study days 3, 6, and 14, as detailed in table 1 , dogs were humanely euthanatized within each treatment group as determined by randomisation completed prior to the start of the trial, and necropsies were performed immediately. gross pathological changes were documented throughout each necropsy and the lungs of each animal were photographed. all experiments involving animals were carried out at a contract research organisation, in compliance with national legislation, and subject to local ethical review. general health observations were performed on each dog, twice daily, and scored for general appearance, breathing, sneezing, coughing, and ocular and nasal discharge as detailed in table 2 . body temperatures were recorded twice daily via implanted microchips, and dogs were weighed on study days à7, à1 and on the day of euthanasia. appetite was recorded according to the quantity of food eaten per room. oropharyngeal viral swabs for rt-pcr analysis (sterilin, uk) and virus isolation (dacron swabs, puritan, in 3 ml virus transport medium (pah)) were collected on study days à1 (pre challenge) 2, 3, 4, 5, 6, 8, 10, 12, and 14 from all dogs on the study. rectal swabs were collected from each dog at necropsy. after sampling, viral swabs were frozen and stored at à70 8c until processed. swab tips for rt-pcr analyses were immersed in 1 ml of rpmi medium (sigma, dorset, uk) and mixed prior to analysis. samples from the following tissues were harvested at necropsy and stored at either à70 8c or fixed in 10% buffered formalin: nasal cavity (included ciliated area), nasal tonsil, palatine tonsil, trachea, apical lung lobe, diaphragmatic lung lobe, and bronchial lymph node. each tissue sample was taken using a new set of sterile instruments. lung lavage fluid samples were collected in dmem cell culture medium and stored at à70 8c. formalin-fixed tissues were processed and stained for histology and were examined by a veterinary pathologist as described in section 2.9. on study days à1, 3, 6, and 14, serum and edta whole blood samples were collected for serological and haematological analysis as described below. swabs and tissue samples were tested for the presence of crcov by rt-pcr and virus isolation. prior to inoculation on to hrt-18 g cells, swab and lung lavage samples were clarified by centrifugation, and filtered (0.2 mm filter). similarly, approximately 1 g of each tissue sample was homogenised in 3.0 ml of virus transport medium (vtm), clarified by centrifugation and filtered (0.2 mm filter). filtered samples were inoculated onto confluent t25 or t150 monolayers of hrt-18 g cells. inoculated cultures were maintained in dmem supplemented with final concentrations of 200 mm l-glutamine, 22.5 mg/ml gentamicin, and 1% fbs. inoculation media was also supplemented with trypsin. trypsin concentrations were determined for each batch (circa 1 mg/ml) based of cell toxicity. cell culture fluids were sampled weekly for 2 weeks to test for the presence of crcov by immunofluorescence assay (ifa) (section 2.5.3). rna was extracted using the rneasy mini kit (qiagen, crawley, uk) as recommended by the manufacturer from 200 ml of the swab fluid or a 0.5 cm 2 piece of tissue. rna was transcribed into cdna using random hexameres (ge healthcare, little chalfont, uk) and improm ii reverse transcriptase (promega, southampton, uk) according to the manufacturer's protocol. all samples were tested for the presence of the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) by pcr as described previously (grone et al., 1996) . results were expressed as a positive or negative outcome to ensure successful nucleic acid extraction. samples were analysed for crcov using the nested spike gene pcr as described previously (erles et al., 2003) . formalin-fixed tissues were processed for histology and sections were stained using haematoxylin and eosin. the histological sections were examined by a veterinary pathologist who was blinded to the study groups. each tissue was ascribed a histological and cilia score as described in table 3 . a single overall histological ''weighted'' score was assigned to each dog, in which scores assigned to the lower respiratory tract (trachea, lungs, bronchial lymph nodes) were considered more noteworthy than those in the upper respiratory tract (nares, tonsils). this weighting of the overall score was used to take into account that histological changes (especially inflammatory reactions or lymphoid hyperplasia) in the upper respiratory tract are not uncommon, especially in young animals, and unless considerable, are considered part of the normal defences of the upper respiratory tract. conversely, such changes in the lower respiratory tract are more likely to be clinically significant. thus moderate to marked lymphoid hyperplasia or neutrophilic inflammation of the palatine tonsil (score 4) would have less effect on the overall histology score for that individual dog than would a similar grading of lymphoplasmacytic or neutrophilic aggregation in the tracheal mucosa or lungs. paraffin-embedded formalin-fixed tissues (4 mm) were mounted on superfrost plus microscope slides (menzel-glä ser, braunschweig, germany). slides were heated at 60 8c for 1 h, deparaffinised and dehydrated. endogenous peroxidase was blocked with 3% h 2 o 2 for 10 min then slides were washed in dh 2 o, then incubated in prewarmed (37 8c) protease xiv 0.05% (sigma) in tbs for 15 min. slides were rinsed in dh 2 o then incubated with blocking serum (2% normal goat serum [vector laboratories, peterborough] in tbs). staining for crcov was performed as described previously (priestnall et al., 2009) . positive cells were identified microscopically by the presence of staining. haematological analysis of edta blood samples was performed using a cell-dyn 3500 automated haematology machine. a 100 cell differential count of the white blood cells was performed on blood smears stained with a hematek 1 automated stainer using modified wright's stain, by a board certified veterinary clinical pathologist blinded to the treatment groupings. the results for day à1 were analysed for normality and reference intervals were estimated (n = 36). total neutrophil, band neutrophil, lymphocyte and monocyte concentrations were calculated using the automated total wbc concentration, and the observed percentages of each leucocyte type determined by the differential count. the results within each treatment group for days 3, 6 and 14 were compared with each other and with those at day à1 to evaluate trends or significant differences. the paired t-test or wilcoxon signed ranks test with repeat measures were used if distribution was normal or non-normal, respectively. statistical significance was set at p = 0.05. hrt-18g cells were cultured to confluency in 96 well plates. monolayers were inoculated with a usa crcov isolate, incubated for 5 days, and fixed with 80% acetone. canine serum samples were diluted 1:40 in pbs supplemented with 1% bovine serum albumin (w/v) (steris corporation, mentor, oh, usa) and 0.09% sodium azide (mallinkrodt chemicals, hazelwood, mo, usa) followed by two-fold serial dilutions to 1:1280. diluted serum was dispensed at 100 ml/well onto the fixed cells, incubated for 1 h, and then washed twice with water. bound antibody was detected with 50 ml/well of fluorescein isothiocyanate-labelled secondary antibody (rabbit anti-dog igg) (sigma-aldrich, jerusalem, israel) diluted 1:250 in pbs supplemented with 1% bovine serum albumin (w/v) and 0.09% sodium azide (w/v), incubated and washed as before. endpoint crcov titres were observed using fluorescence microscopy and defined as the inverse of the last dilution of serum exhibiting definite crcov fluorescence. in instances where no virus-specific fluorescence at the 1:40 dilution was observed, dogs were considered seronegative or non-exposed to crcov. dog sera collected on study day 14 were tested for serum neutralising antibody titres against 1 uk, and 10 usa crcov isolates. hrt-18g cells were grown to confluency in a 96 well plate. cells were rinsed once in serum-free dmem and then pre-treated for 1 hour with 100 ml/well of serum-free dmem containing c1 mg/ ml trypsin. heat inactivated serum samples were diluted 2-fold, 1:10 through 1:1280, in dmem containing 1% fbs. a further 1:2 dilution of the serum was made in 50-300 tcid 50 of virus per 100 ml, to obtain final serum dilutions of 1:20-1:2560. virus:serum mixtures were incubated for 1 h at room temperature. cells were inoculated in quadruplicate with 200 ml/well of the virus: serum mixture, incubated for 5-7 days, and fixed with 80% acetone. virus growth was detected by ifa as described previously (section 2.5.3). the fifty percent neutralisation endpoint for each serum sample was calculated by the statistical methods of spearman-karber. oropharyngeal amies swabs (sterilin, uk) and lung lavage fluid samples were collected from all dogs at necropsy in order to screen for the presence of bacterial pathogens. briefly each swab was plated onto 1â chocolate agar, 1â macconkey agar, and 2â blood agar (1â aerobic and 1â anaerobic) and submitted to the clinical services division at the royal veterinary college for analysis. samples were also plated onto mycoplasma experience agar (mycoplasma experience ltd. surrey, uk) and incubated at 37 8c with 5% co 2 for 3 days. dogs from all six treatment groups were healthy prior to challenge. dogs in t6 (negative control) remained healthy for the duration of the study, the only exception being one dog which had some serous ocular discharge prior to challenge, and throughout the study. all 36 dogs completed the study on the pre-assigned day. following challenge, a number of dogs from t1 to t5 inclusive displayed mild clinical signs of respiratory disease which included nasal discharge, sneezing and coughing. table 4 shows the total scores for each observation by treatment group. there were no significant changes in body temperature all dogs had normal or fair appetites and gained weight over the course of the study (data not shown). no consistent differences between dogs treated with different strains of the virus were observed, although respiratory signs were recorded most frequently and most severely in one dog from t3 and one from t5 (table 4 ). it is worth noting that in both instances these dogs were euthanized on study day 14 and displayed the clinical signs of respiratory disease recorded throughout the 14-day study period, although the scores were highest in these two dogs on study days 10-13. oropharyngeal swabs were analysed for crcov by both rt-pcr and virus isolation (fig. 1) . all dogs from all six treatment groups were negative for crcov on study day à1 (pre-challenge) using both techniques. all dogs in t6 (negative control) remained negative for the duration of the study. shedding of crcov in t1-t5 was consistently detected for up to 6 days post challenge by both rt-pcr and vi. all rectal swabs, collected at necropsy, were positive for the internal pcr control gapdh. all rectal swabs collected from dogs in t6 (negative control) were negative for crcov by both rt-pcr and vi for the duration of the study, as was the case for dogs in groups t3, t4 and t5. in t1, two dogs euthanized on study day 3, and one dog euthanized on study day 6 were positive for crcov, as was one t2 dog euthanized on study day 3. the remaining dogs in these groups were negative. all rectal swabs were negative for crcov by vi (data not shown). tissues collected at necropsy were tested for the presence of crcov by rt-pcr and vi (fig. 2) . crcov was detected in at least five of the eight tissues [diaphragmatic lung lobe, apical lung lobe, trachea, nasal cavity, bronchial lymph node, nasal and palatine tonsil and lung lavage fluid] from groups t1-t4 by rt-pcr. in t5 crcov was detected in all the tissues tested. vi was successful in seven of eight tissue types from groups t1-t5 including the diaphragmatic lung lobe, apical lung lobe, trachea, nasal cavity, nasal and palatine tonsil as well as the lung lavage fluid. only bronchial lymph nodes were negative for crcov by vi. overall, crcov was detected most frequently from the trachea, followed by the lung lavage fluid, nasal cavity and nasal tonsil, in all five treatment groups. histological examination was carried out on the palatine tonsil, external nares, nasal tonsil, trachea, apical and diaphragmatic lung lobes, and bronchial lymph node. the most significant findings were seen in the trachea and nares where inflammation, with notable changes in the length and distribution of the cilia were observed as described below and shown in figs. 3 and 4. in t6 the overall weighted histology score was consistently minimal (average 1.1), with minimal changes in all the tissues examined. in t1 the weighted score indicated minimal to modest abnormalities (average 1.7), including inflammatory aggregates in the nares and inflammation in the trachea which was associated with loss of cilia from the mucosal surface. one dog in particular on day 3 showed marked changes (scoring 3 or 4) in all of the tissues examined, with the exception of the diaphragmatic lung lobe and nasal tonsil where only minimal to modest changes were observed. in t2 and t3 the overall weighted score showed modest to marked histological abnormalities with average scores of 2 and 2.5, respectively. in the nares modest to marked inflammation was seen, which in t3 trailed off in the later stages of the trial period, whilst inflammation in the trachea of dogs from both groups was mild but with moderate shortening and irregularity of the cilia. in t4, with the exception of two dogs (one each on days 3 and 6), marked histological changes in the nares were observed throughout the trial period. in addition marked changes were also observed in the trachea and cilia. one dog on day 6 also had marked changes in the bronchial lymph node and diaphragmatic lung lobe (data not shown). the overall weighted scores indicated consistently marked abnormal histological changes in all dogs in this group with an average score of 3.3. in t5 modest to marked inflammation was observed in the trachea of dogs in t5 which was reflected in the condition of the cilia, whilst only modest changes were observed in the nares. on study day 14 marked changes were also seen in the bronchial lymph nodes. the overall weighted score showed consistently marked abnormal histological changes (average 2.8), with the exception of one of the dogs at the first time point which had a score of 1. in t1-t5 inclusive histological abnormalities in the lungs, although not marked, were variable and tended to be lymphoid aggregates adjacent to airways or blood vessels, an observation which was not seen in the broth control which yielded consistently mild scores. in all groups, including the broth control, the palatine tonsils had medium to high histological scores (data not shown). coronavirus antigen-positive cells were detected within the epithelium of the trachea and bronchioles of the infected dogs (fig. 5) . positive staining was present in the cytoplasm of ciliated columnar epithelial and goblet cells. positive cells were widespread but often found in focal clusters, and often associated with small aggregates on the luminal surface of the trachea. positive cells were surrounded by areas of vacuolation within the epithelium, possibly representing a cytopathic effect of the virus. seroconversion to crcov was measured by ifa. all dogs were seronegative (ifa < 40) for crcov on study days à1, 3 and 6, and all dogs in the t6 control group remained seronegative throughout the study whilst all dogs in the crcov challenge groups seroconverted by study day 14. crcov serum cross-neutralisation antibodies from serum collected on study day 14 were tested against 1 uk and 10 usa crcov isolates (table 5 ). all serum samples tested from dogs in crcov treatment groups were shown to be serum neutralisation positive against all the crcov isolates tested. serum collected from t6 control dogs showed no neutralising activity against any of the crcov strains. the mean concentrations of lymphocytes, neutrophils and monocytes, for each group on study days à1, 3, 6 and 14 were determined (data not shown). in t6, and t3 no statistical differences in the lymphocyte, neutrophil or monocyte concentrations were detected. in t1, t2, t4 and t5 increases in total white blood cell concentrations were observed as a result of lymphocytosis which was significant in t1 (p = 0.002) on day 6 and t5 (p = 0.027) on day 3. in t2 a marked lymphocytosis was seen on day 14. in t4 there was moderate lymphocytosis accompanied by significant changes in monocyte concentrations which peaked on day 6 [>day 3 (p = 0.003), >day 14 (p = 0.006)]. in contrast there was a significant decrease in neutrophil concentration on day 3 in t1, t4 and t5 (p = 0.004, p = 0.001 and p = 0.005, respectively) compared to day à1, which in t1 resulted in 3 of the dogs becoming neutropenic. across the groups seven dogs had rare to occasional toxic neutrophils exhibiting foamy cytoplasm, mild cytoplasmic basophilia or rare dohle bodies, and detectable levels of band neutrophils. in group t1, all dogs had detectable levels of band neutrophils on day 3, which was a significant increase when compared to day à1 (p = 0.031). on day 3 one dog in each of t2, t4, and t5 had band concentrations higher than the estimated upper reference limit; however this did not result in statistically significant changes at a group level and no other significant changes could be detected. oropharyngeal amies swabs collected from all dogs on day à1 and at euthanasia yielded growth of normal respiratory flora. a majority of dogs also yielded scanty growth of either streptococcus canis or staphylococcus intermedius. in the vast majority of dogs the lungs were sterile for bacterial growth, with the exception of the growth of mycoplasma spp. in four dogs, one from each treatment group t2-t5 inclusive (data not shown). previous publications have collectively demonstrated the global distribution of crcov and its association with respiratory disease in dogs under field conditions (erles et al., 2003; decaro et al., 2007; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 yachi and mochizuki, 2006; knesl et al., 2009) . this is the first publication to report experimental infection of dogs using crcov, and the comparison of different crcov isolates from wide geographic origins. each crcov isolate was derived from dogs with respiratory disease in mo, mi, ut and ne in the usa, and one isolate from london, uk. this study builds upon a preliminary experimental challenge study (mitchell et al., unpublished data) . in that study we demonstrated rapid shed-spread of crcov from experimentally infected dogs to sentinel dogs within 4 days of exposure. peak viral loads were detected in oropharyngeal swabs at 4 and 6 days post infection in experimentally infected, and sentinel dogs, respectively; with shedding lasting for 8-10 days in both groups. both inoculated and sentinel dogs displayed clinical signs of mild respiratory disease, and seroconverted to the virus. in the current study prolific shedding of crcov from the oropharynx of dogs in all treatment groups (detected by rt-pcr and vi from oral swabs) was seen by day 2. in a majority of dogs viral shedding ceased after day 6, although crcov was detected up to 10 days post infection by rt-pcr in one dog. small differences in the duration of viral shedding between the two studies are most likely to be explained by differences in the age of the dogs used in each of the studies (preliminary study: 1-3 weeks old, current study: 12-16 weeks old). despite differences, both studies clearly illustrate that crcov is a quick-hit respiratory pathogen, and supports field data in which the rapid spread of crcov throughout a large rehoming centre was observed (erles et al., 2003) . consistent with observations made during naturally occurring infection, dogs in this study also displayed clinical signs of mild respiratory disease following viral challenge (nasal discharge, sneezing, and coughing); whilst the control group remained healthy. there appeared to be no profound difference in the clinical observations made between groups of dogs challenged with the different strains. two dogs however (one each in t3 and t5) showed more severe and prolonged clinical signs compared to the others. this included increased respiratory noise, and more frequent and prolonged coughing and sneezing. the reason for this was unclear. disease in these two dogs did not appear to be associated with any secondary bacterial infections in the lung, nor with notably different histopathological changes when table 5 study day 14 cross neutralisation titres. two dogs remained on the study at day 14 in each treatment group. samples with sn values of 14 were considered negative at the 1:20 starting dilution. serum samples collected at earlier time points were negative for crcov antibody. treatment group (isolate) lu131 lu172 lu189 lu295 lu317 np599 np604 np617 np631 np634 uk4182 t1 (lu295) 320 80 95 113 113 190 135 135 95 113 80 381 135 80 160 160 226 160 135 135 135 113 t2 (uk4182) 369 80 48 57 113 113 95 113 40 135 57 67 34 28 40 40 28 28 17 40 57 57 t3 (np631) 40 28 48 28 28 28 34 17 34 48 28 95 95 80 95 67 48 95 48 67 113 compared to other challenge dogs in the same group (data not shown). given its multifactorial aetiology; modelling cird under controlled conditions, to mimic disease as it appears in the field is difficult to achieve. when studied in isolation dogs infected with other viruses involved in the cird complex, such as cpiv and cav-2, also cause only mild respiratory disease such as a dry cough and nasal discharge (appel and binn, 1987a,b; castleman, 1985; buonavoglia and martella, 2007) . more severe clinical signs, representative of the disease complex, are likely to occur when other viruses or bacterial respiratory pathogens are also present, and when environmental stresses such as those encountered in rehoming facilities are experienced (appel and binn, 1987a,b; buonavoglia and martella, 2007) . overall there appeared to be little difference in tissue tropism, when comparing the different crcov isolates investigated. crcov was detected in at least five of the eight necropsy samples examined (lung lavage, diaphragmatic lung lobes, trachea, nasal tonsils and nasal cavity) in dogs from all five challenge groups. crucially, isolation of crcov was achieved from six of the seven tissues collected (the bronchial lymph node remained negative) as well as the lung lavage fluid. re-isolation of virus from experimentally infected dogs displaying clinical signs of disease, signifies a causal relationship between crcov and respiratory disease, which until now has been best demonstrated through epidemiological surveys (erles et al., 2004 (erles et al., , 2003 decaro et al., 2007; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 knesl et al., 2009) . in all treatment groups crcov was detected most frequently in the trachea, nasal tonsil and lung lavage fluid, and these same tissues also exhibited the highest viral titres (data not shown). this is consistent with previous findings from a small cohort of naturally infected dogs in which the highest viral loads, detected by quantitative rt-pcr, were seen in the trachea and nasal tonsil . importantly, histological analyses were consistent with our molecular and virological findings. ihc revealed coronavirus positive staining in the cytoplasm of ciliated epithelial and goblet cells of the trachea and bronchioles of crcov infected dogs. this pattern of staining is consistent with previous work which identified coronavirus antigenpositive epithelial cells within the trachea of dogs naturally infected with crcov (ellis et al., 2005; priestnall, 2007) , and in crcov infected tracheal sections maintained in culture (priestnall et al., 2009; priestnall, 2007) . furthermore, histopathological analysis showed a clear association between exposure to crcov and inflammation in the nares and trachea, with loss or damage to cilia in the latter. these changes were particularly marked in dogs belonging to t4 and t5, which may indicate a higher degree of pathogenicity for those crcov strains; although, with the exception of one dog in t5 this was not apparent from the clinical signs. ciliary clearance is a key strategy for the removal of pathogens from the respiratory tract. reductions in the efficiency of ciliary clearance would potentiate infection with secondary respiratory pathogens, leading to increased severity and duration of disease. in addition to the trachea, the nasal tonsil may also represent an important site for crcov infection and pathogenicity; given the consistency and duration of crcov isolation and detection in this tissue. preliminary ihc analysis has shown that crcov infection in the nasal tonsil is associated with the epithelium, and with large mononuclear cells which have the appearance of macrophages (priestnall, 2007) . a clear association of crcov with macrophages is yet to be confirmed, but this could present another interesting area given the importance of macrophages in the pathogenesis of disease associated with other coronaviruses, such as feline infectious peritonitis virus (fipv), severe acute respiratory syndrome coronavirus (sars-cov) and human coronavirus 229e (hcov-229e) (desforges et al., 2007; rottier et al., 2005; shieh et al., 2005) . the detection and re-isolation of crcov from the lungs indicates the virus can also establish an infection in the lower airways. gross and histopathological analysis showed that both the control and challenged dogs had focal reddening of the lungs at necropsy, however no significant histological abnormalities were associated specifically with this reddening, and such reddening is assumed to be an artefact of barbiturate euthanasia (lopez, 2001) . nonetheless, in most cases there were microscopic differences between the lungs of these dogs, which could not be appreciated grossly, and therefore the gross appearance of the lungs was not considered an accurate predictor of histopathological changes in these experimental conditions. pulmonary inflammation in crcov challenge dogs was associated with lymphoid aggregates adjacent to the airways or blood vessels. the significance of this is uncertain, but notably these aggregates were not a striking feature of the control dogs. histological changes in the lungs and bronchial lymph nodes of dogs in t6 tended to be more consistent and mild; whilst scores for dogs in crcov challenge groups, although not marked, were more variable throughout the study. histological changes in the lymphoid tissues of most dogs in the study (t1-t6 inclusive) included hyperplasia. lymphoid hyperplasia is not uncommon in dogs at this age, particularly in the palatine tonsil. acute inflammatory responses were evident; however in some dogs this may have been associated with the presence of fragments of foreign material (e.g. hair) in the tonsillar crypts. where marked lymphoid hyperplasia was seen, this was reflected in the histological scores, which were more variable in the challenge dogs compared to those in the broth control animals throughout the trial period. the close genetic relationship between crcov and bovine coronavirus (bcov) (erles et al., 2003 , raised the question as to whether crcov may have an extended tropism which involves the gastrointestinal tract, as seen with some bcov strains (park et al., 2007) . the molecular detection of crcov in the rectal swabs of some dogs from t1 is interesting, and consistent with findings from a number of previous reports of dogs naturally infected with crcov (decaro et al., 2007; yachi and mochizuki, 2006; mitchell et al., 2009) . dogs in the t1 challenge group were unique in that they were the only dogs to be challenged with virus that had not been grown in vitro. the inability to detect crcov from dogs in other challenge groups may therefore be a result of cell culture adaptation; alternatively, this may be a strain specific phenomenon. at present there is no conclusive evidence that crcov displays a true dual tropism for respiratory and enteric tissues. the failure to isolate crcov from enteric samples collected during the peak oropharyngeal shedding period, suggests that crcov may pass through the canine gut as a bystander, without infection. nevertheless, enteric shedding could have implications for managing the spread of disease, and therefore further investigation is warranted. in addition to the histological changes observed in the tissues, significant changes in the leukogram can also be detected in association with crcov infection. this is summarised by a statistically and also clinically relevant lymphocytosis between days 6 and 14; most clearly seen in groups t1, t2, and t5. this observation is not unexpected due to the viral antigenic stimulation, and the lymphocytic reactions found in various tissues. what was somewhat less expected is the high number of dogs with decreased neutrophil concentrations, including a number which developed neutropenia, mild left shift and toxicity, best seen in groups t1, t2, t4, and t5. these changes suggest an acute inflammatory reaction with a high demand for neutrophils and accelerated production in the marrow. transient neutropenia is not uncommon during infections with some viruses; however, there is currently limited data relating to the leukogram profile following infections with other beta coronaviruses. in one report detailing the experimental infection of cows with bcov, significant reductions in neutrophil concentrations were observed at 2 days post infection, followed by neutrophilia at 14 days post infection (traven et al., 2001) . in sars coronavirus infected patients the picture is mixed. neutropenia has been reported in some cases, however, in most cases high blood neutrophil concentrations were observed, and this is often associated with a poor clinical outcome manocha et al., 2003; wong et al., 2003) . neutrophil infiltration of tissues infected with different coronaviruses such as sars, mhv and rat cov has also been described (bhatt and jacoby, 1977; iacono et al., 2006; ding et al., 2003) ; however, their role in viral clearance and possible immune pathology is largely unknown. considering the presence of mostly lymphoid aggregates in crcov infected tissues in this study, the observed changes are intriguing, and a direct effect of crcov on the myeloid series in the bone marrow cannot be ruled out. seroconversion to crcov and the acquisition of neutralising antibodies to heterologous strains from distinct geographical locations within the usa and uk occurred in all challenge dogs remaining on the study at day 14. this degree of serological cross reactivity is not unexpected given the high degree of amino acid similarity seen in the spike protein of different isolates published to date. whilst the correlation between neutralising titres and protection in vivo is yet to be determined, this finding supports published data which demonstrated that the presence of crcov specific antibodies in dogs significantly decreased the risk of developing respiratory disease upon entry to the kennel (erles et al., 2003) . these findings support a possible role for vaccinating against crcov as part of a preventative strategy for respiratory disease in kennelled dogs. moreover, based on the high degree of cross neutralisation and high degree of amino acid identity among the crcov spike proteins described to date, it is anticipated that a single crcov isolate as a vaccine antigen will be sufficient to provide protection against crcov induced respiratory disease. in summary, this is the first study to describe the development of a model for studying crcov pathogenesis; and to fully demonstrate koch's postulates through the successful re-isolation of crcov from experimentally infected dogs with clinical signs of respiratory disease. isolation of crcov was achieved from a wide variety of respiratory and mucosal associated lymphoid tissues, lung lavage fluids and swabs collected over the 2-week period, thus providing clear evidence of tropism for the canine respiratory tract, accompanied by respiratory shedding. moreover, we have shown crcov infection is associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia alongside inflammatory responses. significant effects on the leukogram, in the form of clinically relevant lymphocytosis and neutrophil changes were also documented. strong serological and cross neutralising reactivity between heterologous crcov isolates demonstrates antigenic homogeneity among crcov from the two continents. this study provides vital evidence supporting a role for crcov in the cird complex. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. canine infectious tracheobronchitis short review: kennel cough canine infectious tracheobronchitis short review: kennel cough sv-5-like parainfluenza virus in dogs bordetella and mycoplasma respiratory infections in dogs and cats pathogenesis of canine bordetellosis experimental infection of adult axenic rats with parker's rat coronavirus canine respiratory viruses bronchiolitis obliterans and pneumonia induced in young dogs by experimental adenovirus infection mycoplasmas associated with canine infectious respiratory disease serological and molecular evidence that canine respiratory coronavirus is circulating in italy activation of human monocytes after infection by human coronavirus 229e the clinical pathology of severe acute respiratory syndrome (sars): a report from association of canine adenovirus (toronto a 26/61) with an outbreak of laryngotracheitis (''kennel cough''): a preliminary report detection of coronavirus in cases of tracheobronchitis in dogs: a retrospective study from 1971 to investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex detection of a group 2 coronavirus in dogs with canine infectious respiratory disease longitudinal study of viruses 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development of a quantitative real-time pcr for the detection of canine respiratory coronavirus dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves the role of a novel coronavirus in canine infectious respiratory disease. royal veterinary college serological prevalence of canine respiratory coronavirus serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus quantification of mrna encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (crcov) acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan experimental reproduction of winter dysentery in lactating cows using bcv -comparison with bcv infection in milk-fed calves haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis survey of dogs in japan for group 2 canine coronavirus infection this study was funded by pfizer animal health. the authors would like to thank m. ikeh for technical assistance. rvc manuscript id number: pid_00399. key: cord-002957-gw2cow0d authors: gray, darren w.; welsh, michael d.; mansoor, fawad; doherty, simon; chevallier, olivier p.; elliott, christopher t.; mooney, mark h. title: diva metabolomics: differentiating vaccination status following viral challenge using metabolomic profiles date: 2018-04-05 journal: plos one doi: 10.1371/journal.pone.0194488 sha: doc_id: 2957 cord_uid: gw2cow0d bovine respiratory disease (brd) is a major source of economic loss within the agricultural industry. vaccination against brd-associated viruses does not offer complete immune protection and vaccine failure animals present potential routes for disease spread. serological differentiation of infected from vaccinated animals (diva) is possible using antigen-deleted vaccines, but during virus outbreaks diva responses are masked by wild-type virus preventing accurate serodiagnosis. previous work by the authors has established the potential for metabolomic profiling to reveal metabolites associated with systemic immune responses to vaccination. the current study builds on this work by demonstrating for the first time the potential to use plasma metabolite profiling to differentiate between vaccinated and non-vaccinated animals following infection-challenge. male holstein friesian calves were intranasally vaccinated (pfizer rispoval(®)pi3+rsv) and subsequently challenged with bovine parainfluenza virus type-3 (bpi3v) via nasal inoculation. metabolomic plasma profiling revealed that viral challenge led to a shift in acquired plasma metabolite profiles from day 2 to 20 p.i., with 26 metabolites identified whose peak intensities were significantly different following viral challenge depending on vaccination status. elevated levels of biliverdin and bilirubin and decreased 3-indolepropionic acid in non-vaccinated animals at day 6 p.i. may be associated with increased oxidative stress and reactive oxygen scavenging at periods of peak virus titre. during latter stages of infection, increased levels of n-[(3α,5β,12α)-3,12-dihydroxy-7,24-dioxocholan-24-yl]glycine and lysophosphatidycholine and decreased enterolactone in non-vaccinated animals may reflect suppression of innate immune response mechanisms and progression to adaptive immune responses. levels of hexahydrohippurate were also shown to be significantly elevated in non-vaccinated animals from days 6 to 20 p.i. these findings demonstrate the potential of metabolomic profiling to identify plasma markers that can be employed in disease diagnostic applications to both differentially identify infected non-vaccinated animals during disease outbreaks and provide greater information on the health status of infected animals. introduction bovine respiratory disease (brd) is a multifactorial disease characteristic of a viral-bacterial synergistic infection with predisposition from environmental stressors [1] . the disease constitutes a major source of economic loss through mortality, clinical disease and the associated treatments and long lasting reduced growth performance of infected young stock [2, 3] . the annual cost of brd is estimated at $1billion in the usa, with preventative measures contributing a further $3billion [4, 5] . vaccines are commonly used for controlling brd viral pathogens [6] , but despite seasonal vaccination, animals can become infected with each new outbreak [7] , maintaining the infection within the population. the viral pathogens associated with brd [bovine parainfluenza virus type-3 (bpi3v), bovine respiratory syncytial virus, bovine viral diarrhoea virus and bovine herpes virus-1] impair immune responses in infected animals and damage the respiratory tract allowing the establishment of secondary infections, that may develop further into bacterial pneumonia [8] . however, vaccinated animals can successfully clear viral infections faster than non-vaccinated animals through immune memory response, reducing the associated viral tissue damage or impairment of immune functions preventing the establishment of secondary bacterial and mycoplasma infections [6] . during disease outbreaks, identification of unvaccinated animals at the early stages of infection could provide a window for effective treatment and facilitate the removal of animals that pose a greater risk of becoming infected and transmitting the infection to more susceptible juvenile stock. furthermore, halting viral disease progression to more severe and costly secondary bacterial infections through the identification of vaccine failure animals during infection outbreaks would reduce the level of antibiotic use in the agricultural industry. the only definitive method for successfully identifying vaccinated animals in the presence of an active viral infection is to determine the rate of viral shedding by virus isolation, cytokine/interleukin profiling or virus neutralization assay [9] . these types of analysis require repeated sampling, a period for seroconversion and are expensive compared to serology based elisa, and are therefore not routinely employed during endemic viral infection outbreaks. differentiating infected from vaccinated animals (diva) marker vaccines (e.g. a modified wild type virus with a gene deletion resulting in the absence of a particular diagnostic antigen) can be employed to differentiate vaccine antibody responses from that of wild type virus. companion serology based tests rely on seroconversion, and upon exposure to wild type virus the antibody response to diva vaccines will be masked by that of the wild type virus. vaccine diva functionality is often limited to large viruses with increased potential for gene deletion and removal of redundant expressed antigens. therefore, for viruses with small genomes such as paramyxoviruses (e.g. bpi3v and bovine respiratory syncytial virus of the brd complex) where gene deletion of neutralizing antigens may reduce vaccine efficacy, alternative approaches are required to provide diva functionality. one approach is to design molecular diva vaccines that contain a marker nucleotide sequence differing from the wild type virus that can be employed in combination with pcr-based molecular diagnostics to differentiate between vaccine and wild virus strains [10, 11] . successful differentiation of vaccinated from non-vaccinated animals using this technique requires concurrent vaccination and infection [12, 13] , with a narrow diagnostic window post-infection for detection of diva vaccine and viral genetic material. furthermore, detection of vaccine genetic material only demonstrates exposure to the vaccine and not the successful generation of immune protection, limiting functionality in assessment of herd level immunity. consequently, there is a clear need for alternative diagnostic methods that can assess efficacy of vaccines and vaccination status of animals exposed to brd viral pathogens at the early stages of infection prior to seroconversion and which do not require repeated sampling. additionally, the lower initial exposure rates to viral infections in field settings combined with variation in strain nucleotide sequences and short periods of virus secretion highlights the requirement for a diva approach with a long diagnostic window which is not strain specific. a potential approach that can meet these needs is based on the application of metabolomics to identify metabolites or 'small molecules' in biological samples that are signatures that correlate or provide some evidence of immune protection. these metabolites are often the end stage products of biological processes and therefore provide an accurate representation of an organism's homeostatic status at time of sampling [14, 15] . metabolomic analysis of bio-fluids has provided new insights to the understanding of the patho-physiological processes involved in disease establishment, development and diagnosis [16] [17] [18] [19] . whilst metabolomics has had limited application in the field of veterinary research, several studies have demonstrated the potential of this technique in the prediction of brd disease outcome [20] , differentiation of stress from viral infection responses [21] , and characteristic of immune responses following vaccination [22] . this study focuses specifically on bpi3v due to its endemnicity within cattle populations and absence of clinical symptoms which still predispose animals to more severe bacterial infections [23] . due to its small genome and absence of non-redundant proteins suitable for removal in diva vaccines, bpi3v is an excellent model for assessing the potential of metabolomics to establish vaccination status in infected animals. the aims of the current study were therefore to assess the performance of reverse phase (rp) and hydrophobic interaction liquid chromatography (hilic) separation methods for ultra performance liquid chromatography-mass spectrometry (uplc-ms) metabolomic profiling of bovine plasma and identify plasma metabolomic markers capable of differentiating between vaccinated and nonvaccinated calves following intranasal challenge with bpi3v. this work for the first time reports the metabolomic responses following challenge with bpi3v and demonstrates how the application of metabolomic profiling may help overcome current limitations in diva diagnostics by identifying markers capable of differentiating between vaccinated and non-vaccinated animals, and importantly allow the development of better tools to assess the performance of vaccines. assessment of clinical findings of animals post bpi3v vaccination and challenge have been reported in detail previously [22] . briefly, animals were healthy throughout the duration of the study with no clinical signs of disease in vaccinated or non-vaccinated study groups. calves were sourced from respiratory disease free farms with no history of vaccination against brd. prior to the commencement of vaccination all calves tested seropositive for anti-bpi3v igg. these residual levels of maternally derived anti-bpi3v immunoglobulin are in keeping with other studies employing calves of the same age [24] . at the commencement of vaccination there was no significant difference in anti-bpi3v igg observed between treatment groups. vaccination with rispoval 1 pi3+rsv resulted in a significant increase in anti-bpi3v igg with no significant increase in non-vaccinated controls. following bpi3v challenge vaccinated calves had significantly higher plasma anti-bpi3v igg relative to non-vaccinated animals. post-bpi3v challenge, anti-bpi3v igg remained elevated in vaccinated animals. although no significant differences in anti-bpi3v igg levels were observed in non-vaccinated animals at days 12-20 post-infection (p.i.), igg was elevated in 2/3 animals relative to day 0 levels, with the remaining animal demonstrating elevated levels from days 14 to day 20 p.i. there was a significant increase in lymphocyte counts in non-vaccinated animals from day 0 to 5 p.i., and a significant decrease from days 5 to 12, with no significant variations observed in vaccinated animals. there were no significant differences in neutrophil counts between study groups at sampling points post-infection, however significant (p < 0.05) temporal variations were observed with a decrease from day 0 to 5 in non-vaccinated animals, and an increase from day 5 to 12 in both study groups. at day 6 p.i., 3 animals per group were prepared for gross postmortem analysis. two of 3 animals in the non-vaccinated group showed gross inflammatory lesions, with some peri-vascular cuffing consistent with pneumonia. no significant gross / histological abnormalities were detected in 3/3 of the vaccinated calves at 6 day p.i. at postmortem. preliminary metabolomic analysis was performed to select a suitable method for the comprehensive profiling of metabolites within bovine plasma. rp and hilic chromatographic methods were compared using day 6 p.i. samples (n = 6), which corresponds to peak viral titre [25] . despite improved resolution of poorly retained hydrophobic compounds eluting between 1-2min by hilic separation, increased chromatographic peaks corresponding to plasma derived compounds (relative to blank injections) were observed in rp acquired profiles (fig 1a and 1b) relative to hilic profiles (fig 1c and 1d ). observable differences between plasma samples from vaccinated and non-vaccinated animals within chromatogram profiles were only present using rp (s1 fig) . furthermore, upon data extraction and processing (metabolites with cv > 50% in qc pools), increased numbers of accurate mass retention time pairs (amrtps) were observed following rp (n = 1165) relative to hilic (n = 538) separation of plasma. unsupervised pca analysis of plasma profiles acquired by rp-uplc-ms resulted in clear separation between study groups when assessing principle components (pc) 1 and 2 (57.4% of systemic (r2x) variation within the dataset) as illustrated in fig 2a. in contrast, hilic-uplc-ms metabolomic profiles facilitated only partial separation between study groups based on the pca scores plot (pc1 and pc2, 35.6% of systemic (r2x) variation) ( fig 2b) . due to the small number of chromatographic peaks, fewer amrtps and poor unsupervised pca separation of plasma profiles acquired following hilic-uplc-ms chromatography, rp-uplc-ms was selected as the method of choice for metabolomic profiling of remaining study plasma samples. plasma samples from days 0, 2, 14 and 20 p.i. were extracted, analysed and combined with day 6 p.i. data for multivariate analysis. mass accuracy and retention time deviation of reference compounds between analysis runs was excellent (s1 table) and within marker lynx extraction parameters (0.02da and 0.2min respectively) ensuring accurate peak matching. amrtps (n = 1593) with %cv less than 50% in inter-run quality control plasma pools were used to construct of pca models (pre-filtered from 7690 amrtps extracted from raw data). the effect of bpi3v challenge on the metabolite profile of all animals irrespective of vaccination status, was illustrated in un-supervised pca scores plot ( fig 3a) by separation in pre-(day 0 p.i.) and post-bpi3v challenge samples (days 2-20 p.i.) when observing pc2 and pc3 (14.1% r2x). the greatest separation was observed between day 0 pre-and day 2 p.i. post-challenge stages in vaccinated and non-vaccinated animals. differentiation of vaccinated from non-vaccinated animals based on metabolite profiles following challenge with bpi3v was investigated using un-supervised (pca) and supervised (opls-da) multivariate analysis. prior to bpi3v challenge metabolite profile variation between vaccinated and non-vaccinated animals was low, evidenced by no separation at day 0 ( fig 3b) when observing all pcs. with study progression to post-challenge stages, the variation between vaccinated and non-vaccinated animal metabolite profiles increased from partial separation at day 2 p.i. (fig 3c, 9 .45% r2x with pcs 5 and 7) to clear separation at days 6, 14 and 20, with 19% (fig 2a) , 32.1% ( fig 3d) and 51.1% ( fig 3e) variation (r2x) respectively in pcs 2 and 3. opls-da analysis was employed for the selection of marker amrtps that could discriminate between vaccinated and non-vaccinated animals post-infection. fig 4a-4d illustrates opls-da score (inset) and s-plots for supervised discriminate analysis of plasma metabolite profiles from bpi3v infected vaccinated and non-vaccinated animals at days 2, 6, 14 and 20 p. i. respectively. the amount of variation responsible for differentiation of animals of different vaccination status (r2x) was 7.02%, 8.09%,12.7% and 20.4% in models generated for days 2, 6, 14 and 20 p.i. respectively, with excellent fit (r2y > 98.5%) and good cross-validated prediction (q2 > 95%). as opls-da is known to over-fit, particularly in megavariate datasets where the number of variables are higher than the sample size we employed permutation and false discovery rate testing to reduce the chances of selecting false positives. leave-one-out cross validation was performed to assess the performance of the models generated. briefly, all technical replicates from a single biological sample (test sample) were removed and the remaining dataset employed to generate a predictive opls-da model (vaccinated vs non-vaccinated). this predictive opls-da model was employed to predict the vaccination treatment group of the test sample. this cross-validation was permeated until all biological samples had been assessed for treatment classification. the results (s2 table) indicated that all biological replicates were accurately classified to their respective treatment groups upon cross validation prediction model testing. amrtps which contributed to class discrimination were selected on a criterion of a variable importance score (v.i.p.) score > 1 from opls-da discriminate analysis. amrtps were further filtered to select only those with a fold change (fc) >1.5 and significant difference (anova with bonferroni post-hoc test) p < 0.05 between study groups. final selection of amrtps was performed by assessment of raw mass spectrometric data to determine those with good peak shape and consistent peak intensity (height) in replicate injections. 383 unique potential markers combined from all opls-da analysis (44 at day 2 p.i., 152 at day 6 p.i., 128 at day 14 p.i., and 91 at day 20 p.i.) were selected for further refinement to remove fragments and adducts for parent ion identification. the selected panel of 383 unique amrtps (s3 table) differentiating animals of different vaccination status at various time-points post-bpi3v challenge were deconvoluted to identify parent ion mass, adducts and low energy fragments using low and high energy data (function 1 and 2 respectively), yielding 26 parent ions for elemental composition determination. nonparametric mann whitney was also employed on the selected panel of metabolites from day 0, 2 and 6 p.i. time points, with all markers attaining significance levels within thresholds comparative with anova. the false discovery rate was calculated from the cv (50%) filtered dataset via the benjamini-hochberg procedure with a threshold of 20%. all selected markers passed this threshold and met further selection criteria (v.i.p. score > 1 and fc > 1.5) were selected as potential markers. retention times and accurate masses of potential markers post-bpi3v challenge (days 2, 6, 14 and 20 p.i.) are shown in table 1 this is the first study to report the potential to differentiate between infected animals with differing vaccination status through the use of metabolomic profiling techniques (diva metabolomics). previous work [22] by the authors has demonstrated that metabolomics can identify metabolites associated with immune responses to vaccination, and the current study has advanced this concept by applying metabolomics analysis of plasma to identify unique metabolite marker profiles that are capable of distinguishing between vaccinated and non-vaccinated animals following infection. calves vaccinated (rispoval 1 pi3+rsv) and infection-challenged (bpi3v) demonstrated a significantly stimulated anti-bpi3v igg post-vaccination response which remained elevated post-challenge. lung histology and haematology confirmed that the bpi3v inoculum employed for viral challenge was sufficient to induce some evident respiratory pathology in non-vaccinated animals, with an absence in vaccinated animals. the subsequent challenge in primed vaccinated animals also resulted in maintenance of pre-challenge elevated anti-bpi3v igg (indicating response to inoculum), with absence of respiratory pathology. following preliminary analysis of a subset of samples (day 6 p.i.), rp-uplc-ms analysis demonstrated increased capacity to profile bovine plasma metabolites relative to hilic-based chromatographic separation and was subsequently used for extensive metabolomic plasma profiling. unsupervised pca analysis of acquired metabolomic profiles of preand post-challenge plasma revealed clear and distinguishable variation in plasma metabolites between vaccinated and non-vaccinated animals. considering that elevated anti-bpi3v igg typically occurs at 2 weeks post-challenge [6, 26] , variation in plasma metabolite profiles as early as day 2 p.i. illustrates the capacity of metabolomic profiling methods to detect changes stimulated by immune responses at early post-infection phases. supervised multivariate discriminant analysis of plasma metabolomic profiles yielded 383 potential metabolites (amrtps) that were significantly different in the plasma of vaccinated compared to non-vaccinated animals following bpi3v challenge. following de-convolution (with removal of adducts and fragment ions), 26 parent metabolite ions were identified and shown to be present at different levels within plasma from vaccinated and non-vaccinated animals from days 6-20 p.i. despite no significant differences in the parent ion intensity between treatment groups at day 2 p.i. plasma metabolite profiles differed between vaccinated and nonvaccinated animals through supervised opsl-da. identities of 17 of these 26 parent metabolites were revealed via database searching, in silco fragmentation and spectral matching. divergent plasma metabolite profiles have previously [22] been reported following vaccination, with altered metabolites shown to be associated with primary or secondary immune responses to vaccination. the metabolomic profiling performed here in this study on post-bpi3v challenge acquired samples, has identified a unique panel of plasma metabolites which differ between vaccinated and non-vaccinated animals, and significantly are involved in recognised immune response mechanisms. at day 6 p.i., increased biliverdin (fc = 2.86), bilirubin (fc = 3.25) and decreased 3-indolepropionic acid (fc = -2.40) levels were observed in plasma of non-vaccinated animals. biliverdin is degraded from heme by heme-oxygenases, and is further reduced to bilirubin by bilirubin reductase [27] . heme-oxygenase-1 exerts anti-inflammatory effects as demonstrated by reduced tumour necrosis factor alpha release in lipopolysaccharide-stimulated macrophages [28] . 3-indolepropionic acid is a reactive oxygen species scavenger [29] , produced in the microbiome [30] . phagocyte activation by rna viruses results in both increased reactive oxygen species release and the production of pro-oxidant cytokines (tumour necrosis factor alpha and interleukin-1) which promote iron uptake by the mononuclear phagocyte system [31, 32] . decreasing plasma 3-indolepropionic acid levels in concert with elevated biliverdin and bilirubin levels (via heme-oxygenase-1 action) in non-vaccinated animals at day 6 p.i. maybe indicative of 3-indolepropionic acid scavenging of reactive oxygen species produced by phagocytes, or increased downstream ros production from cytokine signalling. fluctuations in the levels of a number of bile acids (3-oxocholic acid, cholic acid and ndgca) were observed in plasma of animals at various stages in the study. 