key: cord-322585-5gio6ruj authors: lanari, marcello; chiereghin, angela; biserni, giovanni battista; rocca, alessandro; re, maria carla; lazzarotto, tiziana title: children and sars-cov-2 infection: innocent bystanders…until proven otherwise date: 2020-06-25 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.017 sha: doc_id: 322585 cord_uid: 5gio6ruj in this commentary, we focus our attention on what is known about sars-cov-2 infection in the pediatric population. we report literature and national data. the possible and different explanations for understanding why the infection seems to be more benign and less frequent in children are discussed. the possible role of children in the covid-19 viral disease pandemic is also commented. finally, our work suggests to search for future evidence and containment strategies to manage virus spread. children, along with other members of vulnerable populations, e.g. elderly and individuals with 29 pre-existing comorbidities, typically pay a high price in terms of incidence and severity of 30 respiratory tract illnesses. however, the current available data on severe acute respiratory syndrome 31 coronavirus 2 (sars-cov-2) show that, from the beginning of the outbreak until now, there is a 32 low attack rate in paediatrics worldwide. particularly, in madrid region (spain), individuals <18 33 years-old accounted for 0.8% of the laboratory-confirmed cases during the first 2 weeks of the finally, the lockdown imposed until recently by some governments, along with the growing fear of 103 going to hospitals, has led to a significant reduction in the circulation of the other respiratory pathogens and in the number of paediatric er visits. this all could have led to a lack of laboratory screening 158 and severity of coronavirus disease 2019 (covid-19) in children in coronavirus disease 2019 in children -united states european centre for disease prevention and control. paediatric inflammatory multisystem 163 syndrome and sars-cov-2 infection in children -15 angiotensin-converting enzyme in developing lung and 165 kidney a crucial role of angiotensin converting 167 enzyme 2 (ace2) in sars coronavirus-induced lung injury evolution of the immune system in humans from 169 infancy to old age prevalence of human coronaviruses in 171 adults with acute respiratory tract infections in beijing review article: gastrointestinal features in covid 19 and the 173 possibility of faecal transmission children are unlikely to be the main drivers of the covid-19 pandemic -a 175 systematic review school closure and 177 management practices during coronavirus outbreaks including covid-19: a rapid systematic 178 review a case series of children with 2019 novel 180 coronavirus infection: clinical and epidemiological features further studies are needed [10] . to date, child-to-adult transmission seems to be uncommon [3] and large evidence on child-to-child key: cord-275068-yr076sl6 authors: ayoub, fares; sato, toshiro; sakuraba, atsushi title: football and covid-19 risk: correlation is not causation date: 2020-09-03 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.08.034 sha: doc_id: 275068 cord_uid: yr076sl6 nan to the editor, 1 as the world battles the coronavirus disease 2019 (covid-19) pandemic, wading through the deluge of 2 published covid-19 research has become a challenge for clinicians. despite a strong push for the 3 practice of evidence-based medicine over the past decades, a search of covid-19 literature in recent 4 months paints a different picture. the use of hydroxychloroquine has been the 'poster child' for the 5 importance of conducting randomized controlled trials (rcts), initially showing promise in uncontrolled 6 studies [1] and even being recommended by governmental leaders, only to later show no benefit in 7 rcts. [2] similarly, many observational studies have reported on risk factors for covid-19. a recent 8 genome wide association analysis (gwas) has implicated that blood group a patients had a higher risk 9 of severe covid-19 compared to other blood types,[3] while other studies have found associations 10 between vitamin d levels/latitude, and bacille de calmette et guérin (bcg) vaccine and mortality to 11 covid-19. [4] based on the results of these studies, shall we tell our blood group a patients to stay home 12 or tell everyone to take vitamin d or get vaccinated with bcg? what are the mechanisms underlying 13 these relationships? although blood group antigens are known to play a role in infections, the results of 14 the recent gwas study may have been influenced by the control group comprised of blood donors and 15 the lack of adjustment for comorbidities. [5] low vitamin d levels have often been associated with a 16 higher risk of infections, but vitamin d supplementation has not been shown to prevent respiratory 17 1 impact journals during the pandemic, but it is important to remember that correlation does not equal 2 causation. to further demonstrate our point, we studied the correlation between the global ranking of 3 the fédération internationale de football association (fifa) and the ranking of covid-19 cases by 4 country (figure 1 hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, 8 randomised controlled trial genomewide association study of severe covid-19 with respiratory failure letter: low population mortality from covid-19 in 6 countries south of latitude 35 degrees north supports vitamin d as a factor determining severity-7 authors' reply worldometer covid-19 coronavirus pandemic effect of monthly high-dose vitamin d supplementation on acute respiratory infections in older adults: 12 a randomized controlled trial sars-cov-2 rates in bcg-vaccinated and unvaccinated young 14 key: cord-032016-2rzszcke authors: kumar, anil; paulose, roopa; sadasivan, shine; bajad, chandan; ramachandran, arya; nair, priya title: sarcoidosis, steroids and strongyloides -what’s the catch?() date: 2020-09-17 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.012 sha: doc_id: 32016 cord_uid: 2rzszcke nan a 55-year-old male, who is a known case of sarcoidosis, on steroids for the last 13 years, presented with weight loss for the last 2-3 months, loss of appetite, intermittent loose stools, abdominal discomfort, and frequent flatulence. colonoscopy showed aphthous ulcers in the rectum, descending, transverse, ascending colon as well as splenic, and hepatic flexures (see supplementary material, video s1). histopathologic examination of biopsy from the ulcerated mucosa showed dense infiltrate of eosinophils in the lamina propria, associated with patchy ulceration, and s. stercoralis in the mucosal crypts. stool examination showed plenty of rabditiform larvae of s. stercoralis . the patient was diagnosed with hyperinfection syndrome due to s. stercoralis. s. stercoralis is an intestinal pathogen seen both in immunocompetent as well as individuals with defects in cellmediated immunity [1] . three 2-day courses of ivermectin 200 µg/kg/day, every 15 days, resulted in a full symptomatic resolution. chronic gastrointestinal infection with s. stercoralis can have either no symptoms or mild nonspecific gastrointestinal, respiratory, and cutaneous symptoms. the increased larval burden can lead to complications such as ileus, gastrointestinal bleeding, intestinal obstruction, and even death. alteration of immune status results in increases larval load, leading to hyperinfection syndrome. there is exacerbation of gastrointestinal and pulmonary symptoms which is evidenced by demonstration of larvae in the stool and /or sputum [1] . moreover, the migration of larvae outside the gastrointestinal and respiratory tracts can be observed, causing extrapulmonary and extra-intestinal signs/symptoms. among the immunosuppressive drugs, glucocorticoids are the most specifically associated with transforming chronic strongyloidiasis to hyperinfection [1] . hyperinfection can result from high-dose steroids, low-dose steroids, and even locally injected steroids [1] . signs and symptoms usually begin as early as 20 days after the onset of steroid therapy, and as late as several years without an obvious additional immunocompromising condition supervening [1] . it is important for clinicians to rule out gastrointestinal infection with s. stercoralis in patients on long term steroids in order to prevent hyperinfection syndrome. in hyperinfection the diagnosis is easily made by stool examination, while in chronic uncomplicated infection, stool examination might result in a disappointing high proportion of false negatives. hence, screening of at-risk patients should include other specific tests for s. stercoralis (for instance stool culture, baermann test, pcr, and/or serology). with dexamethasone becoming standard of care for severe covid-19, the risk of strongyloidiasis reactivation should be considered. it would be worth investigating whether the recommended low-dose short-course really brings higher risk for hyperinfection syndrome due to s. stercoralis. ak: conceptualization, supervision, validation, investigation, writing -original draft. rp: resources, visualization, investigation, writing -review & editing. ss: resources, visualization, investigation, writing -review & editing. cb: investigation, writing -review & editing ar: investigation, writing -review & editing pn: visualization, investigation, writing -review & editing strongyloides stercoralis infection in the immunocompromised host legend: figure a. colonoscopy showing aphthous ulcers in colon colonic biopsy on histopathology (hematoxylin and eosin) showing eosinophilic colitis with stronglyloides stercoralis in the mucosal crypts stool examination showing rabditiform larvae of stronglyloides stercoralis (400x) video of colonoscopy showing biopsy of the ulcers. key: cord-269345-5tlyy8jp authors: minuz, pietro; mansueto, giancarlo; mazzaferri, fulvia; fava, cristiano; dalbeni, andrea; ambrosetti, maria chiara; sibani, marcella; tacconelli, evelina title: high rate of pulmonary thromboembolism in patients with sars-cov-2 pneumonia date: 2020-06-18 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.011 sha: doc_id: 269345 cord_uid: 5tlyy8jp nan recently, thromboembolic events have been reported in 20/81 patients with severe sars-cov-2 32 pneumonia admitted to intensive care units (icus). about 90% of patients showed an increased 33 coagulation activity, including high d-dimer concentrations, which demonstrated 85% sensitivity 34 and 89% specificity for identifying high-risk groups for thromboembolic events [1] . moreover during the study period, ten patients underwent a ctpa scan. pte was detected in six of them, all 52 displaying d-dimer values >10000 µg/l, and a persistent pao 2 /fio 2 ratio < 150. consistently with 53 the diagnosis of viral pneumonia, in all six patients, ctpa scan showed peripherally distributed 54 bilateral ground-glass opacities, reticular opacities, and areas of consolidation in the posterior-55 basal segments. multiple filling defects involving lobar or segmental and subsegmental branches of 56 pulmonary arteries with subsegmental vessels enlargement were also detected in all patients. 57 these features were bilateral in four patients. one patient showed both principal and segmental 58 pulmonary arteries involvement, with filling defects affecting corresponding pulmonary vein 59 four out of six patients were males and the median age was 75 (range, 55-86). none of the 61 patients had a previous history of thromboembolic events, four had a history of arterial 62 hypertension, and only one had a relevant risk factor for thromboembolic diseases (cancer). the 63 mean time between covid-19 symptom onset and hospital admission was 12 days (range, 9-16 64 days) while the mean time between hospital admission and the diagnosis of pte was 9,3 days 65 (range, 5-17 days). on the day pte was diagnosed, the pao 2 /fio 2 ratio was < 150 in all cases; 66 the leukocyte count ranged between 5970-31480/µl. five patients showed reduced c-reactive 67 protein values compared to the assessment at the hospital admission. il-6 levels were increased 68 in three patients (table 1) . 69 the prevalence of pte was substantially higher than the ones recorded in the previous three years 70 in the same 60-bed unit, admitting mainly patients with infectious diseases: 1.6% (13/ 801) in 2019, 71 1.4% (11/799) in 2018, and 1.8% (16/869) in 2017. although the prevalence in our small cohort of 72 non-icu patients is less than the one recently reported in icu patients [1] , it seems to confirm the 73 increased risk of pte in covid-19 patients. 74 in our case series, the involvement of segmental and subsegmental branches of the pulmonary 75 arteries along with the peculiar multiple and bilateral filling defects distribution, suggest a non-76 embolic origin of the pulmonary arteries thrombosis [4] . furthermore, the contiguity of most filling 77 defects to the parenchymal opacities suggests a link between the sars-cov-2-induced lung 78 inflammation and vascular occlusion, possibly explaining the severe respiratory impairment 79 detected in the patients. 80 compared to the rates previously reported in patients with pneumonia or other severe infections, a 81 higher prevalence of pte in patients with sars-cov-2 pneumonia might be inferred from this 82 small series. the absence of major risk factors for thromboembolic events in 5 out of 6 patients 83 seems to further confirm the role of bilateral sars-cov-2 pneumonia as a risk factor for pte. 84 considering that an undiagnosed thromboembolic process might worsen patients´ outcome, we 86 would suggest including a ctpa scan in the diagnostic assessment of patients with sars-cov-2 87 pneumonia, high d-dimer, and refractory or rapidly deteriorating hypoxemic respiratory failure. prevalence of venous thromboembolism in patients with 119 severe novel coronavirus pneumonia difference of coagulation features between severe pneumonia 121 induced by sars-cov2 and non-sars-cov2 abnormal coagulation parameters are associated with poor 123 prognosis in patients with novel coronavirus pneumonia pulmonary thrombosis: a clinical 126 attention should be paid to venous 129 thromboembolism prophylaxis in the management of covid-19 key: cord-256853-2s31fn04 authors: jin, cheng cheng; zhu, li; gao, chun; zhang, sheng title: correlation between viral rna shedding and serum antibodies in covid-19 patients date: 2020-05-23 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.05.022 sha: doc_id: 256853 cord_uid: 2s31fn04 nan shedding. [2] based on currently available information, the relationship between the 6 dynamic of serum antibodies and viral replication is unclear. in this study, we 7 investigated the correlation between serum antibodies and duration of viral rna 8 shedding. 9 we retrospectively enrolled 89 hospitalized patients (admission date from jan 22 nd 10 to feb 13 rd , 2020) with laboratory confirmed sars-cov-2 infection in tongji hospital 11 of huazhong university of science and technology in wuhan, china. we enrolled 12 patients who had blood tests intended for antibody detection during hospitalization. all 13 patients had mild to moderate illness, and did not required intubation or icu admission. 14 throat and/or nasal swabs collected upon admission and during hospitalizion were 15 analyzed by sars-cov-2 real-time reverse transcription polymerase chain reaction 16 (rt-pcr) according to the manufacturer's protocol (shanghai huirui biotechnology 17 co., ltd). specific antibodies igm and igg to sar-cov-2 were analyzed by 18 chemiluminescent immunoassay (clia) according to the manufacturer's protocol 19 (shenzhen yahuilong biotechnology co., ltd). the kits had two antigens of sars-20 cov-2 coated on the magnetic beads (nucleocapsid protein or n protein, spike protein 21 or s proteins). iflash3000 fully automatic chemiluminescence immunoassay analyzer 22 (shenzhen yahuilong, biotechnology co., ltd) was applied to analyze the samples. 23 serum igm and igg titer (au/ml) was calculated by the immunoassay analyzer. 13.2±4.0 au/ml) declined almost to the reference level (10 au/ml). in prolonged 39 group, serum igg was slightly higher than that in the other group through week 4 to 40 week 8. however, the difference between two groups was not significant (p>0.05). 41 profile of specific antibodies to sars-cov-2: 98 key: cord-256402-b5fel2d3 authors: gevers, s.; kwa, m.s.g.; wijnans, e.; van nieuwkoop, c. title: safety considerations of chloroquine and hydroxychloroquine in treatment of covid-19 date: 2020-05-16 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.05.006 sha: doc_id: 256402 cord_uid: b5fel2d3 chloroquine and hydroxychloroquine are both used to treat covid-19. safety data in this specific population is largely unknown. in particular, cardiologic, gastro-intestinal and neuropsychiatric side-effects of (hydroxychloroquine) needs special attention in covid-19 patients. to the editor: based on a demonstrated in vitro effect on sars-cov-2 and its known safety profile, both chloroquine and hydroxychloroquine (cq/hcq) are currently being used off label to treat covid-19 [1] . however, though safety of cq/hcq is well established in malaria or auto-immune disease, covid-19 patients could be more vulnerable to side-effects because of their advanced age, co-morbidities such as diabetes, obesity and cardiovascular disease, and subsequent co-medication [2] . both agents are metabolized via the liver and the kidney. critically ill patients may have an altered metabolism due to changes in the hepatic and renal function, which could increase the risk of adverse reactions. additionally, there may be interactions with co-medication normally not taken together with cq/hcq. both drugs have long half-lives (approximately 1-2 months) and distribute poorly in fat tissue [3] . therefore, long-term monitoring for adverse reactions is recommended. importantly, cq/hcq have narrow therapeutic ranges and toxic effects are related closely to the ingested dose. an one-time dose of 20 mg/kg cq has been described to be toxic and doses of 30 mg/kg cq have resulted in case fatalities [4] . apart from the general safety profile of cq/hcq, there are adverse reactions that may interfere with the clinical picture of covid-19 due to the similarity with symptoms of illness. in particular, this holds for cardiovascular, neuropsychiatric and gastrointestinal adverse drug reactions. table 1 illustrates the five most commonly reported suspected adverse drug reactions in these system organ classes, as reported to the global pharmacovigilance database of the who (vigiaccesstm) [5] . this global database provides insight in spontaneous postmarketing case safety reports on suspected adverse reactions. the data should be interpreted with caution as the number of reports may be influenced by many different factors, including patients' baseline characteristics, extent of exposure and nature of adverse reactions. reported cardiac side effects of cq/hcq include conduction disturbances (bundle-branch block, incomplete or complete atrioventricular block, qt-prolongation and subsequent torsade de pointes) and cardiomyopathy (hypertrophy and congestive heart failure). due to their systemic infection and comorbidities, covid-19 patients appear to have an higher risk of cardiac arrhythmia, qt-prolongation and myocardial damage a priori [6] . this could result in cardiotoxicity of cq/hcq being of particular importance, especially when given in combination with other qt prolonging agents like azithromycin [7] . neurologic and psychiatric side effects have also been reported following cq/hcq treatment. neurologic side effects include muscular weakness, diplopia, dyskinesia, seizures, myasthenic syndrome, and with long-term use neuromyopathy. psychiatric side effects include sleeplessness, agitation, psychosis, depression, anxiety, aggressiveness and confusion; with psychiatric side effects starting within a few days after beginning of treatment and improving after cessation of treatment. covid-19 patients suffer from dyspnea, which may in turn lead to anxiety and sleeplessness, symptoms that may be aggravated by potential psychiatric side effects of cq/hcq. finally, gastrointestinal symptoms (nausea and diarrhoea) have been reported and are the presenting complaint in some. in 393 patients admitted to two hospitals in new york, diarrhoea and nausea or vomiting were reported in 23.7% and 19.1% of patients, respectively. to these patients, treatment with drugs having potential gastrointestinal side effects could be problematic. [2] in conclusion, it is likely that some of the commonly reported adverse effects of cq/hcq will hamper successful treatment of patients suffering from covid-19. thus, until adequately powered randomized controlled trials (rcts) provide more information on the efficacy and the safety of cq/hcq use in treatment of patients with covid-19, it is very important that the potential benefits of these agents will be weighed against the potential risks. furthermore, clinical trials should also evaluate the long-term (e.g. 3-6 months post-therapy) (side)-effects of covid-19 and the use of cq/hcq, such as cardiomyopathy, muscle weakness, anxiety, sleeplessness and gastro-intestinal disorders. preferably, until data from rcts will become available, the off-label use of cq/hcq should only be reserved for covid-19 patients treated in context of clinical trials in order to improve our knowledge on safety and efficacy. funding: no external funding was received. the text was written by sg, ew and cn. mk collected and added the pharmacovigilance data. all authors reviewed and revised the manuscript. remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro clinical characteristics of covid-19 in current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review antimalarial drug toxicity: a review all data contained in vigiaccesstm is sourced from vigibase®, the who's global database for adrs, maintained by the uppsala monitoring centre association of coronavirus disease 2019 (covid-19) with myocardial injury and mortality risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease key: cord-257696-ybu772zw authors: bartoletti, michele; marconi, lorenzo; scudeller, luigia; pancaldi, livia; tedeschi, sara; giannella, maddalena; rinaldi, matteo; bussini, linda; valentini, ilaria; ferravante, anna filomena; potalivo, antonella; marchionni, elisa; fornaro, giacomo; pascale, renato; pasquini, zeno; puoti, massimo; merli, marco; barchiesi, francesco; volpato, francesca; rubin, arianna; saracino, annalisa; tonetti, tommaso; gaibani, paolo; ranieri, vito marco; viale, pierluigi; cristini, francesco title: efficacy of corticosteroid treatment for hospitalized patients with severe covid-19: a multicenter study date: 2020-09-22 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.014 sha: doc_id: 257696 cord_uid: ybu772zw objectives: to assess the efficacy of corticosteroids in patients with coronavirus disease 2019 (covid-19) methods: multicenter observational study from february 22 through june 30, 2020. we included consecutive adult patients with severe covid-19 defined as respiratory rate ≥30 breath per minute, oxygen saturation ≤93% on ambient air or arterial partial pressure of oxygen to fraction of inspired oxygen ≤300 mmhg. we excluded patients treated with other immunomodulant drugs, receiving low dose of corticosteroids and those receiving corticosteroids after 72h from admission. the primary endpoint was 30-day mortality form hospital admission. the main exposure variable was corticosteroid therapy at dosage of ≥0.5 mg/kg of prednisone equivalents. it was introduced as binomial covariate in a logistic regression model for primary endpoint and inverse probability of treatment weighting using the propensity score. results: of 1717 patients with covid-19 evaluated, 513 patients were included in the study; of these 170 (33%) were treated with corticosteroids. during the hospitalization 166 (34%) patients reached the primary outcome [60/170 (35%) in the corticosteroid group and 106/343 (31%) in the non-corticosteroid group]. at multivariable analysis corticosteroid treatment was not associated with lower 30-day mortality rate [aor 0.59 (0.20-1.74), p=0.33]. after inverse probability of treatment weighting, corticosteroids were not associated to lower 30-day mortality [average treatment effect 0.05 (95% -0.02 to 0.09), p=0.12]. however, subgroup analysis revealed that in patients with po(2)/fio(2) < 200 mmhg at admission [135 patients, 52 (38%) treated with corticosteroids] corticosteroid treatment was associated to a lower risk of 30-day mortality [23/52 (44%) vs 45/83 (54%), aor 0.20 (95%ci 0.04 to 0.90), p=0.036]. conclusion: our study shows that the effect of corticosteroid treatment on mortality might be limited to critically ill covid-19 patients. severe acute respiratory syndrome coronavirus 2 (sars-cov-2)-associated coronavirus 73 disease 2019 (covid-19) is characterized by significant morbidity and mortality. 74 the clinical spectrum of covid-19 is broad with the majority of infected individuals 75 experiencing only a mild or subclinical illness, especially in the early phase of disease [1] . 76 however, approximately 14 to 30% of hospitalized patients diagnosed with covid-19 77 develop a severe respiratory failure requiring intensive care [2] [3] [4] [5] . 78 it has been hypothesized that the main cause of illness progression is a cytokine storm east respiratory syndrome (mers) infections failed to find a benefit of corticosteroids [8] . 85 among covid-19 patients, two randomized trials showed conflicting results [9, 10]. 86 the exposure variable was corticosteroid treatment, defined as treatment with any 119 corticosteroid drug at dosage of ≥0.5 mg/kg of prednisone equivalents initiated within 72h 120 from hospital admission; it was treated as a binomial variable in models. 121 the primary endpoint was 30-day mortality from hospital admission. the effect of steroid treatment on 30-day mortality in two ways. first, univariable and 170 multivariable logistic models were fitted. at multivariable models, clinically relevant 171 variables, and those with p<0.10 at univariable analysis, were included, with no further 172 selection. to take time-dependency of steroid treatment in the analysis, we expanded our 173 dataset with one observation per each day since symptom onset; for each day, a binary 174 indicator for steroid treatment in that day for that patient was created. finally, time since 175 symptom onset was subsequently included in models as cubic splines interacting with the 176 steroid treatment indicator; to take into account the multiple records per patient, robust 177 variance was estimated clustering by patient. as a secondary analysis, logistic models with augmented inverse-probability-weighting 179 (ipw) on propensity score for receiving steroid were also fitted. risk factors for 30-day 180 mortality, besides corticosteroid treatment, were age, diabetes, hypertension, chronic 181 kidney disease, respiratory rate, sofa score, creatinine, and crp. variables contributing 182 to the propensity score of receiving steroid in our model were study site, calendar month 183 into the pandemic, age, crp, days since symptoms onset (as cubic splines). covariate (table 2 and web-only supplementary table s1) , several factors were 214 associated to 30-day mortality. at multivariable analysis (table 2) in this study we were not able to find a lower mortality rate among hospitalized patients characteristics of and important lessons from the coronavirus 333 disease 2019 (covid-19) outbreak in china characteristics, comorbidities, and outcomes among 5700 patients hospitalized with critical care utilization for the covid-19 outbreak in early experience and forecast during an emergency response clinical features of patients infected with 2019 novel 346 coronavirus in wuhan development and validation of 351 a prediction model for severe respiratory failure in hospitalized patients with sars-cov-2 infection: 352 a multicenter cohort study (predi-co study), clinical microbiology and infection : the official tocilizumab in patients with severe covid-19: a retrospective 362 cohort study impact of corticosteroid therapy 364 on outcomes of persons with sars-cov-2, sars-cov, or mers-cov infection: a systematic 365 review and meta-analysis dexamethasone in hospitalized patients with covid-19 -preliminary methylprednisolone as adjunctive therapy for patients hospitalized with covid-19 (metcovid): a placebo-controlled trial key: cord-283411-40ojqv1y authors: ben-shmuel, amir; brosh-nissimov, tal; glinert, itai; bar-david, elad; sittner, assa; poni, reut; cohen, regev; achdout, hagit; tamir, hadas; yahalom-ronen, yfat; politi, boaz; melamed, sharon; vitner, einat; cherry, lilach; israeli, ofir; beth-din, adi; paran, nir; israely, tomer; yitzhaki, shmuel; haim levy; weiss, shay title: detection and infectivity potential of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) environmental contamination in isolation units and quarantine facilities date: 2020-09-10 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.004 sha: doc_id: 283411 cord_uid: 40ojqv1y objectives: environmental surfaces have been suggested as likely contributors to the transmission of covid-19. this study assessed the infectivity of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) contamination on surfaces and objects in hospital isolation units and a quarantine hotel. methods: sars-cov-2 virus stability and infectivity on non-porous surfaces was tested under controlled laboratory conditions. surfaces and air sampling was conducted at two covid-19 isolation units and in a quarantine hotel. viral rna detected by rt-pcr and infectivity was assessed by vero e6 cpe test. results: in laboratory-controlled conditions, sars-cov-2 gradually lost its infectivity completely at day 4 at ambient temperature and the decay rate of viral viability on surfaces directly correlated with increase in temperature. viral rna detected in 29/55 (52.7%) and 16/42 (38%) surface samples from the surrounding of symptomatic covid-19 patients in isolation units of two hospitals and in a quarantine hotel for asymptomatic and very mild covid-19 patients. none of the surface and air samples from all three sites (0/97) were found to contain infectious titers sars-cov-2 in tissue culture assay. conclusions: despite prolonged viability of sars-cov-2 in laboratory-controlled conditions, uncultivable viral contamination on inanimate surfaces might suggest low feasibility for indirect fomite transmission. sampling was performed in covid-19 isolation units in two hospitals and one quarantine 78 facility. hospital-a isolation unit is a 28-bed ward, comprised of secluded rooms occupied by 1unit is a 40-bed ward, comprised of secluded rooms occupied by 1-6 patients each. staff used 84 coveralls, masks, shoe covers and face shields. sop was the use of two pairs of gloves at a time. the outer pair was not consistently changed during patient care. at the time of sampling, the unit 86 contained patients with mild-to-severe disease, including seven ventilated patients. in both 87 hospitals, patients were free to ambulate in the unit if they were fit to walk. in addition, routine 88 cleaning and decontamination was done twice a day using 1000ppm bleach solution in both 89 wards. the quarantine facility was a hotel repurposed for the isolation of patients with 90 asymptomatic to mild disease until becoming negative for viral nasopharyngeal pcr. sampling 91 j o u r n a l p r e -p r o o f was conducted at main public areas and in hotel rooms. patients stayed in private rooms either 92 alone or as a family but were free to move around the hotel and socialize in public spaces. routine cleaning and decontamination was done once daily at best only on communal areas. was tested by seeding quadruplets of 200 µl on vero e6 cells for cpe assay as described 115 above. cpe assay limit of detection has been determined to be 10pfu/ml. finally, we mapped the contamination in a quarantine hotel for asymptomatic (patients found to 155 be infected during contact testing but had no symptoms) and very mild covid-19 patients. sampling was performed at the hotel communal areas and at three hotel rooms that had been 157 j o u r n a l p r e -p r o o f recently (2-4 days) occupied by newly diagnosed patients. we found that 16/42 (38%) samples 158 were positive for viral rna (table 2) . at the communal areas we found viral rna 159 contamination on a water cooler, chairs, most elevator buttons, a kettle and a used cup. in this study, viral rna contamination was found in 46% of the surface and air samples. our study has some limitations. there was a delay between onset of symptoms and the actual 217 sampling in patients' rooms. therefore, at the time of sampling these patients might not have 218 shed viable virus as suggested by studies that showed culturable viruses in respiratory samples 219 up to 8 th -9 th day of illness (18, 24) . for that reason, we have noted new patients with recent 220 disease onset in hospital a and the quarantine hotel and sampled around them. the cpe assay 221 has a 10pfu/ml limit of detection that is comparable to ct value of 34, therefore a very low level j o u r n a l p r e -p r o o f who. coronavirus disease effects of air temperature and relative 255 humidity on coronavirus survival on surfaces stability of sars-cov-2 in 258 different environmental conditions transmission of sars and 260 mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface 261 contamination surface environmental, and 263 personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 264 (sars-cov-2) from a symptomatic patient environmental contamination of sars-cov-2 266 in healthcare premises. the journal of infection detection of severe acute 268 respiratory syndrome coronavirus 2 rna on surfaces in quarantine rooms. emerging infectious 269 diseases protective equipment tests for sars-cov-2 in the isolation room of an infant with infection. annals of 272 internal medicine a single dose of 274 recombinant vsv-δg-spike vaccine provides protection against sars-cov-2 challenge stability and infectivity of 277 coronaviruses in inanimate environments lack of sars-cov-2 rna 279 environmental contamination in a tertiary referral hospital for infectious diseases in northern italy. the 280 journal of hospital infection sars-cov-2 rna detection of hospital isolation 282 wards hygiene monitoring during the coronavirus disease 2019 outbreak in a chinese hospital. 283 international journal of infectious diseases : ijid : official publication of the international society for 284 infectious diseases detection of airborne severe 286 acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak 287 units. the journal of infectious diseases clinical infectious diseases : an official 290 publication of the infectious diseases society of america transmission routes of respiratory 292 viruses among humans. current opinion in virology severe acute respiratory 294 syndrome coronavirus 2 rna contamination of inanimate surfaces and virus viability in a health care 295 emergency unit. clinical microbiology and infection : the official publication of the european society of 296 clinical microbiology and infectious diseases virological 298 assessment of hospitalized patients with covid-2019 sars-cov-2 in 300 environmental samples of quarantined households simulated 304 sunlight rapidly inactivates sars-cov-2 on surfaces. the journal of infectious diseases antiviral activity and 307 increased host defense against influenza infection elicited by the human cathelicidin ll-37 the power of saliva: antimicrobial and beyond. plos 310 pathog clinical and virologic 312 characteristics of the first 12 patients with coronavirus disease 2019 (covid-19) in the united states. 313 nature medicine key: cord-327253-gge6wzly authors: villa, simone; jaramillo, ernesto; mangioni, davide; bandera, alessandra; gori, andrea; raviglione, mario carlo title: stigma at the time of the covid-19 pandemic date: 2020-08-07 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.08.001 sha: doc_id: 327253 cord_uid: gge6wzly nan respiratory infectious disease outbreaks marked by significant morbidity and mortality tend to produce serious distress in the general population. physical distancing is necessary to reduce the chances of transmission of the pathogen. but this practice may engender stigma and discrimination, which can have the counterproductive effect of hindering disease control. people start to hide their symptoms, avoid seeking medical attention and testing until they are seriously ill, and do not collaborate in contact investigation efforts. epidemic outbreaks have historically been accompanied by stigma, discrimination, and xenophobia. tuberculosis, hiv, and leprosy are well known stigmatised infectious diseases. more recently, survivors of the 2013-16 west africa ebola outbreak have faced exclusion and unemployment once they returned to their communities. 1 beginning in late january 2020, when the covid-19 epidemic was still largely limited to china, verbal and physical attacks against chinese or people of asian descent have been documented in many countries. 2 in italy, for example, numerous racial and violent actions have taken place, including physical violence. in the vicenza province a young asian man was beaten and verbally assaulted and a young asian woman was insulted and accused of spreading covid-19; 3 in rome some private stores began to exclude clients of asian origin barring "all people coming from china" from entering. 2 similar incidents were reported in countries like france, where there were cases of people refusing to be served by asian persons in shops and restaurants, and the united states of america, where a single week in march saw around 650 racist acts against asian americans. 4 uses of language by some media, newspapers and political leaders sometimes contribute to fuel stigma. for instance, in january 2020 numerous italian newspapers used the terms "chinese virus" and "chinese syndrome" as if a nationality could be attributed to a virus or a disease. 5 likewise, in france, provocative and imprudent headlines such as "the alert jaune", namely the "yellow alert", appeared in le courrier picard. the president of the united states of america has frequently described covid-19 as the "chinese virus". in italy, some politicians accused chinese people of poor hygiene and unhealthy cultural practices, including that of eating "live mice". 6 in rome, a school principal asked all chinese students to exhibit a formal medical certificate declaring that they were disease-free, if they were to be allowed to attend classes. 2 some regional governors proposed to exclude children of chinese descent from class. counteracting this j o u r n a l p r e -p r o o f language with gestures such as the condemnation of race-based discriminatory behavior by the italian prime minister or the visit of the italian president to a primary school in rome where half of the children are of chinese origin might be insufficient to mitigate the stigma and fear already created. in france, initiatives such as the hashtag #jenesuispasunvirus (i am not a virus), spread on twitter after provocative headlines in french newspapers. once the pandemic reached italian territory, stigma was rapidly redirected towards ethnic italians. 7 the blame went immediately towards people from the north of italy, the area initially affected by the covid-19 epidemic, with (fortunately isolated) threats from people from southern italy of not renting houses to those from the north. stigmatizing a population can also serve political needs. some italian political parties, for example, managed to shift the blame to germany, stating that the italian outbreak originated in that country. 8 when covid-19 became a pandemic affecting more than one hundred countries, stigma and discrimination changed their pattern once again. it is well documented how healthcare workers and ambulance crews in the most affected areas in some latin-american, african, and european countries became the target of stigma and discrimination. 9 the general public started to see them as the "anointers" -those individual perceived to voluntarily favour the spread of plague in the community during the middle ages -and, as a result, deplorable actions have been documented. for instance, in the city of pisa, italy, a medical doctor going back home found a notice in which the neighbours asked her to be careful not to touch anything in the common area of the building where she lives. covid-19 survivors also have had to cope with stigma, especially from neighbours. given the shortage in testing kits and the overwhelming of laboratories, people who survive could not always be retested as proof of final cure. this led to avoidance and social isolation due to the fear of becoming infected. 10 in contrary, uninfected covid-19 people may be facing discrimination when applying for jobs in some countries that may implement covid-19 passport strategies, despite recommendations of the world health organization against such a practice. 11 most countries are struggling to implement an appropriate risk communication strategy to prevent and mitigate covid-19-related stigma. the role played by stigma and discrimination in favouring the spread of infection has been repeatedly highlighted. 12, 13 stigma, for instance, can lead ill people to hide their symptoms in the attempt to avoid marginalization. this reactive behaviour facilitates spreading of infectious pathogens especially among those with mild symptoms who avoid seeking medical attention and act j o u r n a l p r e -p r o o f as usual not to raise suspicion on their condition. besides easing transmission, this behaviour can be conducive to deterioration of clinical conditions and may have psychological consequences. on the other hand, patients with covid-19 diagnosis frequently suffer from anxiety and depression, mainly as a consequence of hospitalization or home quarantine, or due to the sense of guilt towards family members or acquaintances. the world cannot bear a parallel pandemic of stigma, which only serves to boost the spread of infectious diseases and worsen people's health conditions and social behaviours. noteworthy, individuals with covid-19 may develop poor health-seeking behaviours (e.g. avoiding testing) because, by anticipating and fearing stigma, they may perceive the risk of losing their job and being marginalised in the society. this stress, together with the one caused by hiding symptoms, may further exacerbate conditions linked to biological stress response (i.e., elevated cortisol) leading to immune depression and delay in timely and adequate treatment. 12 governments, backed by civil societies, have the responsibility to act urgently and make a definite commitment to fight any form of stigmatization, discrimination and xenophobia fuelled by infectious outbreaks. 13 we need to identify and isolate those who exploit the human tragedies of infectious epidemics for their political aims. interventions to mitigate social stigma should embrace risk communication strategies to fill the knowledge gap in the general population with additional attention to specific, vulnerable segments such as migrants and ethnic minorities, and to prevent the "fake news" from spreading. for instance, medical and scientific societies should encourage health care and public health professionals to develop ad hoc materials to educate patients and the general public. in doing so, public health authorities should seek advice from communication and social media experts especially when developing key messages (e.g., on mask use) and technical guidelines as these can be potentially misunderstood by media and the general public. technical information should also be paired with comprehensible and clear messages for non-technical recipients. ebola virus disease-related stigma among survivors declined in liberia over an 18-month, post-outbreak period: an observational cohort study outbreaks of xenophobia in west as coronavirus spread. the guardian as if we were the disease': coronavirus brings prejudice for italy's chinese workers. the guardian asian americans report over 650 racist acts over last week, new data says politician apologizes for saying coronavirus caused by chinese people eating 'live mice' italiani untori you could lick the benches': life for the first wave of u immunity passports" in the context of covid-19. geneva: world health organization 2020 infectious disease stigmas: maladaptive in modern society social stigma associated with covid-19 world health organization. effective media communication during public health emergencies -a who handbook. geneva: world health organization key: cord-304710-gjb6zo81 authors: khan, s.; nabi, g.; han, g.; siddique, r.; lian, s.; shi, h.; bashir, n.; ali, a.; shereen, m. adnan title: novel coronavirus: how things are in wuhan date: 2020-02-11 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.02.005 sha: doc_id: 304710 cord_uid: gjb6zo81 nan wuhan, a metropolis of 11 million people and home to thousands of foreign students, scientists, teachers and businessmen, is currently facing one of the deadliest outbreaks of a novel coronavirus (2019n-cov). the newly emerged coronavirus, 2019n-cov, was detected in december 2019 and the first fatality was reported at the start of 2020 [1] . during first month of the outbreak, there were 16 500 confirmed cases, 360 fatalities and over 20 000 suspected cases [2] . the government declared an emergency and wuhan and several cities nearby are in lockdown to prevent the rapid spread of the virus. currently, healthcare workers are in a critical stage. there is a great risk of medical and clinical staff (and workers) becoming infected with 2019n-cov because of their direct interaction with infected and suspected individuals. a total of 15 medical staff have been infected, and one doctor has died from 2019n-cov infection in wuhan hospital [3] . at hubei general hospital (one of the most famous hospitals in wuhan, also known as renmin hospital), a large number of clinical workers have been infected and admitted to the hospital; however, some of them have asked to be isolated at home due to the scarcity of sickbeds and clinical supplies. every day the dramatic increase in the number of infected individuals is causing a huge burden on the medical staff. there is a shortage of doctors and nurses, who are compelled to work longer without taking enough rest. working for long hours, disturbed daily routines including eating and sleeping schedules and fear of being infected are key factors that increase the risks of stress and anxiety for doctors and nurses, and may lead to their working less efficiently in terms of providing better treatment and care to patients. under these pressures, some medical staff have experienced an emotional breakdown at the frontline [4] . the situation worsened for medical staff after the death of dr liang wudong, who contracted 2019n-cov. the increasing number of suspected individuals every day and the shortage of laboratory staff could increase the workload and may delay routine clinical tests for infected or suspected individuals. furthermore, the shortage of protective coverings further increases the chances of getting the infection [5] . staff have been asked to use substandard masks, putting them at greater risk [4] . the healthcare authorities have stepped up efforts to overcome the problems and have sent thousands of medical personnel including army medical staff to wuhan. to cope with further shortages, the designated hospitals have transferred medical and clinical testing staff to frontline departments from other departments such as oncology, orthopaedics and medicines. in addition, healthcare workers from several undesignated healthcare units have been transferred to designated hospitals for coronavirus-infected patients. in order to increase the space and availability of sickbeds, regular hospital patients (not infected with 2019n-cov) are being moved out of hospitals, and admissions for new regular patients are being delayed. several hospitals are setting up online medical consultations for these patients to discuss their conditions and seek help. furthermore, some companies are working to make diagnostic kits available on a large scale, but it may take some time. however, the increasing number of patients every day and the expected peak in the coming days [6] may cause a further shortage of medical staff and health and logistic issues for the frontline healthcare provider. specific areas have been set aside inside designated hospitals to provide services according to the severity of the disease. suspected and confirmed cases are given diagnostic and treatment facilities in an isolated and protected environment; however, individuals in a critical condition are given treatment on a priority basis. in some cases, allopathic medicines are advised in combination with chinese medicine such as huoxiangzhengqi (for gastrointestinal problems), jinhua qinggan, lianhua qingjia, shufengjiedu and fangfengtongsheng (for fatigue and fever). psychological counselling is now available to handle the anxiety and fear among patients. confirmed patients have been given access to the internet and provided with healthy food; communication with relatives is through mobile video calls. online assistance using a video chat between doctor and patient (suspected or confirmed) is also used in some hospitals. some doctors and nurses prefer to communicate via mobile phone rather than direct contacting with the admitted patient. patients are discharged and sent back home in protected and disinfected vehicles after they have recovered from the fever, respiratory symptoms are improved and the nucleic acid test is negative. despite these services, new suspected and/or confirmed individuals are facing problems. the scarcity of sickbeds is causing confirmed patients to wait for a long time before they are admitted to a hospital, while the availability of sickbeds depends on recovery of already admitted patients. however, failure to provide admitted patients with protection measures such as goggles and suits, the shortage of medicines and the lack of isolated rooms are negatively affecting the recovery of patients. in some places, rapid measurement kits are not available. a pharyngeal swab-based test needs to be repeated for confirmation, which delays the process of admitting infected patients. this may increase the severity of symptoms and the risk of fatality. some of the infected individuals were found to have mild symptoms and rather than admitting them they were offered therapy and were advised to stay in isolated rooms. to cope with the increasing number of infected people, two new hospitals are nearing completion and will be able to house about 2300 beds [7] . however, the daily the numbers of confirmed and suspected cases are increasing in the thousands, thus raising concern about future treatment and management strategies. scientists in china are working efficiently, as observed by the early detection of the virus, sequencing of its first genome, designing of rapid detection kits, isolation of the virus in the laboratory and developing a vaccine [1, 8, 9] . a group of researchers in the wuhan institute of virology is studying the genome complexity, which may lead to uncovering unique structural features and drug target sites. moreover, research groups from the wuhan institute of virology and academy of military medical sciences have identified some broad-spectrum antiviral drugs that may have potential inhibitory effects against 2019-ncov. researchers and scientists at the wuhan institute of virology, wuhan university, huazhong university of science and technology and several other laboratories in wuhan and across the country are working to find ways of prevention and treatment in order to prevent further spread. broad range combinational therapies including lopinavireritonavir and interferon antiviral peptides are being evaluated for use against 2019n-cov. overall the current measures to control the 2019n-cov are being implemented with care and strictness. the entrances of residential communities, dormitories and public places are restricted and residents entering are monitored for temperature and related symptoms. people from wuhan are registered and kept under medical observation in some cities in china; meanwhile, their suspected contacts may be tracked to isolate them for monitoring purposes. farmers are being directed to maintain hygiene, and seafood markets are being monitored in major cities. the authors declare that there is no conflict of interest. no external funding was received for this article. the continuing 2019-ncov epidemic threat of novel coronaviruses to global health e the latest 2019 novel coronavirus outbreak in wuhan, china coronavirus claims first life outside china as wuhan enforces quarantine for all suspected patients china coronavirus: wuhan medical staff being infected at much faster pace than reported as national death toll hits 26 clinical features of patients infected with 2019 novel coronavirus in wuhan, china wuhan calls for more material help to deal with virus. china: daily dramatic hong kong data predicts coronavirus outbreak infecting 150,000 every day china building two hospitals in just a few days to tackle virus coronaviruses: genome structure, replication, and pathogenesis health workers in wuhan under growing risk as medical supplies run low the authors acknowledge the postdoctoral grant from the second affiliated hospital of zhengzhou university for s. khan. key: cord-297470-lx3xwg92 authors: pan, yunbao; li, xinran; yang, gui; fan, junli; tang, yueting; hong, xiaoyue; guo, shuang; li, jin; yao, dongai; cheng, zhenshun; yuan, yufeng; li, yirong; wang, xinghuan title: seroprevalence of sars-cov-2 immunoglobulin antibodies in wuhan, china: part of the city-wide massive testing campaign date: 2020-10-07 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.044 sha: doc_id: 297470 cord_uid: lx3xwg92 objectives: the outbreak of 2019 coronavirus disease (covid-19) pandemic in wuhan, china, has subsided after a hard hit by the disease and subsequent city lockdown. information on the number of people involved in wuhan is still inadequate. this study aimed to describe the screening results of 61,437 community members in wuchang district, wuhan. methods: in mid-may 2020, wuhan launched a population-scale city-wide sars-cov-2 testing campaign, which aimed to perform nucleic acid and viral antibody testing for citizens in wuhan. here we show the screening results of cluster sampled 61,437 residents in wuchang district, wuhan, china. results: a total of 1470 (2.39%, 95% ci: 2.27-2.52) individuals were detected positive for at least one antiviral antibody. among the positive individuals, 324 (0.53%, 95% ci: 0.47-0.59) and 1200 (1.95%, 95% ci: 1.85-2.07) were positive for immunoglobulin igm and igg, respectively, and 54 (0.08%, 95% ci: 0.07-0.12) were positive for both antibodies. the positive rate of female carriers of antibodies were higher than those of male counterparts (male-to-female ratio of 0.75), especially in elderly citizens (ratio of 0.18 in 90+ age subgroup), indicating a sexual discrepancy in seroprevalence. in addition, viral nucleic acid detection using real-time pcr had showed 8 (0.013%, 95% ci: 0.006-0.026) asymptomatic virus carriers. conclusions: the seroprevalence of sars-cov-2 in wuhan was low. most of wuhan residents are still susceptible to this virus. precautions, such as wearing mask, frequent hand hygiene, and proper social distance, are necessary before an effective vaccine or antiviral treatments are available. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) related coronavirus disease (covid-19) is a respiratory transmissible disease that may cause critical illness to death (1, 2) . various control measures against sars-cov-2 were implemented in wuhan, the first city hit by the coronavirus. after months of endeavor, the viral transmission was largely contained (3). however, sporadically infected cases and asymptomatic carriers were still detected. hence, wuhan launched a population-scale, massive sars-cov-2 testing campaign for detecting viral nucleic acid and antibodies in residents to further prevent viral transmission, screen out infected patients who were in the incubation period or were asymptomatic virus carriers, and map the epidemiological sero-distribution of this infectious disease in the epicenter. wuchang district, one of the 13th administrative divisions in wuhan, is located in the central urban area and is adjacent to the yangtze river. according to the wuhan statistical yearbook 2018, the total population of wuchang district is 1.28 million, accounting for 11.7% of the wuhan population. the present study described the screening results of 61,437 community members in wuchang district, wuhan. j o u r n a l p r e -p r o o f individual blood samples were collected at community sampling stations. the the graphs of age and sex distribution were depicted for the entire tested population. for the seropositive populations, the positive rates in each age and sex group were calculated by dividing the corresponding entire tested population. the 95% confidence interval (ci) was presented. the male-to-female ratio (mfr) was calculated as the male positivity rate divided by the female positivity rate. among 61,437 community members included in the study, 30,032 (48.88%) were male. the age range is from several months to 101 years old and the median age was 48 (interquartile range (iqr): 32-64) years. the sex and age distribution is depicted in figure 1 . as shown in table 1 table 1 ). furthermore, the participants were voluntarily recruited, and hence it was reasonable to speculate that a small proportion of mobility-impaired individuals were reluctant to participate, although they performed fewer activities during the outbreak and had fewer chances to be infected. the present study was conducted in wuchang district, while data in different districts in wuhan hit by covid-19 might vary. however, the observed rate was comparable with that reported in other similar studies. a recent study detected 17,368 individuals from different geographic regions in china, including 1993 residents from different wuhan sub-cohorts. it suggested a seropositivity rate of 3.8%, 3.2%, and 3.8% in healthcare workers, their family members, and their staff members, respectively, from hotels designated for the accommodation of healthcare workers during city lockdown in wuhan between march 30 and april 10, 2020 (5) . as the exposure of healthcare workers and their close contacts to sars-cov-2 was relatively higher than that of most of the other citizens, it was reasonable that the seropositivity from these j o u r n a l p r e -p r o o f subgroups was higher than that from populations with massive testing. another study testing 452 asymptomatic hong kong residents evacuated from hubei province in early march 2020 indicated a 3.76% (17/452) seropositivity rate (6) . all these seropositivity rates indicated that the prevalence of the population carrying the antibody in wuhan was low. eight (0.013%), 324 (0.53%), and 1200 (1.95%) individuals were detected positive for nucleic acid, igm and igg, respectively. as igm is regarded as the first class of immunoglobulins in response to initial exposure, the presence of igm antibody represents an early exposure to the antigen (7). the anti-sars-cov-2 igm antibody could be detected in patients after 4 days of onset, peaking at 2-3 weeks after the onset of symptoms before its level started to decline (8, 9) . however, igm positivity alone may not be a good diagnostic indicator because not all of the people develop a detectable igm antibody (8) . in addition, the igm antibody may still be detectable after several months, although it is considered as an "early infection". most of the recent studies showed detectable sars-cov-2 anti-igm antibodies after one to two months (10, 11) . igg represents the most robust and long-duration antibody against the virus (12) . remarkably, the present study detected more female carriers of asymptomatic antibodies compared with male carriers in most age subgroups and a reverse correlation trend of mfr with the increase in age. another study in wuhan also reported similar findings (13) . indeed, it was suggested that sars-cov-2 affected women less compared with men, due to different innate immunity, steroid hormones, and factors related to sex chromosomes (14) . however, this female over male trend in asymptomatic carriers was not captured in other areas and countries, such as south korea, thailand, iran, spain, and california of usa (15) (16) (17) (18) . whether this trend is observed only in wuhan or can be observed in other parts of china or other countries too needs further investigation. this study had several limitations. first, several groups of people were not included in the study, which might have had different impacts on detecting real seropositivity. second, the rates were affected by the quality of the kit. as (19) . these intrinsic shortcomings of the rapid immunochromatographic kit might inevitably cause false-positive and false-negative results. third, massive tests were conducted within 12 days, and hence the possibility of a more false-positive or false-negative rate due to the labor-intensive work was unavoidable. fourth, a rapid and ready-to-use method, the immune colloidal gold technique, was adopted for the screening test because a large number of samples were needed for handling and the technicians had some limitations. the test provided only a qualitative positive or negative result. a more quantitative result may be obtained using the chemiluminescence enzyme immunoassay. in summary, the majority of the residents in wuhan are still immunologically naive to sars-cov-2, far from herd immunity. although wuhan re-opened since april 8, 2020, proper control measures, such as frequent hand hygiene, wearing face masks, and keeping proper social distance, are necessary before effective vaccines or antiviral drugs are available. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study the effect of human mobility and control measures on the covid-19 epidemic in china strengthening the reporting of molecular epidemiology for infectious diseases (strome-id): an extension of the strobe statement seroprevalence of immunoglobulin m and g antibodies against sars-cov-2 in china seroprevalence of sars-cov-2 in hong kong and in residents evacuated from hubei province, china: a multicohort study. the lancet microbe role of natural and immune igm antibodies in immune responses serological immunochromatographic approach in diagnosis with sars-cov-2 infected covid-19 patients patterns of igg and igm antibody response in covid-19 patients profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) analytical performances of a chemiluminescence immunoassay for sars chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus seroprevalence and epidemiological characteristics of immunoglobulin m and g antibodies against sars-cov-2 in asymptomatic people in wuhan, china coronavirus cov-19/sars-cov-2 affects women less than men: clinical response to viral infection igg seroprevalence of covid-19 among individuals without a history of the coronavirus disease infection in daegu antibody in thai community hospitals seroprevalence of covid-19 virus infection in guilan province covid-19 antibody seroprevalence in sars-cov-2-induced humoral immunity through b cell epitope analysis and neutralizing activity in covid-19 infected individuals in japan key: cord-344027-qghktrm1 authors: fiolet, thibault; guihur, anthony; rebeaud, mathieu e.; mulot, matthieu; peiffer-smadja, nathan; mahamat-saleh, yahya title: 'effect of hydroxychloroquine with or without azithromycin on the mortality of covid-19 patients' – author’s reply date: 2020-10-17 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.10.002 sha: doc_id: 344027 cord_uid: qghktrm1 nan 3) there is a high imbalance between groups for age and comorbidities, factors associated 47 with a poorer outcome. moreover, patients with contraindications to hcq or azi were 48 included in the control group, while they should have been excluded from the comparison. 49 as with all studies at risk of critical bias included in our systematic review, it was excluded 50 from the main analysis. a sensitivity analysis including studies at risk of critical bias was 51 performed, which only marginally modified our results (supplementary table s6 the statement that we used "subjective and specious" inclusion criteria is wrong. the 150 prisma statement for reporting systematic reviews and meta-analyses of studies that 151 evaluate healthcare interventions: explanation and elaboration treatment 154 with hydroxychloroquine, azithromycin, and combination in patients hospitalized with clinical 158 efficacy of chloroquine derivatives in covid-19 infection: comparative meta-analysis 159 between the big data and the real world hydroxychloroquine in nonhospitalized adults with early covid-19. annals of internal 163 medicine 2020 hydroxychloroquine in hospitalized patients with covid-19: preliminary results from a 166 multi-centre, randomized, controlled trial covid-19 177 prevention and treatment: a critical analysis of chloroquine and hydroxychloroquine 178 clinical pharmacology concentration-dependent mortality of chloroquine in overdose utilization 184 of covid-19 treatments and clinical outcomes among patients with cancer: a covid-185 19 and cancer consortium (ccc19) cohort study interventions 188 for treatment of covid-19: a living systematic review with meta-analyses and trial 189 sequential analyses (the living project) mortality outcomes with hydroxychloroquine and chloroquine in covid-19: an 193 international collaborative meta-analysis of randomized trials hydroxychloroquine as pre-exposure prophylaxis for covid-19 in healthcare workers: 203 a randomized trial key: cord-275556-798oed8n authors: piubelli, chiara; deiana, michela; pomari, elena; silva, ronaldo; bisoffi, zeno; formenti, fabio; perandin, francesca; gobbi, federico; buonfrate, dora title: overall decrease of sars-cov-2 viral load and reduction of clinical burden: the experience of a northern italy hospital date: 2020-10-12 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.10.006 sha: doc_id: 275556 cord_uid: 798oed8n objectives: in italy the burden of patients with coronavirus disease 2019 (covid-19) gradually decreased from march to the end of may. in this work, we aimed at evaluating a possible association between the severity of clinical manifestations and viral load over time, during the epidemiological transition from high to low transmission setting. methods: we reviewed the cases of covid-19 diagnosed at the emergency room of our hospital, retrieving the proportion of patients admitted to the intensive care unit. a raw estimation of the viral load was done evaluating the ct (cycle threshold) trend obtained from our diagnostic reverse transcriptase real-time pcr test. results: the proportion of patients requiring intensive care significantly reduced from 6.7% (19/281) in march, to 1.1% (1/86) in april, and to none in may (fisher’s test p-value=0.0067). as for viral load, we observed a trend of ct increasing from a median value of 24 (iqr 19-29) to 34 (iqr 29-37) between march and may, with a statistically significant difference between march and april (pairwise wilcoxon test with stepdown bonferroni adjustment for multiple testing, p=0.0003). conclusions: we observed a reduction over time of the proportion of patients with covid-19 requiring intensive care, along with decreasing median values of viral load. as the epidemiological context changes from high to low transmission setting, people are presumably exposed to a lower viral load, which has been previously associated to less severe clinical manifestations. the proportion of patients requiring intensive care significantly reduced from 6.7% ( we observed a reduction over time of the proportion of patients with covid-19 requiring intensive 46 care, along with decreasing median values of viral load. as the epidemiological context changes 47 from high to low transmission setting, people are presumably exposed to a lower viral load, which 48 has been previously associated to less severe clinical manifestations. of course, other factors might have had an influence on the decrease of severe cases, such as stricter 119 adherence to quarantine of the most fragile groups of people (i.e. those with chronic conditions, the 120 elderly). in our cohort, median age increased from march to april, but older age has been 121 associated to a worse outcome [10], so we suppose that this factor might not have a major role in 122 our findings. conversely, the relevance of the transmission setting seems plausible, and efforts the covid-19 infection: lessons from the italian experience. 148 journal of public health policy an interactive web-based dashboard to track covid-19 in real time. the 150 lancet infectious diseases covid-19 dashboard by the center for systems science and engineering (csse) at johns hopkins 152 university (jhu) laboratory testing for coronavirus disease (covid-19) in suspected 158 human cases, interim guidance detection of 2019 novel coronavirus (2019-ncov) by real-time 162 rt-pcr. euro surveillance : bulletin europeen sur les maladies transmissibles = european 163 communicable disease bulletin analysis of relative gene expression data using real-time quantitative pcr 165 and the 2(-delta delta c(t)) method exposure to sars-cov-2 in a high transmission setting increases 167 the risk of severe covid-19 compared to exposure to a low transmission setting? journal of travel 168 medicine viral dynamics in mild and severe cases of covid-19. the lancet 170 infectious diseases euro surveillance : bulletin europeen sur les maladies 173 transmissibles = european communicable disease bulletin key: cord-286525-0354438s authors: lee, todd c.; butler-laporte, guillaume; chagla, zain; mcdonald, emily g. title: tocilizumab versus the covid19 tempest: all’s well that ends well or much ado about nothing? date: 2020-09-29 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.033 sha: doc_id: 286525 cord_uid: 0354438s nan nearly nine months have passed since the first mention of sars-cov-2, initially described as a mysterious respiratory illness [1] that has gone on to catch the world offguard. since then we have learned a great deal about covid-19 and made important strides in patient care including pivoting away from pre-emptive intubation, which may have inflated early mortality [2] . however, many questions remain unanswered. since the outset, a severe phenotype of the disease associated with elevated markers of inflammation and culminating in substantial lung injury and death has been recognized to develop in a subset of patients [3] . studies of potential treatments aimed at prevention and treatment of this phenotype have focused on medications with antiviral, antithrombotic, and/or anti-inflammatory properties. one of these treatments, dexamethasone, has already demonstrated a mortality benefit in a large randomized controlled trial [4] . analogies with cytokine storm, which may or may not be an apt comparison [5] , have led to an interest in il-6 inhibition as a therapeutic modality for severe covid-19. tocilizumab is a biological il-6 inhibitor therapy that is used for the treatment of rheumatoid arthritis and giant cell arteritis. indeed, several observational cohorts have explored the repurposing of tocilizumab for covid-19 [6] [also cite martinez-sanz et al., cmi when in press] and there are, at present, dozens of registered clinical trials involving tocilizumab or sarilumab. rodríguez-baño et. al [7] tackle the management of patients with an inflammatory presentation by providing observational data in support of tocilizumab for select patients with covid-19. the authors conducted a retrospective cohort study in patients admitted to sixty hospitals in spain between february 2 and march 31, 2020. patients selected for inclusion in this analysis were clinically ill with both fever and oxygen requirements at study enrollment but without immediate need for mechanical ventilation. most importantly, they selected patients with signs of a "hyperinflammatory" response, which the authors defined by presence of an elevation in ferritin, d-dimer, and/or il-6 levels. after adjustment for several potential confounders, using a variety of techniques, the authors found that the receipt of tocilizumab without corticosteroids was associated with j o u r n a l p r e -p r o o f reduced hazard for intubation or death. by contrast, neither steroid therapy alone nor in combination with tocilizumab was convincingly protective for either outcome. of note, a second observational study from spain [cite martinez-sanz et al. when it is in press] was subsequently published in clinical microbiology and infection and reported a decreased risk of death when tocilizumab was given to patients with high levels of creactive protein (>150mg/l). rodriguez-baño et al appropriately adjusted for several comorbidities and used propensity score methods. prior to propensity score matching, the treatment groups differed, with a higher prevalence of cardiac disease and severe renal insufficiency in the untreated group. comparatively, the tocilizumab group also had a longer median duration of illness and hospitalization prior to enrolment as well as lower adverse negative prognostic markers (ferritin and d-dimer). even after propensity score matching, some of these imbalances persisted. the major challenge due to the study design is that we will never know with certainty why in some cases a given therapy was chosen over no therapy at all. this confounding by indication [8] is extremely difficult to eliminate. likewise, it is hard to ascertain through observational data alone whether patients who received tocilizumab monotherapy were systematically treated differently than those who did not. additionally, the authors attempted to address immortal time bias by excluding early primary outcomes and performing a sensitivity analysis with a time dependent covariate. yet, there is likely residual survivor bias as the fundamental definition of day 0 did not correspond to the day of admission, rather to the time of enrollment in the cohort. as such, the tocilizumab group was further along in the illness trajectory with a median of one of the unexpected findings from this study was an observed lack of benefit from steroids in contrast to the recovery randomized controlled trial results [4] or a recent meta-analysis of steroid trials in critically ill patients. [9] there are several ways this can be reconciled. perhaps like in the tale of "goldilocks", the "porridge was too hot": many patients received 10x the dose of steroids used in the recovery trial and less than 10% of the cohort was treated with dexamethasone. while it is tantalizing to assume that the steroid effect is a class effect, dexamethasone may work differently with its lack of mineralocorticoid activity and longer half-life. indeed, a recent randomized controlled trial of methylprednisolone at higher equivalent doses to recovery's dexamethasone failed to demonstrate a mortality benefit [10] and methylprednisolone appeared to have the least benefit in the meta-analysis [9] . finally, the patients in the sam-covid-19 cohort differed from those in recovery with a lower prevalence of heart and kidney disease and fewer mechanically ventilated patients (1-3% vs. 15%) who seem to benefit most from steroids. finally, we must always consider the possibility of residual confounding by indication and/or other differences in the care received by the steroid group in the present trial. it is also important to put this observational data in context with several industrysponsored trials that have examined il-6 inhibiting monoclonal antibodies with disappointing conclusions. the phase iii covacta trial (nct04317092) found tocilizumab did not reduce mortality in hospitalized patients with severe covid-19 pneumonia [11] . similarly a large trial of sarilumab (nct04315298) in severe and critical covid-19 was stopped by the data safety monitoring board due to lack of benefit and a potential signal for harm in non-ventilated patients [12] and a second international trial (nct04327388) also failed to meet its primary or key secondary outcomes [13] . whether or not a specific subgroup of patients with a hyperinflammatory response might benefit as proposed by the authors remains to be seen in future (ideally randomized) trials. j o u r n a l p r e -p r o o f implications early rapid mobilization of research efforts allowed the investigators to collect valuable data through an observational cohort study and important attempts were made to control for confounding by indication and survivor bias in the analyses. the authors appropriately conclude with a proposal to investigate the role of il-6 inhibition as dexamethasone emerges as standard of care, and they caution against widespread use based on observational data alone. reconciling their results with those from randomized control trials raises important questions about the causal effect of the hyperinflammatory response and its role in the development of severe covid-19. given the promising findings associated with the use of corticosteroids, we speculate whether a broader anti-inflammatory approach is the best option for most patients, whereas only a subset of patients might benefit from targeted anti-inflammatory management. we hope that trials like recovery (www.recoverytrial.net) and remap-cap (www.remapcap.org) will soon bring substantial clarity to the role, if any, that drugs like tocilizumab might play in combating the worldwide covid-19 tempest. pneumonia of unknown aetiology in wuhan, china: potential for international spread via commercial air travel ventilation techniques and risk for transmission of coronavirus disease, including covid-19: a living systematic review of multiple streams of evidence the role of il-6 and other mediators in the cytokine storm associated with sars-cov-2 infection dexamethasone in hospitalized patients with covid-19 -preliminary report is a "cytokine storm" relevant to covid-19? efficacy of tocilizumab in covid-19: a systematic review and meta-analysis clinical microbiology and infection : the official publication of the european society of clinical microbiology and infectious diseases confounding by indication in clinical research association between administration of systemic corticosteroids and mortality among critically ill patients with covid-19: a meta-analysis methylprednisolone as adjunctive therapy for patients hospitalized with covid-19 (metcovid): a randomised, double-blind, phase iib, placebo-controlled trial roche provides an update on the phase iii covacta trial of actemra/roactemra in hospitalised patients with severe covid-19 associated pneumonia sanofi and regeneron provide update on kevzara® (sarilumab) phase 3 u.s. trial in covid-19 patients sanofi provides update on kevzara® (sarilumab) phase 3 trial in severe and critically ill covid-19 patients outside the drs. lee and mcdonald receive research salary support from the fonds de recherche québec -santé. dr butler-laporte is supported by a scholarship from the fonds de key: cord-253250-zet48zcl authors: thaden, j.t.; maskarinec, s.a. title: when two for the price of one isn’t a bargain: estimating prevalence and microbiology of bacterial co-infections in patients with covid-19 date: 2020-09-09 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.002 sha: doc_id: 253250 cord_uid: zet48zcl nan in people with viral respiratory tract infections, the presence of a concomitant bacterial infection has been associated with poor clinical outcomes. for example, in patients with influenza, superimposed bacterial infection is present in 20-30% of patients (1, 2) and has been associated with increased rates of shock, mechanical ventilation, and mortality (1, 2) . similarly, in children with severe respiratory syncytial virus (rsv) infection, multiple studies have demonstrated rates of superimposed bacterial pneumonia in excess of 30%, and this has been associated with a longer duration of mechanical ventilation (3) . studying the rates and microbiology of bacterial co-infection in patients with viral respiratory infections can aid in how we determine empiric antibiotic therapy, understand prognosis, and discern pathogenesis in viral-bacterial co-infections. the prevalence and microbiology of concomitant bacterial infections in patients with sars-cov-2 infection are not yet well understood. therefore, we read with interest the study by langford and colleagues in which they performed a rapid systematic review of studies that examined rates of bacterial pneumonia or bloodstream infection in patients with covid-19 (4). in this meta-analysis they identified a large cohort of 3448 patients from 28 studies that primarily consisted of hospitalized adults in asia. there was heterogeneity in the outcomes of the studies that were included in the meta-analysis, as some reported whether bacterial infection was noted on presentation to the hospital (n=20 studies; termed bacterial co-infection), while the remaining studies reported whether a concomitant bacterial infection developed during the course of the patient's hospitalization (n=8 studies; termed secondary bacterial infection). in a random effects meta-analysis, bacterial co-infection was identified in 3.5% of covid-19 patients, and secondary bacterial infection was identified in 15.5% of covid-19 patients. no data on prevalence of bacteremia versus bacterial pneumonia was presented. interestingly, stratification of patients by illness severity showed that bacterial infections were more prevalent in fatal (11.6%) and icu (8.1%) cases relative to non-icu hospitalized patients (5.8%). it is not yet clear if the concomitant bacterial infections are driving poor clinical outcomes or if they are simply more common in sicker patients that are receiving a higher level of care (e.g., ventilator-associated pneumonia in intubated patients). the overall low prevalence of bacterial infection in patients with covid-19 was similar as that noted in another recent meta-analysis (7%) (5) and rapid review (8%) (6) of the literature, though there is significant overlap in the studies included in these reviews. taken together, the overall rate of bacterial has been demonstrated to be dependent upon factors such as disruption of the nox2 inflammatory pathway (7) and increased tlr9 expression (8) . whether sars-cov-2 similarly disrupts such pathways is as yet unknown. despite the lower prevalence of bacterial co-infection in patients with sars-cov-2 infection relative to other viral respiratory pathogens, many patients with covid-19 (71%) were treated with antibiotics. most commonly, these were broad-spectrum agents such as fluoroquinolones or carbapenems. while detailed information on antibiotic use patterns such as timing and duration of antibiotic therapy is not provided, the data from langford at present we do not have enough data to make firm conclusions on the rates and microbiology of bacterial infections in patients with covid-19. yet while the available data is limited, the emerging picture is one of lower bacterial co-infection rates in patients with covid-19 relative to pandemic influenza. despite this, the reported use of broad-spectrum antibiotic therapy in patients with covid-19 is high. at present there is not good evidence to support the broad use of empiric antibiotics in patients with covid-19, particularly in those without critical illness. antibiotic therapy should be limited to those with suspected or proven bacterial coinfection, with frequent re-evaluation based on the clinical course, laboratory findings, and imaging findings. critical illness from 2009 pandemic influenza a virus and bacterial coinfection in the united states bacterial and viral coinfections complicating severe influenza: incidence and impact among 507 u.s. patients, 2013-14 viral bacterial interactions in children: impact on clinical outcomes bacterial co-infection and secondary infection in patients with covid-19: a living rapid review and meta-analysis co-infections in people with covid-19: a systematic review and meta-analysis bacterial and fungal co-infection in individuals with coronavirus: a rapid review to support covid-19 antimicrobial prescribing nox2-derived oxidative stress results in inefficacy of antibiotics against post-influenza s. aureus pneumonia influenza-induced immune suppression to methicillin-resistant staphylococcus aureus is mediated by tlr9 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical and immunological features of severe and moderate coronavirus disease 2019 impact of low dose tocilizumab on mortality rate in patients with covid-19 related pneumonia epidemic and pandemic viral infections: impact on tuberculosis and the lung. a consensus by the world association for infectious diseases and immunological disorders (waidid), global tuberculosis network (gtn) and members(#) of escmid study group for mycobacterial infections (esgmyc) key: cord-331002-7uojryqz authors: valent, francesca; di chiara, antonio title: detection of sars-cov-2 in nasopharynx according to clinical phenotype of affected patients date: 2020-09-06 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.08.041 sha: doc_id: 331002 cord_uid: 7uojryqz objectives: duration of sars-cov-2 in upper respiratory tract is extremely variable, but its relation to disease severity is unknown. we investigated such relation in the 530,000-inhabitant north-eastern italian province of udine. methods: we analysed real‐time reverse‐transcriptase polymerase chain reaction (rt-pcr) tests for sars-cov-2 on upper respiratory specimens conducted at the virology laboratory of the university hospital of udine, italy, serving the whole province, from march 1 to april 30, 2020, on positive subjects of four groups characterized by different disease severity (critically ill patients admitted to intensive care units, patients admitted to infectious disease units, symptomatic patients visited at the emergency department and not hospitalized, and asymptomatic subjects tested during contact tracing or screening activities). duration of viral positivity was assessed from the first positive test to the day of the first of two consecutive negative tests. univariate and multivariate analyses were conducted to investigate differences in the four groups. results: from march 1 to april 30, 39,483 rt-pcr tests for sars-cov-2 were conducted on 23,778 persons. 974 subjects had a positive test result. among those with multiple tests (n=878), mean time to negativity was 23.7 days (standard error 0.3639) (median 23, interquartile range: 16-30 days). mean time to negativity was longer in the icu group than in the others, whereas no difference was observed between asymptomatic patients and those with mild disease. conclusions: disease control measures should not be adjusted to account for differences in viral shedding according to symptomatic status. italian province of udine. after the first positive test were excluded from further analyses. 90 in each group, time of return to negativity was described through the mean and standard error 91 (se) and the quartiles. kaplan-meier curves were used to describe time to negativity in each group. 92 the log-rank test and wilcoxon's test were used to assess whether the groups differed significantly. p-93 values<0.05 were considered statistically significant. finally, cox regression was used to assess table 2 shows the distributions of time to negativity in the 4 groups. mean, median, and 123 maximum values were similar in the asymptomatic, ed, and idu groups, but they were higher in the 124 icu group. figure 1 shows kaplan-meier curves for time to negativity in the 4 groups. the curves 125 were not significantly different according to the log-rank test (p-value 0.1020), but they were 126 significantly different according to wilcoxon's test which is more sensitive to earlier time points (p-127 value 0.0242). 128 the results of cox regression are illustrated in table 3 . after adjusting for sex and age, at any 129 time point, icu patients were less likely to return to negativity than the other patients. female sex and 130 increasing age were associated with a reduced likelihood of returning to negativity. the main finding of our study is the long duration of sars-cov-2 positivity in a population 133 including patients ranging from asymptomatic to acute respiratory distress syndrome requiring icu 134 care. for the whole cohort, the median duration is 23 days (with a range from 2 to 53 days). in addition, a standardized method of np swab collection was adopted. broncho alveolar lavage 172 or gastric tubage were never used to assess the non-infectiveness of the subject in healing phase. 173 this research has also a number of limitations. our source of data did not include information 174 on medications used by patients so we cannot say whether any therapeutic agent might have influenced j o u r n a l p r e -p r o o f world health organization. coronavirus disease 2019 (covid-19) situation report: 28 laboratory testing for coronavirus disease (covid-19) in suspected 209 human cases clinical course of patients infected with sars-cov-2 in singapore clinical course and risk factors for mortality of 215 adult inpatients with covid-19 in wuhan, china: a retrospective cohort study sars-cov-2 viral load in upper 218 respiratory specimens of infected patients early risk factors for the duration of 220 sars-cov-2 viral positivity in covid-19 patients the early phase of the 223 covid-19 outbreak in viral 226 shedding and antibody response in 37 patients with middle east respiratory syndrome 227 viral load kinetics of mers 229 duration for carrying sars-cov-2 in covid-19 231 patients difference is associated with severity of coronavirus disease 2019 infection: an insight from a 234 sars-cov-2 viral load in upper 236 respiratory specimens of infected patients key: cord-031493-w8agvg9g authors: davido, benjamin; seang, sophie; barizien, nicolas; tubiana, roland; de truchis, pierre title: possible therapies of post-covid-19 chronic symptoms() date: 2020-09-06 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.001 sha: doc_id: 31493 cord_uid: w8agvg9g nan responsible for dysautonomia in patients with persistent symptoms following acute sars-23 cov2 infection. physicians must keep in mind that covid-19 is not only a disease 24 responsible for lung injury and its sequel but may affect other organs as olfactory and 25 gustatory dysfunction and as such, should inform the general audience, especially young 26 individuals that are the most concerned [2] . actually, we did not state that these symptoms of autonomic impairment do not require 28 specific treatment, but we believe it must be a case by case management depending on the breathing/hyperventilation syndrome in adults efficacy of 68 therapies for postural tachycardia syndrome: a systematic review and meta-69 analysis key: cord-292296-nocmabcg authors: shang, l.; xu, j.; cao, b. title: fangcang shelter hospitals in covid-19 pandemic: the practice and its significance date: 2020-05-01 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.04.038 sha: doc_id: 292296 cord_uid: nocmabcg nan coronavirus disease 2019 (covid-19), an emerging respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has been spreading across the world rapidly and claiming tens of thousands of lives. on 11 march 2020, the who announced it to be a global pandemic. as of 6 april 2020, more than a million covid-19 cases have been confirmed globally with over 60 000 deaths [1] . the rapidly increasing number of covid-19 cases is posing a huge challenge to medical systems worldwide. in pandemicaffected areas, hospital beds are limited and overwhelmed by the large numbers of patients. given the shortage of medical resources in some countries, only severely ill patients can be admitted to hospitals, leaving many more patients, often with mild-to-moderate symptoms, left unattended at home. home isolation can lead to two problems. first, individuals with sars-cov-2 infection who stay at home contribute to the household and community transmission of sars-cov-2. it is inevitable that those staying at home will have close contact with their family members and easily transmit the virus. also, it may be hard for some countries to strictly manage the large number of patients under home isolation because of the lack of extensive human resources, so the patients might move around and have contact with other people in the community. this could result in community transmission and further increase the number of covid-19 cases [2] . second, leaving patients at home might delay the optimised timing of supportive medical care. individuals with covid-19 can deteriorate quickly from mild/moderate to severe illness [3] . individuals in home isolation often did not receive appropriate symptom monitoring and prompt referral to hospitals when necessary. without appropriate medical care, individuals with rapid disease progression would further increase the burden of medical systems. the abovementioned factors will consistently contribute to the shortage of medical resources and ultimately collapse the medical system, as is happening currently in many disease-stricken areas around the world. during past infectious disease epidemics or natural disasters, mobile field hospitals have been put in place to cope with the shortage of medical resources [4, 5] . however, the capacity of mobile field hospitals is comparatively small, especially in the face of the exponentially increasing number of covid-19 cases. the fangcang shelter hospital, also referred to as 'fangcang hospital' for short, was built in wuhan, china to curb the spread of covid-19 and provide timely basic medical care to patients. fangcang shelter hospitals were transformed from large public facilities such as sports stadia and exhibition centres in a very short time, providing a large number of beds to admit and treat individuals with mild-tomoderate covid-19. as discussed at length in a recent healthpolicy article, the major functions of fangcang shelter hospitals are isolation, triage, basic medical care, frequent monitoring and rapid referral [6] . apart from being a hospital, the fangcang shelter hospital also provided food and shelter, as well as social engagement, for individuals with covid-19 [6] . by obviating the risk of within-household and community transmission, fangcang shelter hospitals were one of the key measures to control the epidemic in wuhan, china [7] , and could be a game changer for other countries as well [8] . in this issue of clinical microbiology and infection, wang et al. described the work flow of fangcang shelter hospitals and reported the clinical characteristics of covid-19 patients in dongxihu fangcang shelter hospital, one of the largest fangcang shelter hospitals in wuhan [9] . dongxihu fangcang shelter hospital was transformed from an exhibition centre to a temporary hospital with more than 1000 beds and began to admit patients on 7 february 2020. the authors retrospectively analysed the clinical data of 1012 individuals admitted to the dongxihu fangcang shelter hospital from 7 to 12 february 2020. all patients had laboratory-confirmed covid-19 with moderate symptoms (respiratory rate <30 breaths/ minute and blood oxygen saturation >93% at resting state), and were tested negative for influenza virus before admission. they were also required to have self-care ability without serious underlying diseases or co-morbidities. this paper was the first to report the clinical characteristics of individuals with mild-to-moderate covid-19 in a fangcang shelter hospital. the most common symptoms (fever and cough), underlying diseases (hypertension and diabetes) and radiological manifestations (patchy opacity and ground-glass opacity) in this cohort were similar to the observations in previous reports [10, 11] . however, the authors reported a relatively high rate of diarrhoea (15%) in the fangcang cohort [12] . it is still not clear whether this is a characteristic of mild disease or related to certain treatments before admission. as faecal samples of some patients were reported to be rt-pcr positive for sars-cov-2, there is the possibility for faecaleoral transmission [3, 13] . therefore, the gastrointestinal symptoms of the individuals with mild-to-moderate disease should be carefully monitored. asymptomatic patients might also transmit the virus and make effective disease control more difficult [14] . this paper reported a low proportion (1.4%) of asymptomatic infection in the fangcang cohort. however, the data should be interpreted carefully because they were derived from hospitalised patients and data from the general population are lacking. large-scale seraepidemiology studies in the general population and follow-up investigation of close contacts may help to confirm the proportion of asymptomatic individuals with sars-cov-2 infection. besides, in the fangcang cohort, the white blood cell counts were normal for most individuals, but the differential counts and many other laboratory findings were absent in many cases. as fangcang shelter hospitals or their equivalents are being built worldwide, it is important to investigate the laboratory characteristics of individuals with mild-to-moderate covid-19 to optimise patient management. of note, the authors identified some risk factors of disease progression by comparing the clinical characteristics of patients with exacerbation and those without. previous studies have identified older age, higher d-dimer, and coexisting diseases, such as hypertension and diabetes, to be associated with higher risk of severe disease or death [15e17]. with dynamic observation of patients with mild-to-moderate disease, this paper provided clues to risk factors for disease progression, such as older age, diabetes and cardiovascular disease, some of which were similar to the risk factors for poor prognosis identified earlier. however, most of the patients in the study were still hospitalised at the final follow up and the results were not adjusted for potential confounding factors, so this might not be the final conclusion and should be interpreted prudently. identifying risk factors for disease progression in individuals with mild-to-moderate disease is important for optimal triage and management of patients in fangcang shelter hospitals or their equivalents around the world. further studies with longer follow up and definite outcomes are needed to confirm the risk factors for disease progression. as the number of confirmed cases is soaring in many countries around the world [1] , fangcang shelter hospitals, or facilities with similar functions, are urgently required. although the names and admission criteria may differ among facilities, the core concept is to completely isolate mild-to-moderate covid-19 patients in fangcang shelter hospitals, not in homes, thus reducing household and community transmission. fangcang shelter hospitals are a novel approach for responding to the covid-19 pandemic and have provided isolation, triage, timely and high-quality medical care, disease monitoring and referral, and social engagement for mild-to-moderate covid-19 patients. wang et al. expanded our knowledge on fangcang shelter hospitals by presenting clinical features of the admitted patients [9] . these data could help to identify the disease characteristics of this mild-to-moderate sub-group and the risk factors for disease progression. other countries can also refer to the experience in combatting covid-19. bc is the corresponding author and conceived the article. ls and jx wrote the original draft and bc, jx and ls were responsible for reviewing and editing the article. world health organization early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical and virological data of the first cases of covid-19 in europe: a case series intensive care in a field hospital in an urban disaster area: lessons from the august 1999 earthquake in turkey formulating and improving care while mitigating risk in a military ebola virus disease treatment unit fangcang shelter hospitals: a novel concept for responding to public health emergencies association of public health interventions with the epidemiology of the covid-19 outbreak in wuhan, china institutional, not home-based, isolation could contain the covid-19 outbreak clinical characteristics of non-critically ill patients with novel coronavirus infection (covid-19) in a fangcang hospital clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of coronavirus disease 2019 in china review article: gastrointestinal features in covid-19 and the possibility of faecal transmission molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes familial cluster of covid-19 infection from an asymptomatic diabetes is a risk factor for the progression and prognosis of covid-19 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study covid-19: risk factors for severe disease and death all authors have no conflicts of interest to declare. no financial support was received for the present work. key: cord-030061-55ntc4wj authors: helleberg, marie; steensen, morten; arendrup, maiken cavling title: invasive aspergillosis in patients with severe covid-19 pneumonia date: 2020-08-05 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.07.047 sha: doc_id: 30061 cord_uid: 55ntc4wj nan intensive care units (icus) due to respiratory failure. in recent years, it has been recognized that rates of 10 invasive aspergillosis are high (19%) among patients admitted to the icu with severe influenza, including 11 those without other risk factors for invasive fungal infections (14%). 1 influenza-associated aspergillosis is 12 diagnosed median 3 days after admission to the icu and is associated with increased mortality (51% versus 13 28%, p=0.0001). 1 other severe viral pneumonias may also increase the risk of invasive aspergillosis 14 significantly. 2-4 recently few case reports of covid-19-associated a. fumigatus infection have been 15 published, but the incidence of invasive aspergillosis in covid-19 patients with critical illness is unknown. 5-6 16 rigshospitalet, is a tertiary referral university hospital, and one of two centers for extracorporeal 17 membrane oxygenation (ecmo) in denmark. we have treated eight covid-19 patients with ecmo in the 18 period 15-03-2020 to 11-04-2020. two of the eight patients were diagnosed with plausible pulmonary 19 aspergillosis due to worsening of respiratory insufficiency, pulmonary infiltrates despite antibacterial 20 therapy, and positive aspergillus cultures and galactomannan antigen (gm) tests on samples of serum or 21 lower respiratory tract secretion. biopsy or bronchoalveolar lavage (bal) was not performed to confirm the 22 diagnosis of pulmonary aspergillosis to reduce risk of aggravation of respiratory failure and risk of 23 transmission of sars-cov-2. thus, the diagnosis of pulmonary aspergillosis was plausible but not proven. 24 however, a therapeutic bal was performed later in the disease course for one of the patients and gm 25 testing of the bal fluid was positive. clinical and mycological characteristics, treatment and outcomes of 26 the two patients are summarized in table 1 . both patients were middle-aged females. one had a history of 27 asthma and was treated with systemic corticosteroids (methylprednisolone 80 mg iv for two days then oral 28 prednisolone 37.5 mg for four days) upon admission, the other patient had no classical risk factors for 29 invasive aspergillosis. the icu algorithm for diagnosis of putative invasive aspergillosis require 1) an 30 aspergillus-positive lower respiratory tract specimen culture, 2) compatible symptoms, 3) abnormal chest 31 x-ray/ct scan and 4) either a host factor or a positive bal culture and microscopy. 7 gilead, novartis, msd, and seges. she is the current chairman of the eucast-afst. 59 60 author contributions 61 all authors contributed to conceptualization, formal analysis, investigation and resources. mh wrote the 62 original draft of the manuscript. all authors edited and reviewed the manuscript and approved the final 63 version. 64 invasive aspergillosis in patients admitted to the 73 intensive care unit with severe influenza: a retrospective cohort study incidence, risk factors, and outcome of pulmonary invasive 76 fungal disease after respiratory virus infection in allogeneic hematopoietic stem cell transplantation 77 recipients clinical features of fatal severe fever with thrombocytopenia syndrome 79 that is complicated by invasive pulmonary aspergillosis lower respiratory tract respiratory virus infections increase 81 the risk of invasive aspergillosis after a reduced-intensity allogeneic hematopoietic sct covid-19 associated pulmonary aspergillosis fatal invasive aspergillosis and coronavirus disease in an 86 immunocompetent patient. emerg infect dis a clinical algorithm to diagnose invasive pulmonary 88 aspergillosis in critically ill patients revision and update of the consensus definitions of 90 invasive fungal disease from the european organization for research and treatment of cancer and 91 the mycoses study group education and research consortium key: cord-264261-98h1bmb2 authors: caruana, giorgia; croxatto, antony; coste, alix t.; opota, onya; lamoth, frederic; jaton, katia; greub, gilbert title: diagnostic strategies for sars-cov-2 infection and interpretation of microbiological results date: 2020-06-25 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.019 sha: doc_id: 264261 cord_uid: 98h1bmb2 background: to face the current covid-19 pandemic, diagnostic tools are essential. it is recommended to use real-time rt-pcr for rna viruses in order (i) to perform a rapid and accurate diagnostic, (ii) to guide patient care and management and (iii) to guide epidemiological strategies. further studies are warranted to define the role of serological diagnosis and a possible correlation between serological response and prognosis. objectives: to guide clinical microbiologists in the use of these diagnostic tests and clinicians in the interpretation of their results. sources: a research of literature was performed through pubmed and google scholar using the keywords sars-cov-2, sars-cov-2 molecular diagnosis, sars-cov-2 immune response, sars-cov-2 serology/antibody testing, coronavirus diagnosis. content: the present review discusses performances, limitations and use of current and future diagnostic tests for sars-cov-2. implications: real-time rt-pcr remains the reference method for diagnosis of sars-cov-2 infection. on the other hand, notwithstanding its varying sensitivity according to the time of infection, serology represents a valid asset (i) to try to solve possible discrepancies between a highly suggestive clinical and radiological presentation and negative rt-pcr, (ii) to solve discrepancies between different pcr assays, and (iii) for epidemiological purposes. in december 2019, numerous cases of pneumonia of unknown etiology were reported in 46 wuhan (china) [1] . in january, the novel causative virus named sars-cov-2 was identified, 47 which spread to other chinese regions and to other countries, causing a world pandemic [2, 3] . 48 the clinical presentation of this disease, named "coronavirus disease 2019 (covid-19)", 49 varied from asymptomatic or mild flu-like symptoms to severe bilateral pneumonia with acute 50 respiratory distress and death. a rapid replication of the virus within the first 24 hours from 51 the infection and the relatively high (about 3) reproduction number were described [4] . 52 the available viral genome sequences allowed to soon recognize the close relationship (recombination), thus donating them some genomic plasticity [5] . furthermore, rna 59 biosynthesis seems to use a virus-specific template switch, which results in transcription of 60 sub-genomic mrnas and eventually leading to homologous rna recombination [5] . 61 nevertheless, by encoding a 3'-5'exoribonuclease within nonstructural protein 14 (nsp14-62 exon), which is required for high-fidelity replication, the mutation capacity of sars-cov-2 63 is debated [6] . in the end, somehow the plasticity allowed coronaviridae to acquire a rich 64 strains biodiversity and the ability to jump species, which already caused previous zoonotic 65 outbreaks, as for mers-cov and sars-cov [7-9]. 66 starting from observed similarities in a short region of rdrp gene between sars-cov-2 and 67 a bat coronavirus (batcovratg13), further sequences were identified to be 96% identical at 68 the whole-genome level, corroborating the hypothesis of animals to humans spill-over [10]. as of june 3, more than 6 million cases of covid-19 have been declared, including more rna extraction methods can generally be classified into i) one-step (with the rt step and the 106 pcr reaction in the same tube) and ii) two-steps rt-pcr (initial creation of dna copies 107 with rt reaction followed by their addiction to the pcr reaction). typically, one-step pcr table 1 ), which can allow to improve sensitivity and 113 tat but also sometimes increase the costs. despite the good performance of the validated naats, there is still a risk of false negative 115 results. most of them concern the pre-analytic setting, such as the timing of the specimen 116 collection (too early or too late in the infection course, including the limit of detection due to 117 late infections with atypical manifestations), the quality of sampling (insufficient material) or 118 type of specimens (bronchoalveolar lavage -bal exhibits the highest sensitivity, followed by 119 induced sputum, naso-pharyngeal -np swab, oro-pharyngeal -op swab, and feces), and finally 120 the sample transport (inappropriate container, exposure to extreme temperatures, etc.) [18] [19] . sensitivity and specificity of serological assays can also be affected by the target antigen. as 146 highlighted by meyer and colleagues, the s protein (produced in more advanced stage of 147 sars-cov-2 infection) showed lower levels of sensitivity and more specificity (especially 148 the s1 subunit) compared to the n protein, [38] . noteworthy, we recently observed (a. coste 149 et al., data not shown) that the antibodies directed against the n protein seems to decrease 150 earlier than the s protein; thus the sensitivity of assays targeting only the n protein may be 151 impaired according to the timing of infection (figure 1 ). for this reason, we recommend to 152 systematically use two tests, one targeting the s protein and one targeting the n protein for 153 diagnostic purposes. for sero-epidmiological studies, a test targeting the s protein is 154 recommended. the added value to target the s protein is that the titers will likely better reflect 155 protection against reinfection. in table 2 we summarized the interpretation of diagnostic assays with sensitivity greater than 95% and specificity superior or equal to 98% should be 165 used. as well as for naats, different platforms can be considered also for serological tests. interestingly, the appearance of igm occurs at the same time than igg, thus, the main 183 advantage to also test igm is to assess the timing of the infection. table 3 . 206 while serological assays can represent a useful epidemiological asset, naats remains the 207 gold standard for diagnosis, due to their high sensitivity even at early stages of the disease. samples like blood and urines were found to be weakly-to-none sensitive, while the virus was 231 also found in feces and perineal swabs of patients with gastro-intestinal symptoms [44] [45] , improved molecular diagnosis of covid-19 by the novel, highly sensitive 316 and specific covid-19-rdrp/hel real-time reverse transcription-polymerase chain 317 reaction assay validated in vitro and with clinical specimens ten years of r&d and full automation 320 in molecular diagnosis letter to the editor: 322 sars-cov-2 detection by real-time rt-pcr potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review false-negative rate of reverse transcriptase polymerase chain reaction-based 330 sars-cov-2 tests by time since exposure sources of pre-analytical, analytical and post-analytical errors in 332 the microbiology laboratory pcr inhibitors-occurrence, 336 properties and removal sars-cov-2 diagnostic pipeline xpert® xpress sars-cov-2 has received fda 340 purification and enrichment of 343 virus samples utilizing magnetic beads on a microfluidic system high-speed rna microextraction technology 346 using magnetic oligo-dt beads and lateral magnetophoresis parallel rna extraction 349 using magnetic beads and a droplet array viral dna/rna 96 kit; c2020 351 ez1 advanced xl; c2020 ideal96 automated extraction robot; c2020 multifunctional device for 360 nucleic acid extraction based on magnetic separation and its co-working with liquid 361 handling system for high throughput sample preparation a simple 364 magnetic nanoparticles-based viral rna extraction method for efficient detection of 365 profile of specific antibodies to the sars-associated 369 coronavirus antibody responses to sars-cov-2 in patients of 373 novel coronavirus disease 2019 longitudinal monitoring of sars-cov-2 igm and igg seropositivity 378 to detect covid-19 antibody 387 responses to sars-cov-2 in patients with covid-19 kinetics of serologic responses to mers coronavirus infection in humans, south 392 korea antigenic relationships amongst coronaviruses. arch gesamte 394 virusforsch emergencies preparedness, response. who guidelines for 396 the global surveillance of severe acute respiratory syndrome (sars): updated 397 recommendations serological assays for emerging 401 coronaviruses: challenges and pitfalls novel antibody epitopes dominate the antigenicity of spike 403 glycoprotein in sars-cov-2 compared to sars-cov potent binding of 2019 novel coronavirus spike protein by a sars 407 coronavirus-specific human monoclonal antibody lateral flow assays sars-cov-2 viral load in 413 upper respiratory specimens of infected patients correlation of chest ct and rt-pcr testing in coronavirus disease 2019 china: a report of 1014 cases fecal specimen diagnosis 2019 novel coronavirus-419 infected pneumonia covid-19: gastrointestinal manifestations and potential 421 fecal-oral transmission respiratory viral infection-424 induced microbiome alterations and secondary bacterial pneumonia the respiratory tract microbiome and 427 lung inflammation: a two-way street key: cord-334835-j6u8t8j2 authors: berenguer, juan; ryan, pablo; rodríguez-baño, jesús; jarrín, inmaculada; carratalà, jordi; pachón, jerónimo; yllescas, maría; arribas, josé ramón title: characteristics and predictors of death among 4,035 consecutively hospitalized patients with covid-19 in spain date: 2020-08-04 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.07.024 sha: doc_id: 334835 cord_uid: j6u8t8j2 objectives: we aimed to analyse the characteristics and predictors of death in hospitalized patients with covid-19 in spain. methods: retrospective observational study of the first consecutive patients hospitalized with covid-19 confirmed by real-time polymerase chain reaction (rt-pcr) assay in 127 spanish centres until march 17, 2020. the follow-up censoring date was april 17, 2020. we collected demographic, clinical, laboratory, treatment, and complications data. the primary endpoint was all-cause mortality. univariable and multivariable cox regression analyses were performed to identify factors associated with death. results: of the 4,035 patients, males accounted for 2,433/3,987 (61.0%), the median age was 70 years, and 2,539/3,439 (73.8%) had >1 comorbidity. the most common symptoms were a history of fever, cough, malaise, and dyspnoea. during hospitalization 1,255/3,979 (31.5%) patients developed acute respiratory distress syndrome, 736/3,988 (18.5%) were admitted to intensive care units, and 619/3,992 (15.5%) underwent mechanical ventilation. viral or host-targeted medications included lopinavir/ritonavir 2,820/4,005 (70.4%), hydroxychloroquine 2,618/3,995 (65.5%), interferon-beta 1,153/3,950 (29.2%), corticosteroids 1,109/3,965 (28.0%), and tocilizumab 373/3,951 (9.4%). overall 1,131/4,035 (28%) patients died. mortality increased with age (85.6% occurring in older than 65 years). seventeen factors were independently associated with an increased hazard of death, the strongest among them included advanced age, liver cirrhosis, low age-adjusted oxygen saturation, higher concentrations of c-reactive protein, and lower estimated glomerular filtration rate. conclusions: our findings provide comprehensive information about characteristics and complications of severe covid-19 and may help to identify patients at a higher risk of death. 8 carried out a block-wise forward procedure allocating the predictor variables into five clusters: 133 sociodemographic characteristics, comorbidities, admission signs and symptoms, vital signs, and 134 laboratory parameters. a multivariable regression analysis was fitted within each block using two 135 criteria to achieve the best set of predictors: relevance to the clinical situation and statistical 136 significance (p<0.10). we used variance inflation factors to detect collinearity among predictors 137 included in the multivariable models. we carried out a sensitivity analysis in which the order of 138 entry of the blocks was inverted. we checked the proportional hazards assumption. variables with 139 more than 25% of missing values have not been considered, and missing values were treated as a 140 separate category for analysis. heterogeneity introduced by different hospitals was accounted for 141 by using robust methods to estimate standard errors and, thus, to calculate 95% confidence 142 intervals (ci) and p-values. statistical analyses were done using stata software (version 15.0; stata 143 corporation, college station, texas, usa. this study is registered with clinicaltrials.gov, 144 the final cohort included 4,035 hospitalized patients (see web-only supplementary figure s1 ) 147 in which sars-cov-2 was detected by , pharyngeal swabs 148 patients' characteristics, categorized by survival, are shown in table 1 . in brief, males accounted 155 for 61.0%, the median age was 70 years, and 25.1% were > 80 years old. most patients were 156 spanish born whites. the age distribution of patients stratified by sex is shown in figure 1a . 157 at least one comorbidity was present in 73.8% and 26.7% had at least three comorbid conditions. 158 the most common comorbidities were arterial hypertension (51.2%), chronic heart disease 159 (23.3%), diabetes mellitus (21.8%), chronic pulmonary disease (not asthma) (17.9%), and obesity 160 (13.8%). only 0.7% of patients had hiv. before admission, 19.4% patients were on angiotensin-161 converting enzyme (ace) inhibitors, and 17.3% were receiving angiotensin ii receptor blockers 162 (arbs) ( table 1) . 163 the median duration of symptoms before hospitalization was 4 (iqr 2 -7) days, and the most 164 commonly reported were history of fever ( (15.4%), presumed bacterial pneumonia (10.6%), heart failure (5.8%), and blood-stream infection 199 (4.9%). during the study period, 28.0% of patients died, 64.1% were discharged, and 7.8% 200 remained hospitalized. the median (iqr) time to death since the beginning of symptoms and since 201 hospital admission was 13 (9-19) days and 10 (6-16) days, respectively. death was particularly 202 high among patients ≥ 80 years (54.9%) ( figure 1b) and those with ≥3 comorbid conditions 203 (47.7%). death was also very high among those with ards (59.3%), those who were admitted to 204 icu (42.4%), and those who underwent mechanical ventilation (45.7%). the median (iqr) length 205 of stay was 4 (1-9) days for patients who were discharged; and 35 (32-38) days for those who 206 remained hospitalized at the censoring date. 207 208 independent predictors of death in the different clusters of variables are shown in table 3 . in the 209 final adjusted analysis, we found 17 factors independently associated with an increased hazard of 210 death: male sex, older age, arterial hypertension, obesity, liver cirrhosis, chronic neurological 211 disorder, active cancer, dementia, dyspnoea, confusion, low age-adjusted sao2 on room air, higher 212 white cell blood count (wbc), higher neutrophil-to-lymphocyte ratio, lower platelet count, 213 prolonged inr, lower egfr, and higher concentrations of crp (figure 2) . no collinearity was 214 detected, the proportional hazards assumption was fulfilled, and the results were not changed 215 when the order of entry of the blocks was inverted. kaplan-meier plots for death according to age 216 and sex are shown in figure 3 . the adjusted hazard ratio (ahr) of death for being admitted early 217 in the epidemic (before 13 march) vs later was 1.07 (95% confidence interval [ci]: 0.90; 1.28), 218 p=0.407. the variable unilateral or bilateral lung opacities had missing values in 29% individuals 219 and was not included in the final model. however, when this variable was included in the model, 220 the ahr (95%ci) of death for bilateral opacities in comparison with unilateral opacities was 1.32 221 (0.11; 1.55) p=0.002. we also carried out two post-hoc analyses (data not shown). in the first one, 222 the predictors of mortality among patients ≤ 65 years were not substantially different from those 223 found in the whole data set. in the second analysis, the mortality hazard did not change depending china (3, 13) and one from uk. the majority of patients in all four cohorts were male. however, in 232 comparison with chinese patients, those from spain and the uk were on average, two decades 233 older and had a prevalence three times higher of comorbid conditions. it is not surprising thus 234 that mortality was substantially higher in spain (28%) and the uk (26%) than china (1.4 and 235 3.2%). presenting features were similar in all cohorts. however, dyspnoea was less frequent in 236 chinese patients suggesting a more severe course in the older spanish and british patients. in our 237 cohort, age was the main determinant of death, as has been in other series of hospitalized patients 238 with 8, 9, 14, 19) . independently of the higher prevalence of comorbidities, it cannot 239 be ruled out that older patients could not have been prioritized to receive icu treatment. death 240 was also significantly higher among men than in women, as has also been described in other 241 cohorts (3, 8, 9, 13, 14) . there are sex differences in innate and adaptive immune responses that 242 might have an impact on the inflammatory response and outcomes of covid-19 and deserve 243 further investigation (20). hypertension was not only the most common comorbidity in our 244 cohort, as in other studies, but also an independent predictor of mortality. the association 245 between hypertension and poor outcomes in covid-19 does not seem to be simply a matter of 246 high prevalence; alternative explanations include pre-existing hypertensive end-organ or 247 endothelial damage and interactions between covid-19 and antihypertensive medications (21). 248 many patients with hypertension were receiving ace inhibitors or arbs, but they did not increase 249 j o u r n a l p r e -p r o o f 14 mortality. obesity was the fifth most common comorbidity in our cohort but one with the highest 250 hazard of mortality. obesity has been found to increase the risk of hospitalization and severe 251 outcomes during influenza seasons (22). recent studies with covid-19 patients indicate that 252 younger hospitalized individuals were more likely to be obese (23) and that obesity is associated 253 with severe pictures (23-25) and increased mortality (14). other underlying conditions associated 254 with an increased hazard of death were active cancer and cirrhosis and as has been reported 255 elsewhere (26, 27); meaning that clinicians should consider patients with these underlying 256 conditions as a high-risk category for . we identified several routine laboratory 257 markers as predictors of mortality, including the neutrophil-to-lymphocyte ratio, an indicator of 258 systemic inflammation that has been found of prognostic utility in sepsis (28), and covid-19 (29, 259 our study is limited by the retrospective design and the high number of sites, which might have 261 jeopardized the quality of the data. we tried to solve this by selecting simple and well-defined 262 variables and by careful monitoring of the data. admission criteria might have differed between 263 the sites; nevertheless, we controlled the site effect in the analysis. we could not include in the 264 multivariable model some potentially interesting laboratory parameters, nor changes in 265 laboratory findings over time. the study's strengths include the large sample size, which allowed 266 the identification of a high number of predictors of death at admission, the analysis of clinical and 267 laboratory variables, and the inclusion of sites from areas with different incidence rates. 268 in summary, here we report the clinical characteristics of a large cohort of patients with covid-19 269 consecutively admitted to hospitals in spain during the first month of the epidemic. our findings ministerio de ciencia, innovación y universidades -co-financed by european development 293 regional fund "a way to achieve europe", operative program intelligent growth 2014-2020. distribution -no./with data (%) abbreviations: iqr, interquartile range; egfr, estimated glomerular filtration rate median (iqr) -cells/ x10 9 <1,000 cells/ μl -no./with data (%) mg/dl median (iqr) /with data (%) pg/ml blood cell count; egfr, estimated glomerular filtration rate; ckd-epi, chronic kidney disease epidemiology collaboration; aptt, activated partial thromboplastin time; inr, international normalized ratio key: cord-283818-4m9p717r authors: yan, chao; cui, jinghua; huang, lei; du, bing; chen, lu; xue, guanhua; li, shaoli; zhang, weiwei; zhao, linqing; sun, yu; yao, hailan; li, nannan; zhao, hanqing; feng, yanling; liu, shiyu; zhang, qun; liu, di; yuan, jing title: rapid and visual detection of 2019 novel coronavirus (sars-cov-2) by a reverse transcription loop-mediated isothermal amplification assay date: 2020-04-08 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.04.001 sha: doc_id: 283818 cord_uid: 4m9p717r objective: to evaluate a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for detection of sars-cov-2, and compare it with rt polymerase chain reaction (rt-pcr). methods: we designed primers specific to the orf1ab and s genes of sars-cov-2. total viral rna was extracted using the qiaamp viral rna mini kit. we optimized the rt-lamp assay. and, this assay was evaluated for its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. results: the primer sets orf1ab-4 and s-123 amplified the genes in the shortest times, the mean (±sd) time was 18 ± 1.32 min and 20 ± 1.80 min, respectively, and 63°c was the optimum reaction temperature. the sensitivity was 2×10(1) copies and 2×10(2) copies per reaction with primer sets orf1ab-4 and s-123, respectively. this assay showed no cross-reactivity with other 60 respiratory pathogens. to describe the availability of this method in clinical diagnosis, we collected 130 specimens from patients with clinically suspected sars-cov-2 infection. among them, 58 were confirmed to be positive and 72 were negative by rt-lamp. the sensiticity was 100% (95% ci 92.3% 100%), specificity 100% (95% ci 93.7% 100%). this assay detected sars-cov-2 in the mean (±sd) time of 26.28 ± 4.48 min and the results can be identified with visual observation. conclusion: these results demonstrate that we developed a rapid, simple, specific, and sensitive rt-lamp assay for sars-cov-2 detection among clinical samples. it will be a powerful tool for sars-cov-2 identification, and for monitoring suspected patients, close contacts, and high-risk groups. (rt-lamp) assay for detection of sars-cov-2, and compare it with rt polymerase 23 chain reaction (rt-pcr). methods. we designed primers specific to the orf1ab and s genes of sars-cov-2. 25 total viral rna was extracted using the qiaamp viral rna mini kit. we optimized 26 the rt-lamp assay. and, this assay was evaluated for its sensitivity and specificity of 27 detection using real-time turbidity monitoring and visual observation. the outbreak of a cluster of respiratory infections designated as coronavirus disease 46 2019 (covid19) , caused by severe acute respiratory syndrome coronavirus 2 47 (sars-cov-2), had a significant impact on both the health and economy of the china 48 [1] [2] [3] [4] . to date (1 april 2020), up to 754, 948 sars-cov-2 cases have been confirmed, 49 including more than 36, 571 deaths (https://www.who.int/home). human-to-human 50 transmission was confirmed through a case of five patients in a family cluster [5] . the 51 virus transmitted rapidly via aerial droplets, contact, and fomites [6] . 52 sars-cov-2 has been classified as a beta-coronavirus of group 2b with higher 53 similarity, over 96% identical at the whole-genome level, to two bat-derived the extracted rnas were stored at -80°c. (xiamen zeesan biotech co., ltd.) were also used. the copy number of the 98 pseudo-viruses was calculated using the following formula: copies/µl = 6.02× 10 23 99 ×10 -9 × concentration(ng/µl) / (fragment length × 340). then, 10-fold serial dilutions 100 of the pseudo-viruses ranging from 1×10 8 copies/µl to 1 copy/µl were prepared. (table s1 ). to screen for the optimum temperature, the reaction was incubated at five different 166 temperatures (60-64°c) for 60 min. as presented in figure 1 , the highest 167 amplification efficiency occurred at 63°c. therefore, 63°c was confirmed as the 168 optimum reaction temperature for this assay. sensitivity test for the rt-lamp assay 170 the sensitivity of the rt-lamp assay using primer sets orf1ab-4 and s-123 was 171 evaluated using turbidity monitoring and visual observation. ten-fold serial dilutions 172 of the pseudo-viruses, ranging from 1×10 8 copies/µl to 1 copy/µl (concentration of 173 template input) were detected by the rt-lamp assays. as illustrated in figure 2 taken for positive detection ranged from 20 min at 1×10 8 copies /µl to 48 min at 177 1×10 2 copies/µl . thus, sensitivity of the assays was 2×10 1 copies and 2×10 2 copies 178 per reaction at 63 within 60 min with primer sets orf1ab-4 and s-123, respectively. the main findings of this study is that we established a rapid, sensitive, and 199 specific assay for sars-cov-2 detection by rt-lamp. to improve the sensitivity, preparation [19] . the sensitivity of this assay was 20 copies per reaction, similar to 216 that reported for the rt-pcr assay in a previous study [20] . the rt-lamp assay 217 showed no cross-reactivity with other respiratory pathogens, so the diagnostic 218 specificity of this method was higher than that reported for the serology test [21] . to assess the applicability of the assay for the clinical diagnosis of sars-cov-2, the main limitation of the current study is that we aligned only 103 complete 235 genomes of sars-cov-2 obtained from four databases when design the primers. with the spread of this virus, the accuracy of this rt-lamp assay will be affected by 237 the mutations occurring in the primers sequence region of target gene. so, it is 238 necessary to monitor the mutant sites of virus genome by whole genome sequencing. the established rt-lamp assay has important implications for clinical practice. the detection was monitored by turbidity using a loopamp real-time turbidimeter, 17 and was judged by the naked eye depending on a color change from orange to green. in b, 1-9: 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 copies/µl. 19 in e, 1-8: 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 copies/µl. in c and f, 1-8: 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 copies/µl. the reaction volume of 25 µl contained 2 µl rna template, and the template 22 concentration was 1ng/µl. in the sensitivity test, 60 min can be used as the cut-off for 23 the visual detection. the first two cases of 278 2019-ncov in italy: where they come from transmission of 2019-ncov infection from an asymptomatic contact in germany washington state 2019-ncov case investigation team. first case of coronavirus in the united states covid-19) in france: surveillance, 287 investigations and control measures a familial cluster of infection associated 290 with the 2019 novel coronavirus indicating potential person-to-person transmission 291 during the incubation period world health organization. novel coronavirus (2019-ncov) situation report-7: 27 293 genomic characterisation and 295 epidemiology of 2019 novel coronavirus: implications for virus origins and receptor 296 binding the global 298 spread of 2019-ncov: a molecular evolutionary analysis divergence of the novel coronavirus (2019-ncov) originating in china laboratory readiness and response for novel coronavirus (2019-ncov) in expert 305 laboratories in 30 eu/eea countries loop-mediated isothermal amplification of dna rapid and sensitive detection of 311 novel avian-origin influenza a (h7n9) virus by reverse transcription loop-mediated 312 isothermal amplification combined with a lateral-flow device a rapid and specific assay 315 for the detection of visual detection of west nile 317 virus using reverse transcription loop-mediated isothermal amplification combined 318 with a vertical flow visualization strip rapid and specific detection of asian-and african-lineage zika 321 viruses survey and visual detection of 323 zaire ebolavirus in clinical samples targeting the nucleoprotein gene in sierra 324 a real-time 326 reverse transcription loop-mediated isothermal amplification assay for the rapid 327 detection of yellow fever virus loop-mediated isothermal amplification 329 (lamp): principle, features, and future prospects detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr molecular 334 diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia molecular and 337 serological investigation of 2019-ncov infected patients: implication of multiple 338 shedding routes clinical features of patients 340 infected with 2019 novel coronavirus in wuhan, china a novel coronavirus 343 from patients with pneumonia in china genetic diagnostic methods for novel coronavirus 2019 (ncov-2019) in japan rna based mngs approach 349 identifies a novel human coronavirus from two individual pneumonia cases wuhan outbreak load in upper respiratory specimens of infected patients hcov-nl63-3; s6: hcov-oc43-1; s7: hcov-oc43-2; s8: hcov-hku1-1 adv-1-2; s29: adv-2-1 a-2; s41: rsv b-1; s42: rsv b-2; s43: hmpv-1; s44: hmpv-2; s45: bov-1 bov-2; s47: rh a-1; s48: rha-2; s49: rh b-1; s50: rhb-2; s51: rh c-1 s56: klebsiella pneumoniae; s57: streptococcus pneumoniae; s58: pseudomonas 45 aeruginosa figure 1 sequence comparison and gene analysis of the orf1b and s genes 49 sequence comparison and gene mutation analysis of the orf1b gene (8084bp) sequence comparison and gene mutation analysis of the s gene (3822bp) f3: outer forward primer; b3: outer backward primer; fip: forward inner primer; bip: 52 backward inner primer; lf: loop forward primer bat severe acute respiratory syndrome like coronavirus 55 mers: middle east respiratory syndrome coronavirus genbank accession no. mg772933; bat-sl-covzc45 genbank accession no. nc_019843.3 key: cord-255971-kamai25b authors: wee, liang en; conceicao, edwin philip; sim, xiang ying jean; ko, kwan ki karrie; ling, moi lin; venkatachalam, indumathi title: reduction in healthcare-associated respiratory viral infections during a covid-19 outbreak date: 2020-07-03 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.027 sha: doc_id: 255971 cord_uid: kamai25b nan healthcare-associated respiratory viral infections (rvi) remain an underappreciated cause of in-40 hospital morbidity and mortality. [1] infection prevention bundles comprising segregation of 41 symptomatic patients, droplet/ contact precautions and visitor screening can potentially reduce the 42 transmission of rvi on high-risk units, [2] though hospital-wide implementation has been limited. 43 the current covid-19 pandemic highlights the importance of strengthening hospital-wide 44 infection control against common rvi. however, the effectiveness of infection control during a 45 covid-19 outbreak on healthcare-associated rvi has yet to be assessed. the impact of these policies on the incidence of healthcare-associated rvi was evaluated by 58 comparing the daily incidence of healthcare-associated rvi amongst hospitalized inpatients over the healthcare-associated rvi and should continue in some form even after the covid-19 pandemic is 108 over. hospital-acquired respiratory viral infections: incidence, morbidity, and mortality in 120 prevention of hospital-acquired respiratory viral infections: 122 assessment of a multimodal intervention program decreased influenza incidence under covid-19 control measures emerg infect dis containing covid-19 outside the isolation ward: the impact of an infection control bundle on 128 environmental contamination and transmission in a cohorted general ward lew, iv conceptualised the study. data was gathered by epc, kkkk, and xyjs. lew prepared the initial 112 draft and the manuscript was critically revised by kkkk, xyjs, mll and iv. 113 the authors report no conflicts of interest. 115 this work was not grant-funded. 117 118 key: cord-254278-awdqguoo authors: khan, suliman; jun, li; nawsherwan; siddique, rabeea; li, yanyan; han, guang; xue, mengzhou; nabi, ghulam; liu, jianbo title: association of covid-19 infection with pregnancy outcomes in healthcare workers and general women date: 2020-04-08 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.03.034 sha: doc_id: 254278 cord_uid: awdqguoo nan viral pneumonia is thought to be the most common non-obstetric infectious disease 1 during pregnancy, which is associated with maternal and neonatal morbidity and mortality 2 during pregnancy [1] . atypical pneumonia known as coronavirus disease (covid-19) caused by 3 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is highly infectious and is 4 currently spreading rapidly around the globe [2] . before leading to the global emergency, sars-5 cov-2 emerged in wuhan, hubei province, china during december 2019 [3, 4] . several studies 6 focusing on infected patients from the general population have been reported, however, limited 7 information is available in the aspects of pregnancy outcomes of covid-19 infected women. general infected women were included in the study. 15 we conducted a case series study on pregnant women (n =17) infected with covid-19 16 admitted to hubei general hospital (renmin hospital) from jan 25 to feb 15, 2020. covid-19 17 pneumonia was diagnosed according to the new coronavirus pneumonia prevention and control 18 program of 5 th and 6 th editions. all the seventeen pregnant women were found positive for 19 covid-19 using either quantitative rt-pcr (qrt-pcr) and/or ct scan imaging or both. to 20 assess the neonatal infection with covid-19, cord blood and neonatal throat swab samples were 21 collected immediately after delivery in the operating room and were tested by using quantitative 22 rt-pcr. all the patients delivered babies by c-section, and the detailed information collected 23 are presented in tables 1-4. we conducted a comprehensive literature search for the current 24 outbreak of covid-19 infection in pregnant women and a thorough search for the impact of 25 sars-cov pregnancy outcomes. the age range of the patients was 24-34 years and the range of gestational weeks at 27 admission was 35 weeks to 41 weeks. moreover, the range of gestational weeks at delivery was 28 35 weeks and 5 days to 41weeks. respectively. in twelve individuals, the nucleic acid test from 29 the throat swab was positive for covid-19 while only in five individuals both the ct scan and nucleic acid test indicated covid-19. we observed fever in three individuals and complications 1 in five. other common symptoms were cough (n = 6), diarrhea (n = 3), nasal congestion (n = 2), 2 shortness of breath (n = 2), and sputum production (n = 1). patients were receiving antibiotics 3 (n=17), hormones (n =8), and antivirals together with chinese medicine (n= 15). a total of 17 4 neonates including three preterm neonates with birthweight ranging from 2300 g to 3750 g and 5 birth length from 45 cm to 52 cm (49.2 cm) were delivered through c-section. there was no fetal 6 or neonatal death. the ultrasound results and fetal heart rate were normal for all the neonates 7 while the apgar score for 16 neonates was in the range of 9-10. only two neonates (case 6 and 8 case 14) after birth were suspected for covid-19 while five neonates were reported with 9 neonatal pneumonia. moreover, three cases were found with preterm delivery (table 1 and table 10 2). based on our findings in these seventeen patients, we suggest that covid-19 infection 12 may lead to the occurrence of neonatal pneumonia and preterm delivery. however, we cannot in summary, we found two neonates suspected for covid-19 infection and five neonates 1 with neonatal pneumonia, suggesting the possibility that adverse pregnancy outcomes may be 2 linked to covid-19 infection. 4/17 (23.5% and 0.63-2.37) 4 out of 17 patients were found with lymphopenia, which could be linked with covid-19 infection elevated alt (>45 u/l) 2/17 (11.7% and 9-46) two out of 17 patients were found with elevated ast and/or alt. however, the majority of the patients had normal alt and ast levels. elevated ast (>35 u/l) 2/17 (11.7% and 12-39) 4/17 (23.5% and 0.63-2.37) 4 out of 17 patients were found with lymphopenia, which could be linked with covid-19 infection elevated alt (>45 u/l) 2/17 (11.7% and 9-46) two out of 17 patients were found with elevated ast and/or alt. however, the majority of the patients had normal alt and ast levels. elevated ast (>35 u/l) 2/17 (11.7% and 12-39) potential maternal and infant outcomes from (wuhan) key: cord-009295-4c0zwhdh authors: bal, a.; destras, g.; gaymard, a.; bouscambert-duchamp, m.; valette, m.; escuret, v.; frobert, e.; billaud, g.; trouillet-assant, s.; cheynet, v.; brengel-pesce, k.; morfin, f.; lina, b.; josset, l. title: molecular characterization of sars-cov-2 in the first covid-19 cluster in france reveals an amino acid deletion in nsp2 (asp268del) date: 2020-03-28 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.03.020 sha: doc_id: 9295 cord_uid: 4c0zwhdh nan in december 2019, a novel coronavirus emerged in china, causing outbreaks of pneumonia [1] . the virus was subsequently identified as a betacoronavirus and named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). sars-cov-2 is responsible for the coronavirus disease 2019 (covid-19) pandemic which includes asymptomatic upper and lower respiratory tract infections. among the first european cases of covid-19, six were associated with a cluster of transmissions in the french alps in late january 2020 [2] . the index case of this cluster travelled from singapore to france and went back to the united kingdom (uk) where he tested positive for sars-cov-2 on february 6th. here, we aimed to investigate the french cases related to this cluster using metagenomic next-generation sequencing (mngs) analysis. of the six contact patients who tested positive for sars-cov-2, the three samples with the highest viral loads (assessed by rt-pcr targeting the rdrp gene) were selected for mngs analysis [3] . one nasopharyngeal swab was collected from a patient with an upper respiratory tract infection on february 7th (sample #1, ct ¼ 31.3). the other two samples were collected from the same asymptomatic patient on february 8th (sample #2, nasopharyngeal swab, ct ¼ 31.1) and 9th (sample #3, nasopharyngeal aspirate, ct ¼ 28.8). a previously described mngs protocol was used, but dnase treatment was performed after nucleic acid extraction in order to increase the sensitivity for the detection of rna viruses [4] . lowquality and human reads were filtered out, and remaining reads were aligned to the sars-cov-2 reference genome (isolate wuhan-hu-1, epi_isl_402125) using the bwa-mem algorithm. a mean of 19 445 767 reads per sample were generated, of which a mean of 605 243 reads per sample were mapped to the sars-cov-2 reference genome. the percentage of genome covered at a minimum depth of coverage of 100x was 38.3% for sample #1, 99.6% for sample #2 and 80.6% for sample #3. the whole-genome sequence (wgs) generated from sample #2 was deposited on gisaid (global initiative on sharing all influenza data) (epi_isl_410486). the phylogenetic analysis using the 571 wgs of sars-cov-2 publicly available (as of march 17th 2020) found that this sequence clustered with a sequence (epi_-isl_408488) collected in jiangsu, china, on january 19th, suggesting a separate introduction from asia (fig. 1) . compared to the reference sars-cov-2 sequence, a three-nucleotide deletion in open reading frame 1a (orf1a) at positions 1607e1609 was identified. this deletion was found in 100% of the reads covering this position with a sequencing depth of 1745x around the deletion. importantly, this deletion was also identified in 100% of the reads of sample #1 and sample #3 with a depth of 54x and 481x, respectively. using the cov-glue resource, we found that this mutation leads to a deletion of amino acid 268 in non-structural protein 2 (nsp2) [5] . this deletion in nsp2 (asp268del) was also characterized in 37/571 (6.1%) of the wgss available on march 17th (england n ¼ 6; the netherlands n ¼ 31). wgs-based phylogenetic analysis found that 15 viruses containing this specific deletion were close to viruses collected in china between december 2019 and early february 2020, while 23 viruses with asp268del collected in the netherlands have slightly diverged (fig. 1 ). the analysis included 571 wgs of sars-cov-2 (>29 000 bp) collected in humans and available on gisaid (global initiative on sharing all influenza data) from march 17th, 2020. the following sequences were excluded from the analysisdepi_isl_406592, epi_-isl_414588, epi_isl_412900, epi_isl_408487, epi_isl_408483 and epi_isl_406595 because they were outliers, and epi_isl_413747, epi_isl_413695dbecause of incomplete sequences in orf1ab. the hcov19/wuhan/ipbcamswh01/2019 strain was used as an outgroup virus. genetic distances were calculated using the kimura's two-parameter model (k80) and pairwise deletion. the tree was constructed by the neighbour-joining method using r seqinr and ggtree packages and validated using 1000 bootstrap pseudo-replicates. sequence from sample #2 (epi_isl_410486) is indicated by the black arrow. nucleotide alignment (1601e1615) is depicted as a heatmap on the right panel with the threenucleotide deletion shown in black. corresponding amino acid sequence (nsp2: 266-270) for the reference sequence is indicated below the heatmap. letter to the editor / clinical microbiology and infection xxx (xxxx) xxx sars-cov-2 sequences were not further compared between the two patients due to largely incomplete coverage of the sars-cov-2 genome in sample #1. nonetheless, the longitudinal samples from the asymptomatic patient (sample #2 versus sample #3) were compared using a minimum depth of coverage of 100x in order to make a preliminary assessment of intra-host genetic variability. three snvs were noticed between the two samples: c366a (nsp1: s34y), a20475g (synonymous mutation in nsp15), and t24084a (protein s: l841h), suggesting intra-host evolution of the virus. for all three positions, nucleotides from sample #2 were still detected in sample #3, but as minor variants. in this short report, we present the first genetic characterization of a covid-19 cluster in europe. despite low viral loads, the mngs workflow used herein allowed us to characterize the wholegenome sequences of sars-cov-2 isolated from an asymptomatic patient in two clinical samples collected 1 day apart. comparison of these sequences suggests viral evolution with development of quasispecies. specific studies using high depth of coverage are needed to explore potential intra-host adaptation. in addition, the present workflow identified a new deletion in nsp2 (asp268del) which was found in all three samples originating from this cluster. the analysis of 571 wgs identified this deletion in 37 other viruses collected in england (february) and in the netherlands (march), suggesting the spread of this deletion in europe. the impact of asp268del on sars-cov-2 transmission and pathogenicity, as well as on pcr performances and antiviral strategies, should be rapidly evaluated in further studies. investigations complied with the general data protection regulation (regulation (eu) 2016/679 and directive 95/46/ec) and the french data protection law (law n 78e17 on 06/01/1978 and d ecret n 2019-536 on 29/05/2019). informed consent concerning the disclosure of information relevant to this publication was obtained from the confirmed cases in france. ab and gd have contributed equally to this work. a novel coronavirus from patients with pneumonia in china first cases of coronavirus disease 2019 (covid-19) in the who european region detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr quality control implementation for universal characterization of dna and rna viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow amino acid analysis for the sars-cov-2 outbreak letter to the editor / clinical microbiology and infection xxx (xxxx) xxx we would like to thank all the patients, clinicians, laboratory technicians and informatics department who contributed to this investigation. we are also grateful to v er ena landel and philip robinson (drci, hospices civils de lyon) for help in manuscript preparation. we thank the authors, the originating and submitting laboratories for their sequence and metadata shared through gisaid on which this research is based. we gratefully acknowledge all the members of cov-glue, nextstrain.org, and virological.org for sharing their analysis in real time. key: cord-314288-6vh7dvad authors: leibovici, l.; allerberger, f.; cevik, m.; huttner, a.; paul, m.; rodríguez-baño, j.; scudeller, l. title: submissions and publications in corona times date: 2020-05-15 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.05.008 sha: doc_id: 314288 cord_uid: 6vh7dvad nan in times of the covid-19 pandemic we struggle at cmi between the urge to bring data to the readers as soon as possible and the necessity to publish trustworthy, robust material. the number of submissions to cmi during the first 4 months of 2020 increased by 60% compared to the same months in 2019. at its peak, over 40 articles were submitted on a single day; most of them concerning covid-19. the majority was rejected, many immediately by the editors without peer-review. the decisions were not easy. we would like to explain our decisions, according to the type of submission. we hope thereby to save authors, editors and peer-reviewers time and avoid unnecessary efforts. letters to the editor: under this format we publish interesting case reports and case series; and as expected we received many letters describing one (or a few) patients with presentations of covid-19 that had not been described before. when infected people are counted in hundreds it makes sense to describe a small number of patients with an atypical presentation. but when counted in millions, clinical or laboratory findings in one or two patients might be just a coincidence (e.g., a non-specific finding that may ultimately be due to another condition in a patient with covid-19; or a false positive result for the virus in a patient with another disease); and we would like to see larger and well described denominators. commentaries: we have received many commentaries claiming that known remedies (vitamins, anti-diabetes treatments, anti-hypertensive and anti-inflammatory drugs, antimicrobials and antiviral treatments) could be effective against covid-19. the chain of evidence was nebulous at best and we have decided not to publish such articles. we are happy to publish commentaries describing how people, hospitals or other healthcare settings have dealt with the challenges of managing covid-19, as well as opinion articles discussing the implications of available evidence on clinical practice. narrative reviews: we have also received many reviews attempting to address all questions and data relevant to the virus and the pandemic, some of which were lengthy monographs. we decided not to publish content that would likely be well-known to an informed practitioner. we are pleased to consider in-depth, updated reviews addressing aspects of the pandemic relevant to our readers [1, 2] . we are happy to publish systematic reviews in which the synthesis is greater than what can be gleaned from the original studies [3] . however systematic reviews of observational studies, and especially meta-analyses of such studies assessing the efficiency of drugs, are problematic. bias by indication is always suspected, and we cannot be sure that the adjustments done in the original studies have taken care of all relevant biases. systematic reviews and meta-analyses of small observational studies, with ingrained biases, are not helpful. critical assessment of such studies, without an artificial attempt to combine the results, might be valuable. observational studies: now we have a good idea from published studies on the clinical course of covid-19, and further descriptions of small groups of patients have little to add. several studies have already been published on risk factors for symptomatic disease, severe disease and death in patients affected by the virus. however large studies will allow us a better look at the sub-groups of interest; on the interaction between risk factors; and permit external validation of prognostic models. multi-centre and multi-national prospective studies can address both the concern that some risk factors are local; and point to true differences between populations. we (and others) see a problem with observational studies comparing one treatment to another treatment or to no treatment. since the choice of treatment was made by the practitioners, we have no way to capture and correct for all factors which influenced this decision. was the new treatment given to the worst patients? or the other way around, to those patients perceived to stand a better chance? or (in some settings) to those who can pay? or to opinionated patients with opinionated families? or prescribed by some physicians, or units, and not by others? it is difficult to assume that the new treatments were given at random. we should also be concerned by publication bias: authors are more likely to submit for publication observational studies with a positive result than those with a negative result. observational studies can be of interest: they can generate hypotheses; serve as the base for randomized controlled trials (rcts); or, if convincingly negative, to lower the priority for rct testing of the treatment. but in order to provide convincing results, we would like to see efforts to compare like to like and to adjust for confounders, in large cohorts; to present data carefully collected in full; outcomes that matter to patients; and a correct ascertainment and counting of outcomes. we expect careful descriptions of the methods and results [4, 5] . the drive to develop better diagnostics for a new disease -faster, more accurate or even cheaper -is understandable. considering our readers, we publish studies on diagnostics only if tested on clinical samples, and preferably in clinical situations [6] . we expect the sample size to be large enough to offer confidence in the results [7] . we believe rcts to be the major building blocks of evidence based medicine, and are happy to consider them for publication [8] . rcts are not free of problems: small sample size, studies stopped before recruitment was completed, or studies that are not up to methodological standards. we wish to honor the goodwill of the patients who agreed to participate in the study and were promised that the results would be published to help other patients. the most telling summary was provided by a cover letter of one of the submissions: "please publish our article within 3-4 days. the data might be falsified (sic) in a few days". we, as editors, do not want to publish data that will be outdated or falsified within a few days (or a few months for that matter). editorial note -not peer-reviewed. cmi readers' survey 2019 cmi readers' survey the cmi welcomes systematic reviews reporting methods of observational cohort studies in cmi observational studies examining patient management in infectious diseases how to: evaluate a diagnostic test sample size calculations for diagnostic studies randomized controlled trials in cmi key: cord-008258-5v55vv4s authors: raoult, d. title: is it the end of the nervous breakdown on avian influenza? date: 2015-06-21 journal: clin microbiol infect doi: 10.1016/j.cmi.2015.06.011 sha: doc_id: 8258 cord_uid: 5v55vv4s nan the nervous breakdown on avian influenza that has affected the who, the different governments and the largest journals in the world may perhaps end with two articles published in the journal of infectious diseases in may 2015 [1, 2] . both avian influenza h5n1 and h9n2 have triggered a catastrophic tornado of information like never before [1] . in france, a plan to fight bird flu with vaccine orders and the description of an apocalyptic vision of the next flu epidemic was observed. indeed, marseille was supposed to reserve 700 beds in case of a flu epidemic! this terror was justified by the memory of the spanish flu, although we now know that most of the patients died as a result of bacterial infections [3] and the fact that, as with any infectious disease, from the beginning the first detected cases are only the severe and fatal cases. with regards to h5n1, which is one of the infectious poultry agents that cause large epidemicsdespite its discovery in 1997, of only 650 cases diagnosed in humans, 60% died [1] . this led to a dramatization, with vaccine orders and the creation of experimental models to try to predict when a mutant may become transmissible between humans and transform this zoonotic disease into a human disease [4] . the echoes of this in scientific journals were huge and disproportionate [5] . this current work [2] has just confirmed what was noted for several years but had been denied, which is the high occurrence of asymptomatic infection in people who are in contact with infected poultry. this prospective survey in egypt over 3 years on 1000 people showed that when h5n1 was endemic, 2% of the exposed population had antibodies compared with 0% of controls, and when the epidemic h9n2 appeared in poultry, seroprevalence increased from 0 to 5.6% and 7.5%, all asymptomatic infections. in total the zoonotic variant of h5n1 avian flu and h9n2 is very common in people in contact with poultry; it is banal and most of the time, asymptomatic. these are not the viruses that will destroy humanity! as i have had several opportunities to say, we do not predict future outbreaks [4] . experimental models may well confirm certain elements but cannot predict the severity, as seen with h1n1 and as we still see with h5n1 [5] . transmissibility in the ferret probably has no relationship to transmissibility in humans, in which the virus is probably passed mainly through inanimate objects and hands. it is necessary to regain some