3-oxocholic acid and ndgca were found to be significantly increased (fc = 3.4 and 1.74 respectively) in the plasma of non-vaccinated animals at day 6 and 14 p.i respectively, whereas cholic acid was significantly higher in vaccinated animals (fc = -2.72) at day 20 p.i. altered plasma bile acid profiles at distinct study phases suggest that bile acid metabolism and conjugation is associated with different immune response mechanisms. bile acid driven farnesoid x receptor activation (expressed in pulmonary endothelial cells [33, 34] ) enables a regulatory immune response as demonstrated by dendritic cell modulation [35] , natural killer t-cell inhibition, reduced proinflammatory osteopontin production [36] and reduced lung permeability, suppressing leukocyte movement to sites of tissue inflammation [37] . when transported into cells ndgca is a potent farnesoid x receptor activator [38] and elevated ndgca at day 14 in non-vaccinated animals suggests an association with the switching and/or suppression of innate inflammatory responses towards adaptive immune responses. cholic acid, the primary metabolite of cholesterol, was found to significantly increase from day 0 to 20 p.i. in vaccinated animals. cholesterol, a component of lymphocyte lipid rafts, supports b-and t-cell receptor signalling [39] . reverse cholesterol transport is associated with immunosuppression via reduction in b-and t-cell receptor signalling, lymphocyte activation and proliferation [40, 41] , and elevated cholic acid may indicate increased cholesterol metabolism via reverse cholesterol transport in proliferating lymphocytes, down-regulating the secondary immune response to viral challenge during later stages of infection (day 20 p.i.). lysophosphatidylcholine produced from phosphatidylcholine [42] [43] [44] stimulates dendritic cell maturation through the action of a g-protein-coupled receptor with further ability to stimulate interleukin-2 and interferon-γ production in t cells [45] indicating a role in innate-toadaptive immune response progression. plasma levels of six lysophosphatidylcholine derivatives were significantly up-regulated (fc > 2.23) in non-vaccinated animals at day 14 p.i. significantly higher plasma levels suggest involvement in systemic immune responses to primary bpi3v antigen stimulation with dendritic cell recruitment to lymph nodes and subsequent dendritic cell driven t-cell activation. decreased plasma peak intensity of enterolactone (fc = -2.35), a lignan metabolite formed in ruminants through the action of colonic bacteria on secoisolariciresinol diglucoside [46] , was observed in non-vaccinated animals at days 14 and 20 p.i. enterolactone crosses the intestinal barrier and exerts anti-inflammatory functions by suppressing nuclear factor-κb signalling, tumour necrosis factor alpha production [47] and interleukin-1β release [48] . as enterolactone is dietary derived it cannot be readily replenished or up-regulated during periods of high demand, therefore decreased enterolactone in non-vaccinated animals during the latter stages of bpi3v infection (days 14 and 20 post-challenge) may reflect its metabolism to induce anti-inflammatory effects associated with the transition of acute to adaptive immune responses. from a diagnostic perspective, those plasma metabolite markers found to be altered at early stages of infection prior to sero-conversion and which persist to the later stages of infection are of particular interest. hexahydrohippurate, despite not showing significantly altered plasma levels until day 6 p.i., remained elevated in vaccinated animals (fc > -2.05) for the duration of the study. significantly lower levels of n-methylhippuric acid (fc = 2.46) and higher n-(cyclohex-1-en-1-ylcarbonyl)glycine (fc = -5.52) were also observed in vaccinated calves at day 6 p.i. n-(cyclohex-1-en-1-ylcarbonyl)glycine, n-methylhippuric acid and hexahydrohippurate are formed through the action of glycine n-acyltransferase on cyclohexanecarboxy-coa, cyclohexene-1-carboxyl-coa or benzoyl-coa, produced during shikimate and phenylalanine metabolism [30, [49] [50] [51] [52] . with no significant variation in the plasma levels of phenylalanine (which would attribute post-bpi3v challenge acyl-glycine conjugate to dietary variations), elevated hexahydrohippurate and n-(cyclohex-1-en-1-ylcarbonyl)glycine levels may be a consequence of an increased demand for coa in the liver due to the need to free coa otherwise sequestered in cyclohexanecarboxy-coa or cyclohexene-1-carboxyl-coa. increased coa demand may therefore be associated with increased rate of b-and t-cell proliferation and antibody production following an adaptive memory response to bpi3v challenge (as illustrated previously by post-parental immunization [22] ). assessment of hexahydrohippurate concentrations in plasma could allow for differentiation of vaccination status in infected calves with a wider diagnostic window to that observable currently through monitoring differences in anti-bpi3v igg levels. hexahydrohippurate occurs at high abundance in plasma offering potential for use as a realistic marker that could be measured using on-site based testing methods. in conclusion, this study highlights the potential of untargeted uplc-ms metabolomics to differentiate the vaccination statuses of virus challenged animals (i.e. diva metabolomics). the differential pre-challenge immune status of vaccinated and non-vaccinated animals resulted in divergent plasma metabolite profiles following bpi3v challenge, evident as early as 2 days p.i., with increasing variation from time post challenge. the metabolites identified were associated with immune cell regulation mechanisms, including t/b-cell proliferation and phagocyte activation and maturation. the wide diagnostic window for hexahydrohippurate combined with metabolite markers altered at distinct time periods associated with specific immune response mechanism (e.g. lysopc, biliverdin, bilirubin or 3-indolepropionic acid), could find application in staging of infection. the effectiveness of these efforts is reflected in the large magnitude fold-change and low intra-group variation of statistically significantly metabolites identified at days 14 and 20 p.i. similar to cytokine profiling, metabolomic based diagnostics are unlikely to match the pathogen specificity of molecular and serological testing but instead provide greater information on physiological health status, with future disease diagnostics potentially employing multiple methods to improve disease management decisions. a limitation of this study, as with many other biomarker discovery investigations involving large bovine animals, is the small cohort size (i.e. n = 6 per group; n = 3 at days 14 and 21 p.i.) which can be incorporated effectively into experimental groups. this potentially may impact on how findings from resulting analysis can be accurately translated to that observed within the wider more variable herd population. to mitigate against such issues, animals were extensively screened with regards to health and maternal antibody status prior to study group inclusion, and post-metabolomics profiling, stringent metabolite selection criteria (fc > 1.5; p < 0.05; v.i.p. score > 1; 20% false discovery rate) were applied to select only the most robust metabolites as marker candidates. as such, further investigation using a larger, more comprehensive sample set (differing sexes, breeds and ages of animals) with alternative routes of vaccination, vaccination failure (degradation or immunosuppression) and challenge (with multiple pathogens) may determine a panel or 'fingerprint' of metabolites with greater diagnostic specificity. a number of unidentified markers showed promising expression profiles and as metabolomics is still a relatively new field, lacking the level of database curation compared to genomics and proteomics for marker identification, these markers may be identified in the future. further studies are required to expand upon this initial proof-of-concept to determine if the observed diva metabolite markers are robust. importantly these studies have identified potential markers that would also be of benefit in screening and assessing new vaccine formulations and allow identification of trails indicative of protective immune responses. hplc grade acetone and analytical standards were purchased from sigma aldrich (dorset, uk). lc-ms grade acetonitrile, water, methanol and chloroform were purchased from fisher scientific (loughborough, uk). all animal studies were carried out in accordance with the uk animals (scientific procedures) act 1986 and with the approval of the agri-food and biosciences institute northern ireland ethical review committee. calves were vaccinated with rispoval 1 pi3+rsv as previously reported [22] . briefly, 12 male holstein friesian calves aged between 20 and 25 weeks were sourced commercially from farms with no history of prior respiratory disease outbreaks. the calves had no prior vaccination for brd and were clinically examined and declared fit for the study on day 0 by the named veterinary surgeon. claves were divided into two study groups (n = 6) and assigned as non-vaccinated and vaccinated calves. vaccinated calves were treated with pfizer rispo-val 1 pi3+rsv intranasal vaccine (designated vaccinated animals) as per manufacturer's instructions, and non-vaccinated calves treated with empty poly-(lactic-co-glycolic) acid nanoparticles (designated non-vaccinated) prepared using standard double emulsification solvent evaporation technique (w/o/w) [53] . calves received two dosages of vaccine formulation at 70 and 35 days prior to intranasal bpi3v challenge (post-infection = p.i.) (inoculation with 2 ml of virus suspension (tcid 50 of 10 6.78 /ml) per nostril). calves were screened weekly following vaccination and at days 0, 5, 12, 14 and 20 post-bpi3v infection (p.i.) for the presence of bpi3v igg in blood serum using svanovir-pi3v-ab kit (boehringer ingelheim svanovir, uppsala, sweden) as per manufacturer's instructions. at day 6 p.i. 3 animals per group were sacrificed for viral isolation and histology after sampling. blood samples drawn via jugular venepuncture at days 0, 2, 6, 14 and 20 p.i. into 6ml plastic k3 edta vacuette tubes (greiner bio-one, stroudwater, uk) were processed at random to platelet poor plasma via a double centrifugation method [54] optimized for metabolomic analysis within 2 hours of initial blood drawing and plasma stored at -80˚c prior to use. samples processing order was randomized to negate processing bias on sample metabolite profiles. 400 μl of plasma was added to 1.6 ml of ice cold acetone, vortexed for 30 sec and placed on ice for 15 min. the sample was then deproteinated by centrifugation at 15,000g at 4˚c for 15 min. 1.6 ml of supernatant was removed and dried under nitrogen for 45 min at 40˚c using turbovap lv (caliper life sciences, hopkinton, usa). resulting residue was reconstituted in 500 μl of ultra-pure h 2 o and liquid/liquid extraction of lipids performed by addition of 500 μl of ice-cold methanol:chloroform (1:1 v/v) and vortexing for 30 sec followed by centrifugation at 16,000g at 4˚c for 15 min. liquid/liquid extraction was repeated and after centrifugation 900 μl of the aqueous layer was removed and dried under nitrogen. the residue was reconstituted in 150 μl ultra-pure h 2 o and filtered by centrifugation at 10,000g, 4˚c using 0.22μm costar spin-x 1 centrifuge tube filter for 5 mins. 100 μl of plasma was added to a well in an ostro protein precipitation and phospholipid removal plate (waters corporation, milford, ma, usa). 100μl of 1% formic acid/acetonitrile (v/v) was added to the sample well, the plate shaken for 30 sec and extracted metabolites drawn through under vacuum into a 96 well 2 ml collection plate. rp-uplc-ms analysis was performed using an acquity uplc system coupled to a xevo g2 q-tof (waters corporation, milford, ma, usa). a test mix of acetominophen, sulfaguanidine, sulfadimethoxine, val-tyr-val, verapmil, terfenadine, leucine-enkephalin, reserpine and erythromycin was injected to ensure calibration of mass spectrometer mass accuracy and uplc performance prior to analysis. pooled samples (comprising a 10μl aliquot from all study samples) were injected 5 times before the start of each run for column conditioning [55] and intermittently throughout the run to validate instrument performance. the run-order of samples entering the mass spectrometer was constructed using a randomized sample list comprising 5 technical replicates per biological sample. no other samples were injected during the analysis run. 8 μl of prepared sample extracts were injected onto an acquity uplc hss-t3 column (100 mm x 2.1 mm i.d., 1.8 μm; waters corporation, milford, ma, usa). column and autosampler temperature were maintained at 50˚c and 10˚c respectively. chromatographic separation was carried out at a flow rate of 600 μl/min with mobile phase consisting of 99.9% h2o/0.1% formic acid (a) and 99.9% acetonitrile/0.1% formic acid (b). the elution gradient was as follows: 0-2 min isocratic at 1% of b, 2-14.5 min linear gradient form 1-100% of b, 14.5-17.5 min isocratic at 100% of b, and finally 17.5-20 min linear gradient at 100-1% of b. mass spectrometry was performed in positive-ion mode (esi+) with the capillary voltage set to 1500 v and the sampling cone voltage 30v. the desolvation and cone gas flows were set at 750 l/h and 100 l/h respectively. source and desolvation temperatures were 120˚c and 400˚c respectively. leucine enkephalin ([m+h] + = 278.1141 da, and [m+h] + = 556.2771 da) was used for accurate lockmass calibration during data acquisition. lockmass acquisition setting were: 0.5 sec scan time, 30 sec interval, 3 scan average, mass window +/-0.5da. centroid data were acquired in positive mode using resolution mode. collision energy was only applied on function 2, with ramping between 15 ev and 30 ev. for hilic-uplc-ms/ms analysis the test mix was cytosine, o-acetyl-l-carnitine and lvaline. pooled samples (comprising a 10μl aliquot from all study samples) were injected 5 times before the start of each run for column conditioning [55] and intermittently throughout the run to validate instrument performance. the run-order of samples entering the mass spectrometer was constructed using a randomized sample list comprising 5 technical replicates per biological sample. no other samples were injected during the analysis run. 1 μl of prepared sample extracts were injected onto an acquity uplc beh hilic column (2.1 mm x 100 mm i.d., 1.7 μm; waters corporation, milford, ma, usa). column and autosampler temperature were maintained at 45˚c and 6˚c respectively. chromatographic separation was carried out at a flow rate of 600 μl/min with mobile phase consisting of 10 mm ammonium formate ph 3.3 (a) and acetonitrile with 0.2% formic acid (b). the elution gradient was as follows: 0-2 min isocratic at 5% of a, 2-10 min linear gradient form 5-30% of a, 10-11 min linear gradient from 30-90% of a, 11-12 min isocratic at 90% of a, 12-12.5 min linear gradient from 90-5% of a, 12.5-16 min isocratic at 5% of a. mass spectrometry was performed using a waters xevo g2 q-tof (milford, ma) operating in positive-ion mode (esi+) with the capillary voltage set to 300v and the sampling cone voltage 20v. the desolvation and cone gas flows were set at 700l/h and 5l/h respectively. source and desolvation temperatures were 120˚c and 450˚c respectively. leucine enkephalin was used for accurate lockmass calibration during data acquisition, with acquisition settings the same as with rp-uplc-ms analysis. centroid data were acquired in positive mode using resolution mode. collision energy was only applied on function 2, with ramping between 20v and 35v. total ion count (tic) chromatograms and spectra were acquired with masslynx version 4.1 (waters corporation, milford, ma, usa) in centroid format and metabolite data was processed using the markerlynx software. the markerlynx method for data extraction and deconvolution was as follows. ions were extracted from function 1 data using peak detection analysis of retention time window 0.30-14.50min, with a mass range of 100da to 1200da. the xic window for data collection was 0.2da and apex peak tracking parameters were set to automatic with no smoothing. data collection parameters consisted of an intensity threshold (counts) of 500, a mass window of 0.02da with a retention time window of 0.20min. a noise elimination level of 6 was applied and isotopes were removed. peak heights for extracted ions were normalized against the total peak height of all extracted ions and standardized to a total ion count of 10,000. the results were exported in .csv format as a two dimensional data table in which rows and columns respectively represented analysed samples and the relative normalized peak heights of each detected mass spectrometric signal, i.e. as an accurate mass (m/z) and retention time (min) pair (amrtp). extracted and processed data submitted for downstream multivariate and statistical analysis for metabolite marker selection can be found in supplementary data (s4 table) . temporal changes in bpi3v antibody titre were analysed using two-tailed paired t-test, and significant differences between treatment groups at sampling stages was assessed using two-tailed heteroscedastic t-test. simca-p+ version 13.0 (umetrics, sweden) was used for multivariate metabolite marker selection. the dataset was pre-filtered to exclude amrtps with coefficient of variation greater than 50% in inter-run quality control pools (generated from equal aliquots of all extracted samples and injected intermittently throughout the analysis run). all centroid data were pareto scaled and analysed by unsupervised principle component analysis (pca) and supervised discriminatory analysis by orthogonal projections of latent structures-discriminant analysis (opls-da). unsupervised pca models were generated at each sampling day to reveal potential relationships between treatment groups. supervised analysis by opls-da was performed to reveal potential markers of response to treatment in vaccinated calves compared to non-vaccinated calves at each sampling day. robustness of final opls-da discriminative models was assessed by setting a predictive model of each case in which 2/3 of the data (known treatment) was used to predict the remaining 1/3 (unknown treatment). significance of the identified markers at all time points was determined using anova with bonferroni post-hoc test and non-parametric mann whitney for day 14 and 21 p.i. time points. the elemental composition of selected parent compounds was determined in masslynx using both positive and negative mode data. mass uncertainty was set to 3 mda, odd and even electron state, carbon isotope filter of +/-5% and elements included were c, h, o, n, p and s. where applicable na and k adduct elemental composition were determined with the respective element included in the analysis parameters. elemental compositions were searched against pubchem and chemspider online databases, and where possible function 2 fragments were matched against metlin, hmdb or massbank databases. where fragmentation spectra for the analyte in question was not available, in silico fragmentation was performed using metfrag and function 2 fragmentation data was validated against potential in silico fragments. identified compounds were validated using mass spectrum and retention time relative to authentic analytical standards analysed under identical experimental conditions (s4 fig). pooled plasma samples and individual analytical standards (1 μm) were analysed under identical uplc and mass spectrometric run conditions as utilized previously [22] , and metabolite identities confirmed by matching retention time and function 1 and function 2 spectra (including low and high energy fragments and adducts). putative annotations were acquired for compounds with spectral similarity to public spectral libraries (metlin, hmcb or massbank). table. cross-validation of opls-da models. leave-one-out (loo) cross validation was performed to assess the performance of the models generated. all technical replicated from a single biological sample were removed and an opls-da model containing the remaining biological and technical replicates employed to predict class representation. (xlsx) s3 table. selected amrtps of plasma metabolomic markers of response bpi3v challenge in vaccinated and non-vaccinated animals. combined list of 383 amrtps selected using opls-da at days 2, 6, 14 and 20 post-bpi3v infection filtered to exclude those with p < 0.05, fc < 1.5 and v.i.p. score < 1. 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metabolism of quinic, cyclohexanecarboxylic and non-phenolic aromatic acids to benzoic acid the preparation and characterization of poly(lactide-co-glycolide) microparticles. 2. the entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display global metabolic profiling procedures for urine using uplc-ms we acknowledge the staff at afbi animal services unit vsd (stormont) for their help in the animal study and the staff at the advanced asset centre igfs (qub) for their excellent technical assistance. key: cord-031315-p7jb4gf2 authors: kong, qing; mo, shuming; wang, wenqian; tang, zihui; wei, ying; du, yijie; liu, baojun; kong, lingwen; lv, yubao; dong, jingcheng title: efficacy and safety of jia wei bushen yiqi formulas as an adjunct therapy to systemic glucocorticoids on acute exacerbation of copd: study protocol for a randomized, double-blinded, multi-center, placebo-controlled clinical trial date: 2020-09-03 journal: trials doi: 10.1186/s13063-020-04669-5 sha: doc_id: 31315 cord_uid: p7jb4gf2 background: systemic glucocorticoids are effective for the management of chronic obstructive pulmonary disease (copd) exacerbation but have serious adverse effects. traditional chinese medicine (tcm) can bring additional benefits to these patients but has few adverse effects. the present study aims to evaluate the efficacy and safety of jia wei bushen yiqi (jwby) formulas in patients who suffer from copd exacerbations and to investigate whether the short-term (5-days) systemic glucocorticoid therapy is non-inferior to the long-term (9-day) regime. methods: in this multi-center, randomized, double-blinded trial, eligible inpatients with copd exacerbation are randomly assigned to four groups (a, b, c, and d). group a will receive placebo plus 5-day prednisone, group b will receive placebo plus 9-day prednisone, group c will receive jwby formulas plus 5-day prednisone, and group d will receive jwby formulas plus 9-day prednisone. the primary outcomes are the time interval to the patient’s next exacerbation during a 180-day following up and the copd assessment test (cat) during treatment. secondary outcomes include lung function, tcm syndrome assessment, laboratory tests, and safety. the changes of the hypothalamic pituitary adrenaline axis (hpa axis) and inflammatory cytokine will be measured as well. discussion: by demonstrating the advantages of utilizing tcm and an appropriate duration of systemic glucocorticoids, this effectiveness comparison trial will provide new references to physicians on how to improve the management of copd exacerbation. the results of hpa axis and inflammation cytokine measurements will shed light on the molecular mechanisms and entail further mechanism studies. trial registration: www.chictr.org.cn chictr1900023364. registered on 24 may 2019. chronic obstructive pulmonary disease (copd) will become the third leading cause of death worldwide in 2030 [1] . 13 .7% of the population over 40 years old suffer from copd in china [2] , creating a large socioeconomic burden [3] [4] [5] . copd exacerbation is defined as the acute worsening of respiratory symptoms that require additional therapy [6] [7] [8] . acute exacerbations of copd impair pulmonary function and exponentially increase the risk of death [9] . therefore, effective management of copd is critical to human health. according to international guidelines and evidencebased reviews, systemic glucocorticoids are recommended to treat copd exacerbation [10] [11] [12] [13] [14] [15] . the advantages include shortened recovery time and hospitalization duration, improved lung function and oxygenation, and reduced relapse risk and treatment failure, which have been demonstrated by numerous randomized clinical trials (rct) [16] [17] [18] [19] [20] [21] [22] . however, the side effects like hypertension, hyperglycemia, gastrointestinal bleeding, psychiatric disease, and hypothalamic pituitary adrenal axis (hpa axis) suppression increase with the extension of treatment duration and the escalation of dose [23] . controversy over the optional duration continues. on one hand, a dose of 40 mg prednisone (a common oral systemic glucocorticoid) daily for 5 days has been recommended by the global initiative for chronic obstructive lung disease (gold) science committee report based on the reduce randomized clinical trial since 2015 [24] . the trial indicated the efficacy of 5-day systemic glucocorticoids is noninferior to 14-day systemic glucocorticoids regarding relapse within a 6-month follow-up, but significantly reduced glucocorticoid exposure. on the other hand, a dose of 30-40 mg prednisone daily for 9-14 days [10, 12, 13] was suggested by another academy of china, korea, and europe in 2017. yet, no clinical trials have determined the difference between the 5-day and 9-day regimes. in addition, treatment individualization brings benefits. for instance, an inhaled corticosteroid (ics) is more efficacious in patients with high blood eosinophils [25] [26] [27] . however, present pharmacotherapy has failed to reverse the downtrend in pulmonary function completely [28] . hopefully, traditional chinese medicines (tcm) can expand copd treatment in terms of syndromic difference, also called zheng [29] . not only has tcm alleviated symptoms such as coughing, shortness of breath, and sputum for thousands of years, but also has demonstrated its efficacy and safety [30] [31] [32] [33] [34] . however, there are rarely studies focused on copd patients during the acute exacerbation period, most of them focused on the relatively stable period. we conducted a randomized and placebo-controlled trial enrolling stable copd patients in 2014, which illustrated that tcm formulas called bushen yiqi (by) formulas can improve the lung function, reduce the frequency of acute exacerbation of copd, and modulate the hpa axis [35] . dr. shen replaced glucocorticoid therapy with tcm formula (by) totally in chronic inflammatory disease [36] . moreover, several ingredients in by can decrease the inflammatory reactions in copd animal models [37] . recently, we have observed that by formulae combined with another two chinese herbs-huang qin (scutellaria) and chi shao (paeoniae rubra radix)-demonstrate more effectiveness on the management of acute exacerbation of copd in clinical practice, such as relieving the symptoms including the cough, sputum, as well as shortness of breath. interestingly, the laboratory experiments showed that the main compound of these two chinese herbs benefits the animal of copd model. for instance, scutellaria baicalensis in huang qin significantly improved lung function, ameliorated the pathological damage, and attenuated inflammatory cytokines infiltration into the lungs [38] . similarly, paeonol in chi shao showed antiinflammatory and antioxidant effects against cs-induced lung inflammation in both in vivo and in vitro experiments [39] . therefore, we propose that jia wei bushen yiqi formulae (jwby)-bushen yiqi formulae combined with huang qin and chi shao-will benefit patients with acute exacerbation of copd. this study aims to demonstrate non-inferiority of a 5-day therapy compared with a 9-day regimen of systemic glucocorticoids based on the copd outcome during the 180-day follow-up period. it also seeks to determine the relative inferiority of jwby formula as an adjunct treatment to systemic glucocorticoids compared with systemic glucocorticoids alone for copd exacerbation. this is a multi-center, double-blinded, placebocontrolled, randomized clinical trial. this trial will be conducted in two stages: a 9-day treatment and then a 180-day follow-up. qualified patients will be randomized to 4 groups: group a will receive placebo plus 5-day prednisone, group b will receive placebo plus 9-day prednisone, group c will receive jwby formulas plus 5day prednisone, and group d will receive jwby formulas plus 9-day prednisone. assessments will be performed on day 6 and on day 10 during treatment and telephone calls will be conducted on day 90 and on day 180 when patients are discharged (fig. 1) . the 9 day-treatment is chosen for two reasons. first, it is because of the two aims that were mentioned above. second, the 9-day treatment period is based on our investigation result that most copd exacerbation symptoms can be alleviated within 10 days. in other words, 9 days are the common hospitalization time in ten subcenters. therefore, the 9 day-treatment is a good time for patients to complete the study during hospitalization, which will promote the compliance of patients and collect as much data as possible. the 180-day follow-up time is based on the results from the reduce randomized clinical trial research published on jama in 2014. it is reported in this trial that the median number of days of follow-up was 180 in both the conventional group (10th percentile, 179; 90th percentile, 181 days) and in the short-term treatment group (10th percentile, 178; 90th percentile, 181 days). this trial will be conducted at ten hospitals located in shanghai, yunnan, xinjiang, and jiangsu province in china. five hospitals are selected because they are attached to universities and another five hospitals are selected because they are experienced in rct. also, these hospitals are spread out throughout china ( table 1 ). the principal investigator (pi) work at huashan hospital and is responsible for the steering committee meeting, which includes protocol training, supervision of safety, quality control, feedback of progress, and study reports. pis of other hospitals will organize their clinical physicians and nurses to carry out recruitment and follow-up. patients that are hospitalized with copd acute exacerbation and meet the inclusion and exclusion criteria ( table 2) will be eligible to be study participants. acute exacerbation of copd with clinical grade 2 is defined as follows: respiratory rate > 30 times/min, application of assisted respiratory muscles, no mental state change, hypoxemia can be improved by the 25%-30% oxygen concentration in the inner cover of the venturi, and hypercapnia or partial pressure of carbon dioxide (paco 2 ) increases to 50-60 mmhg from the baseline value. patients who are diagnosed as having respiratory failure but without the risk of death are appropriate for ordinary hospitalization, as recommended by the chinese expert consensus on the diagnosis and treatment of acute exacerbation of chronic obstructive pulmonary disease (aecopd) (2017 update) [10] . in other words, a moderate degree of copd exacerbation does not indicate the need for intensive care unit (icu) admission according to 2019 gold guideline [24] . tcm syndrome differentiation-fei_shen_qi_xu_yu_ re zheng in chinese-specifies people who have lung and kidney qi deficiency mixed with blood stasis and (table 3) . study centers are selected from level a hospital in china. the investigators will be selected from attending physician who majors in respiratory disease. prior to the trial, all sub-center physicians, nurses, and other staff will be trained to understand the protocol. attending physician who will take charge of the patients obtains consent from potential participants or authorized surrogates. firstly, attending physician will introduce the trial including the origin of tcm formula, the prednisone effect, what they should do, and what will benefit them if they volunteer to participate in this trial. then, physician will reply to the questions that confuse patients. finally, both the physician and patient will sign the informed consent form to indicate the patient's full understanding of the protocol. in the consent form, participants will be asked if they agree to use of their data should they choose to withdraw from the trial, and if they are volunteer to provide another 25 ml blood for storage, which are used to explore their inflammation level, hpa axis function, and the relationship between effectiveness and gene type. participants will also be asked for permission for the research team to share relevant data with people from the hospitals who take part in the research. as we mentioned in background, prednisone of 30-40 mg once daily is recommend for copd exacerbation management since 2014 by golg guideline. the evidence is from a clinical trial that compare the efficacy of 14 days of prednisone treatment with 5 days. the participants come from sweden. as in other countries like china, the duration of prednisone treatment is recommended as 9-14 days. the differences of the outcomes between 5 days of treatment and 9 days are unknown in the chinese patients. since we choose the relatively mild patients with copd exacerbation, the minimum dose of prednisone 30 mg once daily is decided in this trial. in addition, tcm formula has been used for copd therapy for thousands of years. we have observed the superiority of tcm as an adjunct therapy in copd administration. but there is no evidence to show the exact outcomes. the doses of five tcm herbs are decided by a group of experienced tcm physician who used the principle of tcm in treating copd for many years. the control group is placebo that contains 10% true herbs with the same appearance and smelling as the drugs. severe impairment of heart, liver and kidney function (heart function 3-4 degree, aspartate aminotransferase (alt) and/or alanine aminotransferase (ast) exceeds 1.5 times of the upper limit of normal, creatinine (cr) exceeds the upper limit of normal) 6 . received systemic glucocorticoids within 2 weeks or participation in other drug clinical trials within 3 months prior to the trial 7. other conditions that the investigators consider to be improper all the participants will be provided with standard of care (soc) according to the 2019 gold guideline for copd exacerbation during hospitalization and after discharge (table 4) . a 9-day adjunct medication includes systemic glucocorticoids and tcm herbs or their placebo. a basic dose of 30 mg prednisone daily for 5 days will be provided for all participants. the prednisone will be continued in the long-term glucocorticoids arm of the trial in the following 4 days and replaced with the placebo in short-term glucocorticoids arm of the trial. the 9-day treatment period is based on the fact that most copd exacerbation can be relieved within 10 days. meanwhile, participants will be treated with tcm herbs or placebo. participants will be randomized to four groups with different adjunct medication ( fig. 1) . because of the complex and variety in copd exacerbation, variation among patients will be allowed. any variation like another antibiotic used for the indication will be recorded in the case report form (crf). tcm treatment is in accordance with the most common tcm syndromes of copd in a real-world study [40] . the dosage of jwby formula is selected according to the pharmacopeia of chinese medicine, and the effective ingredient of its granules is determined according to the pharmacopeia of pharmacopeia. jwby formulas contain 5 kinds of herbs: huang qi (astragalus) 30 g, yin yang huo (epimedy) 20 g, sheng di huang (radix rehmaniae) 15 g, chi shao (red peony) 30 g, and huang qin (scutellaria) 30 g, concentrated as 20.48 g granules. to use, patients can infuse 10.24 g granules into 125 ml of boiling water and ingest orally after breakfast and supper, twice daily. its placebo is identical in appearance, shape, size, and package with jwby formulas, but only contains 10% real herbs. the granules will be produced and packed by huarui sanjiu pharmaceutical industry in shenzhen, china. granule production will be certified to get the standard certification of the tcm national drug regulatory authority. modification or discontinuation of the intervention will be decided by the pis in each center, according to the requests from participants, or when a participant's disease is worsened to grade 3 which indicates the need for icu admission, or when unexpected adverse effects happen. prednisone and jwby granules are free as study drugs. five-day drugs will be provided to participants at baseline by a sub-center investigator and another 4-day drug at day 6. participants will use patient diaries for recording medication and changes in symptoms. all unused packs of drugs and empty bags will be returned to investigational site on day 6 and on day 10. compliance will be calculated by counting drugs or empty bags for a 9day course. compliance % of medication = [actual dose/ (specified daily dose × days)] × 100%. total medication consistency ranging from 80 to 120% will be eligible for the protocol analysis set. patients enrolled in the trial will be all hospitalized and all the laboratory tests will be performed on standard schedule, which aids in the monitoring of adherence. once the patient is randomized, the investigators will take every reasonable effort to follow the patient for the entire course of the study. all examination and transportation costs in the 30-day will be covered and the results of symptoms and physical exams will be explained at every visit. messages will be sent through wechat or by phone prior to every visit to remind the patients of the follow-up visits. extra copd-related drugs, such as leukotriene receptor antagonists, antihistamines, immunosuppressants, and antioxidants, will be forbidden during the trial. tcm herbs that are tonifying kidney, benefiting qi, clearing away heat, and promoting blood circulation, whose tcm characteristics are like those within jwby formulas, will be avoided. drug combinations will be recorded in the case report form at each follow-up visit. "tonifying kidney" ("bushen" in chinese) is a tcm term of treatment, which aims at the tcm syndrome "deficiency of kidney" ("shen_xu" zheng in chinese). the chinese herbs used in "tonifying kidney" treatment can relieve "deficiency of kidney" syndrome including shortness of breath, deterioration with movement, fatigue, waist and knee area sore, and their weakness, tinnitus, dizziness, incontinence, or heavy urine volume. patients that are enrolled into the study will be covered by indemnity through the standard national health service indemnity arrangements. the pi will provide the compensation to those who suffer due to trial participation. primary outcomes measurements 1) the time to the next exacerbation of copd during the 180-day follow-up is defined as one primary outcome. the definition of exacerbation is deterioration of the cardinal symptom of dyspnea, increased sputum purulence and volume, and purulent sputum. this may be combined with one of the other symptoms: increased cough and wheeze, sore throat, nasal congestion due to cold, fever (oral temperature > 37.5°c), increased cough, and increased wheezing. the above changes should last for ≥ 2 days at least. a minimum of 1 week between two exacerbations is needed in order for them to be considered as separate events. the duration of exacerbation is measured from the onset of acute exacerbation to a significant reduction which is defined as the symptoms return to the level before the exacerbation per the records in patients' dairies. the diaries are distributed to participants during the treatment and after the treatment. participants record changes of their symptoms and their health status by choosing the right description in terms of feeling. the primary symptom is measured with modified british medical research council (mmrc) and copd assessment test (cat) scores. the days of exacerbation are calculated from the onset date of the primary symptom to the date when all symptoms disappear. the degree is classified as mild (treated with short acting bronchodilators only, sabds), moderate (treated with sabds plus antibiotics and/or oral corticosteroids), or severe (patient requires hospitalization or visits to the emergency room). severe exacerbations may be associated with acute respiratory failure. 2) the mean difference of cat scores between day 6 or day 10 and baseline is another primary outcome. the cat involves an 8-dimension measurement of health-status impairment in copd. cat is universally acknowledged as a reliable and valid measurement in evaluating the changes of copd. 1) tcm syndrome assessment will be evaluated from baseline to day 6 and day 10. according to the guiding principles for clinical research of new drugs in traditional chinese medicine, the syndrome score is calculated as efficacy index n = (pre-treatment score − post-treatment score)/ pre-treatment score × 100%. in terms of mild, moderate, and severe symptoms, the primary symptoms are given 2, 4, and 6 points while the secondary symptoms are given 1, 2, and 3 points respectively. total score = scores of the primary symptoms + scores of the secondary symptoms. 2) lung ventilation function will be assessed by forced expiratory volume in 1 s (fev1), forced vital capacity (fvc), and peak expiratory flow (pef) from baseline to day 6 and day 10 with standardized equipment (erich jaeger uk ltd., market harborough, uk jaeger master-screen, germany) and per the standard procedure recommended by american thoracic society (ats) [39] . 3) blood gas analyses including partial pressure of oxygen (pao 2 ), partial pressure of carbon dioxide (paco 2 ), infectious indexes including blood eosinophil count in cells per micrometer (eos), creactive protein (crp), and proclamation will be tested by clinical laboratories in the sub-center from baseline to day 6 and day 10. side effects will be collected at day 30, day 60, and day 180 during follow-up. this specifically refers to (1) the changes in hyperglycemia: fasting plasma glucose ≥ 5.6 mmol/l or random plasma glucose ≥ 7.8 mmol/l or rise ≥ 20% in daily doses of insulin or any increase in oral anti-diabetic drugs or initiation of one or more antidiabetic therapeutics, (2) changes in hypertension: systolic blood pressure ≥ 140 mmhg and/or diastolic blood pressure ≥ 90 mmhg or the addition of one or more anti-hypertensive drugs to previous treatment regimens, and (3) the number of psychiatric symptoms, asphalt, vomiting coffee samples, and new infection. laboratory tests which include routine blood test, routine urine test, electrocardiogram (ecg), kidney and liver function, and x-ray computed tomography (ct scan/x-ray) of the chest will be conducted at baseline, day 10, and day 30 during the follow-up. if the results of ct scan/x-ray and ecg are normal at baseline, it will be skipped in the follow-up. the pathology of copd is relevant to the inflammation and the suppression of the hpa axis that follows the treatment with glucocorticoids. therefore, changes in the hpa axis including corticotropin-releasing hormone (crh), adrenocorticotropic hormone (acth), and cortisol and the inflammation cytokines including interleukin-6, interleukin-8, and interleukin-10 at baseline and on day 6 and day 10 will be measured. there are four groups with two variables in this trial-tcm treatment and systemic glucocorticoid treatment. therefore, according to primary endpoints collected from previous trial [34, 41] , we choose the maximum sample size needed, as calculated by two way on http:// www.powerandsamplesize.com (table 2 ). at the 5% significance level, a total of 67 patients per group will be required for a 2-group, 1-sided calculation to achieve 80% power and the differences of 10.30 ± 6.31 and 12.95 ± 5.99 in cat mean score between the tcm treatment group and placebo group (table 5) . meanwhile, a total of 88 participants will be required for a 2group non-inferiority calculation to achieve the mean difference of the time to next exacerbation (43.5, 29) and a non-inferiority margin of 10, under the condition that the standard deviation of the groups is equal to 12 (table 5) . a loss of 15-20% to follow-up is predicted based on experience-this increases the sample size to 200 participants per group, resulting in 400 in total. all investigators in the sub-center will advertise and distribute posters in their emergency department and nearby communities. in addition, we will set up a hierarchical medical system in shanghai-communities refer the potential patients to huashan hospital directly where the clinical trial is undertaken. participants will be randomized with equal probability (1:1:1:1) to receive one of the four treatments that were mentioned above. as the size of each group is predicted to be 100, the allocation sequence is generated with sample randomization and stratification by trial center. the sequences will be generated by software and in excel format. before the study begins, a series of random numbers will be generated by the computer, and the pharmacists involved in the study place the random numbers in plain, closed envelopes marked with patient numbers. envelopes will be made and stored at the pharmacy and opened by the pharmacist only when the subjects are randomized. the envelopes will be not accessible to individuals directly involved in the study. allocation sequence will be generated by a statistician who will not participate in enrolling participants. participants will be blindly randomized and allocated with an identified number. principal investigators including attending physician and nurses will involve in enrolling participants. pharmacist will distribute an independent emergency envelope for each participant, which contains the treatment assignment. the participants in the placebo group will be given the same number of pills and followed the same medication schedule as the treatment group. to ensure the implementation of the blinding method, the pill and herbs in both the treatment group and the placebo group will be made in the same shapes, smells and tastes. trial participants, care providers including attending physician and nurse, outcome assessors including pi and sub-pi, and data analysts will be blinded after the assignment of interventions. double-grade unblinding will be adopted. first grade unblinding: it will be conducted before the data analysis. after the double input of all the crf data into the computer and blinded review, the data will be locked. afterwards, the personnel who keep the blinded materials will unblind them for the first time, which is to divide the groups corresponding to the case numbers into blinded codes of two groups and to tell the statisticians so as to statistically analyze all the data. second grade unblinding is after the statistical analysis and the completion of clinical trial report. it will be conducted at the wrap up meeting for the clinical trial. the treatment group and control group will be unblinded. place of unblinding will be the unit where the clinical trial is in charged. executive personnel will be the chief researcher and statisticians of the unit that are in charge of the trial. if there is severe adverse event, which impedes the progress of the trial and the selection of the treatment measures, urgent unblinding can be carried out. during the process, all the researcher, sub-pi, and clinical supervisors should take part. the local administrative unit should be informed within 24 h. the reason, time, and place of unblinding should be recorded in detail and all the records should be signed off. afterwards, the clinical supervisors should be informed timely. the case data should be kept intact. prior to the start of the trial, sub-center physicians will be trained. the results of laboratory tests from different hospitals are adjusted per the huashan hospital standards during analysis. demographic information (date of birth, gender, etc.) and medical condition (medical history, concomitant medication, etc.) will be recorded at baseline. all the questionnaires will be answered by patients without inducement. when adverse events that are related to study drugs happen, emergency envelope can be considered as needed to be opened by pis and physician. the investigators will report the reasons and outcome to the pi within 24 h. prior investigation shows that the mean hospitalization duration time is about 8-9 days in these 10 hospitals, which matches the trial requirement of 9 days of treatment. after screening and completing baseline evaluations, participants will visit the physician at day 6 and day 10 during adjunctive treatments and day 30 when patients are discharged (fig. 2) . we will provide free tcm granules and partial examination reimbursement to participants. the participants and their family member will be informed that standardized treatment is beneficial to reduce copd exacerbation, which will reduce medical expenses the benefits. two telephone calls will be conducted on day 90 and day 180. the writing and transfer of case report the case report will be written by the doctor who has participated in the trial. every case should have a complete case report. the case report, once completed, should be checked by the supervisor. afterwards, it will be transferred to the data administrator for data entry and management. all the information in crf table will be recorded in a specialized clinical experimental database that is designed by chinese academy of traditional chinese medicine. the format of the database should be close to that of the crf table so as to facilitate the data entry. the variables in the crf table will be encoded and the codes will be kept unchanged during the whole process of clinical research. the crf data will be entered by highly trained specialists from the research centers. the audit of data can be divided into two forms: manual audit and system audit. the former refers that the administrator checks the consistency and logic of the data so as to find the mistakes and to generate the question list. sas software sets the limit of all variables and rules out automatically the unqualified data by running the system program. the question list is sent to the clinical supervisor who transfers it to the researcher for reconfirmation. the related revision should be signed and dated by the researcher. the researcher will correct the data for the last time after the return of all question lists. all the corrections and updates should be recorded and filed. after the data is verified, the data administration meeting will be held so that the corrections and updates can be summarized. at last, the data administrator will announce the locking of database and keep the cipher code. the statistical analysis prospectus will not be changed after the database lock. the data will be transferred to the statistical department for analysis. all the data should be kept according to the requirements of gcp. after the experiment, all the original copy of case reports and records for the administrations of clinical drugs should be checked, signed, and stamped by the supervisors, head researchers, and representatives from gcp office of each clinical center, and finally, these records will be sent to the leading site where the database will be established and the data will processed. statisticians will analyze the data and materials from the participating centers, and the summary of the clinical trial will be completed in the leading site. case report form (crf) collects all the information throughout the trial for every participant. as soon as verification is completed, data will be securely stored and sent to huashan hospital from the sub-centers. a data management group will be established, and the information will be entered into the database provided by http://www.rilintech.comt through independent doubledata entry. the errors and inconsistencies of data will be checked during the entry process. the user identification code and password will be protected by the data management group. the pis will be given access to the cleaned data sets. sub-investigators will only have access to the data sets in their own hospital. original paper forms will be kept in huashan hospital for 5 years. plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} the process and collection of blood samples separation of 1-2 ml of plasma from 4 ml of whole blood the anticoagulant and aprotinin (for concentration and amount, please refer to the note) will be added to the blood-collection tube, which is placed at 4°c for precooling, and then1.5 ml of whole blood will be collected. the samples will be mixed in the tube slowly, and afterwards, the mixture will be centrifuged at a low temperature (4°c, 4000 r/min, 15-20 min). 0.5 ml of plasma will be collected and kept at a low temperature (− 80°c). if the collected blood cannot be centrifuged immediately, it can only be stored in 4°c freezer for up to 1 h. note: the concentration and amount of anticoagulant and aprotinin. anticoagulant: 0.3medta.2na concentration (20ul/ ml) or 1% heparin (10ul/ml); aprotinin (500 iu/ml). there are two kinds of aprotinin: liquid (the concentration will be noted on the label) and solid (10,800 iu/mg). the solid form of aprotinin can be dissolved in normal saline, so its concentration can be adjusted to 500 iu/20 μl. requirements for sample storage the samples should be kept immediately in − 80°freezer. throughout the transportation, the samples cannot be taken out. in huashan hospital, all of the samples are checked. the samples should be labeled with case codes and collection date. blood serum should be kept in dry ice for transportation. primary and secondary outcomes the tcm intervention arm-jwby (jia wei bushen yiqi formulas)-will be compared against the placebo. the short-term systemic glucocorticoid (ssg) arm will be compared against the long-term systemic glucocorticoid (lsg) arm. four groups will be compared with each other independently. statistical package for social sciences for windows, version 24.0 (spss, chicago, il, usa) will be used for analysis. the tests will be 2-sided, and a p value with alpha ≤ 0.05 level is considered significant. p values will be reported to four decimal places with p values less than 0.001 reported as p < 0.001. the bonferroni method will be used to appropriately adjust the overall level of significance for multiple comparisons, assuming an exchangeable correlation structure. categorical variables will be summarized by absolute numbers and percentages of total. the difference of categorical variables will be assessed with the generalized estimating equations (gee). gee will also be used to assess the impact of potential clustering of participants in the same hospital. safety outcomes will be analyzed with summary statistics (frequency, count, percentage). the method of analysis of each variable are summarized in table 6 . the score of copd assessment test (cat) will be collected at baseline, day 6, day 10, and in the 30 days, 90 days, 180 days after discharge. the mixed effect normal model (menm) will be used to compare each outcome against the tcm intervention group and placebo. the estimate of treatment effect will be presented as unadjusted rate ratio followed by an adjusted ratio with adjustment for a set of pre-specified baseline variables. the list of pre-specified variables is as follows: centers (as a random effect), age (in years), gender (male or female), weight (in kilogram), smoking (pack per year), fev1% predicted, the number of copd exacerbation in the previous 1 year, and home-oxygen therapy. fixed effects will include the visit number, treatment, and all the prespecified variables. participant and visit interaction will be fitted as random effects. an autoregressive correction structure will be used throughout. the difference of interval time to next exacerbation during follow-up in the 30 days, 90 days, and 180 days will be compared between ssg and lsg groups using the generalized linear model (glm) with a log-link function, a propriety over dispersion parameter, and length of time as an offset. the numbers will be described respectively in three gradesoutpatient, inpatient, and icu. durations of copd exacerbation will be compared between each two of the four groups with glm in the similar manner as before. specially, the shortness of breath measured by mmrc dyspnea scale (1-5 degree) in the diary will be undertaken in the logit link function independently. the changes in tcm syndrome score, infectious index, lung function, blood gas analysis, inflammatory cytokine levels, and hpa axis will be collected in baseline, at day 6, and at day 10. glm will be used to analyze the change between each two of the four groups as well. none. in this trial, interventions for participants include 9 days of tcm granules and 5 or 9 days of prednisone. these two interventions will be carried out during hospitalization and they are routine treatments in china, so there are no anticipated problems that will be detrimental to the participant. therefore, there will be no interim analyses and there are not anticipated formal stopping rules for the trial. methods for additional analyses (e.g., subgroup analyses) {20b} subgroup analysis the potential subgroups have been listed in table 3 . the analysis of primary outcomes will be repeated in the subgroups. methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} analysis will be in accordance with the intent-to-treat principles. the safety set (ss) includes participants that are randomized and have received adjunct treatments and one post-treatment safety assessment at least. the full analysis set (fas) includes participants that are randomized and have received adjunct treatment, and their primary outcomes are available at least in one visit. the per protocol set (pps) includes participants in accordance with all the following conditions: valid baseline values, compliance with the program, no violation of the inclusion and exclusion criteria specified in the program, completion of all assessments, and good compliance (defined as participants taking at least 80% of expected doses of study drugs as determined by counting). missing data is predicted to appear on day 30 and during the two telephone calls after discharge. the imputation of all the outcomes will be replaced by the mean of the group. plans to give access to the full protocol, participant level data, and statistical code {31c} full protocol participant level dataset in chinese will be accessible in the register site. and statistical code will be provided by trial statistician. for sharing purpose, data will be available to outside investigators at the end of the trial. the finding of this trial will be published in peerreviewed journals and presented at conferences. the results of the study will be released to the participating physician and patients. composition of the coordinating center and trial steering committee {5d} multi-center trial coordination committee will be established. the huashan hospital affiliated to fudan university will take charge of the committee, and the main researchers of the participating units will serve as the members. the committee will be responsible for the implementation of the whole experiment and resolve problems during the trial process. the head researcher should strengthen quality surveillance of the clinical trial in his own center. composition of the data monitoring committee, its roles and reporting structure {21a} the data monitoring committee is unnecessary in this trial, because the drug duration in this trial is short-9 days. tcm granules and prednisone are routine treatment in china and will be carried out during hospitalization; only minimal risks are anticipated. adverse event report regardless of whether it is related to the study drug or not, any clinically significant abnormalities of medical events or laboratory tests will be defined as an adverse event (ae). for all adverse events, the time, duration, treatment measures and outcomes, the severity of the disease, and the association with the study drug will be evaluated and recorded. it is divided into mild, moderate, and severe according to the following list: conscious symptoms, ability to tolerate, impact on daily activities, duration, whether it is relieved during continued medication, and whether treatment is required. serious adverse events (sae) will be defined as death or life-threatening events. if a sae occurs, the doctor will immediately take emergency measures and report it to the pi and the ethics committee within 24 h. according to the occurrence of adverse events and a reasonable time interval, and alleviation after withdrawal of the study drugs, the correlation between adverse events and study drugs will be evaluated as affirmative (sure), probably related (very likely), may be relevant (possible), may be unrelated (suspicious), and irrelevant (impossible). due to the unsatisfactory treatment effect, the patient will withdraw from the trial. the emergency letter of the case will be opened, and the patient's family will coordinate with the follow-up and report the result to the lead center. the relevant information will be recorded in the case report form. although the formula is optimized to instant granule instead of tcm herbs decoration in our study, some participants who never accepted tcm herbal previously may have gastrointestinal reactions such as nausea and vomiting. they will be suspended for 3 days and evaluated on their abilities to continue to participate in. because the participants are in the acute exacerbation period, their disease may deteriorate to grade 3 at any time with the worsening of clinical symptoms including increase of dyspnea, mental consciousness changes, blood gas analysis of acidosis, and hypoxemia that cannot be improved by oxygen absorption or other treatments. participants will be admitted to the intensive care unit (icu) if it happens. due to the worsening of the disease or the unsatisfactory effect, the emergency letter of the case will be opened. the physician-incharge will communicate with the patient's family if participant needs to withdraw from the study. the relevant information is reported to pi and recorded in the case report form. as for any deterioration syndromes that arise after discharged, participants will be advised to come to the hospital. the investigator will provide free medical services appropriately. the recommend dose of prednisone by 2017 chinese consensus is 30-40 mg daily for 9-14 days. the low dose of 30 mg is chosen. extra management measures were suggested during the initial meeting. first, the participants will be informed that the withdrawal symptoms include fatigue, joint muscle soreness, low mood, poor appetite, and even nausea and vomiting. second, participants discharged from the hospital with adrenal insufficiency will receive instructions on how to take less than 30 mg daily if they cannot tolerate the treatment. finally, participants will be advised to take the following preventive measures against possible adverse events. closely and modulate the number of hypoglycemic agents or insulin. 