common sense for respiratory viruses. it is plausible that the mers (middle east respiratory syndrome) coronavirus problem is of the same nature and that only a subpart of the population respond with a clinical infection, perhaps because of genetic characteristics or environmental parameters. a lesson is to be learned from this episode-it is not necessary to set fire to the planet due to some zoonotic infection, nor to spend billions of euros, nor to create a governmental crisis; ultimately it is 'much ado about nothing' [6] . how low is the risk of influenza a (h5n1) infection? avian influenza a(h5n1) and a(h9n2) seroprevalence and risk factors for infection among egyptians: a prospective, controlled seroepidemiological study microbe interactions undermine predictions modeling in infectious diseases: between haphazard and hazard of ignorance and blindness: for a post-modern science. kindle-amazon emerging respiratory viruses: is it 'much ado about nothing'? (shakespeare) key: cord-279125-w6sh7xpn authors: egli, adrian; schrenzel, jacques; greub, gilbert title: digital microbiology date: 2020-06-27 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.023 sha: doc_id: 279125 cord_uid: w6sh7xpn background: digitalisation and artificial intelligence have an important impact on the way microbiology laboratories will work in the near future. opportunities and challenges lay ahead to digitalise the microbiological workflows. making an efficient use of big data, machine learning, and artificial intelligence in clinical microbiology requires a profound understanding of data handling aspects. objective: this review article summarizes the most important concepts of digital microbiology. the article provides microbiologists, clinicians and data scientists a viewpoint and practical examples along the diagnostic process. sources: we used peer-reviewed literature identified by a pubmed search for digitalisation, machine learning, artificial intelligence and microbiology. content: we describe the opportunities and challenges of digitalisation in microbiological diagnostic process with various examples. we also provide in this context key aspects of data structure and interoperability, as well as legal aspects. finally, we outline the way for applications in a modern microbiology laboratory. implications: we predict that digitalization and the usage of machine learning will have a profound impact on the daily routine of the laboratory staff. along the analytical process, the most important steps should be identified, where digital technologies can be applied and provide a benefit. the education of all staff involved should be adapted to prepare for the advances in digital microbiology. inflammatory response syndrome (sirs) and the presence of a central venous line, the risk of blood culture contamination can be assessed 20 . in the future, the combination of lis and electronic health 2 record (ehr) data may allow more sophisticated feedback loops and provide automated quality 3 assessments reports to the microbiologist and clinician. another important pre-analytical aspect is diagnostic stewardship. diagnostic stewardship 5 incorporates the concept of recommending the best diagnostic approach for a given situation [21] [22] [23] . digital solutions in this field may range from digital twins 24, 25 to machine-learning based algorithms in 7 smartphone app 26 or chatbots 27, 28 . recently, chatbots have been developed to support the diagnostic 8 evaluation and recommending immediate measures, when patients are exposed to sars-cov-2 27 . similarly to a microbiologist consultant, a chatbot may provide helpful diagnostic information and 10 advice e.g. on the correct transport media for a sample, assay costs, the expected turn-around time, and test performance in specific sample types. such an interactive tool may be a first source of 12 information for routine and repetitive questions, and could support the pre-analytical quality test performance and data generation within the laboratory are parts of analytics. as an example, automated microscopy allows to acquire high-resolution images of smears from positive blood 23 cultures and can categorize gram staining with high sensitivity and specificity 30, 31 . besides state-of-24 the-art automated microscopes, smartphones can also be used for image analysis of microscopy 25 data 32, 33 . automated plate reading systems act similarly on pattern recognition and can reliably 26 recognize bacterial growth on a agar plate and could be used to pre-screen culture plates [34] [35] [36] [37] [38] . such 27 automated plate reading systems are currently established in many european laboratories as part of 28 the ongoing automation process. reading of e-tests and inhibition zone diameters around antibiotic-29 impregnated disks can also be automatized with well-developed reading software 39, 40 . clinical decision support systems based on machine learning to provide automated feedback 7 regarding empiric antibiotic prescription adapted to specific patient groups 46 . as a next step, also 8 more complex datasets will be analysed. as physiology and laboratory parameters can rapidly change 9 during an infection, time-series data greatly impact the predictive values of such algorithms -similar 10 to a doctor, who observers the patient during disease progression -machine learning algorithms will 11 also follow the patient's data stream. recently, a series of studies has shown the impact of highfrequency physiological parameters in icus on the prediction of sepsis 47-49 or meningitis 50, 51 . these 13 studies are retrospective analysis and prospective controlled validation studies are largely missing in 14 the field. therefore, although our expectations for digital microbiology may be high, we should remain 15 critical and carefully address the associated challenges. challenges of digitalisation in the microbiology diagnostic process the collection, quality control and cleaning, storage, security and protection, stewardship and governance, interoperability and interconnection, reporting and visualization, versioning, and sharing 20 of data pose considerable challenges for big data in microbiology diagnostic laboratories. some of 21 these data handling aspects may be managed with a profound understanding of the laboratory and due to the increasing quantity of data (explosion of information), it will soon become almost a first step: data structure and interoperability diseases (figure 1 ) [72] [73] [74] . machine learning algorithms require large, structured, interoperable, and 2 interconnected datasets. healthcare data has to be further standardized and annotated with 3 international recognized definitions 75, 76 . ontologies help to structure data in such a way by using a 4 common vocabulary, and allow to determine relations of variables within a data model 77 . the previously mentioned concepts for data handling have been used for a series of large healthcare university hospitals. the goal is to discover digital biomarkers for early sepsis recognition and 8 prediction of mortality using machine learning algorithms (www.sphn.ch/). epidemiological databases can also benefit from structured data. for example, pulsenet is a large 14 15 predictions or decisions without being explicitly programmed to perform that task 9, 112 . machine 18 learning algorithms may be used at each step of the microbiological diagnostic process from pre-to 19 post-analytics, helping us to deal with the increasing quantities and complexity of data 113,114 (table 1) . human analytical capacity has reached its limits to (i) grasp the huge amount of available complex process management is key, (ii) data handling is easiest at the point where the data is actually diagnostic tests. in general, incentives are needed to further support all aspects of data handling in 3 laboratory medicine -including standardization data structures and machine learning algorithms. conclusion 6 digitalisation in healthcare shows already a profound impact on patients. it is expected, that the 7 developments started will further gain momentum. machine learning radically changes the way we 8 handle healthcare-related data -including data of clinical microbiology and infectious diseases. likely, we will move from the internet-of-things environment (interconnected datasets in a patient with in a disease-free time. in addition, developments of molecular diagnostics such as metagenomics will 12 increase the data complexity. current trends indicate, that the importance of laboratory diagnostics we have to develop strategies for the next five to ten years to face the opportunities and challenges 1 2 table s1 . glossary basel) for critically feedback regarding the manuscript. conflict of interest disclosure: none of the authors had a conflict of interest. quality control how reliable is the analytical performance of a test? -surveillance of reagent lots performance with internal and external controls and automated reported in connection to specific used lots of time. imaging are there bacteria on the microscope slide? -automated image acquisition with a microscope and scan for pathogen-like structures and category 30, 32, 33 plate reading is there bacterial growth on the plate? -automated image acquisition and scan for colonies and subsequent identification (telebacteriology). expert system does the detected resistance profile make sense? -medical validation of antibiotic resistance profiles with expert database. public health is there a potential outbreak? -automated screening for pathogen similarities e.g. resistance profile or automated bioinformatics 130,131 is there a potential bacterial phenotype? -detection of resistance by analysing maldi-tof spectra 43, 44 sepsis treatment what is the best treatment for the patient? -prediction of sepsis, and best treatment e.g. volume and antibiotics for the patient 47-49 tracking strains in the microbiome: insights from metagenomics and design and evaluation of a bacterial clinical infectious diseases ontology ontologies for clinical and translational research: 35 introduction semantic data interoperability, digital medicine, and e-37 health in infectious disease management: a review the need for a global language -snomed ct introduction. stud health technol mimic-iii, a freely accessible critical care database the eicu collaborative research database, a freely available multi-center 14 database for critical care research pulsenet and the changing paradigm of laboratory-based surveillance microreact: visualizing and sharing data for genomic epidemiology and 19 phylogeography nextstrain: real-time tracking of pathogen evolution improving the quality and workflow of bacterial genome sequencing and 23 analysis: paving the way for a switzerland-wide molecular epidemiological surveillance 24 platform a comprehensive collection of systems biology data characterizing infectious diseases and associated ethical impacts big data and machine learning in 35 critical care: opportunities for collaborative research privacy in the age of medical big data the compare data hubs. database (oxford) 2019, justice point of view introduction to machine learning machine learning in infection management using routine electronic health machine learning for clinical decision support in infectious diseases: 13 a narrative review of current applications machine learning for healthcare: on the verge of a major shift in 118 supervised machine learning for the prediction of infection on admission 18 to hospital: a prospective observational cohort study using artificial intelligence to reduce 21 diagnostic workload without compromising detection of urinary tract infections unsupervised extraction of epidemic syndromes from participatory influenza stewardship: fair enough? the promise of the internet of 4 things in healthcare: how hard is it to keep? stud wearable devices in medical internet of things: scientific 7 research and commercially available devices key: cord-325186-nq6ay4eo authors: sieswerda, elske; de boer, mark g.j.; bonten, marc m.j.; boersma, wim g.; jonkers, rené e.; aleva, roel m.; kullberg, bart-jan; schouten, jeroen a.; van de garde, ewoudt m.w.; verheij, theo j.; van der eerden, menno m.; prins, jan m.; wiersinga, w. joost title: recommendations for antibacterial therapy in adults with covid-19 – an evidence based guideline date: 2020-10-01 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.041 sha: doc_id: 325186 cord_uid: nq6ay4eo scope: the dutch working party on antibiotic policy constituted a multidisciplinary expert committee to provide evidence-based recommendation for the use of antibacterial therapy in hospitalized adults with a respiratory infection and suspected or proven 2019 coronavirus disease (covid-19). methods: we performed a literature search to answer four key questions. the committee graded the evidence and developed recommendations by using grading of recommendations assessment, development, and evaluation methodology. questions addressed by the guideline and recommendations: we assessed evidence on the risk of bacterial infections in hospitalized covid-19 patients, the associated bacterial pathogens, how to diagnose bacterial infections and how to treat bacterial infections. bacterial co-infection upon admission was reported in 3.5% of covid-19 patients, while bacterial secondary infections during hospitalization occurred up to 15%. no or very low quality evidence was found to answer the other key clinical questions. although the evidence base on bacterial infections in covid-19 is currently limited, available evidence supports restrictive antibiotic use from an antibiotic stewardship perspective, especially upon admission. to support restrictive antibiotic use, maximum efforts should be undertaken to obtain sputum and blood culture samples as well as pneumococcal urinary antigen testing. we suggest to stop antibiotics in patients who started antibiotic treatment upon admission when representative cultures as well as urinary antigen tests show no signs of involvement of bacterial pathogens after 48 hours. for patients with secondary bacterial respiratory infection we recommend to follow other guideline recommendations on antibacterial treatment for patients with hospital-acquired and ventilator-associated pneumonia. an antibiotic treatment duration of five days in patients with covid-19 and suspected bacterial respiratory infection is recommended upon improvement of signs, symptoms and inflammatory markers. larger, prospective studies about the epidemiology of bacterial infections in covid-19 are urgently needed to confirm our conclusions and ultimately prevent unnecessary antibiotic use during the covid-19 pandemic. infection to life-threatening pneumonia. severe disease is frequently associated with high 66 inflammation marker levels. it is therefore challenging to define if a patient fulfilling criteria 67 for cap who is positive for sars-cov-2 has a bacterial co-infection upon admission. during 68 hospitalization it may be difficult to distinguish between severe covid-19 and bacterial 69 secondary infections. 70 in several reports the majority of hospitalized patients with covid-19 were treated with 71 broad-spectrum antibiotics with unknown efficacy [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . as covid-19 patients frequently 72 need prolonged hospitalization and respiratory support, unnecessary antibiotics upon 73 hospitalization may increase the individual risk of subsequent hospital-acquired pneumonia 74 (hap) and other adverse events [12, 13] . on a population level, universal antibiotic 75 prescriptions for all or the vast majority of hospitalized covid-19 patients can lead to a 76 steep increase in antibiotic use during a pandemic and as a result, a potential increase in 77 antimicrobial resistance rates [14] . 78 the dutch working party on antibiotic policy (swab) coordinates activities in the 79 netherlands with the aim to optimize antibiotic use, to contain the development of and acinetobacter baumannii were isolated from respiratory material [19] . in one patient in 178 the netherlands, pseudomonas aeruginosa was cultured from blood, but it was not cov-2 and other respiratory pathogens, jama (2020). j o u r n a l p r e -p r o o f coronavirus disease 2019 in china the first 29 covid-19-patients in a clinic: early experiences from a dutch 416 hospital the first 418 100 covid-19 patients admitted to the elisabeth-tweesteden hospital covid-19 in the emergency 421 department of bernhoven hospital clinical features of patients 423 infected with 2019 novel coronavirus in critically ill patients in the seattle region -case series prediction models for diagnosis and prognosis of covid-19 infection: systematic review and 430 critical appraisal prospective comparison of three validated prediction rules for prognosis in community-433 acquired pneumonia a 435 prediction rule to identify low-risk patients with community-acquired pneumonia 438 defining community acquired pneumonia severity on presentation to hospital: an 439 international derivation and validation study diagnosis 441 and treatment of adults with community-acquired pneumonia. an official clinical practice 442 guideline of the surviving 445 sepsis campaign: guidelines on the management of critically ill adults with coronavirus 446 disease 2019 (covid-19) antimicrobial prescribing. nice guideline antimicrobial 450 de-escalation in critically ill patients: a position statement from a task force of the european 451 society of intensive care medicine (esicm) and european society of clinical microbiology 452 and infectious diseases (escmid) critically ill patients study group (esgcip) covid-19: don't neglect 455 antimicrobial stewardship principles!, clinical microbiology and infection : the official 456 publication of the european society of clinical microbiology and infectious diseases using procalcitonin to guide antibiotic therapy key: cord-298441-77w86l8q authors: lombardi, andrea; consonni, dario; carugno, michele; bozzi, giorgio; mangioni, davide; muscatello, antonio; castelli, valeria; palomba, emanuele; cantù, anna paola; ceriotti, ferruccio; tiso, basilio; pesatori, angela cecilia; riboldi, luciano; bandera, alessandra; lunghi, giovanna; gori, andrea title: characteristics of 1,573 healthcare workers who underwent nasopharyngeal swab for sars-cov-2 in milano, lombardy, italy date: 2020-06-20 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.013 sha: doc_id: 298441 cord_uid: 77w86l8q objectives: the management of healthcare workers (hcws) exposed to confirmed cases of covid-19 is still a matter of debate. we aimed to assess in this group the attack rate of asymptomatic carriers and the symptoms most frequently associated with the infection. methods: occupational and clinical characteristics of hcws who performed a nasopharyngeal swab for the detection of sars-cov-2 in a university hospital from february 24, to march 31, 2020, were collected. for those who tested positive and for the asymptomatic positives we checked laboratory and clinical data as of may 22 to calculate the time necessary to become test-negative and to verify whether symptoms developed thereafter. frequencies of positive tests were compared according to selected variables using multivariable logistic regression models. results: positive tests were 139 among 1,573 hcws (8.8%, 95% confidence interval [ci]: 7.5-10.3), with a marked difference between symptomatic (122/503, 24.2%) and asymptomatic (17/1,070, 1.6%) workers (p<0.001). physicians were the group with the highest frequency of positive tests (61/582, 10.5%), whereas clerical workers and technicians displayed the lowest frequency (5/137, 3.6%). the likelihood of being positive increased with the number of reported symptoms and the strongest predictors were taste and smell alterations (odds ratio [or]= 76.9) and fever (or = 9.12). the median time from first positive test to a negative test was 27 days (95% ci: 24-30). conclusions: a relevant number of hcws can be infected by sars-cov-2 without displaying any symptom. among symptomatic workers, the key symptoms to guide diagnosis are taste and smell alterations and fever. in median, almost four weeks are necessary to achieve negativity of nasopharyngeal swab. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a previously unknown 48 virus which recently jumped from a not yet identified animal host to humans and it is 49 responsible of coronavirus disease 2019 . 1 the virus has now spread worldwide 50 from china, causing the first pandemic of the xxi century, disrupting health-care services in 51 the affected countries and exacting a terrific toll of human lives. 2-3 52 healthcare workers (hcws) are a crucial actor in the pandemic. indeed, they are acting in an 53 emergency situation and are continuously at risk of being infected. at the same time, they are 54 in contact with the most fragile elements of our society, those who need health assistance. it 55 is therefore mandatory to avoid that infected hcws act as spreaders of the disease. 56 unfortunately, it is still unclear which microbiologic investigations and procedures should be 57 adopted toward hcws in covid-19 settings, especially to those exposed to confirmed cases 58 of covid-19 and at risk for infection. to answer this question, we reviewed all the 59 nasopharyngeal swab performed in hcws exposed to confirmed cases of covid-19 at the 60 foundation irccs ca' granda ospedale maggiore policlinico located in milan, the capital 61 of lombardy, by large the italian region mostly affected by we assessed 62 frequency of positive tests among symptomatic and asymptomatic hcws and evaluated the 63 association between occupation, symptoms (type and number), and presence of the infection. furthermore, we also calculated the median time between the day of diagnosis (first positive test) and the day in which the hcw became test-negative. we collected occupational and clinical characteristics of all the consecutive hcws who we tested hcws at risk for infection, which is defined as a contact with a patient or another 74 hcw with (or later diagnosed with) sars-cov-2 infection. all those at risk were, according 75 to the internal protocol, identified and contacted by the hospital infection prevention unit, 76 isolated at home and tested. hcws were subdivided into physicians (including residents), 77 nurses and midwives, healthcare assistants, health technicians, and clerical workers and 78 technicians. all the information was collected by the infectious disease notification form 79 associated to each test. workers were defined as symptomatic if presented any of the 80 following in the 14 days preceding the test: fever, cough, dyspnoea, asthenia, myalgia, 81 coryza, sore throat, headache, ageusia or dysgeusia, anosmia or parosmia, ocular symptoms, men (three physicians and two nurses) and three women (two physicians and a clerical 144 worker) were hospitalized. a minority of the hcws (81/1,537, 5.3%) reported to have had a contact with an infected 146 person outside the hospital (relatives, colleague, or friends). of these, 12/81 (14/81, 8.7%) 147 were found to be positive. in this italian group of hcws exposed to confirmed cases of covid-19, the presence of 150 symptoms, and particularly taste and smell alterations and fever, was associated with interestingly, the auc of a model considering six groups of symptoms (fever, myalgia, 155 asthenia, ocular symptoms, dyspnoea, and taste and smell alterations) was 0.83. based on 156 these results, it seems reasonable to tailor the screening approach of hcws at risk based on 157 the resources available. in low-resource settings we suggest focusing to test those with 158 symptoms to maximize efficacy, especially considering the continuous exposure of hcws to 159 at risk situations, thus requiring repeated testing sessions. nevertheless, it should be 160 underlined that in our study a non-negligible number of workers were infected but displayed 161 no symptoms, meaning that a fraction of those infected can be lost with a symptoms-based 162 screening strategy. therefore, in middle-and high-resource settings a mass screening for all 163 hcws exposed to confirmed covid-19 cases appears the best approach to limit the spread when stratified according to occupation, test-positive frequencies were clearly higher among 177 subsets with direct contact with patients (physicians including residents, nurses and 178 midwives, healthcare assistants and health technicians) than those without (clerical works and 179 technicians). consequently, careful screening of these groups of workers should be 180 mandatory. no differences in terms of infection attack rate were seen between different age 181 groups nor between men and women, suggesting that risk factors for acquiring covid-19 182 among hcws are unrelated to age and sex. another relevant point is the significant number of hcws who were negative at the first test 184 but resulted positive when tested a second time. this might represent a serious concern, as a 185 discrete fraction of those can further spread the virus unnoticed, thus hampering the efficacy 186 of the screening strategy. it should be noted, however, that the second test was performed on 187 a small number of operators and not on a routine basis, making these considerations subject 188 to several potential biases. in addition, in a relevant proportion of our population we could 189 not retrieve information about the most likely date of exposure to a documented case. thus, we cannot exclude a recent contact in which case the first test may have been performed too early (i.e. still in the incubation period which has been estimated to be five 192 days), before a sufficient amount of viral particles is detectable in the nasopharynx. 8 moreover, it has to be considered that hcws employed in covid-19 units/hospitals are at 194 risk of sars-cov-2 exposure on a daily basis and therefore repeated exposures, even 195 unnoticed, can occur also after the first one who motivated the test. moreover, technical 196 limitation can be responsible of falsely negative test, considering that the sensitivity of 197 nasopharyngeal swab for sars-cov-2 detection has been estimated to be around 71%. 9 finally, we observed a median time from first positive test to a negative test of 27 days. this our study has some limitations. first, the surveillance system was quickly set-up in a few 206 days due the virus spread in our region since february 20, when the first italian case was 207 identified in the south-east part of lombardy. therefore, data quality was imperfect and 208 extensive time-consuming data editing (through review of electronical records and, when 209 necessary, of paper forms) was required to retrieve and complete the relevant information. for the same reason, and because we wanted to provide a rapid response to concerns about 211 virus spread in the hospital, we were forced to limit the analyses to only a part of the work world health organization (who), covid-19 definitions key: cord-256429-ntx0eay0 authors: hao, w.; li, m.; huang, x. title: first atypical case of 2019 novel coronavirus in yan'an, china date: 2020-02-20 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.02.011 sha: doc_id: 256429 cord_uid: ntx0eay0 nan a 60-year-old man with a history of travel to wuhan, china, where the 2019 novel coronavirus (2019-ncov) has been spreading [1e3], presented to the emergency department with a 5-day history of unexplained fatigue. the patient's chest ct scan (january 23, 2020) demonstrated a patchy high-density shadow in both lungs (figs. 1a and c). however, the patient's oropharyngeal swab was negative for the 2019 novel coronavirus on the real-time reverse-transcription pcr assay. he was then admitted to the department of respiratory and critical care medicine for treatment. laboratory investigations illustrated that elevated blood levels for c-reactive protein (43.15 mg/l; normal range 0~10 mg/l), highsensitivity c-reactive protein (>5.0 mg/l; normal range 0~3 mg/l), and erythrocyte sedimentation rate (49 mm/h; normal range 0~20 mm/h). the white blood cell count (5.2 â 10 9 /l) and d-dimer (0.26 mg/l; normal range 0~0.5 mg/l) were normal. the lymphocyte count was slightly reduced at 1.16 â 10 9 /l (normal range 2.0~7.0 â 10 9 /l). after 5 days of treatment, a chest ct scan (january 28, 2020) was performed again to show patchy consolidation in the dorsal segment of the right upper lobe and lower lobe of both lungs, surrounded by ground-glass-like shadows, with grid shadows and bronchial inflation signs in the lesion (figs. 1b and d) . on january 29, 2020, another sample of the patient's oropharyngeal swab was taken for the 2019-ncov nucleic acid test, which this time showed a positive result. based on epidemiological characteristics, chest imaging, and laboratory findings, the patient was eventually diagnosed with 2019-ncov pneumonia. however, this case has many special features. first, there were no respiratory symptoms such as fever, cough, and sputum, and the first symptoms were only fatigue. second, the diagnosis of 2019-ncov pneumonia requires repeated nucleic acid testing. moreover, the rapid progression of chest imaging in the short term (<7 days) has critical diagnostic value for patients with negative 2019-ncov nucleic acid tests. the image showed multiple patchy consolidations with high-density shadows in the lower lobe of both lungs. (d) images obtained 5 days later showed partial absorption of the consolidation lesions in the right lower lobe, but fibrosis, bronchiectasis, and vascular thickening occurred. clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia w-dh and x-qh obtained and analysed the clinical data and produced the figure. all authors contributed to editing the figure and writing and editing the manuscript. written consent for publication was obtained from the patient. we declare no competing interests. key: cord-288425-po35m6d9 authors: albert, eliseo; torres, ignacio; bueno, felipe; huntley, dixie; molla, estefanía; fernández-fuentes, miguel ángel; martínez, mireia; poujois, sandrine; forqué, lorena; valdivia, arantxa; solano de la asunción, carlos; ferrer, josep; colomina, javier; navarro, david title: field evaluation of a rapid antigen test (panbio™ covid-19 ag rapid test device) for covid-19 diagnosis in primary healthcare centers date: 2020-11-13 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.11.004 sha: doc_id: 288425 cord_uid: po35m6d9 objectives: to our knowledge no previous study has assessed the performance of a rapid antigen diagnostic immunoassay (rad) conducted at the point of care (poc). we evaluated the panbio™ covid-19 ag rapid test device for covid-19 diagnosis in symptomatic patients (n=412) attended in primary healthcare centers. methods: rad was performed immediately after sampling following the manufacturer’s instructions (reading at 15 min.). rt-pcrs were carried out within 24 h. of specimen collection. samples displaying discordant results were processed for culture in vero e6 cells. presence of sars-cov-2 in cell cultures was confirmed by rt-pcr. results: out of 412 patients, 43 (10.4%) tested positive by rt-pcr and rad and 358 (86.9%) negative by both methods, showing discordant results (rt-pcr+/rad-) in 11 patients (2.7%). overall specificity and sensitivity of rapid antigen detection (rad) was 100% (95% ci, 98.7-100%), and 79.6% (95% ci, 67.-88.8%), respectively, taking rt-pcr as the reference. overall rad negative predictive value for an estimated prevalence of 5% and 10% was 99% (95% ci, 97.4-99.6%) and 97.9% (95% ci, 95.9-98.9), respectively. sars-cov-2 could not be cultured from specimens yielding rt-pcr+/rad-results (n=11). conclusion: the panbio™ covid-19 ag rapid test device performed well as a poct for early diagnosis of covid-19 in primary healthcare centers. more crucially, the data suggested that patients with rt-pcr-proven covid-19 testing negative by rad are unlikely to be infectious. objectives: to our knowledge no previous study has assessed the performance of a 24 rapid antigen diagnostic immunoassay (rad) conducted at the point of care (poc). we 25 evaluated the panbio™ covid-19 ag rapid test device for covid-19 diagnosis in 26 symptomatic patients (n=412) attended in primary healthcare centers. table 1 ). concordance between the two methods was good (κ, table 2 ). overall rad negative predictive value for an estimated prevalence of 5% and 10% (the 106 incidence of covid-19 in our health department during the study period was within 107 that range), was 99% (95% ci, 97.4-99.6%) and 97.9% (95% ci, 95.9-98.9), 108 respectively. rt-pcr c t values were significantly higher and sars-cov-2 rna loads 110 significantly lower (p <0.001) in rt-pcr+/rad-than in rt-pcr+/rad+ specimens 111 ( figures 1b and 1c) . roc curve analyses indicated that rt-pcr c t <25 and sars-range, 1-6 days) patients. all 11 specimens yielding discordant rt-pcr/rad results tested negative by culture, 120 whereas sars-cov-2 could be recovered from all 3 specimens returning rt-121 pcr+/rad+ results (c t : 4, 14 and 16). nevertheless, the overall sensitivity for the rad assay reported herein was much closer 134 to that (86.5%) found by linares and colleagues [3] . sensitivity of sars-cov-2 rad 135 assays has been reported to vary between 45%-97% [3-7], yet direct comparison 136 between studies is hampered by marked dissimilarities in patient clinical characteristics 137 and age, testing sites, type of specimen processed, and time to testing, among others. interestingly, sensitivity was higher in adults than in pediatric patients. previous studies 139 found no age-related differences in sars-cov-2 rna load in the upper respiratory tract [8] . although speculative, dating of symptoms onset could have been more 141 inaccurate in children than in adults. in a setting like ours with an incidence of covid-19 ranging between 5% and 10% at 143 the time of study, the rad npv was 99% (95% ci, 97.4-99.6%) and 97.9% (95% ci, 144 95.9-98.9), respectively. out of 54 rt-pcr positive specimens, 11 tested negative by rad. in line with 146 previous reports [2-4], sars-cov-2 rna load was significantly higher in rt-pcr+/ 147 rad+ specimens than in rt-pcr+/ rad-samples. in our setting, specimens with rt-148 pcr c t >25 (equivalent to sars-cov-2 rna loads < 5.9 log 10 copies/ml) returned 149 discordant rad/rt-pcr results. an important observation of our study was that sars-cov-2 could not be cultured 151 from rt-pcr+ (c t >25)/rad-specimens. along these lines, pekosz and colleagues [2] 152 found 1 out of 27 rad-/culture + specimens, using a highly sensitive cell culture 153 system (veroe6 tmprss2). the sars-cov-2 rna load threshold associated with 154 culture positivity herein (> 5.9 log 10 copies/ml) was remarkably close to other 155 previously published results (around 10 6 copies/ml) [2, [9] [10] [11] university hospital for their unwavering commitment in the fight against covid-19. 179 we would also like to thank maría josé beltrán, pilar botija and ana sanmartín for 180 assistance in organizing rad testing in primary healthcare centers. this work received no public or private funds. our group has received private funds for antigen-based testing but not real-time pcr correlates with sars-cov-2 virus 198 culture panbio antigen rapid test is reliable to diagnose sars-cov-2 202 infection in the first 7 days after the onset of symptoms low performance of rapid antigen detection test as 210 frontline testing for covid-19 diagnosis evaluation of a rapid diagnostic assay for detection 213 of sars-cov-2 antigen in nasopharyngeal swabs evaluation of 216 rapid antigen test for detection of sars-cov-2 virus cov-2 viral load in the upper respiratory tract of children and adults with early 220 acute covid-19 virological assessment of hospitalized patients with covid-2019 based virus isolation to evaluate potential infectivity of clinical specimens 226 tested for covid-19 key: cord-314808-ssiggi2z authors: pappas, g.; kiriaze, i.j.; giannakis, p.; falagas, m.e. title: psychosocial consequences of infectious diseases date: 2014-12-12 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2009.02947.x sha: doc_id: 314808 cord_uid: ssiggi2z historically, there has been an exaggerated fear related to infection compared to other conditions. infection possesses unique characteristics that account for this disproportionate degree of fear: it is transmitted rapidly and invisibly; historically, it has accounted for major morbidity and mortality; old forms re-emerge and new forms emerge; and both the media and society are often in awe. because, in an outbreak, the patient is both a victim and a vector, and because there exists the potential for infringement of personal rights in order to control an outbreak, infection may be viewed (and has been depicted in popular culture) as a foreign invasion. during recent outbreaks, fear, denial, stigmatization and loss have been recorded in the implicated individuals. stigmatization and discrimination may further involve ethical correlations, and attempts to adress these issues through activism may also have unwarranted effects. public health initiatives can address the public's fears by increasing health literacy, which can contribute to reducing stigmatization. a man watches the news and starts feeling anxious. his hands feel sweaty and his heartbeat increases. he experiences a sense of agitation as he hears of a possible bird flu pandemic. the media puts before him scattered images of people rushing to buy flu vaccines, discussions on the utility and potential shortage of antiviral agents, journalists reporting the death toll of the previous influenza pandemics, the hundreds of millions of birds slaughtered in southeast asia, the hundreds of millions of human victims expected worldwide, the extraordinary expense of the control of past outbreaks and the anticipated expense apparently needed to enhance preparedness. the man feels overwhelmed by the amount of information. in a nearby hospital, a nurse in the emergency department thinks of asking for a long-term leave because she wants to be absent when an outbreak emerges; she is thinking of her family and feels she is unqualified to deal with, and not secured against, morbid infection. an infectious diseases specialist is on a plane, returning from an international congress on infectious diseases; several hours earlier, he attended a lecture about the then evolving severe acute respiratory syndrome (sars) outbreak. now, a chinese passenger sits in a nearby seat; the specialist is transiently overwhelmed by fears: what if this person is a carrier; what should one do if this fellow passenger coughs? later, he manages to reassure himself. these three individuals, among many others, experience levels of fear associated with infectious diseases in their everyday lives. they share the anxiety, the uncertainty, and the potential for irrational behavior due to fear of an unknown disease. they suffer from 'germ panic' [1] . infectious diseases have had a significant role in shaping human history, and are responsible for, through the great plagues of the past, more deaths than any other human pathology [2] ; these outbreaks have engraved an automatic response in our subconscious of a fear of infection. in an era of major scientific progress in battling, and even eliminating, certain infections, this fear may seem unwarranted. yet 'germ panic' consistently re-emerges, in contrast to the fear related to more burdensome entities, in terms of mortality, such as cardiovascular disease. why is it that infectious diseases cause the most significant psychological unrest, both in the public and in health professionals alike? infection is: (i) transmissible, (ii) imminent and (iii) invisible. moreover, the field of infectious diseases is ever-expanding. the risk of cardiovascular disease is a recognized entity with predisposing factors that have changed little over the years. on the other hand, numerous new major threats have emerged during the last three decades; the pandemic of aids, the sars outbreak, the ominous scenarios of an avian influenza pandemic, and the threat of biological weapons are just some examples explaining the concern among health authorities, the media, and the public. the evolution of the 'global village' further enhances the fear of contracting exotic diseases that can be imported into metropolitan areas (e.g. the chikungunya virus) [3] , diseases that can be transmitted in the context of air travel [4, 5] , or simply diseases that emerge in new areas as a result of nature's peculiar ways (e.g. the west nile virus new world epidemic) [6] . fear, in strict neuropsychological terms, is a normal reaction to an evolving threat, preparing the individual, both physically and mentally, for an acute response to possible harm. this reaction, however, is triggered both in the cerebral cortex, the outcome of a rational mental approach to the present situation, and by the amygdala, a process generated earlier than the cortical one, which is subconscious and potentially irrational, often crossing the barrier to panic. there are numerous exogenous factors that shape the nature of this subconscious response. the psychological response of both patients and the public to the threat of infection has been evaluated with respect to numerous circumstances in recent years, not only acute outbreaks such as sars, but also gradually evolving pandemics such as aids, threats with marginal risk for humans such as bovine spongiform encephalopathy (bse; mad cow disease), and even threats that are only theoretical such as avian influenza. moreover, inordinate psychological responses to infection have been recorded in the context of epidemics. for which an unidentified, readily transmissible agent with high mortality was responsible. fear, denial and frustration, which comprise three sequential stages of the rational response to fear, have been reported as predominant among patients or quarantined individuals during the sars outbreak in canada and amoy gardens in hong-kong [7] [8] [9] . loss and a conflict between duty to the patient and the will to be with one's family have been recorded in health care workers quarantined during the sars outbreak in canada [10] . anxiety extends (in the case of patients and exposed persons) beyond the physical consequences of infection, to social consequences such as stigmatization, with the latter even extending to asian populations of non-endemic regions such as new york's chinatown [11] . a similar case of stigmatization during an acute outbreak was also racially orientated: in the us 1993 hantavirus outbreak, the native american indians were stigmatized by the term 'navajo disease', a term which ignored the fact that non-navajos were also becoming ill; as a result, 'anti-indian racism mixed with fears of disease' emerged [12] . the potential effect of psychological reactions was also exemplified in the 1994 plague outbreak in surat, india, which led to an extended official and unofficial quarantine, with stigmatization being disproportionate to the extent of the outbreak [13] . in the case of an unknown agent, a lack of preparedness on the part of medical authorities and misleading information reproduced by the media may further aggravate these pathological psychological responses. in the sars epidemic, both these factors have been recognized, and media miscommunications and inconsistent health policies have been highlighted as factors amplifying stigmatization in hong kong [7] . medical authorities can also inadvertently augment a problem by initiating and recycling fear. apart from the awe-inducing isolation procedures, devices and uniforms (with the latter being reminiscent of astronauts and the concept of alien invasion), the medical disputes over preventive and therapeutic strategies may perpetuate fear when made public. 2 psychosocial reactions in gradually evolving epidemics: the case of aids: the aids pandemic was also attributed to a hitherto unknown agent, but significant differences contributed, in part, to shaping the psychological response of both patients and the public. the aids pandemic developed over a period of years, instead of days, and it was related to sexual practices, further influencing public response. the initial stages of the disease, however, were reminiscent of the 'navajo disease', in that a marginalized population was targeted and stigmatized. however, the history of aids highlights the fact that such discrimination continues to exist, and targeted populations are marginalized through germ panic. activism here acts like a double-edged sword; it fights discrimination and augments public health literacy, but may also enhance fear [1] . attempts to raise awareness of an issue may be subject to media misinterpretation; continuous discussion of an issue may raise awareness, but also may raise the sense of threat in individuals who are inadequately informed. although the psychological responses to some extent reflect the epidemic, the aids story exemplifies that responses also reflect the content of public education campaigns and public health efforts, as well as media and news coverage [14] . the surgeon general's aidsrelated campaign in the usa took place in 1988, comprising the first official nationwide effort to promote risk reduction or even explain the mechanics of hiv transmission. it is worth noting here that a pamphlet by callen and berkowitz entitled 'how to have sex in an epidemic', produced by several gay activists, was distributed in 1983, 5 years ahead of the surgeon general's campaign, to help sort through the confusing information concerning the new epidemic and the divergent theories regarding the cause of the syndrome [15] . as a result, the epidemic was better understood among the gay community, regardless of the officials' silence, which left the rest of the population uninformed for a protracted period. a similar observation was made in israel in a region that was affected by poultry avian influenza; the residents of this area had a significantly greater understanding compared to residents of the rest of the country [16] . awareness is a key issue, particularly when there is ample time for it to be enhanced. 3 fear of forthcoming epidemics: the case of avian influenza, mad-cow disease, and more to come: fear may be a physical response leading to individual protection, but, sometimes, protective measures undertaken according to public initiative can lead to increased morbidity because of the protective measures themselves rather than the threat against which they were supposed to be protective [17] . in the case of both bse and the, only now gradually subsiding, avian influenza pandemic scenarios, a common denominator was the climax of the threat, with the mass media capturing the public's attention, classically highlighting the subconscious, and memories of the great epidemics of the past (e.g. the 1918 spanish influenza pandemic). in the case of bse, fear rapidly extended to other countries [18, 19] and continents [20] [21] [22] , aided by coverage of the subject in well respected journals of medical and general interest; in the latter case, with eye-catching titles such as 'can it happen here?'. in one french study [19] , the perceived risk of bse (which is significantly different to the actual risk) modified the public's approach towards meat consumption, although this modification of the peoples' cognitive and affective responses to hazard peaked rapidly and subsided in approximately 1 year. in the case of avian influenza, a similar 'vaccination panic' that rapidly subsided was recorded in greece [23] , underlining the distorted ways in which the public reacts when overwhelmed by information. the psychosocial effect of misconceptions about the disease was also demonstrated in israel, where the public had a distorted perception of the dynamics of human-to-human transmission [16] . the way that the media and scientists present relevant information can also account for this effect [24] . 'scare statistics' and imaginary titles in the news all contribute to arouse the subconscious perception of threat; although some have proposed the use of fear as an educational tool, behavioral effects in this case have not been demonstrated [25] . regarding avian influenza, fear extends to hospital personnel and the public alike [26, 27] , and cannot be underestimated. a study conducted in hong kong showed that the majority of the public would expect panic or other forms of stressrelated responses to emerge [28] , as well as a potential for stigmatization [13] . 4 fear of infection in non-epidemic situations: people continue to use antibiotics, even when advised against doing so, for numerous respiratory tract infections of obvious viral origin. patients fear that they may develop pneumonia and overestimate the morbidity, even the mortality, related to their symptoms. infection is often considered as a social issue that indirectly leads to stigmatization, as in the case of brucellosis, where patients may express denial, because of a correlation of the infection with a lower socio-economic status (i.e. an indirect form of stigmatization) [29] . this has been the case also for outbreak-causing diseases in the aftermath of the outbreak, as with bse, where protective measures have been dismissed by many uk farmers as potentially stigmatizing individual farmers in terms of 'bad practicing' [30] . fear develops in public and refers to the society. its evolution is not a strict medical process of the nervous system, but the result of a complex interplay of medical and social factors and forces. fear of infection is not only engraved in our subconscious as a result of memories of former epidemics, but also because of fictional dramatizations of such potential threats. the way that infectious diseases are presented in the cinema is a typical example and can influence society's perceptions [31] . the concept of an unseen foreign invasion, the numerous apocalyptic views of the end of the world as a result of an unknown virus, and the scenes of panic, are all derived from public fears and they concomitantly, via feedback, shape these fears. mass media is another major factor that shapes the physical and psychological response of the public to an infectious disease threat, as depicted in numerous attack scenarios in the literature [32] [33] [34] . a simulation of a q fever outbreak in spain after deliberate release highlighted such potential: one journalist retrieved a medical report of person-to-person transmission of the disease; the public was already informed that such transmission is not possible; some journalists accused the scientists of hiding the truth; the public felt misinformed by the scientific community. and this was a scenario focusing on an agent of limited mortality [34] . it would be unfair to judge the public as a homogenous group; the public is a coalition of numerous subgroups of individuals, with vastly different social, educational and economical backgrounds. one would expect these subgroups to face threats of infection in different manners. for example, a higher educational background should theoretically be related to lower levels of fear; on the other hand, it may be related to increased access to information in general and to medical advice, and thus to increased individual participation in the development of the perception of 'threat'. these differences in the perception of disease in general, and infection in particular, among individuals of different social, economical and educational status have not been adequately evaluated. a series of ethical dilemmas applies to the control of infectious diseases, and these dilemmas further serve to enhance the fear of infection. the typical ethical dilemma is the conflict between feelings and decisions [35] ; in an outbreak, the patient is a victim, but also a vector, and isolation and quarantine practices may make stigmatization unavoidable. a recent statistical model has focused on the effect of individual psychological responses during the outbreak itself; fear induces a 'fight or flight' response, flight in this case predisposing to outbreak spread [36] . control of a large-scale infectious disease outbreak may often demand the infringement of individual liberties and civil rights [37] . these ethical dilemmas extend beyond the actual nature of the disease and its psychological consequences, and may implicate the means and content of public communications [38] , from authorities and the media, during an outbreak (i.e. how much actual information can the public handle without going into panic, and where does the thin line between the right to know and panic lie in this case). these recently observed psychosocial responses are not unique. we not only have re-emerging diseases, but also re-emerging responses to disease. the equivalent of the famous plague doctor mask of the 1600s in venice is the white surgical mask worn during recent epidemics. public health initiatives can address the public's fears by increasing education about a disease. enhanced health literacy, along with wide-ranging access to health information, can contribute to early case detection and may be useful in reducing stigma and decreasing levels of fear of an illness. the making of a germ panic, then and now emerging infections: a perpetual challenge cases of chikungunya fever imported from the islands of the south west indian ocean to transmission of infectious diseases during commercial air travel contact tracing of passengers exposed to an extensively drug-resistant tuberculosis case during an air flight from beirut to paris west nile virus the experience of sars-related stigma at amoy gardens fear and stigma: the epidemic within the sars outbreak risk perception and compliance with quarantine during the sars outbreak the psychosocial effects of being quarantined following exposure to sars: a qualitative study of toronto health care workers s chinatown: the politics of risk and blame during an epidemic of fear the coming plague: newly emerging diseases in a world out of balance stigma in the time of influenza: social and institutional responses to pandemic emergencies public health communication: evidence for behavior change fatal advice: how safe-sex education went wrong differences in public emotions, interest, sense of knowledge and compliance between the affected area and the nationwide general population during the first phase of a bird flu outbreak in israel suffocation from misuse of gas masks during the gulf war bse fears stir the swiss risk perception of the 'mad cow disease' in france: determinants and consequences fear of bse risks could hit us blood banks can it happen here? panic over mad cow had already infected europe. now it's our turn japan's first bse case fuels fears elsewhere reaction to the threat of influenza pandemic: the mass media and the public avian flu: the creation of expectations in the interplay between science and the media effects of episodic variations in web-based avian influenza education: influence of fear and humor on perception, comprehension, retention and behavior a crisis: fear toward a possible h5n1 pandemic survey of hospital healthcare personnel response during a potential avian influenza pandemic: will they come to work? perceptions related to human avian influenza and their associations with anticipated psychological and behavioral responses at the onset of outbreak in the hong kong chinese general population health literacy in the field of infectious diseases: the paradigm of brucellosis an exploration of the drivers to bio-security collective action among a sample of uk cattle and sheep farmers infectious diseases in cinema: virus hunters and killer microbes smallpox: an attack scenario attack scenarios with rickettsial species: implications for response and management q fever in logrono: an attack scenario are there characteristics of infectious diseases that raise special ethical issues? coupled contagion dynamics of fear and disease: mathematical and computational explorations ethics and infectious disease guilt, fear, stigma and knowledge gaps: ethical issues in public health communication interventions the authors state that there were no sources of funding for the present study. the authors have no conflicts of interest to declare. key: cord-294392-a8s66g96 authors: zhang, shuai; guo, mengfei; wu, feng; xiong, nian; ma, yanling; wang, zhihui; duan, limin; chen, lan; ouyang, haixia; jin, yang title: factors associated with asymptomatic infection in health-care workers with sars-cov-2 infection in wuhan, china: a multi-center retrospective cohort study date: 2020-09-07 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.08.038 sha: doc_id: 294392 cord_uid: a8s66g96 objectives: we aim to describe the fraction of asymptomatic health-care workers (hcws) in two designated hospitals for covid-19 treatment in wuhan and explore the factors associated with asymptomatic sars-cov-2 infection. methods: all hcws in wuhan union hospital and wuhan red cross hospital with either positive sars-cov-2 nucleic acid or antibody test before april 18, 2020 were included. exposure, epidemiologic, demographic information were retrospectively collected by a structured questionnaire. medical records were also reviewed for clinical characteristics and ct images in hcws. results: as of april 18, 2020, a total of 424 hcws were identified. among them, 276 (65.1%) were symptomatic and 148 (34.9%) were asymptomatic. 55 (19.9%) families of the symptomatic hcws and 16 (10.8%) families of the asymptomatic hcws were infected with sars-cov-2. hcws with infected family members tend to be symptomatic cases (odds ratio [or], 2.053 [95% confidence interval (ci), 1.130-3.730]; p=0.018). multivariable logistic regression analysis exhibited that performing tracheal intubation or extubation (or, 4.057 [95% ci, 1.183-13.909]; p=0.026) was associated with an increased likelihood of symptomatic sars-cov-2 infection, while consistent use of n95 respirators (or, 0.369 [95% ci, 0.201-0.680]; p=0.001) and eye protection (or, 0.217 [95% ci, 0.116-0.404]; p<0.001) were associated with an increased likelihood of asymptomatic sars-cov-2 infection. conclusions: asymptomatic sars-cov-2 infection in hcws occupies a considerable proportion during the pandemic of covid-19. those who have performed tracheal intubation or extubation were most likely to develop related symptoms, while those taking aggressive measures including consistent use of n95 masks, and eye protection tended to be asymptomatic cases. the ongoing outbreak of 2019 novel coronavirus disease (covid-19) has been reported in wuhan, china and spread rapidly across the world. 1,2 health-care workers (hcws) have been at high risk for the rna extraction, rt-pcr assay and antibodies test were done according to the previous study. 9 . total rna was extracted from nasopharyngeal and/or oropharyngeal swab samples of hcws within 2 79 hours using the respiratory sample rna isolation kit (xi'an tianlong science and technology co.,ltd. and guangzhou heas biotech co., ltd. ). rt-pcr assay was performed by using detection kit for 81 sars-cov-2 rna (wuhan easydiagnosis biomedicine co., ltd. and daan gene co., ltd. of sun yat-sen university). the serum sars-cov-2 antibodies were detected using the diagnostic kit for igm 83 / igg antibody to sars-cov-2 (zhuhai livzon diagnostics inc.) isolation wards, fever outpatient clinics, emergency departments, icu, medical laboratories, and hypertension, cardiovascular disease, diabetes, cerebrovascular disease, chronic obstructive pulmonary 90 disease (copd), chronic kidney disease, chronic liver disease, rheumatic diseases and malignancy. hair cover and eye protection) were available for the next step. a previous study had shown younger 117 age was associated with asymptomatic sars-cov-2 infection. 8 another study showed that age and 118 comorbidities were predictors for symptom development in the initially asymptomatic carriers at 119 admission. 12 two studies showed that tracheal intubation procedure was related to increased risk of 120 transmission sars-cov-1 and sars-cov-2 to hcws. 13,14 face mask and eye protection especially 121 n95 respirators were most consistently with reduced covid-19 infection among hcws. 14,15 face 122 mask was also reported to be associated with asymptomatic sars-cov-2 infection based on several 123 uncontrolled reports. 16 therefore, we chose age, comorbidities, tracheal intubation or extubation, n95 124 respirators, eye protection as the five variables for our multivariable logistic regression model and were 125 adjusted by using an enter approach. in the analysis, frequency of using ppe and frequency of hands hygiene practice were coded into 2 127 categories: used inconsistently (i.e., "never or sometimes used") or used consistently ("used most or all ci, 1.065-2.704]; p=0.026) while health-care assistants were more likely to be asymptomatic ( the comparison in high-risk procedures and infection protective measures between asymptomatic 163 hcws and symptomatic hcws are shown in table 3 . no significant differences were seen in any 164 high-risk procedures between asymptomatic hcws and symptomatic hcws. compared with 165 symptomatic hcws, asymptomatic hcws more consistently used hand washing, isolation gown, eye 166 protection, n95 respirators, gloves, and hair cover for protection (p<0.001). we also analyzed the factors associated with asymptomatic infection among hcws by using logistic 168 regression model. the univariable logistic regression analysis showed worked in high-risk departments, 169 hand washing consistently and consistent use of isolation gown, n95 respirators, gloves, hair cover, 170 and eye protection were associated with an increased likelihood of asymptomatic infection (table s1) . however, evidences about the differences between symptomatic and asymptomatic individuals with to be associated with an increased likelihood of symptomatic sars-cov-2 infection, while consistent 184 use of n95 respirators and eye protection tended to be asymptomatic infection. all these findings 185 suggested a potential relationship between initial infectious dose and disease severity. masks do more than protect others during covid-19: 286 reducing the inoculum of sars-cov-2 to protect the wearer covid-19: four fifths of cases are asymptomatic, china figures indicate estimating the asymptomatic proportion of coronavirus 291 disease 2019 (covid-19) cases on board the diamond princess cruise ship estimation of the asymptomatic ratio of novel coronavirus 294 infections (covid-19) screening of healthcare workers for sars-cov-2 highlights 296 the role of asymptomatic carriage in covid-19 transmission development and validation of a risk factor-based system to 299 predict short-term survival in adult hospitalized patients with covid-19: a multicenter, retrospective, 300 cohort study factors associated with hospital admission and critical illness we thank all the hcws fighting covid-19 in the front-line. they are putting key: cord-276212-ys5njiw0 authors: wei, l.; chan, k.-h.; ip, d.k.m.; fang, v.j.; fung, r.o.p.; leung, g.m.; peiris, m.j.s.; cowling, b.j. title: burden, seasonal pattern and symptomatology of acute respiratory illnesses with different viral aetiologies in children presenting at outpatient clinics in hong kong date: 2015-05-30 journal: clin microbiol infect doi: 10.1016/j.cmi.2015.05.027 sha: doc_id: 276212 cord_uid: ys5njiw0 respiratory viruses cause acute respiratory diseases with a broad and overlapping spectrum of symptoms. we examined the clinical symptoms and explored the patterns of various respiratory viral infections in children in hong kong. among 2090 specimens collected from outpatient care (2007–2010), 1343 (64.3%) were positive for any virus by the xtag assay, and 81 (3.9%) were positive for co-infection. the most frequently detected viruses among children aged 6–15 years were enterovirus/rhinovirus and influenza virus a, whereas most non-influenza viruses were more frequently detected in younger children. higher body temperature was more common for illnesses associated with influenza viruses than for those associated with non-influenza viruses, but other symptoms were largely similar across all infections. the seasonality pattern varied among different viruses, with influenza virus a being the predominant virus detected in winter, and enterovirus/rhinovirus being more commonly detected than influenza virus a in the other three seasons, except for 2009. acute respiratory illness (ari) represents an important cause of hospitalization and death in all age groups worldwide [1] . although aris can be caused by a wide range of different respiratory viral pathogens, ascertainment of the exact causative agents is rarely clinically indicated, and is thus not routinely performed. among the small proportion of patients needing hospitalization, significant disease burdens have been attributed to adenovirus (adv) in children, respiratory syncytial virus (rsv) and influenza virus a (ifva) in all age groups, and rhinovirus (rhv) and parainfluenza virus 3 (piv 3) in children and the elderly [2] . however, the full spectrum of disease burden among the majority of patients with aris presenting in community outpatient settings has remained largely elusive. seasonal patterns of aris caused by influenza virus [3] and rsv [4] have been better described in some geographical areas, but those of most other respiratory viruses remain poorly understood. the generally overlapping spectrum of non-specific symptoms makes it very difficult to distinguish between infections with different respiratory viruses [5] . a better understanding of their differential symptom patterns may help to identify cases that are more likely to be influenza virus infections, and thus may benefit clinically from specific antiviral treatment. the hong kong special administrative region is situated in the northern hemisphere, and has a subtropical climate, with an annual variation in temperature from 14.5-18.9°c in january and february to 26.2-31.4°c in june and july, and a mean relative humidity from 69-74% in december and january to 83% in march and april. in this study, we aimed to investigate the burden of aris caused by different respiratory viral pathogens among children aged 15 years in a community outpatient setting, to describe their seasonal patterns of occurrence, and to characterize their clinical characteristics at presentation. sources of data as part of a larger study on transmission of influenza viruses in households, we recruited patients from primary-care outpatient clinics in private and public sectors across hong kong who met our inclusion criteria, including: (a) being a hong kong resident; (b) presenting with at least two symptoms of ari, including a body temperature of 37.8°c, headache, sore throat, cough, runny nose, sputum, and myalgia; (c) onset of symptoms within the preceding 48 h; and (d) living in a household with at least two other people, none of whom had reported ari in the preceding 14 days. all consenting subjects completed a short data collection form, and had two sets of pooled nasal and throat swab specimens collected by a trained nurse. one specimen was stored immediately in viral transport medium for subsequent virological testing; the other specimen was tested on site with the quickvue influenza a + b rapid diagnostic test (quidel, san diego, ca, usa). subjects with a positive rapid test result and their household contacts were further followed up [6] , but, in the present analysis, we also analysed laboratory results from the other specimen from all subjects, regardless of their rapid test result. proxy written informed consent was obtained for all participants from their parents or legal guardians, with additional written consent being obtained from those aged 8-16 years. the study protocol was approved by the institutional review board of hong kong university. weekly meteorological data, such as temperature, humidity, and precipitation, were obtained from the hong kong observatory. each pooled nasal and throat swab specimen was stored in viral transport medium (5% bovine serum albumin in earle's balanced salt solution with antibiotic), kept at 2-8°c immediately after collection, and cryopreserved at −70°c within 36 h. the specimens were tested for eight common respiratory viruses (including types and subtypes), namely ifva (subtypes h1 and h3), influenza virus b (ifvb), rsv (subtypes a and b), piv (types 1-4), metapneumovirus (mpv), enterovirus (env)/rhv, adv, bocavirus (bov), and coronavirus (cov) (types nl63, hku1, 229e, and oc43), with the xtag rvp fast version 2.0 multiplex assay (luminex molecular diagnostics, toronto, ontario, canada), and this was followed by product detection and identification with a luminex suspension microarray [7] . total nucleic acid was extracted from the clinical specimens with the nuclisens easymag extraction system (biomerieux, zaltbommel, the netherlands), according to the manufacturer's instructions. the extracted nucleic acid was tested for respiratory viruses. detection rates (and co-detection rates for co-infection) of each virus, stratified by age group (0 -5 years and 6-15 years), were calculated by dividing the number of specimens positive for the corresponding virus by the total of positive specimens in that age group. symptomatology was examined by comparing clinical symptoms of different respiratory virus infections by the use of pearson's chi-square test (χ 2 ) or fisher's exact test (fe). logistic regression was used to examine the association of different symptoms with influenza or non-influenza virus infection. to assess the seasonal pattern, percentages of different positive specimens across different seasons were compared by use of the chi-square test or fe. here, we defined winter as december to february, spring as march to may, summer as june to august, and autumn as september to november. also, logistic regression was used to assess the association between virus detection and meteorological factors, including temperature, absolute humidity, and precipitation. all statistical analyses were performed with r version 2.15.0 (r foundation for statistical computing, vienna, austria). . the detection rate was significantly different across the 4 years, with the highest rate being in 2009, which might due to the pandemic h1n1 ifva. there was no difference in sex distribution across the years, whereas there was a significant difference in age group percentage across years, with a higher percentage of children in the older age group (6-15 years) than in the younger age group (<6 years) for the first 3 years (2007-2009) but not for 2010 (table s1 ). there were 1343 (64.3%) specimens positive for at least one of the respiratory viruses, 81 (3.9%) specimens positive for more than one respiratory virus, and two specimens positive for three respiratory viruses. overall, env/rhv (19.6%) and ifva (23.4%) were the two most frequently detected viruses throughout the 4 years. on comparison of the two age groups, viral aetiology was detected significantly more frequently in the younger age group than in the older age group (table 1) . specifically, ifva and ifvb were detected significantly more frequently in the older age group, whereas most of the other non-influenza viruses were more frequently detected in the younger age group, with the exception of cov, which was detected at similar frequencies in the two age groups. we further stratified the analysis by separating 2009 from the other 3 years, to determine whether the occurrence of the pandemic h1n1 ifva in 2009 had affected the seasonal pattern of other common respiratory viruses. on comparison with the other 3 years with usual seasonal influenza activities, the detection pattern of other respiratory viruses during 2009 was broadly similar, with most non-influenza viruses being more frequently detected in the younger age group. important exceptions included cov and ifva, which were more frequently detected in the older age group, and ifvb and bov, for which no significant difference was seen between the two age groups for 2009 (table s2 ). adv was more frequently detected in the younger age group in 2009, but not in the other 3 years. co-detection of more than one virus was, overall, more frequent in the younger age group than in the older age group (5.6% vs. 2.8%) ( table 1) . for specific viruses, this mainly involved co-detection of env/rhv, rsv, piv, and bov. after stratification by pandemic (2009) and other years, the codetection pattern was generally preserved, with overall codetection and co-detection of bov being significant in 2009 but not in the other years, and co-detection of piv not being significantly different for all years (table s2) . among children with positive viral detection, the most frequently reported clinical symptoms at presentation included cough (81.8%), runny nose (83.4%), fever (59.2%), and sputum (55.2%) (data not shown). on comparison of infections with different viruses detected (bov was excluded here, because there was no detection of bov in the older age group), fever was reported significantly more often for ifva (83.2% and 79.7%), ifvb (80.6% and 82.2%) and adv (82.8% and 75.0%) than for the other viruses, in both age groups (χ 2 , p < 0.001) ( table 2 ). cough was more frequent for mpv (100%), bov (100%) and rsv (96.7%) in the younger age group, and for mpv (97.6%) and ifva (85.5%) in the older age group (fe, p < 0.001). the presence of a runny nose was most frequent for env/rhv in the younger age group (fe, p 0.003), but was not significant in the older age group. sputum was most frequently reported for rsv (72.2%) in the younger age group, and for mpv (81.0%) in the older age group (χ 2 , p < 0.001). results from logistic regression suggested that the occurrence of fever was associated with a significantly increased likelihood of influenza virus (ifva and ifvb) infection vs. infection with other respiratory viruses (or 4.78, 95% ci 2.96-7.74) in the younger age group. cough, runny nose and sputum were also associated with an increased likelihood of influenza virus detection in the older age group (table s2 ). fig. 1 shows the percentages of all positive specimens attributable to each viral pathogen during different seasons in 2009 and other years. ifva and env/rhv were generally the two most frequently detected pathogens in all seasons. ifva was most frequently detected in winter, both for 2009 (57.3%, p < 0.001) and for the other years (31.3%, p < 0.005). in fact, in 2009, when the pandemic h1n1 ifva was predominant, it was responsible for the vast majority of all positive specimens (30.2-57.3%) in different seasons. env/rhv was next most frequently detected virus over the whole year, particularly in spring and autumn. rsv was more frequently detected in nonwinter seasons (12.7%) during all years, but this was less marked in 2009. for other viral pathogens, the seasonal patterns were similar and not significantly different. for various meteorological factors, logistic regression results showed that influenza virus was negatively associated with temperature, whereas rsv was positively associated with temperature. env/ rhv was positively associated with precipitation, and none of the other viruses was significantly associated with any of the three meteorological factors (table s3 ). previous studies have shown the substantial disease burden due to hospitalization or mortality associated with influenza virus in hong kong [8, 9] . our present study further highlighted the important contribution of various non-influenza viruses (env/ rhv, mpv, rsv, piv, etc.) to the disease burden in the local community outpatient setting, especially in the younger children aged <6 years, thus giving a more comprehensive picture of the overall disease spectrum caused by these common respiratory viruses. env/rhv and ifva were the two most frequently identified viruses in both age groups. generally, most non-influenza viruses were more frequently detected among preschool children (aged 0-5 years) than among older children (aged 6-15 years), except for cov, and this is consistent with another study [10] . the co-detection rate was also higher in the younger age group, which is consistent with other studies [11] . co-detection of more than one respiratory virus has been frequently reported; however, the clinical significance of codetection is unclear, with previous studies giving conflicting results regarding whether co-infection is associated with an increase in the severity of related respiratory disease [12] [13] [14] [15] [16] . although it is difficult to differentiate between different respiratory viral infections on the basis of clinical symptomatology alone, our results suggested that a number of symptoms were more commonly reported by patients with influenza virus infections than by patients with infections caused by other respiratory viruses. our study showed that fever was associated with a significantly increased likelihood of influenza virus infection in children aged 0-5 years, whereas fever, cough, runny nose and sputum were also associated with influenza virus infection in children aged 6-16 years. this finding is generally consistent with similar findings of other studies on epidemic influenza [17] . seasonality patterns always vary with different viruses and regions. despite the fact that most previous studies have indicated a predominantly winter and autumn seasonality for influenza virus and env/rhv [18, 19] , we found that both viruses were rather commonly detected over the whole year, with a significant predominance of ifva in the winter for all of the study years. in 2009, when the h1n1 ifva was predominant, it was responsible for the majority of positive specimens in all seasons. rsv was more frequently detected in non-winter seasons, and was less marked in 2009. another study found that rsv activity peaked in july and august, and was positively associated with temperature [20] . the seasonality pattern of an autumn peak for cov-nl63 infections reported by a previous study, however, could not be demonstrated in this study, as all covs were analysed together, without species identification [21] . although the findings regarding associations with temperature or precipitation may help to shed some light on the observed seasonality patterns of different infections, their underlying mechanism remaines largely controversial [19, 22] . in conclusion, our findings suggest that, although the disease burdens of influenza virus infection in terms of hospitalization and mortality are important, they reflect only the tip of the iceberg, and also understanding the wider spectrum of disease burden in terms of outpatient service attendance and morbidity will always be important. the substantial burden of respiratory infection caused by viruses other than influenza viruses shows the importance of improved surveillance and preventive efforts in the community setting. further studies are also important to better characterize the burden of these non-influenza viruses from other perspectives, including morbidity and school absence among children. a better understanding of the seasonality patterns of these common respiratory viruses would also help to inform better disease surveillance, to inform proper healthcare action planning and decisions. the global burden of disease: 2004 update disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (daly) model influenza seasonality: lifting the fog respiratory syncytial virus-a comprehensive review 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bronchiolitis comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children rate and influence of respiratory virus co-infection on pandemic (h1n1) influenza disease predicting influenza infections during epidemics with use of a clinical case definition rhinovirus infection in hospitalized children in hong kong: a prospective study seasonal influenza activity in hong kong and its association with meteorological variations epidemiology of respiratory syncytial virus infection among paediatric patients in hong kong: seasonality and disease impact human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong are meteorological parameters associated with acute respiratory tract infections? the authors declare that they have no conflicts of interest. supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.cmi.2015.05.027 key: cord-009675-utz0iazs authors: madeley, c. r. title: are point‐of‐care (poc) virological tests what is needed? date: 2007-06-05 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2007.01738.x sha: doc_id: 9675 cord_uid: utz0iazs point‐of‐care (poc) tests are becoming more available, although the way in which they should be used is currently undecided. any ‘laboratory’‐based diagnosis of respiratory infections has three components: the specimen taken, the test used, and the interpretation of the results. each of these components needs to be carefully addressed when using poc tests for the diagnosis of respiratory tract infections. given the enthusiasm with which poc tests are being developed, it is likely that they will be used more and more widely. if so, the advantages and limitations of their use should be fully discussed and the implications recognised. in this issue of cmi, weitzel and colleagues from berlin present data concerning the use of a pointof-care (poc) test for the detection of influenza a and b viruses [1] . this and other similar tests are intended to be used by clinicians at the bedside to allow patient management and treatment decisions to be made very rapidly, especially with respect to returning travellers who may be carrying, e.g., new pathogenic strains of pandemic influenza virus. the test evaluated by weitzel et al. [1] shows adequate specificity (the positives were, except for a small number, correct with only one false-positive) for both viruses, but the sensitivity was low. only about two-thirds of the individuals who were positive according to pcr assays or culture were positive according to the poc test. this is worrying if this and other similar tests (http://www.who.int/csr/disease/avian_ influenza/guidelines/rapid_testing/en) are promoted for widespread use as front-line tests for identifying influenza in febrile travellers returning from foreign countries, which is an approach that has been recommended by the who (http:// www.who.int/csr/disease/avian_influenza/guide lines/rapid_testing/en). any 'laboratory-based' diagnosis of respiratory infections, bedside or otherwise, has three components: the specimen taken from the patient; the test used; and the interpretation of the result of the test. all these components must be of a standard good enough for the task, but the quality of the specimen is crucial. a poorly taken specimen containing no or insufficient virus material cannot yield a positive result, even if the patient is, indeed, infected. inevitably, such a specimen will give a false-negative result. where the patient may be bringing a novel infection into a country or community, this is potentially disastrous and negates the purpose of using a poc test. all diagnostic virologists are aware of the difficulties of getting good respiratory specimens. as few patients enjoy having swabs or aspirators inserted into their nose or nasopharynx, it is much, much easier to take a bad specimen than a good one. missing from almost all tests, poc or traditional, is any form of marker to indicate whether the specimen contains sufficient material to make the result reliable, particularly when apparently negative results are obtained. only immunofluorescence (if) provides this vital feedback at present [2] -if there are no ciliated respiratory cells visible in the preparation examined in the microscope, the microscopist knows that the specimen is unsuitable, either because it has been inadequately taken or because it has been mishandled during preparation. poc tests have no such safeguard, and the poor sensitivity reported by weitzel et al. [1] may have been caused, at least in part, by poor specimens having been taken. tellingly, the authors comment that the positivity rate was higher with more floridly ill patients and with children, from both of whom it may have been easier to get a competent specimen. this is not a transient problem. diagnostic virologists know that getting good, consistent respiratory specimens is a battle that is never won. non-virologists rarely understand the problem in detail, and the continuing successful collection of specimens at the bedside relies heavily on (often indignant) feedback from the laboratory. moreover, no sooner is one set of nurses or junior doctors well-trained in how to collect a good specimen than staff rotation takes the situation back to square one. commercially produced tests have not hitherto included a marker to assess specimen quality, and perhaps the manufacturers should be more aware of this deficiency. it does not matter how well the actual test performs if it does not tell the user that time is being wasted on a useless specimen, and that the ensuing result (if negative) cannot be relied upon. dipstick-type tests have been available to detect protein, sugar, haemoglobin, etc. in urine for many years. in contrast to respiratory tract specimens, urine is (comparatively speaking) easy to collect and much more standard than the respiratory equivalents-if it looks like and smells like urine, that is what it is likely to be. a swab, or even an aspirate, is not so informative, and the available quantity is much smaller. bacteriological swabs are less critical because culture is relatively fast and can compensate better for a minimal specimen. another limitation of the specific poc test evaluated by weitzel et al. [1] is that, at present, it detects only influenza a or b viruses; other viruses also cause very similar syndromes [3] . the ability to detect a different aetiological cause (because dual infections are generally rare, at least in adults) is more useful than a test that is simply negative for influenza. not influenza virus? then what is the patient suffering from? any test that concentrates on one virus to the exclusion of others must give a biased perspective on virus diseases generally. it may not be influenza virus, but it could be sars virus, an adenovirus, a parainfluenza virus, a respiratory syncytial virus, a metapneumovirus, or even measles virus. not to mention a common cold virus. finally, there is the matter of interpreting (any and all) results and assessing new tests and variants of old tests. this is, or has been, the province of the professional virologist (http:// www.rcpath.org/index.asp?pageid = 117). training to become proficient takes time and experience; are those clinicians who might use poc tests willing to acquire a similar proficiency in virology, as well as in their own clinical specialty? in english, this is called 'keeping a dog and barking yourself'. does it make sense? both a positive result and a negative result need interpretation, and even positive results are not always significant [4] [5] [6] [7] [8] [9] [10] . this should be a task for an individual who is familiar not only with the test being used, and its limitations, but also with whatever other viruses are currently circulating in the community ⁄ world at large, as well as the medical details of the patient; in other words, someone who can put the result into a wider and proper context. given the enthusiasm with which poc tests are being developed, it is likely that they will be used more and more widely. if so, the advantages and limitations of their use should be fully discussed and the implications recognised. evaluation of a new point-of-care test for influenza a and b in travellers with influenza-like symptoms methods in virus diagnosis: immunofluorescence revisited the principles and practice of clinical virology pitfalls in the diagnosis of enteroviral infection in young children contamination with pcr-detectable virus in a virus isolation quality assurance panel diagnosis of herpes simplex infections of the cns the importance of specific virus diagnosis and monitoring for antiviral treatment total quality management in clinical virology laboratories quality control in nucleic acid testing-where do we stand? key: cord-295479-mcfqs7vf authors: davido, benjamin; seang, sophie; tubiana, roland; de truchis, pierre title: post-covid-19 chronic symptoms: a post-infectious entity?