2) investigators will pay attention to whether the patient has abdominal pain, vomiting of coffee-like substances, or tar-like black stool. if this occurs, the patient should promptly come to the emergency department and be treated with acid-suppressing stomach and other drugs. 3) investigators will observe the patient's neuropsychiatric symptoms closely, such as euphoria, excitement, mania, and insomnia. if necessary, advise patients to seek medical help. the designated monitor will visit each investigational site once a month. the monitor will check that if the regulatory binder is complete and all that associated documents is stored well or not, including crf, informed consent forms, and adverse events reports. and the monitor will help the investigational site resolve the issues happened in the trial. plans for communicating important protocol amendments to relevant parties (e.g., trial participants, ethical committees) {25} any modification to the protocol which may impact the conduct of the study and the potential benefit of the patients will be reported to ethic committee. the amendments will be approved by the ethics committee before it is announced to each investigational site. and an investigator training about new protocol will be held through wechat video meeting. participants will be informed of the new protocol. participant information will not be released outside of the study without the permission of individuals except for monitoring. blood samples, data collection, and administrative forms will be identified with the same code and stored separately in a locked place. all data will be uploaded to the resman original data sharing platform (ipd sharing platform) http://www.medresman. org of the china clinical trial registry, which is available to outside investigators when the trial ends. the result will be published in peer-reviewed journals and shared at conferences. the findings of the trial will be released to the participating physicians and patients. with the design of tcm as an adjunct to systemic glucocorticoids to treat copd exacerbation in this randomized trial, we will test the non-inferiority of two different treatment terms of systemic glucocorticoids in copd exacerbation. the finding will bring new proofs to the controversial applications of glucocorticoids. in addition, we will clarify a pragmatic method to identify the efficacy of classic description based on tcm syndrome differences despite limitations like bias of measurement and the subjectivity of the questionnaire assessment which may be exacerbated by the loss of some participant during follow-up. the difference between the four groups will indicate that tcm reduces the suppression of the hpa axis and strengthens the anti-inflammation effect of glucocorticoids. tcm may strongly support and enrich the management of copd exacerbation. however, there are some limitations in this protocol. firstly, we choose one of the specific tcm syndromes as the criteria. the result is hard to be extended to the whole patients with copd exacerbation. in addition, we use the chinese guideline to evaluate the degree of copd exacerbation, which relies on the subjective assessment of symptoms of the enrolled participants by the physician. hopefully, an objective method will be proposed to assess the copd exacerbation. our trial has enrolled 10 volunteers in shanghai from 07 august 2019 up to today. we have modified protocol according to the practice and standard protocol items [42, 43] . in the meantime, we proposed amendment to ethics commitment and chinese clinical trial registry in july of 2019. the protocol version is ky 2019-299,03,03, july, 2019. the new protocol was reported to all subcenter pis in a group meeting. due to covid-19, we expect to complete the recruitment process around october 2020 and report the results as soon as possible. supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-020-04669-5. additional file 1. burden of copd prevalence and risk factors of chronic obstructive pulmonary disease in china (the china pulmonary health cph study): a national cross-sectional study prevention and management of copd in china: successes and major challenges chronic obstructive pulmonary disease in china: a nationwide prevalence study mortality, morbidity, and risk factors in china and its provinces, 1990-2017: a systematic analysis for the global burden of disease study international variation in the prevalence of copd (the bold study): a population-based prevalence study copd exacerbations: defining their cause and prevention symptom variability in patients with severe copd: a pan-european cross-sectional study acute copd exacerbations expert consensus on acute exacerbation of chronic obstructive pulmonary disease in the people's republic of china treatment with systemic steroids in severe chronic obstructive pulmonary disease exacerbations: use of short regimens in routine clinical practice and their impact on hospital stay copd clinical practice guideline of the korean academy of tuberculosis and respiratory disease: a summary diagnosis, prevention and treatment of stable copd and acute exacerbations of copd: the swiss recommendations use of glucocorticoids in patients with copd exacerbations in china: a retrospective observational study controlled trial of oral prednisone in outpatients with acute copd exacerbation oral corticosteroids in patients admitted to hospital with exacerbations of chronic obstructive pulmonary disease: a prospective randomised controlled trial effect of systemic glucocorticoids on exacerbations of chronic obstructive pulmonary disease oral or iv prednisolone in the treatment of copd exacerbations -a randomized, controlled, double-blind study efficacy of corticosteroid therapy in patients with an acute exacerbation of chronic obstructive pulmonary disease receiving ventilatory support systemic corticosteroids in acute exacerbation of copd: a metaanalysis of controlled studies with emphasis on icu patients prednisone in copd exacerbation requiring ventilatory support: an open-label randomised evaluation global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease: the gold science committee report 2019 susceptibility to exacerbation in chronic obstructive pulmonary disease acute exacerbations of chronic obstructive pulmonary disease identification of biologic clusters and their biomarkers blood eosinophils to direct corticosteroid treatment of exacerbations of chronic obstructive pulmonary disease: a randomized placebo-controlled trial effects of smoking intervention and the use of an inhaled anticholinergic bronchodilator on the rate of decline of fev (1) -the lung health study zheng: a systems biology approach to diagnosis and treatments oral chinese herbal medicine for improvement of quality of life in patients with stable chronic obstructive pulmonary disease: a systematic review efficacy and safety of indacaterol 150 and 300 mu g in chronic obstructive pulmonary disease patients from six asian areas including japan: a 12-week, placebo-controlled study bu-fei yi-shen granule combined with acupoint sticking therapy in patients with stable chronic obstructive pulmonary disease: a randomized, double-blind, double-dummy, active-controlled, 4-centre study effects of comprehensive therapy based on traditional chinese medicine patterns in stable chronic obstructive pulmonary disease: a four-centre, open-label, randomized, controlled study effects of yupingfeng granules on acute exacerbations of copd: a randomized, placebo-controlled study effects of two chinese herbal formulae for the treatment of moderate to severe stable chronic obstructive pulmonary disease: a multicentre, double-blind, randomized controlled trial important action of improving adrenocortical function for certain diseases recovery effect and mechanism of several traditional chinese medicine components on inflammatory response of chronic obstructive pulmonary disease caused by exposure to cigarette smoke scutellaria baicalensis attenuates airway remodeling via pi3k/akt/nf-kappab pathway in cigarette smoke mediated-copd rats model paeonol attenuates cigarette smoke-induced lung inflammation by inhibiting ros-sensitive inflammatory signaling a real-world evidence study for distribution of traditional chinese medicine syndrome and its elements on respiratory disease short-term vs conventional glucocorticoid therapy in acute exacerbations of chronic obstructive pulmonary disease: the reduce randomized clinical trial spirit 2013 statement: defining standard protocol items for clinical trials spirit 2013 explanation and elaboration: guidance for protocols of clinical trials publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank dr. yiyuan zeng and dr. waijiao cai from school of public health, boston university, for their linguistic assistance during the preparation of this manuscript. qing kong and shuming mo drafted the manuscript. wenqian wang, zihui tang, baojun liu, yijie du and lingwen kong participated in the design of the study. zihui tang participated in the statistic plan. ying wei, yubao lv and jingcheng dong conceived the study, participated in its design and coordination, and drafted the manuscript. all authors read and approved the final manuscript. all named authors adhere to the authorship guidelines of trials; no professional writers have been involved. the enrolled participants who sign the informed consent forms about clinical data and bio-sample collection are provided with free medication of jwby formulas and prednisone for 9 days as needed. participants' information will not be released outside of the study without the permission of individuals except for monitoring. blood samples, data collection, and administrative forms will be identified with the same code and stored separately in a locked place. all data will be uploaded to the resman original data sharing platform (ipd sharing platform) http://www.medresman.org of the china clinical trial registry, which is available to outside investigators when the trial ends. the result will be published in peer-reviewed journals and shared at conferences. the findings of the trial will be released to the participating physicians and patients. central ethics committee approval has been obtained from ethics committee of huashan hospital affiliated to fudan university in shanghai, china (id: ky-2019299). the local ethics committee of other ten hospitals has approved the protocol, too. the trial was registered on www.chictr.org.cn (id: chictr1900023364) on may 24, 2019. the investigator will make safety and progress reports to the ethics committee monthly. protocol amendment will be approved by the ethics committee prior to the implementation of amended protocol at the sub-centers. all investigators are trained to carry out the new protocol. there is no conflict of interests among the subcenters. informed consent will be obtained from all study participants. these are available from the corresponding author upon request. there are no competing interests in this work.author details key: cord-275889-4qwp3um1 authors: guarnieri, m.; brayton, c.; sarabia-estrada, r.; tyler, b.; mcknight, p.; detolla, l. title: subcutaneous implants of a cholesterol-triglyceride-buprenorphine suspension in rats date: 2017-04-09 journal: j vet med doi: 10.1155/2017/3102567 sha: doc_id: 275889 cord_uid: 4qwp3um1 a target animal safety protocol was used to examine adverse events in male and female fischer f344/ntac rats treated with increasing doses of a subcutaneous implant of a lipid suspension of buprenorphine. a single injection of 0.65 mg/kg afforded clinically significant blood levels of drug for 3 days. chemistry, hematology, coagulation, and urinalysis values with 2to 10-fold excess doses of the drug-lipid suspension were within normal limits. histopathology findings were unremarkable. the skin and underlying tissue surrounding the drug injection were unremarkable. approximately 25% of a cohort of rats given the excess doses of 1.3, 3.9, and 6.5 mg/kg displayed nausea-related behavior consisting of intermittent and limited excess grooming and self-gnawing. these results confirm the safety of cholesterol-triglyceride carrier systems for subcutaneous drug delivery of buprenorphine in laboratory animals and further demonstrate the utility of lipid-based carriers as scaffolds for subcutaneous, long-acting drug therapy. the challenge of providing long-acting drug therapy to laboratory animals has been managed by adding drugs to the feed or water supplies [1, 2] . the utility of this method decreases when the mixture may be released inadvertently to the environment, or the drug is regulated, such as controlled substance. feed-based drugs also may have limited postsurgical applications because pain can suppress appetite. alternative approaches have focused on long-acting drug implants made by combining a drug with biodegradable envelops composed of lipids or polymers. polymers have been studied as drug carriers for neurooncology [3] . side effects generally have been modest and localized when the polymer is implanted into neural tissue [4] . less is known about biodegradable polymers for subcutaneous (sc) delivery of chemotherapy. moderate to severe inflammatory reactions have been reported for sc implants of polymer-opiate constructs [5] [6] [7] [8] [9] [10] . we investigated the properties of lipid-based delivery vehicles. cholesterol, triglycerides, and phospholipids have been widely used as drug-carriers [11] . kent described an implantable cholesterol matrix that delivered large molecules such as insulin and growth hormone [12] . grant and coworkers demonstrated that a phospholipid-morphine liposome had prolonged activity and greater safety in mice than the free drug [13] . liposomal strategies have been refined for the delivery of several opiates [14] . pontani and misra described a cholesterol-triglyceride matrix for the long-term delivery of drugs to treat chronic pain and opiate addiction [15] . the cholesterol-triglyceride vehicle appeared to provide a promising carrier to examine the delivery of antibiotics, anti-inflammatory drugs, and analgesics in surgically treated animals. to investigate the safety of this system we chose 2 journal of veterinary medicine buprenorphine as a model drug. it has a high therapeutic index [16] and is a front-line analgesic for animals [17, 18] . the present report describes bioequivalence studies demonstrating that a 0.65 mg/kg dose of a lipid-buprenorphine suspension provides blood concentrations of drug greater than 0.7 ng/ml for 2-3 days. this concentration has been associated with clinically effective analgesia in mice, dogs, and humans [19] [20] [21] [22] [23] . a standard analgesiometric test confirmed the efficacy of the intended 0.65 mg/kg dose. safety studies used a target animal safety (tas) trial format [24, 25] . surgically treated male and female rats were injected with multiple overdoses of the drug suspension and monitored for adverse events (ae). the trials included clinical tests, histopathology studies, and clinical observations. the results described in the present report, when taken together with a previous report on the safety of a lipid-buprenorphine suspension in mice [26] , provide further evidence that sc drug implants using lipid envelopes increase options for longterm drug therapy in rats. a preliminary account of this research has been published [27] . studies were approved by the johns hopkins institutional animal care and use committee. the protocol complies with the national institutes of health guide for the care and use of laboratory animals and the requirements of the association for the assessment and accreditation of laboratory animal care international program. guidelines for tas studies specify a minimum number of three animals per group. four rats were used in tas studies to account for potential morbidity during jugular vein phlebotomy. fischer f344/ntac rats, 6-8 weeks old (male 160-180 g; female 120-130 g), were obtained from taconic farms (hudson ny) and housed in an environmentally controlled room which maintained the temperatures of 20 to 26 ∘ c. monthly health surveillance was conducted by a soiled-bedding sentinel system. sentinel rats were considered negative for pneumonia virus of mice, reovirus, sendai virus, lymphocytic choriomeningitis virus, rat coronavirus, sialodacryoadenitis virus, rat parvovirus, kilham rat virus, toolan h1 parvovirus, rat theilovirus, cilia-associated respiratory bacillus, pneumocystis carinii, mycoplasma pulmonis, and pinworms throughout the study. the facility maintained a relative humidity of 30 to 70% with a 12-hour light cycle with lights on from 6 am-6 pm. animals were ear tagged and group housed (up to 3 per cage) during the quarantine and acclimation period based on sex. the rats were quarantined and acclimated for six days prior to dosing. no disease-related signs were noted during the quarantine/acclimation period. prior to being placed on test, a clinical veterinarian approved the animals for study use. all rats appeared normal prior to dosing. the animals assigned to the two tas trials were randomized by body weight into four groups per trial of 4 male and 4 female rats using random numbers generated by excel (microsoft corp., redmond, wa). the weight of each animal was within 10% of the mean weight of the group. on allocation and dosing, rats in the bioequivalence, efficacy, 4-day, and 12-day tas trials were individually housed in ventilated microisolator cages throughout the study. cages were changed daily to reduce redosing by coprophagy. studies used soft fiber bedding from carefresh natural bedding (ferndale, wa) to house the rats. animals were provided ad libitum with access to drinking water (baltimore city water system, baltimore, md) in disposable water bottles. the animals were provided ad libitum with access to harlan teklad certified global rodent diet 2016c (harlan teklad, indianapolis, in). rats were provided with enrichment devices of polycarbonate red tubes (bio services, uden, the netherlands). design. the intended label dose of 0.65 mg/kg, which provides 2-3 days of clinically significant blood levels of drug, was established in bioequivalence trials and efficacy studies to be described using male and female rats. single-and repeat-dose tas trials were performed using excess amounts of the intended dose. in both safety trials, the lowest dose of drug tested was twofold greater than the intended label dose of 0.65 mg/kg. in the single-dose phase of the trials, 4 groups of 8 rats (4 male, 4 female) were dosed after surgery (described below) with 0.0 (vehicle control), 1.3 (2x), 3.9 (6x), or 6.5 (10x) mg/kg drug suspension of buprenorphine on day 0. the volumes of the vehicle control, 2x, 6x, and 10x doses were 1.0, 0.2, 0.6, and 1.0 ml, respectively. as shown in table 1 , blood and urine samples were collected at day 2. on day 4 animals were euthanized and blood and urine collected. in the repeat-dose trials, 4 groups of 8 rats (4 male, 4 female) were dosed after surgery with the vehicle control or drug suspensions containing 1.3, 3.9, or 6.5 mg/kg of buprenorphine on day 0 and following anesthesia on days 4 and 8. surgery was not repeated on days 4 and 8. blood and urine samples were collected at day 6. blood, urine, and histopathology studies were conducted on tissues collected following euthanasia on day 12 (table 1 ). a surgical procedure was performed to mimic the implantation of an implantable pump or a telemetry device. each rat was anesthetized by isoflurane gas at approximately 3% with an oxygen flow rate of 1% during the procedure. the duration of the anesthesia exposure was approximately 2 minutes. following induction of anesthesia, the scapular surface (between the shoulder blades) was shaved, washed with ethanol, and then coated with betadine. the animal was transferred to a clean procedural area where it was assessed to ensure a deep surgical plane of anesthesia using the toe pinch method. once deep anesthesia was verified, breathing rate and capillary fill rate were documented. clean, sterilized forceps were used to gently grasp the skin, and then clean, sterile scissors were used to make a 4-5 mm incision through the skin only. bone and muscle were not penetrated. the clean, sterile scissors were used to separate the skin 2 cm rostral and distal, and 2 cm lateral in the subcutis, and create a subcutaneous pocket (approximately 2 × 4 cm). the incision was then apposed and stapled using a 9 mm autoclip5 (kent scientific, torrington, ct). the drug suspension consisted of buprenorphine, cholesterol, and glycerol tristearate, suspended in a medium-chain triglyceride oil (8 mg/100 ul), trade name animalgesics for mice. control and drug suspensions were supplied by animalgesic labs (millersville, md). to limit stress associated with constraining conscious animals for sc injections, each rat was injected with the designated dose of test article or buprenorphine-free control suspension following surgery before they recovered from anesthesia in the single-dose trial. in the repeat-dose trial, rats were injected with drug following surgery and under anesthesia on day 0 and under anesthesia on days 4 and 8. the dose was administered sc on the middorsal area about 1 cm rostral to the surgical incision using a 25 g needle (bd, franklin, nj) attached to a 1 ml bd tuberculin syringe. following dose administration, animals were transferred to a clean cage on a heating pad until recovered. once the rat regained consciousness and demonstrated normal movement and the absence of signs of distress, it was returned to its home cage. male and female rats were provided with a single dose of drug and sampled at time intervals from 8 hours to 9 days to measure blood concentrations of buprenorphine. rat blood samples were obtained from technician-restrained, unanesthetized animals by jugular vein phlebotomy. one ml disposable syringes with 20gauge needles were used to collect approximately 0.4 ml of blood. the sample was immediately transferred to bd tubes containing dipotassium edta. the samples were stored on ice for approximately 1 hour and then centrifuged to collect plasma. the plasma was stored at −20 ∘ c until it was thawed for analysis. buprenorphine plasma concentrations were measured by the mcwhorter school of pharmacy, samford university (birmingham, al), using a shimadzu lc-20ad (columbia, md) and mass spec applied biosystems 4000 qtrap (carlsbad, ca) assay requiring 0.25 ml of plasma. the sensitivity of the assay was 0.5 ng/ml. mean concentration-time data was used for the pharmacokinetic analysis. noncompartmental-analyses module in phoenix winnonlin version 5.3 (princeton nj) was used to assess the area under the curve (auc) and the maximum plasma concentration ( max) time at which the cmax is realized ( max). max and max were the observed values. the auc was calculated by the log-linear trapezoidal rule to the end of the sample collection (auclast) [28] . 2.6. efficacy. studies were conducted with 18 f344 female rats at two dose levels: 0.65 and 1.3 mg/kg. rats were injected with vehicle, 0.65, and 1.3 mg/kg dose of drug and tested for their tail flick response using a 55 ∘ c water bath. tests were conducted by a female veterinarian who was blinded to the treatment group. female rats were used because mu opioid agonists such as morphine appear to be less sensitive in female than male rats [29, 30] . the rats were injected with drug or vehicle on day 0 and examined for 5 days to monitor tail flick reaction times [31] . the rats were housed 3 per cage and cages were changed daily. during the course of this study, animals were observed at the cage level once daily by a single observer prior to 9 am for morbidity, mortality, and signs of pain or stress: abnormal breathing, tremors, ocular discharge, facial signs (squinting, eyes closed), posture, and movement and overall appearance including condition of the hair coat and grooming. incision sites were examined at the 9 am time for bleeding, swelling, or signs of infection. because pica was not expected, methods to measure it, such as kaolin intake, were not used. animals received "hands-on" detailed clinical observations once daily by a single observer after 2 pm for abnormal clinical signs (ocular discharge, motor activity, and signs of pain or distress). incision sites were examined again at this time for bleeding, swelling, and signs of infection. body surface was inspected for skin lesions. the process used to observe and record nausea-related behaviors has been published [27] . briefly, observers noted in comments on the report form of hair loss and the presence of lesions as evidence of excessive grooming or self-gnawing behavior. they did not grade the amount of hair loss or the degree of biting. we considered any reports of hair loss or lesions on the paws to be signs of nausea-related behavior, and we recorded the number of rats of each sex in each experimental group that showed these signs during each observation period [27] . the observers were blinded to the treatment group. approximately 38,000 data points were recorded in the two trials. in addition, the observers could add comments to each chart. the two male observers were certified (aalas) lab animal technologist and technician. weights of individual animals were taken for randomization (prior to study start), at midpoints (day 2 and day 6) and endpoints (day 4 and day 12) of the safety trials by an observer blinded to the dose. pathology. blood and urine samples were collected at the mid-and endpoint of the each tas trial. blood was collected in the morning via jugular vein puncture. approximately 0.5 ml of blood was collected for the midpoint clinical pathology studies. the midpoint sample was transferred to a collection tube containing dipotassium edta. approximately 0.5 ml of the endpoint sample was transferred to a collection tube containing dipotassium edta, and 1 ml was transferred to a collection tube containing sodium citrate for coagulation factor measurements. the samples were refrigerated before analyses. the hematology examination included red blood cell (rbc) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell (wbc) count, differential blood cell count, and blood smear. clinical chemistry tests included glucose, urea nitrogen, creatinine, total protein, albumin, globulin (as calculation), total cholesterol, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, calcium, sodium, potassium, chloride, and phosphate. in the two tas trials, coagulation factor measurements were prothrombin time, activated partial thromboplastin time, and fibrinogen. because of the amount of blood needed, coagulation factors were analyzed only on endpoint days 4 and 12. expressed urine samples were collected in the afternoon on a clean surface and pipetted into a sterile eppendorf tube. urine dip sticks (bayer multistix 10 sg reagent strips, romeoville, il) were read manually. these tests measured ph, appearance, color, protein, glucose, bilirubin, and blood. after the final collection of blood and urine specimens on endpoint days 4 and 12, animals were humanely euthanized using co 2 inhalation, followed by a thoracotomy. death was confirmed by cessation of heart rate. a comprehensive necropsy was then performed for each animal. once the lungs were examined and weighed they were inflated with formalin to ensure proper fixation. tissues were placed in an individually labeled container containing 10% neutralbuffered formalin, with the exception of testes (males) and eyes with optic nerves, which were preserved in modified davidson's fixative. for short term studies, testes and eyes with optic nerves were transferred from the modified davidson's solution to 70% ethanol 1-2 days following collection. the transfer was performed and documented by the histology lab. containers were labeled with study number, date, group number, and animal number. organ weights included adrenal, brain, epididymis, heart, kidney, liver, lung, ovaries, spleen, testes, thyroid with parathyroid, and uterus with cervix. according to tas protocols, histopathology studies were performed on the vehicle control animals and the animals given the highest dose of drug in the single-and repeat-dose trials, table 1 . unless significant pathology was observed at the highest doses, slides from the two lower doses were not examined. slides from the vehicle control animals and both doses in the long-term study were examined. thirty-three tissues were harvested for histopathology examination of organs including the dorsal skin surrounding the injection site: adrenal gland, large intestine, colon, small intestine (jejunum, ileum, and duodenum), large intestine (cecum), liver, bone with bone marrow, femur, urinary bladder, lung, spinal cord with spine, brain (cerebrum, midbrain, cerebellum, and medulla/pons), lymph nodes including submandibular superficial cervical collected with salivary glands from the neck, mesenteric and pancreaticoduodenal collected with mesentery and pancreas, spleen, epididymis (males), mammary glands (females), stomach, eyes with optic nerve, ovaries (females), ventral skin, gall bladder, pancreas, heart, and parathyroid gland. parathyroid glands were evaluated when present in the plane of section of the thyroid gland, thyroid (with parathyroid), testis (male), kidneys, and skeletal muscle (biceps femoris). a comprehensive statistical analysis (mean, standard deviations, ) was conducted for group mean body weight data comparing treated groups to the control group of each sex using one-way analysis of variance (anova). an additional zero-inflated poisson regression was conducted to estimate the dose and sex differences over time for pica behavior [32] . the zero-inflated poisson regression model provides robust estimates and hypothesis tests for count data with a predominance of zeros. statistical analyses (mean, standard deviations, ) were conducted for organ weight and clinical pathology data comparing treated groups to the control group using one-way analysis of variance (anova). additionally, dunnett's -test was used for control versus treated group comparisons [33] . studies. the bioavailability of buprenorphine and its pharmacokinetic profile in male and female rats at defined time points following a single sc injection of the test article was examined. as shown in table 2 (a), a single dose of 0.65 mg/kg provided at least 2 days of clinically significant drug concentrations, defined as greater than 0.75 ng/ml of plasma buprenorphine. a single female had a significant concentration of plasma buprenorphine at day 7. table 2 (a) also shows the concentrations of blood present in rats dosed with 1.3 mg/kg of drug. both male and female rats had clinically relevant plasma concentrations of buprenorphine throughout the 4 days of the single-dose and repeat-dose trials in which the lowest dose evaluated for adverse effects was 1.35 mg/kg (table 2(a)). the estimated values for the auc, max, and max are given in table 2 (b). max for female rats in both dose groups was 24 hours. max for male rats in the 1.3 mg/kg dose group moved to 48 hours based on a slightly greater mean blood concentration of buprenorphine on day 2. max values in the 0.65 mg/kg group were 3.4 and 1.8 ng/ml for male and female rats, respectively. the estimated max values for male and female rats in the 1.3 mg/kg dose group were 2-fold greater in male rats and 5-fold greater in females. the data in table 3 shows that the extended release preparation of buprenorphine provided significant analgesic effects ( > 0.05) at 0.65 and 1.3 mg/kg dose. all rats survived to the scheduled termination date in both trials. rats dosed with the test article on average appeared with slightly slower movement scores when compared with the vehicle control rats on study day 0, approximately 5 hours after dose administration. minor wounds on the front paw or wrists associated with excess grooming and self-gnawing were noted in the drug treated animals in both trials. excessive grooming and self-gnawing behavior were not observed during the morning observation period that was given to each animal. this behavior was inferred by the absence of hair and the presence of a wound on the paw. the observer noted the findings as a comment on the animal's chart. the amount of hair loss and the degree of biting were not graded. there was no evidence of an open wound. these nausea-related signs were seen in the pm observation cycle when the animals were physically handled during an examination of the surgery site and monitoring of the entire skin surface for lesions. signs of nausea-related behavior were first noted in one male animal 1 day after an analgesic injection of 3.9 mg/kg, in the single-dose trial, table 4 . a single male rat in the 1.3 mg/kg dose group exhibited the behavior on day 2 and on day 3. the rats in the 3.9 mg/kg dose group showed an increasing incidence over time of the behavior and had the highest cumulative number of animals exhibiting the signs with = 1 on day 1, = 2 on day 2, and = 3 on day 3. the highest dose group of 6.5 (10x) mg/kg exhibited a delayed onset and a lower incidence of the behavior compared to the 3.9 mg/kg (6x) group ( = 2 on day 3). a similar pattern was seen in the repeat-dose trial. overall, by day 3, self-licking or paw biting was seen in 6 (25%) of the animals in the drug treated groups (beta = 1.01, se = 0.41, and = 0.01). the behavior consistently focused on the forepaws. male and female rats exhibited similar rates of these types of behavior (beta = −0.406, se = 0.65, and = 0.53). three of the animals in the vehicle control group of the repeat-dose trial exhibited this behavior. no signs of pain or distress were noted in the animals. all males and one female rat treated with 3.9 mg/kg buprenorphine lost weight between days 0 and 4 in the single-dose trial ( table 5) . two of the male rats treated with 3.9 mg/kg of buprenorphine continued to lose weight during the course of the study, while the other two gained weight between days 2 and 4 and had an overall weight gain during the course of the study. all female rats treated with 3.9 mg/kg buprenorphine lost weight between days 2 and 4, but only two of them had an overall weight loss during the course of the study. all male rats treated with 6.5 mg/kg buprenorphine lost weight between days 0 and 2, while all females treated with the same dosage gained weight. alternatively, all female rats treated with 6.5 mg/kg buprenorphine lost weight between days 2 and 4, while all males treated with the same dosage gained weight. as shown in table 5 , a similar pattern of weight changes was seen in the repeat-dose trial. male rats in the 6.5 mg/kg dose group treated with 3 doses in 8 days gained the least amount of weight by day 12. female rats in the 1.3 mg/kg repeat-dose group showed the least weight gain. overall, there were no significant changes in body weights between the vehicle control and drug treated rats. there were no significant changes in organ weight measurements with increasing doses of drug in either male or female rats in the single-dose trial. organ weights for livers of males in the 3.9 mg/kg and 6.5 mg/kg groups in the repeatdose trial showed significant post hoc differences when compared to the vehicle control group, but no treatmentrelated effects were seen upon microscopic examination of the tissues. organ weight measurements for brain, heart, kidneys, spleen, and thyroid remained essentially unchanged. increasing doses of drug also had no effects on the weights for epididymis and testes in males and uterus in females. the average weight of adrenal glands in male rats increased from a control value of 0.050 to 0.074 g in the highest dose group. the change was not seen in female rats. average liver weights decreased in drug treated male and female rats. there were no significant weight changes in the organs, other than the liver, of the rats in the long-term study. tests of expressed urine in the single-dose trial detected urine protein in 11 (34%) and 16 (50%) of 32 rats on day 2 and day 4, respectively. in the repeat-dose trial, protein was detected in all animals in both day 6 and day 12 samples. the finding did not correlate with the dose group or sex. in the single-dose trial trace amounts of blood were detected in 21 (66%) and 10 (31%) rats on day 2 and day 4, respectively. in the repeatdose trial, blood was detected in 7 (22%) rats on day 6 and 2 (6%) rats on day 12. in both trials, values varied from trace to moderate levels. the findings did not correlate with sex or dose group. tests for glucose and bilirubin were negative for all samples. appearance, ph, and color were normal in all samples in both trials. coagulation factor tests were performed on blood from day 4 of the single-dose trial and day 12 from the repeat-dose trial. prothrombin time, activated partial thromboplastin time, and fibrinogen levels were normal in all dose groups in male and female rats. average differences in values between control and drug groups were noted in 9 of 14 hematology values and 14 of 16 clinical chemistry parameters in one or both tas trials. these parameters were examined to determine whether changes with drug treatment varied or increased with increasing dose in male or female rats. in numerous cases, differences between control values and values from animals seen at 1.3 mg/kg dose levels were not seen in the 3.9 and 6.5 mg/kg dose levels. when differences were noted between the controls and the animals receiving drug-challenges, the changing values remained within the normal range or equaled values in the control group. rbc values consistently decreased in day 4 and day 12 endpoint collections compared to the day 2 and day 6 midpoint values in all groups. this change was attributed to blood loss due to the previous blood collection. there was a slight increase in wbc counts after phlebotomy, an increase that we have observed in mouse phlebotomy [34] . among the rbc indices there were no significant differences between the control groups and the animals in the 6.5 mg/kg dose groups. this indicates that the differences noted per group and sexes were random. there was no evidence of leucopenia or cytosis. absolute values for nucleated wbcs were unremarkable, including neutrophils, eosinophils, and basophils. alanine aminotransferase and aspartate aminotransferase are sensitive yet modestly specific indicators of hepatocyte damage. elevations in these serum or plasma enzyme activities are expected in drug-induced hepatotoxicity. in the present study, the enzyme levels in the drug groups closely resembled control values, even at the highest levels of drug tested. in several groups, they were modestly decreased. serum alkaline phosphatase (alp) can be altered by physiologic or pathologic changes in various tissues including kidney, hepatobiliary, intestine, and bone. in the present study, alp values decrease significantly with increasing dose challenges. sustained decreased levels of alp have been associated with loss of appetite and fasting. cholesterol, bun, creatinine, and calcium levels show small but inconsistent, and not significant, changes between the drug and control groups. the values in the drug and control groups remain within established laboratory normal values. electrolyte levels were unremarkable: chloride, sodium, and potassium. as shown in table 6 , total protein levels on average were decreased in the highest dose group compared to controls. the decrease was not significant in the 1.3 and 3.9 mg/kg dose groups. the decrease in total protein levels appeared entirely related to decreased albumin levels with increasing drug exposure, table 6 . primary factors affecting albumin synthesis include protein and amino acid nutrition, colloidal osmotic pressure, the action of certain hormones, and disease states. fasting or a protein-deficient diets cause a decrease in albumin synthesis as long as the deficiency state is maintained. in the long-term study bun values were decreased in the 1.3 mg/kg group compared to the 0.65 mg/kg group and the vehicle controls, 13.3 ± 1.0, 15.5 ± 1.0 and 16.5 ± 1.3 mg/dl, respectively. histopathology. the single-dose study reported a macroscopic observation of the thymus of one male rat in the control dose group presenting as "discolored red." microscopically, this presented with hemorrhage and was considered an incidental finding, possibly related to terminal cardiocentesis. no other microscopic changes were observed. in the repeat-dose trial, macroscopic observations during necropsy included reddened mandibular lymph node in one vehicle control female and one male rat treated with 6.5 mg/kg, subcutaneous hemorrhage below the injection site in one female and two males treated with 6.5 mg/kg, fluid filled uterus in one female treated with 1.3 mg/kg, thickened skin lateral to the site of injection in one female treated with 6.5 mg/kg, and 8 × 5 × 4 mm nodule on the median lobe of the liver in one female in the 6.5 mg/kg dose group. although organ weights for livers of males in the 3.9 mg/kg and 6.5 mg/kg groups in the repeat-dose trial showed significant post hoc differences when compared to the vehicle control group, no treatment-related effects were seen upon microscopic examination of the tissues. inflammatory changes (granulocytic infiltration and mixed cell infiltrates) and hemorrhage were commonly seen at increased severity at or near the dorsal skin injection sites in rats in the 6.5 mg/kg dose group. similar changes were seen in the vehicle control group. no other microscopic changes were observed. the objective of this study was to evaluate the safety of a lipid suspension of buprenorphine for delivery of postprocedural pain relief in f344 rats. blood level measurements demonstrated that a single 0.65 mg/kg sc dose of a cholesteroltriglyceride buprenorphine suspension provided significant blood concentrations of drug for at least two days (table 2(a)). following declining mean plasma concentrations of drug on days 3 and 4, a single female rat in this dose group had an elevated blood concentration at day 7. this secondary peak may be attributed to redosing by coprophagy. studies have shown that more than 75% of an initial dose of buprenorphine is excreted unmetabolized within one week [35] . max for the intended dose of 0.65 mg/kg was 24 hours in male and female rats. the estimated auc for the female rats given single 0.65 mg/kg was about 60% the value for male rats given the same dose but slightly greater than males when given the 1.3 mg/kg dose (table 2(b)). a comparison of the parameters between the two dose groups is difficult because little is known about the pharmacokinetics of sc lipid drug delivery systems. blood was not collected after day 4 from the rats in the 1.3 mg/kg test group to limit potential morbidity associated with jugular phlebotomy. efficacy studies using a potentially painful stimulus confirmed that a 0.65 mg/kg dose in male and female rats provided analgesia for at least three days ( table 4 ). reviews of the specificity of these tests have demonstrated that thermal sensitivity tests provide a good predictor of clinical efficacy in humans [17, 36] . high thermal latency measurements at days 4 and 5 at the 0.65 mg/kg dose level are somewhat surprising because bioequivalence tests on a separate cohort of male and female rats at this dose level (table 3 ) demonstrated that mean blood levels of buprenorphine dropped below 0.4 mg/ml by day 4. yet, the results are consistent with the studies of an extended release buprenorphine depot in human volunteers showing blood level decreases of drug below 0.75 mg/ml at the end of the first week and significant clinical effects maintained for almost 6 weeks [37] . buprenorphine and its metabolite norbuprenorphine are rapidly converted to glucuronide conjugates in rats [38] . both conjugates have biologic activity [39] and may be relatively undetected in standard lc/ms assays. a standard safety trial format in the present study used excess dose challenges to monitor adverse effects that might occur in real world situations where the animal was given a repeat dose of the drug or had morbidities that could enhance drug toxicity. opiates as a class have been associated with respiratory deficiency. studies in rats have demonstrated that compared to morphine, fentanyl, and methadone there is a ceiling effect on the action of buprenorphine [40, 41] . the present study demonstrates that prolonged buprenorphine therapy in a lipid envelop can be safely tolerated in young adult f344 rats, but the effects on other species, older animals, and transgenic rats remain unknown. decreasing efficacy, tolerance, and hyperalgesia have been attributed to opiates including buprenorphine [42, 43] . studies have illustrated a complex association between experimental designs, chronic drug therapy, and hyperalgesia [44] . no significant signs of locomotor activity or hyperalgesia were observed in the studies described here. of interest, questions concerning the clinical significance of "hyperalgesia" appear to have been mooted by the studies of chronic buprenorphine therapy using transdermal skin patches. in all cases, reported hyperalgesic signs have been minimal in rats and humans [45] [46] [47] [48] . in 1977, cowan et al. described the first report of buprenorphine-induced "nausea" in rats: increased stereotyped licking and biting movements [49] . mitchell and coworkers demonstrated the association of nausea with pica by spinning rats to induce motion sickness [50] . yamamoto et al. demonstrated that radiation sickness induced pica [51] . takeda and coworkers confirmed the association by treating rats with opiate inhibitors to prevent nausea [52] . de jonghe et al. demonstrated that pica in rats is an adaptive response to nausea [53] . drugs that block mu receptors such as methylnaltrexone can be used to block the emetic effects of opiates in humans and pica in rats [54] . the two tas trials at 1.3, 3.9, and 6.5 mg/kg doses, which were conducted on soft bedding, reduced a risk of intestinal blockage and allowed a prospective determination of the rate of emetic behavior. as shown in table 5 , the observed cumulative rate of nausea signs in the 4-day, single-dose trial increased to 25%. the observed cumulative rate was 19% in the 12-day repeat-dose trial. the animals were identified by the observers in the pm observation cycle who examined the dorsal skin surfaces of the paws. the actual rate in the 4-day trial may have been higher because animals were removed from the study before the pm observation. the rate did not increase in the 12-day trial with increasing doses of drug. in both trials, the behavior was self-limiting and produced no apparent lasting consequences. this incidence is similar to the incidence of nausea-related behavior reported in human patients treated with opiate therapies [55] . weight loss has been cited as a deterrent to the use of postsurgical buprenorphine analgesia, and it has been linked to significant morbidity secondary to gastrointestinal blockage associated with hardwood bedding [56, 57] . a number of reports between 2000 and 2010 described weight loss in rats treated with buprenorphine without reference to the bedding used in the experiment [58] , or they report using hardwood bedding without reference to previous reports associating hardwood bedding with pica [39] . previous studies have demonstrated that the risk of pica-related gastric distress can be controlled by the appropriate choice of bedding [59] . the studies reported here confirm this observation. there do not appear to be clinically significant treatmentrelated effects following repeated subcutaneous injections of an extended release lipid suspension of buprenorphine at 1.3 mg/kg, 3.9 mg/kg, or 6.5 mg/kg dose. although several clinical pathology findings exceeded normal limits and urinalysis results showed abnormal parameters, there were no correlated changes or findings in body weights, clinical observations, organ weights, or microscopic evaluation of tissues. m. guarnieri owns a significant financial interest in animalgesic labs. the authors declare that they have no conflicts of interest. voluntary ingestion of nut paste for administration of buprenorphine in rats and mice oral self-administration of buprenorphine in the diet for analgesia in mice local delivery of rapamycin: a toxicity and efficacy study in an experimental malignant glioma model in rats brain biocompatibility of a biodegradable, controlledrelease polymer in rats pharmacokinetic comparison of sustained-release and standard buprenorphine in mice duration of action of sustained-release buprenorphine 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buprenorphine: pharmacokinetic, efficacy and skin irritancy studies opioids and the management of chronic severe pain in the elderly: consensus statement of an international expert panel with focus on the six clinically most often used world health organization step iii opioids (buprenorphine, fentanyl, hydromorphone, methadone, morphine, oxycodone) the animal pharmacology of buprenorphine, an oripavine analgesic agent motion sicknessinduced pica in the rat establishment of an animal model for radiation-induced vomiting in rats using pica pica in rats is analogous to emesis: an animal model in emesis research pica as an adaptive response: kaolin consumption helps rats recover from chemotherapy-induced illness methylnaltrexone prevents morphine-induced kaolin intake in the rat opioid-induced emesis among hospitalized nonsurgical patients: effect on pain and quality of life pica behavior associated with buprenorphine administration in the rat adverse effects on growth rates in rats caused by buprenorphine administration correlation between body weight changes and postoperative pain in rats treated with meloxicam or buprenorphine influence of buprenorphine analgesia on post-operative recovery in two strains of rats the authors acknowledge the support of dr. rana rais for the analyses of the pharmacokinetic parameters and dr. greg gorman for the analyses of plasma buprenorphine. funding for this research was supplied by the maryland biotechnology center biotechnology development awards, maryland industrial partnerships (mips), and by animalgesic labs. key: cord-018239-n7axd9bq authors: rusoke-dierich, olaf title: travel medicine date: 2018-03-13 journal: diving medicine doi: 10.1007/978-3-319-73836-9_32 sha: doc_id: 18239 cord_uid: n7axd9bq before travelling to other countries, thorough travel advice should be provided. not only information about diseases of specific countries but also general advice for travelling should be given on this consultation. before travelling to other countries, thorough travel advice should be provided. not only information about diseases of specific countries but also general advice for travelling should be given on this consultation. the following topics should be included in the travel advice consultation: 5 vaccinations (general and country specific) 5 country-specific diseases 5 malaria prophylaxis 5 mosquito prophylaxis (wearing bright long-sleeved clothes, avoiding perfume, staying in air-conditioned rooms, using a mosquito net, using insect repellents, staying inside at dawn and dusk) 5 food consumption and drinking overseas (no consumption of ice cubes, uncooked meals, salads and food, which is exposed to flies, limited alcohol consumption) 5 uv protection (using sun cream, avoiding sun exposure between 11.00 and 15.00 o' clock, remaining in shaded areas, wearing a hat and covering skin) 5 fitness assessment for travelling, flying and diving 5 challenges of different climates and their effects on the personal health (dehydration, hyperthermia) 5 medications 5 thrombosis counselling 5 counselling on symptoms on return, which require review (fever, skin changes, abnormal bleeding, lymphadenopathy, diarrhoea) 5 sexual transmitted diseases 5 contraception 5 rabies the following items should be asked to enable to give the appropriate advice: 5 risk assessment of the travel in a particular country (transport, area of stay/ rural or resort, reason for travelling, appropriate conduct overseas, pre-existing diseases and medications) 5 vaccination status 5 accomodation and stopovers 5 duration of the stay the vast majority of up-to-date travel information and information about tropical disease are available on who (world health organization) or cdc (centres for disease control and prevention) websites. information on these websites are frequently updated. before giving appropriate advice based on these online resources, it should be checked, which medications are available in the particular countries. hence, recommendations need to be adjusted individually. usually, a medication record is required at the customs. however, it might be sufficient, if the original medication box has the patients and prescribing doctors details (. table 32 .1). malaria is a tropical disease transmitted by the female anopheles mosquito. the distribution of malaria is primarily in the tropics and subtropics of africa, central and south america, asia, papua new guinea and the western pacific islands. as popular diving spots are located in these areas, malaria prophylaxis and advice should be given. the who (world health organization) estimates the worldwide number of people affected by malaria with about 198 million and 1,200,000 deaths (2013). the plasmodium parasites need temperatures above 20 °c in order to complete the entire growth cycle. therefore, malaria occurs in some places only seasonal. additionally, there are differences in anopheles species regarding the affinity to the host and their local distribution. some genetic factors are protected against malaria. for example, sickle cell anaemia gives a certain protection against p. falciparum and duffy negative blood group against p. vivax. it appears that after recurrent malaria infections, the body adapts to the disease. this means that an infection is possible, but the symptoms of malaria seem to be reduced. children and pregnant women have an increased risk of being affected by malaria. additionally, children have a high mortality rate. during pregnancy the resistance against malaria is reduced. it also poses an increased risk for the unborn child (low birth weight). anopheles is active especially at sunrise and sunset. different kinds of mosquitoes are rather active during the day and can transmit other diseases such as dengue. especially p. falciparum and p. vivax have resistances against antimalaria drugs. there are different plasmodium pathogens: 5 p. falciparum: worldwide tropical and subtropical distribution, mainly in africa; pathogen of severe malaria causes 1 million deaths per year; rapid growth in the blood with haemolysis and emboli due to cytoadherence of affected erythrocytes; 7-30 days of incubation, irregular fever spikes. 