() date: 2020-07-23 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.07.028 sha: doc_id: 295479 cord_uid: mcfqs7vf nan symptomatic outpatients could not be tested by pcr and stayed home in compliance with the 26 laws in force. surprisingly today, while we are fearing a second wave, we receive more and more of those covid-19 : point épidémiologique du 21 mai assistance publique-hôpitaux de paris' response to the covid-19 88 pandemic anosmia and ageusia: common findings 90 in covid-19 patients patients recovered from covid-19 active epstein-barr virus infection in 95 post-viral fatigue syndrome serological igg antibody response on the abbott architect for established sars-cov-99 2 infection neurologic manifestations in hospitalized patients 102 with covid-19: the albacovid registry cerebrovascular disease in patients with covid-19: 106 neuroimaging, histological and clinical description kawasaki-like multisystem inflammatory syndrome in children during the covid-19 chikungunya-induced arthritis in reunion island: a 113 long-term observational follow-up study showing frequently persistent joint 114 some cases of persistent chikungunya immunoglobulin m positivity, and 115 no anticyclic citrullinated peptide seroconversi key: cord-270947-6e5cw2q9 authors: huang, h.-s.; tsai, c.-l.; chang, j.; hsu, t.-c.; lin, s.; lee, c.-c. title: multiplex pcr system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis date: 2017-12-05 journal: clin microbiol infect doi: 10.1016/j.cmi.2017.11.018 sha: doc_id: 270947 cord_uid: 6e5cw2q9 objectives: to provide a summary of evidence for the diagnostic accuracies of three multiplex pcr systems (mpcrs)—biofire filmarray rp (filmarray), nanosphere verigene rv+ test (verigene rv+) and hologic gen-probe prodesse assays—on the detection of viral respiratory infections. methods: a comprehensive search up to 1 july 2017 was conducted on medline and embase for studies that utilized filmarray, verigene rv+ and prodesse for diagnosis of viral respiratory infections. a summary of diagnostic accuracies for the following five viruses were calculated: influenza a virus (flua), influenza b virus, respiratory syncytial virus, human metapneumovirus and adenovirus. hierarchical summary receiver operating curves were used for estimating the viral detection performance per assay. results: twenty studies of 5510 patient samples were eligible for analysis. multiplex pcrs demonstrated high diagnostic accuracy, with area under the receiver operating characteristic curve (auroc) equal to or more than 0.98 for all the above viruses except for adenovirus (auroc 0.89). filmarray, verigene rv+ and proflu+ (the only prodesse assay with enough data) demonstrated a summary sensitivity for flua of 0.911 (95% confidence interval, 0.848–0.949), 0.949 (95% confidence interval, 0.882–0.979) and 0.954 (95% confidence interval, 0.871–0.985), respectively. the three mpcrs were comparable in terms of detection of flua. conclusions: point estimates calculated from eligible studies showed that the three mpcrs (filmarray, verigene rv+ and proflu+) are highly accurate and may provide important diagnostic information for early identification of respiratory virus infections. in patients with low pretest probability for flua, these three mpcrs can predict a low possibility of infection and may justify withholding empirical antiviral treatments. acute respiratory tract infections (ari) cause high morbidity and mortality [1] . among them, viral aris are one of the leading causes for paediatric and geriatric hospitalization and clinic visits [2, 3] . each year, seasonal influenza causes >200 000 hospitalizations and more than $10 billion direct medical costs in the united states. in specific populations (e.g. immunocompromised patients, neonates, and chronic pulmonary disease patients), the high complication and mortality rates from viral aris is a major concern [4] . moreover, empirical antibiotics are commonly prescribed to patients with viral aris because of the lack of rapid and sensitive diagnostic methods and nonspecific symptoms, which delay proper treatments and precipitate antibiotic resistance [4e6] . traditional diagnostic techniques (e.g. virus culture, haemagglutination inhibition assay, enzyme immunoassay and direct fluorescent antibody) were once the mainstays for pathogen detection. however, these methods were either insensitive, time consuming, labor intensive or operator dependent [7e9] . new technologies have emerged as a result of massive clinical demands, such as melting curve analysis, microfluidic device and nucleic acid amplification technologies [5,6,10e13] . these molecular diagnostic tools have shorter turnaround times and higher sensitivity for viral pathogens [14, 15] . in addition, they allow for detection of a broader panel of viruses and coinfection [15, 16] , and they thus have become more widely used than the conventional virologic assays [8,17e19] . in particular, multiplex pcr (mpcr) is a validated strategy for the rapid detection and precise identification of a large number of respiratory viruses [19e22] by incorporating several primers within one reaction tube to amplify genomic fragments of many pathogens [22, 23] . with the use of a mpcr panel, one study demonstrated a 30% to 50% increase in the diagnostic yield of respiratory viruses compared to direct fluorescent antibody and culture [24] . there are a number of us food and drug association (fda)cleared mpcrs available today for detecting respiratory pathogens, each with pros and cons. the characteristics of the three fdaapproved mpcr systems included in our study are listed in table 1 . the biofire filmarray rp (filmarray) respiratory panel [24] , which utilizes melting curve analysis, is a random-access molecular test using principles of real-time pcr. the verigene rvþ test is based on gold nanoparticle technology and silver signal amplification. lastly, hologic gen-probe prodesse launches several assays with variable run sizes that also utilize melting curve analysis but with limited multiplexing ability. although each prodesse assay can only detect two to three viruses at a time, the prodesse assays are still viewed as mpcr [25] . these three mpcrs were chosen because they have shorter turnaround times and have more available studies for analysis among a number of fda-approved mpcrs. there is also one original study that provided direct comparison of these three mpcrs [25] . to gain insight into the optimal diagnostic tool for routine clinical use, we here provide a summary of evidence comparing the diagnostic accuracies of filmarray, verigene rvþ and hologic gen-probe prodesse assays for the detection of viral respiratory infections. the protocol of our study was based on the prisma (preferred reporting items for systematic review and meta-analysis) statement [26] and the standard guideline for systematic reviews of diagnostic tests by the cochrane collaboration [27] . a comprehensive search of literature was conducted using two databases: pubmed (from inception to april 2015) and embase (from inception to april 2015). the search term combination was: (multiplex and pcr or (multiplex and polymerase and chain and reaction) or filmarray or verigene or prodesse or proflu or profast or proadeno or proparaflu or (pro hmpv)) and ((respiratory and tract and infection) or (respiratory and infection) or (respiratory and virus) or (respiratory and tract and disease) or (respiratory and disease) or (common and cold) or influenza or pneumonia or bronchitis or bronchiolitis or rhinosinusitis or pharyngitis or laryngitis or (otitis and media) or tonsillitis or asthma or copd or (chronic and obstructive and lung and disease)). the detailed search strategy is provided in supplementary materials s1. no language restrictions were applied to the search. the search was then supplemented by bibliographies of retrieved full-text articles and the latest narrative reviews. we also contacted the authors of publications that did not provide required data. an updated search to 1 july 2017 was performed before starting the statistical analysis. studies that evaluated the performance of fda-approved mpcr systems for the detection of viral respiratory infection were included, as follow: (a) they assessed the accuracy of one or more the following systems: filmarray, nanosphere verigene rvþ and hologic gen-probe prodesse assays (profluþ, profast, proparafluþ, proadenoþ and pro hmpvþ) against reference standards and (b) they provided sufficient information to calculate sensitivity and standard to validate other mpcr systems and (c) they used an mpcr assay not approved by fda as reference. in addition, we excluded reviews, guidelines, case reports, editorials, panel discussions, letters, notes and comments. for multiple publications, only the latest available publications with complete data of the same patient group were included. studies that compared more than one fda-approved mpcr systems with the same reference standard, used different reference methods for different viruses, used different samples for different viruses or reported prospective and retrospective samples independently have separate data sets for each comparison. three reviewers independently screened the titles and abstracts from pubmed and embase. disagreements or uncertainties were resolved by discussion. a data extraction form was used in 20 included studies by two reviewers before being finalized. each data set was extracted by two authors independently to avoid bias. the form consisted of the following characteristics: study type, study design, patient age, patient inclusion criteria, specimen type, mpcr systems used, reference standard and 2 â 2 tables. the 2 â 2 tables were further used to calculate sensitivities and specificities of the target assays. for the reference methods, virus culture and direct fluorescent antibody were grouped together because they are universally recognized as the reference standard [28, 29] . reverse transcription (rt) pcr referred to both commercial rt-pcrs and in-house rt-pcrs. composite reference standard was defined as a standard that used more than one comparator assays. discrepant analyses encompassed studies that resolved discrepancies between target assay and comparator assay by bidirectional sequencing, rt-pcr, repeated testing or other methods. studies were also grouped on the basis of whether they incorporated filmarray, verigene rvþ and hologic gen-probe prodesse assays as part of a composite reference standard. studies that had <5% of the samples taken from the lower respiratory tract were categorized as using upper respiratory specimens. children were defined as patients younger than 18 years. studies that included both children and adult population were categorized as mixed. the quality of the eligible studies was independently assessed by two reviewers using the quality assessment of diagnostic accuracy studies 2 tool (quadas-2) [30] . for each diagnostic study, we determined the risk for bias and general applicability in all four domains of quadas-2 and reported them separately. those with low risk of bias or low concern regarding applicability were judged as low. a study would be judged as unclear if there were insufficient data for interpretation. a bivariate model was applied to estimate summary sensitivity and specificity. the positive likelihood ratios (lrþ) and negative likelihood ratios (lrà) were then calculated from summary sensitivity and specificity. the bivariate model approach modelled the logit-transformed sensitivity and specificity simultaneously to account for the inherent negative correlation between sensitivity and specificity that may arise due to different thresholds in different studies [30] . in addition, the bivariate model could also account for between-study heterogeneity. all analyses except for the summary receiver operating characteristic curve (roc) were performed by the 'mada' package in r software (r foundation for statistical computing, vienna, austria; http://www.r-project.org/). the summary roc and area under the roc was calculated by the 'midas' package in stata (stata inc, college station, texas). a twosided p value of <0.05 indicated statistical significance for all tests. our comprehensive search yielded 1900 studies from embase and 1350 studies from pubmed. after inclusion and exclusion following the protocol and an updated search to 1 july 2017 (fig. 1) , a total of 20 studies (25 data sets) were eligible for analysis, encompassing a total of 5510 patient samples. profluþ was the only prodesse assay with enough data sets for quantitative analysis. parainfluenza virus was not analysed because of insufficient data. the laboratory characteristics of filmarray rp, verigene rvþ and prodesse profluþ are illustrated in table 1 . a simplified summary of the characteristics of the included studies is provided in table 2 . details of the characteristics and key results of each individual study are provided in supplementary materials s2. none of the studies specifically recruited adults only, and approximately 30% of the studies obtained their samples from children. discrepant analysis and composite reference standard were the two most commonly applied reference standards. the studies varied in quality. quality assessment by the quadas-2 tool is demonstrated in fig. 2 . for the 'patient selection' domain, some studies were not clear about how the patients were recruited. some studies used refrigerated samples that were precollected without specifying their original sources; others did not avoid a caseecontrol design. for the 'reference standard' domain, most studies did not specify the use of blinding for reference standard. some studies that were of low quality in these regards used the index test as part of the reference method. others did not use the same reference method on all their samples. however, most studies were identified as high quality for flow and timing. overall, the majority of the studies had low concern regarding applicability. the overall lrà were all below 0.1, which suggested a high rule-out value. table 3 provides point estimates for flub, rsv, hmpv and adenovirus. fig. 3 shows the forest plot of sensitivity and specificity of the mpcrs for each virus. fig. 4 illustrates the receiver operating characteristic curve and area under the receiver operating characteristic curve for the detection of each virus. overall, in our analyses including 5510 patient samples, mpcr systems demonstrated high diagnostic accuracy (auroc !0.98) for flua, flub, rsv and hmpv, with the exception of adenovirus (auroc 0.89). filmarray rp, verigene rvþ and prodesse profluþ demonstrated a summary sensitivity for flua of 0.911 (95% confidence interval (ci), 0.848e0.949), 0.949 (95% ci, 0.882e0.979) and 0.954 (95% ci, 0.871e0.985), respectively. the three mpcrs were comparable in terms of detection of flua, and filmarray rp and verigene rvþ were comparable for rsv detection. although prodesse profluþ was found to have a statistically significant higher sensitivity than filmarray rp for flub detection, they demonstrated comparable auroc and thus comparable accuracy. of the five respiratory viruses that these systems detect, the diagnosis of influenza virus infection may have the greatest clinical impact [31] . on the basis of our study, overall, the mpcrs exhibited reasonable sensitivity (0.940; 95% ci, 0.902e0.964) and high specificity (0.987; 95% ci, 0.979,0.992) for flua. in current clinical practice, the immunoassay-based rapid influenza diagnostic test is most widely used for screening influenza infections [8] . although the rapid influenza diagnostic test can detect flua and flub in respiratory specimens in approximately 15 to 30 minutes, its sensitivity is limited, as reported by a previous meta-analysis [32] . this may give false-negative results, which prevents its use as a reliable excluding diagnostic tool in clinical practice. furthermore, commonly used reference standards such as rt-pcr or virus culture may take several days to yield results and have little value for treatment decision. our meta-analysis showed that mpcrs provide highly accurate results in a clinically relevant time frame and may potentially change the current diagnostic and treatment practice for flua infection. as a result of the low sensitivity of the rapid influenza diagnostic tests, the decision of antiviral treatment for flua infection is largely based on clinical grounds alone [5] , which may lead to overprescription of anti-influenza drugs and increasing drug resistance [33, 34] . a study by van wesenbeeck et al. [25] of 171 clinical samples concluded that filmarray rp and prodesse profluþ have better sensitivity for flua than verigene rvþ. this is contrary to our results, which found the three mpcrs to be comparable for flua detection. our meta-analysis synthesized data across different studies encompassing 5510 patient samples and may present a more accurate estimate of the real situation. although these three systems have comparable accuracy for flua, filmarray rp has the shortest hands-on time (2 minutes) and run time (1 hour) among the three mpcrs and includes sample preparation in its panel. furthermore, its reagents can be stored at room temperature, and its assay detects the largest number of targets. therefore, filmarray rp may be the best choice in emergency rooms or during the influenza season. there is less interest in rapid diagnostic tests for rsv, hmpv and adenovirus as a result of a lack of specific treatments available against these viruses and the common practice of supportive care in clinical settings. nevertheless, rsv is one of the leading causes of aris in children [35] and immunocompromised patients, and it may cause severe complications; it therefore requires definitive diagnosis. our results revealed that mpcrs are highly sensitive and specific for rsv and hmpv, and they offer a more rapid and accurate alternative to traditional methods [7, 36] . the current treatment is ribavirin for high-risk infants and young children [37] , with several clinical trials for novel rsv treatments underway [38e41]. in contrast, for adenovirus, filmarray rp had a moderate to low sensitivity (<70%), but its superior specificity (>0.99) makes it a reliable rule-in tool. the literature has demonstrated that commercial filmarray rp (v1.6) has low sensitivity for adenovirus [24,42e44] , but its sensitivity greatly improved in the commercial filmarray 1.7 [42] , developed later. of note, none of our included studies specifically reported using filmarray 1.7. to our knowledge, this is the first report to perform a comparative meta-analysis on different mpcr platforms and different viruses. not only did we provide detailed comparison on the characteristics of the three systems but we also provided quantitative accuracy measure for the different viruses by different systems. there are several limitations to our study, however. firstly, some included studies did not provide sufficient details regarding the version of filmarray rp used (premarket or commercial). we traced their publication dates and contacted the filmarray rp manufacturers as well as the authors of the original study at an attempt to confirm the version of filmarray rp. however, sensitivity analysis showed no clinically significant difference between the sensitivities and specificities of precommercial and fdaapproved commercial assays (data not published). secondly, some included studies have a retrospective study design; they failed to specify their inclusion criteria, and they used only previously stored samples from patients with unknown characteristics. there is also concern regarding diagnostic review bias, in which the interpretation of the result of reference test is made with knowledge of the index test result [45] . however, a test review bias is highly unlikely because the interpretation of the index tests (mpcrs) was objective. of note, five of the included studies adopted the caseecontrol design, which tends to give an overestimation of the accuracy of the index test [45] . in addition, results of this study only showed the accuracy of three commercial mpcr systems for diagnosis of respiratory virus infection. whether they have equal strength in therapeutic guidance remains to be validated. lastly, our study does not provide an answer to whether mpcr systems improve the outcome of patients with respiratory virus infection compared to clinical diagnosis alone. analysis of eligible studies showed that three commercial mpcr systemsdfilmarray, verigene rvþ and genprobe prodesse pro-fluþdmay provide important diagnostic information to help early identification of influenza virus, respiratory syncytial virus, adenovirus and hmpv. the great improvement of the sensitivity of the rapid diagnosis of influenza may have the potential to change current modes of diagnosis and management. however, not all of these systems are equally useful in terms of inclusion or exclusion diagnosis. clinicians should interpret the results on the basis of likelihood ratio and pretest probability. the next step would be to assess whether provision of information from these mpcr tests can modify clinical outcomes. estimates of world-wide distribution of child deaths from acute respiratory infections prevalence and correlation of infectious agents in hospitalized children with acute respiratory tract infections in central china community-acquired pneumonia requiring hospitalization: 5-year prospective study the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital cost-effective use of rapid diagnostic techniques in the treatment and prevention of viral respiratory infections excessive antibiotic use for acute respiratory infections in the united states detection of respiratory viruses by molecular methods current approaches for diagnosis of influenza virus infections in humans current best practices for respiratory virus testing cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients cost analysis of multiplex pcr testing for diagnosing respiratory virus infections thermally multiplexed polymerase chain reaction genotyping of respiratory syncytial virus (rsv) group a in stockholm, sweden, using pcr and two-dimensional melting curve analysis nucleic acid amplificationebased diagnosis of respiratory virus infections evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak emerging molecular assays for detection and characterization of respiratory viruses development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay evaluation of a multiplexed pcr assay for detection of respiratory viral pathogens in a public health laboratory setting respifinder: a new multiparameter test to differentially identify fifteen respiratory viruses respiratory viruses in children admitted to hospital intensive care units: evaluating the clart® pneumovir dna array broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcriptionepcr dna microarray systems the filmarray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens comparison of the filmarray rp, verigene rvþ, and prodesse profluþ/fastþ multiplex platforms for detection of influenza viruses in clinical samples from the 2011e2012 influenza season in belgium the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration cochrane diagnostic test accuracy working group. systematic reviews of diagnostic test accuracy diagnostic virology: from animals to automation role of cell culture for virus detection in the age of technology cochrane diagnostic test accuracy reviews antivirals for treatment of influenzada systematic review and meta-analysis of observational studies accuracy of rapid influenza diagnostic tests: a meta-analysis emerging influenza antiviral resistance threats influenza drug resistance. seminars in respiratory and critical care medicine risk of primary infection and reinfection with respiratory syncytial virus respiratory syncytial virus infection ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children oral gs-5806 activity in a respiratory syncytial virus challenge study discovery of an oral respiratory syncytial virus (rsv) fusion inhibitor (gs-5806) and clinical proof of concept in a human rsv challenge study activity of oral als-008176 in a respiratory syncytial virus challenge study coadministration of the broad-spectrum antiviral, brincidofovir (cmx001), with smallpox vaccine does not compromise vaccine protection in mice challenged with ectromelia virus evaluation and implementation of fil-marray version 1.7 for improved detection of adenovirus respiratory tract infection comparison of the idaho technology filmarray system to real-time pcr for detection of respiratory pathogens in children comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens evaluating bias and variability in diagnostic test reports comparison of the focus diagnostics simplexa flu a/b & rsv direct assay with the prodesse profluþ assay for detection of influenza a virus (iav), influenza b virus (ibv), and respiratory syncytial virus (rsv) in clinical specimens detection of influenza a, b and rsv in respiratory specimens by the profluþtm kit and roche lc 480 performance analysis of pandemic influenza detection methods simultaneous detection of influenza a and its subtypes (h1, h3, 2009 h1n1), influenza b, and rsv a and b in respiratory specimens on an automated, random access, molecular platform comparative evaluation of the nanosphere verigene rvþ assay and the simplexa flu a/b & rsv kit for detection of influenza and respiratory syncytial viruses comparison of the biofire filmarray rp, genmark esensor rvp, luminex xtag rvpv1, and luminex xtag rvp fast multiplex assays for detection of respiratory viruses comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp comparison of xtag respiratory virus panel and verigene respiratory virus plus for detecting influenza virus and respiratory syncytial virus fil-marray, an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection rapid multiplex pcr assay to identify respiratory viral pathogens: moving forward diagnosing the common cold the clinical utility of a near patient care rapid microarray-based diagnostic test for influenza and respiratory syncytial virus infections in the pediatric setting evaluation of three influenza a and b real-time reverse transcriptionepcr assays and a new 2009 h1n1 assay for detection of influenza viruses development of a rapid automated influenza a, influenza b, and respiratory syncytial virus a/b multiplex real-time rt-pcr assay and its use during the 2009 h1n1 swine-origin influenza virus epidemic in performance of the cobas® influenza a/b assay for rapid pcr-based detection of influenza compared to prodesse profluþ and viral culture surveillance of upper respiratory infections using a new multiplex pcr assay compared to conventional methods during the influenza season in taiwan effect of genomic drift of influenza pcr tests we thank the staff of core labs, department of medical research, national taiwan university hospital, for technical support; and we thank our medical librarian, h.-p. chiu, for consultation in formulating the search strategy. research grant ntuh106-p04. all authors report no conflicts of interest relevant to this article. supplementary data related to this article can be found at https://doi.org/10.1016/j.cmi.2017.11.018. key: cord-294546-0otd1heg authors: prendki, v.; huttner, b.; marti, c.; mamin, a.; fubini, p.e.; meynet, m.p.; scheffler, m.; montet, x.; janssens, j.p.; reny, j.l.; kaiser, l.; garin, n.; stirnemann, j. title: accuracy of comprehensive pcr analysis of nasopharyngeal and oropharyngeal swabs for ct-scan-confirmed pneumonia in elderly patients: a prospective cohort study date: 2019-01-12 journal: clin microbiol infect doi: 10.1016/j.cmi.2018.12.037 sha: doc_id: 294546 cord_uid: 0otd1heg objectives: we aimed to assess the accuracy of pcr detection of viruses and bacteria on nasopharyngeal and oropharyngeal swabs (nps) for the diagnosis of pneumonia in elderly individuals. methods: we included consecutive hospitalized elderly individuals suspected of having pneumonia. at inclusion, nps were collected from all participants and tested by pcr for the presence of viral and bacterial respiratory pathogens (index test, defined as comprehensive molecular testing). routine diagnostic tests (blood and sputum culture, urine antigen detection) were also performed. the reference standard was the presence of pneumonia on a low-dose ct scan as assessed by two independent expert radiologists. results: the diagnosis of pneumonia was confirmed in 127 of 199 (64%) included patients (mean age 83 years, community-acquired pneumonia in 105 (83%)). a pathogen was identified by comprehensive molecular testing in 114 patients (57%) and by routine methods in 22 (11%). comprehensive molecular testing was positive for viruses in 62 patients (31%) and for bacteria in 73 (37%). the sensitivity and specificity were 61% (95% ci 53%–69%) and 50% (95% ci 39%–61%) for comprehensive molecular testing, and 14% (95% ci 82%–21%) and 94% (95% ci 86%–98%) for routine testing, respectively. positive likelihood ratio was 2.55 for routine methods and 1.23 for comprehensive molecular testing. conclusion: comprehensive molecular testing of nps increases the number of pathogens detected compared with routine methods, but results are poorly predictive of the presence of pneumonia. hence, comprehensive molecular testing is unlikely to impact clinical decision-making (nct02467192). clinical trials registration: nct02467192. identification of the pathogen causing pneumonia is useful to guide antibiotic therapy, to help with differential diagnosis and for epidemiological reasons. hence, most current guidelines recommend microbiological investigations in patients hospitalized for suspected community-acquired pneumonia (cap) [1e3] . nevertheless the microorganism causing cap is identified in only a minority of patients [4] . the difficulty in obtaining high-quality respiratory samples for microbiological analysis (e.g. sputum cultures) is an important limitation in the elderly [5] . use of molecular biology technology improves the diagnostic yield in suspected pneumonia and is often prescribed by physicians, but it is unclear how it impacts clinical management [6] . oosterheert et al. showed in a randomized controlled trial (107 individuals with lower respiratory tract infections, mean age 65 years) that pcr for viruses and atypical bacteria in nasopharyngeal and oropharyngeal swabs (nps) allowed the identification of additional pathogens but did not reduce antibiotic use or costs [7] . a pragmatic randomized controlled trial (720 individuals with acute respiratory illness, mean age 63 years) showed that molecular point-of-care testing for respiratory viruses did not reduce the proportion of patients treated with antibiotics [8] . finally, in a prospective observational study (147 individuals with lower respiratory tract infection, mean age 78 years), pcr detection of respiratory viruses had no impact on antibiotic use and length of stay [9] . we aimed to assess the diagnostic accuracy of pcr detection of viral and bacterial pathogens on nps for the diagnosis of pneumonia in elderly individuals. individuals admitted to hospital for suspected pneumonia had nps collected at inclusion for the detection of multiple bacterial and viral pathogens using multiplex pcr (comprehensive molecular testing), in addition to routine testing. a chest low-dose computed tomography scan (ldct) was performed as soon as possible. results regarding the diagnostic performance of ldct have been published elsewhere [10] . the present study is a preplanned secondary analysis evaluating the accuracy of comprehensive molecular testing in patients with suspected pneumonia. sample size is based on the power calculation of the original study. this study took place in the geriatric and internal medicine wards of geneva university hospitals, an 1800-bed tertiary-care institution serving approximately 500 000 inhabitants. the study was approved by geneva's institutional review board (cer and registered at clinicaltrials.gov (nct02467192). informed consent was obtained from all patients or next of kin. consecutive hospitalized individuals 65 years old, suspected of having community-, nursing-home-or hospital-acquired pneumonia were enrolled between 1 may 2015 and 30 april 2016. individuals included had at least one respiratory symptom and at least one symptom or laboratory finding compatible with infection [10] . individuals treated for pneumonia during the previous 6 months or treated with antimicrobial therapy for more than 48 h before inclusion were excluded (fig. 1) . demographic data, co-morbidities, vital signs, clinical findings, severity scores, results of standard laboratory tests, blood, sputum and urine cultures, urinary antigen detection, pcr for respiratory viruses on nps, and antimicrobial therapy administered were recorded prospectively. images were interpreted as consistent or inconsistent with pneumonia by two independent radiologists experienced in thoracic radiology. discordant cases were reviewed together to reach a consensus. the radiological diagnoses were taken as the reference standard and patients with a seemingly infectious infiltrate were considered to have pneumonia. the radiologists were blinded to patients' results. the nps were performed on all individuals at inclusion. these were placed in copan ® 305 universal transport medium (copan italia spa, brescia, italy), sent to the central virology laboratory as soon as possible and processed directly (neither frozen nor thawed). nucleic acids were extracted with qiasymphony (qiagen, hombrechtikon, switzerland) using a virus/pathogen kit (937055, qiagen used to detect nine bacterial species/genera: streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, staphylococcus aureus, mycoplasma pneumoniae, chlamydia pneumoniae, legionella spp., klebsiella pneumoniae and pseudomonas aeruginosa. the results of the pcr for viruses were available to the treating physicians whereas the results of pcr for bacteria were performed subsequently as a batch on nucleic acid extraction, which were kept at e80 c and were not reported to the treating physicians. comprehensive molecular testing was defined as the results of both viral and bacterial pcr. routine microbiological tests were performed according to recommendations, including blood cultures, sputum cultures in patients able to expectorate and pneumococcal and legionella pneumophila urinary antigen detection [3] . sputum samples with 25 or more neutrophils and fewer than ten epithelial cells were evaluated after gram-staining, and were subsequently cultured. we used frequencies, percentage, mean with range and median with interquartile range for descriptive purposes. variables including results of comprehensive molecular testing, viral pcr alone, bacterial pcr alone and routine methods, were compared between patients with and without pneumonia in univariate analysis using the mannewhitneyewilcoxon test or the kruskalewallis method for continuous variables, and fisher's exact test or chi-square test for categorical variables, as appropriate. comprehensive molecular testing was the index test. the test characteristics (sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios (lr), and diagnostic odds ratios) of comprehensive molecular testing, viral pcr, bacterial pcr, and routine methods, were computed using two-by-two tables. a p value of <0.05 was considered significant. analyses were performed using the r statistical software package, version 3.1.1 (www.cran.r-project.org). baseline characteristics of included patients are shown in table 1 . nps were performed in 199/200 patients (99.5%) and presence of pneumonia was confirmed in 127/199 (64%). pneumonia was community-acquired in 105/127 (83%). the median delay between inclusion and ldct was 2.2 h (interquartile range 0.9e15.4). among the 72 patients without pneumonia, the most frequent diagnoses were non-respiratory sepsis, viral upper respiratory tract infection, bronchitis or exacerbation of chronic obstructive pulmonary disease, and heart failure. mean duration of antimicrobial therapy was 6.8 days. eighty-six of 199 patients (43.2%) received a combination antimicrobial therapy for a mean of 3.1 days (most frequently a b-lactam and a macrolide, according to institutional guidelines). results are depicted in table 2 and the supplementary material (table s1 ). comprehensive molecular testing was positive in 114/ 199 patients (57%). sixty-two patients (31%) had a positive pcr for at least one virus, 73 patients (37%) had a positive pcr for at least one bacterium, and 21 (11%) had a positive pcr for both virus and bacteria. antimicrobial therapy was stopped in 6/62 patients (9.7%) with a positive viral pcr. results are displayed in table 2 and the supplementary material (table s1 ). blood cultures were performed in 176/199 patients (88%), urinary antigens for legionella spp. and streptococcus pneumoniae in 178/199 patients (89%) and 183/199 patients (92%), respectively. sputum was obtained in 81/199 patients (41%), with only 12/81 (15%) of sufficient quality to warrant further evaluation. routine methods led to the identification of a pathogen in 22/199 patients (11%). twelve patients (6%) had bacteraemia, six (3%) of which had a non-respiratory origin (four urinary and two abdominal). details for each pathogen are available in the supplementary material (table s1 ). comprehensive molecular testing was positive in 78/127 patients (61%) with pneumonia and 36/72 patients (50%) without. at least one viral pcr was positive in 45/127 patients (35%) with pneumonia and 17/72 (24%) without. bacterial pcr was positive in 50/127 patients (39%) with pneumonia and 23/72 (32%) without. routine methods were positive in 18/127 patients (14%) with pneumonia and 4/72 (6%) without. results are depicted in table 3 . sensitivity and specificity were 61% and 50% for comprehensive molecular testing, 14% and 94% for routine methods, 35% and 76% for viral pcr, and 39% and 68% for bacterial pcr, respectively. the positive lr was 2.55 for routine methods and 1.23 for comprehensive molecular testing. negative lrs of routine and molecular methods were 0.91 and 0.77, respectively. our main findings are that results of comprehensive molecular testing of nps are poorly predictive of the presence of pneumonia. positive (1.23) and negative (0.77) lr of comprehensive molecular testing are too low to affect the probability of having pneumonia. comprehensive molecular testing increased the sensitivity to 61% in comparison with 14% with routine methods. gadsby et al. achieved pathogen detection in 87% of patients hospitalized for cap using comprehensive molecular testing [6] . this higher sensitivity is probably explained by collection of sputum in 96% of their patients, who were much younger (median age 67 years). in comparison, sputum of adequate quality was obtained in only 12/199 (6%) of our patients. though sputum culture is recommended in international guidelines [3, 11] , it is hard to obtain in elderly patients [12] . our results compare with those of putot et al., who were able to collect sputum from only 15% of elderly patients hospitalized for pneumonia [5] . obtaining more good-quality lower respiratory tract samples would require an invasive procedure [13, 14] . in our cohort, 31% of the patients had a positive result for a virus, mainly rhinovirus and influenza virus. forty-five (22.6%) had both a positive viral pcr and pneumonia. positive and negative lrs were 1.50 and 0.85, respectively. this is in accordance with previous results in the literature. in another study assessing the performance of pcr on nps for the prediction of ct-scan-confirmed pneumonia, prevalence of positive viral pcr was 28%, and positive and negative lrs were 1.39 and 0.87, respectively [15] . certainly, many viruses, including influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus and rhinovirus, are known causes of pneumonia [16e21]. jain et al. showed that respiratory viruses were more frequently detected than bacteria in patients hospitalized for non-severe cap (median age 57 years) [22] . but viruses are also detected in nps in individuals with bronchitis, exacerbation of chronic obstructive pulmonary disease, and even in asymptomatic patients [23e25]. in the present study, many patients with non-respiratory sepsis or cardiac failure had positive nps. our results confirm that viruses are frequently identified in patients with symptoms of a respiratory illness, including those without pneumonia. finally, antimicrobial therapy was stopped in only 10% of patients with a positive viral pcr, suggesting that comprehensive molecular testing might not be an adequate means of reducing antimicrobial therapy prescription in patients with symptoms of lower respiratory tract infection. we had hypothesized that a positive bacterial pcr in nps might be a surrogate for bacterial pneumonia, allowing us to surpass the aforementioned difficulties to obtain good-quality sputum samples. however, bacterial pcr had poor diagnostic accuracy (positive and negative lrs of 1.23 and 0.90, respectively), probably because they are not able to differentiate between pharyngeal carriage and lower respiratory tract infection. compared with the results of the capita cohort, pharyngeal carriage rate of staphylococcus aureus and streptococcus pneumoniae were lower in our cohort (14% versus 21% and 3% versus 17%, respectively), whereas the carriage rate was similar for haemophilus influenzae (8% versus 7%) and higher for moraxella catarrhalis (14% versus 8%) [26] . this differences may stem from the different population enrolled, the capita cohort including community-dwelling elderly people (mean age 72 years), a far younger and healthier population than ours. finally, the identification of a pathogen with routine methods did not result in a high diagnostic accuracy, although better than with comprehensive molecular testing. our study has several strengths. it was conducted in a consecutive cohort of unselected elderly patients who were submitted to extensive testing. we used a robust reference standard based on ldct scan. the radiologists were blinded to participants' clinical, biological and microbiological results, so incorporation bias could be attenuated. according to recent findings, a reference standard for pneumonia based on chest x-ray may lead to frequent misclassifications, which can flaw the evaluation of microbiological test accuracy [10, 27] . our comprehensive molecular testing included more respiratory bacterial pathogens than previous works [6, 7] . our study also had some limitations. the lack of a control arm prevented the assessment of the impact of comprehensive molecular testing on patient management. we did not perform pcr analyses on a quantitative basis, and multiplex pcr have different analytical sensitivities according to the viruses sought. sputum was not tested with pcr because good-quality sputum could only be obtained in a small minority of patients. finally, the presence of an infiltrate on an ldct scan may be an imperfect reference standard for the diagnosis of infectious pneumonia. however, using microbiological results of the patients in the reference definition would have led to a risk of incorporation bias. the present study highlights the difficulties in identifying a causative agent in elderly patients with suspected pneumonia. viruses and bacteria are frequently isolated by pcr in the upper airway of elderly patients but their presence is not useful for predicting the presence or absence of pneumonia. hence they are unlikely to be helpful in making patient management decisions. further investigation is needed to assess the usefulness of pcr sampling in patients with proven pneumonia to direct treatment. the authors declare no conflict of interest. the study was supported by grants from the geneva university hospitals (hug) (research & development grant, medical directorate, hug) and the ligue pulmonaire genevoise, a non-profit association involved in the care of patients with respiratory diseases. infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults bts guidelines for the management of community acquired pneumonia in adults: update guidelines for the management of adult lower respiratory tract infectionsdfull version evolving understanding of the causes of pneumonia in adults, with special attention to the role of pneumococcus impact of microbiological samples in the hospital management of community-acquired, nursing home-acquired and hospital-acquired pneumonia in older patients comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial a comparison of nasopharyngeal and oropharyngeal swabbing for the detection of influenza virus by real-time pcr low-dose computed tomography for the diagnosis of pneumonia in elderly patients: a prospective, interventional cohort study diagnosis and management of community and hospital acquired pneumonia in adults: summary of nice guidance pneumonia in the very old lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia viral infection in patients with severe pneumonia requiring intensive care unit admission viruses detected by systematic multiplex polymerase chain reaction in adults with suspected community-acquired pneumonia attending emergency departments in france viral pneumonia in older adults respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness human metapneumovirus: review of an important respiratory pathogen radiographic and ct features of viral pneumonia ct findings in viral lower respiratory tract infections caused by parainfluenza virus, influenza virus and respiratory syncytial virus rhinovirusdnot just the common cold community-acquired pneumonia requiring hospitalization among us adults heterogeneous and dynamic prevalence of asymptomatic influenza virus infections respiratory syncytial virus evaluation among asymptomatic and symptomatic subjects in a university hospital in sao paulo, brazil in the period of viral shedding and transmission potential of asymptomatic and paucisymptomatic influenza virus infections in the community the impact of the 13-valent pneumococcal conjugate vaccine on pneumococcal carriage in the community acquired pneumonia immunization trial in adults (capita) study early chest computed tomography scan to assist diagnosis and guide treatment decision for suspected community-acquired pneumonia this work was presented at the 28th eccmid (madrid). we thank the patients and their families for their participation in this study. we also thank the pneumoldct study group, clinicians, radiology technicians, research nurses and case managers who helped us to enrol our participants, and our translator, mr darren hart. we acknowledge the contribution of the escmid study group for infections in the elderly (esgie, www.escmid.org/esgie). vp and js conceived the study, wrote the grant to obtain funding, obtained ethical approval, analysed the results and wrote the manuscript. vp, mpm and am participated in recruitment and followed the participants and the samples. ms and xm were the radiologist experts. bh, ng, cm, am, pef, jpj, jlr and lk helped design the study and contributed to the manuscript. js performed the statistical plan and analyses. supplementary data to this article can be found online at https://doi.org/10.1016/j.cmi.2018.12.037. key: cord-307273-pplky6g4 authors: schrooyen, loïc; delforge, marc; lebout, faustine; vanbaelen, thibaut; lecompte, amaryl; dauby, nicolas title: homeless people hospitalized with covid-19 in brussels date: 2020-08-07 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.08.002 sha: doc_id: 307273 cord_uid: pplky6g4 nan to the editor, compared to the general population, homeless people have higher mortality, both related to communicable and non-communicable diseases, partly explained by higher exposure to risk factors including alcoholism, illicit drug abuse and smoking(1,2). transmissible infectious diseases contribute significantly to the morbidity and mortality of homeless(1). notably, airborne diseases such as tuberculosis, influenza and pneumococcal pneumonia have been reported with increased incidence and severity in homeless population (2) . shelters overcrowding and limited access to hygienic supplies could enhance the transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in this vulnerable population. we assessed the prevalence, incidence and outcome of homeless patients hospitalized in our institution with covid-19 between 3 rd march and 26 th may 2020. sociodemographic features and risk factors were compared with those of non-homeless patients admitted during the same period. only symptomatic hospitalized patients with sars-cov-2 positive rt-pcr or rapid antigen test with evidence of pneumonia on computed tomography were included. nosocomial cases and pregnant women were excluded. demographic data including age, gender, smoke, alcohol abuse, methadone therapy for opioid substitution, human immunodeficiency virus, hepatitis b virus and hepatitis c virus serological status and chronic comorbidities as arterial hypertension, diabetes, obesity, neurological, cardiovascular and pulmonary diseases were collected. homeless patients were retrospectively identified based on systematic social inquiry performed upon admission. in order to assess disease severity and outcome, each case (homeless) was matched to three controls based on sex and age categories. nonparametric wilcoxon's and fisher's exact tests were used for continuous and binomial variables analyses, respectively. between 3 rd march and 26 th may 2020, 14 homeless people were identified among 238 patients hospitalized for a covid-19 pneumonia resulting in a homelessness prevalence of 5.88%. all but 3 resided in homeless shelters. incidences of covid-19 among homeless and non-homeless patients were calculated using homeless census report and our hospital catchment population. according to the last homeless census report, there were 2151 homeless people in brussels in november 2018 (3) . most of them were found to attend homeless shelters located in the downtown area surrounding our hospital. the centre hospitalier universitaire (chu) saint-pierre is a public tertiary hospital, working closely with public social services of the capital and is a referral center for resourcelimited patients in brussels city. the estimated catchment population of our institution was 122.808 people in 2018 (data provided by the federal public health service, food chain and safety environment). for the reporting period, incidences were 650 and 194/100.000 hospitalized homeless and non-homeless patients for covid-19, respectively. the median age was 56.36 (standard deviation ±16.76) and 61.78 (standard deviation ±16.87) years old for homeless and non-homeless patients, respectively. we observed a male predominance in both populations (71.43% and 58.04%). compared to nonhomeless patients, the homeless were more likely to smoke (or 4.14, ic95 1.73-9.85), suffer from alcoholism (9.82, ic95 3.07-30.64), be treated with methadone for opioid substitution (or 37.17, ic95 3.90-538.2) and have neurological diseases (or 5.88, ic95 1.84-18.64). there was no difference between the 2 groups in terms of c-reactive protein and lactate dehydrogenase levels and lymphocytes count at admission, delay of symptoms before admission, intensive care unit (icu) admission, invasive ventilation, dialysis, treatment uptake with hydroxychloroquine, length of stay (los) in icu, total los in hospital and death. (table 1) in the present study, we found an incidence of hospitalization for covid-19 three times higher in homeless as compared to the general population. a recent report in the usa identified a high prevalence (36%) of sars-cov-2 rt-pcr positivity in a homeless shelter (4) . most subjects (88%) with positive rt-pcr in the latter study were asymptomatic, highlighting the risk of spread among residents of homeless shelters. we found a high but similar proportion of comorbidities (arterial hypertension, diabetes and cardiovascular diseases) in both populations hospitalized with covid-19. smoking, opioid substitution and alcohol abuse were highly prevalent among homeless patients as previously reported(1). the high prevalence of comorbidities and the increased exposure to risk factors in the homeless population could increase their risk of more severe disease and mortality following sars-cov-2 infection. although more severe manifestations could explain higher hospitalization rates, the disease severity of the homeless included in this study tended to be reduced as compared to non-homeless with decreased rate of icu admission and mechanical ventilation requirement and shorter hospital and icu los. moreover, a trend of shorter duration of symptoms upon admission in homeless patients was not evoking any delay in the access to care. the main limitation of our study is the small sample size of homeless group and the monocentric design. larger studies are required to properly assess the outcome of covid-19 in homeless patients. in conclusion, we found a high incidence of hospitalization for covid-19 among homeless patients in brussels. they had high but similar proportion of comorbidities as compared to non-homeless. outcome was not worse, although the interpretation is limited by the small sample size of homeless patients. our results illustrate the urgent need for implementing strategies in order to stop the spread of covid-19 in homeless population. strategies based on wide scale preventing, screening and managing covid the health of homeless people in high-income countries: descriptive epidemiology, health consequences, and clinical and policy recommendations. the lancet preventing and controlling emerging and reemerging transmissible diseases in the homeless. emerg infect dis dénombrement des personnes sans-abri et mal logées en région de la strada asbl prevalence of sars-cov-2 infection in residents of a large homeless shelter in boston outbreak among three affiliated homeless service sites -king county, washington, 2020. mmwr morb mortal wkly rep addressing covid-19 among people experiencing homelessness: description, adaptation, and early findings of a multiagency response in boston. public health rep wash dc 1974 key: cord-279111-jaa45kyc authors: ieven, m.; coenen, s.; loens, k.; lammens, c.; coenjaerts, f.; vanderstraeten, a.; henriques-normark, b.; crook, d.; huygen, k.; butler, c.c.; verheij, t.j.m.; little, p.; zlateva, k.; van loon, a.; claas, e.c.j.; goossens, h. title: aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 11 european countries date: 2018-02-12 journal: clin microbiol infect doi: 10.1016/j.cmi.2018.02.004 sha: doc_id: 279111 cord_uid: jaa45kyc objectives: to describe the role of bacteria (including bacterial resistance), viruses (including those recently described) and mixed bacterial–viral infections in adults presenting to primary care with lower respiratory tract infection (lrti). methods: in all, 3104 adults with lrti were enrolled, of whom 141 (4.5%) had community-acquired pneumonia (cap), and 2985 matched controls in a prospective study in 16 primary care networks in europe, and followed patients up at 28–35 days. we detected streptococcus pneumoniae and haemophilus influenzae and assessed susceptibility, atypical bacteria and viruses. results: a potential pathogen was detected in 1844 (59%) (in 350 (11%) bacterial pathogens only, in 1190 (38%) viral pathogens only, and in 304 (10%) both bacterial and viral pathogens). the most common bacterial pathogens isolated were s. pneumoniae (5.5% overall, 9.2% in cap patients) and h. influenzae (5.4% overall, 14.2% in cap patients). less than 1% of s. pneumoniae were highly resistant to penicillin and 12.6% of h. influenzae were β-lactamase positive. the most common viral pathogens detected were human rhinovirus (20.1%), influenza viruses (9.9%), and human coronavirus (7.4%). influenza virus, human parainfluenza viruses and human respiratory syncytial virus as well as human rhinovirus, human coronavirus and human metapneumovirus were detected significantly more frequently in lrti patients than in controls. conclusions: a bacterial pathogen is identified in approximately one in five adult patients with lrti in primary care, and a viral pathogen in just under half, with mixed infections in one in ten. penicillin-resistant pneumococci and β-lactamase-producing h. influenzae are uncommon. these new findings support a restrictive approach to antibiotic prescribing for lrti and the use of first-line, narrow-spectrum agents in primary care. objectives: to describe the role of bacteria (including bacterial resistance), viruses (including those recently described) and mixed bacterialeviral infections in adults presenting to primary care with lower respiratory tract infection (lrti). methods: in all, 3104 adults with lrti were enrolled, of whom 141 (4.5%) had community-acquired pneumonia (cap), and 2985 matched controls in a prospective study in 16 primary care networks in europe, and followed patients up at 28e35 days. we detected streptococcus pneumoniae and haemophilus influenzae and assessed susceptibility, atypical bacteria and viruses. results: a potential pathogen was detected in 1844 (59%) (in 350 (11%) bacterial pathogens only, in 1190 (38%) viral pathogens only, and in 304 (10%) both bacterial and viral pathogens). the most common bacterial pathogens isolated were s. pneumoniae (5.5% overall, 9.2% in cap patients) and h. influenzae (5.4% overall, 14.2% in cap patients). less than 1% of s. pneumoniae were highly resistant to penicillin and 12.6% of h. influenzae were b-lactamase positive. the most common viral pathogens detected were human rhinovirus (20.1%), influenza viruses (9.9%), and human coronavirus (7.4%). influenza virus, human parainfluenza viruses and human respiratory syncytial virus as well as human rhinovirus, human coronavirus and human metapneumovirus were detected significantly more frequently in lrti patients than in controls. community-acquired lower respiratory tract infection (lrti) is one of the commonest reasons for consulting in primary care and accounts for considerable antibiotic use and health-care costs. it is neither feasible nor cost-efficient to identify microbial aetiology in most patients who present with lrti in primary care because of sampling challenges, limited access diagnostics and the limited clinical utility of receiving a result after empirical treatment decision has been made [1] . consequently, little is known about the aetiology of lrti in everyday primary care. in addition, detecting pathogens in both symptomatic patients and contemporaneous controls to distinguish between asymptomatic carriage and the presence of agents causing symptoms has rarely been carried out. nevertheless, despite limited knowledge of the proportion of patients that have an identifiable bacterial aetiology and the sensitivities of these pathogens, and evidence of limited or no clinical benefit from antibiotic treatment, more than half of patients presenting to primary care with lrti/acute cough in europe are prescribed antibiotics [2e4] . this contributes to the selection of antimicrobial-resistant bacteria [5] . improved knowledge of likely pathogens (at the point of care) and the likely susceptibility of bacterial pathogens, could help to guide antibiotic prescribing decisions and so help contain unnecessary antibiotic use and antimicrobial resistance. furthermore, such information could support public health policy on prevention of respiratory illness, including vaccination. our primary objective was to describe the viral and bacterial aetiology in adult patients presenting to primary care with lrti and in those with community-acquired pneumonia (cap). our secondary objectives were to describe the presence of resistance in bacterial infections and of mixed viralebacterial infections. the study was part of the european union fp6 funded network of excellence grace (genomics to combat resistance against antibiotics in community-acquired lrti in europe network of excellence; www.grace-lrti.org). we recruited patients between october 2007 and april 2010 in 16 primary care networks that had a track record of conducting research based in 11 european countries: antwerp and ghent (belgium); barcelona and mataro (spain); bialystok, lodz and szczecin (poland); bratislava (slovakia); cardiff and southampton (uk); jesenice (slovenia); j€ onk€ oping (sweden); milan (italy); nice (france); rotenburg (germany) and utrecht (the netherlands). inclusion criteria for patients were: age !18 years, with an acute or worsened cough ( 28 days duration) as the main symptom, or any clinical presentation considered to be caused by lrti by the general practitioner (gp) and consulting for the first time for this illness episode. patients with presumed cough of non-infective origin, antibiotic consumption in the previous month, and any serious condition associated with an immunocompromised condition were excluded. for each patient, we planned to include a control patient matched for age, maximum 5 years of difference, and gender, consulting at the gp office for any other reason than acute respiratory illness within the same 2-week period. the study was approved by the local ethics committees in all participating centres and by the competent authority in each country. written informed consent was obtained from each patient and control participant before inclusion. symptomatic patients were assessed at first presentation (day 1) and between days 28 and 35. chest radiographs were taken within 1 week after inclusion. cap was considered present if the local radiologist reported lobar or bronchopneumonia; other diagnoses were categorized as 'pneumonia absent' [6] . all recruiting gps received standardized sampling material and a protocol with detailed instructions on the sampling of the patients. within 24 h of first presentation and inclusion, serum and edta blood, sputum, if available, and two nasopharyngeal flocked swabs (nps; copan) were taken. at days 28e35, serum sampling and the two nps were repeated. controls were sampled for edta blood and two nps at baseline. sputum was not obtained from controls and the controls were not followed up. serum, edta and nps were stored frozen in the local laboratories until regular shipment to the central laboratory (university hospital antwerp), where specimens were stored at à80 c until analysis. sputum samples were examined in the local laboratories using direct microscopy to assess the quality (ratio of white blood cells/ epithelial cells !1 as criterion for good quality), then gram-stained, cultured and subsequently frozen at à80 c. streptococcus pneumoniae and h. influenzae were identified using conventional biochemical tests and isolates were frozen in microbanks until shipped in batches to the central laboratory, where nps were cultured for s. pneumoniae and/or h. influenzae. their susceptibility was tested at the karolinska institute and the oxford university, respectively, after frozen transport. the mics of s. pneumoniae to penicillin g, erythromycin, clindamycin, tetracycline and levofloxacin were determined. isolates were classified as sensitive, indeterminate or resistant according to the eucast breakpoints for these species (www.eucast.org/antimicrobial-susceptibilitytesting/breakpoints). haemophilus influenzae isolates were tested for b-lactamase production. pcrs for mycoplasma pneumoniae, chlamydia pneumoniae, bordetella pertussis, legionella pneumophila and respiratory viruses nucleic acid from nps was extracted with the nuclisens easy-mag (biom erieux, marcy l' etoile, france) in antwerp after which aliquots were shipped to three collaborating laboratories for subsequent analysis with their in-house amplification and detection methods, which had been evaluated previously [7] . for the detection of m. pneumoniae-specific and c. pneumoniae specific igg or igm antibodies, m. pneumoniae or c. pneumoniae igg and igm-elisa kits (medac gmbh, wedel, germany) were used according to the instructions of the manufacturer. igg antibodies to b. pertussis toxin (institut virion-serion gmbh, würzburg, germany) were analysed in a convalescent serum sample. the isolation of s. pneumoniae and h. influenzae, and the identification of l. pneumophila or respiratory viruses by use of pcr in respiratory samples were considered to support an aetiological diagnosis. infection with m. pneumoniae or c. pneumoniae was defined as: positive pcr in respiratory samples, the presence of igm antibodies in the acute-phase serum and/or convalescent-phase sample, igg seroconversion or a significant increase in igg between acute and convalescent samples. a patient was considered positive for an acute b. pertussis infection (infection in the last 6 months) if positive by pcr in a respiratory sample and/or the presence of an antibody titre to pertussis toxin of !125 iu/ml in convalescent serum (days 28e35), demonstrated previously as a cut-off with high sensitivity and specificity [8, 9] . generalized estimating equations were used to assess differences in the proportion of potential pathogens between lrti patients' day 1 and days 28e35 samples, and between day 1 samples of lrti patients and controls. the caseecontrol design was applied to assess causality between viral pathogens and lrti (cap). chisquared tests were used to assess differences in the proportion of specific viruses or bacteria between lrti patients with and those without cap. student's t-test was used to assess differences in age between lrti patients with and those without specific viral or bacterial aetiology (ibm ® spss ® statistics, release 20.0.0). a p value of <0.05 was considered to be statistically significant. a total of 3104 adult lrti patients were included by 294 gps from october 2007 to april 2010, 1860 (60.0%) were women ( table 1 ). the mean age was 49.8 years (range 18e92 years) and 141 were diagnosed with cap (4.5%); among elderly patients (>65 years n ¼ 628, 20.2%) 40 patients had a cap (6.4%). we recruited a total of 2985 controls without symptoms of lrti. day 1 nps and blood samples were available from 3085 (99.4%) and 3054 (98.4%) lrti patients, respectively, and sputum samples from 2121 (68.3%). on days 28e35, 2673 patients (86.1%) were seen: in 2552 (95.5%) and 2575 (96.3%) of these, blood samples and nps, respectively, could be collected. only controls who matched with patients according to all criteria (n ¼ 2063) were further included to estimate causality. the proportion of patients with lrti and cap with an identified bacterial, viral or mixed aetiology is presented in fig. 1 . a potential bacterial pathogen was found in 655 (21.1%) lrti patients on day 1, significantly more often in patients with cap compared with those without ( fig. 1 and table 2 ). streptococcus pneumoniae and h. influenzae were significantly more prevalent in patients presenting with cap. only 9.2% of all 3104 patients and 10.6% of cap patients were vaccinated against s. pneumoniae. prevalence of pneumococci in these groups was 4.9% and 0%, respectively. twenty-four of 172 (14.0%) had a reduced susceptibility to penicillin g (one isolate highly resistant, 23 (13.4%) intermediate resistance). thirty-six (20.9%) isolates were less susceptible to erythromycin/clindamycin, 78 (45.3%) had a reduced susceptibility to tetracycline and 3 (1,7%) were resistant to levofloxacin. twentyone of 167 (12.6%) h. influenzae isolates produced b-lactamases. any viral aetiology was identified in 1494 (48.1%) of lrti patients, significantly less often in those with cap compared with those without cap ( fig. 1 and tables 2 and 3 ). the commonest viruses in our cohort of patients were human rhinovirus (hrv), influenza virus and human coronavirus (hcov). a respiratory virus was detected on days 28e35 in 336 patients (12.6%), as well as in 205 (9.9%) of the matched controls. all respiratory viruses, except for human adenovirus, human bocavirus (hbov) and wu polyomavirus and ki polyomavirus, were significantly more frequently detected in day 1 nps of lrti patients than in their days 28e35 nps or in the nps of their matched controls (table 3) . apart from human adenovirus, virus prevalence did not differ significantly between patients with cap or with lrti. in all, 23.6% of all lrti patients and 29.1% of cap patients were vaccinated against influenza virus. prevalence of influenza virus in these groups was 5.3% and 4.9%, respectively. casewise analysis of atypical bacterial agents or viruses detected during illness compared with subsequent detection at follow up is presented in table 4 . none of the patients who were initially pcr positive for m. pneumoniae, b. pertussis, influenza virus or human parainfluenza viruses 1e4 remained positive for these aetiologies at follow up. very few patients positive for hrv, hcov, respiratory syncytial virus, human metapneumovirus (hmpv), polyomaviruses (wuþki) and hbov had the same pathogen detected at follow up. among all 3104 lrti patients, a mixed bacterial, mixed viral or mixed bacterialeviral infection was detected in 51 (1.6%), 118 (3.8%) and 304 (9.8%) patients, respectively. the pathogens involved are described in more detail in the supplementary material. this is the only prospective, large international, caseecontrol study using standardized sampling and comprehensive microbiological work up to provide accurate estimates of the prevalence of both bacterial and viral aetiology in patients consulting with lrti in primary care. the overall microbiological yield was high, mainly due to the high prevalence of viruses. a potential bacterial pathogen was isolated in only one in five patients, and antibiotic-resistant pathogens were rare. previous studies have mainly studied more severely ill patients hospitalized with cap rather than lrti in primary care [1,10e12] , and few of those studies used comprehensive diagnostic methods, including pcr, to detect respiratory viruses [10, 12, 13] . we identified a potential pathogen in about 60% of cap patients. however, comparisons are difficult in that our study is unique in terms of study design, the broad inclusion criteria, the high numbers of patients sampled at baseline and follow up, the inclusion of matched controls, and the comprehensive conventional and molecular microbiological diagnostics used. the prevalence of s. pneumoniae and h. influenzae in our cap subgroup was significantly higher than in the non-cap patients, but lower in comparison to most previous studies. we do not consider that the implementation of pneumococcal vaccine influenced our findings because of the small number of cap patients who had been vaccinated. however, only 5% of patients in the most comprehensive aetiological study of adult patients hospitalized with cap in the usa had pneumococcal pneumonia [14] . highlevel penicillin resistance in pneumococci remains very low in all european countries in this setting, which supports the recommendation that if antibiotics are to be prescribed, amoxicillin should be the first-line agent for lrti [1] . mycoplasma pneumoniae infections occur in epidemics every 4e5 years: we included patients in our study between two epidemic waves, possibly explaining the low m. pneumoniae prevalence observed [15, 16] . this is also the first large european prospective study on the prevalence of pertussis in adults consulting primary care physicians for acute cough [17] . we detected at least one respiratory viral pathogen in almost 50% of patients. nps sampling may have yielded significantly more infected respiratory epithelial cells [18] , with sensitive pcr-based diagnostic techniques augmenting specifically for viruses. influenza virus, human parainfluenza viruses 1e4 and respiratory syncytial virus are recognized causes of cap in hospitalized patients and in the elderly [13] influenza vaccination resulted in lower prevalence of influenza virus in the elderly (data not shown). hrv, hcov and hmpv are rarely detected in cap and other lrti in outpatients. hrv has been associated with outbreaks of severe respiratory disease, including cap, in older people [19e21] and has been isolated in hospitalized patients with cap [10] , but a prevalence of 14.2% in cap in outpatients is high and a novel finding. hcov have recently been identified in small numbers of adults with severe pneumonia [10, 21] , but is not routinely tested for in adult outpatients with cap or lrti. we may have underestimated the prevalence of hcov as hku-1 testing was not performed and hku-1 is generally as prevalent as nl63 and oc43 [22] . infections due to hmpv are mainly described in long-term care facilities [10] . we found hmpv more prevalent in outpatients with cap compared with those with other lrtis, with even greater prevalence in cap patients than respiratory syncytial virus, and similar to the 3%e7% hmpv infection prevalence found in hospitalized adults [23] . although numbers are small, human adenovirus was significantly more prevalent in cap compared with other lrti, a unique finding in immunocompetent outpatients. the high rates of viral detection in outpatients with lrti and cap suggests that comprehensive microbiological assessment is important to guide management and may explain the limited average benefit from antibiotic treatment in the placebo-controlled study that we conducted in a large subset of patients included in the present analysis [2] . our study is the first that compared the prevalence of respiratory viruses in symptomatic adults with that in matched controls without respiratory symptoms. influenza virus, human parainfluenza viruses 1e4 and respiratory syncytial virus were never, or rarely, detected in controls or at follow up in symptomatic patients. this strongly implicates these agents as causative pathogens. similarly, the significantly lower prevalence of hrv, hmpv and hcov in patients at follow up and in controls suggests that asymptomatic carriage of these viruses is uncommon in adults, and indicates that these viruses should also be regarded as causative agents in cap [11, 14, 23] . for hrv, the rates of prolonged shedding (same genotype in 35%) versus re-infection (other genotype) in the grace study have been further investigated [24] . human bocavirus was detected in cap and in <1% of lrti patients at baseline, with similar findings among controls and at follow up of patients. hbov was identified in respiratory specimens from 1.5% of hospitalized adults with no alternative viral aetiology, but controls were not included in that study [25] . as hbov is often found in the presence of other pathogens in respiratory specimens we agree that hbov probably has no relevance or primary role as a causative agent in lrti in primary care [26] . there may be an association between high hbov viral loads and hbov being the only virus detected [27] , suggesting that a quantitative approach should be considered [26] . this also applies to ki polyomavirus and wu polyomavirus. although it is not yet possible to draw firm conclusions on their role in human pathology [28e30], our data show no evidence for a causative role in outpatient cap or lrtidassessment of the viral loads could potentially help to further clarify their significance. sputum was not obtained from all patients, and sputa and follow-up serology were not obtained from control patients. consequently, a valid estimation of the prevalence of bacterial pathogens in controls was not always possible. although the most important elements of this study are the descriptive results, we also performed multiple statistical tests so the finding of statistical significance may reflect type i error. however this is much less likely when supporting prior work on aetiology (e.g. bacterial causes of cap) or when the p-value is very small (e.g. the caseecontrol comparisons of viral aetiology). this unique comprehensive prospective study using modern microbiological methods suggests that the traditional view of aetiology in cap and outpatient lrti should be revised. we have found that viral cap and lrti are also caused by hrv, hcov and hmpv. our high viral detection rates should also inform clinical decision making. better diagnostics are needed to distinguish viral from bacterial cap or lrti at the point of care. the current study provides microbiological evidence for why antibiotics do not help patients with lrti. only approximately one in five lrti patients have a bacterial pathogen isolated and so could conceivably benefit from antibiotic treatment. this evidence should support primary care clinicians' restrictive approach to antibiotic prescribing for lrti. if they consider antibiotics are indeed indicated, the low resistance levels in s. pneumoniae and h. influenzae should support the prescription of narrow-spectrum antibiotics. we declare that we have no conflict of interest. part of this work was presented at the eccmid, berlin, germany, 27e30 september 2013 (s-557). the larger grace observational study was designed by ccb, tv, pl, sc and hg, and sampling protocols by mi, cl, kl and hg. mi, cl, pl, tv and hg supervised the day-to-day management at study sites. pcr and serological analyses were performed by kl, av, cl, kh, kz, ec, fc and avl. data were analysed by mi, kl and cl. statistical analysis was performed by sc.the manuscript was drafted by mi, kl, sc, hg and ccb and was reviewed by all authors. guidelines for the management of adult lower respiratory tract infectionsefull version amoxicillin for acute lower-respiratory-tract infection in primary care when pneumonia is not suspected: a 12-country, randomised, placebo-controlled trial amoxicillin for acute lower respiratory tract infection in primary care: subgroup analysis of potential high-risk groups variation in antibiotic prescribing and its impact on recovery in patients with acute cough in primary care: prospective study in 13 countries impact of amoxicillin therapy on resistance selection in patients with community-acquired lower respiratory tract infections: a randomized, placebo-controlled study use of serum c reactive protein and procalcitonin concentrations in addition to symptoms and signs to predict pneumonia in patients presenting to primary care with acute cough: diagnostic study performance of different mono-and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel specificity and sensitivity of high levels of immunoglobulin g antibodies against pertussis toxin in a single serum sample for diagnosis of infection with bordetella pertussis bordetella pertussis seroprevalence in belgian adults aged 20e39 years viral pneumonia aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods respiratory viruses in adults with community-acquired pneumonia community-acquired pneumonia requiring hospitalization among us adults mycoplasma pneumoniae infection in primary care investigated by real-time pcr in england and wales increased incidence of mycoplasma pneumoniae infections detected by laboratory-based surveillance in denmark in 2010 prevalence, diagnosis, and disease course of pertussis in adults with acute cough in primary care swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs rhinovirus and coronavirus infections two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona-and toroviruses detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness evidence of human bocavirus circulating in children and adults comorbidity and high viral load linked to clinical presentation of respiratory human bocavirus infection human bocavirus: passenger or pathogen in acute respiratory tract infections? the novel ki, wu, mc polyomaviruses: possible human pathogens? no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease the human polyomaviruses ki and wu: virological background and clinical implications we thank the gps, the grace study team, and the patients for taking part in this study. supplementary data related to this article can be found at https://doi.org/10.1016/j.cmi.2018.02.004.funding grace (genomics to combat resistance against antibiotics in ca-lrti in europe, www.grace-lrti.org) was supported by the research foundation flanders (belgium) (g$0274$08n) and the 6th framework program of the european commission, contract no. lshm-ct-2005-518226). the work reported in this publication has been financially supported through the european science foundation (esf), in the framework of the research networking programme trace (http://archives.esf.org/trace; 09-rnp-053). the funding sources were not involved in the design, conduct, analysis and interpretation of the data, nor in the writing and decision to submit the paper. key: cord-318021-4qrf5m8s authors: wolfensberger, a.; jakob, w.; faes hesse, m.; kuster, s.p.; meier, a.h.; schreiber, p.w.; clack, l.; sax, h. title: development and validation of a semi-automated surveillance system—lowering the fruit for non-ventilator-associated hospital-acquired pneumonia (nvhap) prevention() date: 2019-03-25 journal: clin microbiol infect doi: 10.1016/j.cmi.2019.03.019 sha: doc_id: 318021 cord_uid: 4qrf5m8s objectives: conducting manual surveillance of non-ventilator-associated hospital-acquired pneumonia (nvhap) using ecdc (european centre for disease prevention and control) surveillance criteria is very resource intensive. we developed and validated a semi-automated surveillance system for nvhap, and describe nvhap incidence and aetiology at our hospital. methods: we applied an automated classification algorithm mirroring ecdc definition criteria to distinguish patients ‘not at risk’ from patients ‘at risk’ for suffering from nvhap. ‘at risk’-patients were manually screened for nvhap. for validation, we applied the reference standard of full manual evaluation to three validation samples comprising 2091 patients. results: among the 39 519 university hospital zurich inpatient discharges in 2017, the algorithm identified 2454 ‘at-risk’ patients, reducing the number of medical records to be manually screened by 93.8%. from this subset, nvhap was identified in 251 patients (0.64%, 95%ci: 0.57–0.73). sensitivity, negative predictive value, and accuracy of semi-automated surveillance versus full manual surveillance were lowest in the validation sample consisting of patients with hap according to the international classification of diseases (icd-10) discharge diagnostic codes, with 97.5% (ci: 93.7–99.3%), 99.2% (ci: 97.9–99.8%), and 99.4% (ci: 98.4–99.8%), respectively. the overall incidence rate of nvhap was 0.83/1000 patient days (95%ci: 0.73–0.94), with highest rates in haematology/oncology, cardiac and thoracic surgery, and internal medicine including subspecialties. conclusions: the semi-automated surveillance demonstrated a very high sensitivity, negative predictive value, and accuracy. this approach significantly reduces manual surveillance workload, thus making continuous nvhap surveillance feasible as a pivotal element for successful prevention efforts. hospital-acquired pneumonia (hap) is divided into two groups: ventilator-associated pneumonia (vap) and non-ventilator-associated hospital-acquired pneumonia (nvhap). hap and lower respiratory tract infections were shown to be the most common hospital-acquired infections (hais), constituting a proportion of 26% [1, 2] . almost two thirds of haps are nvhaps [1, 2] . although nvhap is more frequent, and has been shown to be comparable in mortality and costs to vap [3] , current research, prevention guidelines, and prevention efforts focus almost exclusively on vap. in 2017, ewan et al. called nvhap a 'neglected disease' among hais [4] . as a fundamental first step to preventing nvhap, they argued for obtaining accurate estimates of prevalence and incidence through prospective surveillance [4] . apart from point prevalence studies in which 0.5e1.1% of all included patients are reported to be affected by nvhap on a given day [2, 5, 6] , literature on nvhap prevalence or incidence is scarce. weber et al. reported an hap incidence of 0.37%, of which 44% were nvhaps, by performing a time-consuming full manual surveillance from using the centers for disease control and prevention (cdc) surveillance definitions of 1988 [7] . three authors reported nvhap incidence rates between 0.12 and 2.28 per 1000 patient days by applying the 2013 cdc surveillance definition to patients with international classification of diseases (icd-9-cm) codes for pneumonia not present on admission [8e10] . these studies may reflect incidence of nvhap only very roughly, as positive predictive value and sensitivity of discharge diagnostic codes for identifying patients with nvhap were shown to be as low as 35% and 59%, respectively [11] . manual surveillance is resource-intensive, and can be applied to only a limited patient population. therefore, automated or semiautomated surveillance algorithms based on electronically available patient data represent a promising alternative to manual surveillance. the ipc-team of the university hospital zurich (uhz) saw the need for continuous surveillance of nvhap as part of their hospitalwide hai prevention programme. the primary aim of this study was to develop and validate a semi-automated nvhap surveillance system, and to assess its sensitivity, negative predictive value and accuracy. second, we aimed to describe incidence and aetiology of nvhap to mitigate data scarcity in this field. the study was conducted at uhz (switzerland), a 950-bed tertiary-care teaching hospital covering all medical specialties (n ¼ 21) except paediatrics and orthopaedics. the study period was january 1st to december 31st 2017. we included all patients who were discharged or passed away during this period. we excluded neonatology patients and newborns in the obstetrics department due to differences in the nvhap surveillance definition for this patient population. routinely collected electronic data were used. in our hospital, all patient data are charted electronically via an electronic medical records (emr) system. selected data are stored in a clinical data warehouse. the necessity for a formal ethical evaluation was waived by the zurich cantonal ethics commission (req-2016-00623), based on the swiss law on research on humans. we used the european centre for disease prevention and control (ecdc) definition criteria for pneumonia, as also applied in the european point prevalence study [12] . the definition comprises radiological criteria, systemic symptoms (fever >38 c, leucopenia or leucocytosis) and pulmonary symptoms (e.g. cough, sputum production). pneumonia is defined as hospital-acquired when symptoms start 48 h after admission. if an invasive respiratory device was present in the 48 h preceding symptom onset, the pneumonia is considered a vap. for this study, we categorized aetiology in three groups according to microbiological sampling site: first, sputum, tracheal aspirate, or upper respiratory tract specimen; second, bronchoalveolar lavage, endobronchial aspirate, or tissue sample; and third, blood culture or antigen detection in blood or urine. we defined 'good-quality sputum and tracheal aspirate' as sputum or tracheal aspirate with <10 squamous epithelial cells (secs) per low power field (lpf) in microscopic examination [13] . 'possible fungal pneumonia' was defined when host factors and clinical criteria were met [14] . 'place of acquisition' was determined as affiliation to department and ward 48 h before the first symptoms of nvhap, unless a shorter incubation period was evident from patient history. the automated aspect of the surveillance was developed by the infection prevention and control (ipc) team (aw, spk, ps, hs) in collaboration with a data warehouse analyst (wj) based on availability of electronic data and nvhap definition. we applied an automated classification algorithm ( fig. 1) [5] to distinguish patients 'not at risk' from those 'at risk' for nvhap. based on the ecdc case definition, patients were defined as 'at risk' if they had undergone at least one radiological procedure (x-ray or ct scan) of the chest fulfilling the following criteria: (a) radiological procedure performed 48 h after admission or at any time during hospitalization for patients readmitted within 28 days; (b) systemic symptoms in temporal relationship to radiological procedure; (c) pneumonia not a-priori excluded in the radiologists' report; (d) no invasive respiratory device constantly present during 48 h preceding radiological procedure. as microbiological criteria are not required for the ecdc nvhap definition, we refrained from including microbiological criteria in the classification algorithm. the 'at-risk' patients were then manually screened for nvhap, strictly applying the ecdc case definition [12] . manual surveillance was performed by two skilled nurses of the ipc team (am, mf) and double-checked by an experienced infectious disease physician (aw). investigators manually collected data comprising 'possible fungal pneumonia', date of onset, and 'place of acquisition'. to further reduce the number of patients and radiological procedures to manually evaluate, we adapted the algorithm during an iterative process. after a 3-month period with about 650 'at-risk' patients, the temporal relationship of systemic symptoms to radiological procedure was reduced from e5 and þ3 days to e3 and þ1 day. additionally, radiological procedures whose reports contained key phrases ruling out pneumonia (see supplement 1) were excluded. after adaptation, the number of 'at-risk' patients was reduced by 8% and sensitivity analysis showed no difference in the number of patients with nvhap. to validate the semi-automated surveillance, we chose full manual surveillance by a skilled ipc nurse, double-checked by an experienced infectious disease physician (both blinded to the results of the algorithm) as the reference standard. as resource considerations prevented the review of all emrs, full manual surveillance was applied on three validation samples (vs1 to vs3): a random sample of 700 patients of the study population (vs1), all 637 patients of the study population having hap according to icd-10 discharge diagnostic codes (vs2), and 754 patients of the year 2016 from four distinct departments, comprising 165 patients with and 589 patients without hap according to icd-10 (vs3). rootcause analysis was conducted on patients with nvhap not classified as 'at-risk' patients. to identify hospital areas associated with increased nvhap rates, we grouped the 21 specialty departments into eight department groups: internal medicine and subspecialties; oncology and haematology; abdominal and urogenital surgery; cardiac and thoracic surgery; traumatology and plastic surgery; eye, ear, head and neck surgery; neurology and neurosurgery; gynaecology and obstetrics. the calculation of sensitivity, negative predictive value, and accuracy of the semi-automated surveillance system was executed according to standard epidemiological methods. accuracy was defined as the proportion of true positives and true negatives in all evaluated cases. the c-square test was used to test differences in categorical variables. all calculations were performed with stata version 15 (stata corp., college station, tx, usa). a total of 39 519 patients who were discharged or passed away during the 1-year study period were included. the algorithm reduced the number of patients to screen manually by 93.7% (95% ci: 93.5e94.0%) to 2454 'at-risk' patients. every patient 'at risk' had a mean of 2.3 radiological procedures to be evaluated. a total of 251 patients were identified as having nvhap according to ecdc criteria. the number of emrs needing to be screened to detect one nvhap was 9.8 (95%ci: 8.7e11.4). on average 4.4 min were required to screen one emr. this would translate to a calculated workload for nvhap surveillance in our 950-bed tertiary care centre of approximately 40 min per workday when using the semiautomatic surveillance system. table 1 shows the cross-tabulations of full manual surveillance against semi-automated surveillance for the three validation samples (vs1evs3), and the respective analyses of sensitivity, negative predictive value, and accuracy. four additional patients were identified to have nvhap in vs2 (comprising only patients with icd-10 hap) that were not identified as 'at risk' by the algorithm and hence missed. consequently, compared to full manual surveillance, sensitivity, negative predictive value, and accuracy of semi-automated surveillance were lowest in vs2 with 97.5% (ci: 93.7e99.3%), 99.2% (ci: 97.9e99.8%), and 99.4% (ci: 98.4e99.8%), respectively. root-cause analysis showed why the algorithm did not identify the four patients as 'at risk': (a) a patient on the icu with fever but normal leucocyte count, not identified because temperature measurements for icu patients are not yet included in emrs for software reasons; (b) a patient with fever beyond the defined 24 h after a radiological procedure; (c) a patient with pneumonia diagnosed in a positron emission tomography scan only, a radiological procedure not taken into account by our algorithm; and (d) a patient with an x-ray that was not correctly entered in the emr system. to describe incidence and incidence density, medical specialty attribution, and microbiology of nvhap, the four patients with nvhap identified only during validation were included, totalling 255 patients with nvhap. the mean in-hospital incidence of nvhap was 0.65% (95%ci: 0.57e0.73%) and the overall incidence rate was 0.83/1000 patient days (95%ci: 0.73e0.94) ( table 2 ). the majority of nvhaps (72.5%, n ¼ 185) were acquired on general wards, whereas 11.8% (n ¼ 30) and 15.7% (n ¼ 40), were acquired on icu and imc, respectively. a few nvhaps (4.3%, n ¼ 11) were acquired during a previous hospitalization in our hospital and readmitted. table 3 provides an overview of microbiological aetiology of nvhap. the vast majority of patients (84%, n ¼ 215) were sampled, and blood culture was the most common sampling technique. bacterial and viral pneumonias were found in 36% (n ¼ 91) and 5% (n ¼ 13) of patients, respectively. when only including samples of 'good quality' (i.e. sputum or tracheal aspirate with <10 sec/lpf), the percentage of pneumonias with identification of a bacterial pathogen dropped to 20% (n ¼ 54). possible fungal pneumonia was found in 5% of patients (n ¼ 13). we developed and validated a semi-automated surveillance system for nvhap to allow continuous outcome monitoring as a cornerstone of an infection prevention programme. by applying a classification algorithm, mirroring radiological and systemic criteria of the ecdc definition, the number of patients for manual screening was reduced by more than 90%. the semi-automated surveillance had a very high sensitivity, negative predictive value and accuracy. during 1 year, 255 patients (or one in 154 patients) acquired an nvhap. semi-automated surveillance systems have already been applied and validated to investigate hais, most frequently for surgical site infections [15e17] and catheter-related bloodstream infections [18, 19] . case finding often relies on diagnostic codes, antimicrobial prescription, and/or microbiology. sensitivity of these algorithms varied from 26% to 95% [5] . for nvhap, we were not able to identify a study validating a (semi-)automated surveillance system. instead, two investigations assessed discharge diagnostic codes to substitute conventional nvhap surveillance with an overall poor sensitivity of 42% and 59% [11, 20] , which declassifies this approach for epidemiological surveillance purposes. by challenging our algorithm by full manual evaluation of the patient population diagnosed and coded to have hap, we identified four additional patients with nvhap. knowing that the sensitivity of icd-10 coded hap for hap according to ecdc definition in our hospital is around 60% [11] , we anticipate having potentially missed another three patients maximumda small number compared to the 251 patients identified by our semi-automated surveillance system. by closely mirroring the ecdc surveillance criteria in our classification algorithm, the sensitivity of our semi-automated surveillance system is close to 100%, higher than the sensitivity of (semi-)automated surveillance systems for various hais summarized in the 2013 review by van mourik et al. [5] . the incidence rate of nvhap in our hospital was similar to that found in previous studies basing their surveillance on coding data [8, 9, 21 ], but incidence was higher than reported by weber et al., table 1 validation of semi-automated surveillance who used the former cdc definitions of 1988 [7] . we found the highest nvhap incidence rates in departments with a presumably high proportion of immunocompromised, multi-morbid, and elderly patients. among surgical departments, nvhap was most common in cardiac and thoracic surgery. these results are consistent with the known risk factors for nvhap outside the icu: i.e., malnutrition, chronic renal failure, anaemia, compromised consciousness, and patients undergoing thoracic surgery [21] . for uhz, university hospital zurich. number of patients in 2017: þ, <100 patients; þþ, 100e500 patients; þþþ, 500e1000 patients; þþþþ, 1000e2000 patients; þþþþþ, >2000 patients. a internal medicine (patients in 2017: þþþþ), angiology (þþþ), cardiology (þþþþþ), dermatology (þþþþ), emergency medicine (þ), endocrinology (þþ), gastroenterology (þþþþ), geriatrics (þþ), immunology (þ), infectious diseases (þþ), nephrology (þþ), pneumology (þþþþ), and rheumatology (þþþ). b haematology (þþþþ), nuclear medicine (þþ), oncology (þþþþ), and radio-oncology (þþ). c visceral surgery (þþþþþ) and urology (þþþþ). d cardiac surgery (þþþþ) and thoracic surgery (þþþ). e traumatology (þþþþþ) and plastic surgery (þþþþ). f ophthalmology (þþþþ), oral and maxillofacial surgery (þþþ), and otorhinolaryngology (þþþþþ). g neurology (þþþþ), neuroradiology (þþ), neurosurgery (þþþþ), and psychiatry (þþ). h gynecology (þþþþþ) and obstetrics (þþþþþ). surgical patients, known risk factors are immobilization, aspiration, gastric retention and vomiting, and abdominal surgery [22] . the proportion of patients with an established aetiology was 36%, consistent with the results of other studies [7, 21] . in our hospital, patients only rarely undergo bronchoscopic sampling, partially explaining the low percentage of microbiologically confirmed pneumonias by ecdc definition [12] . on the other hand, viral diagnostics are readily used, leading to higher rates of viral pneumonia as compared with other studies [7] . oral flora were the only detected pathogens in sputum and tracheal aspirates of 'good quality' in one of three patients. even though aspiration pneumonia is considered to be often caused by anaerobic and facultatively aerobic bacteria of the mouth [23] , the relevance of this finding is not clear, and contamination may be an alternative explanation. some limitations of our study should be considered. first, due to limited resources, we were unable to apply our reference standard of manual surveillance to the whole patient population. we aimed to address this limitation by conducting full manual surveillance on three different validation populations, including a validation sample comprising all patients with hap according to icd-10 coding data. second, even careful manual case evaluation is associated with an inherent risk for interrater variability. we addressed this issue by double-checking all borderline cases and all cases with nvhap by a second observer. third, both semi-automated and full manual surveillance rely on radiological procedures demonstrating a pneumonic infiltrate. patients without x-ray (e.g. due to restrictively performed diagnostic procedures) are missed by both surveillance systems, and so are patients who were transferred to another hospital or discharged home. fourth, due to our selected algorithm criteria, patients with delayed radiological procedure and patients whose pneumonia was diagnosed in a radiological procedure of the abdomen or in a pet scan can be omitted, as shown by our rootcause analysis. lastly, our surveillance system may require modification before it is apt for implementation in hospitals with differing characteristics of emrs and data warehouses. adaptations would also be needed when cdc instead of ecdc nvhap definitions are to be used, as cdc definitions include special diagnostic criteria for immunocompromised and elderly patients. this semi-automated surveillance system opens up various possibilities, such as timely feedback of infection rates, a key element in hai prevention programmes [24, 25] . further, we identified medical specialties with high nvhap rates, allowing targeting of prevention efforts to the departments concerned. the resulting dataset identified the aetiological spectrum of nvhap, revealing a surprisingly high percentage of viral infections, which should be considered in future prevention strategies. the continuous application of this semiautomated surveillance system will allow monitoring of the effectiveness of our ongoing nvhap prevention bundle. in conclusion, this novel classification algorithm proved highly successful in filtering out a large majority of patients 'not at risk' for nvhap, thus drastically reducing manual surveillance workload. this made the establishment of a state-of-the-art semi-automated continuous surveillance of nvhap feasible, facilitating timely feedback of infection rates, identifying departments with high nvhap rates, and monitoring the effectiveness of nvhap prevention measures. this surveillance system constitutes a breakthrough and central building block of nvhap prevention, a previously neglected hai. implementation and validation of this semi-automated surveillance system (or adaptations of it) in other hospitals would be of special interest to investigate its overall validity and generalizability. aw, wj, hs, spk, and ps designed the study. wj, am, mf, and aw acquired the data, and aw and spk performed statistical analysis. aw, wj, hs, spk, and ps analysed and interpreted the data. aw drafted the manuscript, and wj, mf, am, spk, ps, lc and hs provided a critical review of the manuscript for important intellectual content. all authors agree with the content and conclusions of this manuscript. all authors declare no conflicts of interest. aline wolfensberger is supported by the academic career programme 'filling the gap' of the medical faculty of the university of zurich. development of the algorithm was supported by 'innovation pool', a university hospital zurich funding programme for developing new approaches in medical diagnostics and treatment. the funding source was not involved in the study design, collection and interpretation of the data, or manuscript submission. health care-associated infections: a meta-analysis of costs and financial impact on the us health care system multistate point-prevalence survey of health care-associated infections the breadth of hospital-acquired pneumonia: nonventilated versus ventilated patients in pennsylvania hospital-acquired pneumonia surveillancedan unmet need automated surveillance for healthcare-associated infections: opportunities for improvement point prevalence survey of healthcare associated 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hospital information system at lyon university hospital enhanced surgical site infection surveillance following hysterectomy, vascular, and colorectal surgery improved surveillance for surgical site infections after orthopedic implantation procedures: extending applications for automated data development of trigger-based semi-automated surveillance of ventilator-associated pneumonia and central line-associated bloodstream infections in a dutch intensive care automated surveillance for central line-associated bloodstream infection in intensive care units frequency of hospital-acquired pneumonia in electronic and paper-based patient record risk factors for hospital-acquired pneumonia outside the intensive care unit: a case-control study incidence and potential risk factors for hospital-acquired pneumonia in an emergency department of surgery geriatric oral health and pneumonia risk the efficacy of infection surveillance and control programs in preventing nosocomial infections in us hospitals feeding back surveillance data to prevent hospital-acquired infections supplementary data to this article can be found online at https://doi.org/10.1016/j.cmi.2019.03.019. key: cord-328499-d6cvaxm9 authors: matzkies, lucie-marie; leitner, eva; stelzl, evelyn; assig, karoline; bozic, michael; siebenhofer, david; mustafa, maria e.; steinmetz, ivo; kessler, harald h. title: lack of sensitivity of an ivd/ce-labeled kit targeting the s gene for detection of sars-cov-2 date: 2020-07-08 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.036 sha: doc_id: 328499 cord_uid: d6cvaxm9 objectives: new molecular tests for sars-cov-2 are rapidly launched in response to the covid-19 pandemic. the aim of this study was to evaluate the analytical and the clinical performance of the viasure sars-cov-2 s gene rt-pcr kit on the bd max™ system and to compare results with those obtained with the cobas® sars-cov-2 test on the cobas® 6800 system. methods: for testing the analytical performance, reference material was used. clinical samples (n=101) obtained from patients with symptoms compatible to covid-19 were studied. oroand nasopharyngeal swabs were collected by using either eswab™ or utm™ collection systems. results: when the analytical performance was evaluated, the sample containing the lowest sars-cov-2 concentration tested negative with the viasure test while results obtained with the cobas® test were found to be concordant with the results expected. six out of the 101 clinical samples (5.9%) showed an inhibition with the viasure test. when analyzing the remaining 95 clinical samples, 27 were found to be negative with both assays. of 68 samples positive with the cobas® test, the viasure test missed 21 (30.9 %) samples. all of those 21 samples had shown ct values ≥ 31 with the cobas® 6800 system. none of the samples tested positive with the viasure test and negative with the cobas® test. conclusions: the viasure test was impaired by a lack of sensitivity and a relatively high number of invalid results. when using the viasure test for routine testing, a significant number of covid-19 positive samples would have been missed. objectives: new molecular tests for sars-cov-2 are rapidly launched in response to the covid19 19 pandemic. the aim of this study was to evaluate the analytical and the clinical performance 20 of the viasure sars-cov-2 s gene rt-pcr kit on the bd max™ system and to compare results 21 with those obtained with the cobas® sars-cov-2 test on the cobas® 6800 system. detection of 2019 novel 177 coronavirus (2019-ncov) by real-time rt-pcr clinical evaluation of 179 the cobas sars-cov-2 test and a diagnostic platform switch during 48 hours in the midst of the 444212 br. viasure sars-cov-2 s-gene real time pcr detection kit comparison of the bd max(r) enteric bacterial panel assay 183 with conventional diagnostic procedures in diarrheal stool samples a microbiological study to 186 investigate the carriage and transmission-potential of clostridium difficile spores on single-use and 187 reusable sharps containers virological 189 assessment of hospitalized patients with covid-2019 detection of sars-cov-2 in different types of 191 sars-cov-2 viral load in upper 193 respiratory specimens of infected patients directive 98/79/ec of the european parliament and of the council of 27 195 october 1998 on in vitro medical devices extraction of viral nucleic 197 acids: comparison of five automated nucleic acid extraction platforms pooling of nasopharyngeal swab specimens for sars-cov-2 199 detection by rt-pcr guidelines on covid-19 in vitro diagnostic tests and their performance brussel: communication from the commission key: cord-291729-4l4v9jxd authors: de salazar, adolfo; aguilera, antonio; trastoy, rocio; fuentes, ana; alados, juan carlos; causse, manuel; galán, juan carlos; moreno, antonio; trigo, matilde; pérez-ruiz, mercedes; roldán, carolina; josé pena, ma; bernal, samuel; serrano-conde, esther; barbeito, gema; torres, eva; riazzo, cristina; cortes-cuevas, jose luis; chueca, natalia; coira, amparo; sanchez-calvo, juan m.; marfil, eduardo; becerra, federico; gude, maría josé; pallarés, ángeles; pérez del molino, maría luisa; garcía, federico title: sample pooling for sars-cov-2 rt-pcr screening date: 2020-09-10 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.008 sha: doc_id: 291729 cord_uid: 4l4v9jxd objective: to evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of covid-19, by using different commercial platforms for nucleic acid extraction and amplification. methods: 3519 nasopharyngeal samples received at 9 spanish clinical microbiology laboratories were processed individually and in pools (342 pools of 10 samples and 11 pools of 9 samples) according to the existing methodology in each of the centres. results: we found that 253 pools (2519 samples) were negative, and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 pcr tests. for 29 pools (made out of 290 samples) we found discordant results when compared to their correspondent individual samples: in 22/29 pools (28 samples), minor discordances were found; for seven pools (7 samples), we found major discordances. sensitivity, specificity, positive and negative predictive values for pooling were 97.10% (ci95%; 94.11-98.82), 100%, 100% and 99.79% (ci95%; 99.56-99.90) respectively; accuracy was 99.80% (ci95%; 99.59-99.92) and kappa concordant coefficient was 0.984. the dilution of samples in our pooling strategy resulted into a median loss of 2.87 (ci95%; 2.46-3.28) cts for e gene, 3.36 (ci95%; 2.89-3.85) cts for rdrp gene and 2.99 (ci95%; 2.56-3.43) cts for n gene. conclusion: we show a high efficiency of pooling strategies for sars-cov-2 rna testing, across different rna extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity, and positive and negative predictive values. sars-cov-2 pandemic has posed an immense challenge for the national health systems of the affected countries. in the absence of a vaccine or effective treatment, molecular diagnosis is the only tool to contain the pandemic, identifying the transmitting infected patients, and proceed to their isolation to avoid new infections. due to its high demand, testing opportunities may have been hampered in some scenarios, as a consequence of the lack of reagent supplies, and their limited production. the current challenge for sars-cov-2 diagnosis is the great demand of testing that we are facing in the new test and trace era. clinical laboratories must plan to increase their analytical capacity to face these new public health challenges that will allow, by means of massive analysis, to identify all infected persons, proceed with their isolation and trace their contacts. this undoubtedly constitutes a challenge due to the high number of diagnostic processes required and the limited resources available in the face of a disease with a variable incubation period, an uncertain viral dynamic [1, 2] and an unknown number of asymptomatic carriers who can transmit the infection. dorfman in 1943 [3] introduced the strategy of mixing samples in a single test into clinical diagnosis, and this strategy has been helpful in correctly identifying all infected individuals using fewer diagnostic tests [4, 5] . the diagnosis of sars-cov-2 infection is fundamentally based on real-time rt-pcr, which is the reference technique (6, 7) . this is a robust technology with high sensitivity and specificity and has already been used in the pooling strategy of samples for the screening of hiv, hbv and hcv (8) (9) (10) (11) , where it has proven to be cost-effective and efficient in surveillance and diagnosis (detection) for prevalence below 30%, regardless of the population studied. therefore, the combination of pooled tests and patients with low risk of infection is considered a practical and effective method to analyse large quantities of samples without compromising precision, especially when it comes to centralized models with automated systems. in sars-cov-2 infection, the data on the best strategy for the detection of cases by grouping of samples and how they influence the sensitivity of the rt-pcr analysis are limited, therefore, it is necessary to investigate the effect of the number of samples, especially with commercially available assays [12] [13] [14] [15] [16] [17] . our objective in this study has been to evaluate the efficacy of sample pooling in a multicentre way compared to the individual analysis for the detection of covid-19 by using different commercial platforms available for genomic extraction and amplification by rt -pcr in real time. j o u r n a l p r e -p r o o f between march and may 2020, nasopharyngeal swabs (n = 3519) were collected from patients or health professionals and sent to the virology laboratories of the participating centres (supplementary table 1) . nasopharyngeal and pharyngeal swab were collected at a time and both swabs were placed in the same tube with transport media. several viral transport mediums were used: vircell transport medium (vircell -(granada, spain)), δswab® transport medium (deltalab -(rubí, spain)) and utm®: universal transport (copan -(brescia, italy). samples were processed on the first 24 hours upon reception. nine or ten individual samples were pooled, and screening was performed using reverse transcriptase-polymerase chain reaction targeting the same target as for individual samples. pooled testing was performed at each of the participating sites in the study. pooling was performed by hand, after inactivation of each sample that made part of the pool. samples for pooling were selected randomly, according to availability at each site during the study period. for both individual testing and pooled analysis, samples were inactivated 1:1 in lysis buffer and processed according to the existing methodology in each laboratory, which is summarized in table 1 . major discordance was defined as a negative pool result when at least one of the individual samples showed ct values <35 for one or more sars-cov-2 genes. minor discordant calls were considered when at least one individual sample had a ct value >35 in one or two of the sars-cov-2 genes assayed and the pool scored negative. the performance characteristics such as sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and relative efficiency was calculated comparing the individually analysed sample (gold standard) with respect to the sample result analysed within the pool. statistics were performed on r studio and graph pad prism v8 software. this study was approved by "comité de ética de la investigación con medicamentos de galicia (ceim-g)" review board. given the deidentified nature of testing, individual patient consent was not required for this study. the study included 3519 samples from 9 different sites in spain. we analyzed all samples individually and also in parallel, pooled into 353 groups of samples (342 pools of 10 samples and 11 pools of 9 samples). two hundred and forty-one (6.85%) of the samples were positive. we found that 253 pools, made up of 2519 samples, were negative (242 pools of 10 samples and 11 pools of 9 samples); 99 pools, made up of 990 samples, were positive (99 pools of 10 samples). one pool, made of 10 samples, was invalid. therefore, our pooling strategy would have saved 2167 (86%) pcr tests. a description of positive and negative pools can be found in table 2. overall, 323 pools with 3219 samples showed concordant results with the individual samples analyzed (224 pools with 2229 samples negative and 99 pools with 990 samples that included at least one positive sample). for 29 pools (290 samples) we found discordant results compared to individual samples. in 22 pools minor discordances were found and in 7 pools we found major discordances. a detailed description of the discordances can be found in supplementary table 2 , where it can be seen that the pools with minor discordances included 24 samples from patients who had a prior positive sars-cov 2 test and were submitted for pcr testing at least 20 days after to evaluate rna clearance. when only major discordances were considered for the analysis, sensitivity, specificity, positive and negative predictive values for pooling were 97.10% (ci95%; 94.11-98.82), 100%, 100% and 99.79% (ci95%; 99.56-99.90) respectively. accuracy was also 99.80% (ci95%; 99.59-99.92) and kappa concordant coefficient was 0.984. these data are detailed in table 3 . when all discordances were considered for the analysis, sensitivity, specificity, positive and negative predictive values for pooling were 85.48% (ci95%; 80.39-89.67), 100%, 100% and 98.94% (ci95%; 98.57-99.22) respectively. accuracy was 99.00% (ci95%; 98.62-99.30) and kappa concordant coefficient was 0.916. these data are detailed in table 4 . supplementary table 3 shows the number and rate of discrepancies across the different tests and targets used at the participating sites. the lowest rate of discrepancies was observed for the viasure sars-cov-2 real time pcr (certest) (n=3; here we report on the high efficiency of pooling strategies for sars-cov-2 rna testing, across different rna extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity, and positive and negative predictive values. we believe that our results may help clinical laboratories to respond to the clinical need on sars-cov-2 testing. although sample pooling strategy works for other pathogens that are diagnosed by rt-pcr, for sars-cov-2 infection, there is still limited data in the literature regarding surveillance and detection strategies by grouping samples [12] [13] [14] [15] [16] [17] . in our study we have evaluated the efficacy of sample pooling in a multicentre way compared to the individual analysis for the detection of covid-19 by using different commercial platforms available for genomic extraction and amplification by rt-pcr in real time. within a 6.8% positive rate, we have obtained excellent numbers in sensitivity, specificity, and positive and negative predictive values, both in a scenario for which only major discordances were considered (97.10%, 100%, 100%, 99.79%, respectively) and also when minor discordances were counted (85.48%. 100%, 100% and 98.94%). as expected, and as other authors have also shown [18] , the dilution of samples in our pooling strategy resulted into a median loss of 2.87 cts for e gene, 3.36 cts for rdrp gene and 2.99 cts for n gene. this drop in the sensitivity was responsible for most of the discordances found in our study, that were mainly observed for samples with the lowest positivity signals, always with cts very close to 40, and very frequently in only one gene of the two-three that were included in the tests. although special attention on rt-pcr false negative results must be paid [19] , it is also known that most of the positive results obtained from just one gene targeted and with cts >35 correspond to non-viable/noninfectious particles that are still detected by rt-pcr [20] . in addition, false positive results yielding cts>35 may also be expected. our study's main limitation was the variability upon extraction and amplification methods used, and the number of samples included in the different pools tested; however, this limitation in the design may turn into its main strength given the consideration that even in this scenario our results were excellent. in the sample pooling strategy, it is a priority to determine the group size in which the maximum precision is maintained in the analysis performed, since, due to the dilution of the sample, this procedure can decrease the sensitivity of the molecular assays of rt-realtime pcr "optimization of group size in pool testing strategy for sars-cov-2: a simple mathematical model" [21] . for this reason, before systematically implementing a sample grouping strategy, it is important to consider these characteristics (detection limit, sensitivity and specificity of the test) together with the expected prevalence; in this regard, there are already applications that allow it to be calculated (https://www.chrisbilder.com/shiny/); in addition, and in depth mathematical analysisof pool testing by cherif et al. is also available [22] . the main advantages of the pooling strategy are that it allows using the same standard protocols of commercial reagents, with no need of additional training, equipment or materials, and consequently it can be implemented immediately to expand the detection and surveillance capabilities of covid-19. another limitation to our study is that samples were pooled by hand. we are currently evaluating the automation of pooling samples, as this is a pre-analytical critical step to be solved to handle the great number of samples that the pooling strategy will allow to process. result reporting is another critical step that needs to be addressed. finally, we did not collect information on clinical data/symptoms; we believe that a great number of patients, if not all, would be symptomatic, as asymptomatic screening has not been a part of clinical practice in spain during the pandemic first wave. it should be noted that pooling as a screening strategy will not completely eliminate the need for individual diagnostic tests, which will be essential when community transmission intensifies. in our study, even in the setting of a 6.86% prevalence, out of 3519 samples analysed, we would have saved a total of 2167 pcr tests, with a great saving in time, costs and personnel. within the current epidemiological situation, in which prevalence has significantly decreased but the high demand on pcr testing continues, this efficiency measures would help clinical laboratories in alleviating the workload in order to prepare for future outbreaks. in summary, we show a high efficiency of pooling strategies for sars-cov-2 rna testing, across different rna extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity, and positive and negative predictive values. whether pooling may be used highly relies on sars-cov-2 prevalence, rather on the fact that patients are asymptomatic (prevalence may be high if community transmission has been reached) or symptomatic. specific recommendations for pooling testing have become available very recently from fda (https://www.fda.gov/newsevents/press-announcements/coronavirus-covid-19-update-fda-issues-first-emergencyauthorization-sample-pooling-diagnostic). as we believe our findings may be essential to expand clinical laboratories capabilities in the very near future, we recommend to validate this strategy in each specific setting of extraction and amplification reagents before its introduction for clinical management, specially to ensure that sensitivity of the assay and especially the false negative rates are acceptable. sars-cov-2 viral load in upper respiratory specimens of infected patients viral load of sars-cov-2 in clinical samples the detection of defective members of large populations improved matrix pooling a methodology for deriving the sensitivity of pooled testing, based on viral load progression and pooling dilution detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr covid-19 diagnosis and management: a comprehensive review screening tests: can we get more by doing less pooling strategies to reduce the cost of hiv-1 rna load monitoring in a resource-limited setting pooled nucleic acid testing to detect antiretroviral treatment failure in mexico occult hbv infection in hiv-infected adults and evaluation of pooled nat for hbv assessment of specimen pooling to conserve sars cov-2 testing resources pooled-sample analysis strategies for covid-19 mass testing: a simulation study sample pooling as a strategy to detect community transmission of sars-cov-2 evaluation of covid-19 rt-qpcr test in multi-sample pools pooling of nasopharyngeal swab specimens for sars-cov-2 detection by rt-pcr evaluating the efficiency of specimen pooling for pcr-based detection of covid-19 pooling of samples for testing for sars-cov-2 in asymptomatic people false negative tests for sars-cov-2 infection -challenges and implications predicting infectious sars-cov-2 from diagnostic samples optimization of group size in pool testing strategy for sars-cov-2: a simple mathematical model simulation of pool testing to identify patients with coronavirus disease 2019 under conditions of limited test availability the authors declare that they have no conflicts of interest in relation with the topic of this paper. key: cord-343090-dsjq98ks authors: fragkou, paraskevi c.; belhadi, drifa; peiffer-smadja, nathan; moschopoulos, charalampos d.; lescure, françois-xavier; janocha, hannah; karofylakis, emmanouil; yazdanpanah, yazdan; mentré, france; skevaki, chrysanthi; laouénan, cédric; tsiodras, sotirios title: review of trials currently testing treatment and prevention of covid-19 date: 2020-05-23 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.05.019 sha: doc_id: 343090 cord_uid: dsjq98ks background: as covid-19 cases continue to rise globally, evidence from large randomised controlled trials is still lacking. currently, numerous trials testing potential treatment and preventative options are undertaken all over the world. objectives: we summarised all registered clinical trials examining treatment and prevention options for covid-19. additionally, we evaluated the quality of the retrieved studies. data sources: clinicaltrials.gov, the chinese clinical trial registry and the european union clinical trials register were systematically searched. study eligibility criteria: registered clinical trials examining treatment and/or prevention options for covid-19 were included. no language, country or study design restrictions were applied. we excluded withdrawn or cancelled studies and trials not reporting therapeutic or preventative strategies for covid-19. participants: and interventions: no restrictions in terms of participants’ age and medical background or type of intervention were enforced. methods: the registries were searched using the term “coronavirus” or “covid-19” from their inception until 26(th) march 2020.additional manual search of the registries was also performed. eligible studies were summarised and tabulated. interventional trials were methodologically analysed, excluding expanded access studies and trials testing traditional chinese medicine. results: in total, 309 trials evaluating therapeutic management options, 23 studies assessing preventive strategies and 3 studies examining both were retrieved. finally, 214 studies were methodologically reviewed. interventional treatment studies were mostly randomised (n=150, 76%) and open-label (n=73, 37%) with a median number of planned inclusions of 90 (iqr 40-200). major categories of interventions that are currently being investigated are discussed. conclusion: numerous clinical trials have been registered since the onset of the covid-19 pandemic. summarised data on these trials will assist physicians and researchers to promote patient care and guide future research efforts for covid-19 pandemic containment. given the steep upsurge of covid-19 cases worldwide within an unprecedented short period 60 9 azvudine, an azidocytidine analogue that inhibits viral reverse transcriptase, has been 153 effective against hiv, hepatitis b and c viruses [29] . its efficacy against sars-cov-2 is 154 being tested in 3 ongoing clinical trials (table 1, table s1 ). another nucleoside analogue 155 undergoing investigation for covid-19 pneumonia is emtricitabine/tenofovir alafenamide. 156 157 chloroquine and hydroxychloroquine are currently licensed for the treatment of malaria and 159 autoimmune diseases [30] . however, they have also been studied against several viruses with 160 promising in vitro results, never confirmed in humans [31] [32] [33] . as weak bases, they are 161 concentrated in acidic intra-cellular organelles, leading to alkalization and disruption of the 162 low ph-dependent steps of viral replication, including viral-cell fusion and uncoating [30, 32] . 163 moreover, they impair the terminal glycosylation of ace2 receptor in golgi apparatus, thus 164 inhibiting the viral penetration into the host cells [34] . 165 as they are accumulated in lymphocytes and macrophages, these drugs reduce secretion of 166 proinflammatory cytokines, and particularly of tumour necrosis factor alpha (tnf-α) [33] . 167 experimental data demonstrated that chloroquine is highly effective in vitro against sars-168 cov-2 in an estimated effective concentration that is easily achievable with standard dosing 169 regimens [25] . however, the efficacy of anti-malaria drugs in clinical practice is still much 170 debated. some preliminary reports from ongoing trials supporting their effectiveness, alone or 171 in combination with azithromycin [35, 36] , have been called into question on the basis of their 172 methodology. moreover, these results were challenged by new trials that did not find 173 any substantial benefit from hydroxychloroquine administration [37] [38] [39] . . therefore, clinical 174 trials with a control group are needed to provide reliable answers for clinicians; antimalaria 175 drugs are being tested in 30 randomised controlled trials (table 1, table s1 ). virus-induced immune response leading to cytokine storm syndrome (css) and secondary 179 haemophagocytic lymphohistiocytosis (hlh) is probably the underlying pathogenetic 180 mechanism that leads to critical and often fatal covid-19 infection [40, 41] . (table 1, table s1 ); 189 tocilizumab, in particular, improved symptoms and laboratory parameters in a small immunomodulators licensed for haematological and rheumatological conditions (such as 197 leflunomide and thalidomide), as well as colchicine that counteracts the assembly of the 198 nlrp3 inflammasome, are also being studied for their therapeutic use against sars-cov-2 199 (table 1,table s1 ) [44] . the immunomodulatory effects of macrolide antibiotics, as well as their pharmacodynamic 201 property to achieve at least 10-fold higher concentrations in epithelial lung fluid than in 202 serum, have led researchers to repurpose them against sars-cov-2 (table 1, table s1 observations the antifibrotic agent pirfenidone is being evaluated in at least three randomised 215 clinical trials for its efficacy in the prevention of post-covid-19 pneumonia fibrosis (table 216 1, table s1 ). pirfenidone targets collagen synthesis by inhibiting transforming growth factor 217 beta (tgf-b), diminishing extracellular matrix deposition and reducing the activity of lung 218 fibroblasts in vitro [53] . 219 finally, immunostimulatory molecules that enhance the hosts' immune response against the 220 invading pathogen, like ifn-α, interferon beta (ifn-β), the recombinant protein produced by (table 1, table s1 ). (table 1, table s1 ). (table 2, table s2 ). 293 many studies are currently evaluating the efficacy of tcm in covid-19 prevention in 294 china. importantly, at least 4 vaccines are under development. among them, an mrna-based 295 vaccine encoding the s-protein is being assessed for its safety, reactogenicity and efficacy 296 against sars-cov-2 (table 2, table s2 ). besides the registered trials, other large companies 297 have also announced the initiation of vaccine development [81, 82] . 298 other preventative molecules include hydroxychloroquine and the recombinant human 299 interferon α1b spray. in the usa, exposed individuals are randomised to hydroxychloroquine 300 or placebo, evaluating the agent's potential as post-exposure prophylaxis (nct04308668, 301 table s2 ). furthermore, another randomised clinical trial evaluates the efficacy of a 3-month 302 course of chloroquine in at-risk healthcare personnel (nct04303507 , table s2 ). finally, the 303 live attenuated strain of mycobacterium bovis is expected to be tested as a preventative 304 strategy against covid-19 among healthcare professionals, in australia and france. 305 306 in total, 198 interventional treatment and 16 prevention trials were included in the 309 methodological analysis respectively (table 3) . among the eligible treatment studies, 310 children recruitment (i.e.< 14 years old) was reported in 7 clinical trials in total: 1 testing 311 darunavir with cobicistat (nct04252274); 2 on human stem cells transfusion 312 (chictr2000029606, chictr2000030944); 1 testing hydroxycholoroquine (eudract 313 number: 2020-000890-25); 1 using tocilizumab (nct04317092); and 1 assessing nutritional 314 supplements (nct04323345) ( table s1 ).with respect to relevant prevention studies, children 315 were included in 2 vaccine trials (nct04276896, nct04299724) as shown in table s2 . 316 317 phase iv and phase iii treatment trials were the most commonly reported interventional study 319 types (n=40, 20% and n=35, 18% respectively) as demonstrated in table 3 . nonetheless, the 320 majority of registered trials do not disclose the study phase (n=83, 42%). 321 in terms of blinding, 73 open-label (37%), 31 double-blinded (16%), and 16 single-blinded 322 (8%) studies were retrieved. most trials were randomised (n=150, 76%) with a parallel 323 assignment between arms. the median (iqr) number of planned inclusions is 90 (40-200) 324 with a range of 5 to 6000 participants. 325 phase iii and phase i prevention studies were the most commonly reported ones (n=6, 38% 326 and n=3, 19% respectively, table3). as with treatment trials, many prevention trials do not 327 report the study phase (n=4, 25%). 328 regarding prevention studies' blinding, 6double-blinded (38%), 5open-label (31%), and 2 329 single-blinded (13%) were found. most studies were randomised (n=10, 63%) with a parallel figure 5 shows 341 the total number of planned inclusions and the number of clinical trials for the ten most 342 frequently studied treatments, with hydroxychloroquine being the treatment associated with a clinical primary outcome was defined in 128 out of 198 therapeutic trials (65% ; table 3) . (6%) studies respectively (table 3) . 354 regarding prevention studies,10 out of 16 (62%)disclosed a clinical primary outcome , such 355 as confirmed symptomatic covid-19 for 3studies, severe covid-19 for 2 studies, 356 confirmed or suspected covid-19 for one study, and safety for 4 (studies evaluating 357 vaccines. the other prevention studies had a virological outcome (confirmed sars-cov-2 358 infection with or without symptoms, n=5) or a biological outcome (n=1, routine blood tests) 359 (table 3) . analyse clinical trials testing these agents. 394 firstly, study design data and details on the interventions being assessed were often lacking. 397 this hampers the available information to researchers and relevant stakeholders, and 398 potentially influences the discovery of successful treatments. 399 secondly, most trials, and especially those registered at the beginning of the pandemic, 400 disclosed low participant numbers, which may impact the robustness of their future results. 401 however, these numbers should be cautiously interpreted, as they represent the anticipated, the progression of computed tomographic (ct) 592 images in patients with coronavirus disease (covid-19) pneumonia the ct progression of covid-19 pneumonia integrating mechanisms of pulmonary fibrosis induction of pro-598 inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 covi-19 or sars-cov-2): anti-inflammatory strategies pirfenidone attenuates 602 lung fibrotic fibroblast responses to transforming growth factor-β1 discovery french steering committee. type 1 interferons as a potential treatment 606 against covid-19 angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers in tmprss2 and adam17 cleave ace2 differentially and only proteolysis by 617 tmprss2 augments entry driven by the severe acute respiratory syndrome renin-angiotensin system blockers and the covid-19 pandemic: at present there is no evidence to abandon renin camostat mesilate, pancrelipase, and rabeprazole combination therapy improves 627 epigastric pain in early chronic pancreatitis and functional dyspepsia with arbidol as a broad-spectrum antiviral: an update antiviral activity of arbidol 639 against influenza a virus, respiratory syncytial virus, rhinovirus, coxsackie virus and 640 adenovirus in vitro and in vivo pharmacokinetics, metabolism, 643 and excretion of the antiviral drug arbidol in humans arbidol combined with lpv/r 648 versus lpv/r alone against corona virus disease 2019:a retrospective cohort study evaluation of convalescent whole blood for treating ebola virus disease in freetown treatment of severe acute respiratory 654 syndrome with convalescent plasma use of 656 convalescent plasma therapy in sars patients in hong kong retrospective 659 comparison of convalescent plasma with continuing high-dose methylprednisolone 660 treatment in sars patients the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the 664 treatment of severe acute respiratory infections of viral etiology: a systematic review 665 and exploratory meta-analysis feasibility 668 of using convalescent plasma immunotherapy for mers-cov infection disappearance of antibodies to sars-associated coronavirus after recovery therapeutic 674 implications of human umbilical cord mesenchymal stromal cells in attenuating 675 influenza a(h5n1) virus-associated acute lung injury mesenchymal stem cells improves the outcome of patients with covid-19 exosomes derived from mesenchymal stem cells exosomes from mesenchymal stem/stromal cells: a new 683 therapeutic paradigm moderna's covid-19 vaccine could reach healthcare workers this fall | fiercebiotech 693 n johnson identifies lead covid-19 vaccine candidate n.d who r&d blueprint novel coronavirus covid-19 therapeutic trial synopsis drug treatment options for the 2019-new coronavirus (2019-ncov) covid-19 infection: the 703 perspectives on immune responses early antiviral treatment contributes 706 to alleviate the severity and improve the prognosis of patients with novel coronavirus 707 disease (covid-19) temporal 709 profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study clinical features of patients 713 infected with 2019 novel coronavirus in wuhan, china covid-19: 716 combining antiviral and anti-inflammatory treatments nih clinical trial of investigational vaccine for covid-19 begins | national institutes 721 of health (nih) n.d chloroquine/ hydroxychloroquine prevention of coronavirus disease (covid-19) in the healthcare setting -full text view -clinicaltrials molecular immune pathogenesis and diagnosis 727 of covid-19 xpress sars-cov-2 has received fda emergency use (covid-19) globally key: cord-265363-xw56intn authors: gautret, p.; yong, w.; soula, g.; gaudart, j.; delmont, j.; dia, a.; parola, p.; brouqui, p. title: incidence of hajj-associated febrile cough episodes among french pilgrims: a prospective cohort study on the influence of statin use and risk factors date: 2014-12-12 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2009.02816.x sha: doc_id: 265363 cord_uid: xw56intn a prospective epidemiological study was conducted to evaluate the incidence of febrile cough episodes among adult muslims travelling from marseille to saudi arabia during the hajj pilgrimage and to assess if use of statin had an influence on this incidence. in total, 580 individuals were presented with a questionnaire. a significant proportion of individuals had chronic medical disorders, e.g. diabetes mellitus (132, 22.8%) and hypertension (147, 25.3%). pilgrims had a low level of education and a low employment rate. sixty (10.3%) were treated with statins for hypercholesterolemia. four hundred and fourty-seven pilgrims were presented a questionnaire on returning home. a total of 74 travellers (16.6%) experienced fever during their stay in saudi arabia (67 attended a doctor) and 271 (60.6%) had cough (259 attended a doctor); 70 travellers with cough were febrile (25.9%). seventy per cent of the travellers who suffered cough episodes developed their first symptoms within 3 days, suggesting a human to human transmission of the responsible pathogen, with short incubation time as evidenced by a bimodal distribution of cough in two peaks at a 24 h interval. none of demographical and socioeconomic characteristics, underlying diseases or vaccination against influenza significantly affected the occurrence of cough. diabetes correlated with an increased risk of febrile cough (or = 2.02 (1.05–3.89)) as well as unemployment (or = 2.22 (0.91–5.53)). use of statins had no significant influence on the occurrence of cough and/or fever during the pilgrimage. this result suggests that while treatment with a statin has been demonstrated to reduce the mortality from severe sepsis associated with respiratory tract infections, it probably does not play a role in the outcome of regular febrile cough episodes as observed in the cohort studied here. each year, approximately 2000 muslims travel from marseille to participate in the hajj, gathering with over two million pilgrims from all over the world. health risks during the hajj are a critical issue due to the extreme congestion of people [1] . infectious diseases represent a major problem during the pilgrimage with acute respiratory infections (ari) as the most common cause of admission to hospital [2] [3] [4] . hajj pilgrims during their 1-month stay in saudi arabia experience relatively homogeneous accommodation conditions, and undertake identical rituals, while retracing the footsteps of the prophet mohammed, thus being very likely exposed to the same risk of ari. in recent years, several non-randomized studies have linked statin use with decreased risk of severe sepsis or death from severe infections, including pneumonia [5] [6] [7] . recent prospective cohort studies confirmed previous observations [8] [9] [10] [11] [12] while another suggested that the apparent beneficial effect of statins probably reflected a 'healthy user' effect, as statin users appeared to be younger, healthier, better educated, and socially and economically more privileged compared to non-statin users [13] . these controversial findings also raised questions about the potential role of statins in the prophylaxis of infectious diseases such as pandemic influenza [14] . muslims departing from marseille to participate in the hajj have been found to have a median age of 61 years, with more than one third being over 64 years old [15] , and are therefore likely to use statins in a significant proportion. we conducted a prospective epidemiological study to evaluate the incidence of febrile cough episodes among hajj pilgrims from marseille and to assess if statin use could have an influence on this incidence. the socio-economic situation and health characteristics of the travellers were not consistent with the hypothesis of a 'healthy user' effect. a prospective cohort study was carried out in the marseille travel medicine centre (hô pital nord) from 4 november to 8 december 2006 . participants in the survey were pilgrims in preparation for the hajj pilgrimage enrolled in the meningococcal vaccination campaign to satisfy compulsory vaccination requirements. pilgrims older than 18 years were included on a voluntary basis and participants were asked to give written consent. pre-travel questionnaires were presented orally, before vaccination, in french, in arabic or in french and arabic, depending on the language fluency level of the participants. post-travel questionnaires were presented by telephone. the pre-travel questionnaire included demographic factors (age, gender, location of residence), indicators of immigration status (country of birth and duration of stay in france), socio-economic indicators (level of education, employment, type of housing, rooms per person and household, complementary health insurance modalities), health status indicators (diabetes, hypertension, chronic respiratory diseases, statin use, vaccination coverage against influenza) and number of previous travels to saudi arabia. the post-travel questionnaire included travel indicators (duration of stay, food and housing conditions) and data about travel-associated diseases (medical consultation, hospitalization, occurrence of cough with or without fever, time of manifestation and duration of symptoms). cough was defined as occurrence of cough with or without sputum in an individual without chronic cough and subjective aggravation of cough in individuals suffering from chronic respiratory diseases. fever was defined as subjective feeling of fever. pilgrims were considered as lost in follow up after three failed attempts to reach them by phone. data were recorded anonymously in a microsoft access database and transferred to epiinfo 6.0 software (cdc, atlanta, ga, usa) for univariate statistic analysis. differences in proportions were evaluated using the chi-square test. as selection procedure, a two-tailed p value £0.25 was considered as significant [16] . multivariate analysis was performed using the spss version 15 software program (spss, inc., chicago, il, usa). factors with a p value <0.25 in univariate models were included in a multivariate model, as suggested in the classical work of mickey and greenland [16] . sex, age and statin use were also included in the model. a stepwise procedure based on likelihood ratio criteria was used in order to obtain the best criteria with the lowest akaike criteria (aic) [17] [18] [19] . for the final model, a two-tailed p value £0.05 was considered as significant. among 650 vaccinees preparing for the hajj pilgrimage, 580 voluntarily participated in the study, yielding a response rate of 89.2%. respondents had an average age of 58 years (range 20-85 years) with a sex ratio (m/f) of 1.32 (table 1) . a total of 217 travellers were living in marseille (37.4%), 357 in other parts of southern france (61.6%); information was not available in six cases (1.0%). most of the pilgrims were born outside of france, with 88.8% having been born in north africa. the mean duration of stay in france was 32 years (range 0-72 years). a proportion of 83.1% of travellers had a primary school education or below. thirty-four per cent of individuals were retired. among those under 65 years which is the age of retirement in france, only 10.9% were employed. a proportion of 47.1% was living in state-subsidized housing and 49% received state subsidies for payment of rent. only 19.8% were property owners. among 41.2% of individuals, the household allocation was less than one room per person. a proportion of 26.6% of travellers was covered by the state-financed complementary health insurance which is accessible to insolvent individuals and 45.2% had a self-financed private complementary health insurance. a proportion of 7.4% were covered under the statefinanced full health insurance coverage in cases of chronic and debilitating disease. forty-three per cent of the pilgrims declared to suffer from chronic diseases, including 22 a total of 447 pilgrims (77.1%) were presented a questionnaire upon returning home, six individuals renounced travel (1.0%) and the remaining (21.9%) were lost to followup. the mean time between return and presentation of the questionnaire was 27 days (range 1-98 days). no significant variation was observed between the 447 travellers who answered the questionnaire and the 580 enrolled pilgrims regarding demographic, immigration and socio-economic characteristics, as well as underlying chronic diseases. the mean duration of the pilgrimage was 30 days (range 14-63 days). the vast majority of pilgrims declared to have been housed and to have eaten together (99.8% and 96.4%, respectively). as shown in table 2 , a proportion of 53.9% of travellers attended a doctor during travel and 6.5% did so after travel. nine individuals were hospitalized (two in saudi arabia, one in algeria and six upon returning to france). among the six patients hospitalized in france, two had a respiratory tract infection. haemophilus influenzae was identified as the responsible pathogen in one of these two patients who was also suffering from diabetes. among the four other hospitalized patients, two had unstable diabetes mellitus and two had haematological disorders. a total of 74 travellers (16.6%) experienced fever during their stay in saudi arabia (67 attended a doctor) and 271 (60.6%) had cough (259 attended a doctor). just over 25% of the travellers with cough were febrile. dates of beginning of fever and cough are shown in fig. 1 . a first peak was observed on 28 december, followed by a second peak on 30 december. the mean duration of fever was 3 days (range 1-15 days) while the mean duration of cough was 11 days (range 2-30 days). none of demographical and socio-economic characteristics of pilgrims significantly affected the occurrence of cough. similarly, previous travel to saudi arabia, diabetes, hypertension and chronic respiratory diseases, as well as vaccination against influenza had no significant influence on the occurrence of cough during the pilgrimage (table 3 ). when considering only the cases of cough associated with fever, travellers with diabetes appeared to have an increased risk compared to other patients in univariate analysis (or = 2.02 (1.1-3.7), p 0.02). similarly, individuals of <65 years and unemployed had a greater risk of cough associated with fever (or = 2.22 (0.98-5.03), p 0.05). several factors appeared to be related to febrile cough, without reaching statistical significance. none of the other factors influenced the risk of febrile cough ( in the present study, we observed that hajj pilgrims from marseille represent a specific population of travellers with more than one third being geriatric patients, mainly originating from north africa. this is consistent with previous findings [15] . of particular concern was the finding that a significant proportion of individuals had chronic medical disorders, e.g. diabetes mellitus and hypertension. similarly, high rates of diabetes and hypertension were found in patients [20] . we also observed that the level of education of hajj pilgrims was particularly low, with a proportion of 83.1% of individuals with a level of education below that of a certificate of primary school education compared to 42.3% in the total immigrant population and 24.1% in the general population of south eastern france (paca) [21] . the pilgrim employment rate was seven-times lower and the proportion of pilgrims living in social housing in state-owned property was twice that of the total immigrant population in the same region [21] . these results, together with an overall low rate of vaccination against tetanus, diphtheria, poliomyelitis and influenza [15] , suggest that hajj travellers departing from marseille represent a category of travellers particularly at risk for travel-related diseases and that their socio-economic conditions should be considered during the pre-travel visit regarding cost-effective vaccines. in our survey, we observed a very high attack rate of cough episodes (60%), higher than that described in other studies. one study reported an incidence of ari of 40% within a group of pilgrims from riyadh [22] . a study based on clinical criteria of influenza-like illness among pilgrims from pakistan reported rates of 36% in influenza-vaccinated pilgrims and 62% in pilgrims not vaccinated against influenza [23] . another study involving english pilgrims, based on seroconversion rates, showed an attack rate of 30% among the vaccinated and 41% among the non-vaccinated participants [24] . finally, an ari attack rate of 26% was recently observed among medical team members treating pilgrims in saudi hospitals [25] . vaccination coverage against influenza did not influence the occurrence of ari in our experience, which strongly suggests that influenza virus was not the pathogen responsible for the observed symptoms. when investigating the pathogens causing respiratory tract infections in hospitalized patients during the hajj, h. influenzae, klebsiella pneumoniae and streptococcus pneumoniae appeared to be the most common pathogens (30%) in one study [26] , while mycobacterium tuberculosis was the most common pathogen (20%) identified in a study on communityacquired pneumonias during the 1994 hajj [27] . viral pathogens are also commonly identified during the hajj, representing 11-20% of pathogens responsible for upper respiratory tract infections in hospitalized pilgrims with influenza a and b virus, rhinovirus and adenovirus being the most common [26] [27] [28] [29] . seventy per cent of the travellers who developed cough episodes in our study developed their first symptoms within 3 days, suggesting human to human transmission of the responsible pathogen, with short incubation time as evi-denced by the bimodal distribution of cough in two peaks at a 24 h-interval. statin use in this study was not associated with a reduction in the occurrence of travel-associated infections during the hajj pilgrimage. occurrence of cough episodes, duration of cough and association with fever were similar in travellers treated with statins and control travellers. to our knowledge, this is the first prospective study investigating a potential role of statins in the outcome of cough episodes in a cohort of individuals exposed to the risk. this result suggests that, while treatment with statin has been demonstrated to reduce the mortality of severe sepsis associated with respiratory tract infections [5] [6] [7] , it does not play a medically significant role in the outcome of regular cough episodes as observed in the cohort studied here. however, the study involved limited numbers of statin users so that no definitive conclusions should be made. in this study, we observed that statin users were older compared to non-users, but the level of education and socio-economic characteristics were similar in both groups. none of the demographic and socio-economic characteristics of travellers affected the incidence of febrile cough in our experience. however, the study does not have the sufficient size for the examination of several risk factors, e.g. a chronic respiratory condition. diabetes mellitus appeared to be correlated with febrile cough in the cohort studied here. it remains uncertain whether diabetes is an independent risk factor for increased incidence or severity of common upper or lower respiratory tract infections [30] ; however infections caused by certain micro-organisms (staphylococcus aureus, gram-negative organisms and m. tuberculosis) occur with increased frequency. infections due to other micro-organisms (s. pneumoniae and influenza virus) are associated with increased mortality and morbidity [31] . our study highlights the fact that respiratory tract infections are very likely to occur during the hajj pilgrimage independently of vaccination coverage against influenza. overcrowding and continuous close contact, notably in the desert plains of mina and arafat where accommodation in collective tents is necessary, greatly increases the spread of respiratory tract infections. under these conditions a single case of severe acute respiratory syndrome during the hajj may cause an epidemic of unprecedented scale. during pre-hajj consultation such an event should be considered in counselling travellers. hand disinfection with alcohol-based scrubs should be recommended as it was proven to protect from ari development; it should be acceptable to most pilgrims given the religious insistence on ritual purity before the five daily prayers [32] . the saudi arabian ministry of health has recommended that masks be used to minimise droplet spread [33] . however, regular use of surgical facemasks was recently shown to offer no significant protection against ari, and intermittent use of surgical-type masks is associated with increased risk of infection [25] . furthermore, many muslims consider covering of the face during the hajj to be prohibited; therefore general compliance with this advice is unlikely. vaccination against h. influenzae and pneumococcus should be recommended to travellers suffering from chronic respiratory disease and diabetes mellitus conditions. vaccination against diphtheria, tetanus, poliomyelitis and pertussis should be updated when required and vaccination against influenza systematically proposed. health risks at the hajj pattern of admission to hospitals during muslim pilgrimage (hajj) causes of hospitalization of pilgrims in the hajj season of the islamic year influenza and the hajj: defining influenza-like illness clinically statins: panacea for sepsis? statins and the risk of pneumonia: a population-based, nested case-control study statin treatment and reduced risk of pneumonia in patients with diabetes the effect of statin therapy on infection-related mortality in patients with atherosclerotic diseases statin use and hospitalization for sepsis in patients with chronic kidney disease influenza and copd mortality protection as pleiotropic, dose-dependent effects of statins sørensen ht. preadmission use of statins and outcomes after hospitalization with pneumonia: population-based cohort study of 29 900 patients prior statin use is associated with improved outcomes in community-acquired pneumonia statins and outcome in patients admitted to hospital with community acquired pneumonia: population prospective cohort study pandemic influenza: a potential role for statins in treatment and prophylaxis pilgrims from marseille, france to mecca: demographics and vaccination status the impact of confounder selection criteria on effect estimation an introduction to model selection categorical data analysis applied logistic regression pattern of medical diseases and determinants of prognosis of hospitalization during 2005 muslim pilgrimage hajj in a terciary care hospital. a prospective cohort study les populations immigrées en provence-alpes-cô te d'azur. insee-falsid hajjassociated acute respiratory infection among hajjis from riyadh the incidence of vaccine preventable influenza-like illness and medication use among pakistani pilgrims to the hajj in saudi arabia influenza among u. k. pilgrims to hajj acute respiratory tract infections among hajj medical mission personnel, saudi arabia bacteria and viruses that causes respiratory tract infections during the pilgrimage (hajj) season in makkah, saudi arabia tuberculosis is the commonest cause of pneumonia requiring hospitalization during hajj (pilgrimage to makkah) influenza a common viral infection among hajj pilgrims: time for routine surveillance and vaccination viral respiratory infections at the hajj: comparison between uk and saudi pilgrims infections in patients with diabetes mellitus pulmonary complications of diabetes mellitus: pneumonia hajj and the risk of influenza health conditions for travelers to saudi arabia pilgrimage to mecca (hajj) we are very much indebted to the conseil géneral of provence-alpes-cô te d'azur for providing vaccines against diphtheria, tetanus and poliomyelitis. we thank t. j. marrie for critical review and editing of the manuscript. the authors state that they have no conflicts of interest. key: cord-326341-egtnqlov authors: liotti, flora marzia; menchinelli, giulia; lalle, eleonora; palucci, ivana; marchetti, simona; colavita, francesca; la sorda, marilena; sberna, giuseppe; bordi, licia; sanguinetti, maurizio; cattani, paola; capobianchi, maria rosaria; posteraro, brunella title: performance of a novel diagnostic assay for rapid sars-cov-2 antigen detection in nasopharynx samples date: 2020-09-23 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.030 sha: doc_id: 326341 cord_uid: egtnqlov nan to date the standard method in many clinical virology laboratories [1] . however, rt-pcr based 27 assays are labor-intensive and, when not completely automated, take hours to yield results. 