5 p. vivax: mainly in asia, latin america and some countries in africa; the disease can be activated after months or years. incubation period of 12-18 days; fever spikes every 2 days. 5 p. ovale: mainly west africa and the western pacific islands. similar to the p. vivax, it can also infect people with duffy-negative blood group; incubation period of 12-18 days; fever spikes every 2 days. 5 p. malariae: worldwide distribution; typical 3-day cycle, untreated can lead to lifelong chronic malaria; incubation period 16-50 days; fever spikes every 3 days. 5 p. knowlesi: southeast asia, mainly infected animals. after the anopheles mosquito aspirates with gametocytes infected blood, the gametocytes develop to gamete in the mosquito's intestines. in the blood of the mosquito, the microgametes (male) penetrate the macrogametes (female), forming zygotes. then cells are changed to an elongated, motile ookinete. this evolves into an oocyst. after the oocyst bursts, sporozoites are released and get in the saliva of the mosquito. the entire cycle inside the mosquito takes 8-16 days. if sporozoites enter the human bloodstream through the saliva of the . symptoms of malaria appear after the incubation period. the incubation period varies depending on the pathogen. it can be between a few weeks and also takes up to several months or even a year (p. vivax or occasionally p. ovale). malaria can be divided in three different forms: 5 malaria tertiana: pathogen: p. vivax and p. ovale; fever every second day with one day without fever, spontaneous remission after max. 5 years 5 malaria quartana: pathogen: p. malaria; fever every third day with 2 days without fever, no spontaneous remission 5 malaria tropica: pathogen: p. falciparum, irregular fevers due to the lack of synchronisation of the parasite reproduction, severe form of malaria (malaria maligna) with high fatality, recurrence up to 2 years the fever has a specific pattern. in the first hour, strong rigors and increasing fever typically develop. the fever can reach 40 °c and more for duration of about 4 h. it is often associated with flushing, vomiting and nausea. the fever stage is followed by an approximately 3-h stage of severe sweating with decreasing fever. severe forms of malaria can be fatal in within few days. causes of death are cerebral malaria, respiratory failure with adrs and kidney failure. the main reason of these complications is the cyto-adherence ("bonding") of the erythrocytes. it results in a failure of the microcirculation followed by ischaemia of vital organs. . the treatment depends on the severity and the pathogen. in complicated malaria, admission to the intensive care should be considered, if more than one of the following criteria exists: 5 inability of the oral intake of medication 5 parasite load of erythrocytes >2% 5 severe symptoms of malaria (see table above) the treatment options of complicated malaria are: 5 artesunate (allowed only in some countries): 2.4 mg/kg/bw iv; first dose on admission, repeated after 12 and 24 h, minimum duration of therapy 24 h and then once a day, till oral therapy is tolerated. or 5 combination of quinine + doxycycline or clindamycin. 5 quinine: ȥ first dose: 20 mg/kg/bw iv over 4 h or 7 mg/kg/bw iv over 30 min with subsequent administration of 10 mg/kg/bw iv over 4 h. ȥ maintenance therapy: 10 mg/kg/bw iv over 4 h three times a day, beginning 4 h after the completion of the first dose. ȥ exemption: if the patient received three or more doses of quinine in the last 48 h or had an mefloquine prophylaxis in the last 24 h or received a mefloquine treatment in the last 3 days. + 5 doxycycline: 100 mg iv twice daily for 7 days (iv or oral) or 5 clindamycin: ȥ initial dose: 10 mg/kg/bw ȥ maintenance dose: 5 mg/bw every 8 h for 7 days (iv or oral) 5 after clinical improvement medication can be changed to a complete cycle of the oral therapy of an uncomplicated malaria (riamet ® or quinine with doxycycline or clindamycin). uncomplicated malaria can be handled on the normal ward. outpatient therapy with close supervision can be considered under the following conditions: 5 parasite load of erythrocytes <1%. 5 age > 12 months. 5 no co-morbidity. 5 pregnancy is excluded. 5 ability of oral medication intake. 5 p. falciparum is excluded. 5 clinically stable under medical therapy for the last 24 h. a daily blood smear is necessary during treatment to follow the process of the disease. the patient can be discharged from the hospital and continue treatment at home; if oral therapy is tolerated, a clinical improvement is achieved and the parasite count decreases. a week and a month after discharge, blood smears should be repeated. primaquine as eradication therapy is approved in some countries. it is the only drug that can be used to eliminate hypnozoites, which are the dormant forms of the malaria parasites that occur with p. ovale and p. vivax. because primaquine causes haemolysis in g-6-pd deficiency, g-6-pd status prior therapy needs to be established. if an eradication with primaquine is required in patients with g-6-pd deficiency, a dose up to 45 mg weekly for 8 weeks, with monitoring for haemolysis, could be considered. in children methaemoglobinaemia can be provoked by giving primaquine. a single dose of primaquine 45 mg for p. falciparum, p malaria and p. knowlesi can be given to sterilise the gametocytes. if malaria caused by p. vivax or p. ovale or co-infection with these parasites is suspected, a 14-day treatment with 15 mg of primaquine twice a day is recommended. before commencing holidays overseas, medical advice should be given in order to assess the malaria risk of the particular country. in nearly all tropical areas, there is a risk of getting infected with malaria. in some tourist areas, this risk might be small, but infection is still possible. in particular day trips to more remote areas pose a risk. some areas have malaria outbreaks and therefore should be avoided. in general, mosquito bites should be avoided to minimise the risk of any mosquitoborne infections. mosquitoes transmitting malaria are mainly active at night, sunrise and sunset. however, mosquito bites are also possible throughout the day. long-sleeved shirts, long pants and closed shoes cover the skin and provide protection against insect bites. insect repellent for the skin and clothes offer additional protection. higher concentrations offer better and longer protection. the protection period of a normal insect repellent lasts usually only 1-2 h. slow release products can prolong the effect. mosquitoes avoid air-conditioned rooms. so staying in air conditioned rooms itself provides certain protection. spraying insecticides in rooms and surroundings can be helpful to repel and minimise the quantities of mosquitoes. the bed should be covered with a mosquito net (. fig. 32 .2). chemoprophylaxis is important, because the main cause of malaria deaths is still inadequate chemoprophylaxis. there are different drugs for chemoprophylaxis available. they are subject to the travel location and the parasite's resistances to certain drugs. in addition, they differ in side effects, dosage and cost. except malarone ® , all other drugs for the chemoprophylaxis against malaria have to be taken 4 weeks after leaving the country as they aren't sufficiently effective against the primary liver stages of malaria. mefloquine (lariam ® ) is the only malaria prophylaxis without absolute contraindication in pregnancy. diving (decrease in vigilance); 2-3 weeks (at least 1 week) before entering the malaria-endemic country and 4 weeks after return; lariam ® is a category b medication and is the only medication against malaria without absolute contraindication in pregnancy. the use in the first trimester should only be considered, if the expected benefits justify the potential risk to the foetus. however, recent studies suggest that even in the first trimester this medication is safe to take. the dengue virus is an arbovirus. it has four different serotypes (denv 1-4). dengue has a worldwide distribution in the tropics and subtropics, especially in asia and south america. approximately 50-100 million cases and about 100,000 with serious complications per year occur. there is a 10% mortality, which can be reduced to 1% with timely diagnosis and appropriate treatment. it has an increased risk for children under 15 years and persons with previous dengue infections. the dengue virus is transmitted by the aedes aegypti mosquitoes. these mosquitoes mainly bite at day and in twilight (. fig. 32 .3). z symptoms the incubation period is 2-10 days. there is a wide range in severity of dengue symptoms. the majority of infections cause minor symptoms. but dengue infections can be also quite severe (. table 32 .3). in particular recurrent infections with dengue are associated with complications and severity of the disease. it is important for the treating doctor to remember that after the initial fever, the critical phase follows. therefore, the patient must be monitored closely during this time. the disease goes through three stages: 5 fever phase (day 1-3): sudden high fever 40 °c occasional associated with bradycardia; myalgia mainly in the spine, arms and legs ("breakbone fever"), headache; retrobulbar pain; rigors; metallic/bitter taste; vomiting; and dehydration. . 5 critical phase (day 4-5): normal temperature with possible mild fever later on, leucopenia, exanthema, petechiae and lymphadenopathy. severe dengue: abdominal pain, spontaneous bleeding, volume shift in to the peritoneal space ("plasma leak"), pleural effusion, hepatomegaly (≥2 cm), rapid increase in haematocrit and decreasing thrombocytes, shock (dengue haemorrhagic shock = dhs or dengue shock syndrome = dss), increased bleeding (dengue haemorrhagic fever = dhf) and organ failure (particularly liver). 5 remission (after 6 days lasting sometimes for weeks): risk of hyperhydration is given when extravascular fluid is reabsorbed without reducing the intravenous fluid administration. in particular in long remissions, fatigue and depression may be present. normally there are no long-term damages after a dengue infection, and the vascular changes recover completely. z treatment there is no medication available to treat dengue directly. the diagnosis of dengue can be demonstrated by pcr in the initial phase and using igm and igg a few days later. due to severe complications, the haematocrit, coagulation parameters, leukocytes and platelets have to be tested daily. thrombocytes <100,000 cells/mm 3 can rise the suspicion of dhf. if pleural effusion is suspected, a cxr should be obtained. by tightening a blood pressure cuff petechiae can be provoked (medium pressure of the systolic and diastolic pressure for 5 min). this can be used as a diagnostic tool. an increase of the haematocrit of >20%, pleural effusion, ascites or hypoproteinaemia could be a sign for extravascular fluid loss. the extravascular fluid loss is typically found in the initial phase. hence, fluid replacement therapy is crucial in this phase. as the extravascular fluid loss can come to an end quite quickly, a complication of the fluid replacement therapy is hyperhydration. decrease of haematocrit of >20% after fluid administration can represent a fluid excess and hyperhydration. hence, careful monitoring of the fluid balance and weight are necessary. the therapy is adjusted according to its severity. if necessary, dic, blood loss or shock require specific treatment. like dengue, chikungunya is a mosquitoborne disease. the species transmitting the chikungunya virus (chikv) are aedes aegypti in the tropics and subtropics and aedes albopictus in colder regions (. fig. 32 .4). these mosquitoes bite day and night, but mainly in the early morning hours and late afternoon. the incubation period is between 2 and 12 days. the symptoms are similar to that of dengue. patients suffer from sudden fever with headache, skin rash, fatigue, strong limbs and muscle pain. affected joints often are swollen. the symptoms generally last for few days but can persist for weeks and years. the disease has no long-term effects. for diagnosis rt-pcr and virological methods can be used in the initial phase. later, it can be diagnosed by igm and igg. igm peaks after 3-5 weeks and can be detected up to 2 months. the treatment requires analgesia only. . yellow fever is a disease transmitted mainly by the aedes aegypti mosquito but also by other mosquitoes or ticks. the pathogen is a rnacontaining flavivirus. it has approximately 200,000 infections with approximately 30,000 deaths annually. 90% of cases occur in africa and the remaining 10% in south america. the risk of getting infected with yellow fever is with 1:200-2000 in africa and higher than 1:20000 in south america (. fig. 32 .5). the transmission occurs in rainforest areas (jungle or sylvatic cycle), where mosquitoes transfer the virus from monkeys to humans, in endemic areas of the savannah (savannah or intermediate cycle) either transferred from monkeys or human to humans via mosquitoes or in urban areas from human to human via mosquitoes. the incubation period is 3-6 days. the disease has two phases. the acute phase comes with fever, headache, myalgia, headand backache, loss of appetite, nausea, vomiting and diarrhoea. the second phase occurs only in approx. 15% of infected humans within the next 24 h. jaundice, abdominal pain and vomiting are rapidly developing, followed by diffuse bleeding (epistaxis and gi bleeding) and multi-organ failure (mainly kidneys). if symptoms of the more severe second phase develop, 50% of the patients die within the next 10-15 days. patients who survive usually recover without significant organ damage. the diagnosis can be made via a blood or tissue biopsy of the liver. there is no cure for yellow fever and only supportive measures can be taken. however, a very effective life-vaccination (stamaril ® ) is available. only authorised doctors are authorised to prescribe and give the vaccine. severe side effects of these vaccinations are severe allergic reaction (1:55000), vaccine-associated neurotropic disease/post-vaccinal encephalopathy (1:125000) and vaccine-associated viscerotropic disease/ multi-organ failure (1:400000). for travelling into countries where yellow fever is endemic, vaccination is mandatory. the side effects seem to be age-related and occur increasingly with progressive age or in young children. the vaccination is contraindicated in children 32.2 · other mosquito-borne diseases below 9 month and during pregnancy. analysis of yellow fever vaccines adverse events demonstrated an increased frequency of serious adverse events in persons age 60 years and older. the risk of viscerotropic side effects in <65 years is 1:400000, in a population of 65-75 years of age 1:40000 and in >75 years of age 1:4000. a failure to be vaccinated or being documented can lead to a refusal of entry into other countries or to a certain time in quarantine when leaving the area where yellow fever occurs. if there is a clinical indication against receiving yellow fever vaccine (e.g. children <9 month or poor immune status), a written medical exemption can be granted, to enable to travel to these countries without vaccination. absolute contraindications for a yellow fever vaccination are: 5 allergy against the vaccine or egg protein 5 age < 6 months 5 immunodeficiency 5 neoplasia 5 transplantations 5 immunosuppressive therapies relative contraindications for a yellow fever vaccination are: 5 age 6-9 months 5 age > 60 5 asymptomatic hiv infections and cd4+ t lymphocytes 200-499/mm 3 (15-24% of the total in children <6 years of age) 5 pregnancy 5 lactation aedes aegypti spreads also the zika virus. however, it is also sexually, intrauterine and perinatal transmitted. currently the main distribution is countries in south and north america as well as the caribbean islands, singapore and some countries in south pacific islands. symptoms of zika infection may be fever, rash, arthralgia, myalgia, headache and conjunctivitis. but in most cases, an infection is asymptomatic (~80%). these symptoms are lasting for several days to a week. the incubation period is 3-14 days but is likely to be a few days to a week. the diagnosis can be made via pcr or serology. blood pcr can be detected only in the first week of the disease. urine pcr can detect the virus up to 2 weeks. there is no specific treatment available. deaths are unlikely. there is a potential risk during pregnancy, as microcephaly or other birth defects (~20%) may develop. the zina virus cane be also transferred via semen and can affect unborn life. ross river virus (rrv) is transmitted by the bites of culex annulirostris, aedes vigilax, aedes normanensis and aedes notoscriptus in australia, papua new guinea, parts of indonesia and the western pacific islands. the main transmission time is in the humid summer month from december till march. the main symptoms are fever, rash, headache, myalgia, arthralgia and fatigue. the initial symptoms with fever last usually for 1-2 weeks. myalgia and arthralgia usually last longer. symptoms of fatigue and depression can be late complications. the incubation time is between 3 days and 3 weeks. the diagnosis is made with igm. there is only symptomatic treatment available. barmah forest virus (bfv) is transmitted by the same species as the rrv. it mainly can be found in australia. many people don't develop any symptoms. the incubation time is 3-11 days. if symptoms appear, they are similar to the one of rrv. the initial symptoms last for 1-2 weeks, and the arthralgia and myalgia may last for 6 months. the diagnosis is made with igm. there is only symptomatic treatment available. sindbis virus (sinv) is related to the chikungunya virus. it is mainly transmitted via the culex and culiseta mosquitoes. it can be found in europe, africa, asia and oceania. the symptoms and the duration of the symptoms are quite similar to rrv and bfv. the diagnosis is made with igm. there is only symptomatic treatment available. the o'nyong-nyong virus (onnv) is related to the chikungunya virus but is restricted to africa. it has similar symptoms as the chikungunya virus but has additionally mainly cervical lymphadenopathy, and the affected joins rarely show signs of an effusion. most of the gastrointestinal tract infections are caused by poor hygienic conditions of the travel destination. occasionally ingested seawater can cause intestinal infections too. the main transmission routes are either food-borne or by contact. however, the most common cause for gastrointestinal infections is eating contaminated food. old, warmed up food, salads, unpeeled fruits, poorly cooked food, contaminated water (ice and already opened bottles with refilled water) and ice cream often have substantial quantities of pathogens and pose a risk. hence, the best protection against gi infections is avoiding contaminated food or drinks. usually gastrointestinal infections last for a few days and are self-limiting. if diarrhoea contains blood or mucus in combination of high fever for more than 2 days, more thorough assessment is required. blood and mucus without fever are most likely related to a parasitic disease. if fever is present, it's most likely a bacterial or viral disease. but also climate change by itself or dehydration may be caused by autonomic dysregulation gastrointestinal symptoms such as nausea, weakness, vomiting and diarrhoea. with dehydration the dci risk increases. rehydration and supply of certain electrolytes such as sodium, chloride and potassium are the most important treatments for gastroenteritis. fatigue is a common associated symptom. tannins of black tea boiled for more than 10 min might be beneficial for diarrhoea. the consumption of bananas is recommended because of the high content of potassium. but the best options are rehydration preparations in form of drinks, powders or icy poles. loperamide may slow down the peristaltic and give some relief from diarrhoea. probiotics may support recovery. a low fibre diet is rec-ommended in the active phase of diarrhoea. administration of antibiotics is rarely necessary and indicated. it only is used for serious illnesses or symptoms. 5 reservoir: poultry or meals prepared with egg 5 incubation: 8-48 h 5 symptoms: fever, vomiting nausea, diarrhoea, occasionally blood and mucous in the stool 5 duration: 4-7 days 5 treatment: symptomatic; azithromycin 1 g od for 5 days or ciprofloxacin 500 mg bd for 7 days or ceftriaxone 2 g od 5 reservoir: water and food 5 incubation: 1-2 weeks 5 symptoms: headache, myalgia, bradycardia, roseola in the abdominal area, continuous fever 39-40 °c, porridge -like diarrhoea, intestinal bleeding and decrease of the fever after 4 weeks 5 treatment: symptomatic; azithromycin 1 g od for 5 days or ciprofloxacin 500 mg bd for 7 days or ceftriaxone 2 g od; vaccination available 5 reservoir: human, flies, food and faeces 5 incubation: 2-5 days 5 symptoms: fever, diarrhoea, sometimes with blood and mucus in the stool and severe abdominal pain 5 treatment: symptomatic; ciprofloxacin 500 bd for 5 days, norfloxacin 400 mg bd for 5 days or bactrim 160/800 mg bd for 5 days 5 reservoir: food and water 5 incubation: 0-2 days 5 symptoms: mild to severe diarrhoea with fever and blood and mucous in the stool, most common cause for diarrhoea overseas 5 treatment: symptomatic; norfloxacin 400 mg od and ciprofloxacin 500 mg od 5 reservoir: food (particular strawberries) and water 5 incubation: 1-4 weeks 5 symptoms: diarrhoea like raspberry jelly, no fever! blood and mucous in the stool, risk for developing a liver abscess 5 treatment: symptomatic, asymptomatic carrier, paromomycin 500 mg tds for 7 days; invasive, tinidazole 2 g od for 3 days or metronidazole 400 mg tds for 7 to 10 days 32.3.6 cholera (vibrio cholerae) 5 reservoir: contaminated food and water 5 incubation: 0-5 days 5 symptoms: often mild gi symptoms, 5-10% develop severe symptom with nausea vomiting, rice water-like diarrhoea and severe dehydration, mortality risk of 25-50% 5 treatment: rehydration, electrolyte substitution; vaccination available; azithromycin 1 g single dose, ciprofloxacin 1 g single dose 5 reservoir: food (in particular sea food) and water 5 incubation: 15-50 days 5 symptoms: initial phase (2-7 days)flulike symptoms, gastrointestinal, hepatomegaly; hepatic manifestation (4-8 weeks), no jaundice (approx. 70%), jaundice (30%) with dark urine, jaundice, pruritus; hepatitis a has no chronic form, rarely fatal (fatality is age dependent) 5 treatment: symptomatic, bed rest, avoidance of liver toxic substances (alcohol, medication); vaccination available japanese encephalitis is caused by a flavivirus, which is transmitted by mosquitoes (culex particularly c. tritaeniorhynchus). the hosts are usually pigs and water birds. in humans there are usually not sufficiently high concentrations of virus to serve as a host. the distribution is the asia, especially in rural areas. epidemics occur every 2-15 years (. fig. 32.6 ). the transmission can occur throughout the year but frequently peaks in the rainy season. there are about 68,000 cases per year. only about 1% of the patients are symptomatic. however, if symptoms develop, the mortality rate is 20-30%. approx. 30-50% of patients who survive have long-term neurological or psychiatric complications. mild courses of japanese encephalitis may be accompanied by mild fever and headache. severe cases show high fever, neck stiffness, photophobia, headache, disorientation, coma, convulsions, spastic paralysis or death. consequential damages may be behavioural disorders, convulsions, paralysis and speech disorders. the diagnosis can be established with blood tests and lumbar puncture. there is currently no treatment option. the vaccination is usually well tolerated and available for prophylaxis. there are various tropical diseases, which are present in poorer countries causing more or less severe symptoms. these diseases are termed "neglected tropical diseases" (ntd). the more common ntds are summarised in this chapter. there are three main conditions caused by these pathogens. the african trypanosomiasis (sleeping sickness) is transmitted by the tsetse fly. the distribution is only in some countries of the sub-saharan africa. seventy percent occur in the democratic republic of congo. tsetse flies are mainly found in rural areas. there are two forms causing sleeping sickness, t. brucei rhodesiense and t. brucei gambiense. t. brucei gambiense has an incubation period of months to years and t. brucei rhodesiense weeks to months. the initial phase is the haemolytic-lymphatic phase, in which pathogens replicate in tissues, blood and lymphatic tissues. symptoms are intermittent fever, headache, myalgia and pruritus. additionally, a painless, indurated chancre on the skin 5-15 days after the bite and lymphadenopathy (axillary and inguinal) can be associated. in the second phase, the cns affected causes continuous headache, behavioural disorders (mood swings and depression), delirium, sensitivity disorders, coordination problems and disruptions of the sleeping cycle (daytime somnolence). the diagnosis is mainly made clinically. only for the t. b. rhodesiense, a blood test (centrifuged or wet preparation) to data . detect the parasite is available. examination of buffy coat increases sensitivity. a biopsy of the lymph node to detect the pathogens can be diagnostic for t. brunei gambiense or be used for a culture and pcr. the card agglutination test for trypanosomiasis (catt) is a field test suitable for mass population screening in endemic areas for t. b. gambienses but has a low specificity and is hence only used for identifying suspected cases. all diagnosed patients need to have their cerebrospinal fluid examined for staging, which influences treatment options (. table 32 .4). the treatment is dependent on the pathogen and the staging. if untreated, infections of both forms lead to coma and death. leishmaniasis has three forms: visceral, cutaneous and mucosal (kala-azar). there are about 30 different pathogens, from which approx. 20 are held responsible for these diseases. the disease is transmitted by mosquitoes or sandflies (phlebotomus and lutzomyia). the cutaneous form is the most common one, which causes skin ulcerations. typically this form appears weeks to months after the initial mosquito bite. initially papules are formed, which later ulcerate. they can be painful or painless. the visceral form affects organs, especially the liver, spleen and bone marrow. therefore, this form can be quite dangerous. the changes occur within months and years. hepatosplenomegaly and pancytopenia develop. the mucus form is rare. ulcerative changes of the mucous membranes (e.g. nose, mouth and throat) are typical for this. endemic areas for leishmaniasis are east africa, some arabic countries, india, bangladesh, brazil and some other south american countries. historically, the diagnosis was made by taking a biopsy (skin, bone marrow or other tissues) for culture. now pcr or serological testing with high sensitivity replaced biopsies for making diagnosis. as the visceral disease is fatal without treatment, it needs to be treated in any case. all other forms require normally no treatment. following medication is available: 5 pentavalent antimonial (sb v ) compounds (20 mg per day iv or im for 28 days) 5 liposomal amphotericin b (3 mg od iv on day 1-5, 14 and 21) 5 miltefosine (in adults > 45 kg 50 mg 3 times daily for 28 days) 5 azoles (fluconazole 200 mg od for 6 weeks, itraconazole 200 mg bd for 28 days, ketoconazole 600 mg od for at least 28 days) 5 paromomycin (uncommonly used) 5 pentamidine isethionate (uncommonly used) the chagas' disease is transmitted via an insect bite ("kissing bug") or by contaminated food. it occurs in central and south america. it has . an acute and chronic phase. in the acute phase within 1-2 weeks after the infection, localised swelling of the area of the insect bite (skin or mucous membranes), lymphadenopathy, bilateral orbital oedema, meningoencephalitis and myocarditis can occur. 20-30% of all infections become chronic, causing arrhythmias with risk of "sudden death", cardiomyopathy and enlargement of the oesophagus (megaoesophagus) or of the colon (megacolon) even after years or decades. the cardiomyopathy consists of fibrosing myocarditis, causing arrhythmia (rbbb, left anterior fascicular block, st changes, premature ventricular beats and bradycardia) and ventricular failure. the diagnosis in the acute phase is made by a blood smear (thick and thin) to visualise the parasite. a serological test is also available. treatment is recommended in the acute phase and in patient up to the age of 50 and no advanced cardiomyopathy with chronic chagas' disease (. table 32 .5). in age groups above 50, benefits and risk need to be outweighed. worm infections are a major problem in underdeveloped countries. they occur mainly in rural areas. these conditions may cause insignificant symptoms but also lead to serious consequences or even cause death. because some dive sites are located far away from tourist centres, these infections should be discussed before travelling. this kind of roundworm is found in the tropical and subtropical regions of africa and southeast asia. the transfer follows on oral intake of eggs by contaminated food. the larvae are entering the bloodstream after hatching in the intestine. they reach the lungs via the blood and penetrate the lung tissue, and the larvae can be coughed up. if the sputum is swallowed again, the larvae reach the intestine, mature there within the next 2-3 months and lay eggs, which are then excreted via the faeces. the adult worms live about 1-2 years. infection is usually asymptomatic. however, abdominal pain, flulike symptoms, allergic skin manifestations, malnutrition, productive cough and a stridor can occur. the diagnosis can be made by examining the faeces (eggs, worms) or sputum (larvae). hookworms are found in tropical and subtropical regions of africa and latin america. the transmission is percutaneously or orally by ingestion of contaminated soil. in contaminated soil the larva is able to survive for about 3-4 weeks. larvae can penetrate the skin and enter the blood and reach the alveoli in the . lungs. from there they ascend in the airways, are swallowed again and finally get into the intestines. there larvae mature to adult worms. the worms attach themselves to the wall of the intestine and feed on blood. the eggs are excreted in the faeces and reach again the soil. the eggs can survive up to 2 years. common symptoms are pruritus and rash at the entry site, abdominal pain, diarrhoea, weight loss, anaemia and extreme fatigue. the diagnosis can be made of the faeces. z filariasis filariasis has a worldwide distribution in tropical and subtropical regions. it is caused by wuchereria bancrofti and brugia malayi. it is transmitted by mosquitoes. the infective filariform grow inside mosquitoes and enter via its saliva during the bite. they migrate to the lymphatic vessels and lymph nodes where they develop into adults. they can live there for about 6 years. the female worms produce microfila, which are circulating in the blood. absorbed by mosquitoes they develop within 1-2 weeks to the infective filariform. initially there are no symptoms. later lymph oedema in extremities or genitals is a common symptom. in men hydrocele can develop. the skin typically swells and hardens ("elephantiasis"). the diagnosis is made via the blood. detection in the blood smear has to be performed at night, as larvae only circulate in the blood at night. there is also a serological detection of anti-filaria igg4 available for diagnosis. the treatment with dec is the drug of choice. concurrent disease of loa loa or onchocerciasis is a contraindication for dec, because of the serious side effects (encephalopathy and deaths). ivermectin is used as a prophylaxis, but not as a therapy. z schistosomiasis (bilharziose) schistosomiasis can be found in tropical and subtropical regions worldwide. in addition to malaria, it is the most common parasitic disease. the parasite schistosoma is housed in freshwater snails. by being exposed to freshwater in these regions, infections can occur. the eggs are excreted in urine or faeces of the host. they hatch under optimal conditions and release miracidia. these miracidia infect freshwater snails and develop into sporocysts. these develop into cercariae and get released into the water, where they can penetrate the skin of the host. there, they shed their tail and become schistosomulae and migrate to the liver. in the liver they mature into adults. the paired adult worms migrate to the bowel and bladder, where they lay the eggs. a rash ("swimmers itch") may develop at the entry site on the skin. suprapubic pain and haematuria, abdominal pain, myalgia, fever, swelling of the lymph nodes, liver and spleen enlargement and eosinophilia can be additional symptoms. the risk of bladder cancer is increased with schistosomiasis. the diagnosis can be made in the stool and urine. the maximum excretion of eggs in the urine is between 12 and 3 pm. z trichuriasis (whipworm) whipworms have a worldwide distribution in the humid tropics. the eggs are orally absorbed via soil or unwashed vegetables or fruits. the whipworm grows in the large intestine. the eggs are excreted via the faeces. in the soil the eggs pass through various stages before getting absorbed again. the symptoms are abdominal pain, chronic diarrhoea, nausea, vomiting, inflammation of the intestine, anaemia and eosinophilia. the diagnosis is made with a stool sample. the treatment on the infection is dependent on the parasite (. table 32 .6). leptospirae are long, motile spirochetes. they have a worldwide distribution, but infections occur more commonly in tropical and subtropical regions. they spread through infected urine, which enters water or soil. leptospires can survive for several weeks and months. infections can be caused by contact with either direct contact with the urine or other body fluids except saliva as well as with contaminated soil and water. the bacteria enter the body through the skin or mucous membranes. a broken skin increases the risk of infection. increased risk is after heavy rainfall or flooding. the incubation period is usually 5-14 days, but can range from 2-30 days. symptoms vary greatly. usually sudden onset of headaches, fever, chills, myalgia, nausea and vomiting, diarrhoea, rash and jaundice are common signs of the first phase for 3-8 days. if the patient doesn't recover the second phase (weil's disease) develops, with renal failure, ards, hepatomegaly, jaundice, haemorrhage and meningitis. this has a fatality rate of 1-5%. untreated symptoms can persist for several months. treatment is either doxycyclin 100 mg bd or benzylpenicillin 1.2 g qid or ceftriaxone 1 g od for 7 days. infections caused by rickettsia, orienta, ehrlichia, neorickettsia, neoehrlichia and anaplasma are summarised as rickettsial infections. rickettsias are divided into the typhus group and the spotted fever group. orienta make up the typhus group. the reservoir is found in mainly animals, like rodents, but some species are found in fish. the vector is commonly ticks. in scrub typhus the vectors are larval mites. others have fleas and lice as a vector. infection occurs either by bites of the vectors or by direct contact, inoculation or inhalation of contaminated fluids or faeces. the clinical presentation varies. mild symptoms are headache, myalgia, abdominal pain, cough and rash. some rickettsial infections, . q-fever is a zoonosis caused by the protozoa coxiella burnetii. the bacterium is quite resilient due to its sporelike life cycle and remains virulent for months even up to more than a year. the primary reservoir is cattle, goats, sheep and other wildlife like kangaroos, rats and cats. rarely is it transmitted by tick bites or by ingestion of unpasteurised milk or dairy products. the incubation time is usually 2-3 weeks but can range from 2 days to 6 weeks. the initial acute q-fever comes with sudden onset of high fever up to 40 °c, headache (retrobulbar), myalgia, chills, non-productive cough and sweats. the symptoms settle within 5-14 days. 50% of all infections are however asymptomatic. often thrombocytopenia and abnormal lfts are found. complications are ards, endocarditis and meningoencephalitis. the diagnosis is based on detecting phase ii and phase i antibodies (igg) 4 weeks apart. the initial test (phase ii) should be taken at the end of the first week of illness. igm and igg rise almost at the same time. a fourfold rise is diagnostic. an initial negative titre doesn't rule out q-fever. seroconversion occurs usually between days 7 and 15 but is almost always present by 21 days. pcr testing can be used in the first 2 weeks but before antibiotic administration. however, a negative pcr result doesn't rule out q-fever. chronic q-fever develops in 0.2-4%. it can result in endocarditis, aneurysms, osteomyelitis, hepatitis, neurogic (mononeuritis, optic neuritis), pulmonary (interstitial fibrosis, pseudotu-mor) and renal (glomerulonephritis) disease. chronic q-fever usually develops shortly after the infection. however, chronic endocarditis may not come apparent until 2-4 years or even longer. chronic fatigue syndrome is described in approx. 10%. typically in chronic q-fever, the initial igg titre is increasing (>1:800). the treatment for acute q-fever is doxycyclin 100 mg bd for 14 days or for at least 3 days after fever subsides and until clinical improvement. as serological confirmation takes time, treatment should not be delayed. early treatment is effective at preventing severe complications. for chronic q-fever, 18 months of doxycyclin 100 mg bd and hydroxychloroquine 200 mg tds is recommended as standard treatment. rabies has an almost worldwide distribution. more than 95% of deaths occur in africa and asia. about 40% are children under 15 years of age. dogs are the main vectors. in asia, there is also a risk of transmission through monkeys. in addition to other diseases, like the lyssavirus, bats or flying foxes can transfer rabies. it is transmitted by bites or scratch wounds but also by inoculation of saliva onto mucous membranes or eye of an infected animal. thorough cleaning of the wound and vaccination within hours can prevent the disease. the incubation period is usually 1-3 months but can be less than 1 week and more than a year. initial symptoms include paraesthesia in the wound area. the disease can pass in two forms. the hyperactive form (70%) shows up with hyperactivity, manic behaviour, paranoia, hallucinations, delirium, hydrophobicity and occasionally aerophobia (triggered by the extremely painful spasms in the larynx area). the paralytic form (30%) is characterised by a slow but steady increasing paralysis. the paralysis begins in the area of the infection. the diagnostics can be established on the animal that has inflicted the wound. the tissue samples of the animal are taken from the brain (brainstem and cerebel-lum). the diagnosis in humans is difficult and unreliable. investigation of blood (antibodies), saliva (pcr), spinal fluid (antibodies) and skin biopsies (rabies antigen) are available. the vaccine and the immune globulin can be given during pregnancy. typical side effects of the vaccine are headache, myalgia, malaise, fatigue and nausea. treatment after potential infection (postexposure prophylaxis pep) includes: 5 irrigation of the wound for a minimum of 15 min and washing of the wound with water, soap, iodine or other disinfecting substances 5 rabies vaccine 5 rabies immunoglobulin into the wound area within 7 days after the first vaccination following data should be recorded when a rabies vaccine is given overseas: 5 address, email and telephone of the practice or hospital 5 date of vaccinations 5 batch number, name of the vaccine and manufacturer 5 how many vaccinations are given 5 application: subcutaneous or intramuscular injection who recommends the following approach with potential rabies after animal contact: vaccination against rabies is recommended for: 5 travellers, who for more than 1 month in areas, in which rabies is present 5 professions that deal with bats or fruit bats 5 professions, in which might get with rabies in contact (e.g., veterinary surgeon or nurse) 5 laboratory workers who handle objects with rabies or lyssavirus 5 after animal contact category 2 + 3 pre-exposure prophylaxis (prep) includes three vaccinations on day 0, 7 and 21-28. the dose is 0.1 ml intramuscularly or subcutaneously. the vaccination lasts for 10 years. follow-up vaccinations (post-exposure prophylaxis = pep) include four vaccinations on day 0, 3, 7 and 14. the dose is 1.0 ml intramuscularly. immunocompromised patients should receive five vaccinations with an additional vaccination on the 28th day. with previous vaccinations, two vaccinations are recommended on day 0 and 3 after exposure. it is not recommended to change the brand or the manufacturer during the course of vaccinations. however, it is possible, if that particular vaccine is not available. immunoglobulin should be administered with the first vaccination. the dose is 20 iu/kgbw. the immunoglobulin preferably should be given in proximity of the wound. the immunoglobulin can be diluted, if the wounds is large, to enable to cover the entire wound area. the immunoglobulin is not recommended, if the first vaccination was given more than 7 days ago, if prep or pep was completed or if an adequate serologic detection of vnab titres (≥0.5 iu/ml) is present. to avoid infection, no animals should be fed. bringing your own food or carrying items like handbags, water bottles, etc. should be avoided, if you stay in the range of monkeys. distance should be maintained to stray cats and dogs. the middle east respiratory syndrome (mers) is caused by a corona virus. corona viruses can cause mild flulike symptoms but also severe symptoms like the severe acute respiratory syndrome (sars). the mers-cov occurs 32.7 · mers mainly on the arabian peninsula (iran, jordan, kuwait, lebanon, oman, qatar, saudi arabia, united arab emirates and yemen). but through international travel, it can spread worldwide. recently it resulted in some cases in korea. mers has 37% mortality. the disease is transmitted through droplets or direct contact. the mers-cov also has a wide range of symptoms, from mild common cold symptoms and infections of the upper respiratory tract to a rapidly progressive pneumonitis, respiratory failure, septic shock and multi-organ failure. it seems the mers-cov has a low virulence, since the transmission occurs usually only through close contact by human to human, such as the care of a person suffering from mers. camels seem to be the original reservoir. mild forms with fever and mild respiratory symptoms, mers should be considered, if close contact with infected people existed prior to these symptoms. mers can be asymptomatic but also lead to respiratory failure and death. typical symptoms include fever, cough and shortness of breath. pneumonia or pneumonitis is often associated with mers. sometimes gastrointestinal symptoms such as diarrhoea and vomiting can occur. it has a high mortality of 36%. the treatment depends on the severity of the disease. caution in contact with camels in affected countries should be taken. eating insufficient heated camel meat and milk should be avoided. a suspicion of mers should be considered in individuals with the following risk profile: 5 fever and pneumonia/pneumonitis and stay in endemic areas or contact with a symptomatic person from an endemic area within 14 days before onset of symptoms 5 fever and pneumonia/pneumonitis and hospitalisation in endemic areas or contact with camels and camel products in an endemic area within 14 days before onset of symptoms 5 fever and pneumonia/pneumonitis and contact with a mers diseased person within 14 days before onset of symptoms 5 cluster of patient (especially medical personnel) with severe respiratory symptoms with unclear aetiology tuberculosis is caused by an acid-resistant mycobacterium. m. tuberculosis is responsible for tuberculosis in more than 95%. it has global distribution but occurs more frequently in countries with low hygienic standards. tuberculosis spreads around the globe through international travel and immigration. it also shows a rising rate of resistances to conventional therapies. the time between the initial infection and tuberculin conversion takes approx. the diagnosis can be made with the tuberculin skin test (tst/mendel mantoux). 3 days after the strictly intradermal injection of the substance, the induration at the injection site is measured. an induration of >5 mm may be suggestive of tuberculosis. it is considered a positive test if either the patient has a radiological proof, had close contact with someone with tuberculosis, and has symptoms of tuberculosis, is hiv positive or suffers from immunodeficiency. an induration >10 mm is considered as positive, when the patient who travelled to a country with high tb prevalence is an iv. drug user, homeless and a resident of nursing home or prison and has diabetes mellitus, silicosis, m. hodgkin's or end-stage renal failure. an induration >15 mm is considered as evidence of tuberculosis without any risk factors or symptoms. the tst can be negative in the first 8 weeks after an infection as well as in patients suffering from miliary tuberculosis, m. hodgkin, sarcoidosis, viral infections, and lowered immunity, receiving an immunosuppressive therapy or at high age. a false-positive test can occur after multiple tsts, after vaccination against tuberculosis and infection of other mycobacteria. the interferon-γ test (quantiferon ® tb gold) offers an alternative testing method. this test has the same sensitivity as the tst but a higher specificity. moreover, this test is a confirmation test and isn't affected by previous bcg-immunisations. it consists of three parts, the control (to determine the baseline-interferon-γ), mitogen control (determining the ability of an immune response) and antigen detection (detection of prior infections). a cxr may demonstrate caverns or hilar lymph nodes, but is not a diagnostic tool to exclude tuberculosis. the treatment duration of uncomplicated tuberculosis is 6 months, of complicated tuberculosis 9-12 months (. table 32 .7). it's a combination treatment of different drugs. medications for the tuberculosis treatment are: 5 isoniazid: 5 mg/kgbw, max. 300 mg /d; side effects: elevated serum transaminases, polyneuropathy, prophylaxis to avoid side effects of pyridoxine 40-80 mg/d a vaccination bcg vaccine is not recommended due to its side effects and the lack of efficacy. all vaccinations should be given 28 days before travelling. minimum time for a sufficient protection is 2 weeks (. divers alert network (dan) is a non-profit organisation for divers. they provide medical information and articles, diving insurance, life insurance and travel insurance. they also offer courses, support and research. dan has an international hotline for support and coordination of diving accidents but also for general medical advice overseas. european underwater and baromedical society (eubs) is a european organisation for diving and hyperbaric medicine. they provide guidelines for hyperbaric treatment and training of medical professionals for the hyperbaric medicine. the german organisation for diving and hyperbaric medicine is the "gesellschaft für tauch-und überdruckmedizin" (getüm). . . single dose certificate is valid for 10 years, a new vaccination may be required after 10 years to renew the certificate they provide guidelines for hyperbaric treatment and training of medical professionals for the hyperbaric medicine. brazil; office: tel: +1-919-684-2948, emergency-hotline: +1-919-684-9111. japan: japan marine recreation association, kowa-ota-machi bldg office: tel: +81-45-228-3066, f ax southern africa: private bag x197, halfway house, midrand 1685 eubs: webmaster@eubs.org 5 gtuem: c/o bg-unfallklinik, professor-kuentscher-str. 8, d-82418 murnau spums: 630 st kilda road uhms: 631 us highway 1, suite dan: 7 www. diversalertnetwork. org 5 dan europe: 7 www. daneurope. org 5 emedicine yellow fever chikungunya virus middle east respiratory syndrome (mers) parasites -african trypamosiasis (also known as sleeping sickness) parasites -american trypanosomiasis (also known as chagas disease) parasites -trichuriasis (also known as whipworm) air embolism of the brain in rabbits pretreated with mechlorethamine an examination of the critical released gas concept in decompression sickness accessed 12 middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus (mers-cov) key: cord-033791-q0wizf2n authors: kavirayani, akhila; charlesworth, james e g; segal, shelley; kelly, dominic; wilson, shaun; qureshi, amrana; blanco, esther; weitz, james; o'shea, deirdre; bailey, kathryn title: the lazarus effect of very high-dose intravenous anakinra in severe non-familial cns-hlh date: 2020-10-15 journal: lancet rheumatol doi: 10.1016/s2665-9913(20)30361-1 sha: doc_id: 33791 cord_uid: q0wizf2n nan bal negative bal -aspergillus (month 2 of admission), candida in urine, pseudomonas stenotrophomonas, maltophilia and bacillus species line infections soluble cd25 (<2500 pg/ml) not processed control perforin cd56+ cells -90.3 % patient perforin cd56+ cells -79.5 % suboptimal perforin expression. percentage is normal but the patient has slightly less bright perforin than would normally be expected. perforin gene normal (homozygous polymorphism); felt to be consistent with clinical condition. control gra cd8+cd107a+ -2.4 % patient gra cd8+cd107a+ -0.9 % control gra nk cells cd107a+ -11.1 % patient gra nk cells cd107a+ -unable to analyse normal cytotoxic granule release assay as detected by cd107a expression in response to cd3 (t cells) stimulation. too few nks for analysis. this suggests that this patient does not have hlh due to a defect in this pathway (including syntaxin 11, munc 13-4 and 18-2) excludes fhl2,3,4,5*, chediak-higashi syndrome and griscelli syndrome *including a novel gene recently described in vienna 'tiger' primary immunodeficiency panel all known genes for fhl negative traps mutation (tnfrff1ap.1334v) (unknown significance) abbreviations: bal -bronchoalveolar lavage, crp -c-reactive protein, traps -tumour necrosis factor receptor-associated periodic syndrome, wcc -white cell count, fhl -familial haemophagocytic lymphohistiocytosis necrotic lesion to the left arm of the patient is shown, taken 3 weeks following discharge, reproduced with consent. 