28 conversely, rapid antigen detection assays-intrinsically less laborious and requiring few minutes 29 to results-have the potential to satisfy the pressing demand for an early sars-cov-2 infection 30 here, we evaluated the performance of the standard f covid-19 ag fia (sd biosensor, 32 suwon, south korea) assay, a fluorescent immunoassay detecting sars-cov-2 nucleoprotein 33 antigen, on nasopharynx swab samples. it consists of a test device on which a pre-extracted sample 34 is allowed to react with a monoclonal anti-sars-cov-2 antibody and, after a 30-min incubation, a 35 standard fx 2400 analyzer instrument reads the intensity of fluorescence following the antibody-36 antigen complex formation. initially, we determined the limit of detection (lod) of the 37 standard f covid-19 ag fia assay by analyzing replicates of a dilution series containing 38 vero e6 cell-cultured sars-cov-2 (inmi-1 strain) spiked in rt-pcr negative nasopharynx swab 39 samples at a 1.0 × 10 3 50% tissue culture infective dose (tcid 50 )/ml (4 × 10 6 rna copies/ml) to 40 62.5 tcid 50 /ml (2.5 × 10 5 rna copies/ml) concentration range. the lod was 5 × 10 2 41 tcid 50 /ml (2 × 10 6 rna copies/ml) at 95% detection probability ( supplementary fig. s1 our study shows that the standard f covid-19 ag fia assay had a good specificity for 65 sars-cov-2 detection in nasopharynx swab samples but had a good sensitivity only for samples 66 with ct values lower than 25 (corresponding to higher viral loads). thus, we believe that the 67 standard f covid-19 ag fia (or similar) assays. this scenario would also encompass "new" 75 patients who begin their sars-cov-2 infection course with a low viral load (resulting in ct values 76 of ≥35). in the light of these observations, it is presently difficult to envisage the correct, fruitful 77 and safe use of these assays unless they are integrated in laboratory diagnostic algorithms based on 78 both molecular and serological testing for sars-cov-2 infection. 79 assay. probit analysis revealed a lod of 5 × 10 2 tcid 50 /ml (2 × 10 6 rna copies/ml) at 95% 110 detection probability. 111 world health organization. laboratory testing for coronavirus disease (covid-19) in laboratory-2020.5-eng evaluation of a rapid diagnostic assay for detection of sars-cov-102 2 antigen in nasopharyngeal swabs evaluation of rapid 104 antigen test for detection of sars-cov-2 virus first case of 106 2019 novel coronavirus in the united states key: cord-326703-akn92p1r authors: bartoletti, michele; giannella, maddalena; scudeller, luigia; tedeschi, sara; rinaldi, matteo; bussini, linda; fornaro, giacomo; pascale, renato; pancaldi, livia; pasquini, zeno; trapani, filippo; badia, lorenzo; campoli, caterina; tadolini, marina; attard, luciano; puoti, massimo; merli, marco; mussini, cristina; menozzi, marianna; meschiari, marianna; codeluppi, mauro; barchiesi, francesco; cristini, francesco; saracino, annalisa; licci, alberto; rapuano, silvia; tonetti, tommaso; gaibani, paolo; ranieri, vito marco; viale, pierluigi title: development and validation of a prediction model for severe respiratory failure in hospitalized patients with sars-cov-2 infection: a multicenter cohort study (predi-co study) date: 2020-08-08 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.08.003 sha: doc_id: 326703 cord_uid: akn92p1r objectives: we aimed to develop and validate a risk score to predict severe respiratory failure (srf) among patients hospitalized with coronavirus disease-2019 (covid-19). methods: we performed a multicentre cohort study among hospitalized (>24 hours) patients diagnosed with covid-19 from february 22 to april 3 2020, at 11 italian hospitals. patients were divided into derivation and validation cohorts according to random sorting of hospitals. srf was assessed from admission to hospital discharge and was defined as: spo2<93% with 100% fio2, respiratory rate (rr)>30bpm, or respiratory distress. multivariable logistic regression models were built to identify predictors of srf, β-coefficients were used to develop a risk score. trial registration nct04316949. results: we analyzed 1113 patients (644 derivation, 469 validation cohort). mean (±standard deviation)age was 65.7(±15) years, 704 (63.3%) were male. srf occurred in 189/644 (29%) and 187/469 (40%) patients in derivation and validation cohort, respectively. at multivariate analysis, risk factors for srf in the derivation cohort assessed at hospitalization were age ≥70 years [or 2.74 (95%ci 1.66-4.50)], obesity [or 4.62 (95%ci 2.78-7.70)], body temperature ≥38°c [or 1.73 (95%ci 1.30-2.29)], rr ≥22bpm [or 3.75 (95%ci 2.01-7.01)], lymphocytes ≤900/mm(3) [or 2.69 (95%ci 1.60-4.51)], creatinine ≥1 mg/dl [or 2.38 (95%ci 1.59-3.56)], c-reactive protein ≥10mg/dl [or 5.91 (95%ci 4.88-7.17)], and lactate dehydrogenase ≥350iu/l[or 2.39 (95%ci 1.11-5.11)]. assigning points to each variable an individual risk score (predi-co score) was obtained. area under receiver-operator curve (auroc) was 0.89 (0.86-0.92). at score of >3, sensitivity, specificity, positive and negative predictive values were 71.6%(65-79%), 89.1% (86-92%), 74%(67-80%), and 89%(85-91%), respectively;. predi-co score showed similar prognostic ability in the validation cohort: auroc 0.85 (0.81-0.88). at score of >3, sensitivity, specificity, positive and negative predictive values were 80% (73-85%), 76 (70-81%), 69%(60-74%) and 85% (80-89%), respectively. conclusion: predi-co score can be useful to allocate resources and prioritize treatments during covid-19 pandemic. severe acute respiratory syndrome coronavirus 2 (sars-cov-2)-associated coronavirus disease 108 2019 (covid-19) has gripped the world in a pandemic, challenging its culture, economy and 109 healthcare system. the virus was first reported in china in december 2019 and has subsequently 110 spread worldwide. 111 the clinical spectrum of covid-19 is broad with the majority of infected individuals experiencing 112 only mild or subclinical illness, especially in the early phase of disease [1] . however, approximately 113 14 to 30% of hospitalized patients diagnosed with covid-19 develop a severe respiratory failure 114 (srf) requiring intensive care [2] [3] [4] . 115 to date, no therapy has proven effective, thus supportive care aimed to protect multi-organ 116 function represents the main resource to reduce mortality [5] . unfortunately, the capacity of the 117 system is limited prompting the need of rationing decisions [6] . on the other hand, a number of 118 promising innovative drugs and treatment strategies are under investigation [7] . thus, we deemed 119 that an early identification of patients at risk of developing srf, could support the planning of 120 resources and help to set up organizational and clinical interventions, including early 121 pharmacological treatment to prevent icu admission. 122 the objectives of the study were therefore (a) develop a risk model to identify patients at high risk 123 of developing srf on hospital admission using a cohort of hospitalized patient with 124 microbiologically confirmed diagnosis of and (b) to validate this risk model in an 125 external multicenter cohort. 126 we performed a retrospective multicenter cohort study of prospectively collected data from patients 130 with laboratory-confirmed sars-cov2 virus infection, hospitalized from february 22 through april 131 3, 2020. last follow-up date was april 23, 2020. 132 eleven hospitals from four italian regions, including four tertiary teaching hospitals, five non-133 teaching tertiary hospitals and two secondary hospitals, participated in the study (see 134 supplementary figure 1) . 135 diagnostic testing for covid-19 and hospitalization were performed according to local policy and 136 clinical judgment, and were not dictated by a study protocol. the local microbiology laboratory 137 information and management systems were used to identify patients. clinical charts and hospital 138 electronic records were used as data sources. de-identified data were collected and managed 139 using redcap electronic data capture tools, alma mater university of bologna [8, 9] . 140 the study was approved by the ethic committee of the promoting center (comitato etico 141 indipendente di area vasta emilia centro, n.283/2020/oss/aoubo). a waiver of informed consent 142 was granted by the ethic committee due to safety risk. to develop the risk score (predi-co score), variables in the multivariate logistic regression model 215 regardless of their significance were assigned a point value corresponding to the β-coefficient 216 (fixed effects) rounded to the nearest integer; the total score was obtained by summation of 217 individual variables scores. 218 the discrimination of predi-co score towards srf was then analyzed by nonparametric analysis 219 of roc curve under covariates, using bootstrap (1000 replications), with clustering per hospital. an 220 optimal cut-point was then assigned using the youden's j statistic, and performance 221 characteristics at the cut-point (sensitivity, specificity, positive and negative likelihood, diagnostic 222 accuracy, positive and negative predictive values) were calculated with the corresponding 95% 223 confidence intervals. 224 in the validation cohort, the slope and intercept of the linear predictor were also assessed. the 225 results of multivariable analysis in the validation cohort was not used to change the model obtained 226 in the derivation cohort. 227 all statistical tests were two-sided. stata computer software version 16.0 (stata corporation, 4905 228 lakeway drive, college station, texas 77845, usa) was used for statistical analysis. 229 the initial population consisted of 1265 patients: 739 in the derivation and 526 in the validation 232 cohort. one-hundred fifty-two patients were excluded according to eligibility criteria. of the 1113 233 patients analyzed: 644 were in the derivation and 469 in the validation cohort ( figure 1 ). the 234 median number of patient included per hospital was 40 (iqr 11-84, range 4-384). 235 the mean age of included patients was 65.7±15 years, and 704 (63.3%) were male. the median 236 time from onset of symptoms to hospital admission was 6 (iqr 3-9) days. the two cohorts were 237 different in several patients' characteristics (table 1) . 238 three-hundred seventy-six patients (33%) developed srf after ≥ 24 hours of admission. median 239 time to srf in this group was 4 (iqr 2-7) days from hospital admission and 10 (7-13) days from 240 onset of symptoms. the rate of srf was 29% (189/644) and 40% (187/469) in the derivation and 241 validation cohort, respectively. 242 there were several differences between patients with and without srf in derivation (table 2) and 243 validation (table 3) in the derivation cohort, multivariate analysis showed that age ≥70 years, obesity, fever at 245 hospitalization (body temperature ≥ 38°c), respiratory rate ≥22 breaths per minute, lymphocytes 246 ≤900/mm3, creatinine ≥ 1 mg/dl, c-reactive protein (crp) ≥10 mg/dl, and ldh ≥350 ui/l were 247 independent risk factors for developing srf (table 4) assignment of points on the basis of β coefficient for these 8 independent variables generated an 255 individual risk score for each patient ranging from 0-9 (table 4) table 1) . 265 finally, according to the roc curve analysis the prediction ability for srf of our score was higher we developed and independently validated a simple individual risk score (the predi-co score) to 275 identify at the time of hospitalization patients with covid-19 at high risk of developing srf during 276 hospitalization. we found that of the patients hospitalized with covid-19 on the wards for at least 277 j o u r n a l p r e -p r o o f period. a predictive model was built and validated, using age>70 years, obesity, fever at 279 hospitalization, respiratory rate ≥22 breaths per minute, lymphocytes count ≤900 cells per mm3, 280 creatinine ≥1 mg/dl, crp ≥10 mg/dl, and ldh ≥350 iu/l. our model and risk score performed 281 similarly even in different cohorts, as defined by different hospitals, providing independent 282 validation. 283 the rate of srf in our cohort of hospitalized patients with covid-19 was higher than that in initial 284 reports [4, 13] , but in line with more recent findings [14, 15] . demographic characteristics of 285 population, socio-cultural issues and local strategies for diagnostic testing have been appointed 286 among the factors contributing to the different severity of covid-19 across countries [14] . indeed, 287 the mean age of our patients was 65.7 years compared with 47 and 49 years in the cohorts from 288 singapore and china, respectively [4, 13] . 289 it is worth mentioning that in most of the published prognostic studies on covid-19 demographic 290 characteristics (older age and male sex), underlying comorbidities, and altered laboratory tests 291 (e.g. crp, ldh and lymphocytes counts) correlated with poor outcome as in our study [16, 17] . 292 the strongest underlying condition influencing outcome in our analysis was obesity as observed for 293 other severe viral pneumonia, like h1n1 flu [18] . recently, a similar score was developed and 294 validated in chinese hospitals [19] . this score compared to ours requires online calculator so it 295 could be less applicable in emergency situations and some of the included variables like 296 hemoptysis were very rarely reported in our cohort. this may represent differences between 297 population and settings. 298 our study has a number of limitations. first, being a retrospective study, several variables were not 299 systematically collected across all centers, especially in these times of great clinical duties and 300 stress of the healthcare system. this might introduce bias if patients in more severe clinical 301 conditions had a higher chance of missing information. for example, interleukin-6 and d-dimer 302 previously showed a significant correlation with disease progression [20], but were not available in 303 this study. however, the strict correlation between interleukin-6 and all acute phase proteins, 304 including crp is well known [21] . additionally, interleukin-6 is not available in most laboratory 305 chemistry panels of emergency rooms or wards of non-tertiary hospitals. the inclusion of such 306 parameters in our score could reduce the applicability of our score. second, we included only 307 patients with sars-cov-2 positive nasopharyngeal swab; this could contribute to a selection bias. 308 in fact, the testing algorithm may have been affected by local policies [14] . additionally, some 309 patients could have been excluded from the study considering the suboptimal sensitivity of 310 nasopharyngeal swabs [22] . third, patients with srf within the first 24 hours from admission, were 311 excluded: we made this choice because we aimed to identify patients at risk of unfavorable clinical 312 evolution, rather than discriminating between those already in severe clinical conditions at 313 admission. fourth, our score has been developed and validated in italian hospitals; even if 314 restricted to single country analysis, local care practices might have strong impact on srf rates. 315 however, the predi-co score performed similarly in different cohorts, providing external 316 validation. lastly, one risk factor for srf (respiratory rate) may overlap with its definition. being 317 aware that this may constitute a bias we preferred to maintain this parameter as is commonly used 318 in other clinical score (qsofa and curb-65)to increase the applicability of our model. 319 to conclude, we developed and validated an individual risk score including eight strong predictors 320 of srf to identify at hospital admission patients with covid-19 diagnosis deserving a high level of 321 care and a prompt medical treatment. in particular, in our setting with high frequency of respiratory 322 failure (as was seen in the first phases of the pandemic in italy) the negative predictive values was 323 good, and therefore our score might be useful to identify patients which might not need icu or high 324 intensity care. if furtherly validated in a prospective study our score might serve for both rationing 325 abbreviations: bmi body mass index; copd chronic obstructive pulmonary disease esld end-stage liver disease; gcs glasgow coma scale; hrct high-resolution computed tomography ldh lactate dehydrogenase; map mead arterial pressure; pr pulse rate * for each year/day, point or unit increase abbreviations: bmi body mass index; copd chronic obstructive pulmonary disease; crp c-reactive protein; esld end-stage liver disease gcs glasgow coma scale; hrct high-resolution computed tomography ldh lactate dehydrogenase; map mead arterial pressure; pr pulse rate key: cord-278807-p1crrb8n authors: antón, a.; marcos, m.a.; torner, n.; isanta, r.; camps, m.; martínez, a.; domínguez, a.; jané, m.; jiménez de anta, m.t.; pumarola, t. title: virological surveillance of influenza and other respiratory viruses during six consecutive seasons from 2006 to 2012 in catalonia, spain date: 2016-03-02 journal: clin microbiol infect doi: 10.1016/j.cmi.2016.02.007 sha: doc_id: 278807 cord_uid: p1crrb8n most attention is given to seasonal influenza and respiratory syncytial virus outbreaks, but the cumulative burden caused by other respiratory viruses (rv) is not widely considered. the aim of the present study is to describe the circulation of rv in the general population during six consecutive seasons from 2006 to 2012 in catalonia, spain. cell culture, immunofluorescence and pcr-based assays were used for the rv laboratory-confirmation and influenza subtyping. phylogenetic and molecular characterizations of viral haemagglutinin, partial neuraminidase and matrix 2 proteins were performed from a representative sampling of influenza viruses. a total of 6315 nasopharyngeal samples were collected, of which 64% were laboratory-confirmed, mainly as influenza a viruses and rhinoviruses. results show the significant burden of viral aetiological agents in acute respiratory infection, particularly in the youngest cases. the study of influenza strains reveals their continuous evolution through either progressive mutations or by segment reassortments. moreover, the predominant influenza b lineage was different from that included in the recommended vaccine in half of the studied seasons, supporting the formulation and use of a quadrivalent influenza vaccine. regarding neuraminidase inhibitors resistance, with the exception of the 2007/08 h275y seasonal a(h1n1) strains, no other circulating influenza strains carrying known resistance genetic markers were found. moreover, all circulating a(h1n1)pdm09 and a(h3n2) strains finally became genetically resistant to adamantanes. a wide knowledge of the seasonality patterns of the rv in the general population is well-appreciated, but it is a challenge due to the unpredictable circulation of rv, highlighting the value of local and global rv surveillance. respiratory viruses (rv) cause significant morbidity and mortality in the human population. most attention is given to the impact of seasonal outbreaks by human respiratory syncytial (hrsv) and influenza viruses, but the cumulative burden caused by more than 200 other known rv (picornaviruses, paramyxoviruses, coronaviruses and adenoviruses, among others) is not widely appreciated [1] . in the present study the circulation and seasonality of rv from 2006 to 2012 in catalonia (spain) are described. periods, demographic characteristics (gender and age) and nasopharyngeal samples were systematically collected for virological diagnosis from outpatients with influenza-like illness (ili) (two first ili consultations per week per physician), through the pidirac (daily information on acute respiratory illness plan of catalonia) sentinel surveillance network. ili is defined as acute respiratory tract infection presenting with sudden onset of symptoms; and at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath, according to the european centre for disease prevention and control's clinical criteria of ili [2] . the pidirac sentinel surveillance network is based on a medical sentinel network at primarycare centres coordinated by the public health agency of catalonia, that covers all seven health regions into which the catalan territory is divided. primary-care centres involved in the sampling varied from the 2006/07 season to the 2011/12, ranging from 27 in the former to 38 in the latter, and covered approximately 1% of the total population in catalonia. two independent nested multiplex rt-pcr were used to detect human influenza a (fluav), b (flubv) and c (flucv) viruses, hrsv, human adenoviruses (hadv), human parainfluenza viruses (hpiv) 1-4, human coronaviruses (hcov) 229e and oc43, human enteroviruses (hev) and human rhinoviruses (hrv) a, b and c [3, 4] . subtyping (seasonal h1, h1pdm09 and h3) of influenza a viruses isolated on mdck or mdck-siat1 (vircell, granada, spain) cell culture was performed by using the annual who influenza immunofluorescence assay, or directly from laboratory-confirmed clinical samples using a one-step multiplex real-time rt-pcr assay [5] . influenza laboratory-confirmed samples collected from patients belonging to different age groups (0-4, 5-14, 15-65, > 65 years old), from different geographical sites and in different weeks were selected for a good representativeness of the phylogenetic and molecular characterizations of circulating influenza viruses in catalonia throughout the period of study. the coding sequences of complete domain ha1 of viral haemagglutinin (ha) protein, and the partial neuraminidase (na) and matrix 2 (m2) proteins from fluav and flubv laboratory-confirmed specimens were sequenced, as well as, the coding region of the haemagglutinin-esterase (he) protein from flucv laboratory-confirmed specimens, as previously described [6] . updated amplification and sequencing protocols are available on request. phylogenetic analyses of sequences from the present study together with sequences from clade reference strains downloaded from the gisaid database (global initiative on sharing avian influenza data, available at: www.platform.gisaid.org) were carried out with mega v5.2 [7] . sequences were aligned using the muscle program, and the molecular evolutionary models of nucleotide substitutions were fitted to the multiple sequence alignments using evolutionary analyses conducted in mega v5.2 [7] . the phylogenetic trees were reconstructed using the neighbour-joining (nj) distance method as implemented in mega v5.2 [7] with the evolutionary model with the lowest bayesian information criterion score. reliability for the internal branch was assessed using the non-parametric bootstrap analysis with 1000 replicates. the amino acid substitutions of predicted influenza protein sequences were studied using mega v5.2 [7] relative to the homologous sequences of the corresponding recommended vaccine strains [8] . the potential n-linked glycosylation sites in ha1 amino acid sequences were tracked using the n-glycosite tool [9] . statistical analyses were performed using spss v17 (spss inc., chicago, il, usa). numeric variables were compared using the non-parametric mann-whitney u-test for comparisons between more than two groups. chi-squared test, fisher test, and the or and their 95% ci were calculated to assess associations between categorical variables. values of p <0.05 were considered to be statistically significant. the nucleotide sequences from the present study were submitted to the gisaid database. no ethical approval was required for this study. the seasonal hrsv outbreaks usually started early every season, before the seasonal influenza circulation. when fluav and flubv were co-detected during a season, fluav was first, with the predominance of a particular fluav subtype, and was followed by flubv later (fig. 1 ). the only exception was the 2009 influenza pandemic. a(h1n1)pdm09, which was first noted in june 2009 and which circulated showing a biphasic pattern. a first peak was detected during weeks 24-35 (summer months), and a second peak during weeks 41-49 (autumn months), before hrsv circulation and outside the usual months of influenza outbreaks (from december to march). during the first two 2009 pandemic peaks, other fluav subtypes (seasonal h1 and h3) and flubv remained almost undetected despite the large sampling done to strengthen the a(h1n1) pdm09 surveillance. during these six consecutive seasons other rv than influenza viruses were mainly detected during the cold months, often just before and after the seasonal influenza epidemics ( fig. 2) , with scarce circulation during the inter-seasonal periods. differences between the rv detection rates (see supplementary material, table s1 ) were observed just before and after the 2009 pandemic (p <0.05). the detection rates of hcov, hrv, hpiv 1-4 and hev increased after the pandemic table s2 ) [10] . phylogenetic analyses (ha1 and na) of 111/117 seasonal a(h3n2) strains (table 2; phylogenetic analyses (ha1 and na) of 121/123 a(h1n1) pdm09 strains (table 2; and supplementary material, fig. s3 ), showed that 116 strains were carrying the genetic features (s203t in ha1, and v106i and n248d in na) of strains belonging to the clade 7 described by nelson et al. [11] . strains collected during the 2009/10 season remained genetically close to those first described at the beginning of the pandemic. most of 2010/11 strains genetically evolved and fell within four different genetic subgroups based on ha sequences. in addition, at least ten strains without key genetic features of nelson's clade 7 were detected during the first two pandemic seasons, of which the latest strains (2010/11 season) showed genetic drift from the early 2009 isolates. phylogenetic analysis of ha1 sequences of 126 flubv strains revealed the co-circulation of b/victoria (54) and b/ yamagata-lineage (72) strains. an alternance in the predominant lineage, which was different from what was included in the recommended vaccine composition, was shown in three out of the six studied seasons ( table 2) . but this alternance did not seem to affect the flubv detection rates (see supplementary material, table s1 ). in fact, the highest flubv detection rate (19%) was reported during the 2010/11 season, when the predominant circulating lineage was well-matched with the lineage included in the vaccine ( table 2) . phylogenetic analyses (ha1 and na) of 51/54 b/victoria strains ( table 2; (2) in case of different phylogenetic variants, the most frequent is marked in bold letters. 1 the partial na sequences for phylogenetic and molecular characterization could not be obtained from some of these strains. 2 one intra-clade reassortant strain (iowa/19 ha; england/259 na). 3 one intra-clade reassortant strain (england/259 ha; stockholm/18 na). 4 intra-clade reassortant (brisbane/60 ha; malaysia/2506 na). for a(h3n2) subtype during the 2011/12 season (see supplementary material, fig. s2 ). phylogenetic analysis of 19 flucv strains (see supplementary material, fig. s6 ) detected during the 2009/10 and 2011/12 seasons, revealed that strains belonged to the c/ kanagawa/1/76-related and to c/sao paulo/378/82-related lineages [5] , remaining genetically similar. regarding na mutations related to neuraminidase inhibitors (nais) resistance, known genetic markers were not found in the influenza strains studied, with the only exception being the h275y mutation [10] in some 2007/08 seasonal a(h1n1) strains, as described above. some mutations within the enzyme active site or its surroundings were found in the characterized strains (see supplementary material, table s2 ), which might be associated with decreased or reduced susceptibility to nais [10, 12] , but are not yet characterized. in m2 sequences, the predominance of the genetic adamantanes-resistant a(h3n2) strains during the 2006/07 season (13/15, 87%) and later (100%) by acquiring the s31n mutation [12] were observed. all characterized a(h1n1) pdm09 strains were also carrying s31n mutation as described at the beginning of the pandemic. double mutations, s31n/ v27a and s31n/v27f, in one 2011/12 a(h3n2) strain and in one 2009/10 a(h1n1)pdm09 strain were also found, respectively. no mutations in m2 protein sequences related to antiviral resistance were found in seasonal a(h1n1) strains. our results show the significant burden of viral aetiological agents in acute respiratory infections, particularly in the youngest patient group, as well as the decline in rv detection rates as the age increases. in the adult population, viral respiratory infection might be underestimated because it is usually mild and self-limiting. gender did not seem to be related to an increased infection susceptibility, except in hrsv or hev. overall, the most frequently detected rv were fluav, hrv, hadv, flubv and hrsv, although hrsv and influenza viruses mostly circulated as seasonal outbreaks, and not continously throughout the year, such as hadv and hrv. differences in age distribution among rv were found. statistical differences between the age of patients infected by the several fluav subtypes were not found, although the means and iqr suggest that patients infected by seasonal a(h1n1) or a(h1n1)pdm09 cases were younger than those infected by a(h3n2). variations in the pattern of age-specific positive proportions between different subtypes were previously described [13, 14] . more a(h1n1)pdm09 susceptibility in younger patients was attributed to the little or no pre-existing immunity to the virus among children and young adults [15] . hrv or hcov were commonly detected in all age groups, which might be explained by their high genetic diversity or the incomplete cross-reactive immune response, leading to continuous re-infections throughout life. differences in the rv detection rates and in the ages of infected patients after the 2009 pandemic were observed as noted by other authors [16, 17] , although these have not been reported in other studies [18] . this might be a result of specific and non-specific cross-reactive immunity against other rv following the a(h1n1)pdm09 infection [17] . but a consequence of the larger sampling cannot be discarded. continuous evolution through either progressive amino acid substitutions (with changes on potential n-glycosylation sites) or by segment reassortments [19, 20] can affect (a) the antigenicity and tropism features by changes in the protective antigenic epitopes or in the receptor binding site of ha protein [21] [22] [23] [24] [25] , or (b) the susceptibility to the available antivirals through changes in the na and m2 proteins [10, 12] . driftedstrains with substantial antigenic changes, driven by the host immune response acting as an evolutionary selective pressure, lead to the annual vaccine composition update [8] . a(h3n2) strains, which circulated at varying levels throughout the study period, despite the wide community protection acquired by the natural infection and the seasonal vaccination since its appearance in 1968, belonged to several genetic subgroups, showing a great genetic heterogenity. a(h1n1)pdm09 has been genetically evolving into several phylogenetic groups since 2009, but remains antigenically similar [8] . however, close attention should be paid to future antigenic drift events in response to an increased natural or vaccine-induced immunity. regarding flubv and vaccine composition, the predominant lineage did not match the recommended vaccine lineage in half of the studied seasons. the inaccurate prediction of the predominant flubv lineage in trivalent influenza vaccines supported the formulation of a quadrivalent influenza vaccine [26] [27] [28] to enhance protection. however, flubv lineage alternance has not been reported so far, with a high predominance of b/yamagata lineage since the 2011/12 season [29] . some intra-clade reassortant strains were found. it is wellknown that viral segment reassortment is a powerful genetic mechanism for influenza evolution. as these genetic events are uncommon, these findings highlight the importance of studying at least the envelope ha and na sequences to monitor their emergence and spread, as well as, the value of local surveillance to detect these minor viral populations. throughout these six consecutive seasons the picture of the available antiviral drugs to fight against influenza infection changed considerably, and our results also showed the trends clinical microbiology and infection, volume 22 number 6, june 2016 reported worldwide [8, 30] . during the 2006/07 season both adamantane and nais were the two antiviral family drugs available. in catalonia, the first genetic oseltamivir-resistant seasonal a(h1n1) variants, which carried h275y mutation in na associated with antiviral resistance [10] , were detected in a percentage of 7% during the 2007/08 season. these strains also carried the compensatory mutations, that favoured the global spread of h275y variants despite the absence of drug selective pressure [10, 30] . according to who data, an average of approximately 24% of characterized strains in europe were shown to possess high-level oseltamivir-resistance, ranging from no detection in some countries to 68% in norway [30] . in addition, circulating a(h1n1)pdm09 and a(h3n2) strains are also resistant to adamantanes, since they carry the s31n mutation in m2 protein [12, 30] , remaining susceptible to nais. indeed, adamantanes cannot now be considered suitable for seasonal influenza treatment. in the present study, with the exception of h275y seasonal a(h1n1) strains that did not circulate since the 2009 pandemics, no other circulating influenza strains carrying genetic markers related to nais resistance were found [10] . changes within the enzyme active site or its surroundings were found in na sequences, but further phenotyping studies should be performed. there is public health concern that the antiviral resistance genetic markers could become fixed in the viral genome, as detected in a low percentage (<1%) among circulating viruses [31] . the rapid global spread of oseltamivir-resistant seasonal a(h1n1) influenza viruses without drug pressure should serve as a reminder for close local and global surveillance. a wide knowledge of the seasonal patterns of rv in the general population contributes to a better diagnosis and management of respiratory infections, but it is considered a challenge because of the unpredictable nature of rv circulation. indeed, continuous local and global surveillance of influenza and other rv must be carried out to monitor their prevalence, their genetic diversity and the emergence of antiviral resistance. the authors have no conflicts to declare. viral pneumonia influenza surveillance: influenza case definitions. european centre for disease prevention and control simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays selection and viral load kinetics of an oseltamivirresistant pandemic influenza 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a cross-sectional serological study impact of the 2009 influenza a(h1n1) pandemic wave on the pattern of hibernal respiratory virus epidemics relationships between a(h1n1)pdm09 influenza infection and infections with other respiratory viruses circulation of other respiratory viruses and viral co-infection during the 2009 pandemic influenza the evolution of human influenza viruses orthomyxoviridae: the viruses and their replication crystal structure of unliganded influenza b virus hemagglutinin cross-protective potential of a novel monoclonal antibody directed against antigenic site b of the hemagglutinin of influenza a viruses networks link antigenic and receptor-binding sites of influenza hemagglutinin: mechanistic insight into fitter strain propagation structural basis of preexisting immunity to the 2009 h1n1 pandemic influenza virus glycans on influenza hemagglutinin affect receptor binding and immune response the rationale for quadrivalent influenza vaccines the need for quadrivalent vaccine against seasonal influenza public health impact of including two lineages of influenza b in a quadrivalent seasonal influenza vaccine medical research council's national institute for medical research (mimr) oseltamivir-resistant influenza a(h1n1)pdm09 viruses the authors would like to thank the working group of influenza surveillance network in catalonia. this work was partially supported by fondo key: cord-257248-aii0tj9x authors: o'grady, k.f.; grimwood, k.; sloots, t.p.; whiley, d.m.; acworth, j.p.; phillips, n.; goyal, v.; chang, a.b. title: prevalence, codetection and seasonal distribution of upper airway viruses and bacteria in children with acute respiratory illnesses with cough as a symptom date: 2016-02-22 journal: clin microbiol infect doi: 10.1016/j.cmi.2016.02.004 sha: doc_id: 257248 cord_uid: aii0tj9x most studies exploring the role of upper airway viruses and bacteria in paediatric acute respiratory infections (ari) focus on specific clinical diagnoses and/or do not account for virus–bacteria interactions. we aimed to describe the frequency and predictors of virus and bacteria codetection in children with ari and cough, irrespective of clinical diagnosis. bilateral nasal swabs, demographic, clinical and risk factor data were collected at enrollment in children aged <15 years presenting to an emergency department with an ari and where cough was a symptom. swabs were tested by polymerase chain reaction for 17 respiratory viruses and seven respiratory bacteria. logistic regression was used to investigate associations between child characteristics and codetection of the organisms of interest. between december 2011 and august 2014, swabs were collected from 817 (93.3%) of 876 enrolled children, median age 27.7 months (interquartile range 13.9–60.3 months). overall, 740 (90.6%) of 817 specimens were positive for any organism. both viruses and bacteria were detected in 423 specimens (51.8%). factors associated with codetection were age (adjusted odds ratio (aor) for age <12 months = 4.9, 95% confidence interval (ci) 3.0, 7.9; age 12 to <24 months = 6.0, 95% ci 3.7, 9.8; age 24 to <60 months = 2.4, 95% ci 1.5, 3.9), male gender (aor 1.46; 95% ci 1.1, 2.0), child care attendance (aor 2.0; 95% ci 1.4, 2.8) and winter enrollment (aor 2.0; 95% ci 1.3, 3.0). haemophilus influenzae dominated the virus–bacteria pairs. virus–h. influenzae interactions in ari should be investigated further, especially as the contribution of nontypeable h. influenzae to acute and chronic respiratory diseases is being increasingly recognized. the importance of virus-bacteria interactions in childhood acute respiratory infections (ari) remains uncertain [1] . molecular methods enabling simultaneous detection of bacteria and viruses from a single specimen [2] allow these relationships to be investigated. until recently, however, studies have focused mainly on either viruses [3] or bacteria alone [4] . studies that have examined both viruses and bacteria during an ari have typically been limited in their scope, including short duration (e.g. 1 year during the 2009 influenza a/h1n1 pandemic [5] ), linkage to specific diagnostic criteria [6] and small sample sizes [7] . there is a substantial body of work in the literature that has examined aetiologic associations between respiratory microbes and ari that has used upper airway specimens, particularly lower ari [8] . while upper airway specimens (nasopharyngeal swabs) are controversial because they cannot reliably distinguish between carriage and disease [8] , they continue to be widely used in observational and experimental studies of ari in children, including those attempting to identify associations between organisms and clinical symptoms and/or severity. notably, the association between viral ari and the later development of asthma has been based on upper airway specimens [9] . while a causal association between human rhinovirus (hrv) and respiratory syncytial virus (rsv) infections in early life with future asthma has been proposed by cohort studies [9] , these studies did not report on upper airway bacteria that are likely to be important in early immune development and viral infections [10, 11] . indeed recent larger studies which examined for both bacteria and viruses found that after adjustment for potential confounding factors it was the number of respiratory episodes rather than hrv or rsv infections in early life that were associated with future asthma [12] . in the context of the above limitations, we focused on relating the virus and bacteria detections with epidemiologic data instead of attempting to assign causal associations between upper airway microbes and clinical disease. we describe the upper airway bacteria and viruses in 817 children presenting to a tertiary paediatric emergency department (ed) with an ari that included cough as a symptom. we sought to describe the frequency and child-specific predictors of virus and bacteria codetection in this population and to describe the seasonal distribution of each organism and its codetections. the royal children's hospital (rch), brisbane, australia (now the lady cilento children's hospital), is the largest tertiary pediatric hospital in the state. its ed annually services over 25 000 children. brisbane has a subtropical climate with maximum temperatures averaging 30°c in summer and 17°c in winter. the average monthly rainfall is almost 100 mm, with summer the wettest season. we conducted a prospective study of children aged <15 years presenting to the rch ed with an ari including cough as a symptom between 11 december 2011 and 30 august 2014. the primary objective of the overall cohort study was to determine the prevalence and predictors of chronic cough after ari in children; its full study protocol has been published previously [13] . here we focus on the microbiologic aspects of that primary study. the children's health queensland (hrec/11/qrch/83) and queensland university of technology research ethics committee (2012000700) approved the study. children were excluded if they had known chronic medical conditions (excluding asthma); were immunocompromised or receiving immunomodulating drugs (other than short-course (<2 weeks) oral or inhaled steroids) in the preceding 30 days, or had insufficient english. written informed consent was obtained from parents/guardians of the child and from adolescents (aged >12 years). bilateral anterior nasal swabs were obtained using the virocult specimen collection system (medical wire and equipment, wiltshire, england, uk). protocol-specific criteria with respect to the adequacy of collection technique helped assess sampling quality [13] . swabs were stored at −80°c within 24 hours of collection and were transferred to the research laboratory for virus and bacteria identification by validated pcr assays, as described previously [14, 15] . viruses of interest included hrv, rsv a and b, influenza a and b, parainfluenza 1-3, adenovirus, human metapneumovirus, human coronaviruses (oc43, 229e, nl63 + hku1), human bocavirus, enterovirus and human polyomaviruses ki and wu. respiratory bacterial pathogens of interest included streptococcus pneumoniae, nontypeable haemophilus influenzae (nthi), moraxella catarrhalis, staphylococcus aureus, bordetella pertussis, mycoplasma pneumoniae and chlamydia pneumoniae. descriptive analyses were performed with data expressed as proportions and/or means of the selected characteristics with the corresponding 95% confidence intervals (cis). where continuous data were not normally distributed, medians with accompanying interquartile ranges are presented. logistic regression was used to assess the relationship between codetection of virus and bacteria and specimen quality, age, gender, length of illness (days), antibiotics in the past 7 days, oral steroids in the previous 30 days, household tobacco smoke exposure, child care attendance, siblings, household pets, breastfeeding history and season of enrollment. validated vaccination histories were not available and hence are not included in the analysis, although parent-reported influenza vaccination in the preceding 12 months was included. factors in univariable analyses with p <0.1 were entered into a backwards selection regression model to identify characteristics independently associated with virus and bacteria codetection; adjusted odds ratios (aor) and their corresponding 95% cis were calculated; p <0.05 was considered statistically significant. model goodness of fit was assessed by the pearson chi-square likelihood ratio test. all analyses were performed in stata v12se (statacorp, college station, tx, usa). of the 2594 children screened for participation, 876 (33.8%) were enrolled. reasons for nonenrollment included ineligibility (24.8%), refusal (33.2%) and other reasons (42.0%) (e.g. ed staff workload, discharged before consent obtained, critically ill children). nasal swabs were collected from 827 (94.4%) enrolled (60.2% male) children; median age was 27.7 months (interquartile range 13.9-60.3 months). parent-reported receipt of an influenza vaccine in the preceding 12 months occurred in 8% of children in the study. ten swabs were not tested because of poor sample quality; hence, 817 children were included in this analysis. overall, 740 (90.6%) of 817 specimens were positive for any organism. four hundred ninety-seven (60.8%) of the 817 swabs had at least one virus; 411 (50.3%) of 817 had only one virus and 86 (10.5%) of 817 had two or more viruses detected. the most commonly detected viruses were hrv (27.0%) and rsv (16.5%) (supplementary table 1 ). six hundred sixteen (75.4%) of the 817 swabs had at least one bacterial pathogen identified; 232 (28.4%) of 817 tested positive for only one, while 384 (47.0%) of 817 had two or more bacteria detected. the most commonly detected bacteria were m. catarrhalis (53.4%), s. pneumoniae (46.5%) and nthi (29.6%) (supplementary table 1 ). in contrast, only ten specimens were positive for m. pneumoniae and two for c. pneumoniae; these were not considered further in the analysis. both viruses and bacteria were codetected in 423 (51.8%) of 817 specimens, and of these, 67 swabs (15.8%) tested positive for two or more bacteria and two viruses together. univariate analyses of associations between child characteristics and virus-bacteria codetections identified age, gender, child care attendance, siblings and enrolling season for inclusion in regression models ( table 1 ). factors that remained significantly associated with codetection in the final model (likelihood ratio χ 2 = 0.32, p 0.569) were age (aor for age <12 months = 4.9, 95% ci 3.0, 7.9; age 12 to <24 months = 6.0, 95% ci 3.7, 9.8; age 24 to <60 months = 2.4, 95% ci 1.5, 3.9), male gender (aor 1.46, 95% ci 1.1, 2.0), child care attendance (aor 2.0, 95% ci 1.4, 2.8) and winter enrollment (aor 2.0, 95% ci 1.3, 3.0). table 2 presents the unadjusted and adjusted (for age, season and antibiotics in the past 7 days) associations between individual viruses and codetection with respiratory bacterial pathogens. of note was that rsv was significantly associated with nthi, s. pneumoniae, m. catarrhalis and s. aureus (table 2) . given the associations between rsv and each bacterium, we constructed a model to identify predictors of rsv that included all four bacteria of interest, age, season and antibiotics in the past 7 days. in the final model, nthi (aor 1.9 (95% ci 1.2, 2.8), age (<12 months: aor 7.5, 95% ci 3.5, 16.3; 12 to <24 months = 5.9, 95% ci 2.7, 12.8; 24 to <60 months = 3.6, 95% ci 1.7, 7.7) and autumn enrollment (aor 4.3, 95% ci 2.4, 7.7) remained significantly associated with rsv detection. associations with season are presented in fig. 1 we investigated the child characteristics associated with nasal codetection of viruses and bacteria in children with ari with cough using molecular methods. in 817 children presenting to a tertiary paediatric ed with an ari and cough, at least one virus or bacterium was detected in nasal swab specimens from 90.6% of cases, while viruses and bacteria were codetected in 51.8%. identification of organisms in isolation was uncommon. factors significantly associated with virus-bacteria codetections were young age, male gender, child care attendance and winter season. differences emerged between clinically important viruses [16] . for example, rsv was associated with age, the autumn months and nthi, while influenza was associated with winter and s. pneumoniae. other than for s. pneumoniae with influenza or rsv, little published data exist describing the frequency of virus-bacteria coinfections in nasal specimens during an ari [17] . h. influenzae dominated the virus-bacteria pairs in our study despite not being the most common bacteria detected. indeed, the role of nthi in the pathogenesis of ari may be underestimated [18] . there is a growing body of evidence suggesting both beneficial and detrimental synergies [19] that may play important roles in regulating host responses, clinical severity [20] and treatment responses [21] . further, the availability of vaccines, now and in the future, that may affect h. influenzae necessitates the need for it to be a focus of ari research. risk factors associated with virus-bacteria codetections are similar to those observed for ari elsewhere [22] . a study of 3181 hospitalized children with ari in china found those aged 1 to 4 years and boys were more likely to have virus-bacteria codetection [23] . however, the prevalence of any bacteria identified in that study was only 22%, and codetection was just 17%. it also did not include b. pertussis, s. aureus or m. catarrhalis and relied solely on culture for identifying bacteria. while our overall detection rate of at least one organism in >90% of subjects was substantially higher than some studies reporting on upper airways organisms in association with disease, our high detection rate is similar to other studies. a community-acquired pneumonia study [21] and an american indian child cohort of ari [5] described detection rates of 97% in the former [21] and 88% in the latter [5] . the seasonal distribution of most organisms correlated with the patterns of childhood ari in subtropical climates, particularly over autumn and winter months, although all organisms were identified year round. of note in our study is the relatively low frequency of rsv (16.5%) and influenza (3.9%) detected despite significant rsv and influenza seasons reported by state surveillance systems over the study period (https://www.health.qld.gov.au/ph/cdb/ sru_data.asp). similarly, only seven children (0.8%) were found to be positive for b. pertussis despite the presence of a waning pertussis epidemic in the first year of the study [24] . the low prevalence of rsv may partly be related to the relatively low number of enrolled children aged <12 months, in whom rsv is a dominant ari pathogen [25] , and those who were critically ill. the prevalence of influenza virus in our study is consistent with other australian paediatric studies that included both low and high influenza activity seasons [26, 27] . an australian observational study of influenza vaccine effectiveness in children aged 6 months to <3 years in 2010 (a low influenza activity season) found 5 (4.3%) of 117 influenza-like illnesses with specimens were influenza virus positive [26] . similarly, in an australian cohort study of aris in children aged <5 years followed for 12 months that coincided with increased influenza activity, influenza virus was detected in 4% of 543 aris where parent-collected nasal specimens were obtained [27] . however, a study from the united states of paediatric ed visits over a 10-year period found the proportion of ari/fever visits involving confirmed influenza infections ranged from 10 to 48% (median 15%) [28] , while a french study of a rapid influenza diagnostic tests in a paediatric ed setting reported 57% of children presenting with fever without source were positive for influenza virus during an epidemic period [29] . the difference between these and our study are likely to reflect differences in case definitions and that the study was not conducted during an influenza pandemic, during which parents may be more likely to present to an ed if their child is ill. with respect to b. pertussis, the ratios of notifications of pertussis cases against the 5-year means in queensland for 2012, 2013 and 2014 were 1.4, 0.6 and 0.2 respectively (https://www.health.qld.gov.au/ph/cdb/sru_data.asp), and the declines were evident in all age groups (lisa mchugh, personal communication, 2015); hence, it is likely that the study period incorporated an interepidemic period, consistent with pertussis trends over time. a strength of our study is the detail of potential factors associated with detection rates. we accounted for prior antibiotic and oral steroid use and illness duration at presentation and did not limit recruitment to children with specific clinical entities, such as pneumonia or wheezing illnesses. the lack of an effect of prior antibiotic exposure likely reflects our reliance on pcr assays rather than culture for bacteria identification. the major limitation of the study is the proportion of children with cough who were screened but not enrolled, particularly those in the younger age groups and those with mild or severe disease, and our results may thus not reflect the population of children with ari attending an ed. further limitations include the inability to investigate causality, given the absence of controls and the cross-sectional design, meaning that temporal relationships between codetected organisms could not be examined. however, that was not the intent of our study. the use of anterior nasal rather than nasopharyngeal swabs may have led to an underestimation of bacteria species. however, anterior nasal swabs are less traumatic in young children and facilitate bilateral sampling; a loss in sensitivity for bacteria was considered acceptable for the purposes of the overall study [13] for which the samples were collected. the infrequency of detecting just a single bacterium with a virus precluded investigating the interactions between organisms in greater detail. our study in children with cough highlights several things. firstly, relating the upper airway microbial epidemiology in relation to ari in children is complex and suggests that reports focusing solely on either bacteria or virus should be interpreted cautiously. secondly, studies that relate upper airway pathogens to clinical data should take into account factors that influence bacteria and virus detection, such as age, gender, season, child care attendance, duration of illness, specimen quality and prior antibiotic and steroid use. thirdly, our finding of the significant association of nthi with rsv should be further investigated in the context of the increasing appreciation that the contribution of nthi to acute and chronic respiratory diseases is receiving [8] . we are now in an era of recognizing these complexities and the interactions of individual constituents and how this might influence host-specific factors, including immune responses and clinical severity [19] . making substantial inroads into reducing the ari burden in children therefore requires high-quality studies in several different population settings. viral bacterial co-infection of the respiratory tract during early childhood cough formation in viral infections in children is virus coinfection a predictor of severity in children with viral respiratory infections? staphylococcus aureus colonization is associated with wheeze and asthma among us children and young adults a prospective study of agents associated with acute respiratory infection among young american indian children three-weekly doses of azithromycin for indigenous infants hospitalized with bronchiolitis: a multicentre, randomized, placebo-controlled trial upper airway viruses and bacteria detection in clinical pneumonia in a population with high nasal colonisation do not relate to clinical signs epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries evidence for a causal relationship between respiratory syncytial virus infection and asthma does the microbiota regulate immune responses outside the gut? bacterial colonization dampens influenza-mediated acute lung injury via induction of m2 alveolar macrophages association between respiratory infections in early life and later asthma is independent of virus type the development of chronic cough in children following presentation to a tertiary paediatric emergency department with acute respiratory illness: study protocol for a prospective cohort study successful application of a simple specimen transport method for the conduct of respiratory virus surveillance in remote indigenous communities in australia mailed versus frozen transport of nasal swabs for surveillance of respiratory bacteria in remote indigenous communities in australia aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis the co-pathogenesis of influenza viruses with bacteria in the lung non-typeable haemophilus influenzae, an under-recognised pathogen airway microbiota and acute respiratory infection in children nasopharyngeal microbiota in healthy children and pneumonia patients viruses and bacteria in sputum samples of children with community-acquired pneumonia acute respiratory infection in children from developing nations: a multi-level study. paediatr int child health detection of viral and bacterial pathogens in hospitalized children with acute respiratory illnesses australian vaccine preventable disease epidemiological review series: pertussis lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics epidemiology of respiratory viral infections in children enrolled in a study of influenza vaccine effectiveness community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens influenza-related hospitalization and ed visits in children less than 5 years impact of rapid influenza diagnostic test on physician estimation of viral infection probability in paediatric emergency department during epidemic period we thank the following for their support with study implementation and recruitment: m. lang, p. key: cord-276399-omjfyog0 authors: to, k.k.w.; chan, k.-h.; ho, j.; pang, p.k.p.; ho, d.t.y.; chang, a.c.h.; seng, c.w.; yip, c.c.y.; cheng, v.c.c.; hung, i.f.n.; yuen, k.-y. title: respiratory virus infection among hospitalized adult patients with or without clinically apparent respiratory infection: a prospective cohort study date: 2019-04-18 journal: clin microbiol infect doi: 10.1016/j.cmi.2019.04.012 sha: doc_id: 276399 cord_uid: omjfyog0 objectives: to determine the viral epidemiology and clinical characteristics of patients with and without clinically apparent respiratory tract infection. methods: this prospective cohort study was conducted during the 2018 winter influenza season. adult patients with fever/respiratory symptoms (fever/rs group) were ageand sex-matched with patients without fever/rs (non-fever/rs group) in a 1:1 ratio. respiratory viruses were tested using nxtag™ respiratory pathogen panel ivd, a commercially-available multiplex pcr panel. results: a total of 214 acutely hospitalized patients were included in the final analysis, consisting of 107 with fever/rs (fever/rs group), and 107 ageand sex-matched patients without fever/rs (non-fever/rs group). respiratory viruses were detected in 34.1% (73/214) of patients, and co-infection occurred in 7.9% (17/214) of patients. the incidence of respiratory virus was higher in the fever/rs group than in the non-fever/rs group (44.9% (48/107) versus 23.4% (25/107), p 0.001). influenza b virus, enterovirus/rhinovirus and coronaviruses were detected more frequently in the fever/rs group, whereas parainfluenza virus 4b and adenovirus were detected more frequently in the non-fever/rs group. among the non-fever/rs group, chest discomfort was more common among patients tested positive for respiratory viruses than those without respiratory virus detected (44% (11/25) versus 22% (18/82), p 0.04). conclusions: respiratory viruses can be frequently detected among hospitalized patients without typical features of respiratory tract infection. these patients may be a source of nosocomial outbreaks. respiratory viruses are the leading cause of respiratory tract infection [1, 2] . influenza virus and respiratory syncytial virus have been associated with high morbidity and mortality [3e5]. rhinovirus has been found to be the most frequently detected respiratory virus among patients with community-acquired pneumonia, and has been associated with significant morbidity and mortality [1,6e8] . adenovirus and parainfluenza virus (piv) were reported to have the highest hospitalizationefatality ratio [9] . defining of the burden of respiratory viruses is one of the agendas in the who battle against respiratory viruses (brave) initiative [10] . accurate data on the burden of respiratory virus infection is critical for patient management, infection control measures and public health policies. current clinical guidelines suggest that respiratory virus should be tested in patients with respiratory symptoms, or those with myocarditis or encephalitis [2] . as a result, epidemiological data on respiratory virus infection mostly rely on studies analysing patients with clinical evidence of respiratory tract infection [1,9,11e15 ]. however, non-respiratory symptoms and extrapulmonary complications are common among patients with respiratory virus infection [15e18] . renal failure is particularly common among patients with middle east respiratory syndrome coronavirus infection [19, 20] . myocardial infarction and stroke can be triggered by respiratory virus infection [12, 14, 21] . hence, epidemiological studies excluding patients without symptoms or signs of respiratory tract infection may underestimate the true burden of respiratory virus infection. here, our objective was to reveal the hidden burden of respiratory virus infection among patients requiring hospitalization without clinical suspicion of respiratory virus infection. we explored the differences in the incidence of different respiratory viruses between patients with or without clinical suspicion of respiratory tract infection. we analysed the clinical features associated with the detection of respiratory viruses among patients without fever or respiratory symptoms. to reduce patient discomfort, we collected saliva specimens for respiratory virus testing. we and others have demonstrated that there is a high concordance (>90%) between saliva and nasopharyngeal specimens in the detection of respiratory viruses [22e24]. this was a prospective cohort study conducted in queen mary hospital of hong kong, an acute-care university-affiliated teaching hospital with 1700 beds. all adult patients admitted to the acute medical wards from 9 january to 12 february, 2018 were screened for eligibility. inclusion criteria were age !18 years, admission to any hospitals for <24 hours, competent and agreed to provide written informed consent. patients were excluded if they could not provide adequate saliva. written informed consent was obtained from all recruited patients. saliva specimens were collected from all eligible patients. eligible patients with fever !38.0 c or any respiratory symptoms (rs) upon admission were classified as the fever/rs group, whereas those without fever or respiratory symptoms in the preceding 7 days before hospitalization were classified as non-fever/rs group. respiratory symptoms included runny nose, sore throat, cough (including haemoptysis), sputum and shortness of breath. a saliva specimen was collected from all eligible patients. each patient in the fever/rs group was matched with an age-matched (within 5 years) and sex-matched patient in the non-fever/rs group in a 1:1 ratio. all unmatched patients were excluded from further testing. saliva specimens were tested for respiratory viruses using nxtag™ respiratory pathogen panel ivd (luminex, austin, tx, usa) as described previously [22, 25] . differentiation of rhinovirus and enterovirus was performed by sequence analysis of the vp4/ vp2 gene region. data on fever or respiratory symptoms were collected by research nurses using a standardized questionnaire. final diagnosis, outcome and laboratory investigation results were obtained from the clinical management system. this study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw 13-265). this study is reported using the strengthening the reporting of observational studies in epidemiology (strobe) guidelines [26] . please refer to supplementary material (appendix s1)for details on respiratory virus testing and sequencing, and specimen collection. all statistical analysis was performed using spss 23.0. mannewhitney u test and fisher's exact test were used for the a total of 385 patients were included in the study, and 214 patients were included for respiratory virus testing in their saliva and final analysis, including 107 patients in the fever/rs group and 107 age-and sex-matched patients in the non-fever/rs group (table 1 and fig. 1 ). the median age was 67.5 years (range 18e93 years), and 42.1% (90/214) were female. there was no significant difference in the demographics and co-morbidities between the fever/rs group and the non-fever/rs group, except that chronic lung disease was more common among the fever/rs group than the non-fever/ rs group (25.2% (27/107) versus 6.5% (7/107); p < 0.001). the number of patients in each week was not statistically different between the fever/rs group and the non-fever/rs group (p 0.489) (see supplementary material, table s1 ). among the fever/rs group, the most common respiratory symptom was cough (61.7%; 66/107) ( table 1) . sixteen patients (15.0%) only had fever but no respiratory symptoms. among patients in the non-fever/rs group, the most common symptom was chest discomfort (27.1%; 29/107), followed by dizziness (20.6%; 22/ 107) and gastrointestinal symptoms (18.7%; 20/107) ( table 1) . three patients in the non-fever/rs group developed fever (n ¼ 2) or respiratory symptoms (n ¼ 1) during hospitalization after collection of saliva. there was no significant difference in the frequency of non-respiratory symptoms between the fever/rs group and the non-fever/rs group, except that palpitation was significantly more common in the non-fever/rs group (17.8% (19/107) versus 7.5% (8/ 107); p 0.038). significantly more patients in the fever/rs group required oxygen supplementation when compared with those in the non-fever/rs group (25.2% (27/107) versus 3.7% (4/107); p < 0.001). oseltamivir was only given empirically in the fever/rs group, not in the non-fever/rs group. respiratory viruses were detected in 34.1% (73/214) of patients (table 2; and see supplementary material, table s2 ). the respiratory virus detection rate in the fever/rs group (44.9%; 48/107) was significantly higher than that in the non-fever/rs group (23.4%; 25/ 107) (p 0.001). for the 16 patients with fever only in the fever/rs group, seven (43.8%) had respiratory virus detected. co-infection with two or more respiratory viruses was detected in 17 patients, including 10.3% (11/107) in the fever/rs group and 5.6% (6/107) in the non-fever/rs group, but the difference was not statistically significant (p 0.312). there was no significant difference in the proportion of patients with co-infection among different respiratory viruses (see supplementary material, fig. s1 ). overall, the most frequently detected respiratory viruses were adenovirus (7.9%; 17/214) and influenza b virus (7.9%; 17/214) ( table 2 , and see supplementary material, fig. s1 ). for the fever/rs group, the most frequently detected respiratory viruses were influenza b virus (15.0%; 16/107) and enterovirus/rhinovirus (12.1%; 13/107) ( table 2 ). for the non-fever/rs group, the most frequently detected viruses were adenovirus (10.3%; 11/107) and piv-4b (9.3%; 10/107). the detection rates of influenza b virus (15.0% (16/107) versus 0.9% (1/107), p < 0.001), enterovirus/rhinovirus (12.1% (13/ 107) versus 2.8% (3/107); p 0.017) and coronaviruses (5.6% (6/107) versus 0% (0/107); p 0.029) were significantly higher in the fever/rs group than in the non-fever/rs group (fig. 2) . adenovirus (10.3% (11/107) versus 5.6% (6/107); p 0.312) and piv-4b (9.3% (10/107) versus 2.8% (3/107); p 0.082) were more common in the non-fever/ rs group than in the fever/rs group, but not reaching statistical significance. we have further compared patients with and without virus detection among patients in the non-fever/rs group (table 3) . for co-morbidities, there were no significant differences between patients with respiratory virus detected than those without (17.2%) were the most common respiratory viruses detected in patients with chest discomfort (see supplementary material, table s3 ). this study showed that respiratory viruses could be frequently detected among acutely hospitalized patients without fever or respiratory symptoms (23.4%), although less frequently than in patients with fever or respiratory symptoms (44.9%). for the fever/ rs group, influenza b virus and enterovirus/rhinovirus were the most commonly identified viruses, which was similar to the surveillance data from the public health laboratory service in hong kong during the same period (see supplementary material, fig. s2 ). however, for the non-fever/rs group, adenovirus and piv-4b were the most frequently detected viruses. notably, adenovirus and piv-4b were more common among patients in the non-fever/rs group than those in the fever/rs group. among patients in the non-fever/ rs group, chest discomfort was more frequently reported by patients with respiratory virus detected than those without respiratory virus detected. previous studies have reported that respiratory symptoms predominated in patients with symptomatic infection, whereas non-respiratory symptoms were only present in a minority of patients [7, 15] . many retrospective studies have demonstrated an association between respiratory virus infection and extrapulmonary complications [12, 14, 21] . however, because of the bias in testing patients with fever or respiratory symptoms, these reported results may not be accurate. our results suggest that patients without respiratory symptoms should be enrolled in future studies on the role of respiratory viruses in extrapulmonary complications. adenovirus and piv-4b were more frequently detected in the non-fever/rs group than in the fever/rs group. adenovirus and piv are generally perceived to be less important than influenza virus or respiratory syncytial virus because of the low incidence. however, a retrospective study showed that adenovirus and piv had the highest hospitalizationefatality rate among different respiratory viruses [9] . it is possible that these respiratory viruses are transient, colonizing the patients' upper respiratory tract but not actually causing the symptoms or disease. however, although asymptomatic infections due to adenovirus and piv are often found in children [11, 27, 28] , adenovirus and piv are rarely detected among healthy asymptomatic adults [11, 29] . this study was designed to minimize potential bias. first, we have recorded the respiratory symptom data from study participants using a standardized questionnaire. this is important because mild symptoms may not be actively reported by patients. second, the fever/rs group and non-fever/rs group were matched for age and sex. the matching of age is particularly important because previous studies have shown that the proportion of patients with symptoms varies according to age [28] . another unique feature in this study is that we have included patients with fever but without respiratory symptoms in the fever/rs group because many clinicians test respiratory viruses in febrile patients without respiratory symptoms. in the current study, patients in the non-fever/rs group were admitted to hospital for acute non-respiratory symptoms. hence, our patient cohort differs from studies involving asymptomatic adult individuals who were not clinically affected by the respiratory viruses [11, 27, 29] . further studies are required to determine whether there is a causal relationship between the identified respiratory virus and the symptoms among patients in the fever/rs group. this study was conducted during the influenza b epidemic. as expected, influenza b virus was the most commonly detected respiratory virus among the fever/rs group. interestingly, influenza b virus was only found in one patient in the non-fever/rs group. the ratio in the detection rate of influenza b virus in the fever/rs group and non-fever/rs group was greatest among all viruses. our results suggest that patients with influenza b virus infection are more likely to present with respiratory symptoms than those with other respiratory virus infections. first, we only recruited patients from acute adult medical wards. therefore, adult patients hospitalized with surgical conditions or paediatric patients would not be included. second, we enrolled patients within 24 hours of hospitalization. the role of respiratory viruses for nosocomial complications should be further explored. third, this study was conducted over a 1-month period during the influenza b winter season, and therefore the respiratory viruses detected in this study are not representative for the entire year. a long-term study should be conducted to capture the peak seasons of other respiratory viruses. fourth, some patients in the non-fever/ rs group may have prolonged viral shedding from a previous respiratory illness that occurred more than 7 days before admission. finally, we studied acutely hospitalized patients, who would have more severe symptoms or complications when compared with patients seeking help in the community. therefore, further studies should be performed at general practice in the community. this study showed that respiratory viruses, especially noninfluenza viruses, can be detected in a substantial proportion of acutely hospitalized adult patients without clinically apparent respiratory tract infection. for future studies, the inclusion of hospitalized patients without clinically apparent respiratory tract infection will lead to a better understanding of the non-respiratory symptomatology and extrapulmonary complications of respiratory viruses. the high rate of detection of respiratory viruses among the nonfever/rs group may be important for infection control and clinical management. respiratory viruses are responsible for many nosocomial outbreaks. patients without clinical evidence of respiratory illness may represent an 'occult' source of respiratory viruses for nosocomial outbreaks, and therefore these patients might have to be included in outbreak investigations. furthermore, the detection of respiratory viruses in these patients may affect antiviral treatment decisions, especially when novel antivirals against noninfluenza viruses become available [6, 30] . all authors declare no conflict of interest. community-acquired pneumonia requiring hospitalization among u.s. adults practical guidance for clinical microbiology laboratories: viruses causing acute respiratory tract infections the emergence of influenza a h7n9 in human beings 16 years after influenza a h5n1: a tale of two cities global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study estimates of global seasonal influenza-associated respiratory mortality: a modelling study rhinovirusdfrom bench to bedside rhinovirus respiratory tract infection in hospitalized adult patients is associated with th2 response irrespective of asthma the burden of viruses in pneumonia associated with acute respiratory failure: an underappreciated issue hospitalization incidence, mortality, and seasonality of common respiratory viruses over a period of 15 years in a developed subtropical city research needs for the battle against respiratory viruses (brave) respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia acute myocardial infarction after laboratory-confirmed influenza infection parainfluenza virus in hospitalized adults: a 7-year retrospective study laboratoryconfirmed respiratory infections as predictors of hospital admission for myocardial infarction and stroke: time-series analysis of english data for concurrent comparison of epidemiology, clinical presentation and outcome between adult patients suffering from the pandemic influenza a (h1n1) 2009 virus and the seasonal influenza a virus infection clinical and virological factors associated with gastrointestinal symptoms in patients with acute respiratory infection: a two-year prospective study in general practice medicine delayed clearance of viral load and marked cytokine activation in severe cases of pandemic h1n1 2009 influenza virus infection pulmonary and extrapulmonary complications of human rhinovirus infection in critically ill patients mers coronavirus induces apoptosis in kidney and lung by upregulating smad7 and fgf2 middle east respiratory syndrome laboratory-confirmed respiratory infections as triggers for acute myocardial infarction and stroke: a self-controlled case series analysis of national linked datasets from scotland additional molecular testing of saliva specimens improves the detection of respiratory viruses saliva as a diagnostic specimen for testing respiratory virus by a point-of-care molecular assay: a diagnostic validity study comparison between saliva and nasopharyngeal swab specimens for detection of respiratory viruses by multiplex reverse transcription-pcr evaluation of nxtag respiratory pathogen panel and comparison with xtag respiratory viral panel fast v2 and film array respiratory panel for detecting respiratory pathogens in nasopharyngeal aspirates and swine/avian-origin influenza a subtypes in culture isolates the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies viral shedding and transmission potential of asymptomatic and paucisymptomatic influenza virus infections in the community frequent asymptomatic respiratory syncytial virus infections during an epidemic in a rural kenyan household cohort asymptomatic summertime shedding of respiratory viruses antiviral treatment of severe non-influenza respiratory virus infection we acknowledge mr chin-ki ng and mr joy-yan lam for their assistance in processing clinical specimens. kkwt, khc, jh, vccc, ifnh and kyy designed the study. kkwt, khc, jh, pkpp, dtyh, achc, cws and ccyy acquired the data. kkwt carried out the statistical analysis. all authors interpreted the data, revised the manuscript critically for important intellectual content and approved the final report. the investigators will share data used in developing the results presented in this manuscript on request to the corresponding author. anonymized record-level data will be made available on proposal for analysis by those who have received ethical clearance from their host institution. supplementary data to this article can be found online at https://doi.org/10.1016/j.cmi.2019.04.012. key: cord-010162-hfo35gsq authors: saikku, pekka title: atypical respiratory pathogens date: 2014-12-29 journal: clin microbiol infect doi: 10.1111/j.1469-0691.1997.tb00464.x sha: doc_id: 10162 cord_uid: hfo35gsq the main atypical pathogens in respiratory tract infections are classified on the basis of their ability to cause atypical pneumonia. this is not a well-defined clinical entity, and it is evident that atypical pathogens can sometimes cause ‘typical’ pneumonias and vice versa. this emphasizes the need for microbiological diagnosis, since it affects the selection of proper treatment, in which β-lactam antibiotics and aminoglycosides are not effective. moreover, mixed infections caused by atypical and typical pathogens together are common. at this moment rapid and sensitive diagnostic methods are lacking. besides numerous viruses, the main bacterial pathogens causing atypical pneumonias are mycoplasma pneumoniae, two chlamydial species, chlamydia pneumoniae and c. psittaci, one rickettsia, coxiella burnetti, and several legionella species. the majority of these pathogens cause upper respiratory tract infections more often than overt pneumonias. an atypical agent, chlamydia pneumoniae, has also been associated with chronic inflammatory conditions in the cardiovascular system. the most recently discovered pathogen in atypical pneumonias is a hantavirus causing hantavirus pulmonary syndrome. 'atypical pathogens' in community-acquired respiratory tract infections are purely microbiological entities: apart from pneumonias, where the pneumococcus is the leading causative agent, the overwhelming majority of respiratory infections are caused by 'atypical pathogens' (table 1 ). the list of atypical pathogens is long, and the more microbial diagnostic methods are used, the more pathogens are found. a recent example comprises the hantaviruses recently found in hantavirus pulmonary syndrome [l] . although pneumonia and pneumonic symptoms had earlier been described in hantavirus infections, as had pulmonary edema in rickettsioses [2] , no one expected a hemorrhagic fever virus to be the causative agent in severe pneumonias. the use of advanced microbial diagnostic methods established a new disease syndrome. the history of atypical pathogens starts with psittacosis, caused by chlamydia psittaci [3], influenza viruses [4] and q fever, caused by rickettsia, coxiella burnetti [5] . influenza virus continues to be a great scourge of all mankind, and a possible appearance of a new killer strain is in every autumn a greater menace than the feared ebola virus. the other two agents also continue to be important respiratory tract pathogens, but being zoonoses, are limited in their occurrence mostly to cases with animal contacts. luckily, 'atypical pneumonias' are usually not clinically severe; rather, the converse is the case. some clinical features of atypical pneumonias are presented in table 2 . in the patient history, family cases and the epidemiologic situation can aid in the diagnosis, as well as contact with a sick parrot when there is suspicion of psittacosis. the onset can be delayed and insidious. sputum production can be nlinimal and polymorphonuclear leukocytes are not present. in the chest x-ray, no lobar infiltrates, but more diffuse alterations 'typical of atypical pneumonia', are seen. leukocytosis can be absent in cases without massive pulmonary destruction, the erythrocyte sedimentation level is usually elevated, but c-reactive protein does not reach the levels found especially in severe pneumococcal pneumonias. however, none of these symptoms and signs readily differentiates an 'atypical' disease from a 'typical' one. a u the values overlap, and even a lobar infiltrate in the x-ray can be caused by an atypical pathogen [7] . without proper microbial diagnosis, the first clue to the etiology can too often be only the lack of response to the standard antimicrobial treatment used. in the following sections the main bacterial agents causing atypical respiratory tract infections are discussed, with a special emphasis on the latest bacterial addition, chlamydia pneumoniae. mycoplasmal pneumonias concentrate in the younger age groups, and this is illustrated in figure 1 , comparing the prevalence of antibodies against m . pneumoniae and chlamydia pneumoniae in the finnish population. the clinical description of mycoplasmal infections is classical but there are some open questions. one is the lack of reliable diagnostic methods, since conventional serologic methods, cold-agglutinin and complementfixation tests, are neither sensitive nor specific, although the former is easy to perform. a second question is, how effective is the antibiotic treatment in mycoplasmal pneumonias? mycoplasmal diseases in general are a neglected area, and we should not rely on serology only in the diagnosis of mycoplasmal pneumonias [8] . the epidemic of a curious disease named afterwards as 'legionellosis' in pittsburgh in 1976 brought a special bacterial genus to our attention [9] . although its first member had been discovered over thirty years earlier [lo], only then was it discovered to be the causative agent of several serious epidemics of respiratory infections. relatively new also is its association with environmental constructions and air-conditioning techbeen associated with clinical diseases, especially with severe pneumonias, but the importance of these environmental bacteria varies considerably between different regions and settings. in some places they are causing a considerable proportion of all pneumonias [i21 and are listed among the three major causes. in other areas, e.g. finland, they are, despite an intensive search, rarities, usually imported by tourists. respiratory tract infections caused by chlamydia psittaci are directly dependent on exposure to birds carrying the pathogen. 'therefore, cases are seen in connection with curkcy and duck farming (chickens seem not to be iziportant), pigeon breeding, and sick pet birds. casual contacts with synanthropic birds are common, and ruling out a bird contact is much more difficult than finding one. there has been a debate over whether chlamydia pneumoniae is a more common agent than chlamydia psittaci. even in the microimmunofluorescence (mif) test it is sometimes difficult to differentiate these two chlamydial species from each other [13] , and it demands expertise [14] . fortunately, the treatment is the same in both chlamydial pneumonias. according to seroepidemiologic surveys, chlamydia pneumoniae infections are 20-50 times more common than chlamydia psitfari infections [15,16]. there is a possibility that the severity of chlamydia psittaci infections could be due to the sensitization of the patient to this species by an earlier, mild chlamydia pneumoniae pulmonary infection. the most recent addition to the long list of important atypical bacterial pathogens is chlamydia przeumoniae. like the first legionella strains, the first strains of this new chlaniydial agent were isolated [17] years before an epidemic in northern finland brought them to our attention [i 81. in the beginning, chlamydia ptzeumoniae seemed to be an agent causing beiiign and mild disease, but later it was shown to cause-the typical feature of all chlamydia-silent, slowly creeping infections, gradually leading to severe tissue damage 1191. the pathogenesis of chlamydia przeumoniae infections has been studied using mouse models 120,211. when chlamydia pneumoniae is given intranasally, acute infection with polymorphonuclear leukocytes in the lungs is seen only after massive challenge doses. otherwise a silent pneumonitis with a clear histologic picture of mononuclear inflammation around bronchioles and vessels develop without overt illness. repeated inocula-tions aggravate this inflammation temporarily, but the presence of the agent is difficult to demonstrate by isolation [22] . the demonstration of nucleic acids by polymerase chain reaction (pcr) gives, however, a positive finding and cortisone treatment, after alleviating inflammation, makes isolation of the agent possible [23] . chlamydia pneumoniae is also demonstrable after intranasal challenge in the blood circulation, alveolar and peritoneal macrophages, and the liver and spleen, pointing to a disseminated infection 1241. upper respiratory tract carriage has been described [25, 26] , and there is a possibility that carriers do not develop antibody responses or, if they do, only after a prolonged period. pneumonias due to chlamydia pneumoniae haw been described in infants [27] , and from japan there is a report of an epidemic in daycare centres [28] . however, in industrialized countries antibodies usually start to appear when children enter school [29, 301 . in a recent finnish study on childhood pneumonias, the youngest patient with a chlamydia pneumoniae infection was 7 years old, and the majority of patients were over 10 years of age (korppi et al, unpublished data). other respiratory syndromes associated with chlamydia pneumoniae are rhinitis, sinusitis, pharyngitis, otitis, and bronchitis. a recent review of chlamydia peumoniae infections in children concentrated on respiratory tract infections [31] . however, during a chlamydia pneumoniae epidemic in northern finland, only a third of the children presenting a seroconversion were hospitalized because of respiratory tract symptoms (uhari et al, unpublished data). the disease picture in children can thus be quite variable and demands further study. primary infection in young adults leads to pneumonia in about 10% of cases [32] . this is usually a mild disease, but can be prolonged with a long convalescence. in young age groups, reinfections seem not to lead to pneumonias (321. however, reinfection pneumonias, especially in elderly patients with underlying diseases, can be very severe 1331. one possibility is that when the resistance to infection has decreased enough to allow the agent to invade the lungs, or the invading strain is different enough to be able to colonize the lungs, partial immunity can lead to hypersensitivity reactions typical of all chlamydia1 infections. the role that chlamydia pneumoniae plays in other acute respiratory tract infections is still under study. in adults, it has been associated with 0.5-7'% of pharyngitis 134,351 cases (the last figure during an epidemic), and 5-100/;, of acute bronchitis cases. sinusitis and otitis in adults have also been reported, but wider studies are so far lacking. mixed infections are coninion in chlamydia p i mrnoniae infections. in the studies on finnish children, half or even the majority of patients have had a concomitant infection caused by another pathogen; virus, mycoplasma or bacterium. similarly, during an epidemic in north finland, nearly half of the chlamydia pneumoniae pneumonias were combined with invasive pneumococcal infections [36] . chlamydia pneumoniae has a ciliostatic effect [37] , which in these cases may help pneumococci carried in the upper respiratory tract to invade deeper layers. these double infections were more severe than usual and the response to antibiotics effective against the pneumococcus only was poor [38] . one should not be satisfied when one pathogen is diagnosed, but always keep in mind the possibility of mixed infection. serology has traditionally been used to diagnose infections caused by atypical pathogens. antigens and complete kits for antibody assays are commercially available from several sources. the most serious disadvantage is the need to demonstrate a seroconversion in the majority of the cases. this delays the diagnosis and is then of no aid to the clinician treating the acutely ill patient. attempts have been made to overcome this delay by the demonstration of igm antibodies in the acute phase or by using 'diagnostic' high titers in the first serum sample. the pitfalls are the lack of igm in reinfections and the uncertainty of the diagnostic value of high titers in acute diseases caused by several atypical agents. serology, even though inadequate, has remained the main diagnostic tool in mycoplasmal and chlamydia1 pneumonias as well as in q fever. culture of the atypical pathogens demands special media not widely used in microbiology laboratories or, in the case of obligatory intracellular pathogens, cultured living cells in specialized units. moreover, coxiella burnetti and chlamydia psittaci present dangers to laboratory workers handling the agent, and even chlamydia pneumoniae has caused laboratory-acquired pneumonias [39] . in legionellosis, however, culture is a standard diagnostic procedure [40] . it should also be attempted in chlamydia pneumoniae infections in order to obtain information on disease associations and strain variability of this newly recognized pathogen. antigen detection in respiratory tract infections has been utilized mainly in viral infections, with good success. its use in the case of atypical bacterial pathogens has not been as rewarding. moreover, techniques based on immunofluorescence demand experience and patience from the reader, since numbers of pathogens are often limited and their reliable identification is difficult. lack of commercially available reagents and luts is a problem in the diagnosis of uncommon atypical pathogens. however, in legionellosis, detection of antigen in urine seems to be a reliable diagnostic method [11, 41] . in pneumonias, the presence ofbacterial components in the circulation, alone or in immune complexes, has been used successfully in the diagnosis of pneumococcal pneumonias [42] , but has remained unstudied in the case of atypical pathogens. these types of complex are commonly, seen, however, in chronic chlamydia pneumoniae infections, which lessens their diagnostic value in acute infections, especially in elderly males with arteriosclerotic lesions [43] . nucleic acid (na) detections seems to be the diagnostic method of the future for atypical respiratory pathogens. the commercial kit for m . pneumoniae direct na detection is no longer available, but diagnostic companies are developing kits based on na amplification for legionella spp., m . pneumoniae and chlamydia pneumoniae. these, whether based on the polymerase or ligase chain reaction, would provide a sensitive and specific diagnosis for these pathogens in 24 h. this would finally give a firm basis for a rationally targeted therapy. table 3 shows the recommended treatment for infections caused by atypical pathogens. the therapeutic response of bacterial atypical pathogens to p-lactam antibiotics and aminoglycosides is lacking or marginal. the drugs used are tetracyclines, macrolides, and azalides. time will tell how much the advent of newer quinolones will alter these recommendations. prolonged treatment of 2-4 weeks has been used in legionellosis and in chlamydia pneumoniae pneumonias. the possibility of mixed infections should always be kept in mind. appropriate therapy can be efficient in preventing long-term sequelae caused by some of these atypical pathogens. q fever endocarditis is a feared complication, which often leads to valvular operations or drug therapy for the rest of the patient's life 1441. recently discovered chlamydia pneumoniae seems to be associated with complications of unexpected severity and ubiquity. signs of chronic chlamydia pneumoniae infection have been found in both childhood [45] and adult-onset [16] asthma, chronic bronchitis [47] , and sarcoidosis [48] . most surprising is its association with arteriosclerosis, the leading cause of death in industrialized countries. markers of chronic chlamydia yneumoniae infections are found in acute myocardial infarction [49, 50] , and they have repeatedly been shown to be a risk factor for cardiac events [51, 52] ; and, finally, the pathogen has been demonstrated in atherosclerotic lesions 153,541. animal experiments have been positive [55, 56] , as have preliminary intervention trials [57, 58] . it may be possible that appropriate treatment of chlavnydin pneumoniae respiratory infection in the acute phase prevents infection from progressing to a chronic state, which can be much more difficult to cure. this would demand acute diagnosis and targeted therapy for atypical pneumonias. hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized hsease lower respiratory tract involvement in rocky mountain spotted fever a virus obtained from influenza patents studies on the etiology of primary atypical pneumonia: a filterable agent transmissible to cotton rat, hamsters, and chick embryo comparative radiographic features of community acquired legionnaires' disease, pneumococcal pneumoniae, mycoplasma pneumonia, and psittacosis detection of mycoplarma pneurtioniae in clinical samples from pediatric patients by polymerase chain reaction legionnaires' disease: description of an epidenlic of pneumonia new and emerging etiologies for community-acquired pneumonia with implications for therapy: a prospective multicentre study of 359 cases specificity of the niicronnmunofluorescence assay for the serodiagnosis of chlaniydia pneurnoniae infections utility of complement fixation and microinimunofluorescence assays for detecting serologic responses in patients with clinically diagnosed psittacosis a comparison of the seroepidemiology of chlamydia1 infection in pigeon fanciers and farmers in the uk acute lower respiratory trxt infection associated with chlamydia pneumoniae in germany a new chlartiydiii prithici strain, twar, isolated in acute respiratory tract infections an epidemic of mild pneumonia due to an unusual strain of chlamydia psittaci the epidemiology and significance of chlamydia pneumoniac experimental infection of chlamydia pnrumoriiac in mice a mouce model of chlamydia pneumonia strain twak pneumonitis experimental chlaniydia prieirmoniae infection in mice: effect of reinfection and passive protection by immune serum saikku 't? keactivation of clilanzydia pueumoniae infection in mice by corticone treatment syrtemic dissemination of chlamydia pneumoniae following intranasal inoculation in mice asymptomatic respiratory tract infection with chlamydia pnerrnzoriiae twar prevalence of asymptomatic nasopharyngeal carriage of chlamydia pnenmoniae in subjectively healthy adults: assessment by polymerase chain reaction-enzyme immunoassay and culture chlamydia pneumoniae (twar) in neonates an outbreak of chlamydia pneumoniae infection in households and schools seroepidemiology of chlamydia pneumoniae twar infection in seattle families, 1966-1979 a new respiratory tract pathogen: chlamydia pnenmoniae strain twar chlamydia pneumoniae (twar) infections in children epidemics of pneumonia caused by twar, a new chlamydia organism, in military trainees in finland chlamydia pneumoniae as a new source of infectious outbreaks in nursing homes chlamydia pnenmoniae, strain twar, mycoplasma pneumoniae and viral infections in acute respiratory disease in a university student health clinic population pharyngitis in adults: the presence and coexistence of viruses and bacterial organisms shemer-avni y. lieberman d. chlamydia pnrumoniaeinduced ciliostasis in ciliated bronchial epithelial cells clinical picture of cominunity-acquired chlamydia pncumoniae requiring hospital treatment: a comparison between chlamydia1 and pneumocozcal pneumonia cell rnediated immunity to chlanzydia prirunzoniaf measured as lymphocyte blast transformation in uifw diagnosis of legionnaires' disease; an update of laboratory methods with new emphasic on isolation by culture frequency and diagnosis of legionella pneumonia: a 3 year prospective study with emphasis on application of urinary antigen detection demonstration of pneumolysin antibodies in circulating immune complexes-a new chagnostic method for pneumococcal pneumonia presence of chlamydia pnenmoniae specific antiboches in circulating immune complexes in coronary heart disease the association of chlamydia pneumoniae infection and reactive airway disease in children association of chlamydia pneumoniae (strain twar) infection with wheezing, asthmatic bronchitis, and adult-onset asthma chlamydia pneumoniae infection in acute exacerbations of copd antibodies to twar-a novel type of chlamydia-in sarcoidosis sarcoidosis and other granulomatous disorders serologic evidence of an association of a novel chlamydia, twar, with chronic coronary heart disease and acute myocarchal infarction circulating immune complexes containing chlamydia lipopolysaccharide in acute myocardial infarction chronic chlamydia pneumoniae infection as a risk factor for coronary heart disease in the helsinki heart study chlamydia pnenmoniae strain twar antibody and angiographically demonstrated coronary artery chsease. arteriosclerosis thrombosis 53. shor a, kuo cc, patton dl. detection of chlamydia pneumoniae in coronary arterial fatty streaks and atheromatous plaques demonstration of chlamydia pneumoniae in atherosclerotic lesions of coronary arteries chlamydia pneumoniae multiplies in human endothelial cells in vitro chlamydia pneumoiziae infection induces inflammatory changes in the aorta of rabbits elevated chlamydia pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of myocardial infarction roxis study group. randomised trial of roxithromycin in non-q-wave coronary syndromes: roxis pilot study key: cord-317918-pl625ela authors: ripa, marco; galli, laura; poli, andrea; oltolini, chiara; spagnuolo, vincenzo; mastrangelo, andrea; muccini, camilla; monti, giacomo; de luca, giacomo; landoni, giovanni; dagna, lorenzo; clementi, massimo; querini, patrizia rovere; ciceri, fabio; tresoldi, moreno; lazzarin, adriano; zangrillo, alberto; scarpellini, paolo; castagna, antonella title: secondary infections in patients hospitalized with covid-19: incidence and predictive factors date: 2020-10-24 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.10.021 sha: doc_id: 317918 cord_uid: pl625ela objectives: aim of our study was to describe the incidence and predictive factors of secondary infections in patients with covid-19. methods: cohort study on patients hospitalized with covid-19 at irccs san raffaele hospital between february 25(th) and april 6th, 2020 (nct04318366). we considered secondary bloodstream (bsis) or possible lower respiratory tract infections (plrtis) occurred after 48 hours since hospital admission until death or discharge. we calculated multivariable fine-gray models, to assess factors associated with risk of secondary infections. results: among 731 patients, a secondary infection was diagnosed in 68 patients (9.3%): 58/731 patients (7.9%) had at least one bsi and 22/731 patients (3.0%) at least one plrti. overall 28-day cumulative incidence was 16.4% (95% ci 12.4% 21.0%). the majority of bsis was due to gram-positive pathogens (76/106 isolates, 71.7%), specifically coagulase-negative staphylococci (53/76, 69.7%), while among gram-negatives (23/106, 21.7%) acinetobacter baumanii (7/23, 30.4%) and escherichia coli (5/23, 21.7%) predominated. plrtis were mainly caused by gram-negative pathogens (14/26, 53.8%). eleven patients were diagnosed with putative invasive aspergillosis. at multivariable analysis, factors associated with secondary infections were low baseline lymphocyte count (<0.7 vs >0.7 per 10(9)/l: subdistribution hazard ratios (sdhrs) 1.93 [95% ci 1.11-3.35]), baseline pao(2)/fio(2) (per 100-points lower: sdhrs 1.56 [95% ci 1.21-2.04]), and intensive-care unit (icu) admission in the first 48 hours (sdhr 2.51 [95% ci 1.04-6.05]). conclusions: patients hospitalized with covid-19 had a high incidence of secondary infections. at multivariable analysis, early need for icu, respiratory failure, and severe lymphopenia, were identified as risk factors for secondary infections. the pandemic caused by sars-cov-2 has affected more than thirty-two million patients worldwide as of patients with no microbiology specimens requested were considered without secondary infections. we compared characteristics and outcomes of patients who had at least one secondary infection during 144 hospitalization and those who did not using chi-square test or fisher exact test for categorical variables and 145 mann-whitney u test for continuous variables. in the analysis, we used three scores (the cytolysis score, the coagulation score, and the inflammation score), 147 defined as the number of laboratory parameters with markedly elevated values (values at or above the 75th 148 percentile). absolute lymphocyte counts were stratified on the 25th percentile (at or below we subsequently performed inverse probability-weighted (ipw) competing risks multivariable analyses to 159 simultaneously account for indication bias associated with the treatment with biologic immunosuppressive 160 drugs and competing death for the estimation of the cumulative incidence of patients with secondary 161 infections, to provide a more accurate estimate of secondary infections burden [15, 16] . a logistic regression analysis was applied to estimate the propensity of biologic immunosuppressive drugs use, 163 conditioned on a pre-specified list of baseline covariates; the predicted probabilities of biologic 164 immunosuppressive drugs treatment (propensity-score) were used to calculate the stabilized ipw in order to 165 account for non-randomization to biological drugs in this observational study. the inverses of these propensities were used as weights in multivariable fine-gray models assessing the 167 association between demographic and other clinical or laboratory factors and the risk of secondary infections. all statistical tests were two-sided at 5% level and were performed using sas 9.4 (statistical analyses system 169 inc, cary, nc, usa). further details on the cytolysis score, the coagulation score, and the inflammation score definitions and on the 171 statistical analyses are provided in the supplementary material. in our cohort, the incidence rate of bsis appears significantly higher compared to previous reports concerning 230 nosocomial and icu-related bsis in european countries [21, 22] (ranging from 0.5 to 1.3 and 0.7 to 6.6 per 231 1000-pdfu, respectively). similarly, the incidence rate of plrtis among patients with covid-19 admitted to 232 the icu appears to be higher compared with historical european cohorts [22] clinical characteristics of coronavirus disease 323 2019 in china comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new 326 clinical course and outcomes of critically ill patients 328 with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. the 329 covid-19 illness in native and immunosuppressed states: a clinical-therapeutic 331 staging proposal covid-19: consider cytokine 333 storm syndromes and immunosuppression should we stimulate or suppress bacterial and fungal coinfection among european centre for disease prevention and control. incidence and attributable mortality of 370 healthcare-associated infections in intensive care units in europe associated pulmonary aspergillosis prevalence of putative invasive pulmonary 377 aspergillosis in critically ill patients with covid-19. the lancet respiratory medicine diagnosing 380 covid-19-associated pulmonary aspergillosis covid-19: don't neglect antimicrobial 382 stewardship principles! pathological findings of covid-19 associated with 384 acute respiratory distress syndrome an interpretable mortality prediction days since hospitalization gray's test: p=0.052 bl absolute lymphocyte count ≤0.7 per 10 9 /l bl absolute lymphocyte count >0.7 per 10 9 /l follow-up (days) patients with bl pao 2 /fio 2 ≤200 (95% confidence interval) patients with bl pao 2 /fio 2 >200 key: cord-351028-p5cq2is5 authors: yang, jia-wei; yang, ling; luo, rong-guang; xu, jin-fu title: corticosteroid administration for viral pneumonia: covid-19 and beyond date: 2020-06-27 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.020 sha: doc_id: 351028 cord_uid: p5cq2is5 background: corticosteroids are commonly used as adjuvant therapy for acute respiratory distress syndrome (ards) by many clinicians due to their perceived anti-inflammatory effects. however, for patients with severe viral pneumonia, the corticosteroid treatment is highly controversial. objectives: the purpose of this review is to systematically evaluate the effect and potential mechanism of corticosteroid administration in pandemic viral pneumonia. sources: we comprehensively searched all manuscripts on corticosteroids therapy for influenza, sars, mers and sars-cov-2 viral pneumonia from the pubmed, embase, web of science and cochrane library databases. content: we systematic summarized the effects of corticosteroids therapy for pandemic viral pneumonia and the potential mechanism of corticosteroid worked in covid-19. implications: observational studies showed that corticosteroid treatment was associated with increased mortality and nosocomial infections for influenza and delay virus clearance for sars-cov and mers-cov. limited data on corticosteroid therapy for covid-19 were reported. corticosteroids were used in about a fifth of patients (670/2995, 22.4%). although clinical observational studies reported the improvement in symptoms and oxygenation for the severe covid-19 patients received corticosteroids therapy, case fatality rate in the corticosteroid group was significantly higher than that in the non-corticosteroid group (69/443, 15.6% vs 56/1310, 4.3%). compared with non-severe patients, severe patients were more likely to receive corticosteroid therapy (201/382, 52.6% vs 201/1310, 15.3%). although there is no evidence of corticosteroid therapy reduce the mortality of covid-19 patients, some improvements in clinical symptoms and oxygenation were reported in some clinical observational studies. excessive inflammatory response and lymphopenia might be critical factors associated with disease severity and mortality of covid-19. sufficiently powered randomized controlled trials with rigorous inclusion/exclusion criteria and standardized dose and duration of corticosteroids are needed to verify the effectiveness and safety of corticosteroid therapy. background corticosteroids are commonly used as adjuvant therapy for acute 23 respiratory distress syndrome (ards) by many clinicians due to their perceived 24 anti-inflammatory effects. however, for patients with severe viral pneumonia, the 25 corticosteroid treatment is highly controversial. coronavirus" or "2019-ncov" or "covid-19". no language restrictions were set. 106 the references of involved studies were also searched. 107 two investigators independently extracted useful information and data from original 108 studies. disagreements were resolved by discussion and consulting statistician. due to 109 data processing and conversion analysis, some of the results may differ slightly from 110 those published original articles. 111 the initial search identified 19227 potential studies. 18445 articles were excluded by 112 screening of the titles and abstracts due to irrelevance or redundancy. ultimately, 782 113 full-text articles were reviewed, 212 of which were related to corticosteroid about 114 influenza, 196 were related to corticosteroid about sars, 33 were related to 115 corticosteroid about mers, and 341 were related to corticosteroid about covid-19. 116 the details of the screening process are shown in figure 1 . that methylprednisolone was the most frequently used corticosteroid. the median 128 daily dose was equivalent to 80 mg of methylprednisolone (interquartile range: iqr 129 60-120) for a median duration of 7 days (iqr 5-10). after propensity score matching, 130 corticosteroid application for influenza pneumonia was associated with icu mortality 131 in cox regression analysis (hr 1.32, 95% ci 1.08-1.60) and competing risks analysis 132 (shr 1.37, 95% ci 1.12-1.68) in this study [15] . moreover, studies from (table s1 ). they showed that corticosteroid therapy was 139 significantly associated with mortality ( figure 2a the outcomes of corticosteroid therapy in sars were divergent based on the 166 published researches. we cannot conclude a definite conclusion. 167 reports on corticosteroids administration for mers were relatively rare. arabi treatment of severe acute respiratory 379 syndrome with glucosteroids the use of corticosteroid as treatment in 381 sars was associated with adverse outcomes: a retrospective cohort study corticosteroid treatment of severe acute 384 respiratory syndrome in hong kong corticosteroid therapy for 386 critically ill patients with the middle east respiratory syndrome: a multicenter 387 retrospective cohort study effects of early corticosteroid treatment on 389 plasma sars-associated coronavirus rna concentrations in adult patients clinical evidence does not support 392 corticosteroid treatment for 2019-ncov lung injury on the use of corticosteroids for 394 2019-ncov pneumonia effect of infectious diseases society of america: 2018 update on diagnosis, treatment, 418 chemoprophylaxis, and institutional outbreak management of seasonal influenza high-dose pulse versus nonpulse corticosteroid 421 regimens in severe acute respiratory syndrome severe acute respiratory syndrome: report of 424 treatment and outcome after a major outbreak fatal aspergillosis in a 426 patient with sars who was treated with corticosteroids steroid therapy and the risk of osteonecrosis 429 in sars patients: a dose-response meta-analysis epidemiological and clinical characteristics of 432 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive 433 study clinical characteristics of 138 hospitalized patients with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: 439 retrospective case series clinical characteristics and imaging manifestations 443 of the 2019 novel coronavirus disease (covid-19): a multi-center study in 444 wenzhou city clinical characteristics of imported cases of covid-19 jiangsu province: a multicenter descriptive study clinical course and outcomes of critically ill patients 449 with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, 450 observational study potential benefits of precise corticosteroids therapy 452 syndrome and death in patients with coronavirus disease analysis of epidemiological and clinical features in 463 older patients with coronavirus disease 2019 (covid-19) out of wuhan characteristics of and important lessons from the 466 coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 467 72314 cases from the chinese center for disease control and prevention pathogenic human coronavirus infections: causes 470 and consequences of cytokine storm and immunopathology plasma inflammatory cytokines and 473 chemokines in severe acute respiratory syndrome a pneumonia outbreak associated with a new 479 coronavirus of probable bat origin another decade, another coronavirus genomic characterisation and epidemiology of 2019 483 novel coronavirus: implications for virus origins and receptor binding clinical features predicting mortality risk in 486 patients with viral pneumonia: the mulbsta score preexisting influenza-specific cd4+ t 489 cells correlate with disease protection against influenza challenge in humans key: cord-307213-i8yijbiu authors: ip, jonathan daniel; kok, kin-hang; chan, wan-mui; wing-ho chu, allen; wu, wai-lan; chik-yan yip, cyril; to, wing-kin; tak-yin tsang, owen; leung, wai-shing; shiu-hong chik, thomas; chan, kwok-hung; fan-ngai hung, ivan; yuen, kwok-yung; kai-wang to, kelvin title: intrahost non-synonymous diversity at a neutralising antibody epitope of sars-cov-2 spike protein n-terminal domain date: 2020-11-02 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.10.030 sha: doc_id: 307213 cord_uid: i8yijbiu objectives: sars-cov-2 has evolved rapidly into several genetic clusters. however, data on mutations during the course of infection are scarce. this study aims to determine viral genome diversity in serial samples of covid-19 patients. methods: targeted deep sequencing of spike gene was performed on serial respiratory specimens from covid-19 patients using nanopore and illumina sequencing. sanger sequencing was then performed to confirm the single nucleotide polymorphisms. results: a total of 28 serial respiratory specimens from 12 patients were successfully sequenced using nanopore and illumina sequencing. a 75-year-old patient with severe disease had a mutation, g22017t, identified in the second specimen. the frequency of g22017t increased from ≤5% (nanopore: 3.8%; illumina: 5%) from first respiratory tract specimen (sputum) to ≥60% (nanopore: 67.7%; illumina: 60.4%) in the second specimen (saliva; collected 2 days after the 1(st) specimen). the difference in g22017t frequency was also confirmed by sanger sequencing. g22017t corresponds to w152l amino acid mutation in the spike protein which was only found in <0.03% of the sequences deposited into a public database. spike amino acid residue 152 is located within the n-terminal domain, which mediates the binding of a neutralizing antibody. conclusions: a spike protein amino acid mutation w152l located within a neutralizing epitope has appeared naturally in a patient. our study demonstrated that monitoring of serial specimens is important in identifying hotspots of mutations, especially those occurring at neutralizing epitopes which may affect the therapeutic efficacy of monoclonal antibodies. a total of 28 serial respiratory specimens from 12 patients were successfully sequenced 49 using nanopore and illumina sequencing. a 75-year-old patient with severe disease had a 50 mutation, g22017t, identified in the second specimen. the frequency of g22017t increased 51 from ≤5% (nanopore: 3.8%; illumina: 5%) from first respiratory tract specimen (sputum) to 52 ≥60% (nanopore: 67.7%; illumina: 60.4%) in the second specimen (saliva; collected 2 days after 53 the 1 st specimen). the difference in g22017t frequency was also confirmed by sanger 54 sequencing. g22017t corresponds to w152l amino acid mutation in the spike protein which 55 was only found in <0.03% of the sequences deposited into a public database. spike amino acid 56 residue 152 is located within the n-terminal domain, which mediates the binding of a 57 neutralizing antibody. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has spread rapidly, 66 resulting in more than 28 million laboratory-confirmed covid-19 cases globally as of 67 september 14, 2020. sars-cov-2 mainly causes respiratory tract infection, although 68 extrapulmonary manifestations have been reported [1] . the efficient person-to-person 69 transmission may be related to the high viral load shortly after symptom onset and the large as an rna virus, the genome replication of sars-cov-2 is prone to error, and gene 73 mutations arise frequently. whole genome sequencing showed that the viral genomes may differ 74 between family members [4] . major genetic diversity has already been seen [5] . phylogenetic 75 analysis has demonstrated that even patients within the same geographical region were infected 76 with genetically-diverse sars-cov-2 strains [6] . previously [4] . full-length spike gene was amplified using superscript™ iii one-step rt-pcr system with platinum™ taq high fidelity dna polymerase (thermo fisher scientific, 111 waltham, ma, usa) using primer set 1, or primer set 2, and 3 (supplementary table s1) illumina adapters were removed from the reads. any reads with length of at least 100 bp 158 and at least 90% of bases with quality score of ≥30 were retained during the quality filtering 159 process using fastp [21] . pair-end reads were aligned with the reference genome sars-cov-2 we are grateful to the centre for panoromic sciences (cpos) of the university of hong 295 kong for providing illumina sequencing. we gratefully acknowledge the originating and 296 submitting laboratories who contributed sequences to gisaid (supplementary table s5 gastrointestinal 306 manifestations of sars-cov-2 infection and virus load in fecal samples from the hong kong 307 cohort and systematic review and meta-analysis temporal profiles of 310 viral load in posterior oropharyngeal saliva samples and serum antibody responses during 311 infection by sars-cov-2: an observational cohort study sars-cov-2 shedding 314 and seroconversion among passengers quarantined after disembarking a cruise ship: a case 315 series minionqc: fast and 352 simple quality control for minion sequencing data fast and accurate long-read alignment with burrows-wheeler transform bcftools/roh: a 356 hidden markov model approach for detecting autozygosity from next-generation sequencing 357 data alignment/map format and samtools varscan 2: 362 somatic mutation and copy number alteration discovery in cancer by exome sequencing fastp: an ultra-fast all-in-one fastq preprocessor high prevalence of four 367 novel astrovirus genotype species identified from rodents in china a neutralizing human antibody 370 binds to the n-terminal domain of the spike protein of sars-cov-2 broad 373 neutralization of sars-related viruses by human monoclonal antibodies structures of human antibodies bound to sars-cov-2 spike reveal common epitopes 377 and recurrent features of antibodies human 379 coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-380 binding site in spike protein domain a cov spike glycoprotein in complex with sialoside attachment receptors structure of mouse coronavirus 385 spike protein complexed with receptor reveals mechanism for viral entry nextstrain: 388 real-time tracking of pathogen evolution triple combination of 390 interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to 391 hospital with covid-19: an open-label, randomised, phase 2 trial saliva or nasopharyngeal swab specimens for detection of sars-cov-2 viral load dynamics and disease 397 severity in patients infected with sars-cov-2 in zhejiang province, china key: cord-286544-ipmcqz8n authors: cheng, biao; hu, jiahao; zuo, xiuran; chen, jian; li, xiaochao; chen, yuchen; yang, guoliang; shi, xiaowu; deng, aiping title: predictors of progression from moderate to severe covid-19: a retrospective cohort date: 2020-07-02 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.033 sha: doc_id: 286544 cord_uid: ipmcqz8n objective: most coronavirus disease 2019 (covid-19) cases were identified as moderate, which is defined as having a fever or dry cough and lung imaging with ground-glass opacities. the risk factors and predictors of prognosis in such cohorts remain uncertain. methods: all adult patients with covid-19 of moderate severity diagnosed using qrt-pcr and hospitalized at the central hospital of wuhan, china, from jan 1 to mar 20, 2020 were enrolled in this retrospective study. the main outcomes were progression from moderate to severe or critical condition or death. results: among the 456 enrolled patients with moderate covid-19, 251/456 (55.0%) had poor prognosis. multivariate logistic regression analysis identified higher nlr on admission (or =1.032, 95%ci 1.042-1.230, p = 0.004) and higher crp on admission (or =3.017, 95%ci 1.941-4.690, p < 0.001) were associated with increased odds ratios of poor prognosis. the area under the receiver operating characteristic (roc) curve (auc) for nlr and crp in predicting progression to critical condition was 0.77 (95% ci 0.694-0.846, p < 0.001) and 0.84 (95% ci 0.780-0.905, p < 0.001), with a cut-off value of 2.79 and 25.95 mg/l, respectively. the auc of nlr and crp in predicting death was 0.81 (95% ci, 0.732-0.878, p < 0.001) and 0.89 (95% ci 0.825-0.946, p < 0.001), with a cut-off value of 3.19 and 33.4 mg/l, respectively. conclusions: higher levels of nlr and crp at admission were associated with poor prognosis of moderate covid-19 patients. nlr and crp were good predictors of progression to critical condition and death. as of apr 19, 2020, there have been 2,241,359 confirmed cases of covid-19 worldwide, including 152,551 deaths reported by who [1] . the outbreak of covid-19 has become an international public health emergency [2, 3] . the prognosis of covid-19 patients with different severities at admission is significantly different. most mild or moderate patients that receive basic medical care at fangcang shelter hospitals, which are large-scale, temporary hospitals rapidly built since feb 5 in china, have better prognosis [4] . relative to the moderate cases, severe or critical patients have a higher probability of being admitted to intensive care units (icu), have longer stays [5, 6] , and are more likely to die [7, 8] . identification of which initially mild or moderate patients will deteriorate into severe or critical illness is useful, as it would allow for earlier treatment to prevent worsening outcomes and save medical resources for other patients. in this study, we focus on the clinical features and outcomes of patients with moderate covid-19 treated at a single institution and explore the factors and indicators associated with their prognosis. all adult patients with moderate cases of covid-19 hospitalized at the central hospital of wuhan from january 1 to march 20, 2020, were enrolled in this retrospective cohort study. this is a tertiary hospital located in the central area of wuhan, china, and is one of the designated hospitals for treating covid-19 patients. the data cutoff for this study was march 31, 2020. the flowchart of confirmed patients enrolled in this study is shown in fig s1. all patients were diagnosed with covid-19 based on positive sars-cov-2 qrt-pcr using throat swab samples, in accordance with the diagnosis and treatment protocol for novel coronavirus pneumonia recommended by the national health commission (nhc) of china (version 7.0) [9] . this study was approved by the central hospital of wuhan hospital ethics committee (no. 2020-75). written informed consent was waived by the ethics commission of the designated hospital for emerging infectious diseases. epidemiological, demographic, clinical, laboratory, treatment, and outcome data (progression to severe/critical/death) were reviewed and extracted from electronic medical records using a standardized data collection form by experienced clinicians and independently reviewed by two researchers. fever was defined as an axillary temperature of at least 37.3°c. disease severity grading (mild, moderate, severe, or critical) of covid-19 was defined according to the diagnosis and treatment protocol for novel coronavirus pneumonia. mild grade was defined as few symptoms (low fever, fatigue) and without lung computed tomography (ct) findings. moderate grade was defined as fever, respiratory symptoms (dry cough, chest distress, and shortness of breath after activities), and lung ct findings (i.e. ground glass opacity, multiple small patchy shadows, and pulmonary consolidation). severe grade was defined as respiratory frequency ≥ 30/min, blood oxygen saturation ≤ 93%, oxygenation index < 300 mmhg, and/or lung infiltrates > 50% within 24 to 48 hours. critical grade was defined as respiratory failure, septic shock, and/or multiple organ dysfunction or failure. poor prognosis refers to progression from moderate to severe grade, critical grade, or death. categorical variables are reported as number (%). normally distributed continuous data were reported as mean ± standard deviation (sd) and non-normally distributed continuous data were reported as median (interquartile range [iqr] ). categorical data were compared using the χ2 test or fisher exact test. independent t-tests were used to compare normally distributed continuous data, while the mann-whitney u-test or exact mann-whitney rank sum test was used to compare non-normally distributed continuous data. to adjust for the risk factors associated with illness progression inhospital, univariable and multivariable logistic regression models were used. considering the total number of prognoses (n=251) in our study and to avoid overfitting of the model, 12 variables were chosen for multivariable logistic analysis on the basis of univariable logistic analysis results and clinical significance. multivariable cox proportional hazards regression analyses were used to further adjust the risk factors associated with survival. considering the total number of deaths (n=46) in our study and to avoid overfitting of the model, four variables were chosen for cox regression analysis on the basis of multivariable logistic analysis results and clinical significance. receiver operating characteristic (roc) curves were used to evaluate the potential predictive value of risk factors on prognoses in-hospital. the hosmer-lemeshow test was used to calibrate the roc curves. the net reclassification index (nri) was used to determine which indicators of roc curves analysis were better at predicting outcomes, in line with previously published methods [10] . p value less than 0.05 was considered statistically significant. statistical analysis was performed using spss (version 19.0) and graphpad prism (version 8.0) software. a total of 456 (100%) moderate cases were recruited in this study (table 1) the laboratory data of all moderate cases on admission are shown in table 1 . numerous variables were significantly associated with outcome, and cases with poor prognoses generally had lower lymphocyte counts, and higher levels of c-reactive protein (crp), neutrophil/lymphocyte ratio (nlr), and procalcitonin. treatment and outcome data are presented in table 2 . as indicated, antiviral treatment (i.e. ribavirin, arbidol and lopinavir/ritonavir) was the most common treatment method for moderate cases (437/456, 95.83%), followed by antibiotic treatment (i.e. ephalosporins and quinolones; 369/456, 80.92%) and glucocorticoid treatment (226/456 patients, 49.56%). glucocorticoid treatment and intravenous immunoglobin were more commonly used for patients with poor prognoses than patients that did not progress. the median time of illness onset to admission was 7 days (iqr 4.25-14) in all moderate patients and did not differ significantly between two groups (p > 0.05). were associated with increased odds ratios of poor prognoses. furthermore, we calculated the odds ratio for the different of prognoses in more detail (table s1) . briefly, older age, male gender, and nlr and crp levels at admission greater than 6.0 mg/l were associated with increased odds ratios of severe progression. male gender, nlr, crp greater than 6.0 mg/l on admission were associated with increased odds ratios of progression to critical condition. older age, male gender, nlr, procalcitonin greater than 0.5 ng/ml, and crp greater than 6.0 mg/l on admission were associated with increased odds ratios of death. these results are consistent with our cox regression analysis (table s2) . to explore risk factors that can predict prognosis of patients with moderate covid-19, we used roc curve analysis. the roc curve of nlr and crp in predicting the total poor prognoses and severe progression is shown in figure 1a table 4 . in this retrospective study, the major symptoms of moderate covid-19 were fever and cough and these symptoms did not differ between the two outcome groups (table 1 ). therefore, predicting prognosis based on symptoms is not possible. using comparative and multivariable analyses of basic patient characteristics, we found that comorbidities in moderate cases are not a risk factor for poor prognosis, which is consistent with recent studies [11] . however, older age, male gender, and nlr and crp levels on admission were significantly associated with poor prognoses in patients with moderate covid-19. in our study, the auc of both nlr and crp in predicting progression to critical condition and death was more than 0.75 (table 4) , which suggests that nlr and crp may act as predictors of progression. compared with nlr, the nri of crp was greater than 0 in predicting progression to critical condition and death, indicating that crp is a better predictor, which is consistent with auc results. additionally, although the auc of pct in predicting death was also more than 0.75, the p value of roc curve of the hosmer-lemeshow test for pct was less than 0.001 (table 4) , which suggests poor calibration of the roc curve. hence, the difference between the predicted value and the true value cannot be explained by chance. thus, these results indicate that pct is not a good predictor of death in moderate covid-19 cases in our study. additionally, multivariable logistic analysis revealed that antibiotic, intravenous immunoglobin, and glucocorticoids treatments were not associated with prognosis (table s3 ), suggesting that these medications did not improve prognosis when given to patients with moderate covid-19. as most covid-19 cases are mild or moderate and medical resources are limited, these findings are clinically significant for taking appropriate treatment options and utilizing medical resources in a cost-effective way. however, randomized controlled trials (rcts) are required to confirm the impact of drug treatment on moderate covid-19 patients. there are several limitations of the study. first, this is a single center, retrospective study. second, most moderate covid-19 patients that were enrolled in this study were older and had multiple comorbidities, and thus were more likely to have adverse outcomes. hence, the rate of disease progression in our study may not reflect the true rate. in conclusion, age, gender, and nlr and crp levels at admission are associated with poor prognoses of patients with moderate covid-19. nlr and crp levels on admission tend to be a good predictor of critical progression and death. the authors declare that they have no conflicts of interest. total poor prognoses, moderate cases progress to severe, critical cases or death; nlr, neutrophil-lymphocyte ratio. crp, c-reactive protein. coronavirus disease (covid-19) outbreak situation characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention fangcang shelter hospitals: a novel concept for responding to public health emergencies clinical progression of patients with covid-19 in shanghai clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on 4-week follow-up diagnosis and treatment protocol for novel coronavirus pneumonia recommended discrimination and calibration of clinical prediction models: users' guides to the medical literature prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis none. this work was supported by the natural science foundation of hubei province of china (2019cfa426). key: cord-293542-o0zspgrk authors: ippolito, g.; fusco, f.m.; caro, a. di; nisii, c.; pompa, m.g.; thinus, g.; pletschette, m.; capobianchi, m.r. title: facing the threat of highly infectious diseases in europe: the need for a networking approach date: 2014-12-12 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2009.02876.x sha: doc_id: 293542 cord_uid: o0zspgrk in recent years emerging and re-emerging infections, as well as the risk of bioterrorist events, have attracted increasing attention from health authorities because of the epidemic potential that renders some of them a real public health challenge. these highly infectious diseases (hids) are occurring more and more frequently in europe, and despite the many initiatives in place to face them, many unsolved problems remain, and coordinated efforts for dealing with hids appear mandatory. whereas uncoordinated measures would lead to only partial and poor responses to these emerging threats, networking represents a valuable approach to these diseases, in order to: (i) ensure a rapid and effective response; (ii) stimulate complementarity and prevent duplication; (iii) promote international cooperation, exchange of experience, good practice and protocols; and (iv) support the less prepared countries in the european community. despite hopes to the contrary, infectious diseases appear far from being defeated and continue to claim the attention of public health authorities. particularly in recent years, yet to be fully understood changes in the environment, increased movement of goods and persons, and the local influence of global warming and other phenomena concerning vectors and hosts, seem to have promoted and accelerated changes in the presentation of old infectious diseases and the development of new ones [1] [2] [3] . the relevance of the 'emerging and re-emerging' infectious diseases, usually defined as 'infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range', is further noted by the who in its recent world health report 2007 [4] . the who stressed that infectious diseases are spreading faster and emerging more quickly than ever before. some emerging and re-emerging diseases represent a real challenge because of their epidemic potential. recently, many global alarms involving infectious diseases-such as the anthrax crisis in the usa, the emergence of sars, the pandemic threat posed by the highly pathogenic avian influenza a (h5n1), and the cases of imported or autochthonous viral haemorrhagic fever (vhfs) in europe-have highlighted the need to improve preparedness for these highly infectious diseases (hids), also in order to increase certain aspects of what is perceived in many areas as an issue of collective and national security [5] . emerging hids are of particular concern because they usually hit relatively unprepared public health systems, and appropriate diagnostic tests, vaccines, drugs, containment and mitigation measures are frequently not available or not immediately so. a similar situation could occur if a pandemic strain of influenza virus emerges: several surveys conducted in european countries and in the usa have revealed many gaps in their preparedness plans, in particular in terms of making such plans truly operational, in stepping up prevention measures against seasonal influenza, in ensuring essential services, in enhancing collaboration with adjacent countries, and in extending and better directing influenza research [6] [7] [8] . research on emerging infectious diseases has been funded since the inception of the european union (eu) framework programmes (fp) for research in 1985. in 2002 the eu developed recommendations for early diagnosis and management of bioterrorism-related infections, with the aim of providing member states with a common basis for dealing with these diseases [9] . among activities/projects covered in the fp6 2002-2006, more than half are focused on various aspects of influenza, which makes the commission's fps arguably the single largest funding source for influenza research in europe. the other topics covered include: vhfs, sars, transmissible spongiform encephalopathies, food-and water-borne diseases, other zoonoses, as well as issues such as preparedness and capacity building for different diseases in a more generic fashion. in total, both influenza research and research on other emerging infectious diseases have received more than €100 million of eu funding each since 2002. a complete searchable list and short descriptions of all projects, grouped into different categories (as well as a downloadable pdf version) is available online [10] . concurrent with the increasing awareness of the threat of a new influenza pandemic, the current fp7 2007-2013 introduces for the first time a specific area dedicated to 'potentially new and re-emerging epidemics', specifying that its 'focus will be on confronting emerging pathogens with pandemic potential including zoonoses'. the term 'potentially new and re-emerging epidemics', which is uncommon in the scientific literature, refers mainly to those emerging viral diseases of current or future relevance for europe. this new mandate to cover research systematically in the area of emerging epidemics establishes a focal point within the fp from which calls for proposals in this area can be strategically planned. the past calls in the area of emerging and re-emerging infectious diseases were frequently published ad hoc, in response to specific threats, and the lack of a dedicated area was responsible for the limited coordination and long-term planning. the new mandate in fp7 specifically dedicated to diseases should overcome these problems. although research on influenza will continue to receive support in view of the magnitude and likelihood of an influenza pandemic, future calls will increasingly build a strategic european research capacity for other emerging and re-emerging hids. a definition of hids and the agents/diseases included are summarized in table 1 . several cases of these diseases have been reported in europe since 2000: 32 cases of sars were imported in eight countries, and approximately 15 imported confirmed or suspected cases of vhfs have been reported, mainly lassa fever [11] [12] [13] [14] . very recently, two isolated cases of lassa fever have been diagnosed in london in travellers who returned to the uk from nigeria and mali [15, 16] , and several cases of autochthonous crimean-congo haemorrhagic fevers have been reported in the european region (in turkey and in some states in the balkans) and in some countries within the eu (bulgaria and greece) [17, 18] . no human cases of highly pathogenic influenza a (h5n1) virus have occurred in europe, but two suspected cases were managed in the netherlands and belgium, and public health authorities in greece faced a pseudo-outbreak [19] [20] [21] . moreover, several recent cases of cowpox infections have been reported recently in europe: 18 confirmed cases in germany, one suspected case in the netherlands, five confirmed and seven suspected cases in france. although human cowpoxvirus infections are not classified as hid, these cases are worth mentioning here as an example of how an unexpected agent can disseminate rapidly. some of the cases described above were proven to be caused by the same virus, indicating exposure to a common source of infection related to an international trade in pet rats by a czech rat breeder [22] . two cases of human infection with an orthopoxvirus, similar to but distinct from cowpox, have been identified in north-eastern italy in two veterinary doctors who had been exposed to infected cats [23] . this finding, and the fact that the two infections occurred independently of one another, underscore the need to enhance awareness of zoonotic poxvirus transmission (possibly endemic) also in regions where this problem has not been addressed so far, e.g. the southern alps. almost all of the cases requiring isolation were first admitted to a general hospital without adequate isolation capabilities, and later transferred to a high-level isolation unit. despite the fact that no outbreaks occurred in europe, these experiences exposed weaknesses in terms of recognition, public health response, and diagnostic and clinical management. indeed, despite the wide availability of national and international plans and guidelines, their application in 'real-life' scenarios remains poor. not surprisingly, public health policies and diagnostic and clinical approaches to hids differ widely among european countries, and a common platform that would enable scientists to respond in a quick and powerful manner is still lacking. hids require multidisciplinary expertise. experts in microbiology (especially virology), public health, epidemiology, infectious diseases, and communication need to work together to respond to such incidents. for hids in particular, because of the rarity of their occurrence, strong collaboration and exchange of data, and attention to lessons learned from previous episodes, are advisable. for these reasons, creating new networks and enhancing those functioning well should be strongly promoted, in order to: 1 ensure a rapid and effective response to health threats deriving from natural infection by or deliberate release of hid agents; 2 stimulate complementarity and prevent duplication; 3 promote international cooperation, exchange of experience, good practice and protocols; 4 support the less prepared countries in the european community. a continuous effort is necessary for sustaining and promoting research on hids, whether basic or translational, in order to promote the increase of general knowledge concerning on these issues and to support the development of new tools for facing them in an effective manner. the development and refinement of new diagnostic tests, new therapeutics and innovative vaccines is mandatory as never before. the use of networking and international partnership could represent the successful strategy in this context. the traditional boundaries between basic science and clinical medicine should be dropped, and through effective networks the few hid events that occur worldwide should be studied thoroughly. a network of top-quality scientists and clinicians will provide the complementarity required for the development of these new approaches. moreover, improved funding for research on hids could come from the involvement of networks in the private sector, which could be encouraged to invest in this area because of the epidemic potential and the possible large-scale economic consequences. early recognition and prompt reaction to hids rest upon adequate preparedness, which should include the availability of adequate infrastructures and specific training for healthcare workers. several differences exist among european countries, due to government policies, as well as to pre-existing conditions, and these may result in delayed and dissimilar public health interventions and non-standardized training programmes. a better coordination of public health approaches to hids may lead to standardization of interventions and protocols, such as the prompt isolation within structures with adequate technical and logistic features, to the development of a common core-curriculum, and substantial improvement in the application of international health regulations. moreover, from a practical perspective, a network involving the main public health institutes may play a key role in the management of: 1 a returning hid patient travelling through more than one country (e.g. in the case of one or more connecting flights), in order to coordinate public health interventions; 2 an hid patient admitted in a country without adequate healthcare settings for isolation (in this case, a cross-border transport by ground or air may be the most appropriate solution); 3 multi-country outbreaks. due to the current perceived international security threats, several eu member states are considering establishing biosafety level (bsl)-4 diagnostic facilities. to improve and sustain the existing initiatives and networks aimed at promoting collaboration among the existing bsl-4 laboratories appears mandatory, as well as to provide assistance, through these networks, to other european countries not equipped with such sophisticated and costly facilities [24] . moreover, among the critical points identified in the context of the laboratory diagnosis of hid agents are the scarcity of biological samples to validate the diagnostic methods and the fact that few commercial diagnostic tests are available for these pathogens. thus, a well-functioning network is essential for: 1 the sharing of diagnostic and research experience of the currently operating bsl-4 european laboratories, as well as diagnostic protocols, samples, reagents, and personnel for training; 2 the review of current laboratory diagnostic capability for hid agents; 3 the development of new hazard-free diagnostic tests suitable to be transferred to other non-bsl-4 laboratories; 4 the standardization of procedures for biosafety and biosecurity. intra-hospital procedures for clinical assistance and infection control for hid cases represent 'the core' of managing these diseases, and represent effective measures for hid containment. on the other hand, hospitals may play an important role in the amplification of an outbreak if infection control measures are inconsistently applied. consequently, common protocols for infection control and biosafety during the clinical and diagnostic management of hids patients, based on the available evidence and on 'reallife' experiences, are strongly advisable [25] [26] [27] [28] . moreover, in order to offer to these patients the best available standards of care, a set of specific skills is required, and thus, given the scarcity of these events, a functioning network for expert consultation, second opinion, and scientific support is needed. as more and more persons, animals and goods move within europe, the need for improved and coordinated responses to hids continues to grow. furthermore, it is increasingly recognized that hids can pose a significant threat to each country's national security. innovative research and coordinated efforts, through the establishment of well-functioning networks, are the only way to deal with these issues, in order to improve preparedness and to react quickly: in short, to be 'prepared for the unknown'. a key role may be played by the european centre for disease prevention and control, whose mission is, among others, to 'coordinate the european networking of bodies operating in the fields within the centre's mission' [29] . uncoordinated measures can lead to only partial and poor responses, and different approaches to similar health threats in various eu countries are likely to negatively affect the compliance by health professionals, and the perception of the population. although well-functioning networks are already in place, many gaps still exist, as well as opportunities for future collaboration. fortunately, new scientific developments, and new perceptions of these health threats, make this field one of the most stimulating research areas with a direct impact on the health of millions of people. global trends in emerging infectious diseases climate change and infectious disease: a dangerous liaison? european lab network prepares for high-risk pathogen threat world health organisation: geneva infectious diseases and national security how prepared is europe for pandemic influenza? analysis of national plans challenges remain in preparedness european centre for disease prevention and control gouvras g task force on biological and chemical agent threats, public health directorate, european commission, luxembourg. bichat clinical guidelines for bioterrorist agents european commission quality assurance for the diagnostics of viral diseases to enhance the emergency preparedness in europe no authors listed e-alert 24 july: case of lassa fever imported into germany from sierra leone marburg hemorrhagic fever -the netherlands ex uganda a fatal case of lassa fever in london the first case of lassa fever imported from mali to the united kingdom crimean-congo hemorrhagic fever in greece: a public health perspective probable cases of crimean-congohaemorrhagic fever in bulgaria: a preliminary report management of potential human cases of influenza a/h5n1: lessons from belgium a dutch case of atypical pneumonia after culling of h5n1 positive ducks in bavaria was found infected with chlamydophila psittaci a pseudo-outbreak of human a/h5n1 infections in greece and its public health implications european centre for disease prevention and control. cowpox in germany and france related to rodent pets cat-to-human orthopoxvirus transmission, northeastern italy networking for infectious-disease emergencies in europe risk management of febrile respiratory illness in emergency departments the initial hospital response to an epidemic framework for the design and operation of highlevel isolation units: consensus of the european network of infectious diseases infection control in the management of highly pathogenic infectious diseases: consensus statement from the european network for infectious diseases the authors wish to thank c. schmaltz (european commission, research directorate general, brussels) for the data about eu-funded research projects, and for his invaluable suggestions and critical reading of the manuscript. all authors declare no dual or conflicting interests. key: cord-329350-qrxl5o1e authors: pan, angelo; matteo, giorgi-pierfranceschi; giancarlo, bosio; lorenzo, cammelli; laura, romanini title: suggestions from cremona, italy two months into the pandemic at the frontline of covid-19 in europe date: 2020-06-09 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.05.038 sha: doc_id: 329350 cord_uid: qrxl5o1e nan the covid-19 pandemic is hitting hard even the most advanced health care (1). we have had to care for high numbers of severely ill patients with limited resources, i.e. ventilators and specialists in respiratory failure management, often with a lack of health-care workers (hcw): a terrible situation. the hospital of cremona, italy, is a 500-bed facility and was the second hospital hit with this tsunami-like disease in europe, on february 21st. rapidly the number of patients with covid-19 induced pneumonia reached 540. during the first eight weeks of pandemic the emergency room evaluated 1706 patients, with 1542 admissions; 242 patients were intubated, 419 underwent noninvasive ventilation (niv), and 342 died. home care was activated in 58 cases. at two months into the pandemic and in the phase of descent, we are offering advice -useful tips derived from real life experience -to our colleagues facing this disease. indications regarding preparedness are available, but a view from the "battlefield" may help in everyday practice (see factual summary) (2,3). the indications here described should be managed by a group of clinicians and management experts, in charge of the organization of the hospital in this war-like setting, this being point zero. 1. education first: it is difficult to organize continuing hcw education in an emergency setting, but it is necessary to implement courses on infection control and prevention (icp) and on covid-19 management. three main points need to addressed (4-7): a. correct use of personal protective equipment (ppe): many hcw will be displaced from their routine work to a new task, the treatment of a transmissible infection. hcw need to be rapidly updated on necessary competencies required to manage highly infectious patients with respiratory failure. rapid and thorough courses on the correct use of ppe is the first thing that should be done to protect both hcw and patients. doffing procedures are critical, due to a high risk of contamination (4, 8) . while hcw are often placing stress on the use of face masks, meticulous hand hygiene (hh) is probably the most important prevention strategy, and adherence to this is instrumental (8, 9) . b. proper nasopharyngeal swab taking is fundamental to obtain the best sensitivity/specificity of this test. c. covid-19 management: "fast and dirty" courses on should be organized on general principles of respiratory insufficiency, blood gas analysis, oxygen therapy, venous thromboembolism prevention, antivirals and anti-inflammatory drugs use (7) . intensive care patients management retraining for hcw should be performed. since indications evolve rapidly, courses should be repeated regularly. 