2mg/kg/day (60mg/day) over 12 hours, 12mg/kg/day (360mg/day) over 36 hours, escalated to 48mg/kg/day (1440mg/day) over 72 hours, stepped down to 24mg/kg/day (720mg/day) over 6 days, then 12mg/kg/day (360mg/day) over 6 days before converting to sc. 100mg/day for 15 months, then reduced by 20mg weekly and discontinued. iv methyl prednisolone, 1 dose of iv immunoglobulin, empiric antibiotics and antivirals-iv aciclovir and iv ceftriaxone, switched to meropenem/teicoplanin upon deterioration (doxycycline/clindamycin/co-amoxiclav also administered subsequently with co-trimoxazole/fluconazole prophylaxis) inotropes for profound hypotension. iv methylprednisolone was substituted with dexamethasone with cns symptoms, ciclosporin added when renal impairment allowed. dexamethasone later changed to oral prednisolone. candida and pcp prophylaxis., 1 dose of low dose etoposide alongside highest anakinra dose. tpn. fluconazole for pulmonary aspergillosis. antibiotics for line infection. multiple transfusions of red cells, platelets, cryoprecipitate and ffp. appendicectomy, haemofiltration, mechanical ventilation. abbreviations: ffp -fresh frozen plasma, picu -paediatric intensive care unit, pcp -pneumocystis pneumonia, tpn -total parenteral nutrition interleukin 1 receptor antagonist to treat cytophagic histiocytic panniculitis with secondary hemophagocytic lymphohistiocytosis interleukin-1 receptor antagonist penetrates human brain at experimentally therapeutic concentrations the effect of intravenous interleukin-1 receptor antagonist on inflammatory mediators in cerebrospinal fluid after subarachnoid haemorrhage: a phase ii randomised controlled trial benefit of anakinra in treating pediatric secondary hemophagocytic lymphohistiocytosis successful treatment of severe paediatric rheumatic disease-associated macrophage activation syndrome with interleukin-1 inhibition following conventional immunosuppressive therapy: case series with 12 patie nts continuous intravenous anakinra infusion to calm the cytokine storm in macrophage activation syndrome therapeutic role of anakinra, an interleukin-1 receptor antagonist, in the management of secondary hemophagocytic lymphohistiocytosis/sepsis/multiple organ dysfunction/macrophage activating syndrome in critically ill children*: pediatric anakinra treatment in macrophage activation syndrome: a single center experience and systemic review of literature salvage therapy of refractory hemophagocytic lymphohistiocytosis with alemtuzumab: alemtuzumab for refractory hlh central nervous system-restricted familial hemophagocytic lymphohistiocytosis responds to hematopoietic cell transplantation pediatric cns-isolated hemophagocytic lymphohistiocytosis neurological associations of covid-19 key: cord-034257-kl2ccmz5 authors: de jonge, jeroen c.; woodhouse, lisa j.; reinink, hendrik; van der worp, h. bart; bath, philip m. title: precious: prevention of complications to improve outcome in elderly patients with acute stroke—statistical analysis plan of a randomised, open, phase iii, clinical trial with blinded outcome assessment date: 2020-10-26 journal: trials doi: 10.1186/s13063-020-04717-0 sha: doc_id: 34257 cord_uid: kl2ccmz5 rationale: aspiration, infections, and fever are common in the first days after stroke, especially in older patients. the occurrence of these complications has been associated with an increased risk of death or dependency. aims and design: prevention of complications to improve outcome in elderly patients with acute stroke (precious) is an international, multi-centre, 3 × 2 factorial, randomised, controlled, open-label clinical trial with blinded outcome assessment, which will assess whether prevention of aspiration, infections, or fever with metoclopramide, ceftriaxone, paracetamol, respectively, or any combination of these in the first 4 days after stroke onset improves functional outcome at 90 days in elderly patients with acute stroke. discussion: this statistical analysis plan provides a technical description of the statistical methodology and unpopulated tables and figures. the paper is written prior to data lock and unblinding of treatment allocation. trial registration: isrctn registry isrctn82217627. registered on 22 september 2015. the trial was prospectively registered. in the first days after stroke, about half of all patients develop one or more complications, including aspiration, infections, or fever. the risk of developing these events is greater in patients of higher age or with more severe stroke [1] [2] [3] . these complications can impede functional recovery, prolong hospital admissions, and are independently associated with an increased risk of death or longterm dependency [1, 2, [4] [5] [6] [7] [8] [9] [10] [11] . the risk of developing these complications can be reduced by very simple, safe, and inexpensive measures, such as metoclopramide for the management of dysphagia, antibiotics for the prevention of infections, and paracetamol for the prevention of fever, but it is uncertain whether these measures also improve functional outcome [12] [13] [14] [15] . in some generally small, randomised trials, preventive treatment with these drugs not only convincingly reduced the risks of aspiration, infections, or fever by one third to one half, but was also associated with clear trends towards a lower risk of death or poor outcome [12] [13] [14] [15] . however, in two large randomised clinical trials, preventive treatment with antibiotics did not improve functional outcomes [16, 17] . guidelines of the european stroke organisation concluded that there is insufficient evidence from randomised trials to make strong recommendations on whether, when, and to whom preventive antibiotic or antipyretic treatment should be given after ischaemic stroke or intracerebral haemorrhage [18, 19] . the prevention of complications to improve outcome in elderly patients with acute stroke (precious) trial will assess whether prevention of aspiration, infections, or fever with metoclopramide, ceftriaxone, paracetamol, or any combination of these in the first 4 days after stroke onset improves functional outcome at 90 days in older patients with acute stroke. the current paper describes the statistical analysis plan (sap) of the trial and conforms to the guidelines set by gamble et al. [20] . the details of the study protocol of the precious trial have been published earlier [21] . precious has received funding from the european union's horizon 2020 research and innovation programme under grant agreement no. 634809. precious is an international, multi-centre, multifactorial, randomised, controlled, phase iii, open-label clinical trial with blinded outcome assessment (probe). the primary objective is to assess whether prevention of aspiration, infections, or fever with metoclopramide, ceftriaxone, paracetamol, or any combination of these in the first 4 days after stroke onset improves functional outcome at 90 days in older patients with acute stroke. patients will be randomly allocated in a 2 × 2 × 2 factorial design to any combination of open-label oral, rectal, or intravenous metoclopramide (10 mg thrice daily); intravenous ceftriaxone (2000 mg once daily); oral, rectal, or intravenous paracetamol (1000 mg four times daily); or usual care, started within 24 h after symptom onset and continued for 4 days or until complete recovery or discharge from hospital, if earlier. in patients with moderate to severe renal impairment or with severe hepatic impairment, the dose of metoclopramide is reduced to 5 mg thrice daily, and in patients with end-stage renal disease to 2.5 mg thrice daily. patients will be stratified according to country (estonia, germany, greece, hungary, italy, the netherlands, norway, poland, uk), and there will be 5 minimisation factors: age (66-75 years; > 75 years), sex (male vs. female), stroke type (ischaemic stroke vs. intracerebral haemorrhage), stroke severity (nihss 6-12 vs. > 12), and diabetes mellitus (yes vs. no).a total of 3800 patients will be recruited, based on the sample size calculation described in the previously published protocol [21] . an independent data and safety monitoring board (dsmb) will conduct unblinded interim analyses after 600, 1200, 1800, 2400, and 3000 patients have completed follow-up to assess the safety of the interventions in the trial. with respect to efficacy, the dsmb will conduct unblinded interim analyses after 2400 patients had their final follow-up. dsmb members will receive listings of all sae reports as well as unblinded aggregate summaries of data by treatment groups for review in closed meetings. the results of these interim analyses are confidential and limited to the members of dsmb. this statistical analysis plan (sap) will be signed off by the trial steering committee and then submitted for publication prior to data lock and final analysis. the final statistical analysis will be performed once recruitment has ceased, final follow-up and final outcome adjudication have been completed, final data have been checked and any errors corrected, and the database has been locked. the analyses will be carried out according to the current statistical analysis plan. the statistical analyses will be performed by the nottingham stroke trial unit (nstu) at the university of nottingham (unott) in collaboration with the umc utrecht. the study population will consist of patients aged 66 years or older who are hospitalised with moderately severe to severe (national institutes of health stroke scale (nihss) ≥ 6) acute ischaemic stroke or intracerebral haemorrhage. patients will only be included if treatment can be started within 24 h of stroke onset. for a complete overview of the inclusion and exclusion criteria, we refer to the study protocol [21] . patients are planned to be recruited in about 80 hospitals in 9 european countries over a period of about 4 years. to increase the generalisability of the findings, these countries are distributed across europe and include estonia, germany, greece, hungary, italy, the netherlands, norway, poland, and the uk. for the same reason, the trial will recruit patients both in academic and regional hospitals ( table 1 , fig. 1 ). the primary outcome measure is the score on the modified rankin scale (mrs) at 90 days (± 14 days). the mrs is an ordinal scale ranging from 0 to 6 [22] . the mrs assessment at 90 days will be during a hospital/home visit or by telephone, and the assessment or a report thereof will be recorded using a digital video camera. three blinded raters will view the videotape and adjudicate a score on the mrs. pre-stroke method of food intake oral softened food or fluids only paracetamol acute stroke treatment (%) data are n (%) or median [iqr] . mrs modified rankin scale, nihss national institutes of health stroke scale, bp blood pressure for each patient, a median mrs score will be calculated from the three mrs scores obtained through centralised adjudications by raters who are blinded to treatment allocation. the use of three scores increases the precision in scoring and statistical power as compared to a single mrs assessment [23] . the primary effect estimate will be the difference in the mrs scores between the active treatment group and controls assessed using ordinal logistic regression, and will be expressed as an odds ratio with 95% confidence interval [24] . the primary analysis will be performed on all randomised patients with a valid mrs score at 90 days. the distribution of the mrs scores will be shown as a figure (fig. 2 ). three separate primary analyses will be performed for each intervention vs. their respective controls (e.g. metoclopramide vs. nonthe primary analyses will be adjusted for stratification (country), minimisation (age, sex, stroke type, stroke severity, diabetes), and other baseline prognostic (e.g. premorbid mrs, atrial fibrillation, reperfusion treatment [alteplase and/or thrombectomy], time from onset to randomisation) factors, and treatment allocation for the other two strata of the trial (table 2) . comparison of the effect of the three intervention groups vs. their respective controls on the primary outcome will be performed in the following pre-specified subgroups (assuming sufficient numbers in each subgroup) with assessment of interaction between treatment and the minimisation factors (these subgroup analyses are considered hypothesis-generating) ( table 3) : age (≤ 75, > 75 years); sex (male, female); stroke type (ischaemic stroke, intracerebral haemorrhage); stroke severity (nihss 6-12, > 12); diabetes mellitus (yes, no). in addition, the interaction between treatment and other baseline factors will be assessed: presence of atrial fibrillation (yes, no); pre-stroke mrs score (0, > 0); reperfusion treatment (alteplase and/or mechanical thrombectomy); time to treatment (< 6, ≥ 6 h < 12 h, ≥ 12 h); treatment allocation for the other two trial strata (paracetamol-active, control; ceftriaxone-active, control; metoclopramide-active, control). since the study is not powered to detect interactions between the three interventions, these interactions will be investigated in secondary analyses. four sensitivity analyses of the mrs will also be performed: unadjusted ordinal logistic regression, adjusted analysis of mrs following regression imputation of missing data, multiple linear regression on the mean mrs score for each participant, and binary logistic regression on mrs > 2. the following secondary outcomes will be assessed at 7 days (± 1 day) or at discharge, if earlier: infections in the first 7 days (± 1 day; frequency, type, and clostridium difficile infections). infections will be categorised as diagnosed by the clinician and as judged by an independent adjudication committee (masked to treatment allocation); third generation cephalosporin resistance in the first 7 days (± 1 day), detected as part of routine clinical practice; antimicrobial use during the first 7 days, converted to units of defined daily doses according to the classification of the who anatomical therapeutic chemical classification system with defined daily doses index; serious adverse events (saes) in the first 7 days; in a subgroup of patients: presence of extended-spectrum beta-lactamase (esbl)-producing bacteria as detected by pcr in a rectal swab at day 7 (± 1 day, or at discharge, if earlier). the following secondary outcomes will be assessed at 90 days (± 14 days) ( table 4 ): death; unfavourable functional outcome, defined as mrs 3 to 6; disability assessed with the score on the barthel index (bi); cognition assessed with the montreal cognitive assessment (moca); quality of life assessed with the euroqol 5d-5l (eq-5d-5l) and eq-visual analogue scale (eq-vas); home time: the number of nights among the first 90 since stroke onset that are spent in the patient's own home or a relative's home. resource use will be censored at 90 days. where final follow-up occurs earlier, the last known placement will be extrapolated to 90 days; patient location over first 90 days (± 14 days): hospital, rehabilitation service, chronic nursing facility, and home. binary logistic regression will be used for binary outcomes (e.g. mrs > 2). cox proportional hazards regression will be used for time to events (e.g. death). ordinal logistic regression will be used for ordered categorical data (e.g. mrs). multiple linear regression will be used for continuous outcomes (e.g. bi, eq-vas). patients with missing outcome data will be excluded from the analysis. patients without a primary outcome assessment at 90 ± 14 days will be considered as a lost to follow-up. the total amount of patients who are lost to follow-up will be recorded and calculated for each treatment arm. the primary analysis will be performed on all randomised patients with a valid mrs score at 90 days. in a sensitivity analysis, missing mrs data will be imputed using multiple regression-based imputation. for the secondary outcome measures (barthel index, moca, eq-5d-5l, eq-vas), patients who die will be assigned a value one unit worse than any living value. this way, patients who die cannot be given a score similar to the worst score of patients who are alive, and it ensures that all patients will be included in the analysis. potential scores, with worst with dead added, are as follows: -modified rankin scale (mrs), 0 to 5 with death = 6; -barthel index (bi), 100 to 0 with death = − 5; -euroqol 5d-5l (eq-5d-5l), − 0.5 to 1 with death = 0; -euroqol visual analogue scale (eq-vas), 0 to 100 with death = − 1; -montreal cognitive assessment (moca), 0 to 30 with death = − 1. in the first 7 days after randomisation, all saes will be reported and described by duration (start and stop dates), severity, outcome, treatment, and relation to the investigational medical product (imp), or if unrelated, the cause. all saes will be tabulated per treatment stratum. in addition, any sae occurring between day 7 and the end of follow-up on day 90 (± 14 days) for which a causal relationship between the imp and the sae is considered at least a reasonable possibility (i.e. sars and susars) should be reported as other saes. the presence of any treatment restriction will be recorded at baseline and during the hospital phase, and classified as (1) do not resuscitate, (2) do not intubate and ventilate, (3) withhold other treatments that may prolong life, (4) withhold food, (5) withhold fluids, and (6) palliation (e.g. with morphine or a benzodiazepine). any combination of these strategies is possible. the primary study will report on the frequency of each treatment restriction, and further analyses on this topic will be published in future subgroup analyses. precious is an open-label clinical trial, and both patients and treating physicians are therefore aware of the assigned treatment. knowledge of treatment allocation can influence outcome assessment, and unblinded trials like precious are therefore at risk of detection bias. in addition, despite its apparent simplicity, assessment of the score on the mrs has been associated with considerable inter-observer variability, especially in multicentre studies, and may therefore affect trial power and treatment effect size. in precious, these two major issues are minimised through (1) online training and certification of outcome assessors via a link on the precious website and (2) central outcome assessment by three blinded adjudicators based on digital video recordings of the 90-day outcome interviews. this central adjudication by trained adjudicators offers several benefits [23] : 1. blinding is assured; 2. standardisation is possible across multiple regions and cultures; 3. statistical power is enhanced through the use of three repeated assessments; 4. the estimate of treatment effect size is restored (since statistical noise leads to underestimation); 5. it provides independent validation of the information that is collected, thereby minimising the risk of fraud; 6. site staff perform to a higher standard when aware that there will be review or audit of their activity. in addition, the risk of bias is reduced by performing the statistical analyses according to the intention-totreat principle and adjusting for the minimisation factors, other relevant baseline characteristics, and treatment allocation for the other two strata of the trial. analyses will be two-sided p < 0.05 with 95% confidence intervals presented. the trial is testing the effect of the interventions on mrs, and analyses in subgroups and on other outcomes are considered hypothesis-generating. hence, no adjustment will be made for multiplicity of testing. the data monitoring committee performs safety assessments using the haybittle-peto boundary rule (p < 0.001); hence, no significant spending of alpha will occur during the trial. all analyses will be two-tailed, and p values of < 0.05 will denote statistical significance; 95% confidence intervals will be provided. adjustment for multiple comparisons will not be performed, but all contrasts will be declared. compliance with allocated treatment will be tabulated. for each of the three study drugs, the number of received dosages will be calculated (maximum of four for ceftriaxone, twelve for metoclopramide, and sixteen for paracetamol). the number of patients who received the first dosage within the time window of 24 h will also be presented; if the dosage was not given within 24 h, the reason will be given (withdrawn informed consent, death, human error, other reason). all efficacy analyses will be performed on the intentionto-treat population. the robustness of the primary and key secondary analyses will be assessed in the perprotocol population. safety analyses will be performed on the safety population. the following population definitions will be used: ▪ intention-to-treat in primary efficacy analysis: all randomised participants who received any study medication and with a valid mrs score recorded at 90 days. ▪ intention-to-treat in primary safety analysis: all randomised participants with a vital status recorded at 90 days. ▪ per-protocol: all participants in the intention-to-treat population who are deemed to have no major protocol violations that could interfere with the objectives of the study. patients with protocol violations in trial eligibility will be included in the intention-to-treat population, but excluded in the per-protocol analysis. patients who withdrew informed consent before initiating treatment will be excluded from analysis. if (per accident) multiple randomisations are performed for a single patient, the result of the first randomisation will be used. the trial received approval from the central medical ethics committee of the university medical center utrecht, the netherlands, on 3 february 2016. the dutch national competent authority (centrale commissie mensgebonden onderzoek (ccmo)) declared to have no objection against the execution of the clinical trial within the netherlands on 17 november 2015. in addition, the national (and local, if applicable) medical ethical committees and competent authorities of the other 8 participating countries have approved the trial. the first patient was included in may 2016. the analysis and reporting of the trial will be in accordance with consort guidelines. after publication of the trial, to promote the independent re-use of precious data, a coded dataset will be made available in a public data repository within 18 months of the final follow-up of the last patient. coded data will also be included in the virtual international stroke trials archive (vista). supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-020-04717-0. additional file 1: table s1 . protocol violations in eligibility. data are n (%). mrs, modified rankin scale. additional file 2: table s2 . compliance and cross-over in first 7 days. data are n (%). comparisons made by binary logistic regression. additional file 3: table s3 . secondary outcomes and treatment restrictions at 7 days. mrs, modified rankin scale. data are n (%) or median [iqr] . aor: adjusted odds ratio. comparison by adjusted ordinal logistic regression (aolr) or binary logistic regression (ablr). * converted to units of defined daily doses according to the classification of the who anatomical therapeutic chemical classification system with defined daily doses (ddd) index. additional file 4: table s4 . overview of safety. data are n (%). sae, severe adverse event; sar, severe adverse reaction; susar, severe unexpected serious adverse reaction. comparisons made by binary logistic regression. medical complications after stroke characteristic adverse events and their incidence among patients participating in acute ischemic stroke trials development and internal validation of a prediction rule for post-stroke infection and poststroke pneumonia in acute stroke patients post-stroke infection: a systematic review and meta-analysis therapeutic hypothermia in acute ischemic stroke impact of fever on outcome in patients with stroke and neurologic injury: a comprehensive meta-analysis effect of hyperthermia on prognosis after acute ischemic stroke dysphagia after stroke: incidence, diagnosis, and pulmonary complications temporal profile of body temperature in acute ischemic stroke: relation to infarct size and outcome poststroke dysphagia: a review and design considerations for future trials route of feeding as a proxy for dysphagia after stroke and the effect of transdermal glyceryl trinitrate: data from the efficacy of nitric oxide in stroke randomised controlled trial an early rise in body temperature is related to unfavorable outcome after stroke: data from the pais study antibiotic therapy for preventing infections in patients with acute stroke the paracetamol (acetaminophen) in stroke (pais) trial: a multicentre, randomised, placebo-controlled, phase iii trial safety and effect of metoclopramide to prevent pneumonia in patients with stroke fed via nasogastric tubes trial the preventive antibiotics in stroke study (pass): a pragmatic randomised open-label masked endpoint clinical trial prophylactic antibiotics after acute stroke for reducing pneumonia in patients with dysphagia (stroke-inf): a prospective, cluster-randomised, open-label, masked endpoint, controlled clinical trial european stroke organisation (eso) guidelines for the management of spontaneous intracerebral hemorrhage european stroke organisation (eso) guidelines for the management of temperature in patients with acute ischemic stroke guidelines for the content of statistical analysis plans in clinical trials precious: prevention of complications to improve outcome in elderly patients with acute stroke. rationale and design of a randomised, open, phase iii, clinical trial with blinded outcome assessment contemporary outcome measures in acute stroke research improving the efficiency of stroke trials statistical analysis of the primary outcome in acute stroke trials publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none. hbvdw is the precious coordinating investigator. all authors contributed to the design of the statistical analysis. jdj wrote the first draft of the manuscript, and all authors reviewed the manuscript carefully. all authors read and approved the final version of the manuscript. the details of the study protocol have been published earlier [20] . after publication of the trial, to promote the independent re-use of precious data, a coded dataset will be made available in a public data repository within 18 months of the final follow-up of the last patient. coded data will also be included in vista. the primary ethics approval for the precious trial has been provided by the medical ethics committee of the university medical center utrecht, utrecht, the netherlands (nl54304.041.13). we have obtained informed consent from all participants in the study. key: cord-007456-acbo4zs2 authors: thomas, l.h.; howard, c.j.; parsons, k.r.; anger, h.s. title: growth of mycoplasma bovis in organ cultures of bovine foetal trachea and comparison with mycoplasma dispar date: 2002-11-13 journal: vet microbiol doi: 10.1016/0378-1135(87)90044-7 sha: doc_id: 7456 cord_uid: acbo4zs2 inoculation of tracheal organ cultures from bovine foetuses with mycoplasma bovis resulted in a loss of cellular structure of the lamina propria, followed 20–22 days later by lifting and detachment of overlying epithelium. the effect was associated with large numbers of m. bovis, identified by immunoperoxidase labelling and electromicroscopy, infiltrating between the epithelial cells and amassing in the lamina propria, especially in the region of the basement membrane of the epithelium. ciliary activity was undiminished for up to 18 days following inoculation and little or no cytopathic effect on the ciliated epithelium was seen in spite of the close proximity of large numbers of organisms. in contrast, m. dispar was restricted to the margin of the ciliated epithelium where, as previously reported, it caused pyknosis, sloughing and flattening of the epithelium with consequent loss of ciliary activity. the cytopathology observed for each mycoplasma bore a close similarity to the behaviour of the two mycoplasmas in vivo and it is suggested that the organ culture system may be a useful and relevant system to elucidate the pathogenic mechanisms for each mycoplasma. studies of microorganisms isolated from the respiratory tract have been greatly advanced by the use of the organ culture technique first described by i-ioorn and tyrrell (1965) . since then the technique has been used by collier et al. (1969 collier et al. ( , 1971 to investigate the effects of m. pneumoniae in organ cultures of hamster trachea, by cherry and taylor-robinson (1970) to study m. mycoides var capri in chicken tracheal organ cultures, by pijoan et al. (1972) to investigate four porcine mycoplasmas in pig tracheal organ cultures and by thomas and howard (1974) to study four bovine mycoplasmas in foetal calf tracheal organ cultures. m. bovis is a significant pathogen for the bovine respiratory tract thomas et al., 1986) ; it was thought therefore that a useful insight into its pathogenicity might be obtained by its inoculation into tracheal organ cultures and by comparison with another pathogen of the bovine respiratory tract, m. dispar (thomas and howard, 1974; howard et al., 1976) . this paper describes the results of this investigation. m. bovis strains were grown in broth (gourlay and leach, 1970) or solid media (howard et al., 1977) , except that ampicillin (1 mg m1-1) was substituted for penicillin, and thallium acetate was omitted from broth used to grow inocula. numbers of organisms were measured as colony forming units (cfu). the ab/1 strain has been shown to be pathogenic for the calf respiratory tract thomas et al., 1986) , strain ab/3 was isolated from an outbreak of mastitis and strains sm1018/c and minr1 were isolated from two separate outbreaks of calf pneumonia. m. dispar strain gri226 was grown in broth as described above. numbers of organisms were measured as colour change units (ccu) (gourlay and leach, 1970) as this species frequently fails to produce colonies on agar and ccu give a more accurate assessment of the number of viable organisms present. this strain has also been shown to be pathogenic for the calf respiratory tract gourlay et al., 1979) . tracheal organ cultures were prepared from four bovine foetuses at 5-6 months gestation (thomas and howard, 1974) . they were maintained as rings and rolled singly (experiments 1 and 3) in 4-oz bottles with 5 ml of medium , except that mycostatin was omitted from the medium and 5% heated, foetal calf serum was added. cultures were inoculated with 0.2 ml or 0.5 ml of mycoplasmas and the maintenance medium was sampled 1--2 h later (day 0 sample). thereafter medium was sampled and changed on days 2, 4, 10 and 14 post-inoculation (experiment 1) and on days 1, 2, 4, 5, 6, 7, 8, 11, 13, 15, 18, . rings were removed for sampling on days 2, 4 and 14 (experiment 1) and days 2, 4, 8, 11, 15 and 22 (experiment 3) . for experiment 2 six tracheal rings from one of two foetuses were placed in a bottle in 10 ml of medium. duplicate cultures from each foetus were inoculated with 0.1 ml of one of the four strains of m. bovis. the maintenance medium was sampled on day 1 following inoculation and thereafter medium and tissue were sampled at 5-6 day intervals until the termination of the experiment on day 28. the maintenance medium was also changed on the day of sampling. sampled rings were divided into segments and pieces taken for estimation of mycoplasma numbers, histology, including immunoperoxidase labelling, and electron microscopy (experiment 3 only). uninoculated control cultures were maintained in parallel with inoculated cultures in all three experiments and sampled at the same time for comparison. all rings were examined for ciliary activity at 2--3 day intervals. pieces of tracheal tissue were fixed in formol-sublimate (mercuric formalin) for up to 18 h and then transferred to 70% alcohol before embedding in paraffin wax and sectioning. serial sections were then stained by haematoxylin/eosin, giemsa or by the unlabelled antibody immunoperoxidase method. paraffin sections of formol-sublimate fixed tissue were stained by the peroxidase-antiperoxidase method [sternberger et al., 1970 , adapted by parsons et al., (1984 ] using primary antiserum to m. bovis and m. dispar prepared in rabbits (thomas et al., 1986) . samples of tracheal tissue were diced and fixed in fresh 3% phosphate buffered glutaraldehyde for 2 h followed by 2 h fixation in 1% buffered osmium tetroxide (millonig, 1961) . dehydration was performed in ascending grades of methanol and completed in propylene oxide. the tissues were embedded in araldite and polymerized overnight at 60°c. sections, 50--60 nm thick, were cut on a cambridge huxley ultra microtome, using glass knives, and stained with uranylacetate and lead citrate (venable and coggeshall, 1965) for examination in a philips 300 electron microscope using an accelerating voltage of 80 kv. in this preliminary experiment lasting 14 days, three organ cultures were inoculated. titres in the maintenance medium rose from 102"s cfu ml-1 at day 0 to a maximum of l0 s cfu m1-1 at day 4 and fell to 106.7 cfu m1-1 on day 14. no effect on ciliary activity was detected. microscopically, little or no cytopathic effect was detected in the epithelial layer, apart from a slight lifting and detachment by day 14. however in the lamina propria loss of cell nuclei was associated with large numbers of m. bovis located by ipx labelling. by day 4 m. bovis was progressively infiltrating between cells of the epithelium and accumulating in the lamina propria. organisms were also detected in large numbers in the peritracheal connective tissue surrounding the convex margin of the ring but with little or no apparent cytopathic effect. mean titres obtained from maintenance medium and in tissue for the duplicate cultures are shown in table i . titres in medium at day 1 reflected the relative titre of the four inocula and ranged between <101"6 and 10 s cfu m1-1, but following the first change of medium at day 5 numbers of mycoplasmas in both medium and tissue varied by <101 cfu ml -~ for all four strains of m. bovis for the duration of the experiment. it should be noted that numbers in tissue were approximately 20-fold higher than those shown in table i due to the initial 5% dilution involved in the trituration of the tissue. ab/1 3.3 medium <1.6 8.0 7.0 6.6 6,8 6.4 tissue nd 7,2 6.9 6.4 6,4 6.5 ab/3 4.8 medium 8.0 7.7 6.6 6.5 7,0 6.2 tissue nd 7.2 6.5 6.2 6.7 6.4 sm1018/c 4.5 medium 3.2 8.1 6.8 6.7 6,5 6.5 tissue nd 7.3 6.7 6.5 6,6 6.3 minr1 3.9 medium 2.1 8.2 6.4 6.1 6,4 6.1 tissue nd 7.8 6.7 6.9 6,7 6.3 anumber of organisms cfu m1-1 (10 n) at time zero. bnumbers in tissue estimated as a 5% suspension in mycoplasma broth. cmean titre of duplicate cultures, one from each foetus, each containing six tracheal rings at start of experiment. nd, not done. microscopically, changes closely resembled those seen in experiment 1: little or no cytopathic effect was detected in the epithelial layer but a loss of cellular structure in the lamina propria was associated with the presence of large numbers of m. bovis. some difference was seen in the distribution of the 4 strains of m. bovis as detected by immunoperoxidase labelling, in that strain minr1 showed less propensity to colonise the lamina propria. this same observation was made on all samples from day 5 through to conclusion of the experiment at day 28. were first re-isolated on or after the seventh day (medium or tissue). on day 7, 10 ~'7 ccu m1-1 were isolated from the medium of one culture and on day 11, 10 s'7 and 102.7 ccu m1-1 were isolated from the medium and tissue respectively of the same culture. a second culture sampled on day 15 contained 103.7 and 102.7 ccu ml -t in medium and tissue, respectively. no mycoplasmas had been isolated from this culture previously. the remaining three cultures were concluded on days, 2, 4 and 8, and no mycoplasmas were isolated. titres were essentially similar to those obtained in the first two experiments. mycoplasmas were isolated from the medium of two of the five organ cultures on the second day after inoculation with 107 m. bovis. by day 4 mycoplasmas were isolated from the medium of all four remaining cultures at titres of 10s'°--107"s cfum1-1. titres remained at this level until the last culture was sampled on day 22. numbers of mycoplasmas in the tissue were similar to those in the maintenance medium sampled on the same days (105":--107.6 cfu ml-1). activity was normal in cultures inoculated with m. dispar compared to control cultures up to day 11 but declined sharply to very faint activity by day 15. by this time the ciliated margin of the cultures was ragged and uneven due to the presence of extravasated cells. no reduction in ciliary activity could be detected in cultures inoculated with m. bovis up to 18 days after inoculation. by 22 days, however, ciliary activity had declined to one third of that of uninoculated control cultures. in contrast, m. bovis had virtually no cytopathic effect on the ciliated epithelium for 18 days following inoculation in spite of large numbers of organisms infiltrating between the columnar epithelium, accumulating in the lamina propria and amassing in the region of the basement membrane (fig. 3) . infiltration could be detected by day 8 following inoculation and, although pleomorphic organisms were seen by electron microscopy in spaces between the columnar epithelial cells, the cell membranes of the adjacent cells were apparently normal (fig. 4) . by day 22, some parts of the epithelial layer were still intact but in other areas there was lifting and detachment of the epithelium. the effect of m. bovis on the connective tissue cells of the lamina propria was striking: by day 8 a significant loss of cell nuclei was apparent and by day 15 little or no cellular structure, excepting a few epithelial cells lining the secretory glands, was discernible (fig. 5) . no comparable effect was seen in control cultures (fig. 6) or in cultures inoculated with m. dispar. as described in experiments 1 and 2, the cytopathic effect was associated with large numbers of m. bovis and organisms were also present in large numbers in the peritracheal connective tissue investing the outer, convex margin of the culture but had no apparent effect. findings from the three experiments with m. bovis in organ cultures of bovine trachea are in close agreement. the capacity of m. bovis to penetrate between the cells of the respiratory epithelium without causing damage to those cells is remarkable and is in sharp contrast to the action of m. dispar, which, as described in an earlier paper (thomas and howard, 1974) and confirmed in the present study acts, entirely at the ciliated margin of the epithelial layer to produce its cytopathic effect. the ability of all four strains of m. bovis used to penetrate the respiratory epithelium and enter the intercellular spaces resembles to some extent the reported action of m. pneumoniae in organ cultures of hamster trachea (collier et al., 1971) . however, m. pneumoniae attaches, forms clumps and is restricted to the ciliated margin of the epithelium; furthermore this organism appears to possess a special modification for attachment at this site, none of which was observed for m. bovis. subsequent studies have also shown that m. bovis, in contrast to m. pneumoniae, is non-motile (w. bredt and l.h. thomas, unpublished observations) . the eventual and relatively insignificant ciliastatic effect of m. bovis is probably attributable to the extensive necrosis in the underlying tissues, rather than to a direct effect as seen in the cultures inoculated with m. dispar. the relatively slow onset of the cytopathic effect due to m. dispar compared with that seen earlier (thomas and howard, 1974 ) may be attributed to the low titre of inoculum used. this was designed to ensure that the onset of the cytopathic effect for each mycoplasma might coincide; a high titre inoculum for m dispar (104--106.7 ccu ml -~) produced a marked cytopathic effect within 48 h (thomas and howard, 1974) . compatible with their behaviour in vivo. m. dispar causes an exudative bronchiolitis with peribronchiolar and alveolar round cell infiltration whereas m. bovis appears to be a more invasive pathogen, causing extensive coagulative necrosis in lung parenchyma with apparently little effect on the respiratory epithelium (thomas et al., 1986) . some caution should be used, however, in interpreting in vivo effects for these mycoplasma from their behaviour in organ culture; although a progressive penetration of m. bovis from the epithelial layer to the lamina propria was apparently demonstrated, the cut, transverse surface of the tracheal ring does expose the lamina propria for direct penetration by the mycoplasmas. the colonisation of the peritracheal connective tissue by m. bovis is probably an artefact but nevertheless the lack of cytopathic effect in this location is surprising. the work described here supports the view that m. bovis is second only to m. mycoides subsp, rnycoides in its pathogenicity for bovine tissue. the mechanism whereby m. bovis produces its cytopathic effect remains to be elucidated. a toxin has been described for m. bovis (geary et al., 1981) and the relevance of such a toxin to its pathogenicity in lung tissue discussed (thomas et al., 1986 ). however we have, to date, been unable to demonstrate any toxic effect for organ cultures using a cell free filtrate of medium from cultures infected with m. bovis (thomas and howard, unpublished observations) . the apparent similarity of action in vitro and in vivo suggests that organ cultures may be a convenient way of investigating mechanisms of pathogenicity in m. bovis and m. dispar. large quantity production of chicken embryo tracheal organ cultures and use in virus and mycoplasma studies biologic effects of mycoplasma pneumoniae and other mycoplasmas from man on hamster tracheal organ culture mycoplasma pneumoniae in hamster tracheal organ culture: immunofluorescent and electron microscopic studies inflammatory toxin from mycoplasma bovis: isolation and characterization a new mycoplasma species isolated from pneumonic lungs of calves (mycoplasma dispar sp. nov.) pneumonia and arthritis in gnotobiotic calves following inoculation with mycoplasma agalactiae subsp, bovis pathogenicity of some mycoplasma and acholeplasma species in the lungs of gnotobiotic calves on the growth of certain "newer" respiratory viruses in organ cultures induction of pneumonia in gnotobiotic calves following inoculation of mycoplasma dispar and ureaplasmas (t-mycoplasmas) induction of immunity in calves to mycoplasma bovis infection of the respiratory tract advantages of a phosphate buffer for osmium tetroxide solution in fixation localisation of enteropathogens in paraffin-embedded tissue by immunoperoxidase the effect of porcine mycoplasmas on pig tracheal organ cultures the unlabelled antibody-enzyme method of immunohistochemistry. preparation and properties of soluble antigen-antibody complex --horseradish peroxidase --and its use in the identification of spirochaetes replication of a bovine coronavirus in organ cultures of foetal trachea effect of mycoplasma dispar, m. bouirhinis, acholeplasma laidlawii and t-mycoplasmas on explant cultures of bovine trachea the pathology and microbiology of mycoplasma bouis infection in gnotobiotic calves, including combination with respiratory syncytial virus a simplified lead citrate stain for use in electron microscopy we acknowledge the technical assistance of miss n. rolley and mr. paul sopp with the isolation of mycoplasmas. mr. p.f. dennis and mr. brian turfrey prepared the histological sections. mr. h. kay, environmental health officer at the fmc abattoir, salisbury, gave assistance in obtaining the organ culture tissues. key: cord-283257-rh3bxvv7 authors: andrejčáková, zuzana; sopková, drahomíra; vlčková, radoslava; kulichová, lucia; gancarčíková, soňa; almášiová, viera; holovská, katarína; petrilla, vladimír; krešáková, lenka title: synbiotics suppress the release of lactate dehydrogenase, promote non‐specific immunity and integrity of jejunum mucosa in piglets date: 2015-12-21 journal: anim sci j doi: 10.1111/asj.12558 sha: doc_id: 283257 cord_uid: rh3bxvv7 the aim of our experiment was to study how synbiotics are able to deal with the problems of post‐weaning piglets. lactobacillus plantarum – biocenol(tm) lp96 (ccm 7512), lactobacillus fermentum – biocenol(tm) lf99 (ccm 7514) and flaxseed (rich in n‐3 polyunsaturated fatty acids) were administered to 36 conventional piglets from a problematic breed with confirmed presence of enterotoxigenic escherichia coli and coronavirus. the experimental piglets were supplied with probiotic cheeses and crushed flax‐seed in the period starting 10 days before weaning and lasting up to 14 days post‐weaning. piglets in the control group were supplied only control cheese. the impact of such additives on the release of lactate dehydrogenase (ldh; spectroscopic and electrophoretic assay), alteration of immunity (index of metabolic activity), jejunum histology (light microscopy), and health of conventional piglets from a problematic breed (monitoring of hematology, consistency and moisture of feces and body temperature) were examined. we found significant decrease in ldh leakage in the blood serum and tissue extracts, indicating better cell membrane integrity in the individual organs of animals. probiotics and flaxseed applied together seem to be a good source of nutrients to improve the immune status and the integrity of jejunum mucosa during infection. © 2015 japanese society of animal science diarrheic syndrome in weanlings is a serious health and economic problem in livestock production due to high morbidity and mortality. nutrition and feeding of pigs, together with other environmental factors (breeding, reproduction, health status, rearing management) are among the economically most important factors influencing animal performance parameters and the overall economics of pig farming. health and survival of neonatal piglets depend on the ability of intestinal mucosa to act as an effective barrier against various infectious agents. enterotoxigenic escherichia coli (etec) is the major reason for diarrhea in sucklings and weanlings, leading to excessive loss of fluids and electrolytes, resulting in profuse diarrhea, usually without histological lesions (wilson & francis 1986; francis 2002) . infections caused by coronavirus (porcine epidemic diarrhoea virus, pedv; mole 2013) are among the other diseases causing diarrhea and vomiting in this breed of piglets. inflammation caused by etec may be effectively inhibited by the application of suitable immunomodulatory agents demonstrated in numerous experimental studies of piglets (shirkey et al. 2006; tohno et al. 2006; walsh et al. 2008; mizumachi et al. 2009; chytilová et al. 2013) . diet supplementation with probiotics has been shown to: (i) reduce the frequency of post-weaning diarrhea (manner & spieler 1997) ; (ii) play an important role in the intestinal barrier against inflammatory bowel disease (laukoetter et al. 2008) ; and (iii) improve growth performance (scheuermann 1993) in piglets. furthermore, a strong immunomodulatory effect is also assigned to the n-6 and n-3 polyunsaturated fatty acids (pufas). the group of n-6 pufas have the ability to activate the immune system and act more as proinflammatories, whereas n-3 pufas have a significant anti-proliferative and anti-inflammatory effect (yaqoob 2004) . the growth of pigs may be enhanced by the intake of extra n-3 pufas (nguyen et al. 2003) , which have the capability to improve cell membrane fluidity (stulnig et al. 2001; russo 2009 ). the recent ban on the use of natural feeds has put pressure on the development of new feeding strategies and formulations to support performance and gut health. many studies have demonstrated mostly microbiological and immunological improvements of ongoing infections caused by the additives mentioned above. the aim of our study therefore was to examine the effect of synbiotics through flaxseed feed supplementation in combination with lactobacillus plantarum -biocenol tm lp96 (ccm 7512) and lactobacillus fermentum -biocenol tm lf99 (ccm 7514), on the activity of lactate dehydrogenase (ldh), alteration of immunity, jejunum histology and health of conventional piglets from a problematic breed. the experiments with piglets (weanlings) were performed at the institute of microbiology and gnotobiology, university of veterinary medicine and pharmacy (uvmp) in košice, slovak republic. the experiments were approved by the state veterinary and food administration of the slovak republic (approval no. 2519/10-221) and the animals were handled in a humane manner in accordance with the guidelines established by the relevant commission. the experimental animals were housed in stainless steel cages fitted with a slatted floor strewn with ¾ insulating rubber and ambient temperature of 20-22°c. the animals were divided into two groups: control c (n = 18, control cheese, sprinkle on the surface of feed) and lf group (n = 18, probiotic cheeses + crushed flaxseed). throughout the study, the animals were fed diets mixed for early weaning of piglets oš-02 (spišské vlachy, slovak republic; table 1 ) and had ad libitum access to water. the feed mixture was supplemented with crushed flaxseed (cultivar flanders, agritec, czech republic) as a source of pufas at a concentration of 10%. the fatty acid composition (percentage) of flaxseed was as follows: lipids (dry matter (dm) basis) -45.78; palmitic fa (c16:0) -5.1; stearic fa (c18:0) -3.7; oleic fa (c18:1) -18.4; linoleic fa (c18:2) -16.1; linolenic fa (c18:3) -56.8. in the period starting 10 days before weaning and lasting up to 14 days post-weaning, the experimental piglets in group lf were supplied with probiotic cheeses at a dose of 4 g/animal/day for each cheese, and in the same period the feed of group lf was supplemented with crushed flaxseed. piglets in control group c were supplied control cheese at a dose of 8 g/animal/day. the lactobacillus probiotic strains were isolated in the laboratory of the institute of microbiology and gnotobiology, uvmp in košice, slovak republic. the lactobacillus plantarum -biocenol tm lp96 (ccm 7512) strain originated from the gut contents of healthy suckling piglets. this strain was characterized by strong adherence to the epithelial cells from the porcine intestine, by inhibitory activity against e. coli o8:k88ab:h9 under in vitro conditions, and by production of hydrogen peroxide (nemcová et al. 1997) . the lactobacillus fermentum -biocenol tm lf99 (ccm 7514) strain was isolated from the gastrointestinal tract of adult chickens. this strain was characterized by the growth in the presence of bile acids and gastric juice, sensitivity to antibiotics, inhibitory activity against salmonella enterica serovar enteritidis and salmonella enterica serovar düsseldorf, and aggregation and co-aggregation ability (nemcová et al. 2003) . cheddar cheese (chemical composition per 1 kg: proteins 23.8%, sugars 2.8%, lipids 30.1%, metabolisable energy 1.62 mj) was used as a vehicle for the probiotic strains. the probiotic cheeses contained probiotic strains (each cheese contained one probiotic strain) at 1 × 10 9 cremoris) during typical cheddar cheese production. the cheese that was used as a control was similar to cheddar cheese, but without the probiotic strains (referred to as control cheese). during the experimental period the piglets all underwent clinical observation. health data were recorded twice daily (at 08.00 and 15.00 hours), specifically, body temperature (bt), consistency of feces (f) and moisture of feces (mf). samples of feces were assessed visually using a scale from 1 to 5, where the number 1 meant solid feces, 2 paste, 3 sparse, 4 hydrous and 5 feces with an admixture of blood or mucus. the moisture of feces was determined by drying a sample of feces at 80°c to constant weight. blood samples from piglets were taken from the plexus venosus suborbitalis on day 0 (day of weaning; n = 6), day 7 (n = 6) and day 14 (n = 6) after weaning. blood serum was separated from blood samples by centrifugation at 4°c and 1095 × g for 10 min for determination of total ldh (tldh) and its isoenzymes (ldh 1 -ldh 5) expressed in μkat/l and for measurement of leukocytes. on sampling days, the piglets were humanely sacrificed (euthanased) using t61 a.u.v. (intervet international bv, boxmeer, netherlands) intracardiac administration of 1 ml/kg/head. heart, liver and skeletal muscle were collected from each pig and processed (tissue homogenates or extracts) until other analyses were done. the samples were cut into small pieces, and washed in buffered saline to remove excess blood and connective tissue. two grams of tissue were then homogenized in 20 volumes of cold buffer (0.05 mol/l tris-hcl buffer, ph 7.3) in line with heinová et al. (1999) . protein concentration was measured in accordance with bradford (1976) and tldh and its isoenzymes (ldh 1 -ldh 5) were expressed in international units per gram of protein (iu/g). blood plasma was collected into tubes containing menadione ethylenediaminetetraacetic acid (k 3 edta) for hematological analysis using a bc-2008 vet automatic analyzer (mindray, shenzhen, china). the concentration of total ldh (μkat/l or iu/g) activity was assessed using commercial diagnostic kits (randox, crumlin, uk) with an alizé automatic biochemical analyser (lisabio, pouilly-en-auxois, france) at laboratory temperature. the measurement was conducted using light absorbance at 340 nm. for electrophoretic study, 10 μl of the blood serum and culture supernatant was used for each separation. a hydrasys device (sebia, lisses, france) was used for the determination of ldh isoenzyme activities. the samples were separated using commercial hydragel 7 iso-ldh electrophoretic kits (ecomed, žilina, slovak republic) on alkaline-buffered (ph 8.4 ± 0.05) agarose gels (0.8g/dl). the dried gels were prepared for visual examination and densitometry to obtain accurate relative quantification of individual zones. the image of the electrophoretic migration was scanned by light transmission and automatically converted into an optical density curve presentation. then photographs of the gels were taken. qualitative evaluations of the gels were done directly from the electrophoretograms and the densitometric curves of the separations were created by means of epson perfection v 700 photo densitometer scanning at 570 nm and evaluated using phoresis software (version 5.50, 2009, sebia, lisses, france) . the iodonitrotetrazolium test (int) -2-(4-iodophenyl)-5-fenyltetrazolium chloride -int (erba lachema, brno, czech republic)was carried out on microscale in accordance with the modifications made by procházková et al. (1986) . the functional ability of phagocytes of metabolic processes occurring in phagocyting leukocytes (production of microbicidal substances, particularly h 2 o and o 2 ) based on the ratio between spontaneous activity and the activity after stimulation by zymosan (sigma, st. louis, mo, usa), that is, the index of metabolic activity (ima), was assessed. excisions from the jejunal mucosae (collected from piglets on day 0 and day 7) of 1 mm 3 size were fixed by immersion in 3% glutaraldehyde and postfixed in 1% osmium tetraoxide (both in 0.15 cacodylate buffer, ph 7.2-7.4). after dehydration in acetone, the excisions were transferred to propylene oxide, and embedded in durcupan acm (fluka chemie ag, buchs, switzerland). semi-thin sections (250 nm) of specimens processed for transmission electron microscopy were cut on an lkb nova ultramicrotome, stained with toluidine blue (sigma-aldrich, bratislava, slovak republic) and examined under an axio lab. a1 light microscope (zeiss, jena, germany) at 400× magnification. the data were assessed using one-way analysis of variance with tukey's post hoc analysis (graphpad prism 3.0 for windows; graphpad software, san diego, ca, usa). values listed in the tables and figures are the average values obtained from six samples. all data are means with standard error of mean (sem). differences within the group are marked with superscript letters ( a, b, c ) and considered to be significant at levels of a = p < 0.05, b = p < 0.01 and c = p < 0.001. before the experiment was done, diagnostic analysis was conducted in the herd as kept by the animals' owner and samples of biological material were collected for laboratory analyses (bacteriological examination of rectal swabs, virological and parasitological examination of feces, hematological and biochemical examination of blood) which allowed us to diagnose: hypoproteinemia, lymphocytic leucocytosis and increased activity of bilirubin and enzymes (alanine aminotransferase, gammaglutamyltransferase). infection with enterotoxigenic e. coli (institute of microbiology and immunology, uvmp, kosice, slovak republic) and coronavirus (vetservis, s.r.o., nitra, slovak republic) was confirmed in fecal samples. during the experiment selected health parameters of the piglets were recorded as shown in table 2 . the consistency of feces (f) in the control group of piglets deteriorated significantly (p < 0.001) during the experimental period, whereas in the experimental group the consistency had a more settled character. moreover, the piglets with addition of synbiotics had better progress of infection in general. we noticed significant increase in the leukocyte count and hemoglobin (p < 0.01) after 14 days of the supplementation period. in the blood serum in the experimental group of piglets, significant decrease in tldh concentration was noticed on day 7 (p < 0.001) and day 14 (p < 0.01) compared to day 0 (table 3 ). in the control group, without addition of probiotics and flaxseed, significant decrease was recorded on day 14 (p < 0.001) compared to day 7 after weaning. average values of tldh activity on day 7 were significantly lower (p < 0.01) in the experimental group compared to control. significant differences were also observed in the concentration of ldh 1 to 5 isoenzymes. all isoforms exhibited a decreasing trend at the end of the feeding period (day 14) in comparison with the beginning of the supplementation period, and at different significance levels (see table 3 ). tissue extracts from the piglets' hearts (table 4 ) showed significant differences between the observed groups in the concentrations of tldh (p < 0.05) and ldh 1 (p < 0.01) on day 14 after weaning. overall, the excretion of ldh (tldh and ldh 1-5) was markedly reduced on day 14 in the experimental group compared to control. ns ns ns note: c, control group; lf, group treated with probiotics and flaxseed; day 0-14, study day after weaning; data are means ± sem (iu/g); a mean values in rows with same superscript letters are statistically significant at the level of a = p < 0.05. the piglets' livers (table 5 ) exhibited markedly decreased activity of tldh and its isoenzymes on day 14 after weaning at different levels of significance (p < 0.05; p < 0.01; p < 0.001). in the skeletal muscles of the piglets (table 6) , we noticed significant decrease in tldh, ldh 2, 3 and 5 (p < 0.05) on day 14 after weaning when comparing the observed groups. the activity of ldh exhibited a decreasing trend at the end of the feeding period (day 14). in our experiment, significant increase in non-specific immunity (ima) was noticed on day 7 (p < 0.01) and day 14 (p < 0.05) after weaning when comparing the observed groups (fig. 1 ). significant increase (p < 0.05) was recorded in the index of metabolic activity in the experimental group of piglets. at the beginning of sample collection (day 0 of the experiment) in the control group, the mucous layer of intestinal villi (fig. 2) consisted of a single layer of columnar epithelial enterocytes (e) and goblet cells (gc). in the apical part of intestinal villi, small groups of dying enterocytes of irregular shape and with dark cytoplasm were present. goblet cells did not show significant morphological changes. in the epithelium of intestinal villi numerous intraepithelial lymphocytes were present. significant leukocyte infiltrations were observed mainly in the connective tissue of lamina propria mucosae. these findings contrasted with the histological sections of jejunum from the experimental piglets, where dying enterocytes occurred sporadically only in the apical part of intestinal villi. microscopic changes in the mucous layer of the small intestine epithelium indicated persistent inflammation in the control group of weanlings (day 7; fig. 2 ). intestinal villi were broad, and low and had strongly deformed shape. the presence of dying enterocytes was enormous, and the sparse connective tissue of lamina propria mucosae was significantly infiltrated by leukocytes, in contrast to the jejunum of the experimental group, where intestinal villi were high, of regular shape, and mucosa of the jejunum did not show any structural changes. probiotics are live microorganisms which may contribute to the health of the host, when they are administered in appropriate amounts (fao/who 2002) . lactic acid bacteria regulate intestinal microbial homeostasis and the stabilizing function of the gastrointestinal barrier, while expressions of bacteriocins (mazmanian et al. 2008 ) have immunomodulatory effect (salzman et al. 2003) . they may inhibit procarcinogen enzymes and the ability of pathogens to colonize and infect the gut note: c, control group; lf, group treated with probiotics and flaxseed; day 0-14, study day after weaning; data are means ± sem (iu/g); a,b,c mean values in rows with same superscript letters are statistically significant at the levels of a = p < 0.05; b = p < 0.01; c = p < 0.001. mucosa (gill 2003) . probiotic microorganisms act against infections of the gastrointestinal tract by means of their own production of bioactive molecules, such as organic acids, carbon dioxide, hydrogen peroxide, other low molecular weight substances and bacteriocins (gomes et al. 2012) . probiotics are able to displace pathogens such as salmonella spp., clostridium spp. and e. coli in the gastrointestinal tract of pigs through the mechanism of competitive inhibition for binding sites (biernasiak et al. 2011) . the efficacy of probiotics may be potentiated by components of natural origin (so-called synbiotics) such as the essential pufas, which have impact on the nutrition of piglets (tanghe & de smet 2013; tanghe et al. 2014) . the presence of pufas may positively improve the adaptation of piglets to the rapidly changing diet at weaning (bomba et al. 2005; marcinčák et al. 2009; li et al. 2014) . they have direct regulatory action on leukocytes by binding to intracellular receptors or by modifying the release of second messengers (klasing 1998) , and on red blood cell membrane fatty acid composition (boehm et al. 1996) . it is known that ldh is a highly sensitive, but not specific marker of cell membrane disruption. intracellular ldh (ec. 1.1.1.27), l-(+)-lactate: nad + oxidoreductase leakage is a possible indicator of cell membrane integrity and cell viability (legrand et al. 1992) . ldh catalyses the intraconversion of pyruvate and lactate and is involved in both the catabolism and anabolism of carbohydrates. in addition to metabolic roles in the cells, it includes a number of other biological processes (powers et al. 1991) . the function of specific indicators is performed by ldh isoenzymes, which allow us to identify the damage to cells and tissues by different agents. ldh leakage from various cells (e.g. hepatocytes, monocytes, lymphocytes) into the environment is used as one of the markers of cytotoxicity for monitoring tissue and cell damage in in vitro conditions (kopperschläger and kirchberrger 1996; šutiaková et al. 2004) . moreover, mean values between columns with the same superscript letters are statistically significant at levels a = p < 0.05; b = p < 0.01. lactic acid level reflects the quantitative transformation of glycogen, and indicates typical or atypical processes of meat ripening (koréneková et al. 2009 ). in our experiment, leakage of ldh into the interstitial space of organs was monitored in blood serum and tissue extracts from the heart, liver and skeletal muscle in piglets with persistent infection. in the group fed with synbiotics (combination of probiotics and pufas in the form of flaxseed) we noticed significant decrease in tldh leakage and isoenzymes typical for individual tissues. ghanem et al. (2005) experimentally infected mice with schistosoma mansoni, and they found that after 14 days of probiotic administration (yogurt containing l. casei, l. plantarum, l. reuteri, l. acidophilus), the activity of ldh decreased. similar results were recorded in our experiment with weanling pigs. the authors described the protective effect of probiotics through non-specific stimulation of the immune system. otherwise, rajput et al. (2013) described significant increase in concentration of ldh in the blood of pigs given feed supplemented with bacillus subtilis. the concentration of tldh and its isoenzymes in the liver of our piglets significantly decreased at the end of the feeding period, which indicated the bioprotective effect of synbiotics on hepatocytes. new studies (imani fooladi et al. 2013) have confirmed that probiotics have significant effect on the health status of the liver and the ability to treat various liver diseases. based on our results, it could be assumed that the temporary increase in tldh and its isoenzymes in the skeletal muscle of piglets, which otherwise continuously decreased, was probably due to the ongoing infection. daugschies et al. (2000) noticed subsequent decrease in the activity of tldh in muscles of calves after experimental infection with sarcocystis cruzi, which may also be affected by enhancing the release of ldh into the bloodstream during ongoing infection. ldh is also produced by the parasites themselves, even though their presence in the blood of the host was not confirmed in this study. the impact of infection on serum ldh release was observed in the study by nussinovitch et al. (2009) , who noticed an increase in tldh and its isoenzymes ldh 4 and 5 in the blood of people with bacterial meningitis. it is well known that probiotics (delcenserie et al. 2008; trasino et al. 2013) and pufas (harbige 2003) have the ability to improve the immune status of the organism. the index of metabolic activity (ima) seems to be appropriate for testing the characteristics of nonspecific immunity in animals (spišáková et al. 2009; haladová et al. 2011) . in our study, the ima index significantly increased in the group fed with lactobacilli and flaxseed, similar to the study of wen et al. (2011) , who applied probiotics alone against rotavirus disease. the histological sections of jejunum from experimental piglets fed with synbiotics did not show any structural changes, in contrast to the control group of weanlings, where significant damage to intestinal villi was recorded, followed by lymphocyte proliferation. many authors (schroeder et al. 2006; chandran et al. 2013; núñez currently, several studies aimed at improving the problems of pigs after weaning are based on adding healthpromoting substances or additives into animal feed. in our study, we point out the beneficial effect of probiotics (lactobacillus plantarum and lactobacillus fermentum) in combination with pufas (in the form of flaxseed) on animal health. better integrity of cell membranes in the individual organs of animals was recorded by monitoring ldh release into the bloodstream and tissue extracts. probiotics and flaxseed significantly improved the immune response to ongoing infection. in terms of 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lactobacillus paracasei to ossabaw pigs on a high-fat diet prevents cholesteryl-ester accumulation and lps modulation of the liver x receptor and inflammatory axis in alveolar macrophage predominance of a bacteriocin-producing lactobacillus salivarius component of a five-strain probiotic in the porcine ileum and effects on host immune phenotype development of γδ t cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli fimbriae and enterotoxins associated with e.coli serogroups isolated from clinical cases of porcine colibacillosis fatty acids and the immune system: from basic science to clinical applications this study was supported by the vega grant of the ministry for education, science, research and sport of the slovak republic (no. 1/0613/13 and 1/0009/15). we would like to thank ing. m. kozackova for the technical assistance at electrophoresis and mr. a. billingham for english correction of the manuscript. key: cord-258696-01wj76es authors: decaro, nicola; campolo, marco; lorusso, alessio; desario, costantina; mari, viviana; colaianni, maria loredana; elia, gabriella; martella, vito; buonavoglia, canio title: experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia date: 2008-04-30 journal: vet microbiol doi: 10.1016/j.vetmic.2007.10.008 sha: doc_id: 258696 cord_uid: 01wj76es a pantropic canine coronavirus (ccov) strain (cb/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. we report the clinical, virological and serological findings in dogs experimentally infected with strain cb/05. the dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral rna with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. considering the severity of the cb/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8–9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. at postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for ccov by real-time rt-pcr and virus isolation on cell cultures. all pups seroconverted for ccov, as shown by the high optical density values and antibody titres detected by elisa and virusneutralisation tests, respectively. the present study confirms that strain cb/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions. coronaviruses (covs) are enveloped, singlestranded rna viruses which included in three different antigenic groups. covs infecting dogs comprise canine enteric coronavirus (ccov) (enjuanes et al., 2000) and www.elsevier.com/locate/vetmic available online at www.sciencedirect.com veterinary microbiology 128 (2008) [253] [254] [255] [256] [257] [258] [259] [260] the newly recognised canine respiratory coronavirus (crcov) (erles et al., 2003; decaro et al., 2007a) , belonging to group 1 and group 2 covs, respectively. two ccov genotypes have been identified so far, namely ccov type i and ccov type ii, which are responsible for the occurrence enteritis in dogs and are frequently associated in mixed infections decaro et al., 2005c) . although its tropism is restricted to the gastroenteric tract, ccov has been recently associated to systemic disease followed by fatal outcome in pups . severe clinical signs were observed in the affected pups, whereas necropsy examination revealed remarkable gross lesions in lungs, liver, spleen and kidneys. virological and bacteriological investigations failed to detect common canine pathogens. unexpectedly, ccov type ii rna was detected at very high titres in the internal organs of the dead pups and the virus (strain cb/05) was isolated on canine cell cultures. the association of strain cb/05 to a severe, sometimes fatal disease of dogs, together with the isolation of the virus from organs with severe lesions, strongly suggests that ccov has changed its tropism, acquiring the ability to spread from the enteric tract to the internal organs (decaro et al., 2007b) . in this study, we report the results of the experimental infection with isolate cb/05 in pups with different age, showing that in contrast with classical ccovs, this virus is able to cause systemic disease followed by fatal outcome in younger pups. canine fibroma a-72 cells were grown in dulbecco's minimum essential medium supplemented with 10% foetal calf serum. strain cb/05 was isolated from the lungs of a dead pup (117/05-c) and adapted to growth on a-72 cells. at the 3rd passage, the virus was titrated on cell cultures and inocula containing 10 6.25 tcid 50 /ml of viral suspension were stored at à70 8c. contaminations by other canine pathogens, such as canine parvovirus type 2 (cpv-2), canine distemper virus (cdv) and canine adenoviruses (cadvs), were ruled out by specific molecular assays (hu et al., 2001; decaro et al., 2005b; elia et al., 2006) . the experimental study was performed according to the animal health and well-being regulations and was authorised by the ministry of health of italy (authorization no. 53/2005-c) . six mixed-bred female dogs including four 2.5-month-old (n = 1-4) and two 6-month-old (n = 5, 6) pups were housed at the ''infectious disease unit'' of the animal hospital, faculty of veterinary medicine of bari. the dogs had tested negative for ccov rna by a real-time rt-pcr assay carried out on the faeces and for ccov antibodies by an elisa test (pratelli et al., 2002) carried out on serum samples. all dogs were housed individually in separate boxes, fed twice daily with a commercial dry dog food and provided water ad libitum. after an acclimatization period of 5 days, 5 animals (n = 1, 2, 3, 5, 6) were administered oronasally 3 ml of a viral suspension of strain cb/05, with a titre of 10 6.25 tcid 50 and 7.85 â 10 7 rna copies per ml, whereas one dog (n = 4), 2.5-monthold, was maintained uninfected by oronasal administration of 3 ml of sterile saline solution. the clinical condition of each dog was monitored daily for 21 days. a scoring system was devised taking into account rectal temperatures, total white blood cell (wbc) counts, appearance of clinical signs (vomiting, diarrhoea, depression, loss of appetite, dehydration), following the scheme adopted in a previous study (decaro et al., 2005a) and derived by nakamura et al. (2001) , with some modifications (table 1 ). due to ethical reasons, dogs whose total clinical score reached a value !15 were euthanized by intravenous administration of 10 mg/ kg of body weight of zoletil 100 (virbac s.r.l., italy) followed by 0.5 ml/kg body weight of tanax (intervet italia, italy). edta-treated blood samples were collected daily for total and differential wbc counting and for testing for ccov rnaemia by real-time rt-pcr . the presence of the viral rna in the blood was also evaluated at hours 3, 6, 9, 12 and 18 after inoculation. plasma samples were prepared daily to evaluate free viral rnaemia and weekly to determine ccovantibody titres by virus neutralisation (vn) and elisa tests (pratelli et al., 2002) . to evaluate the viral shedding in the faeces, the rectal swabs collected daily from the control dog and from the dogs inoculated with strain cb/05 were subjected to rna extraction using qiaamp 1 viral rna mini kit (qiagen s.p.a.). in addition, tissue samples from parenchymatous organs were withdrawn from the two dead pups (table 2) . rna was extracted from the wbc pellets using qiaamp 1 rna blood mini kit (qiagen s.p.a.), from the plasma samples using qiaamp 1 viral rna mini kit and from the tissue samples using qiaamp 1 rneasy mini kit. attempts to isolate the virus in a-72 cells were carried out on rectal swabs of all infected pups and on organs of the sacrificed animals as described previously (decaro et al., 2007b) . real-time rt-pcr targeting the m gene of ccov type ii (genbank accession number d13096) was carried out on the rna extracts as described elsewhere (decaro et al., 2005c) . the genotypespecific rt-pcr assays were undertaken in an i-cycler iq tm real-time detection system (bio-rad laboratories srl, milan, italy) and the data were analyzed with the appropriate sequence detector software (version 3.0). after reverse transcription, triplicates of the ccov type ii standard dilutions and rna templates were simultaneously subjected to realtime analysis. the 50 ml reaction mixture contained 25 ml of iq tm supermix (bio-rad laboratories srl), 600 nm of each primers ccovii-f (tagtgcat-taggaagaagct) and ccovii-r (agcaatttt-gaacccttc), 200 nm of probe ccovii-pb (fam-cctcttgaaggtgtgcc-tamra) and 20 ml of c-dna. the thermal profile consisted of activation of itaq dna polymerase at 95 8c for 10 min, followed by 45 cycles of denaturation at 95 8c for 15 s, annealing at 48 8c (type ii-specific assay) for 30 s and extension at 60 8c for 1 min. plasma samples from inoculated dogs were tested in parallel by virus neutralisation (vn) and elisa tests (pratelli et al., 2002) . for vn test, duplicates of serial twofold dilutions of heat-inactivated plasmas (starting from dilution 1:2) were mixed with 100 tcid 50 of the isolated strain cb/05 in 96-well microtitre plates. after preincubation at room temperature for 90 min, 20,000 a-72 cells were added to each well. the plates were read after 4 days of incubation at 37 8c. vn titres were calculated with the karber method and expressed as the highest plasma dilution that was able to neutralise the virus. for elisa, microtitre plates were coated with ccov antigen (enteric strain s-378) and, after treatment with blocking solution (0.2% gelatin in carbonate buffer [15 mm na 2 co 3 , 35 mm nahco 3 , ph 9.6]) and repeated washing, the 1:50 dilutions of the plasma samples were added to each well. then the plates were incubated for 90 min at 37 8c, washed positive and negative controls were used as described previously (pratelli et al., 2002) . neither clinical signs or viral shedding were observed in the control dog, whose leukocyte counts did not draw away from the baseline values. all the inoculated animals displayed severe clinical signs similar to those observed in dogs infected naturally, although the outcome of the disease was different on the basis of the age (fig. 1) . in fact, two out of the three youngest pups (dogs 1 and 3) had to be euthanized for ethical reasons, at days 8 and 9 postinfection (p.i.), respectively, while both the 6-month-old pups recovered from the disease, albeit very slowly. irrespective of the final outcome, i.e, euthanasia or recovery, clinical signs were remarkably similar in all inoculated animals. the 2.5-month-old dogs that underwent a fatal outcome (fig. 1a) showed fever at days 1-2 p.i., with a peak of 39.9 8c at day 2 p.i. (dog 1), and from days 1 to 6 p.i., with a peak of 40.0 8c at day 3 p.i. (dog 3). depression (days 3-8 p.i.), anorexia/dysorexia (days 3-9 p.i.), haemorrhagic diarrhoea (days 2-7 p.i.) and vomiting (days 4-5 p.i.) also occurred. leukopenia appeared at day 3 (dog 1) or 2 p.i. (dog 3), with total wbc counts remaining below 60% of the baseline values until euthanasia. acute lymphopenia was observed in both dogs, with lymphocyte numbers dropping below 60% of the initial counts from day 3 p.i. until death (mean, 16.3%; 0.9 â 10 3 lymphocytes/ ml at day 8 p.i.). postmortem examination revealed severe changes in the intestines and major organs, which were very similar to those observed in dogs infected naturally (data not shown). the 2.5-month-old dog that survived (fig. 1b ) displayed less severe symptoms, consisting of fever (up to 39.2 8c) from days 1 to 5 p.i. and at days 11-12 p.i., and mucoid diarrhoea (days 2-6 p.i.). total wbc counts dropped below the 60% of the initial counts from days 3 to 6 p.i., whereas severe lymphopenia (below 60% of baseline values) was registered from days 1 to 8 p.i., with a peak at day 5 (19%; 1.6 â 10 3 lymphocytes/ml). subsequently, the total number of peripheral blood lymphocytes started to rise again and the clinical signs subsided. a mild loss of appetite occurred from days 1 to 6 p.i. in the two 6-month-old pups (fig. 1c) , fever showed a biphasic course, with a first peak at day 2 p.i. of 39.8 and 40.1 8c in dogs 5 and 6, respectively. a transient remission was observed from days 5 to 8 p.i. (dog 5) and at day 5 p.i. (dog 6), and a second episode of pyrexia occurred from days 9 to 14 p.i. with a peak of 39.5 8c at day 10 p.i. (dog 5), and from days 6 to 8 . dogs inoculated oronasally with cb-05 strain were monitored for up to 21 days for total wbc, lymphocyte and polymorphocyte counts (top graphs). in addition, fever, viral rna shed in faeces and clinical score where determined (bottom graphs). total wbc, lymphocyte and polymorphocyte counts are presented as percentages of the cell counts determined at day 0. viral rna titres as determined by real-time rt-pcr are expressed as log copy numbers (log 10 ) per ml of template. clinical scores were calculated as shown in table 1 and are reported for each day in correspondence of the temperature curves. p.i. with a peak of 39.4 8c at day 7 p.i. (dog 6). depression was observed between days 3 and 8 p.i., whereas vomiting appeared only sporadically in the same period (days 3, 4, 8 p.i. in dog 5; days 1, 3, 8 p.i. in dog 6), with 1-4 episodes per day. both dogs displayed anorexia (days 3-7 p.i.), mucoid or fluid diarrhoea (days 3-10), and leucopenia, with wbc values dropping below 60% of the baseline from days 3 to 6 (dog 5) or 3 to 8 p.i. (dog 6). lymphocytes dropped below 60% of the initial cell counts from days 3 to 6 p.i. in dog 5 and from days 3 to 8 p.i. in dog 6 (mean, 32.2%; 1.6 â 10 3 lympholymphocytes/ml at day 3 p.i.). starting from day 10 (dog 5) or 9 p.i. (dog 6), the clinical conditions of the dogs improved progressively, with a complete recovery at days 15-16 p.i. the control (uninfected) pup (fig. 1d ) remained in a good clinical status during the entire observation period and no variations in total wbc and lymphocyte counts were observed. the uninfected dog tested constantly negative for ccov rna. all the ccov-infected dogs tested negative for other common pathogens of dogs, including ccov type i (decaro et al., 2005c) , cdv , cadvs (hu et al., 2001) and cpv-2 (decaro et al., 2005b) . the faecal shedding of the infected dogs followed the similar pattern, although higher viral rna titres were obtained from the two sacrificed animals in comparison to survivors (fig. 1) . the pups that succumbed shed virus starting between days 1 and 3 p.i. and lasting until the day of death, with a peak at day 6 (titre of 4.97 â 10 5 rna copy numbers/ml of template) or 9 (titre of 8.72 â 10 5 rna copy numbers/ ml of template). after their death, ccov type ii rna was detected in the organs at titres slightly lower than those observed in the dogs of the natural outbreak ( table 2 ). the 2.5-month-old pup that recovered shed ccov rna starting from day 1 p.i. (titre of 2.97 â 10 1 rna copy numbers/ml of template) and lasting for the entire observation period (21 days), with a peak at day 6 p.i. (3.24 â 10 4 rna copy numbers/ml of template). shedding of ccov in the faeces of the two 6-month-old dogs was observed from day 2 p.i. (mean titre, 1.40 â 10 3 rna copy numbers/ ml of template) to day 21 p.i. (last day of observation) reaching the maximal mean value of 6.79 â 10 5 rna copy numbers/ml of template at day 10 p.i. surprisingly, ccov rna was never detected in the blood of the 6-month-old pups, as well as in the euthanized animals, in whose organs remarkable viral rna titres were found. traces of ccov rna were detected only in the blood of the survived 2.5-monthold pup between days 7 and 10 p.i., with plasma viral titres ranging from 5.54 â 10 0 to 9.30 â 10 1 rna copies/ml of template. the virus was successfully isolated on cell cultures from the rectal swabs of all inoculated dogs (data not shown) and from some organs of the euthanized pups ( table 2) . all the infected animals seroconverted for ccov, whereas antibodies were not detected in the control dog (fig. 2d ). in the dogs that were euthanized the antibody titres were determined only at day 7 p.i., with vn titres of 1:8 and od values of 0.089 (geometric means, fig. 2a ). in survivors, the maximal antibody titres were reached at days 14 and 21 p.i. by vn and elisa test, respectively ( fig. 2b and c) . in a previous study, a pantropic variant of ccov was associated to a fatal disease of dogs, characterised by leucopenia, gastroenteritis and severe changes in the internal organs . the disease induced by strain cb/05 in pups of the natural outbreak was reproduced in dogs infected experimentally. all inoculated dogs were successfully infected, as shown by the occurrence of faecal shedding, seroconversion and severe clinical signs. the viral excretion was similar to those observed during enteric ccov infection (decaro et al., , 2005c , but the course of disease was more severe, as clinical signs characteristic of systemic infection were observed in the infected dogs. the pantropism of the virus was confirmed by the presence of gross lesions in the internal organs of the dead dogs, as well as by the detection of viral rna in those tissues. interestingly, ccov rna was detected also in the brain of the dead dogs. in contrast, enteric ccov has been never associated to systemic infection, although the virus has been isolated previously from some tissues (tonsils, lungs and liver) of experimentally infected pups (tennant et al., 1991) . two of the three inoculated 2.5-month-old pups had to be euthanized after few days of postinfection, whereas the other dogs (the remaining dog of the same age and the two dogs 6-month of age) recovered after a severe disease. considering that dogs infected naturally were all between 45 and 56 days of age, it could be hypothesised that the age of the infected animals plays a role in determining the fate of cb/05 infection, with a very severe clinical course in the youngest pups. moreover, in the experimental infection, the organs of the dead dogs contained lower ccov rna titres with respect to dogs infected naturally, so that virus isolation was not obtained from all pcr-positive tissues. despite the drop of the wbc counts registered in all infected dogs and the detection of the viral rna in the internal organs of the sacrificed dogs, free or cell-associated ccov rnaemia was not found at any time either in euthanized or survived dogs, with the exception of the recovered 2.5-month-old pup which showed very low rna viral titres in the plasma between days 7 and 10 p.i. thus, at this moment, the mechanisms of lymphopenia and viral spread to internal organs remained unknown. albeit strange, our findings are compatible with those obtained from cats experimentally infected with feline infectious peritonitis virus (fipv). cats succumbed to fipv display very high-viral titres in the haemolymphatic tissues (kipar et al., 2006) , in contrast with the low loads detected in the blood (de groot-mijnes et al., 2005) . in our study, cb/05-infected dogs were found to contain lower viral loads in the lymphoid tissues in comparison to fipv-infected cats, thus likely accounting for the undetected viral rnaemia in euthanized dogs. in conclusion, we have confirmed the pantropism of strain cb/05, reproducing the natural disease even in experimental conditions. further studies will contribute to better understand the epidemiological distribution and the pathogenetic mechanisms of the virus, including the possible involvement of the different lymphocyte classes. canine coronavirus highly pathogenic for dogs natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr maternally-derived antibodies in pups and protection from canine parvovirus infection a real-time pcr assay for rapid detection and quantitation of canine parvovirus type 2 dna in the feces of dogs genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs serological and molecular evidence that canine respiratory coronavirus is circulating in italy molecular characterisation of the virulent canine coronavirus cb/05 strain detection of canine distemper virus in dogs by real-time rt-pcr family coronaviridae detection of a group 2 coronavirus in dogs with canine infectious respiratory disease detection and differentiation of cav-1 and cav-2 by polymerase chain reaction natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats pathogenic potential of canine parvovirus types 2a and 2c in domestic cats prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of italy two genotypes of canine coronavirus simultaneously detected in fecal samples of dogs with diarrhea canine coronavirus infection in the dog following oronasal inoculation key: cord-271298-7vk3wgw1 authors: sato, tomoi; meguid, michael m; quinn, robert h; zhang, lihua; chen, chung title: feeding behavior during sialodacryoadenitis viral infection in rats date: 2001-04-30 journal: physiol behav doi: 10.1016/s0031-9384(01)00420-6 sha: doc_id: 271298 cord_uid: 7vk3wgw1 sialodacryoadenitis (sda) is a highly contagious common viral infection in rats, akin to mumps in humans. anorexia occurs during such viral infection. but the pattern of the decrease in food intake (a decrease in either meal size and meal number or both) during spontaneous viral infection has not been previously characterized. we observed the onset of anorexia and an abnormal feeding pattern during an opportunistic sda viral infection in our rat colony. we thus studied seven male rats. before the viral infection there was a positive association between food intake and meal number (p<.05). after infection food intake decreased by 68%. this occurred via a significant decrease in meal size (by 69%) (p<.05); and a nonsignificant decrease in meal number (p=.71). this pattern of decreased food intake is similar to that occurring during indomethacin-induced ulcerative ileitis, where we previously measured an increase in plasma tumor-necrosis factor (tnf)-α. anorexia in response to bacterial lipopolysaccharide administration, which is also linked to plasma tnf-α, is however, caused only via a decrease in meal number. the differences in the decrease in the feeding pattern between the sda viral and a bacterial infection suggest that factors other than tnf-α alone play a significant role in the mechanism of anorexia during a viral infection. anorexia occurs during acute and chronic diseases. in an acute infectious disease, anorexia can be rationalized to provide some beneficial effect: metabolic switch to hepatic acute phase protein synthesis, rest of gastrointestinal tract or prevention of bacterial growth via reduced availability of nutrients essential for microorganisms [1] , resulting in better prognosis [1] . in a chronic disease such as cancer, the chronicity of anorexia leads to malnutrition, body weight loss and eventually cachexia [2] . the mechanism(s) of anorexia is multifactorial and includes both peripheral and central factors [3] , which await further identification. daily food intake (fi) is a function of meal size (mz) and meal number (mn; fif mz â mn), which constitute feeding pattern. under normal conditions, the constancy of daily food intake is maintained via a reciprocal change in meal size and meal number. this suggests that meal size and meal number be regulated independently via closely coordinated systems [4 ±6 ]. under pathological conditions, a reduction of food intake occurs via a reduction of either meal size or meal number or both, thus providing insights into the etiology and the possible mechanisms of the pathogenesis of eating behavior [6] . an automated computerized rat eater meter (acrem; [7] ), which measures individual meal size and meal number as well as food intake for prolonged periods, provides us with the ability to characterize the biological manifestations associated with feeding behavior including anorexia. sialodacryoadenitis (sda) is a common short-lived acute infection in rats caused by a coronavirus, and is akin to mumps in humans. it is highly contagious among rats and spreads by the respiratory route [8] . sda has a high morbidity but usually a low mortality with mild clinical signs: squinting, photophobia, blinking, sneezing, and/or swelling under the neck caused by either edema, enlarged cervical lymph node, or inflamed salivary glands. normally rats recover within 1 week [8] . anorexia during an influenza virus-induced infection was previously documented [9± 11], but the feeding pattern during a similar viral infection has not been previously reported. an opportunistic sda viral infection occurred in our male fischer-344 rat colony and provided the spontaneous opportunity to measure and document changes in the feeding pattern with the ensuring anorexia. our data provide further insight for understanding the mechanism(s) of anorexia and its associated changes in feeding pattern during an acute viral infection. male fischer-344 rats (taconic, georgetown, ny), with a purchase weight of 230 g, were housed in holding cages for 7 days to acclimate them to the constant study surroundings; 12-h light/dark cycle (lights on 0500 ± 1700 h), 26 1°c room temperature, and 45% relative humidity. rats had free access to fresh coarsely ground chow (diet #5008; ralstonpurina, st. louis, mo) and tap water. when the outbreak of an sda viral infection in the rat colony was first suspected, seven acclimated and apparently healthy rats were placed in the acrem cages whose function was previously described in detail [7] . the acrem continuously measures meal size, meal number, and food intake for a prolonged period, without preconditioning or pretraining the rats. food access, via a feeding tunnel, is monitored via photocells. food consumption is measured via an electronic scale. both data are integrated in real-time and continuously recorded during successive light/ dark cycles. a meal was defined as a bite or a series of bites preceded and followed by at least 5 min of feeding inactivity. body weight was measured daily. the rats were studied until they appeared to have clinically recovered from the sda viral infection. during 10 days of feeding pattern measurement, food intake dramatically decreased when the rats become clinically symptomatic. at the same time, three other rats (the same age and the same body weight) in the same colony room were randomly selected and were euthanized using carbon dioxide. blood was immediately collected via cardiac puncture, and serum was obtained and stored in à 20°c. serologic testing (charles river lab, wilmington, ma) was performed via enzyme-linked immunosorbent assay (elisa) for the following viruses: sda virus/rat coronavirus, sendai virus, pneumonia virus of mice, kilham rat virus, toolan's h-1 virus, mouse polio virus, reovirus type 3, mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse adenovirus fl/k87, and rat parvovirus ns-1. in addition, because the specificity of elisa is slightly low, immunofluorescent antibody testing was performed for the sda virus. we also performed bedding contact surveillance to confirm the presence of the sda viral infection in the colony. because the time at which initial infection occurred in each rat could not be detected, data were synchronized on the day (defined as day 0) when their food intake decreased by 30% of their average daily food intake before clinical infection. data are shown from days à 5 to 7 for body weight and from days à 4 to 6 for food intake, meal size, and meal number. data of food intake, meal size, and meal number were analyzed via one-way anova and as a post hoc test using paired t test between the average value and each value on each day. we calculated the correlation between food intake and meal size or food intake and meal number before and after infection using pearson correlation coefficient to determine their association. the p value is calculated to test the null hypothesis that there is no correlation between food intake and meal size or food intake and meal number. because body weight continuously increased from days à 5 to 0 and then started to decrease from day 1, body weight after day 1 was compared with that on day 0 via paired t test. data indicate mean standard error. a p value less than .05 was accepted as significant. enlarged lymph nodes were palpated in the neck in all rats. no rat died. as shown in fig. 1 , body weight from days à 5 to 0 continuously increased, but from days 1 to 7, body weight was significantly decreased as compared with that on day 0. as shown in fig. 2a , food intake was significantly decreased from days à 2 to 5. this decrease in food intake occurred primarily via a decrease in food intake during light phase, followed thereafter by a decrease in food intake during dark phase after day 1. food intake during light phase recovered faster than that during dark phase (data not shown). food intake reached nadir on day 1 (32.2 11.8% relative to baseline). finally, food intake returned to the average value on day 6. before the viral infection, there was a positive association between food intake and meal number ( p < .05). as shown in fig. 2b , meal size started to decrease on day 0, and significantly decreased from days 1 to 4. after infection, food intake decreased by 68%. this occurred via a significant decrease in meal size ( p < .05), while meal number decreased nonsignificantly (p = .71). similarly to food intake data, meal size reached nadir on day 1 (31.5 5.2% relative to baseline). there was no difference in meal size findings between the light and dark phases. as shown in fig. 2c , meal number from days à 4 to 6 did not significantly decrease. the maximum decrease in meal number occurred on day 0 (78.0 13.8% relative to baseline; p = .06). meal number during light phase on days à 2 and à 1 decreased significantly, but it was offset by an increase in meal number during dark phase. during recovery, meal number slightly increased predominantly via an increase in meal number during light phase (data not shown). bedding contact surveillance of rats showed positive results for the sda virus. as summarized in table 1 , serologic testing also showed positive results only for the sda virus both in elisa and immunofluorescent antibody testing and showed negative results for all the other viruses. after confirmation of the presence of the sda viral infection, all rats in our colony were euthanized using carbon dioxide. although we measured serologic testing in only three rats in the same colony, the highly contagious nature of the sda virus and the fact that all rats eventually developed to our knowledge, these data presented here are the first demonstration of abnormal feeding patterns during an sda viral infection. our data showed that anorexia during the sda viral infection occurred predominantly via a decrease in meal size. meal number did not decrease significantly, and did not simultaneously increase in a compensatory manner, in an attempt to maintain constancy of food intake, so consequently, food intake decreased, which was associated with body weight loss. an approximation of the amount of weight loss due to the decreased food intake can be determined based on the following calculations in which the metabolizable energy of the food is 3.31 kcal/g (purina formulab diet #5008). the average decrease in food intake per rat was about 6.4 g/day. this calculates to an average decrease of 21.2 kcal/day/rat. for maintenance, a rat needs approximately 110 kcal/kg 0.75 / day [12] , which corresponds to 40.6 kcal/day for the average rat in this study which weighed 265 g. the rats started out eating an average of 17 g/day, which equals 37.3 kcal/day. the difference is probably attributable to errors inherent in averaging both the animal weights and food intake. over the 10-day period of decreased intake, this was reduced by 21.2 kcal/day to an average intake of 16 kcal/day. since they require about 40 kcal/day and they were taking in 16 kcal/ day, there was a net loss of 24 kcal/day. conversion of protein, fats, and carbohydrates produces an average gain of approximately 6.4 kcal/g [13] . this roughly corresponds to an average weight loss of about 3.75 g/day or 37.5 g/rat over the 10-day period of decreased intake. it appears that the average rat lost approximately 30 g; the difference being attributable to averaging variable of animals, imprecision in the calculations, and other factors that make this only an estimation. it would appear that the entire loss of body weight could be accounted for by the decrease in food intake, although other unmeasured factors (such as changes in metabolic requirements) were not examined. furthermore, there was a difference in the pattern of the decrease and the recovery in food intake and meal number between light and dark phases. the different pattern in the dynamics of meal size and meal number suggests that meal size and meal number be regulated independently via different systems [6] . this pattern of food intake decrease is different from that which occurs during the anorexia of bacterial lipopolysaccharide (lps)-induced infections [14 ± 16] or in cancer [17] . but, it is similar to that of anorexia of indomethacin-induced ulcerative ileitis, where we previously measured an increase in plasma tumor-necrosis factor (tnf)-a [18] . the sda virus belongs to the coronavirus family, which has been documented to increase tnf-a [19] . pentoxifylline (an anti-tnf-a agent) inhibits tnf-a induced hypophagia [20] . it is interesting to note that with recovery from the sda viral infection, meal number slightly increased. this pattern of recovery may represent the process of normalization of food intake during the recovery period after human viral infection, such as mumps or influenza. during an sda viral infection, inflamed salivary glands and/or swollen neck lymph nodes occur. hence, the observed decrease in meal size could be influenced via mechanical obstruction or diminished salivary secretion. however, under normal conditions, a decrease in either meal size and meal number is offset by an increase in the other to maintain the constancy of daily food intake. thus, the lack of a compensatory increase in meal number during the sda viral infection might indicate abnormal and impaired feeding behavior. in anorexia of acute and chronic diseases, various cytokines [e.g., tnf-a, interleukin (il)-1, il-2, il-6, il-8, interferon (ifn), etc.] alone or synergistically play a significant role [3, 21, 22] . during bacterial and viral infections, microbial products such as bacterial cell wall compounds [e.g., lps and muramyl dipeptide (mdp)], microbial nuclei acids (e.g., bacterial dna and viral double-stranded rna) and viral glycoproteins stimulate the host's acute phase response [14,15,23± 25] . these cytokines act directly or indirectly on the hypothalamus [21, 26, 27] . previously we reported that in food-deprived rats, changes in dopamine concentration in the lateral hypothalamic area (lha) positively correlated with changes in meal size [28, 29] , while those in the ventromedial nucleus in the hypothalamus (vmn) negatively corresponded to intermeal interval, and thus influenced meal number [30, 31] . in the septic rat model, induced by cecal ligation and puncture, dopamine concentration in the vmn progressively decreased and this reduction of dopamine concentration was associated with anorexia [27] . hence, cytokines, induced by microbial products, may influence anorexia and feeding patterns via changes in the concentration of hypothalamic dopamine [32] , among other neuromediators. anorexia, both induced by peripheral il-1a injection and in methylcholanthrene-induced sarcoma-bearing male fischer-344 rats, was produced by predominantly an initial decrease in meal number, followed a day later by a decrease in meal size [17, 33] . while anorexia, induced by lps or mdp, derived from the cell wall of gram-negative orpositive bacteria, respectively, was produced by only a decrease in meal number; meal size was not affected [14 ± 16] . the mechanisms of anorexia induced by these bacterial products are similar [14, 15] , and tnf-a has been repeatedly shown to play a significant role in the mechanisms of anorexia during bacterial infection [10, 16] . it appears that gender differences exist in the acute phase response [34] . previously, we reported that in female lewis rats with indomethacin-induced ulcerative ileitis, food intake decreased mainly via a decrease in meal size and to a lesser extent via a decrease in meal number [18] . this decrease in meal size correlated negatively with plasma tnf-a [18] . even when considering difference in gender and rat strain, the changes in food intake and feeding pattern between the sda viral infection and the indomethacininduced ulcerative ileitis model is similar. these changes have not been previously observed in the other rat models in both genders (e.g., in mca sarcoma-bearing female fischer-344 rat model, food intake decreases only via a decrease in meal number; [35] ). thus, the similarity in pattern of decreased food intake during the sda viral infection and that during indomethacin-induced ulcerative ileitis, suggests a mechanistic link between viral infection, tnf-a, and anorexia. furthermore, data that an infection with the influenza virus is associated with increased tnf-a activity in lung lavage fluid of male swiss ± webster mice [11] , and the manifestation of severe anorexia even in il-1bdeficient mice [10] , suggest a significant and limited role of tnf-a and il-1b in anorexia during a viral infection, respectively. although the mechanism of anorexia during bacterial and viral infection is linked to tnf-a, the difference in feeding pattern between the sda viral infection and a bacterial infection suggests involvement of factors, other than tnf-a and il-1b during these infections. thus, during an sda virus infection, additional cytokines, such as il-2, il-6, il-8, and/or ifn, may play a contributory role in decreasing food intake [3, 11] . particularly, a recent study suggests a role for ifn-g in causing hypophagia and hypermetabolism during infection [36] . since these cytokines act on the hypothalamus to modulate food intake and feeding behavior, they might have a direct or an indirect influence on the lha to decrease meal size and on the vmn to inhibit a compensatory increase in meal number during the sda virus infection. anorexia of infection as a mechanism of host defense mechanisms of cancer cachexia anorexia during acute and chronic disease inhibitory controls of feeding by the ventromedial hypothalamus feeding patterns of rats in response to fasts and changes in environmental conditions food intake equals meal size times meal number automated computerized rat eater meter: description and application the biology and medicine of rabbits and rodents behavioral thermoregulation in mice inoculated with influenza virus thermal and behavioral effects of lipopolysaccharide and influenza in interleukin-1b-deficient mice cytokines and the acute phase response to influenza virus in mice biology and diseases small animal clinical nutrition iii comparison of the effects of bacterial lipopolysaccharide and muramyl dipeptide on food intake differential feeding responses to bacterial lipopolysaccharide and muramyl dipeptide tnf-a tolerance blocks lpsinduced hypophagia but lps tolerance fails to prevent tnf-a-induced hypophagia an analysis of temporal changes in meal number and meal size at onset of anorexia in tumor-bearing male rats mode of food intake reduction in lewis rats with indomethacin-induced ulcerative colitis comparative lung pathology of inbred strain of mice resistant and susceptible to sendai virus infection inhibition of tnf-alpha production contributes to the attenuation of lpsinduced hypophagia by pentoxifylline tumor necrosis factor and interleukin-1 beta: suppression of food intake by direct action in the central nervous system synergistic effect of rhtnf-a and iril-1a in inducing anorexia in rats bacterial dna causes septic shock spontaneous release of stable viral double-stranded rna into the extracellular medium by influenza virus-infected mdck epithelial cells: implications for the viral acute phase response induction of interferonalpha by glycoprotein d of herpes simplex virus: a possible role of chemokine receptors correlation between food intake and csf il-1a in anorectic tumor bearing rats vmn hypothalamic dopamine and serotonin in anorectic septic rats eating induced rise in lha-dopamine correlates with meal size in normal and bulbectomized rats eating-related increase of dopamine concentration in the lha with oronasal stimulation meal size and number: relationship to dopamine levels in the ventromedial hypothalamic nucleus eating-associated vmn-dopamine levels of rats: comparison of oral and intragastric feeding interleukin-1a injection into ventromedial hypothalamic nucleus of normal rats depresses food intake and increases release of dopamine and serotonin. pharmacol temporal changes in meal number and meal size relationship in response to rh-il-1a gender differences in neutrophil function and cytokine-induced neutrophil chemoattractant generation in endotoxic rats estradiol modulates tumor-induced anorexia and feeding pattern a predominant role for ifn-g in infection induced cachexia in comparison to tnf-a (abstract key: cord-003533-8m0vyxq8 authors: jayathilaka, p. g. n. s.; mendis, a. s. v.; perera, m. h. m. t. s.; damsiri, h. m. t.; gunaratne, a. v. c.; agampodi, suneth buddhika title: an outbreak of leptospirosis with predominant cardiac involvement: a case series date: 2019-03-18 journal: bmc infect dis doi: 10.1186/s12879-019-3905-7 sha: doc_id: 3533 cord_uid: 8m0vyxq8 background: severe leptospirosis is known to cause multi organ dysfunction including cardiac involvement. in the clinical setting with limited resources, high degree of suspicion is needed to diagnose cardiac involvement including myocarditis. although myocarditis is not reported as a common complication due to lack of diagnostic facilities, there are evidence to support myocarditis is more prevalent in post mortem studies of patients died due to leptospirosis. we present a case series of severe leptospirosis with cardiac involvement observed during a period of one month at colombo-north teaching hospital, sri lanka. case presentation: we report here five patients with severe leptospirosis complicated with cardiac involvement, admitted to a single medical ward, colombo-north teaching hospital, sri lanka during a one-month period. out of six suspected leptospirosis patients admitted during that period, five in a raw developed severe leptospirosis with cardiac involvement. in this case series, four patients were confirmed serologically or quantitative pcr and one patient had possible leptospirosis. all patients developed shock during their course of illness. two patients developed rapid atrial fibrillation. one patient had dynamic t wave changes in ecg and the other two had sinus tachycardia. two patients had evidence of myocarditis in 2d echocardiogram, whereas other two patients had nonspecific findings and one patient had normal 2d echocardiogram. all five patients had elevated cardiac troponin i titre and it was normalized with the recovery. all five patients developed acute kidney injury. four patients needed inotropic/vasopressor support to maintain mean arterial pressure and one patient recovered from shock with fluid resuscitation. all patients were recovered from their illness and repeat 2d echocardiograms after recovery did not show residual complications. one patient had serologically proven dengue co-infection with leptospirosis. conclusions: myocarditis and cardiac involvement in leptospirosis may be overlooked due to non-specific clinical findings and co-existing multi-organ dysfunction. atypical presentation of this case series may be due to micro-geographic variation and unusual outbreak of leptospirosis. co-infection of dengue with leptospirosis should be considered in managing patients especially in endemic areas. leptospirosis is a well-known zoonosis which causes outbreaks particularly in tropical countries. the causative organism is a spirochete of the genus leptospira. history of leptospirosis is likely to extend to ancient times which is evident by chinese texts describing "rice field jaundice" [1] . in 1886, adolph weil describes a syndrome consists of jaundice, splenomegaly, renal dysfunction, conjunctivitis, and skin rash [2] and few years later, inada described the causative organism of spirochetosis icterohaemorrhegica [3] now known as leptospirosis. the classical untreated disease is described as a biphasic illness with initial acute leptospireamic phase followed by immune phase. most cases are self-limited, but some patients develop fatal complications with severe disease. jaundice and renal failure ("weil's disease"), pulmonary hemorrhage, acute respiratory distress syndrome (ards), uveitis, optic neuritis, peripheral neuropathy, myocarditis, and rhabdomyolysis are well known complications [4] . after the resolution of febrile phase with the clearance of leptospiremia, the immune phase can occur in less than 10% of patients. however, atypical presentations are reported more frequently in the recent history [5] . in sri lanka, these differences of clinical presentations has been observed and attributed to micro-geographic changes [6] . there are more than 250 serovars of leptospira which have been classified in to more than 31 serogroups and the different clinical manifestations are partially attributed to specific serovars. understanding and identifying the varying clinical presentations of leptospirosis mimicking other diseases is important in clinical practice for early treatment and management. in this case series, we describe a series of male patients with severe leptospirosis with cardiac involvement, presented to a single medical ward during a period of one month. here we define cardiac involvement as positivity of at least one of following criteria. they are, 1) transient echocardiogram abnormalities during the illness 2) elevated troponin i titer which came down with the recovery of illness, 3) transient electrocardiogram changes during the illness. we present five patients who were treated for leptospirosis with complications. all are male patients admitted to a single medical ward at north colombo teaching hospital, sri lanka during a one month period starting from 31 to 01-2018. data were collected by direct interview of patients, during admission and follow up visits, and from hospital records. fifty-eight years old previously healthy mason admitted to the hospital on 01/02/2018 with fever for three days. fever was associated with chills, rigors, headache, body aches, faintishness, mild cough producing whitish sputum for two days, dysuria, two episodes of loose stool on day3 of illness, and loss of appetite with poor intake. urine output was normal up to the day of admission. he had a history of cleaning a drainage system one week prior to onset of symptoms. on examination, he was febrile (101°f) and dehydrated. he had low volume pulse with a rate of 104 bpm, blood pressure of 84/50 mmhg. examination of other systems were unremarkable except, few basal crepitations in the right lung. inward uss abdomen was performed and there was no free fluid indicative of dengue hemorrhagic fever. initial investigations revealed neutrophil leukocytosis with thrombocytopenia (table 1) , high c-reactive protein level (table 2) , high serum creatinine with marginally elevated liver transaminases (ast > alt). urine analysis showed microscopic hematuria and ecg showed sinus tachycardia. he was resuscitated with intravenous crystalloids. despite adequate resuscitation he remained in shock and oliguric acute renal failure. after five hours of admission he was started on intravenous noradrenalin infusion and later dobutamin also added to the therapy (table 3) . clinical diagnosis was made as leptospirosis and intravenous cefotaxime was started in the meanwhile. urine output was improved with the rise of mean arterial pressure. but patient was dependent on ionotrope and vasopressor. 2 d echocardiogram showed mild global hypokinesia with ejection fraction 50-55% and concluded as possible myocarditis. troponin i titre became positive. on day 4 of illness, patient developed rapid atrial fibrillation with shock requiring electrical cardioversion to achieve sinus rhythm. by day five of illness, he became heamodyanemically stable without inotropic/ vasopressor support. during the recovery, he developed asymptomatic hypokalemia and potassium was replaced. by day eleven of illness he was completely recovered clinically and full blood count, liver function tests, renal function tests and ecg were normal. c-reactive protein and troponin i titer were coming down and patient was discharged. after three weeks of illness, 2d echocardiogram was performed and it was completely normal. leptospira was detected in qpcr (quantitative polymerase chain reaction) performed on day five of illness and leptospirosis antibody test on day seven of illness (mat) was positive. (titre-1:2560) his urine and blood cultures, dengue antigen were negative. a 67 years old previously healthy male, a retired clerk presented to the medical casualty with a history of fever for three days. it was associated with arthralgia, myalgia, headache and loss of appetite. he did not have respiratory, urinary symptoms and bowel habits were normal. he denied any history of exposure to leptospirosis or contact history of fever. on admission, his general examination was normal with a heart rate of 80 bpm and blood pressure of 100/70 mmhg. other system examination was unremarkable. after admission it was noted that his urine output is low while he was on maintenance fluid. initial investigations revealed neutrophilia with normal white blood cell count, thrombocytopenia, elevated blood urea, serum creatinine, c-reactive protein and ast. urine analysis showed 4-6 pus cells, 1-2 red cells with granular casts. clinical diagnosis of leptospirosis was made on high index of suspicion although there was no significant history of exposure to leptospirosis. patient was started on intravenous cefotaxime. by the day five of illness, he developed confusion (gcs-14/15), low blood pressure (80/40 mmhg) with tachycardia (117 bpm), high fever spike (103 f), and mild dyspnea with spo2 98% on air. ecg showed sinus tachycardia, non-contrast ct brain was normal, 2d echocardiogram revealed ejection fraction of > 60%, chest x ray-pa was normal, and troponin i titer was marginally positive. ultrasound abdomen showed renal parenchymal changes with normal sized kidneys. serum creatinine was rising. patient was started on inotropic and vasopressor support to maintain blood pressure. even after achieving mean arterial pressure > 65 mmhg patient went in to anuric acute renal failure. meanwhile he developed rapid atrial fibrillation which was settled with electrical cardioversion. he was given hemodialysis on day 6 of illness. on day 7 of illness again patient developed rapid atrial fibrillation and it did not respond to electrical cardioversion and started on iv amiodarone infusion and patient regained sinus rhythm and could tail off inotrope and vasopressor. since day 8, he gradually improved clinically with good urine output, hemodynamic stability and confusion settled. but he did not recover from acute kidney injury and renal functions remained rising again. he was given another hemodialysis on day 12 of illness. then his renal functions slowly improved and discharged on day 17 of illness with a follow up plan at nephrology clinic. on discharge patient had normal platelet count, c-reactive protein, liver transaminases, ecg. serum creatinine was static around 250 micromol/l. repeat 2 d echocardiogram which was done three weeks after recovery was normal. leptospirosis antibody titre (mat) on day 7 of illness was positive. (1:10240). a 17 year old male patient presented with fever for two days. fever was associated with chills, rigors, arthralgia, myalgia, frontal headache, faintishness, lower back pain, loss of appetite, vomiting, loose stool 3-4 times/day for two days. patient denied a significant exposure to leptospirosis. there was no contact history of fever. he was a manual worker. on admission he was ill looking, febrile ultrasound scan of abdomen showed acute renal parenchymal changes and there was no evidence of free fluid in the abdomen. initial investigations revealed neutrophil leukocytosis with thrombocytopenia, high c-reactive protein (360 mg/l), high blood urea (172 mg/dl) and serum creatinine (355 micromol/l), marginally elevated liver transaminases (ast > alt), microscopic hematuria, ecg showed sinus tachycardia with mild t inversions in v4-v6. chest x ray was normal. possible diagnosis of leptospirosis was made on clinical grounds and he was started on intravenous cefotaxime. his blood pressure was improved after fluid resuscitation and he had good urine output. his 2d echocardiogram was normal, but his troponin titer increased and then came down. patient was discharged from the ward on day 7 of illness with complete recovery and normal full blood count, renal and liver function tests. crp and trop i titer was coming down. 2 d echocardiogram which was performed after three weeks of recovery was normal. his dengue antigen test, blood and urine cultures were negative. the leptospirosis qpcr test performed on day three of the illness was reported as not detected though one out of triplicate samples was positive. patient was clinically diagnosed as a "possible" case of leptospirosis. a 55 year old male laborer presented with fever for four days duration. he was previously diagnosed to have diabetes mellitus, but he was not taking treatments. fever was associated with arthralgia, myalgia, headache, lower back pain, dysuria and reduced urine output for two days, cough for one week producing scanty amount of whitish sputum. he had a history of muddy contact within one week prior to symptom onset. on admission, patient was febrile (temp-102f), ill looking, mildly dehydrated and had conjunctival suffusion. his pulse rate was 124 bpm with blood pressure of 90/50 mmhg. other system examination was unremarkable. initial laboratory work up showed neutrophilia with normal white cell count, thrombocytopenia, high c-reactive protein (250 mg/l), high serum creatinine (146 micromol/l) and normal liver transaminases. ecg showed sinus tachycardia and chest x ray-pa was normal. depending on clinical grounds, diagnosis was made as leptospirosis and started on intravenous cefotaxime while fluid resuscitation is being carried out. despite adequate fluid resuscitation patient developed shock with low urine output on the same day of admission. (day 4 of illness-pulse rate-130 bpm, bp-85/60) then vasopressor support was given and small dose of frusemide infusion was started after achieving normal blood pressure with noradrenalin. 2d echocardiogram was performed on d5 of illness and it showed mild global hypokinesia with ejection fraction 45-50%, dilated left ventricle with concentric left ventricular hypertrophy and concluded as hypertensive heart disease with or without myocarditis. cardiac troponin i titre became positive and had rising titre when repeated and then came down by the time of recovery. us scan of abdomen revealed bilateral renal parenchymal changes with normal sized kidneys. noradrenalin was tailed off within 24 h and urine output was improved with maintenance fluid therapy. patient had rising serum creatinine till day 6 of illness and then started to come down. serum electrolytes were normal throughout and there was no acidosis. patient was improved dramatically and was discharged from the hospital by day 9 of illness. on discharge he had rising platelet count, normal serum creatinine and dropping troponin i titre and crp. 2 d echocardiogram was repeated after 4 weeks of discharge and his ejection fraction was improved to 60% and there was mild left ventricular hypertrophy with grade i diastolic dysfunction. his diabetes was controlled with soluble insulin during acute illness and changed to oral hypoglycemic treatment with the recovery. leptospirosis antibody titre (mat) done on day 7 of illness was positive (1:5120). a 73 years old male patient presented with fever for 4 days. it was high fever associated with arthralgia, myalgia and mild difficulty in breathing. he also complained of reduced urine output and loose stool (two episodes) for one day. there were no other respiratory or urinary symptoms. he denied a significant exposure to leptospirosis. he had a past history of hypertension for which he was not taking treatment and past history of renal calculi for which he has undergone surgery several years back. on admission he was ill looking, febrile (temp-102 f), and anicteric. pulse rate was 112 bpm and blood pressure 96/66 mmhg. other system examination was unremarkable. initial investigations revealed marked thrombocytopenia, neutrophilia with low normal white blood cell count, high c-reactive protein (236 mg/l), high serum creatinine (267 micromol/l), elevated liver transaminases (ast > alt), urine analysis showed pus cells 65-70, red cells 15-20 and albumin 2+ (urine culture became negative). chest x ray-pa was normal. possibility of dengue fever could not be excluded with his full blood count and clinical presentation, but all other initial investigations were supportive towards leptospirosis although there was no history of significant exposure to leptospirosis. on admission ultrasound scan of the abdomen was performed inward and there was no evidence of fluid leakage. therefore, patient was started on intravenous cefotaxime in addition to hydration with maintenance fluid. patient had low urine output and went in to shock (pr-114, bp-78/41 mmhg) despite of adequate fluid resuscitation (on day 4 of illness). he was started on iv noradrenalin to maintain blood pressure. ultrasound scan of the abdomen revealed right side scarred kidney with left side renal parenchymal changes with normal size kidney. there was no evidence of leaking by the time of developing shock. 2d echocardiogram showed severe mitral regurgitation with and there was no evidence of myocarditis. troponin i titer became marginally positive and later came down. ecg showed sinus tachycardia. histological diagnosis or cardiac mri to diagnose cardiac involvement was not accessible due to lack of resources in the hospital. noradrenalin could be tailed off within 24 h. (on day 5 of illness). by day five of illness urine output was gradually improved but serum creatinine remained rising with normal serum electrolytes. dengue ns1 antigen was negative, but igm and igg antibodies were positive with dropping platelet count and white cell count (neutrophilia persisted). dengue pre-critical monitoring was continued while giving maintenance fluid therapy. daily ultrasound scans were performed to exclude fluid leakage. patient remained hemodynamically stable and platelet and white cell count started to increase by day 7 of illness and serum creatinine started to come down by day 10 of illness. he was discharged from the hospital on day 11 of illness with a plan to be followed up in nephrology clinic for possible chronic kidney disease. 2d echocardiogram was repeated after three weeks of recovery and it was normal other than trivial mitral regurgitation. leptospirosis antibody titer done on day 7 of illness was positive. (1:2560). severe leptospirosis is characterized by multiple organ dysfunction including liver, kidney, lungs and brain. it is also known to cause cardiac involvement as well. cardiac manifestations range from non-specific electrocardiographic changes and arrhythmias to myocarditis, pericarditis, endocarditis and cardiogenic shock [7] [8] [9] . but the pathophysiology behind it is less well understood and the magnitude of the problem is under-reported [10] . all five patients included in this case series had evidence of acute kidney injury. the most striking feature of these five patients admitted to a single unit within a month was cardiac involvement. all five patients developed shock with low blood pressure during their course of illness. except case number 3, all other patients needed vasopressor/inotropic support to maintain blood pressure. case number 1 showed evidence of myocarditis in 2 d echocardiogram at the time of shock. case number 4 had possible evidence of myocarditis whereas case number 2, 3 had normal echo findings. case number 5 had severe mitral regurgitation in his 2d echocardiogram. all these echocardiogram were performed while the patients were in shock. repeat 2d echocardiograms performed after three weeks of recovery were completely normal except in case number 4 and 5. number 4 had mild left ventricular hypertrophy with grade 1 diastolic dysfunction and number 5 had trivial mitral regurgitation. in addition to these various echo findings all of these five patients had more or less positive cardiac troponin i titre which came down with the recovery of illness. case number one and two developed atrial fibrillation which needed intervention for normalization. case number three had mild t wave inversions in anterior leads which was dynamic in serial electrocardiograms. case number 4 and 5 had only sinus tachycardia. all five patients had shock by definition and the most probable explanation is cardiogenic shock due to cardiac involvement of leptospirosis. though not commonly reported, myocarditis in severe leptospirosis may not be a rare complication. the european society of cardiology working group on myocardial and pericardial diseases has developed clinical and diagnostic criteria, when present myocarditis should be suspected. presence of unexplained cardiogenic shock, positive cardiac troponins, variable ecg changes are included for these criteria in addition to several other criteria [10] . definitive diagnosis of myocarditis ideally should be established by histopathological, immunological and immunohistochemical criteria for which myocardial biopsy is required. this is not practical in most settings as these investigations are not routinely done and not required for patient management. in this case series none of the patients underwent histopathological or cardiac mri diagnosis of cardiac involvement due to lack of resources in the hospital. due to wide variability in presentation and non-specific clinical findings, many cases of myocarditis likely to go undetected. as an example, study conducted examining 24 hearts from patients who had died due to leptospirosis has revealed myocarditis in 96% of cases histologically. endocardial inflammation had been observed in 50% of cases [11] . in sri lanka, myocarditis has been reported previously as a complication of leptospirosis [12, 13] and around 7-15% of confirmed cases are being reported as having this complication [6, 14, 15] . however, in most of the previous studies, the details of diagnosis of myocarditis was not clearly given. in our case series, histological diagnosis or cardiac mri to diagnose cardiac involvement was not possible due to lack of resources in the hospital. there is another phenomena coming up in the recent literature to explain the shock in leptospirosis. according to julie cagliero et al. dys-regulation of inflammatory mechanisms in severe leptospirosis can lead to cytokine storm causing sepsis like picture [16] . systemic inflammatory response syndrome (sirs) is supposed to occur in severe leptospirosis [17] . sirs itself can cause elevated cardiac troponins [18, 19] . therefore, pure cardiac involvement in leptospirosis becomes more difficult to diagnose. all these five patients presented during one-month period in a raw and we had only six total suspected (notified) cases of leptospirosis during that month. observing cardiac involvement in five out of six probable cases of leptospirosis may be due to an outbreak caused by a different strain of a leptospira. as previously observed, outbreaks of leptospirosis with uncommon complications such as pancreatitis [5] needs more investigations and explanations. however these patients did not have evidence of pulmonary involvement which is a known complication to occur in severe leptospirosis. case number 5 patient had serological evidence of leptospirosis and co-infection with dengue virus. co-infection of leptospirosis and dengue is a known phenomenon in endemic countries with subtropical and tropical climates. a study conducted in malaysia has concluded that there is a considerable prevalence of leptospirosis and dengue co-infection with overlapping demographic, clinical and laboratory presentations [16] . in sri lanka [17] as well as in many other places [18] [19] [20] , a co-infection of these two had been reported earlier and possible due to high endemicity of both diseases. it is crucial to consider co-infection with dengue where clinical suspicion arise even in the presence of enough supportive evidence for leptospirosis. because close monitoring and fluid management are the lifesaving principles of management of dengue hemorrhagic fever which must be done timely. developing severe leptospirosis in five out of six cases during same period may be due to outbreak of uncommon strain of leptospirosis. cardiac manifestations of leptospirosis are possibly under-diagnosed due to co-existence with other multi-organ involvement. diagnosis of myocarditis is difficult due to lack of imaging facilities, lack of specificity of available tests as well as unavailability of non-invasive gold standard diagnostic test. to assess the significance of cardiac troponins in diagnosing cardiac involvement in leptospirosis further studies are required. co-infection of dengue in a patient with leptospirosis should be considered especially in endemic areas. über eine eigenthümliche mit milztumor, icterus und nephritis einhergehende acute infectionskrankheit areport on the discovery of the causative organism (a new spesies of spirochete) of weil's disease. tokyo ijishinshi leptospirosis in humans severe leptospirosis and pancreatitis; a case series from a leptospirosis outbreak in anuradhapura district, sri lanka regional differences of leptospirosis in sri lanka: observations from a flood-associated outbreak in 2011 cardiac manifestations in leptospirosis. apropos of 15 cases observed in new caledonia cardiac involvement in severe leptospirosis cardiac and pulmonary involvement in leptospirosis current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases cardiac findings in leptospirosis myocarditis causing severe heart failure -an unusual early manifestation of leptospirosis: a case report co-existent facial palsy and myocarditis in a 50-year old farmer diagnosed with probable leptospirosis: a case report predictors of the development of myocarditis or acute renal failure in patients with leptospirosis: an observational study leptospirosis outbreak in sri lanka in 2008: lessons for assessing the global burden of disease demographic, clinical and laboratory features of leptospirosis and dengue co-infection in malaysia fatal co-infection with leptospirosis and dengue in a sri lankan male fatal leptospira spp./zika virus coinfection-puerto rico sero-epidemiology study of leptospirosis in febrile patients from terai region of nepal clinical predictors of dengue fever co-infected with leptospirosis among patients admitted for dengue fever -a pilot study we acknowledge the staff of ward 15 of colombo-north teaching hospital, ragama in making this study a success.funding sba is supported through u.s. public health service grants u19ai115658. the funders have played no role in the research.availability of data and materials all data contained within the article.authors' contributions nj perceived the study and prepared the first draft of the manuscript. nj, asvm, mhmtsp, hmtd, avcg provided patient care, followed up the patients, collected and interpreted clinical data. sba involved in design, analysis, interpretation of data and preparing the manuscript. all authors contributed, read and approved the final manuscript.ethics approval and consent to participate not applicable. written informed consent was obtained from all patients for publication of their individual details. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-275427-00bwhaga authors: aydogdu, ugur; coskun, alparslan; atas, ahmet duran; basbug, onur; agaoglu, zahid tevfik title: the determination of treatment effect of chitosan oligosaccharide in lambs with experimentally cryptosporidiosis date: 2019-11-30 journal: small ruminant research doi: 10.1016/j.smallrumres.2019.09.021 sha: doc_id: 275427 cord_uid: 00bwhaga abstract in this study, it was aimed to investigate the efficacy of chitosan oligosaccharide administrations in different doses of experimental infected lambs with cryptosporidium parvum. 32 male lambs were used in the study and the lambs were divided into 4 groups with 8 lambs in each group. groups 1, 2 and 3, twice a day, were administered chitosan oligosaccharide at a dose of 100, 500, and 1000 mg/kg for 7 days, respectively, with milk replacer. in group 4, lambs with cryptosporidiosis were subjected to normal feeding as control without drug administration. clinical examinations of lambs were made before treatment (day 0) and on days 1, 3, 5 and 7 after treatment and 5 ml of blood was collected from vena jugularis for blood analysis of all lambs. weight changes of lambs were recorded at 0, 7, 14, and 21 days. stool specimens were collected pre-treatment (day 0) and on days 1, 3, 5, 7, 14 and 21 post-treatment to determine oocyst excretion of lambs with cryptosporidiosis. lambs with a mean oocyte counts >10 after stool examination were included to the treatment. changes in clinical hematology, blood gases and biochemical parameters were observed during the course of treatment, but these changes were limited. weight loss was observed at 7th day according to 0th day the lambs with experimental cryptosporidiosis but gradually weight increase was observed at 14th and 21st days and these changes were similar in all groups. oocyst excretion decreased in all groups during treatment. according to 0th day, there was a significant (p < 0.05) decrease in oocyte excretions in the third day in group 1 and 2, and in day 5 in the group 3 and 4. significant changes (p < 0.05) were observed in oocyst excretions on the third and fifth days among the groups. as a result, in lambs with experimental cryptosporidiosis, chitosan oligosaccharide improved in clinical signs and stool character shorter than the positive control group and the administration of chitosan oligosaccharide at doses of 100, 500 and 1000 mg/kg for 7 days significantly reduced oocyst excretion but not enough to remove cryptosporidiosis completely. infection caused by cryptosporidium parvum is common among young ruminants and is seen in various mammalians including humans. the disease has a high prevalence across the world and is among the important causes of diarrhea in neonatal farm animals (scott, 2007; paraud and chartier, 2012; constable et al., 2017) . c. parvum is one of the primary causes of diarrhea seen in young lambs and goats. diarrhea can develop as a result of c. parvum infection alone; however, it mostly develops due to mixed infection. infection can occur as a severe diarrhea outbreak which can cause high mortality in lambs aged 4-10 days (zorana et al., 2006; goma et al., 2007; paraud and chartier, 2012; constable et al., 2017) . there is no specific treatment for cryptosporidiosis. it has been reported that paromomycin, lasalocid, halofuginone lactate, sulfoxinoxalin, azithromycin and toltrazuril have partial or demonstrable effect against in neonatal ruminants with cryptosporidiosis (viu et al., 2000; wright and coop, 2007; giadinis et al., 2008; navarre et al., 2012; yagci et al., 2017; aydogdu et al., 2018) . chitosan is a natural polysaccharide produced by deacetylation of chitin. it is non-toxic, biocompatible and biodegradable (huang et al., 2004; zhao et al., 2018) . chitin is the main component of crustaceans (crab, shrimp, etc.) and is also found in skeletons of insects and cell walls of fungi. there are many derivatives of chitin and the most important one is chitosan (kumar, 2000) . chitosan and chitosan oligosaccharide (cos) have important uses in the medical field, such as controlled drug release, artificial blood vessels, antidiabetic, antibacterial, antifungal and hemostatic effect (baldrick, 2010; xia et al., 2011; muanprasat and chatsudthipong, 2017) . in a study on the treatment of calves with diarrhea, oral chitosan oligosaccharide administration has been reported to be successful in treatment (alam et al., 2012) . in studies conducted, chitosan has been reported to inhibit the in vitro development of c. parvum (luzardo álvarez et al., 2012; adjou et al., 2014; mammeri et al., 2018) . there is no study found in which the efficacy of chitosan oligosaccharide in the treatment of cryptosporidiosis in neonatal ruminants has been determined. according to this information, this study is the first to determine the efficacy of chitosan oligosaccharide in the treatment of cryptosporidiosis in lambs. considering that chitosan inhibits the development of c. parvum in in vitro studies, we hypothesized that oral chitosan administration may be used in the treatment of lambs with experimentally cryptosporidiosis. the aim of this study was to determine the treatment efficacy of oral chitosan oligosaccharide in lambs with experimental cryptosporidiosis. ethics committee approval of the project was taken from sivas cumhuriyet university animal experiments local ethics committee (31.08.2015/69). the study was conducted between 2016-2018. experimental procedure was started simultaneously in all groups. in our study, a total of 32 male lambs younger than 5 days of age, 8 lambs in every 4 groups, were used. stool specimens of lambs were collected and tested using rapid pen-side test (bio-x diagnostics s.a. belgium) in terms of cryptosporidium parvum, rotavirus, escherichia (e) coli and clostridium perfringens. stained stool samples were also examined for cryptosporidium detection. positive ones were excluded from the study. to prevent contact between lambs, each was housed separately in individual compartments. c. parvum oocysts needed for the formation of infection were obtained from rectal stool samples taken from naturally infected calves with diarrhea. stool samples of the calves were examined using a rapid pen-side test (bio-x diagnostics s.a. belgium) in terms of the presence of rotavirus, coronavirus and e. coli and negative ones were used. the oocysts required for infection formation were obtained as reported in a study conducted on goat kids (koudela and jiri, 1997) . for the formation of infection in lambs, 10 ml distilled water containing 10 6 c. parvum oocysts were given using an intraorally catheter (nasogastric catheter 10 ch, 1210 mm, bicakcilar co. ltd., istanbul, turkey). the development of the infection was monitored through the collection of rectal stool samples daily and microscopic examination of the stained preparations. the stained preparations from stool samples was made according to koudela and jiri (1997) . lambs with an average of > 10 oocysts in stained stool preparations were considered to be infected, and treatment was initiated. when infection developed the following treatment protocols were applied to 4 groups, each including 8 lambs. oocyst shedding started in the lambs on the 7th and 8th days, and it had reached to peak level between on day 10 and 13. as a treatment procedure, doses of 100, 500 and 1000 mg/kg chitosan oligosaccharide were given to the lambs in 1st, 2nd, and 3rd groups at intervals of 12 h. no drug was given to the infected lambs in the 4th group and they were fed normally for positive control. considering dehydration degree and blood gas analysis findings, required fluid treatments were given to the lambs in which diarrhea caused dehydration developed (oral fluid treatment was applied to the lambs with mild or moderate dehydration and metabolic acidosis (ph = 7.20-7.35), intravenous 40 ml/kg/hour isotonic sodium bicarbonate was applied to the lambs with severe dehydration and metabolic acidosis (ph < 7.20), 5% dextrose was applied in case of hypoglycemia). during the study, lambs were fed with lamb food (optimilk, optima, kirklareli, turkey) at an amount of 10-12% of their body weight at a proper temperature in two meals. in addition, adlibitum water was provided to lambs. in the study, water-soluble (99%) chitosan oligosaccharide with a deacetylation degree > 90% and a molecular weight < 2000 daltons was used as the treatment material (glycobio company, dalian, china). routine clinical examinations of the lambs (body temperature, heart frequency, respiratory rate, capillary refill time, assessment of dehydration degree) were conducted before the treatment (day 0) and on 1st, 3rd, 5th and 7th days of the treatment. during the treatment, lambs were examined daily clinically and in terms of the stool analysis. 2.5. collection of blood samples and analyses 5 ml of blood samples were collected from lambs before the treatment (day 0) and on 1st, 3rd, 5th and 7th days during treatment for blood gas analysis, hematological analyses (into k 3 edta tubes) and biochemical analyses (into 5 ml anticoagulant free gel tubes). blood hydrogen ion concentration (ph), partial carbon dioxide pressure (pco 2 ), partial oxygen pressure (po 2 ), bicarbonate (hco 3 − ), total carbon dioxide (tco 2 ), base excess (be), oxygen saturation (o 2 sat), glucose and lactate levels were measured using blood gas analyzer (epoc, canada) immediately after blood sample collection without adding anticoagulant agents. using blood samples collected in k 3 edta tubes, white blood cell (wbc), red blood cell (rbc), hemoglobin (hgb), hematocrit (hct), mean corpuscular volume (mcv), mean cell hemoglobin concentration (mchc), red cell distribution width (rdw) and platelet (plt) values were determined by automatic cell counter (bc-2800vet, mindray, china). hematological analyses were conducted within 15 min after blood collection. the tubes without anticoagulant were kept at room temperature for clotting and centrifuged at 4000 rpm for 5 min to obtain serum. serum samples were stored at -80°c until the analysis. urea, creatinine, total protein, albumin, aspartate aminotransferase (ast), gamma-glutamyltransferase (ggt) and alkaline phosphatase (alp) levels in serum samples were determined using autoanalyzer (bs 200, mindray, china). stool samples of lambs were collected from the rectum into sterile stool container before the treatment (day 0) and on the 1st, 3rd, 7th, 14th and 21st days of the treatment. stool samples were evaluated in terms of physical [normal(solid), loose and formless, semi-fluid, watery] and oocysts. the presence of c. parvum oocysts in stools was determined using kinyoun acid-fast staining method (korkmaz and ok, 2011) . stained preparations were examined using immersion oil at 100x magnification in the light microscope. the concentration of c. parvum oocysts in the stool was averaged by counting the oocysts in 20 different microscope fields in each preparation, and semi-quantitative scoring was made as follows; the absence of oocysts (0), oocyst < 1 (1) oocysts between 1-10 (2), oocysts > 10 (3). lambs with a mean number of oocysts > 10 were included in the study (koudela and jiri, 1997) . examination of stool samples were made by the same personnel. lambs were weighed using a digital scale before treatment (day 0) and on post-treatment 7th, 14th and 21st days and weight changes were recorded. the data were expressed as mean and standard error of the mean (sem). kolmogorov-smirnov test was used for normality. one-way anova and tukey multiple range tests were used to evaluate differences between each treatment group during the experiment and significance levels of variation. the statistical significance level was accepted to be p < 0.05. the spss software program (version 15.0, spss inc., chicago, il, usa) was used for statistical analysis. after oral administration of oocysts, as of 3rd day, it was observed that there were changes in the characteristics of stool, that there was a decrease in the stool consistency and that oocyst excretion started with the presence of mucus. after oral administration of oocysts in lambs, as of 4th-5th days, diarrhea ranging from the pasty to liquid in consistency containing a high number of oocysts was observed and lambs that were suitable to the experimental procedure were taken to treatment protocols. infection occurred in all lambs in 4 groups, and experimental procedure was applied to all lambs (n = 32). stools with mucus were observed in 5, 4, 5 and 6 lambs in groups 1, 2, 3, and 4, respectively. when intense oocysts began to be excreted, stools were in yellowgreenish color and sometimes brown color and had a condensed mucus appearance. there was no blood observed in stools. there was no death in groups 1-3 for 7 days; however, 1 lamb in group 4 died on the 3rd day and 1 lamb on the 7th day. both lambs died in the 4th group had condensed mucus and liquid diarrhea and dehydration were observed in these lambs. inappetence, abdominal distension, severe diarrhea and hypoglycemia were observed on the last day in the dead lamb. the lambs did not respond to fluid treatment applications and died. lambs that developed cryptosporidiosis generally had a dynamic appearance, normal mucosa, and good sucking reflex. however, depression, reluctance to stand up, dehydration (max. 6% level), prolonged capillary refill time (3 s) and a decrease in sucking reflex were observed in some lambs. dehydration occurred only in 5 of 32 lambs in a maximum level of 6%. changes in the body temperature, heart and respiratory frequencies of lambs are presented in table 1 . the body temperature of the lambs was observed to range from 38.2 to 40.2 ᵒc. there was a significant difference (p < 0.05) observed on day 0 between the groups in terms of body temperature and no significant difference was found for the other days. moreover, the body temperature of lambs decreased in all groups compared to day 0 and this decrease was statistically significant except for the 4th group (p < 0.05). a significant difference was found on the 1st day between the groups in terms of respiratory frequency (p < 0.05). changes in the oocyst excretion of the lambs before the treatment (day 0) and on post-treatment 1st, 3rd, 5th, 7th, 14th and 21st days are given in table 2 . it was found that oocyst excretion in group 1 and 2 significantly decreased from the 3rd day compared to day 0 (p < 0.05). on the other hand, it was found that oocyst excretion in group 3 and 4 significantly decreased from the 5th day compared to day 0 (p < 0.05). as between groups, a significant decrease was observed in group 2 on the 3rd day and in group 3 on the 5th day compared to group 4 (p < 0.05). body weight measurements of lambs were performed before the treatment (day 0) and on post-treatment 7th, 14th and 21st days. the changes in the body weight of the lambs by days are given in table 3 . it was found that lambs in all groups had weight loss on the 7th day and that there was a gradual increase in the weight on 14th and 21st days. there was no significant difference observed between groups in terms of weight changes of lambs. when the in-group differences between the days were examined, significant differences were found in the groups except for the third group (p < 0.05). the blood gas analysis results of the lambs before and during treatment by days are given in table 4 . in all groups, the mean blood ph was within the normal range (7.35-7.45) during the treatment. ingroups, there was no statistically significant change in blood ph between the days; however, there was a statistically significant difference between the groups on the 5th and 7th days (p < 0.05). metabolic acidosis was observed in 3 cases in the group 1 on day 1 and 3; 1 case in the group 2 on day 0; 2 cases in the group 3 on day 0 and 1; 2 cases in the group 4 on day 5 and 7. while pco 2 levels were similar between groups, increases and decreases were observed in-groups between the days. however, these increases and decreases were statistically significant only in 1st and 2nd groups (p < 0.05). blood po 2 and o 2 sat levels did not show significant change between groups and in-groups between days. the lactate level of group 4 was significantly higher than the other groups on day 0 and the lactate level of group 2 was significantly higher than those of group 1 and group 4 on the second day. there were increases and decreases in lactate level in-group between days and a statistically significant difference was observed in 1st and 4th groups (p < 0.05). there were increases and decreases in hco 3 , be and tco 2 levels of lambs during the treatment. however, these changes were not significant to affect ph. the difference in hco 3 , be and tco 2 levels in-group between days was significant (p < 0.05) only in the 2nd group. there were significant differences determined between the groups in terms of hco 3 , be and tco 2 levels on 5th and 7th days (p < 0.05). changes in the hematological parameters of the lambs by days before and during the treatment are given in table 5 . although there were increases and decreases in hematological parameters in-group between days, there was no statistically significant difference found. there was significant (p < 0.05) difference in wbc levels on the 1st day between the groups and there was no significant difference found on the other days. there was a similarity between the groups in terms of rbc, hgb, hct, mchc and plt levels. in addition, between the groups, mcv levels were different at a statistically significant level on 0th, 1st and 3rd days and rdw levels on the 1st day (p < 0.05). changes in the biochemical parameters of the lambs by days before and during the treatment are given in table 6 . while ast levels of the lambs increased during the treatment compared to pre-treatment (day 0), alp, tp and ggt levels decreased. however, only changes in ast and alp activities of the 1st group and the change in total protein level of the 2nd group were statistically significant (p < 0.05). while there were increases and decreases in urea and creatinine levels of the groups by days, changes in the creatinine levels only in the 1st and 2nd groups were significant (p < 0.05). when the glucose levels of the groups were examined by days, glucose levels of the 1st, 2nd and 4th groups decreased on the 2nd day compared to day 0 and increased in the following days. the glucose level of the 3rd group decreased on the 1st and 3rd days compared to day 0 and increased gradually in the following days. statistical significance was determined only in the 1st group (p < 0.05). significant differences (p < 0.05) between the groups were determined only in tp and creatinine levels on the 7th day. a major problem about cryptosporidiosis is the lack of an effective tool for the prevention and treatment of this disease. more than 200 substances have been tested to treat cryptosporidiosis (dinler and ulutas, 2017). some have shown promising effects; however, none of them have been reported to control clinical findings consistently or eliminate the infection completely. on the other hand, the use of certain drugs may reduce the oocyst excretion and thus probably the environmental pathogen level, the subsequent exposure, and infection of susceptible hosts (shahiduzzaman and daugschies, 2012) . it has been reported that paromomycin, halofuginone lactate, lasalocid, and sulfaquinoxaline have partial or demonstrable effect against cryptosporidium in infected neonatal ruminants (wright and coop, 2007; aydogdu et al., 2018) . however, knowledge about the treatment of cryptosporidiosis in sheep is limited. halofuginone exhibits 2-fold toxicity of the recommended doses of lactate, and its use is contraindicated in severely dehydrated or inappetent animals (wright and coop, 2007) . in addition, many drugs such as paromomycin, azithromycin, and sulfaquinoxaline that have been determined to reduce oocyst excretion in cryptosporidiosis have a risk to leave antibiotic residues. in recent years, serious concerns about antibiotic residues have emerged worldwide. in recent years, there have been studies carried out about the effects of chitosan and its derivatives on the underlying mechanism of antimicrobial activity of chitosan microparticles in the medical field and on the treatment of infectious diseases (jeon et al., 2014) . kim et al. (2001) reported that oral chitosan oligosaccharide applications at doses of 500, 1000 and 2000 mg/kg/day did not cause any side effects different letters in the same rows (a, b) and columns (a, b) are statistically significant (p < 0.05). group 1: 100 mg/kg cos, group 2: 500 mg/kg cos, group 3: 1000 mg/kg cos at intervals of 12 h, group 4: no drug was given to the infected lambs. 3.00 ± 0.00 a 3.00 ± 0.00 a 2.86 ± 0.14 a,a 2.43 ± 0.30 a,b 1.86 ± 0.14 c 1.00 ± 0.00 d 1.00 ± 0.00 d different letters in the same rows (a, b, c, d, e) and columns (a, b) are statistically significant (p < 0.05). group 1: 100 mg/kg cos, group 2: 500 mg/kg cos, group 3: 1000 mg/kg cos at intervals of 12 h, group 4: no drug was given to the infected lambs. in rats. in a study conducted, protective activity of chitosan oligosaccharide has been determined in mice in which colitis was formed experimentally (yousef et al., 2012) . chung et al. (2012) , found that oral administering of chitosan oligosaccharide with a low molecular weight reduced the allergic inflammation in mice in which experimental asthma model was formed. abdel-latif et al. (2016) reported that it had "anticoccidial" effects on eimeria papillata-infected mice. in the same study, it has been reported that both excreted oocysts and developmental stage parasites were reduced and chitosan had anti-inflammatory activity (abdel-latif et al., 2016) . in a study, oral chitosan oligosaccharide administering in the treatment of diarrheal calves has been reported to be very successful (alam et al., 2012) . in addition, chitosan has been used to create excipients in a microparticle system and it has been reported that it might play a role as a physical barrier against cryptosporidium parvum (blanco-garcía et al., 2016) . in the studies conducted, it has been reported to inhibit the in vitro development of c. parvum (luzardo álvarez et al., 2012; adjou et al., 2014; mammeri et al., 2018) . in addition, mammeri et al. (2018) reported that chitosan nag and chitosan mix demonstrated in vivo anticryptosporidial properties in cd-1 mice, a highly sensitive animal model against c. parvum infection. therefore, it has been stated that both of these compounds have a promising potential in therapeutic and preventive applications against c. parvum infection. in this study, oocyst excretion decreased significantly (p < 0.05) in the 1st and 2nd groups by the 3rd day compared to pre-treatment and in the 3rd and 4th groups by the 5th day. as between groups, a significant decrease was observed in group 2 on the 3rd day and in group 3 on the 5th day compared to group 4 (p < 0.05). although stool characteristics became normal on the 14th and 21 st days, it was observed that oocyst excretion still continued in the lambs. while all the lambs in the positive control group had oocyst excretion at the level of +1 on the 14th and 21st days, this level was found to be lower in treatment groups compared to the positive control group. however, there was no statistical difference found. these results showed that chitosan oligosaccharide significantly reduced oocyst excretion compared to the positive control group; however, it was not efficacious to eliminate cryptosporidiosis completely. according to the findings of this study, the use of chitosan oligosaccharide to be used in the treatment of experimental cryptosporidiosis at doses of 100 and/or 500 mg/kg was found to provide an earlier reduction in oocyst excretion compared to 1000 mg/kg dose. the use of higher doses had no negative effect; however, it increases the costs and does not show more positive affects compared to lower doses. considering this, a dose of 100 mg/kg was thought to be the most appropriate dose. the results indicated that low dose of chitosan oligosaccharide was better to reduce oocyst excretion and improve clinical findings in lambs with cryptosporidiosis. studies ph: concentration of hydrogen ions, pco 2 : partial pressure of carbon dioxide, po 2 : partial pressure of oxygen, hco 3 − : bicarbonate, tco 2 ; total amount of carbon dioxide, be: base excess, o 2 sat: oxygen saturation. different letters in the same rows (a, b) and columns (a, b) are statistically significant (p < 0.05). group 1: 100 mg/kg cos, group 2: 500 mg/kg cos, group 3: 1000 mg/kg cos at intervals of 12 h, group 4: no drug was given to the infected lambs. (mammeri et al., 2018) on the exact mechanism of action of chitosan continue. it has been reported that it is possible for chitosan to cause effects on the production of trophozoites and oocysts via various mechanisms (direct effects on parasitic viability and/or infectivity and/or parasitic growth in intestines and/or oocyst formation) (mammeri et al., 2018) . however, further studies are required to determine the exact mechanism of action. the limitations of this study are the absence of a negative control group without infection and treatment and the absence of groups treated with cos without cryptosporidiosis infection. generally, if there is no mix infection, the clinical findings in cryptosporidiosis are not severe and are transient and mild levels. in mix infections, clinical findings are more severe and mortality rates are higher. morbidity rates are high and mortality is low in cryptosporidiosis infections alone (constable et al., 2017) . in this study, it was observed that the changes in the clinical findings were not severe in all groups; however, there were some individual differences. the characteristics of diarrhea improved day by day in the treatment groups and in the positive control group. in the treatment groups (g1-3), stools became normal or pasty in consistency on the 7th day, while there was still diarrhea in liquid level in 2 cases in the positive control group on the 7th day. these results showed that chitosan oligosaccharide provided faster recovery in clinical findings and stool characteristics in lambs with cryptosporidiosis compared to the positive control group. the most important clinical finding in cryptosporidiosis is diarrhea in varying levels. endogenous forms of cryptosporidium lead to loss of mature enterocytes, shortening, and fusion of villus. they disrupt the microvilli boundary which causes the elongation of crypts caused by increased cell division and edema. this causes the loss of membranebound digestive enzymes, reduces intestinal absorption capacity and reduces intake of liquids, electrolytes, and nutrients from the intestinal lumen (foster and smith, 2009; constable et al., 2017) . in this study, it was found that although some significant in-group and/or intragroup changes were observed in blood gases, hematological and biochemical parameters in both treatment groups and in the positive control group, these changes remained limited and progressed generally within the reference limits. metabolic acidosis was observed in 3 cases in the group 1 on day 1 and 3; 1 case in the group 2 on day 0; 2 cases in the group 3 on day 0 and 1; 2 cases in the group 4 on day 5 and 7. however, it was determined that metabolic acidosis was at moderate and severe levels in only 1 st and 4th groups in 1 case in each. in other cases, the severity of metabolic acidosis remained mild. these results show that experimentally developed cryptosporidiosis alone in lambs leads to changes in blood gases and hematological and biochemical parameters; however, these changes were not at a level to disrupt the general condition. in conclusion, it was found that experimental cryptosporidiosis can be formed successfully through the oral inoculation of the solution containing 10 6 oocysts in lambs aged < 5 days, that oral administering of chitosan oligosaccharide in lambs at doses of 100, 500 and 1000 mg/ kg had no side effect, that chitosan oligosaccharide provided faster recovery in clinical findings and stool characteristics in lambs in which cryptosporidiosis was formed experimentally compared to positive control group and that administering of chitosan oligosaccharide at doses of 100, 500, 1000 mg/kg for 7 days significantly decreased oocyst excretion; however, it was not efficient enough to completely eliminate cryptosporidiosis. in this study, the appropriate chitosan oligosaccharide dose to treat cryptosporidiosis in lambs was observed to be 100-500 mg/kg, whereas new studies on this subject are required. we think that this study will provide a basic knowledge for further studies on the use of chitosan and its derivatives in the treatment of cryptosporidiosis in veterinary medicine. the authors declare that they have no conflict of interest. rdw: red cell distribution width; plt: platelet. different letters in the same rows (a, b) and columns (a, b) are statistically significant (p < 0.05) anticoccidial activities of chitosan on eimeria papillata-infected mice efficacy of chitosan, a natural polysaccharide, against cryptosporidium parvum development in infected hct-8 and caco-2 enterocytic cells effects of chitosan-oligosaccharide on diarrhoea in hanwoo calves comparison of the effectiveness of halofuginone lactate and paromomycin in the treatment of calves naturally infected with cryptosporidium parvum the safety of chitosan as a pharmaceutical excipient development of particulate drug formulation against c. parvum: formulation, characterization and in vivo efficacy anti-inflammatory effects of low-molecular weight chitosan oligosaccharides in ige-antigen complex-stimulated rbl-2h3 cells and asthma model mice veterinary medicine. a textbook of the diseases of cattle, horses, sheep, pigs and goats pathophysiology of diarrhea in calves efficacy of halofuginone lactate for the treatment and prevention of cryptosporidiosis in goat kids: an extensive field trial the prevalence and molecular characterisation of cryptosporidium spp. in small ruminants in zambia uptake and cytotoxicity of chitosan molecules and nanoparticles: effects of molecular weight and degree of deacetylation underlying mechanism of antimicrobial activity of chitosan microparticles and implications for the treatment of infectious diseases subacute toxicity of chitosan oligosaccharide in sprague-dawley rats laboratory in parasitology. the turkish society for parasitology experimental cryptosporidiosis in kids a review of chitin and chitosan applications in vitro evaluation of the suppressive effect of chitosan/poly(vinyl alcohol) microspheres on attachment of c. parvum to enterocytic cells efficacy of chitosan, a natural polysaccharide, against cryptosporidium parvum in vitro and in vivo in neonatal mice chitosan oligosaccharide: biological activities and potential therapeutic applications diseases of the gastrointestinal system cryptosporidiosis in small ruminants sheep medicine therapy and prevention of cryptosporidiosis in animals field trial on the therapeutic efficacy of paromomycin on natural cryptosporidium parvum infections in lambs cryptosporidiosis and coccidiosis biological activities of chitosan and chitooligosaccharides the efficacy of a combination of azithromycin and toltrazuril for the treatment of calves naturally infected with cryptosporidiosis: a randomised, double-blind, placebo-controlled comparative clinical trial chitosan oligosaccharide as potential therapy of inflammatory bowel disease: therapeutic efficacy and possible mechanisms of action biomedical applications of chitosan and its derivative nanoparticles cryptosporidium infection in lambs and goat kids in serbia this project supported by the scientific and technological research council of turkey (project number: 116o587). ast: aspartate aminotransferase, alp: alkaline phosphatase, ggt: gamma-glutamyltransferase, tp: total protein. different letters in the same rows (a, b) and columns (a, b) are statistically significant (p < 0.05). group 1: 100 mg/kg cos, group 2: 500 mg/kg cos, group 3: 1000 mg/kg cos at intervals of 12 h, group 4: no drug was given to the infected lambs. key: cord-027811-vk3qnumx authors: freedberg, daniel e.; messina, megan; lynch, elissa; tess, monika; miracle, elizabeth; chong, david h.; wahab, romina; abrams, julian a.; wang, harris h.; munck, christian title: impact of fiber-based enteral nutrition on the gut microbiome of icu patients receiving broad-spectrum antibiotics: a randomized pilot trial date: 2020-06-11 journal: crit care explor doi: 10.1097/cce.0000000000000135 sha: doc_id: 27811 cord_uid: vk3qnumx objectives: dietary fiber increases the abundance of bacteria that metabolize fiber into short-chain fatty acids and confers resistance against gut colonization with multidrug-resistant bacteria. this pilot trial estimated the effect of fiber on gut short-chain fatty acid–producing bacteria in the icu. design: randomized, controlled, open label trial. setting: medical icu. patients: twenty icu adults receiving broad-spectrum iv antibiotics for sepsis. intervention: 1:1 randomization to enteral nutrition with mixed soyand oat-derived fiber (14.3 g fiber/l) versus calorieand micronutrient-identical enteral nutrition with 0 g/l fiber. measurements: rectal swabs and whole stools were collected at baseline and on study days 3, 7, 14, and 30. the primary outcome was within-individual change in the cumulative relative abundance of short-chain fatty acid–producing taxa from baseline to day 3 based on 16s sequencing of rectal swabs. the secondary outcome was day 3 cumulative short-chain fatty acid levels based on mass spectrometry of whole stools. analyses were all intent to treat. main results: by day 3, the fiber group received a median of 32.1 g fiber cumulatively (interquartile range, 17.6–54.6) versus 0 g fiber (interquartile range, 0–4.0) in the no fiber group. the median within-individual change in short-chain fatty acid producer relative abundance from baseline to day 3 was +61% (interquartile range −51 to +1,688) in the fiber group versus −46% (interquartile range, −78 to +13) in the no fiber group (p = 0.28). whole stool short-chain fatty acid levels on day 3 were a median of 707 μg short-chain fatty acids/g stool (interquartile range, 190–7,265) in the fiber group versus 118 μg short-chain fatty acids/g stool (interquartile range, 22–1,195) in the no fiber group (p = 0.16). conclusions: enteral fiber was associated with nonsignificant trends toward increased relative abundance of short-chain fatty acid–producing bacteria and increased short-chain fatty acid levels among icu patients receiving broad-spectrum iv antibiotics. larger studies should be undertaken and our results can be used for effect size estimates. u p to a third of icu patients have gastrointestinal colonization with multidrug-resistant organisms (mdros) such as vancomycin-resistant enterococcus or mdr gram-negative bacteria (1) . once colonized, icu patients are at increased risk for subsequent infection with the same organisms (2) (3) (4) (5) . if gut colonization could be prevented, many high-mortality icu infections would be avoided. loss of commensal gut bacteria facilitates colonization with mdros. nonpathogenic colonic anaerobes compete with mdros for shared resources and in some cases directly antagonize them by producing antibacterial small molecules (6) . among the commensal microbiota, the bacteria that ferment fiber into short-chain fatty acids (scfas) have drawn attention. in animal models, scfa-producing bacteria confer protection against mdro colonization (7, 8) . administration of fiber, either by increasing the abundance of scfa producers or by raising scfa levels themselves, confers similar protective effects (9) (10) (11) (12) . fiber also may attenuate the damage caused by antibiotics on the commensal microbiota (13) . fiber therefore appears to be a suitable therapy to test for the prevention of mdro gut colonization in the icu. yet the effects of fiber on the gastrointestinal microbiota of icu patients, and whether such effects can still be observed in the face of broadspectrum antibiotics, is unknown. this pilot study was designed to test the hypothesis that fiber-based enteral nutrition increases the levels of scfa-producing bacteria and scfa levels in icu patients receiving broad-spectrum iv antibiotics, with a goal of generating effect size estimates that could be used as the basis for future studies involving fiber. adults 18-years-old or more at the time of medical icu admission were eligible for the study if they were expected to receive 3 or more days of enteral nutrition and had received a broad-spectrum iv antibiotic for sepsis within the previous 24 hours. empiric antibiotics were accepted, and subjects were enrolled without a requirement for positive cultures. the following antibiotic classes were considered broad spectrum: β-lactam/β-lactamase inhibitor combinations, carbapenems, cephalosporins, fluoroquinolones, and clindamycin. patients were excluded if they lacked capacity and had no health surrogate, had surgery involving the intestinal lumen within 30 days, or had limited treatment goals (i.e., do not resuscitate/do not intubate). this study was approved by the institutional review board of columbia university medical center and registered on clinicaltrials.gov (nct03509753). patients were randomized in blocks of four with 1:1 assignment to one of two forms of complete enteral nutrition. they received either mixed soy-and oat-derived enteral nutrition with 14.3 g fiber/l (brand name: promote 1.0 with fiber; abbott nutrition, chicago, il) or calorie-and micronutrient-identical nutrition with 0 g/l fiber (brand name: promote 1.0). enteral nutrition higher in fiber is available but these formulas were selected because they are identical aside from fiber. the formula manufacturer, abbott nutrition, had no involvement in the study. after patients were randomized, icu teams were instructed to continue the assigned formula as long as possible, but not to withhold ad lib nutrition once patients were ready to transition to oral diets. enteral feeding rates were individualized based on estimated energy requirements using the mifflin-st. jeor equation and the penn state university 2003b and 2010 equations, as recommended by the american society of parenteral and enteral nutrition (14) . patients were assessed at baseline/day 0 (i.e., before starting enteral nutrition) and subsequently on days 3, 7, 14, and 30. assessments continued until withdrawal from the study, hospital discharge, day 30, or death (whichever came first). at each study assessment, a deep flocked rectal swab (copan diagnostics, murrieta, ca) was collected with fecal soilage to verify sample adequacy. interval spontaneous stools were also collected because rectal swabs do not provide enough material to directly test scfa levels. swabs and stools were flash frozen at −80°c immediately after collection. to ascertain the amount of fiber actually received, the hourly enteral nutrition infusion rate was multiplied by the fiber content of the formula after accounting for interruptions in the feeding. for patients taking food by mouth, the type of meals, number of meals, and percentage of meal consumption was recorded by the patient's nurse. nutritional values for each meal type were obtained from the department of nutrition, including fiber content, so that fiber intake could be calculated even among patients who had transitioned to an oral diet. the primary outcome was the within-individual change from baseline (preintervention) to day 3 in the relative abundance of scfa-producing bacteria from rectal swabs. at the end of the study, rectal swabs were thawed and dna was batch extracted for sequencing of the v4 region of the 16s rrna gene (additional details in supplemental methods, supplemental digital content 1, http://links.lww.com/ccx/a194) (15) . using this data, operational taxonomic units (otus) were classified as scfa-producing or non-scfa-producing based on the study by vital et al. (16) , which identified 17 taxa that account for 85% of colonic butyrate production. the relative abundance of these scfa-producing otus was summed within each patient and calculated as [(day 3 − baseline)/baseline]. differences between study groups were assessed as an intent-to-treat wilcoxon rank-sum test. later study assessments were used to investigate durability of effect using similar methods. sequencing fastq data files are publicly available within the short read archive under bioproject prjna603980. the secondary outcome was scfa levels, measured from whole stool samples. because baseline stools were unavailable, scfa levels were determined from the stool sample closest to day 3 rather than as change from baseline. at the end of the study, homogenized fecal samples were thawed and assessed using liquid chromatographymass spectrometry (supplemental methods, supplemental digital content 1, http://links.lww.com/ccx/a194). the concentrations of eight scfas including butyrate were summed within each sample to yield a single total scfa concentration in μg/g of stool. the clinical effects of fiber administration were assessed focusing on caloric intake, stool frequency, and stool consistency. stool frequency was measured by asking icu nurses at each study assessment to report the number of stools during the prior 24-hour period. stool consistency was measured on a 5-point likert scale analogous to the bristol stool scale (0 = watery; 1 = loose/mucousy; 2 = loose with solid elements; 3 = formed, soft; 4 = formed, hard) (17) . nutritional intake was measured as the proportion of goal calories consumed. adverse effects were assessed in two ways. first, daily medical progress notes were reviewed to ascertain untoward health events, which were graded in terms of severity and relatedness to the study intervention. second, because untoward health events are so common in the icu and because it is challenging to determine relatedness (18) , three types of adverse events were prespecified that could be ascertained objectively: death, culture-proven infection, and electrolyte abnormalities. death was ascertained from the electronic medical record, which interfaces with the social security death index; culture-proven infection was operationalized as previously described (2); and electrolyte abnormalities were recorded as the maximum and minimum values for serum potassium, sodium, calcium, and phosphate. all analyses were performed intent-to-treat, among the patients who provided baseline and day 3 samples. primary analyses were conducted using the baseline and day 3 data, with the later study assessments used to assess for durability of effects. categorical data were compared using fisher exact text and continuous data were compared using rank-sum tests. a power calculation was performed before the study was begun as a two-sample test of means. it was estimated that a sample size of 10 patients per group would yield 80% power to detect a difference of 1.4 sd between groups. all testing was two-sided with alpha 0.05 considered statistically significant. from august 2018 to june 2019, 22 patients were enrolled all of whom had received either a third-generation cephalosporin, carbapenem, or β-lactam/β-lactamase combination antibiotics within the preceding 24 hours (for antibiotics received and duration, see supplemental table 1 , supplemental digital content 2, http://links.lww.com/ccx/a195). one patient was randomized to fiber with surrogate consent but self-extubated the next day and declined to continue the study. another was randomized to no fiber and died before day 3. this left 20 patients who provided baseline and day 3 samples and were analyzed. clinical characteristics were similar for the fiber and no fiber groups ( table 1) . four patients crossed over, two in each group. two patients assigned to fiber had delays initiating of enteral nutrition and did not receive any within 3 days of enrollment. two patients assigned to no fiber transitioned to oral diets more rapidly than anticipated and received small amounts of fiber before day 3. these four patients were analyzed based on their original study assignment (i.e., intent-to-treat). overall, the fiber group received a median of 10.7 g/d (iqr, 5.9-18.2; maximum, 27.2) fiber by day 3 versus 0 g/d (iqr, 0-1.3) in the no fiber group (p < 0.01). by the day 7 study assessment, there was no difference in observed fiber intake between study groups (fig. 1) . within-individual change in scfa-producing bacteria from baseline to day 3 was compared between study groups. there was a median +61% (iqr −51 to +1,688) in the fiber group versus −46% (iqr, −78 to +13) in the no fiber group (p = 0.28) (fig. 2) . this nonsignificant trend toward increased scfa producer relative abundance in the fiber group remained for subsequent study assessments. when scfa-producing otus were considered as individual data points (i.e., with a given patient contributing a data point for each otu), the difference in scfa producers seen with fiber became statistically significant (supplemental fig. 1 the median day 3 fecal scfa concentration was 707 μg/g stool (iqr, 190-7,265) in the fiber group versus 118 μg/g stool (iqr, 195) in the no fiber group (p = 0.16) (fig. 3) . there were no significant differences between scfa levels during subsequent assessments, although the trend remained higher in the fiber group (supplemental fig. 3 , supplemental digital content 6, http://links.lww.com/ccx/a199; legend, supplemental digital content 10, http://links.lww.com/ccx/a203). there were no differences in alpha diversity between study groups, with both groups declining in diversity as the study progressed (supplemental fig. 4 , supplemental digital content 7, http:// links.lww.com/ccx/a200; legend, supplemental digital content 10, http://links.lww.com/ccx/a203). when sequencing data were assessed in a hypothesis-free manner, there were day 3 declines in the non-scfa-producing otus classified as finegoldia genus (p = 0.01) and erysipelotrichaceae family (p = 0.03), comparing fiber versus no fiber. through day 3, a median of 58% (iqr, 24-84%) of goal calories were provided in the fiber group versus 33% (iqr, 2-52%) in the no fiber group (p = 0.24). there was a median of 1 stools/d (iqr, 0.33-3.33) in the fiber group versus 1.67 stools/d (iqr, 0.67-2.67) in the no fiber group (p = 0.85). stool consistency was 1.7 (iqr, adverse effects were monitored through study day 30. there were no differences in the number or severity of untoward health events based on study group (supplemental table 3 , supplemental digital content 8, http://links.lww.com/ccx/a201). there were also no differences in deaths (two fiber vs. four no fiber, p = 0.63), culture-proven infections (three fiber vs. three no fiber, p = 1.0), or electrolyte abnormalities (supplemental table 4 , supplemental digital content 9, http://links.lww.com/ccx/a202). power calculations were performed to guide sample size calculations for future studies. the observed mean change between baseline and day 3 in scfa producers was +26% (sd = 58%) for fiber and −0.33% (sd = 48%) for no fiber. the fiber group had +0.44 sd increase in scfa producers, whereas the study was powered to detect a 1.4 sd or greater change. assuming similar effect size and variance, future studies would require 132 patients (66 per group) to achieve 80% power. this pilot study of 20 icu patients receiving antibiotics found that a median dose of 11 g/d of mixed fiber given as enteral nutrition was associated with a 61% gain in putatively beneficial scfaproducing bacteria over a 3 day time span, whereas patients who were not randomized to fiber had a 46% decline in the same bacteria. actual scfa levels paralleled the changes in scfa producers and were six-fold higher in the fiber group. despite these large differences, neither the scfa producer result nor the scfa level result was statistically significant. all observations were intent-totreat and there was a 20% rate of crossover, a common challenge for icu nutrition studies (19) . this result provides a valuable effect size estimate for future studies. such studies will require 100-150 patients to be adequately powered to assess effects of fiber on scfa producers, although this will presumably depend on fiber amount and type. prior studies have tested prebiotic interventions in the icu. results of these studies are mixed and, because the interventions tested have been heterogenous, hard to interpret. o'keefe et al (20) looked at scfa producer relative abundance and fecal scfa levels in response to supplementation with 18-36 g wheat dextrin/d in 13 icu patients (20) . with fiber, there was a dramatic increase in firmicutes and other scfa producers and substantial increases in scfa levels including a doubling of fecal butyrate concentrations. this result contrasts sharply with the decline in scfa producers and scfa levels usually observed in icu patients (21, 22) . it also accords well with our own retrospective study of 129 icu patients, which found that observed mixed fiber intake correlated well with scfa producer relative abundance during the 72 hours after icu admission (23) . in that study, patients in the highest tertile of fiber received had a two-to figure 1 . actual amount of fiber received during the trial, stratified by study group. box-and-whisker plots depict the mean amount of fiber received for each study period (between baseline and day 3, between day 3 and day 7, etc.), stratified by study group. patients were enrolled in the study if they were expected to receive enteral nutrition for a minimum of 3 days. after 3 days, as patients transitioned off enteral nutrition and onto oral diets, there was substantial crossover between study groups. based on original study assignment, there was a statistically significant difference in the actual amount of fiber received between enrollment and day 3 (p <0.01), but not at later study timepoints. these values were then compared between the two study groups as a wilcoxon rank-sum test. none of the differences between groups were statistically significant. three-fold increase in the relative abundance of scfa producers compared with patients in the lowest tertile of fiber received. other trials have had contradictory results. a study testing 7 days of 7 g/d inulin versus maltodextrin supplementation in 22 icu adults initiating enteral nutrition found no difference in fecal abundance of faecalibacterium prausnitzii or bifidobacteria, or in fecal scfa levels (24) . across these studies, differences in the type and amount of fiber, mode of delivery (supplementation vs. fibercontaining enteral nutrition), and outcome ascertainment could account for the differences in findings (25) . in this study, there were trends toward clinical benefits associated with fiber, some of which reached statistical significance. with fiber, there was a 25% absolute increase in goal calories consumed through day 3 (equivalent to an additional 400 kcal/ patient/d), which was not statistically significant. also, with fiber there was firmer stool consistency and a decrease in stool frequency by about 1 stool/patient-day. this last finding was significant despite the small study size and some crossover. improved stool consistency is not likely to impact survival in the icu but probably does impact patient comfort, hygiene, and nursing care. importantly, fiber did not cause diarrhea, bezoars/intestinal obstruction, or other adverse effects in the icu as has been suggested in the past (26) . this study has strengths but also limitations. by randomizing patients to one of two forms of complete enteral nutrition that were calorie and micronutrient identical other than fiber, it allowed us to reasonably attribute any observed differences to fiber itself. on the other hand, the difference in actual fiber intake between study groups-11 g/d for 3 days-was neither as high in dose nor as long in duration as we would have wished. indirect calorimetry was not performed to measure energy consumption, and we instead relied upon estimating equations (27) . the fiber was integrated within enteral nutrition rather than supplementation with a specific fiber type (e.g., inulin, psyllium, wheat dextrin). this improves generalizability but might obscure biological effects that would only be seen with monosupplementation using a specific fiber type. we have initiated a follow-up trial testing up to 32 g/d of inulin for 7 days in the icu in part to address these limitations (nct03865706). last, the trial was small but rigorously prespecified the primary outcome and a supporting secondary outcome, and carefully assessed relevant clinical and adverse effects. in summary, this randomized icu pilot trial found that mixed fiber, given as part of enteral nutrition, was associated with nonsignificant increases in fecal scfa-producing bacteria and in fecal scfa levels. the study did not seek to investigate the clinical consequences of these microbiome changes. fiber improved stool consistency and was apparently safe up to a maximum of 27 g/d. the results of this trial provide effect size estimates that can be the basis for future trials testing whether fiber, by increasing scfa producers and/or scfa levels, might confer benefit in the icu. supplemental digital content is available for this article. direct url citations appear in the html and pdf versions of this article on the journal's website (http://journals.lww.com/ccejournal). short-chain fatty acid (scfa) levels measured from whole stools on day 3, stratified by study group. baseline preintervention whole stools were not available so scfa levels were compared between study groups at day 3. the sum total of eight scfas was measured using an aliquot of homogenized stool: 2-methylbutyrate, acetate, butyrate, hexanoate, isobutyrate, isovalerate, propionate, and valerate. world health organization: antimicrobial resistance: global report on surveillance. who library cataloguing-in-publication data pathogen colonization of the gastrointestinal microbiome at intensive care unit admission and risk for subsequent death or infection associations between enteral colonization with gram-negative bacteria and intensive care unitacquired infections and colonization of the respiratory tract entry of bacteria into the urinary tracts of patients with inlying catheters the pathogenesis of catheter-related bloodstream infection with noncuffed short-term central venous catheters a gut commensal-produced metabolite mediates colonization resistance to salmonella infection bifidobacteria can protect from enteropathogenic infection through production of acetate depletion of butyrate-producing clostridia from the gut microbiota drives an aerobic luminal expansion of salmonella a dietary fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility dietary supplementation with nonfermentable fiber alters the gut microbiota and confers protection in murine models of sepsis dietary cellulose supplementation modulates the immune response in a murine endotoxemia model dietary xanthan gum alters antibiotic efficacy against the murine gut microbiota and attenuates clostridioides difficile colonization recovery of the gut microbiota after antibiotics depends on host diet, community context, and environmental reservoirs nutrition support core curriculum: a case-based approach: the adult patient. silver spring, md, american society for parenteral and enteral nutrition the human microbiome project colonic butyrate-producing communities in humans: an overview using omics data stool form scale as a useful guide to intestinal transit time serious adverse events in academic critical care research the intensive care medicine research agenda in nutrition and metabolism effect of fiber supplementation on the microbiota in critically ill patients rapid and sustained long-term decrease of fecal short-chain fatty acids in critically ill patients with systemic inflammatory response syndrome rapid gastrointestinal loss of clostridial clusters iv and xiva in the icu associates with an expansion of gut pathogens relationship between dietary fiber intake and short-chain fatty acid-producing bacteria during critical illness: a prospective cohort study additional oligofructose/inulin does not increase faecal bifidobacteria in critically ill patients receiving enteral nutrition: a randomised controlled trial guide to designing, conducting, publishing and communicating results of clinical studies involving probiotic applications in human participants intestinal obstruction from cecal bezoar; a complication of fiber-containing tube feedings society of critical care medicine; american society for parenteral and enteral nutrition: guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition for information regarding this article, e-mail: def2004@cumc.columbia.edu or cm3297@cumc.columbia.edu key: cord-033453-557obi3r authors: bretscher, lorenzo; hsu, alex; simasek, peter; tamoni, andrea title: covid-19 and the cross-section of equity returns: impact and transmission date: 2020-09-24 journal: rev asset pricing stud doi: 10.1093/rapstu/raaa017 sha: doc_id: 33453 cord_uid: 557obi3r using the first reported case of covid-19 in a given u.s. county as the event day, we find that firms headquartered in an affected county experience, on average, a 27-bps lower return in the 10-day post-event window. this negative effect nearly doubles in magnitude for firms in counties with a higher infection rate (-50 bps). we test a number of transmission channels. firms belonging to labor-intensive industries and those located in counties with a large mobility decline have worse stock performance. firms sensitive to covid-19-induced uncertainty also exhibit more negative returns. finally, more negative stock returns are associated with downward revisions in earnings forecasts. deepening economic depression started to set in as grim gross domestic product (gdp) growth predictions for the second quarter are updated. in this paper, we document that the domestic spread of in the united states has a negative and significant impact on firm equity valuations. in particular, we show that the pandemic negatively affects firms through its impact on labor supply and the uncertainty it generates. furthermore, the shock propagates through the equity market by putting downward pressure on analyst earnings forecasts. we attempt to address two questions: (1) how does the sudden collapse in economic activity caused by affect equity valuation in the cross-section of firms? and (2) what are the channels through which the covid-19-induced economic downturn propagates and affects firm-level outcomes? the first question has important implications for investors' portfolio choices, banks' lending policies, and managers' investment decisions. the second question is key to understanding the effectiveness of policy interventions to stabilize the economy. using geographical dispersion of firm headquarter locations and the fact that the pandemic spread through the united states with variable timing, we perform a difference-in-differences estimation to understand the effect of the pandemic on equity returns. we find that, on average, daily returns of public firms are 27 basis points (bps) lower in the 10day window right after the first case of is recorded (event day) in the county where the firm is headquartered. when we consider counties with high infection rates (100 or more cases reached in less than 20 days), the negative effect of the shock nearly doubles to 50 bps. that is, returns are lower relative to days before the event day as well as to returns of firms residing in counties that have never experienced a covid-19 case during our sample period. 1 the transmission of from a health care crisis to a corporate slump may be driven by several different economic mechanisms. to investigate the transmission mechanism, we focus on four potential, and not mutually exclusive, channels: labor supply, uncertainty, government spending and monetary policies, and cash flow expectations. in particular, the stay-at-home orders resulting from the spread of the virus may depress workers' ability to travel to work, while the largely unknown factors surrounding the disease generated considerable uncertainty about future economic activity. some firms may be less affected, thanks to existing government contracts providing stable demand. ultimately, given the widespread and rapid advance of , we anticipate analysts to frequently revise their earnings expectations. various recent studies and many economists predominantly describe the impact of the pandemic on the economy as a demand shock. indeed, because of social distancing measures and closure of public gatherings, consumer demand dropped significantly during march 2020. 2 the resultant momentous rise in unemployment further suggests that demand will be depressed for the foreseeable future. however, this does not rule out the pandemic as a supply shock. in fact, guerrieri, lorenzoni, straub, and werning (2020) suggest that negative supply shocks can turn into negative demand shocks. using a multisector model in incomplete markets, they show that a negative supply shock can trigger changes in aggregate demand as firms exit and jobs disappear. we believe that our natural experiment combining the geographical dispersion of firms with the staggered impact of provides a unique setting to examine the relative strength of various demand and supply channels through which the shock affects firm valuations. in our empirical setting, we rely on large public firms which sell their goods and services across the entire continental united states, and in many instances, internationally. hence, our county-level valuation effect due to the presence of confirmed virus cases is unlikely to be a pure demand-side phenomenon. 3 in fact, our baseline regression results suggest that the crisis negatively affects the supply-side and labor productivity. specifically, to study the labor supply channel of the propagation, we estimate difference-in-differences regressions on a subsample of "labor-intensive" firms. we classify as labor-intensive firms within the mining, construction, and manufacturing sectors. the regression results show that the average daily return of a labor-intensive firm residing in a high intensity county is 1% lower in the 10-day post-event window. this suggests that the negative effect of shocks on equity returns is considerably stronger for laborintensive firms. in other words, the pandemic negatively affects economic output through its impact on labor supply. to further tie return dynamics to labor supply, we use the university of maryland mobility data to document that the countylevel percentage of people staying at home increased following the first reported case of coronavirus in the county. the decline in mobility due to the virus spread suggests that the pandemic negatively affects labor supply. next, we sort counties according to changes in the percentage of people staying at home before and after the first reported case. we find that firms with headquarters located in those counties with large increases in the percentage of people staying at home have more negative stock returns in the days following the first reported case compared to firms residing in counties with small changes in the percentage of people staying at home. taken together, results using mobility data provides evidence that the virus spread causes the population to stay at home more, which worsens the financial performance of local firms. using the date of the first reported case as opposed to another threshold allows us to separate out the effect stemming from the second moment (uncertainty) of the shock from its first moment (level). for example, the productivity level may possibly not fall immediately after the first case is reported, but business activities still can be negatively affected in expectation of a fall due to the uncertainty. to tease out the impact of covid-19-induced uncertainty on equity returns, we employ data from hassan, hollander, van lent, and tahoun (2020) . these authors use textual analysis of earnings call transcripts to obtain firmlevel measures of risk exposure. the risk measure is related to the mentioning of words synonymous to "risk" or "uncertainty" in transcripts, and by construction it captures the management's attitude toward we find that firms concerned with the elevated uncertainty induced by exhibit particularly negative returns after the event day. moreover, we examine whether firm-level exposure to government spending and monetary policies can produce the heterogeneous return reaction we observe in the data. for government spending, goldman (2019) shows that government contractor firms weathered the 2008 financial crisis relatively better compared with noncontractor firms. we apply the goldman (2019) definition of government contractors to our sample of firms and check whether their stock outperformed the noncontractors after the first case of the coronavirus was reported in the county the firm is headquartered. in contrast to goldman (2019), we do not find this to be the case, which highlights a fundamental difference between the pandemic and the financial crisis. large government contractors are primarily in labor-intensive manufacturing industries leaving them more susceptible to the negative labor supply shock induced by the pandemic. focusing on a subsample of labor-intensive firms, we find that government contractors' stock returns are relatively more negative than noncontractors following the first reported case. hence, contractors enjoy stable demand due to contractual relationships with the government, but their stocks perform worse. this result suggests that government contractors might be particularly reliant on the presence of the labor force they employ. furthermore, we do not find evidence that heterogeneous sensitivity to monetary policy drives the cross-sectional return dynamics following the covid-19 news. the last channel of transmission we investigate concerns how the spread of covid-19 affects expected corporate earnings. to this end, we use analysts' forecast data from the i/b/e/s database and document that the first reported coronavirus case results in downward revision of earnings estimates of firms located in the same county. moreover, we find that firms with negative earnings revisions experience greater stock return declines relative to firms with no revisions or positive revisions in the 10-day window after the first case of is reported in a county. taken together, we deduce that the spread of covid-19 leads to financial market fragility partially by depressing the expected cash flows of affected firms. our work is related to a fast-growing literature on the asset price response to the pandemic in the first quarter of 2020. 4 gormsen and koijen (2020) look at the aggregate equity market and dividend futures during the outbreak to infer bounds on future gdp growth. baker et al. (2020b) use text-based methods to study the u.s. stock market reaction to the covid-19 pandemic. cejnek, randl, and zechner (2020) provide evidence that index-level and singlestock near-maturity dividend futures reflect expectations of dividend cuts. pettenuzzo et al. (2020) analyze the signaling role of dividend announcements during the pandemic by looking at the stock market reaction of firms that changed their dividend policy. alfaro, chari, greenland, and schott (2020) show that aggregate equity market returns respond to daily unanticipated changes in predicted cases. toda (2020) uses a sir model to study the impact of the epidemic on the stock market. 5 in a cross-country setting, ru, yang, and zou (2020) find that stock markets reacted more quickly and strongly in countries that had a 2003 sars outbreak, whereas gerding, martin, and nagler (2020) show that stock price reactions were stronger in countries with higher debt-to-gdp ratios. in a cross-country and cross-asset-class setting, croce, farroni, and wolfskeil (2020) study how epidemic news identified from twitter are reflected in asset prices. they conclude that the market price of contagion risk is very high. distinct from these studies, we do not focus on the aggregate u.s. equity market, but rather we rely on geographical heterogeneity of firm headquarters to directly 4 a large number of papers investigate the effect of pandemics on economic growth, rather than asset prices. see barro, ursua, and weng (2020) , correia, luck, and verner (2020) , and ludvigson, ma, and ng (2020) . several papers also model the interaction between economic activity, economic decisions, and epidemic dynamics (see, e.g., alvarez et al., 2020; eichenbaum et al., 2020; jones et al., 2020; van binsbergen and opp, 2020) . 5 more generally, various streams of papers extend the canonical epidemiology sir model to an exploration of the covid-19 epidemic (see, e.g., atkeson, 2020; favero, 2020) . identify the consequences of the pandemic in the cross-section of u.s. equity returns. in this respect, our work contributes to a large number of contemporaneous papers that investigate the firm-and industry-level price responses to covid-19. albuquerque, koskinen, yang, and zhang (2020) focus on the performance of firms with high environmental and social ratings during the covid-19 outbreak. ramelli and wagner (2020) focus on the importance of trade (e.g., china-oriented stocks) and financial policies for firm value. these authors document that firms with more leverage and minimal cash holdings suffered severely in the period from february 24 through march 20, even if these firms did not have international activities. they interpret this finding as evidence that business uncertainty caused by is amplified through certain financial channels. interestingly, hassan, hollander, van lent, and tahoun (2020) document that financing concerns are mentioned relatively rarely in earnings conference transcripts as covid-19 spreads globally. on the contrary, these authors find evidence for decreasing demand, disruption of the supply chain and closure of production facilities, and increased uncertainty as major concerns for firms. papanikolaou and schmidt (2020) construct an industry-level measure of exposure to work disruptions, and investigate whether the cross-sectional differences are predictive of differential economic outcomes during the pandemic. pagano, wagner, and zechner (2020) investigate how companies' exposure to social distancing not only during the outbreak but also prior to and after the outbreak affects asset prices. despite sharing a similar interest on the cross-sectional response of stock prices to the pandemic, we differ from all these papers in two respects. first, our econometric analysis combines the geographical dispersion of firms with the staggered impact of and is akin to the classical difference-in-differences approach commonly used in corporate finance. second, and related, a unique advantage of our natural experiment is that it allows us to examine the relative strength of various channels (specifically, the labor supply channel, the uncertainty channel, the government policy channel, and the cash flow news channel) through which the covid-19 shock affects firm valuations. 6 we also contribute to broader streams of literature in finance and economics. first, our analysis of the labor supply channel speaks to the literature studying how labor-induced operating leverage affects asset prices. for example, donangelo, gourio, kehrig, and palacios (2019) show that high labor-share firms have operating profits that are more sensitive to shocks and have higher expected asset returns. moreover, donangelo (forthcoming) shows that operating leverage due to labor is important in explaining the value premium. belo, lin, li, and zhao (2017) , belo, lin, and bazdresch (2019) , and belo, donangelo, lin, and ding (2020) study how labor market frictions affect asset prices, and favilukis and lin (2016a,b) study the consequences of rigid wages on asset prices and return predictability. our analysis of the labor supply channel also contributes to the recent theoretical contributions. for example, in the eichenbaum, rebelo, and trabandt (2020) model, an epidemic has both aggregate demand and aggregate supply effects. guerrieri, lorenzoni, straub, and werning (2020) develop a theory of keynesian supply shocks that trigger changes in aggregate demand larger than the shocks themselves. these authors argue that the economic shocks associated to the covid-19 epidemic may have this feature. second, our analysis of uncertainty as a possible propagation channel through which the news of the virus's spread onto asset prices speaks to a large literature on second moment shocks. starting with bloom (2009) , an increasing body of research studies how uncertainty shocks influence economic activity and asset prices. veronesi (2012, 2013) examine the response of the equity risk premium to government-induced (political) uncertainty. croce, nguyen, and schmid (2012) show that a reduction of model uncertainty can come at the cost of depressing growth for the long-run. finally, bianchi, kung, and tirskikh (2018) evaluate the effect of different sources of uncertainty (i.e., demand and supply uncertainty) on macroeconomic and financial outcomes via structural estimation. third, our analysis of the possible effects of fiscal and monetary policies during the pandemic complements the literature that has investigated the effectiveness of various policy measures during the great recession (e.g., adelino et al., 2017; chodorow-reich et al., 2012; goldman, 2019; wilson, 2012) . lastly, our investigation of the reaction of earning forecasts of analysts and their relation to returns during the pandemic contributes to a vast literature that analyzes the roles of cash flow expectations and discount rates for asset prices. gormsen and koijen (2020) and landier and thesmar (2020) are two contemporaneous studies that look at these two channels during the covid-19 outbreak exploiting aggregate s&p 500 dividend strips and ibes forecasts, respectively. similar to landier and thesmar (2020) , we also use ibes forecasts and focus on the crosssection of returns. we believe that our staggered difference-in-differences setup, and our analysis of returns reaction over a tight window around the event day, complements their analysis of cumulative returns response to the covid-19 crisis. we obtain public firm ticker symbols from compustat at the end of 2019. using these tickers, we download daily closing prices from bloomberg from december 31, 2019, to march 20, 2020. we stop the sample on march 20th to minimize the effect of the cares act on stock prices. the cares act was passed on march 26, 2020, and, by design, affects specific firms and industries in a heterogeneous manner. we then calculate daily returns for all firms in the sample using the end of day prices. we then drop firms that have at least one missing price or if the price dips below $2 a share on any given trading day since the beginning of 2020. the number of headquarter firms in our data set located in a particular u.s. county firm headquarter locations are also obtained from compustat. we convert zip codes to geographic identifiers (geoid) using a crosswalk from the u.s. census bureau. geoid's are unique to u.s. counties, which allows us to merge firm headquarter locations with the case data from the new york times. figure 1 reports the density of firms in our data set headquartered in a particular county. the completed data set matches trading day-firm observations (firm characteristics as of the end of 2019) and the calendar day on which the first reported case appears in the county where the firm is headquartered. table 1 illustrates the geographical distribution of firms in our sample by state. although california, new york, and texas dominate in having the highest number of firm headquarters, in that together they account for roughly one-third of the sample, substantial heterogeneity in headquarters locations across the united states is evident. in the appendix, as a robustness check, we repeat our baseline tests excluding the three populous states and document similar findings. using the day on which the first covid-19 case is reported as the event day (covid-19 (0) dummy), we examine firm-level returns before and after the event day. we truncate our data at 10 calendar days postevent day such that our post-event window is from day 1 to day 10 (post. this choice aims to minimize the confounding effect due to other news arrivals. figure 2 displays the evolution of cases by county. the figure reports time windows that contain the first reported case by county. the first window spans a longer time period as testing was initially sparse. table 2 presents summary statistics of firms in our study. statistics (in millions of dollars) of total assets (compustat variable: at), capital expenditure (capx), sales (sale), property, plants, and equipment (ppent), and operating income (oibdp) for the baseline sample are shown in panel a. the sample comprises a total of 99,729 trading day-firm observations. these are large firms on average with mean total assets of $14.78 billion and median of $2.09 billion. they have substantial sales with mean of $5.75 billion and median of $973 million. these firms are also highly profitable with average operating income of more than $1 billion. taken together, table 2 suggests the firms in our sample represent the largest companies in our economy. this matters for our identification strategy for the supply channel of the shocks. because these companies are so large, it is unlikely their sales are highly concentrated in the same county where the firm is headquartered. therefore, if the first reported case of in the county where the headquarters resides affects stock returns, the effect is more likely to be transmitted through the supply channel. table 2 panels b, c, d, and e are firm summary statistics broken down by industry, defined by their fama-french 17 (ff17) industry classification. for labor-intensive firms (ff17 industry 1 to 13) firms in panel b, the average total assets is $9.91 billion and the average sales is $7.35 billion. for retail (ff17 industry 15) firms in panel c, the average total assets is $10.09 billion and the average sales is $15.79 billion. for financial (ff17 industry 16) firms in panel d, the average total assets is $38.49 billion and the average sales is $5.01 billion. for services (ff17 industry 17) firms in panel e, the average total assets is $10.62 billion and the average sales is $6.14 billion. the average operating income across all major industries is well over $1 billion. we conclude that large firms with high sales volume are well distributed throughout various industries in our sample. in this section, we examine the impact of the covid-19 pandemic on firm-level equity returns. we devise a natural experiment by combining the geographical heterogeneity of firm headquarter location with the staggered spread of the virus to identify the causal relationship between and firm performance. the date on which the first case of is reported in a given county in the united states defines our event. the differential timing of the first reported case across counties allows us to pin down the treatment effect of covid-19 on the crosssection of firms. employing difference-in-differences estimations and spline regressions, we compare stock performance of firms before and after the first reported case as well as between firms located in and non(no reported covid-19 case during our sample period) counties. to establish our baseline result, we perform a diff-in-diff estimation of firm-level returns in the event window around the first confirmed case of , or event day, in the county a given firm headquarter is located. specifically, we regress the panel of daily log returns on the dummy and the postdummy as shown in equation (1): (1) coefficient loadings î² and î³ are meant to capture the difference in returns on the day of the event and in the 10-day post-event window, respectively, compared with returns prior to the event day. returns of firms residing in a county which never reported a covid-19 case in the sample period also serve as part of the control group. to control for unobserved heterogeneity along several dimensions, we include firm (ï� i ), industry (ï� j ), county (î´ c ), and day (ï� t ) fixed effects in the analysis. standard errors are double clustered at the county and trading day levels. the full sample comprises 99,729 trading day-firm observations. column 1 of table 3 presents the results for the baseline diff-in-diff regression. although the estimated coefficient for the (0) dummy is insignificant, the estimated coefficient for the postdummy is negative and statistically significant at the 5% level. the point estimate of -0.00274 suggests that returns are on average 27 bps lower in the 10-day post-event window relative to returns in the control group. in column 2, instead of using raw returns, we use total returns as the dependent variable. total returns are obtained from bloomberg, and adjust for near-term cash dividends. the point estimate on the post-covid-19 dummy is -0.00256, very similar to the counterpart in column 1. using excess return over the effective fed funds rate leaves the results unchanged. this also holds true for all results discussed below. given the baseline finding showing a localized impact of covid-19 on firm-level returns, we postulate that this effect should be stronger in counties where the spread of the virus is more intense. we then construct a growth intensity measure for each affected county using the number of days for reported cases to go over 100 (see figure 3 ). employing only observations in high growth intensity counties (20 days or less), we repeat the diff-in-diff regression in equation (1) and tabulate results in columns 3 and 4 of table 3 . for raw returns, the î³ coefficient is still negative and significant at the 5% level; more importantly, the magnitude has increased to -0.00499, almost doubling relative to the estimate in column 1. for total returns, the coefficient loading strengthened from -0.00256 in column 2 to -0.00465 in column 4. see figure 4 for a graphical representation. the divergence between cumulative returns in high growth versus low growth intensity counties widens dramatically after the first reported case. these findings validate our hypothesis that in locations considered "hot zones" (likely more densely populated), returns are more negatively affected by the spread of coronavirus. table 3 difference-in-differences regression results for firm-level returns on the shock this table presents difference-in-differences regression results. the sample period is between january 1, 2020, and march 20, 2020. the regression equation is log(return)ijct = î±+î²covid-19 (0) ct +î³post+ï�i +ï�j +î´c +ï�t +îµijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients î² and î³ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the postdummy encompasses the 10 days after the event day. the high ( growth subsample includes only firms residing in counties where the growth between 1 and 100 reported cases took less than 20 days. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth (1) (2) (3) (4) raw return total return raw return total return (0) -0.000841 -0.000809 -0.000699 -0.000789 (-0 .49) (-0 .49) (-0 .28) (-0.34) post.00274** -0.00256* -0.00499** -0.00465** (-2.08) (-1.97 ) (-2 .14) (-2 .13) -0.00744*** -0.00609*** -0.00821*** -0.00675*** (-37 .04) (-33 .49) (-20 .92) (-20 growth intensity of covid-19 cases by county. growth intensity is defined as the number of days before the county reaches 100 reported cases measured from the day the initial case was reported in a county. to further dissect the response of firm-level returns to the shocks, we utilize spline regressions around the day the first coronavirus case is reported in a county. in particular, we regress firm level returns on 15 dummies assigned to each day around the event. we assign individual dummies to each of the days in the post-event window, to . in the pre-event window, we assign individual dummies to each of the 5 calendar days leading up to (0) ( to ), as well as one dummy to capture all the days prior to the pre-event window (covid-19 < (-5) ). the regression specification is shown here: where the dummies capture days leading up to the event day, the covid-19 (0) dummy represents the day of the first reported case, and the dummies are assigned to individual days following the event. the and dummies are dropped so the returns on those days serve as the benchmark. 7 as mentioned previously, all trading days from the beginning of the sample up to 6 days before the event day are designated by the covid-19 < (-5) dummy. we also truncate the data at 10 days post-event. similar to equation (1), firm, industry, county, and day fixed effects are included to control for unobserved heterogeneity. table 4 documents spline regression results. column 1 represents the full sample using raw returns; column 2 represents the full sample using total returns; column 3 represents the high covid-19 growth intensity sample using raw returns; and column 4 represents the high covid-19 growth intensity sample using total returns. across the four columns, none of the coefficients for the dummies denoting days immediately before the event day, to , is statistically significant. this suggests there is no pre-trend leading up to the event and thus satisfies the parallel trend assumption of the diffin-diff estimation. the second item of interest is that the (0) dummy is not significant across the four columns of table 4 . there does not appear to be an immediate impact of reported virus cases to stock returns on the same day. in the post-event window, estimated coefficients for the dummies are generally negative. in table 4 , columns 1 and 2, for the full sample, these coefficients are not statistically significant until . although the coefficient loadings on the post-covid-19 dummy in table 3 , columns 1 and 2, are negative and significant, it takes about a week for the stock valuation impact to be realized. however, when the sample is narrowed to high growth intensity counties, results shown in columns 3 and 4 of table 4 demonstrate that the impact of the shock is concentrated on day 2 and after from the initial reported case. the estimated coefficient of -0.00642 and -0.00653, in columns 3 and 4, respectively, are significant at the 5%. this implies the average return is roughly 65 bps lower 2 days after the event day relative to the 2 days immediately prior to the event day. we conclude from the spline exercise that the first reported case of in the county where a firm is headquartered has a significant negative effect on its stock return, but there is a delay between the reporting date and the stock reaction date. this delay is minimized in counties which have high rates of virus spread. table 4 spline regression results for firm-level returns on the this table presents spline regression results. the sample period is between january 1, 2020, and march 20, 2020. the regression specification is shown in equation (2). both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the ) dummy encompasses all trading days prior to the 5-day window leading up to the event day. the high ( growth subsample includes only firms residing in counties where the growth between 1 and 100 reported cases took less than 20 days. all regressions include firm, industry, county, and day fixed effects. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth (1) (2) (3) (4) raw return total return raw return total return covid-19 < (-5 (-0 .37) (-0 .16) constant -0.00715*** -0.00554*** -0.00664*** -0.00511** (-5 .57) (-4 .01) (-3 .80) (-2 3. how does affect the cross-section of returns? the pandemic is all encompassing. for the financial markets, other than generating unprecedented uncertainty (or risk), it also changes investors' expectations about the future level of earnings and dividends. in this section, we examine a number of potential channels through which the pandemic potentially affects firms. in what follows, we focus on four aspects of the covid-19 crisis: the labor supply channel, the uncertainty channel, the government spending and monetary policies channel, and the cash flow expectations channel. we start by investigating whether our evidence of localized negative firm returns is consistent with the pandemic having a negative effect on local labor productivity. if this is indeed the case, we would expect firms relying more on labor input in the production function to be asymmetrically affected. to test this hypothesis, we focus on "laborintensive" firms and repeat the diff-in-diff analysis. labor-intensive firms are defined to encompass fama-french 17 industry codes between 1 and 13, which excludes firms in utilities, retail, financial, and services industries. table 5 shows the results. +ï�i +ï�j +î´c +ï�t +îµijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients î² and î³ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post-covid-19 dummy encompasses 10 days after the event day. the labor-intensive subsample includes only firms in the fama-french 17 industries 1 to 13 (excluding utilities, retail, financial, and services). robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth and labor intensive (1) (2) (3) (4) raw return total return raw return total return (0) -0.0000996 0.000150 -0.00606 -0.00541 (-0.04) (0.06) (-1 .32) (-1.29) post.00451* -0.00386* -0.0126** -0.0108** (-1.68) (-1.71 ) (-2 .23) (-2 .38) -0.00834*** -0.00705*** -0.00852*** -0.00723*** (-18 .63) (-19 .55) (-9 .07) (-9 for both raw returns and total returns, coefficient loadings on the postdummy in the labor-intensive subsample are negative and statistically significant at the 10% level in columns 1 and 2. in terms of magnitude, â��0.00451 and â��0.00386 respectively, these estimates are larger than the full sample estimates in tables 3, columns 1 and 2. furthermore, when we focus on labor-intensive firms headquartered in high growth intensity counties, the î³ coefficients are significant at the 5% level. this can be seen from columns 3 and 4 in table 5 . when compared with columns 1 and 2 in table 3 , the point estimates suggest that labor-intensive firms located in high growth intensity counties experience a much larger drop in equity value. to further examine how labor supply is affected by the outbreak of the pandemic, we use data from the university of maryland covid-19 impact analysis platform. 8 specifically, we obtain the county-level variable "% staying home" to investigate how the first reported case of coronavirus in a given county affects the population's ability to go to work. similar to our analysis of firm-level returns, we perform a difference-in-differences estimation and a spline regression at the county level employing "% staying home" as the dependent variable. table 6 shows the results. the sample period is from january 1, 2020, to march 20, 2020. in column 1, the difference-in-differences estimates on the covid-19 (0) dummy and the post-covid-19 dummy are both positive and significant at the 1% level. for example, after the first case of is reported in a given county, the percentage of people staying home goes up by 1.609% in the 10-day post-window relative to all days prior to the news. from the spline regression results displayed in column 2 of table 6 , we see that there is no parallel trend violation as all dummies before are insignificant, and all indicator dummies from up to +10 are positive and highly statistically significant, which validates the difference-in-differences finding in column 1. thus, we conclude that the first reported case of coronavirus in a county has a noticeably negative effect on labor supply in the same county as the population stay at home more. next, we link the results documented in table 6 to firm-level returns. to this end, we conjecture that firms located in counties with a large increase in the percentage staying home should have worse stock performance in comparison to firms residing in counties with small changes in percent staying home. that is, if labor supply is indeed a factor in the transmission of the news to the financial market, firms located in areas in which labor is most negatively affected by the pandemic should be penalized more by investors. to test this hypothesis, we sort counties into bins according to the change in percent staying table 6 percentage of people staying home after first reported this table regresses the percentage of people staying home by county on date dummy variables around the event day. data for the percentage of each county staying home come from the maryland transportation institute. the sample period is between january 1, 2020, and march 20, 2020. the dummy denotes the day on which the first case of is reported in the county. the post-covid-19 dummy encompasses 10 days after the event day. the covid-19 < (-5 ) dummy encompasses all trading days prior to the 5-day window leading up to the event day. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. (1) (2) % staying home % staying home covid-19 < (-5) -0.0928 (-0 .65) -0.207 (-1.40 ) (-4) -0.114 (-0 .90) (-3) -0.112 (-1 .10) (0) home in a window around the first reported case of coronavirus. 9 then we assign a staying home dummy to all firms headquartered in counties with large increases in percent staying home. in the return regression, we interact this staying home dummy with the covid-19 (0) dummy and the post-covid-19 dummy, as defined previously. the full regression specification is where i sh is the staying home dummy. î² and î³ are estimated coefficients for firms located in counties experiencing large staying at home increases following the covid-19 news. table 7 reports the results. table 7 columns 1 and 2 report regression results when the countylevel change in percent staying home is measured from 5 days prior to the first reported case to 10 days after, â�� % stay home (-5 ,+10), whereas columns 3 and 4 report results when the change is measured from 10 days before the first reported case to 10 days after, â�� % stay home (-10,+10) . in all scenarios, we find that the post-covid-19 dummy by itself is no longer statistically significant. on the other hand, all coefficient loadings on the interaction between the post-covid-19 dummy and the staying home dummy are negative and statistically significant at the 5% level. firms headquartered in counties experiencing large rises in percent staying home earn 50 bps lower returns relative to those firms residing outside of these counties in days following the first reported case of coronavirus. overall, the findings documented in this section verify our conjecture that the labor supply channel plays an important role in the transmission of the covid-19 shock to firm performance. with no known cure or vaccine as of may 2020, the economic outlook around the world is highly variable. citizens are left wondering when state-by-state shelter-in-place orders will be lifted, when social distancing measures will be relaxed, and when eating and drinking establishments will be allowed to open up again. medical experts have also warned about the risk of a second wave of contagion as the lockdown measures are gradually relaxed (see, e.g., xu and li, 2020) . at the same time, because of the lack of historical precedence of comparable events, economists and market participants are perplexed by the magnitude and length with which output, demand, employment, earnings, etc., are expected to decline. 10 in a recent paper, baker, bloom, davis, and 10 the largely unanticipated nature of the current pandemic is confirmed by the widely read global risk report: the world economic forum did not report a global pandemic among the most likely risks. the first five most likely risks were related to the environment, with climate change listed as the main threat to the planet in 2020 (cf. wef global risks perception survey 2019-2020). however, we cannot rule out that alert investors may have table 7 interaction results for firm-level returns on shock and percentage of people staying home this table presents interaction regression results. the sample period is between january 1, 2020, and march 20, 2020. we interact and dummies with indicators for whether a county has experienced an above-median change in the percentage of the population staying home. we use windows of both 5 and 10 days prior to the event day to a period of 10 days after the event day. where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients î², î³, î², and î³ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the covid-19 0 dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post-covid-19 dummy encompasses 10 days after the event day. i sh c is a dummy variable that equals one if a county is designated as having an above-median change in the percentage of people staying home. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. â�� % stay home (-5,+10) â�� % stay home (-10 -0.007*** -0.006*** -0.007*** -0.006*** (-48 .50) (-37 .75) (-46 .02) (-36 terry (2020a) use three different forward-looking uncertainty measures to document the enormous rise in economic uncertainty as of march of 2020. further, their economic model attributes 60% of the forecasted output contraction by q4 of 2020 (mean of 11%) to covid-19induced uncertainty. figure 5 plots the quarter-on-quarter gdp forecast taken into account pandemic concerns in their portfolio choices in advance of the current covid-19 event. see, for example, pagano et al. (2020) for an analysis of firm returns before the disaster. dispersion from professional forecasters between 1968 and 2020. 11 the spike at the end of this series corresponds to a dramatic increase in uncertainty unlike anything we have experienced in the postwar period. we attempt to tease out the impact of covid-19 on equity returns due to the uncertainty it produces. to that end, we employ data from the work of hassan, hollander, van lent, and tahoun (2020) (hhvlt herein). these authors use textual analysis of earning call transcripts to obtain firm-level measures of covid-19 exposure as well as sentiment and risk associated with that exposure. the sentiment measure is related to the conditional mean of the shock, whereas the risk measure is related to the variance of the shock. by construction, the risk measure captures the management's attitude toward we merge our return data set with the hhvlt data set from 2020q2. the two variables of interest are risk. net sentiment can be positive or negative depending on the 11 according to bloomberg data, as of april 16, 2020. historical data from philadelphia fed. 12 the text classification approach used to construct the risk and sentiment measures has been validated in recent work by hassan et al. (2019) in the context of measuring a firm's exposure to, for example, political risk. firm's outlook due to the shock. risk can be zero or a positive value if the firm expresses an uncertain outlook following the shock. after merging the data, we construct a risk dummy i risk , which we define as any firm in our sample that has a nonzero risk measure in the first quarter of 2020. we then eliminate all firms with a positive net sentiment value since these firms expect to benefit from the pandemic. to see whether uncertainty is associated with the decline in equity following the first reported case of coronavirus in the same county where the firm is headquartered, we extend the diff-in-diff regression from section 2.1 to include interaction terms with i risk . in particular, the regression specification is where î² and î³ are coefficient loadings on the interaction between the covid-19 0 dummy and the postdummy, respectively, with the covid-19 risk dummy. similar to the baseline diff-in-diff regressions, firm, industry, county, and day fixed effects are used as controls. table 8 presents the results. table 8 shows results for the full sample and the high covid-19 growth intensity sample. notice the î³ coefficients in the fourth row are negative and statistically significant across the columns for both raw and total returns. this means that firms affected by risk > 0) experience even lower returns after the event day (covid-19 0) relative to those firms not affected by this uncertainty (covid-19 risk = 0). overall, we find confirmation in these interaction regressions that covid-19-induced uncertainty drives firm-level equity returns after the shock is realized. policies in this section, we investigate whether the stability of recurrent government purchases makes firms more resilient to the economic shock induced by the pandemic. indeed, in the context of the [2008] [2009] financial crisis, goldman (2019) shows that government contractorsdefined as firms deriving more than 10% of sales from the federal government-experienced smaller declines in sales, profitability, and market values than otherwise similar firms. to assess the (direct) effect of government purchases at the firm level, we compare the stock performance of government contractors table 8 interaction regression results for firm-level returns on the this table presents interaction regression results. the sample period is between january 1, 2020, and march 20, 2020. we merge our sample with the hassan, hollander, van lent, and tahoun (2020) data set from 2020q1. their data using textual analysis contains firm-level exposure to . in particular, we use variables net sentiment and risk to measure the first (level) and second (uncertainty) moment, respectively, of the covid-19 shock to each firm. all firms with a positive net sentiment with respect to the shock are dropped. the regression equation is log(return)ijct = î±+î²covid-19 0ct +î³post+î² 0ct ã�i risk i +î³ post+ï�i +ï�j +î´c +ï�t +îµijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients î², î³, î² , and î³ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the covid-19 0 dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the dummy encompasses 10 days after the event day. i risk i is a dummy variable that equals one if a firm has a covid-19 risk value greater than zero in 2020q1. the high ( growth subsample includes only firms residing in counties where the growth between 1 and 100 reported cases took less than 20 days. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth (1) (2) (-2 .83) (-1.82 ) (-2.27 ) .00736*** -0.00610*** -0.00821*** -0.00681*** (-39.73 ) (-37 .76) (-21.15 ) (-20 (gc) and other firms (non-gc) during the pandemic. 13 the regression 13 government purchases directly affect the performance of government contractors that derive a large portion of their revenues from those purchases; in turn, government contractors' performance may also spill over onto other firms, for example, along the supply chains. we study only the direct effect. specification is is a dummy variable that flags government contractor firm i. 14 the parameters of interest are î² and î³ -the difference-in-differences estimate of the effect of being a government contractor during the pandemic. table 9 presents the regression results. columns 1 and 2 summarize findings in the full sample. we observe that none of the coefficient loadings on the interaction terms is statistically significant. this implies that following the first reported case of coronavirus in a county, government contractors' financial performance is not better compared to noncontractor firms. this result suggests that the current economic contraction and the one observed in the financial crisis are fundamentally different phenomena. indeed, while the stability of government purchases provided government contractors with a hedge during the [2008] [2009] financial crisis (goldman, 2019) , this effect is largely absent in the current pandemic. most large government contractors belong to the manufacturing industry (defined as belonging to fama-french industry codes 1 to 13). hence, it is possible, that they are particularly susceptible to negative labor supply shocks. as a result, despite the fact that gc's benefit from the stability of having government contracts during economic downturns, the covid-19 pandemic adversely affects these firms' productivity through the labor supply channel, as discussed in section 3.1. to test this hypothesis, we focus on the subsample of labor-intensive firms and reestimate the regressions including the gc dummy. results are presented in columns 3 and 4 of table 9 . within the labor-intensive subsample, estimated coefficients for the covid-19 0 ã� i gc and the post-covid-19 ã� i gc terms are all negative and statistically significant. in particular, the interaction between the post-covid-19 dummy and the gc dummy exhibits high statistical significance at the 1% level in columns 3 and 4. gc's have lower equity returns than non-gc's in labor-intensive industries in the aftermath of the first reported covid-19 case. hence, this evidence table 9 interaction regression results for firm-level returns on the shock and government contractors this table presents interaction regression results. the sample period is between january 1, 2020, and march 20, 2020. we merge our sample with the goldman (2019) data set, where the author identifies government contractors from compustat. these are firms deriving more than 10% of sales from the federal government. the regression equation is log(return)ijct = î±+î²covid-19 0ct +î³post where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients î², î³, î², and î³ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the covid-19 0 dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post-covid-19 dummy encompasses the 10 days after the event day. i gc i is a dummy variable that equals one if a firm is a designated government contractor. the labor-intensive subsample includes only firms in the fama-french 17 industries 1 to 13 (excluding utilities, retail, financial, and services). robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. labor intensive (1) (2) (-0 .82) (-1.78 ) (-1.94) post-covid-19 ã� i gc 0.000277 -0.0000736 -0.00223*** -0.00180*** (0.09) (-0 .03) (-2 .70) (-3 .11) -0.00737*** -0.00610*** -0.00805*** -0.00681*** (-39 .50) (-37 .48) (-16 .76) (-17 suggests that the strain on labor supply due to the pandemic appears to outweigh the benefits of stable demand for the gc firms. 15 next, we investigate whether heterogeneous exposure to monetary policy can generate the return reaction to news. to the extent that the pandemic causes the federal reserve to implement accommodative monetary policy for the foreseeable future, it is possible that some firms benefit from this (expected) monetary policy easing more than others. fo