2. implement home care: collaborating with gps to correctly manage patients at home, limiting access to the hospital only to patients with possible pneumonia, is of paramount importance. webinars on covid-19 icp strategies and management should be implemented: one hour courses on one-two items are very appreciated. 3. re-organize the emergency room (er): we saw up to 70 covid-19 patients per day: a reorganization of the er will be necessary. consider: how and where to perform triage, and to receive patients into the er -clean and covid-19 triage areas may be necessary. you may rapidly be struggling for beds and even for oxygen therapy points, since most covid-19 patients have respiratory failure. 5. extend intensive care unit and ventilation capacity: we had to increase our intubation capacity from 10 to 52 beds in three weeks. early intubation is recommended to manage covid-19 patients (7) and very rapidly you may run out ventilators. since ventilation weaning takes often over two weeks, a rapid saturation of icu is easily foreseeable, and early intubation may become a difficult problem to solve. you should program in advance when to convert areas with ventilators (i.e. operating theaters) to covid-19 intensive and semi-intensive care units. consider to prone patients to improve respiratory function. a re-organization of the staff is also fundamental since high level skills are needed to manage these patients. 6. re-organize diagnostic services: organize high throughput nasopharyngeal sars-cov-2 swabs and define which exams have to be performed to manage these patients, including d-dimer, ferritin, and il-6 determination. the need for high resolution computed tomography (hrtc), the best diagnostic exam for interstitial pneumonia will rapidly grow. (10, 11) we performed over 2400 pulmonary hrtc in march, as compared to a standard of 200. a dedicated ct service has to be organized. antithrombotic prophylaxis should not be overlooked due to increased risk of venous thromboembolism. to improve knowledge all efforts should go to treat all patients within randomized controlled trials. patients are so numerous that almost any utilized drug will rapidly go out of stock. 8. program work with shortage of hcw: it is likely that a certain number of hcw, will already be infected at the beginning of the epidemic, thus others will become infected. an emergency plan on how to reorganize services and how to re-allocate hcw to continue to offer high level services, is of primary importance. infected hcw should be visited through dedicated internal services and treated following standard procedures. 9 . check facility needs: ensure that all you need for patients with respiratory failure is in place. oxygen consumption will rapidly increase and it may become insufficient: in our hospital oxygen use skyrocketed from 3 to over 80 m3/day. drug use will increase similarly: norepinephrine and midazolam passed from 2,500 and 800 vials/month to 21,000 and 7,000, respectively. blood gas analysis syringe use will increase: in our hospital consumption passed from 1,900 in january 2020 to 12,900 in march. ppe use will be critical: mask use, i.e. surgical masks and ffp2/ffp3 respirators, increased from 5,000 to 41,000/week, impermeable gowns from 1,300 to 11,700/week, goggles/face shields from 30 to 1200/week. adequate supplies have to be organized. 10 . take into account the needs and stress of patients and hcw: patients are scared of the disease and visits, at least in our country, are forbidden. time individually spent with patients is not enough, and the whole team -doctors, nurses, nurses aids -should try to stay as close to them as possible. in our experience this is exactly what every hcw is willing to do, limiting the sense of anxiety and fear that is common during covid-19. on the hcws' side, working with covid-19 patients is an incredible stressful duty since it is a highly transmittable disease. furthermore, the level of uncertainty in management is high, the mortality is dreadful, and patients' social life within the hospital is extremely difficult. additionally, bringing home the stresses from work and worrying about the risk of transmitting sars-cov-2 infection to family members is a source of anxiety to the extent that normal marital relationship may be altered. psychological support from the very beginning of the outbreak would be very useful both for patients and hcw. the latin motto estote parati -be prepared -is what we learnt from this terrible pandemic: while waiting for possible new waves, we are working on education on ppe, hh, and ventilation, and programming how to dedicate general ward and icu to manage new covid-19 patients. finally, once the tsunami is passed you will need to have re-habilitation services to manage patients discharged after long icu stays: be prepared (12) . to conclude, we have rapidly proposed what we think could be of help to our colleagues facing covid-19 pandemic (see table 1 ). this experience has so far taught us that even in these extremely difficult situations you have to struggle for collaboration and discussion. we think that aid to coordinate such a strenuous situation could be sourced form experts in medicine of catastrophe or war medicine: the needs of the hospital, its patients and hcw undergo a rapid and dramatical change over only a few days, similar to what is observed during war. an interactive web-based dashboard to track covid-19 in real time european centre for disease prevention and control. checklist for hospitals preparing for the reception and care of coronavirus 2019 (covid-19) patients. ecdc: stockholm world health organization. critical preparedness, readiness and response actions for covid-19. interim guidance european centre for disease prevention and control. infection prevention and control for covid-19 in healthcare settings -third update how to obtain a nasopharyngeal swab specimen interim clinical guidance for management of patients with confirmed coronavirus disease (covid-19) infectious diseases society of america guidelines on the treatment and management of patients with covid-19 personal protective equipment for preventing highly infectious diseases due to exposure to contaminated body fluids in healthcare staff. cochrane database syst rev air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient coronavirus disease (covid-19): spectrum of ct findings and temporal progression of the disease high-resolution chest ct features and clinical characteristics of patients infected with covid-19 in jiangsu postacute care preparedness for covid-19. thinking ahead transparency declaration • conflict of interest disclosure: that should be identical to the content of the coi form that is submitted • funding: no external funding was received acknowledgments: we want to thank pantelis tsoulfas for his thoughtful review and allegra della ragione for the language review access to data: not applicable. • contribution: 1. all authors gave substantial contributions to the conception of the work, literature search and analysis and discussion and interpretation of data all authors revised it critically for important intellectual content all authors gave final approval of the version to be published all authors agreed to be accountable for all aspects of the work. all authors ensure that all questions related to the accuracy or integrity of any part of the work have been appropriately investigated and resolved all authors have nothing to disclose.the study did not receive any external. key: cord-291272-srt08jh8 authors: peters, e.j.g.; collard, d.; van assen, s.; beudel, m.; bomers, m.k.; buijs, j.; de haan, l.r.; de ruijter, w.; douma, r.a.; elbers, p.w.g.; goorhuis, a.; gritters van den oever, n.c.; knarren, g.h.h.; moeniralam, h.s.; mostard, r.l.m.; quanjel, m.j.r.; reidinga, a.c.; renckens, r.; van den bergh, prof, j.p.w.; vlasveld, i.n.; sikkens, j.j. title: outcomes of persons with covid-19 in hospitals with and without standard treatment with (hydroxy)chloroquine date: 2020-10-14 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.10.004 sha: doc_id: 291272 cord_uid: srt08jh8 objective: to compare survival of subjects with covid-19 treated in hospitals that either did or did not routinely treat patients with hydroxychloroquine or chloroquine. methods: we analysed data of covid-19 patients treated in 9 hospitals in the netherlands. inclusion dates ranged from february 27(th) 2020, to may 15(th), when the dutch national guidelines no longer supported the use of (hydroxy)chloroquine. seven hospitals routinely treated subjects with (hydroxy)chloroquine, two hospitals did not. primary outcome was 21-day all-cause mortality. we performed a survival analysis using log-rank test and cox-regression with adjustment for age, sex and covariates based on premorbid health, disease severity, and the use of steroids for adult respiratory distress syndrome, including dexamethasone. results: among 1949 included subjects, 21-day mortality was 21.5% in 1596 subjects treated in hospitals that routinely prescribed (hydroxy)chloroquine, and 15.0% in 353 subjects that were treated in hospitals that did not. in the adjusted cox-regression models this difference disappeared, with an adjusted hazard ratio of 1.09 (95%ci 0.81-1.47). when stratified by actually received treatment in individual subjects, the use of (hydroxy)chloroquine was associated with an increased 21-day mortality (hr 1.58; 95%ci 1.24-2.02) in the full model. conclusions: after adjustment for confounders, mortality was not significantly different in hospitals that routinely treated patients with (hydroxy)chloroquine, compared with hospitals that did not. we compared outcomes of hospital strategies rather than outcomes of individual patients to reduce the chance of indication bias. this study adds evidence against the use of (hydroxy)chloroquine in hospitalised patients with covid-19. the spread of sars-cov-2, leading to the current pandemic of covid-19, has a profound global 128 impact on daily life, morbidity and mortality. several preliminary studies have reported that the 129 antimalarial agents hydroxychloroquine and chloroquine, or (h)cq, alone or in combination with the 130 antibiotic azithromycin, can have a suppressive effect on the viral replication, and might decrease the 131 mortality of covid-19 1-5 . so far, clinical studies have been hampered by confounding by 132 indication 1,2,4,5 , monocentre setup 2,3 , and small numbers of included subjects 3 . a recently published 133 systematic review 6 , a published randomized controlled trial 7 and an rct only available in pre-print 8 , 134 suggested that hydroxychloroquine is not effective in patients admitted to hospital. side effects of 135 (h)cq are well-known, and include fever and cardiac arrhythmias. while we are awaiting definite 136 results from more rcts, cohort studies can provide quick closure of existing knowledge gaps. when 137 treatment assignment in cohort studies is based on prescriber discretion, the risk of indication bias 138 (even after covariate adjustment) remains high. however, our database of dutch hospitals contains 139 data of subjects from hospitals that either routinely prescribed (h)cq or did not prescribe it at all, 140 offering a unique opportunity to compare both strategies. the comparison of different treatment 141 strategies among hospitals leads to a significant reduction of (indication) bias. the objective of this 142 study was to compare the effect of hospital-wide covid-19 treatment strategies with or without 143 routine (h)cq use on all-cause 21-day mortality. we used data from the ongoing covidpredict clinical course cohort containing over 2,000 persons 150 with covid-19 9 , from 9 hospitals in the netherlands, including two university hospitals. included in 151 the database were all subjects admitted to hospital with positive sars-cov-2 pcr of nasopharynx, 152 throat, sputum or bronchoalveolar lavage samples, or ct-scan abnormalities that were typical for 153 covid-19 (co-rads 4 and 5) 10 , without another explanation for the abnormalities than inclusion dates ranged from the first admitted case in the netherlands on february 27 th 2020, to may 155 15 th , when the dutch national guidelines no longer advised the use of (h)cq. we excluded patients < 156 18 years and patients who were transferred to or from another hospital. dosage of chloroquine base 157 was: loading dose of 600 mg, followed by 300 mg twice a day for a total of 5 days. dosage of 158 hydroxychloroquine sulphate was 400 mg twice daily on the first day, followed by 200 mg twice daily 159 on days 2 to 5. among the seven (h)cq-hospitals, the timing of start of (h)cq treatment differed; 160 three hospitals started at the moment of covid-19 diagnosis, four started after diagnosis but only 161 when patients clinically deteriorated e.g., when there was an increase in respiratory rate or increase 162 in use of supplemental oxygen. the two hospitals that did not routinely treat subjects with (h)cq 163 (i.e., the non-(h)cq-hospitals), offered best supportive care, including oxygen therapy and 164 potentially antibiotic therapy, according to local guidelines and prescriber discretion. participating 165 hospitals did not routinely prescribe other experimental medication (e.g., lopinavir/ritonavir, 166 remdesivir or steroids, see table 1 ). subjects who were incidentally treated with these drugs were 167 included in the study. primary outcome was 21-day all-cause mortality, defined as hospital mortality, 168 or discharge to a hospice care facility. a waiver for the use of hospital record data was obtained 169 through the institutional review board of amsterdam umc; however, patients were given the 170 opportunity to opt out. we collected data according to the collection protocol of the world health 171 organization. missing covariates were imputed using multiple imputation with the mice package 172 (version 3.8.0) and the outcomes were determined by pooling the results of 25 imputed datasets 11 . 173 j o u r n a l p r e -p r o o f we performed a regression analyses and determined the pooled effect. missing data range for all 174 covariates was less than 2.8%, except for obesity (missing data 6.2%) and use of corticosteroids 175 (22.3%). in the primary analysis, we compared effectiveness of (h)cq versus non-(h)cq hospital 176 strategies, irrespective of actual individual (h)cq treatment. we performed a survival analysis using 177 log-rank test and cox-regression with adjustment for age, sex, time in the pandemic (i.e., the number 178 of elapsed days after march 1 st 2020 at hospital admission),and covariates based on premorbid 179 health (i.e., history of lung, kidney and cardiovascular disease, diabetes mellitus, obesity, and 180 neoplasms or hematologic disease), disease severity during presentation (respiratory rate, oxygen 181 saturation) and the use of steroids, including dexamethasone, for adult respiratory distress 182 syndrome (ards) 12,13 . we repeated the analyses comparing actually received treatment, with (h)cq. 183 in a secondary analysis, we used a composite endpoint (either mechanical ventilation or all-cause 184 mortality) at 21 days. as a sensitivity analysis, we performed a complete case analysis using inverse 185 probability weighting of propensity scores (determined using the same covariates). we performed a 186 subgroup analysis in (h)cq hospitals that started (h)cq directly from the moment of diagnosis versus 187 outcomes in non-(h)cq hospitals. all statistical analyses were performed using r versions 3.6.3 (r 188 table 1 . follow-up data were missing for 20 (1.0%) subjects. the patients with missing 195 outcome data were included table 1 saturation during admission were similar in both hospital groups (see table 1 ). in (h)cq-hospitals, 208 9.6% of subjects received corticosteroids for ards and 4.0 were in a study protocol of an 209 experimental sars-cov-2 directed antiviral (e.g., lopinavir/ritonavir) or immunomodulatory drug trial 210 (e.g., imatinib, anti-complement c5), versus 2.3% and 11.3% in non-(h)cq-hospitals, respectively. 211 table 2 ). when 215 stratified by actually received treatment, the use of (h)cq was associated with an increased 21-day 216 mortality (hr 1.58; 95%ci 1.24-2.02, table 3 ) in the full model. in the secondary analysis with either 217 mechanical ventilation or all-cause mortality at 21 days, there were no statistically significant 218 differences between the (h)cq and non-(h)cq hospitals (crude p=0.055, adjusted hr 0.87 (95%ci 219 0.68-1.10), online supplement 1). the complete analysis using propensity scores for treatment 220 strategy and actual treatment showed similar results (see table 4 ). an overview of the distribution of the strength of this study is that data were collected in nine hospitals, including two university 248 hospitals, in the netherlands during the covid-19 epidemic. data collection was set up prospectively 249 and the database included data on all consecutive subjects admitted to general medicine and 250 pulmonology wards, and to intensive care units. the database was set up according to the who 251 standards, which enabled data comparison and uniformity of data among the different participating 252 centres. the comparison of hospital-defined treatment strategies rather than the treatment actually 253 received led to a lower risk of indication bias compared with previous studies 1,2,4,5 . we roughly 254 estimate the extend of the effect of indication bias to be the difference in outcome between the 255 uncorrected and the corrected model. further strengths include the multicentre setup 2,3 , as 256 mentioned above, and the relatively large numbers of included subjects 3 . 257 there are some limitations we need to address. although health care in the netherlands has a 259 homogeneous setup, there was some variability in standard protocols among the hospitals that could 260 j o u r n a l p r e -p r o o f have led to residual confounding. the two non-(h)cq-hospitals were tertiary (university) centres, 261 whereas the (h)cq-hospitals comprised both secondary and tertiary care hospitals. before the 262 covid-19 pandemic, the tertiary care hospitals and their intensive care units function as referral 263 centres for local secondary care hospitals. since we excluded subjects transferred to and from other 264 hospitals, the referral role of the tertiary care hospitals, including the university hospitals, was 265 minimized. furthermore, subjects in the (h)cq hospitals were more likely to receive steroid 266 treatment, while subjects in the non-(h)cq hospitals were more likely to receive other experimental 267 immunomodulatory drugs. the numbers of the individual types of medication were small, making it 268 impossible to draw conclusions from these differences. the results of the recovery trial, suggested 269 a lower mortality in patients treated with dexamethasone 15 . treatment with dexamethasone could 270 therefore have resulted in a lower mortality in the group of (h)cq hospitals. we did not find such an 271 effect, even after correction in the full model. we also used extensive covariate adjustments, using 272 various methods to minimize influence of differences in patient population among hospitals, and the 273 similarity in outcomes between these methods is reassuring in this regard. show a benefit of (h)cq treatment. this may be explained by the timing of the administration of the 282 drug and its specific working mechanism. chloroquine binds in silico and in vitro with high affinity to 283 sialic acids and gangliosides of sars-cov-2. these bindings inhibit the interaction at non-toxic plasma 284 levels with ace-2 receptors and could hypothetically stop the cascade from formation of pulmonary 285 infiltrations to full blown ards and death [17] [18] [19] . the antiviral activity might be more effective in the 286 pre-clinical setting as the deterioration in the hospital is more an effect of the cytokine storm 287 provoked by sars-cov-2 than an effect of the viral infection itself. this hypothesis might explain why 288 the clinical benefit for admitted subjects was absent in our study, although we did not observe a 289 difference in outcome among subjects treated early (at diagnosis) and among those treated later 290 upon clinical deterioration. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f association of treatment with hydroxychloroquine or 347 azithromycin with in-hospital mortality in patients with covid-19 in observational study of hydroxychloroquine in hospitalized 349 patients with covid-19 hydroxychloroquine and azithromycin as a treatment of 351 covid-19: results of an open-label non-randomized clinical trial use of hydroxychloroquine in hospitalised covid-19 354 patients is associated with reduced mortality: findings from the observational multicentre italian 355 corist study low-dose hydroxychloroquine therapy and 357 mortality in hospitalized patients with covid-19: a nationwide observational study of 8075 358 participants effect of 360 hydroxychloroquine with or without azithromycin on the mortality of covid-19 patients: a 361 systematic review and meta-analysis hydroxychloroquine in patients with mainly mild to moderate 363 coronavirus disease 2019: open label, randomised controlled trial effect of hydroxychloroquine in hospitalized patients 365 with covid-19: preliminary results from a multi-centre, randomized, controlled trial co-rads -a categorical ct 369 assessment scheme for patients with suspected covid-19: definition and evaluation multiple imputation by chained equations in praxis: guidelines and review clinical course and outcomes of critically ill patients with sars-cov-2 374 pneumonia in wuhan, china: a single-centered, retrospective, observational study factors associated with hospital admission and critical 377 illness among 5279 people with coronavirus disease a practical guide to propensity score analysis for applied clinical research dexamethasone in hospitalized 382 patients with covid-19 -preliminary report structural and molecular modelling studies reveal a 387 new mechanism of action of chloroquine and hydroxychloroquine against sars-cov-2 infection connecting hydroxychloroquine in vitro antiviral activity to in vivo 390 concentration for prediction of antiviral effect: a critical step in treating covid-19 patients in vitro antiviral activity and projection of optimized dosing 393 design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 394 2 (sars-cov-2) key: cord-287256-hgqz1bcs authors: magurano, fabio; baggieri, melissa; marchi, antonella; rezza, giovanni; nicoletti, loredana title: sars-cov-2 infection: the environmental endurance of the virus can be influenced by the increase of temperature date: 2020-11-05 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.10.034 sha: doc_id: 287256 cord_uid: hgqz1bcs objectives the goal of the current study is to evaluate whether the increase of temperature can influence the environmental endurance of sars-cov-2. methods the virus was inoculated on plastic surface and harvested at predefined time-points in parallel at 20-25°c (rt) and at 28°c (jt). samples collections were tested by tcid50 titers on vero cells. samples collections were tested by tcid50 titers on vero cells. results our results confirm that fomite transmission of the emerging sars-cov2 is possible: the virus reserved its ability to infect cells up to 84 hours at both rt and jt on plastic surface, with a tcid50 viral titre of 0,67 and 0,25 log10 respectively. at rt, an important reduction in the viral titre, from 4 log10 to 3 log10 tcid50 was observed during the first 24-36 hours. at jt the same decay was observed more rapidly (between 8 and 12 hours), the rate of viral inactivation by d-value was 24.74 at rt and 12,21 hours at jt. conclusions this remarkable difference between the two temperatures suggests that virus vitality can be influenced by the environmental temperature and that the hot season could reduce the probability of covid-19 transmission. objectives. the goal of the current study is to evaluate whether the increase of temperature can influence the environmental endurance of sars-cov-2. parallel at 20-25°c (rt) and at 28°c (jt). samples collections were tested by tcid 50 titers on vero cells. samples collections were tested by tcid 50 titers on vero cells. our results confirm that fomite transmission of the emerging sars-cov2 is possible: the virus reserved its ability to infect cells up to 84 hours at both rt and jt on plastic surface, with a tcid 50 viral titre of 0,67 and 0,25 log10 respectively. at rt, an important reduction in the viral titre, from 4 log10 to 3 log10 tcid50 was observed during the first 24-36 hours. at jt the same decay was observed more rapidly (between 8 and 12 hours), the rate of viral inactivation by d-value was 24.74 at rt and 12,21 hours at jt. can be influenced by the environmental temperature and that the hot season could reduce the probability of covid-19 transmission. introduction corona virus disease 19 (covid-19) pandemic has been caused by the enveloped betacoronavirus sars-cov-2, transmitted from person to person through respiratory droplets and direct contact, and potentially by indirect contact through fomites (1) . several studies have shown that viral spread could be influenced by climatic conditions since enveloped viruses tend to reduce their circulation in summertime due to high temperature and solar radiation (2, 3) . no wonder, the current spread of covid-19 along the equator and tropics was shown to be significantly less (4), leading to the hypothesis that the increase of temperature will influence the environmental endurance of sars-cov-2. in order to try to predict the effect on the epidemic dynamic of covid-19 during the summer months, we decided to test sars-cov-2 environmental stability in parallel at room temperature (rt, 20°c-25°c) and at average maximum temperature of june (jt) estimated at 28°c in italy. the strain betacov/italy/cdg1/2020|epi isl 412973|2020-02-20 (5) was used to test sars-cov-2 stability on plastic surface (polypropylene). that strain had an initial viral titre of 10 6.8 tcid 50 /ml, a comparable viral load of symptomatic, asymptomatic or minimally symptomatic patients (6) . the viral preparation was spotted in droplets of 10 µl on 24-well plates and let 30 minutes to dry. then, plates were incubated at both rt and jt for 7 days at relative humidity of 35-45% ethical approval was not need for this study. viral titre of the daily collections of 0, 24, 48 and 72 hours at rt were determined also by plaque assay in vero e6 cells. briefly, 12-well plates were plated with vero e6 cells (150,000/well in mem +10% fcs) and inoculated with logarithmic dilutions of each sample. plates were incubated for 1 hour at 37°c, and 4 ml/well of a medium containing 2% gum tragacanth + mem 2.5% fcs were added. after 5 days at 37°c with 5% co2, titres were calculated by crystal violet dyeing in plaqueforming units per milliliter (pfu/ml). all the experimental procedures were conducted under biosafety level-3 conditions. to approximate a normal distribution viral titer of each well was log-transformed. performing standard deviation with a 95% confidence interval, the results mostly followed a normal distribution j o u r n a l p r e -p r o o f of a set value. further, the ratio of the standard deviation to the mean was investigated by calculation coefficient of variance. analysis of data obtained by tcid50 titration showed that an important reduction in the viral titre, from 4 log 10 to 3 log 10 tcid 50 per milliliter of medium, was observed during the first 24-36 hours at rt (figure) with a d-value of 24.74 hours.. this trend is confirmed by titration by plaque assay (appendix). at jt, the same decay was observed more rapidly (between 8 and 12 hours) indicating that viral infectivity can be influenced by higher temperature with a d-value of 12,21 hours. this decay trend continues until 84 hours showing a remarkable difference between the two temperatures. in both the experimental conditions, the virus is no longer detectable at 96 hours. the present study confirms that fomite transmission of the emerging sars-cov-2 is possible (7) in conclusion, the increase of temperature observed in summer may influence the environmental endurance of the sars-cov-2 but do not influence the need of maintaining social distancing measures. world health organization. modes of transmission of virus causing covid-19: implications for ipc precaution recommendations. who scientific brief association between viral seasonality and meteorological factors survival of coronaviruses in water and wastewater an interactive web-based dashboard to track covid-19 in real time whole genome and phylogenetic analysis of two sars-cov-2 strains isolated in italy in viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china aerosol and surface stability of sarscov-2 as compared with sars-cov-1 possible transmission by fomites of respiratory syncytial virus virus survival in the environment stability of sars-cov-2 and other coronaviruses in the environment and on common touch surfaces and the influence of climatic conditions: a review the authors wish to thank dr. paola stefanelli for providing the virus sars-cov-2 betacov/italy/cdg1/2020|epi isl 412973|2020-02-20. the authors wish to thank alessia caratelli, ambrogio carlei, marina sbattella and eugenio sorrentino for their technical support. the authors declare no competing interests. no external funding was received key: cord-324148-bllyruh8 authors: loubet, paul; mathieu, pauline; lenzi, nezha; galtier, florence; lainé, fabrice; lesieur, zineb; vanhems, philippe; duval, xavier; postil, deborah; amour, sélilah; rogez, sylvie; lagathu, gisèle; l'honneur, anne-sophie; foulongne, vincent; houhou, nadhira; lina, bruno; carrat, fabrice; launay, odile title: characteristics of human metapneumovirus infection in adults hospitalized for community-acquired influenza-like illness in france, 2012-2018: a retrospective observational study date: 2020-04-10 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.04.005 sha: doc_id: 324148 cord_uid: bllyruh8 objectives: to describe the prevalence, clinical features and complications of human metapneumovirus (hmpv) infections in a population of adults hospitalized with influenza-like illness (ili). methods: this was a retrospective, observational, multicenter cohort study using prospectively collected data from adult patients hospitalized during influenza virus circulation, for at least 24h, for community-acquired ili (with symptom onset <7 days). data were collected from five french teaching hospitals over six consecutive winters (2012-2018). respiratory viruses were identified by multiplex rt-pcr on nasopharyngeal specimens. hmpv+ patients were compared to hmpv– patients, influenza+ and respiratory syncytial virus (rsv)+ patients using multivariate logistic regressions. primary outcome was the prevalence of hmpv in patients hospitalized for ili. results: among the 3148 patients included (1449 (46%) women, 1988 (63%) aged 65 and over; 2508 (80%) with chronic disease), at least one respiratory virus was detected in 1604 (51%, 95%ci [49-53]), including 100 cases of hmpv (100/3148, 3% 95%ci [3, 4]), of which 10 (10%) were viral co-infection. in the hmpv+ patients, mean length of stay was 7 days, 62% (56/90) developed a complication, 21% (14/68) were admitted to intensive care unit and 4% (4/90) died during hospitalization. in comparison with influenza+ patients, hmpv+ patients were more frequently > 65 years old (aor=3.3, 95%ci[1.9-6.3]) and presented more acute heart failure during hospitalization (aor=1.8, 95%ci[1.0-2.9]). compared to rsv+ patients, hmpv+ patients had less cancer (aor=0.4, 95%ci[0.2-0.9]) and were less likely to smoke (aor=0.5, 95%ci[0.2-0.9]) but had similar outcomes especially high rate of respiratory and cardiovascular complications. conclusions: adult hmpv infections mainly affect the elderly and patients with chronic conditions and are responsible for frequent cardiac and pulmonary complications similar to those of rsv infections. at-risk populations would benefit from the development of antivirals and vaccines targeting hmpv. during winter, community-acquired influenza-like illness (ili), mostly caused by respiratory 74 viruses, is very common. the most frequent viruses seen in primary care are influenza 75 viruses a/b, rhinovirus, coronavirus, respiratory syncytial virus (rsv) and human 76 metapneumovirus (hmpv) [1, 2] . in the hospital setting, adults with ili are commonly tested 77 only for influenza, resulting in limited data concerning other respiratory viruses. the use of 78 multiplex rt-pcr allows identification of multiple viruses simultaneously but remains a 79 second-line test in non-immunocompromised patients in emergency departments because of 80 its cost and the limited therapeutic options [3] . 81 human mpv, discovered in 2001, is phylogenetically similar to rsv and has been frequently 82 found associated with respiratory tract illnesses [1, 4, 5] . its circulation occurs with a seasonal 83 distribution from january to march in the northern hemisphere, often overlapping or following 84 rsv infection season [6, 7] . human mpv is a major pediatric respiratory pathogen at least one of the following respiratory symptoms: cough, sore throat or dyspnea. patient 105 with contra-indication for influenza immunization, those who had previously tested positive 106 for influenza virus in the same season and those without french social security affiliation 107 were excluded. each participant was interviewed, and nasopharyngeal samples were 108 obtained at enrolment to screen for influenza and other respiratory viruses. 109 in the present study, we included all the patients from the first six fluvac seasons 110 (2012/13, 2013/14, 2014/15, 2015/16, 2016/17, 2017/18) patients with multiple viral infections were excluded from the analyses. univariate analysis 143 was used to asses risk factors for the detection of hmpv infection, influenza infection, rsv 144 infection and acute heart failure. we performed two multivariate analyses using a backward 145 stepwise logistic regression model using hmpv test result (positive/negative) and acute heart 146 failure (yes/no) as the dependent variable in the first and second model respectively. 147 covariates with a p-value <0.2 in univariate analysis were tested in the multivariate model. 148 results from regression models are expressed as crude odds ratios (or) and adjusted ors 149 (figure 1 and table s1 ). ten of the 100 hmpv infections (10%) were coand 41% (37/90) hospitalized in the 12 months preceding the study (table 1) . 174 the median time from symptom onset to admission was similar between hmpv+ and hmpv-175 patients (2 days (iqr, 1-3)) as well as the main symptoms at inclusion except for 176 weakness/malaise that was less frequent in hmpv+ patients (13/90 (14%) vs 27%, p<.007). 177 there was no difference between hmpv+ and hmpv-groups in terms of median length of 178 stay, number of complications during hospitalization, intensive care unit (icu) admission and 179 death. however, hmpv+ patients were more likely to have an acute heart failure during 180 hospitalization (25% (22/89) vs 14% (377/2722), p<.004). 181 there was no difference in sociodemographic characteristics, clinical presentation or 182 outcomes between hmpv and viral coinfection and patients with hmpv infections alone. 183 in the multivariate analysis, when comparing hmpv+ patients to all hmpv-patients, age > 65 184 years (aor 95% ci 3.3 [1.9;6.1], p<0.001) was significantly associated with hmpv detection. 185 in contrast, the sudden onset of symptoms, defined as the occurrence of malaise/weakness, 186 was associated with the absence of hmpv infection (aor 95% ci 0.4 [0.2;0.8], p=0.008) 187 (table 1) . 188 after adjustment for chronic heart disease, age, gender, smoking status and influenza 189 vaccination, hmpv infection was significantly associated with occurrence of acute heart 190 failure during hospitalization (aor 95% ci 1.8 [1.1;3.0], p=0.02). 191 in univariate analysis, in comparison to influenza+ patients, hmpv+ patients were older 193 in our post-hoc analysis of 3148 hospitalized adult patients with community-acquired ili, 211 hmpv was found in 3% of the samples. these patients were older, had chronic conditions, 212 frequent respiratory and cardiac chronic diseases, and frequently presented complications. 213 this prevalence is consistent with several studies that found hmpv in 3 to 6% of adult 214 patients with lower respiratory tract infection in primary care [1, [14] [15] [16] and in 6% of patients 215 hospitalized for acute respiratory infection (ari) [17] . this frequency may vary according to 216 the inclusion criteria, especially temperature cut-off, as hmpv infection frequently causes 217 non-febrile illness [5] . 218 our hospitalized hmpv+ adults were mostly older and/or high risk patients as previously 219 described in the literature [5, 10, [18] [19] [20] . complications were frequent (62%), as well as icu 220 admission (17%) and death (4%). these rates were similar to those of the 91 hospitalized 221 patients with hmpv from the walsh et al. study in 2008 in the usa [10], but lower than those 222 of the 128 critically ill adults with hmpv infection (31% required icu admission and 8% died) 223 from the hasvold et al. study in 2016 [20] . 224 the majority of hmpv+ patients presented several respiratory signs (cough, dyspnea), 225 whereas sudden onset of symptoms was associated with the absence of hmpv infection. 226 there were differences in clinical presentation between hmpv+ patients and influenza+ 227 patients (less frequent constitutional symptoms (headache, weakness, myalgia) but more 228 dyspnea) but not between hmpv+ patients and rsv+ patients. these points emphasize the 229 difficulty of distinguishing respiratory viruses based on clinical signs alone and question the 230 relevance of the current ili definition to detect hmpv infection. 231 interestingly, we also found that hmpv+ patients were older and presented more chronic 232 cardiac conditions and acute heart failure during the hospitalization than influenza+ patients. 233 although, influenza [21] and rsv [22] are known to worsen heart failure, no study has 234 specifically assessed hmpv [21, 22] aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 273 11 european countries baseline 275 characteristics and clinical symptoms related to respiratory viruses identified among patients 276 presenting with influenza-like illness in primary care practice guidelines by the infectious diseases society of america treatment, chemoprophylaxis, and institutional outbreak management of 282 seasonal influenzaa van den hoogen bg, osterhaus ad, fouchier ra. clinical impact and diagnosis of 310 human metapneumovirus infection respiratory virus infections in hospitalized children and adults in lao pdr. influenza 313 other respir clinical features, epidemiology, 315 and climatic impact of genotype-specific human metapneumovirus infections: long-term 316 surveillance of hospitalized patients in south korea human 320 metapneumovirus in patients hospitalized with acute respiratory infections: a meta-analysis human metapneumovirus infections on the 323 icu: a report of three cases exacerbation of chronic obstructive pulmonary disease the role of human 328 metapneumovirus in the critically ill adult patient seasonal trends 330 of heart failure hospitalizations in the united states: a national perspective from with cardiovascular disease in adults mortality in adults hospitalized for respiratory syncytial virus infections clinical 340 characteristics and outcome of respiratory syncytial virus infection among adults hospitalized 341 with influenza-like illness in france onset of symptoms > 7 days: n=124no consent obtained n=6) key: cord-306083-juysx6yo authors: choe, young june; park, sangshin; michelow, ian c. title: co-seasonality and co-detection of respiratory viruses and bacteraemia in children: a retrospective analysis date: 2020-09-10 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.09.006 sha: doc_id: 306083 cord_uid: juysx6yo objectives: the aim of this study was to assess the co-seasonality and co-detection of respiratory viral infections and bacteraemia in children since the introduction of the 13-valent pneumococcal conjugate vaccine (pcv13). methods: children <18 years were eligible for inclusion if they had a respiratory infection and a positive pcr-based assay for respiratory viruses as well as a positive blood culture from 2010 to 2018 at a single referral centre in the united states regardless of their underlying medical condition or antibiotic treatment history. monthly incidence rates of respiratory viruses and bacteraemia were analysed with a seasonal-trend decomposition procedure based on loess (stl) and cross-correlation functions using time series regression modelling. results: we identified 7,415 unique positive respiratory virus tests, including 2,278 rsv (31%), 1,825 influenza viruses (24%), 1,036 parainfluenza viruses (14%), 1,017 hmpv (14%), 677 seasonal coronaviruses (9%), and 582 adenoviruses (8%), and a total of 11,827 episodes of bacteraemia. significant co-seasonality was found between all-cause bacteraemia and rsv (or=1.76, 95% ci 1.50-2.06, p<0.001), influenza viruses (or=1.38, 95% ci 1.13-1.68, p=0.002), and seasonal coronaviruses (or=1.18, 95% ci 1.09-1.28, p<0.001), respectively. analysis of linked viral-bacterial infections in individual children indicated that the rate ratio (rr) of bacteraemia associated with hmpv (rr=2.73, 95% ci 1.12-6.85, p=0.019) and influenza (rr=2.61, 95% ci 1.21-6.11, p=0.013) were more than double that of rsv. staphylococcus aureus and streptococcus pneumoniae were the most commonly identified pathogens causing bacteraemia. conclusions: there is a significant association between hmpv and influenza viruses, and bacteraemia of all causes in hospitalised children at a single paediatric centre in the united states. large multicentre studies are needed to confirm these findings and to elucidate the mechanisms by which hmpv potentiates the virulence and invasive capacity of diverse bacteria. children with respiratory viral infections are susceptible to infection with bacteria that may 2 cause pyogenic complications such as empyema, necrotising pneumonia and bacteraemia [1] . 3 since the influenza pandemic of 1918, streptococcus pneumoniae and staphylococcus aureus 4 have been recognized as the predominant causes of invasive bacterial infections complicating 5 influenza infections [2] . 6 the direct relationship between respiratory viruses and bacteraemia in children remains 7 poorly defined, especially since the introduction of the 7-valent pneumococcal conjugate 8 vaccine (pcv7) in the united states in 2000 [3, 4] . a study conducted in children before the 9 implementation of pcv13, demonstrated significant associations between invasive 10 pneumococcal disease (ipd) and influenza viruses and respiratory syncytial virus (rsv), as well 11 as human metapneumovirus (hmpv), which was a novel observation [5] . 12 the deployment of pcv13 in the united states in 2010 has led to a substantial decline in 13 ipd but it is not currently known which bacteria complicate respiratory viral infections [3] . we 14 hypothesised that respiratory viruses detected in hospitalised children are associated with 15 multiple causes of bacteraemia. to test this hypothesis, we analysed the relationship between 16 16 respiratory viruses and all-cause bacteraemia in children at a single paediatric centre in the 17 united states over 8 years since the introduction of pcv13. 18 care clinic or during hospitalisation from june 2010 to may 2018. blood cultures and 1 respiratory viral pcr assays were obtained at the discretion of attending physicians according to 2 usual local practise for children with fever and respiratory symptoms. no systematic changes 3 were made during the study period. based on the knowledge that respiratory viruses incubate for 4 up to 1 week and are shed for 14 days or longer [6, 7] , we made an a priori assumption that 5 detection of a virus 2 weeks before or up to 1 week after a positive blood culture could 6 potentially be causally associated with bacteraemia. children were eligible regardless of their 7 underlying medical condition or antibiotic treatment history. aggregated laboratory results, 8 season and patients' age were collated using theradoc (premier, charlotte, north carolina), an 9 infection control software system. we constructed a longitudinal database to track monthly incidence of respiratory viruses and 23 bacteraemia. a filtering procedure, called a seasonal-trend decomposition procedure based on 24 loess (stl) was conducted separately for respiratory viruses and bacteria in order to decompose 25 and smooth time series data with seasonal, trend and remaining components [8] . cross-correlation functions were applied using time series regression modelling to determine the 1 highest correlation between overall incidence of various respiratory viruses and bacteraemia. we 2 calculated the incidence of cases with viral-bacterial co-detections as well as a rate ratio (rr) of 3 various respiratory viruses relative to that of rsv. rr was calculated using the median unbiased 4 estimator method. statistical analyses were performed using r (ver. 3.4.3; r development core 5 team, vienna, austria). 6 the institutional review board at rhode island hospital provided ethics approval for this 8 study and exemption from informed consent. table 1 ). there were no significant seasonal 22 associations between adenovirus, hmpv, or parainfluenza viruses and bacteraemia. 23 we used rsv as a reference for computing rr because it had the lowest proportion of 24 bacteraemia episodes. children with hmpv had the highest proportion of bacterial co-detections 25 with an rr of 2.7 relative to rsv (p = 0.019). similarly, the rr for bacteraemia associated 26 with influenza viruses was 2.6 compared with rsv (p = 0.013). adenoviruses, seasonal 1 coronaviruses and parainfluenza viruses had respective rr of 0.92, 1.90 and 2.20 relative to 2 rsv, but the differences were not statistically significant (table 1) . 3 s. aureus (n = 15) and s. pneumoniae (n = 12) were the mostly commonly identified 4 pathogens causing bacteraemia (supplementary table) . 5 6 discussion 7 we observed that the seasonality of coronaviruses, influenza viruses and rsv strongly 8 correlated with that of bacteraemia among hospitalised children. these findings corroborate 9 those of other investigators [5, 9] and indicate that the co-seasonality of respiratory viruses and 10 bacteria is conducive to concurrent host colonisation. after we linked episodes of viral and 11 bacterial infections in individual children, we found that the proportion of co-detections was 12 generally low, ranging from 0.4% for rsv to 1.1% for hmpv, which is within the range of 13 other recent reports (0.4-1.6%) [10] [11] [12] [13] [14] . however, children with hmpv or influenza viruses had 14 more than double the rate of bacteraemia compared with rsv. 15 this report validates the association between influenza and hmpv, and bacteraemia that was 16 reported by ampofo et al [5] before the introduction of pcv13. in addition to s. pneumoniae, 17 we identified s. aureus and a variety of other bacteria, which emphasizes the importance of 18 emerging non-vaccine preventable pathogens. in addition to influenza, hmpv is known to cause 19 degenerative changes in the lower respiratory epithelium, potentially permitting colonising 20 bacteria to invade, and to impair signalling at the immunological synapse between dendritic cells 21 and t cells, potentially disrupting host defences, which may explain its virulence [4, 15] . 22 this study is limited by its retrospective design and lack of detailed patient-level clinical 23 data, such as evidence of upper versus lower respiratory tract infection, underlying 24 comorbidities, antibiotic treatment history, and evidence of prior immunisations. also, the role 25 of multiple viruses detected simultaneously and possible role of presumed contaminants was not 26 assessed due to the aggregated nature of our data. furthermore, the small number of patients 1 with co-detections derived from a single institution limits the generalisability of these findings. 2 on the other hand, this is the first study to appraise the association between respiratory 3 viruses and bacteraemia in children since the deployment of pcv13. despite the small sample 4 size, we employed a rigorous statistical approach to account for seasonal and secular trends, and 5 our findings are consistent with those of a previous larger study conducted before the 6 introduction of pcv13 [5] . 7 in conclusion, we found a strong association between both hmpv and influenza, and 8 bacteraemia in children. large multicentre studies are needed to confirm these findings and to 9 elucidate the mechanisms by which hmpv potentiates the virulence and invasive capacity of 10 diverse bacteria. empiric antibacterial treatment of severely ill children infected with these 11 viruses appears to be warranted. 12 13 j o u r n a l p r e -p r o o f yjc and icm developed the study concept and design. all authors had full access to the data and take responsibility for the integrity of the data and accuracy of the data analysis. yjc was responsible for data collection. yjc and sp performed the statistical analyses. all authors assisted with data interpretation. yjc and icm performed the literature search. yjc wrote the first draft of the manuscript. all authors have critically read and commented on draft versions of the report, and approved the final version. the authors declare no competing interests. this work was supported by a grant from national institute of allergy and infectious diseases at the national institutes of health (grant number r25ai140490 to icm). j o u r n a l p r e -p r o o f epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children the mother of all pandemics is 100 years old (and going strong)! effect of use of 13-valent pneumococcal conjugate vaccine in children on invasive pneumococcal disease in children and adults in the usa: analysis of multisite, populationbased surveillance global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance seasonal invasive pneumococcal disease in children: role of preceding respiratory viral infection patterns of shedding of myxoviruses and paramyxoviruses in children incubation periods of acute respiratory viral infections: a systematic review stl: a seasonal-trend decomposition procedure based on loess seasonality of infectious diseases influenza virus infection and the risk of serious bacterial infections in young febrile infants clinical characteristics of children and adults hospitalized for influenza virus infection prevalence of serious bacterial infections in febrile infants with respiratory syncytial virus infection bacteraemia and antibiotic use in respiratory syncytial virus infections secondary bacteremia following adenovirus infection modulation of host immunity by the human metapneumovirus key: cord-297625-eby014gm authors: l'huillier, a.g.; tapparel, c.; turin, l.; boquete-suter, p.; thomas, y.; kaiser, l. title: survival of rhinoviruses on human fingers date: 2014-12-11 journal: clin microbiol infect doi: 10.1016/j.cmi.2014.12.002 sha: doc_id: 297625 cord_uid: eby014gm rhinovirus is the main cause of the common cold, which remains the most frequent infection worldwide among humans. knowledge and understanding of the rhinovirus transmission route is important to reduce morbidity as only preventive measures are effective. in this study, we investigated the potential of rhinovirus to survive on fingers. rhinovirus-b14 was deposited on fingers for 30, 60, 90 and 120 min. survival was defined as the ability of the virus to grow after 7 days, confirmed by immunofluorescence. rhinovirus survival was not dependent on incubation time on fingers. droplet disruption had no influence on survival. survival was frequent with high rhinovirus concentrations, but rare with low-concentration droplets, which corresponded to the usual rhinovirus concentrations in mucus observed in children and adults, respectively. our study confirms that rhinovirus infectiousness is related to the viral concentration in droplets and suggests that children represent the main transmission source, which occurs only rarely via adults. it confirms also that rhinovirus hand-related transmission is possible and supports hand hygiene as a key prevention measure. rhinoviruses are non-enveloped, positive-stranded rna viruses belonging to the enterovirus genus within the picornaviridae family and the main causative agent of the common cold [1] , the most frequent infection worldwide. although usually a selflimited viral disease, it remains a source of significant morbidity in the community. rhinovirus is associated also with asthma/wheezing and chronic obstructive pulmonary disease exacerbations, as well as several complications, such as acute otitis media, sinusitis, bronchitis and, in some cases, lower respiratory tract diseases. pre-school children seem to be the main reservoir [2] , as approximately six rhinovirus infections are observed per year and per child [3] . there are more than 150 different rhinovirus types with almost no cross-protection, which explains the frequency of rhinovirus infections and the absence of an effective vaccine or antiviral treatment. only preventive measures are currently effective against these highly prevalent viruses and understanding their mode of transmission is important to reduce the number of infected patients. the nasal mucosa and posterior nasopharynx have been documented as the main sites of viral replication and therefore the main shedding site [4, 5] . it is reported that person-to-person transmission is most likely due to the contamination of hands by the nasal secretions of the infected person passed to a susceptible individual, either directly to the fingers or via an environmental intermediary; infection then follows from self-inoculation to the upper nasal airways or eyes [6] [7] [8] [9] . the required infecting virus dose is below one median tissue culture infectious dose/ml (tcid 50 ) [8, 10] . three possible transmission routes have been described: via aerosols of respiratory droplets, direct contact by hands, or indirect contact with environmental objects (fomites). aerosols produced by coughing or sneezing originate mainly from saliva [11] in which the viral load is approximately 30 times lower than in nasal secretions [4, 8] . as rhinovirus transmission depends on the concentration of virus in secretions [4] , this supports expert opinion that aerosol or oral transmission is a rare event [12] [13] [14] . direct contact appears to play a major role in transmission. rhinoviruses have been shown to transiently survive on human skin [4, 6, 13, 15] , leading to the hypothesis that hand-related transmission is the main transmission mechanism [6, 13, 16] . although less frequently than on skin [13, 17, 18] , rhinovirus has been shown to survive on fomites. in an experimental study, 50% of volunteers who touched their nasal mucosa or conjunctiva after handling a contaminated fomite developed infection [15] . however, many authors consider that indirect transmission is unlikely because of the important loss of infectivity during the process [17, 19, 20] . our study was designed to test rhinovirus stability on fingers under experimental conditions, which aimed to reproduce natural conditions as far as possible. we conducted a series of experiments to assess the duration of human rhinovirus infectiousness duration on fingers, as well as the impact of viral concentration on survival rates. survival was defined as the ability of the virus to grow on helaoh cells after 7 days, confirmed by immunofluorescence. experimental conditions aimed to reproduce natural conditions as far as possible. all experiments were performed using the rv-b14 strain and helaoh cells (kindly provided by f.h. hayden, university of virginia, charlottesville, va, usa) for viral culture. rv-b14 stock (1 × 10 e8 tcid 50 /ml) was diluted with respiratory mucus to obtain three different concentrations: 1 × 10 e5 tcid 50 /ml (high concentration (hc)); 1 × 10 e4 tcid 50 /ml (average concentration (ac)); and 1 × 10 e2 tcid 50 /ml (low concentration (lc)). each hc and ac droplet contained 1.1 × 10 e5 and 2.8 × 10 e4 viral rna copies (5.5 × 10 e7 and 1.4 × 10 e7 copies/ml), respectively. lc droplet viral copies were below the limit of detection by realtime rt-pcr assay, but they were expected to represent 200 viral copies given their equivalence to 100 dilutions of the ac. hc represents the average viral load of paediatric nasopharyngeal swabs in our laboratory, whereas lc corresponds to the average measured adult concentration. these values also correlate with epidemiological findings in the literature for paediatric and adult patients [6, 13, 17, 21] . respiratory mucus was obtained by mixing clinical samples sent for routine testing that were rt-pcr and cell culture negative for the usual human respiratory viruses (influenza virus a/b, human metapneumovirus, coronavirus 229e/hku1/oc43/nl63, respiratory syncytial virus a/b, picornavirus and parainfluenza virus 1/2/ 3). a further 20 min of ultraviolet radiation ensured inactivation of putative undetected viruses. to guarantee optimal growth, only mucus with a ph between 6.5 and 7 was retained. participants and finger contamination procedure six specialized laboratory collaborators (technicians and md/ phd graduates) were recruited on a voluntary basis as previously described [22] . the protocol was approved by the institutional review board of the university hospitals of geneva. determination of infectiousness a 2-μl drop of viral suspension of human rv-b14 mixed with respiratory secretions was deposited on the fingertips of each participant. this volume represents the mean size of a large respiratory droplet and can be easily reproduced [22] . for each subject, nine drops containing rhinovirus at different concentrations (three hc, three ac, three lc) were deposited and one negative control (mucus only). each contaminated finger was kept untouched for a defined period of time at room temperature before testing for the presence of infectious rhinovirus. participants' fingers were then immersed in wells (becton dickinson and co., franklin lakes, nj, usa) containing 1 ml of mccoy's 5a medium (1 ×) with 2% serum (gibco, new york, ny, usa) for 60 seconds. then, 400 μl of this eluate was used to immediately inoculate helaoh cells. this represents an additional 2.5-fold dilution of the viral load present in droplets before inoculation onto cell cultures (4.4 × 10 e4 viral copies for hc, 1.1 × 10 e4 viral copies for ac, and <100 copies for lc). after 1 h of adsorption at 33°c, 1 ml of mccoy's 5a medium (1 ×) with 2% serum (gibco) was added and cells were incubated in 5% co 2 at 33°c for 7 days. for each 24-well plate, a negative control as well as a mock-infected control finger was included. the cytopathic effect was read daily until day 7. cells were collected after 7 days and submitted to an immunofluorescence assay. a j2 mouse monoclonal antibody [23] that recognizes doublestranded rna and an anti-mouse monoclonal igg fluorescein isothiocyanate-conjugated antibody were used to confirm the presence of viral infection (chemicon-millipore, zug, switzerland). based on preliminary pilot experiments, we determined that rhinovirus survival on fingertips was equivalent across different incubation times as it remained infectious on all fingers after 30, 60, 90 and 120 min. immediately after deposition, half of the droplets were disrupted and spread on the surface of the fingertip using a pipette tip to determine whether disrupting the integrity and environment of the droplet decreased virus survival. as all intact and disrupted droplets yielded positive culture results, we decided to continue experiments with disrupted droplets only so as to reproduce real-life conditions as much as possible. one hour after the deposit of disrupted droplets on the fingers of the six volunteers, infectious viruses could be detected by culture in all subjects contaminated with hc droplets (6/6), in four of the six volunteers with ac droplets, and none of the six volunteers with lc droplets, which confirmed the influence of concentration on survival (fig. 1) . of note, when droplets were directly incubated without a passage on fingers, the virus survived in 100% (4/4) of tested fingers at hc compared with 25% (1/4) at lc, despite being below the limit of detection by pcr (data not shown). overall, the proportion of fingers with detectable viruses was 16/18 fingers at hc, compared with 6/18 and 0/18 for the ac and lc droplets, respectively (fig. 1) . laboratory room (mean ± standard deviation 24.6 ± 0.7°c) and hood (26.4 ± 1.8°c ) temperature, as well as humidity (44.5 ± 5.6%), were similar for all experiments with all subjects. we aimed to investigate rhinovirus transmission by person-toperson contact, the main transmission route for the most prevalent human respiratory infection worldwide. experiments were designed to reproduce, as much as possible, conditions that could lead to rhinovirus contamination of fingertips in the community. our study showed that rhinovirus can survive on hands for several hours, similar to previous reports of virus survival on human skin [4, 6, 13, 15, 17] , emphasizing that handrelated transmission is the main transmission route. there was no influence of drying time on virus survival under 2 h, in contrast to the study of ansari et al. where virus survival decreased during the first hour [24] . our study showed that virus survival, and therefore infectiousness, was related to the viral concentration in droplets. this correlates well with d'alessio et al. who found that the secondary attack rate was related to the viral concentration in the nose [4] . inoculum seems to be a restrictive factor for transmission, with infectiousness rapidly dropping below a given concentration. as infected children appear to have a higher viral load than adults, this may explain why children are considered to be the main transmission vector. the fact that the viral load in lc droplets was below the level of detection explains why the virus could not be recovered at these concentrations, except in one case without a passage on fingers. lc droplets correspond to the viral concentration recovered in rhinovirus-infected adults and this suggests that transmission via adults occurs rarely. a recent study investigating the transmission of cold-like illnesses between siblings showed that younger children tended to become infected first in most cases. however, the secondary attack rate was greater for older siblings, probably because of a higher viral load in younger siblings' secretions. as younger children tend also to touch nasal secretions directly with their fingers, it is probable that this enhances transmission. the viral load in the mucus of more than 1000 rhinovirus-infected children below 1 year of age was 5.79 × 10 e6 tcid 50 /ml, which is 10 to 100 times higher than our hc of 1 × 10 e5 (regamey et al., private communication). it is very probable that the difference in virus survival between adults and children is even higher than in our results. we showed that virus survival increased at lc when there was no passage on hands. the loss of infectiousness during interhuman contact or fomite manipulation has already been described [20, 24] , highlighting again the importance of viral load for transmission. similarly, rhinovirus was more frequently recovered on fingers from subjects with a high nasal viral load compared with a low nasal viral load [17] . our study confirmed that droplet disruption had no influence on survival at a given concentration. we have previously shown that influenza virus survival on fingers was not related to virus concentration in a study using a similar methodology to the present experiments [22] . survival of influenza virus on fingers declined rapidly, with less than 15% of the fingers remaining positive after 30 min [22] . the influenza envelope, which is known to be a determinant factor decreasing virus survival, may explain why survival was shorter and affected by droplet disruption, which was not the case for rhinovirus [22] . in a similar study, rv-14 in 10-μl droplets at 2.9 × 10 e4 to 1.4 × 10 e5 tcid 50 /ml survived for 1 h on almost 40% of fingers [24] . the fact that virus survival was lower compared with our results, despite the use of bigger droplets and higher viral concentrations, may be explained by the fact that our volunteers did not wash their hands or use an alcohol-based hand rub before experiments as hand rubbing has been shown to decrease virus survival even several hours after use [25] . the fact that disinfecting hands or objects with an iodine or alcoholbased solution reduces the secondary illness rate of rhinovirus infections emphasizes also the importance of hand-related transmission [8, 15, 26] . in conclusion, these laboratory results confirm that handrelated transmission of rhinovirus is possible and support hand hygiene as a key measure to prevent transmission, particularly in children who represent the main transmission source. the authors declare that they have no conflicts of interest. viruses and bacteria in the etiology of the common cold temporal relationships for cold-like illnesses and otitis media in sibling pairs picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season transmission of experimental rhinovirus colds in volunteer married couples sites of rhinovirus recovery after point inoculation of the upper airway hand-to-hand transmission of rhinovirus colds effect of route of inoculation on experimental respiratory viral disease in volunteers and evidence for airborne transmission mechanisms of transmission of rhinovirus infections inoculation of human volunteers with a strain of virus isolated from a common cold relation between naturally acquired immunity and infectivity of two rhinoviruses in volunteers experiments on the spread of colds. 1. laboratory studies on the dispersal of nasal secretion production of tracheobronchitis in volunteers with rhinovirus in a small-particle aerosol transmission of rhinovirus colds by self-inoculation rhinovirus infections in an industrial population. iv. infections within families of employees during two fall peaks of respiratory illness transmission of experimental rhinovirus infection by contaminated surfaces rhinovirus transmission: one if by air, two if by hand an investigation of the possible transmission of rhinovirus colds through indirect contact survival of human rhinovirus type 14 dried onto nonporous inanimate surfaces: effect of relative humidity and suspending medium near disappearance of rhinovirus along a fomite transmission chain role of infectious secretions in the transmission of rhinovirus quantitative rhinovirus shedding patterns in volunteers survival of influenza virus on human fingers an rna replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus 3 and rhinovirus 14 interruption of experimental rhinovirus transmission efficacy of organic acids in hand cleansers for prevention of rhinovirus infections we thank all volunteers who participated in the experiments as well as rosemary sudan for editorial assistance. this study was supported by grants from the swiss national science foundation (me 9580, 310030_146151 and me 9575, 32003b_146991/1) and by the laboratory of virology of the university hospitals of geneva. key: cord-344581-h7ikjgic authors: ong, david s.y.; de man, stijn j.; lindeboom, fokke a.; koeleman, johannes g.m. title: comparison of diagnostic accuracies of rapid serological tests and elisa to molecular diagnostics in patients with suspected covid-19 presenting to the hospital date: 2020-06-02 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.05.028 sha: doc_id: 344581 cord_uid: h7ikjgic objectives: to assess the diagnostic performance of rapid lateral flow immunochromatographic assays (lfas) compared to an enzyme-linked immunosorbent assay (elisa) and nucleic acid amplification tests (nats) in suspected coronavirus disease 2019 (covid-19) patients. methods: patients presenting to a dutch teaching hospital were eligible between march 17 and april 10, 2020, when they had respiratory symptoms that were suspected for covid-19. the performances of six different lfas were evaluated in plasma samples obtained on corresponding respiratory sample dates of nats testing. subsequently, the best performing lfa was evaluated in 228 patients and in 50 sera of a historical patient control group. results: in the pilot analysis sensitivity characteristics of lfa were heterogenous ranging from 2/20 (10%; 95% confidence interval (ci) 0-23) to 11/20 (55%; 95% ci 33-77). in the total cohort, orient gene biotech covid-19 igg/igm rapid test lfa had a sensitivity of 43/99 (43%; 95% ci 34-53) and specificity of 126/129 (98%; 95% ci 95-100). sensitivity increased to 31/52 (60%; 95% ci 46-73) in patients with at least seven days of symptoms, and to 21/33 (64%; 95% ci 47-80) in patients with c-reactive protein (crp) >100 mg/l. sensitivity and specificity of wantai sars-cov-2 ab elisa was 59/95 (62%; 95% ci 52-72) and 125/128 (98%; 95% ci 95-100) in all patients, respectively, but sensitivity increased to 38/48 (79%; 95% ci 68-91) in patients with at least seven days of symptoms. conclusions: there is large variability in diagnostic test performance between rapid lfas, but overall limited sensitivity and high specificity in acutely admitted patients. sensitivity improved in patients with longer existing symptoms or high crp. lfas should only be considered as additional triage tools when these may lead to the improvement of hospital logistics. in december 2019 the outbreak of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) started in wuhan in china [1] , but the coronavirus disease 2019 rapidly spread to other countries as well [2] . the first infected patient in the netherlands was detected on the 27 th of february 2020 [3] . accurate diagnostics are fundamental in the fight against this increasing pandemic. moreover, hospitals would benefit from rapid detection of this virus infection in patients who acutely present to hospitals with respiratory symptoms suspected for covid-19. time delay in the establishment of diagnosis increases logistic challenges and causes stagnation of patient flow in emergency departments as these patients are unable to be transferred to appropriate hospital wards or intensive care units (icus) when results of the diagnostic tests are still pending [4] . nucleic acid amplification tests (nats) are the gold standard because of the high specificity, although sensitivity may depend on the timing of disease presentation, sampling location and severity of illness [5] . nevertheless, it usually takes about 4 to 24 hours before laboratory-based results become available depending on specific nat platforms and laboratory organisation. therefore, numerous lateral flow immunochromatographic assays (lfas) have been introduced into the market, and some countries have stocked up on such rapid tests. these lfas detect the presence of igm and igg against sars-cov-2. this study aimed to assess the diagnostic performance of lfas, and compare these to an enzyme-linked immunosorbent assay (elisa) and nats in suspected covid-19 patients. patients presenting to a teaching hospital in the netherlands were eligible between march 17, 2020 and april 10, 2020 when they had respiratory symptoms that were suspected for respiratory tract infection. patients were sampled from the oral cavity and subsequently from the nasal cavity using the same nasopharyngeal swab, which was tested by nats. in some cases, sputum samples were tested, because of persisting clinical suspicion on covid-19 despite a negative nat on nasopharyngeal swabs. nats were performed according to the national reference method that was established after international collaboration [6] , or by the ce-ivd kit genefindertm covid-19 plus realamp kit using the sample to result platform elite ingenius®. the institutional review board waived the need for informed consent as tests were performed on samples which had been acquired for routine clinical care (irb protocol number 2020-034), and according to hospital procedure all patients were informed about the possibility of an opt-out if they had objections against the use of left-over material for research to improve or validate diagnostic testing procedures. the study was conducted in accordance with helsinki declaration as revised in 2013. first, in a pilot phase 20 nat-positive and 5 nat-negative patients were retrospectively selected for which six lfas were performed on heparin plasma samples obtained upon hospital presentation ( figure s1 ), which corresponded to the dates of molecular testing. lfas were included from boson biotech, cellex, dynamiker biotechnology, orient gene biotech, prometheus bio, and wantai rapid test. any visible band for either igg, igm or unspecified ig was indicative for a positive result. second, based on the sensitivity and specificity results in the pilot study, the best performing lfa was further evaluated in an extended cohort of randomly selected patients. third, this lfa was prospectively tested in consecutive patients between april 6 and april 10. fourth, specificity was additionally tested in a historical control group of randomly selected sera of 50 adult patients in september 2019 as sars-cov-2 was not circulating at that time. finally, samples were also analysed by the wantai sars-cov-2 ab elisa kit, which detects total antibodies, and interpreted according to manufacturer's instructions. both clinical information and reference standard results were unavailable to the performers of lfas and the elisa. all analyses were performed using sas 9.2 (cary, north carolina). we compared groups using non-parametric tests for continuous variables and chi-square test or fisher's exact test for categorical variables as appropriate. p-values <0.05 were considered to be statistically significant. in the pilot study sensitivity characteristics of lfa were very heterogenous ranging from 2/20 (10%; 95% confidence interval (ci) 0-23%) to 11/20 (55%; 95%ci 33-77%)) ( table 1) . we decided to continue with the orient gene biotech covid-19 igg/igm rapid test (ogbrt) as it had the highest sensitivity. a total of 111 patients (including the 25 from the pilot study) were retrospectively selected between march 16 and march 29. subsequently, 117 consecutive patients were prospectively included between april 6 and april 10. in total, 228 patients were included with a median age of 61 years (interquartile range (iqr) 46-74), 117 (52%) were male, 21 (9%) were admitted to the icu within 24 hours and median c-reactive protein (crp) upon hospital presentation was 31 (iqr 7-95) mg/l (table s1 ). median time from symptom onset to sample collection was 7 (iqr 4-14) days. ogbrt had an overall sensitivity of 43/99 (43%; 95%ci 34-53%) and specificity of 126/129 (98%; 95%ci 95-100%) ( table 2) . sensitivity increased to 31/52 (60%; 95%ci 46-73) in patients with at least seven days of symptoms, and to 21/33 (64%; 95%ci 47-80) in patients with crp >100 mg/l upon presentation. however, there was no significant difference between patients requiring icu figure s2 ). in the randomly selected historical control sera, the lfa and the elisa specificity was 49/50 (98%; 95%ci 94-100) and 50/50 (100%; 95%ci 100-100), respectively; lfa showed a very weak igg line in one sample. this study shows that the sensitivity of lfa was low in patients suspected for covid-19 presenting to the hospital, but it improved in patients with at least seven days of symptoms and in those with crp levels >100 mg/l upon presentation. specificity of lfas and the elisa was very high, and fulfilling a frequently used criterium of at least 98%. the elisa had a higher sensitivity compared to lfas. several countries, including spain and the united kingdom, have purchased one or more of these lfas. however, our study findings underline that cautiousness is required when considering implementation of such tests. interestingly, cellex rapid test, which is currently the only rapid diagnostic test that is fda approved, performed less than ogbrt in our pilot study. another rapid test was reported to have a sensitivity below 20% in acute patients referred to emergency department [7] . other studies showed higher sensitivities of lfas up to 90% in unspecified patient groups with more time between disease onset and testing or missing information regarding timing of sampling [8, 9] . test performance characteristics as provided by manufacturers were higher than those observed in our study, which is related to different selection of positive and negative controls. in our study we primarily included consecutive patients presenting to the hospital, which represents clinical practice and clinical sensitivity (i.e. diagnosing covid-19 upon hospital presentation) rather than analytical sensitivity (i.e. detecting the presence of antibodies at that moment). the observed higher sensitivity in patients with at least seven days of symptoms is in line with findings from other studies [10, 11] . there are some study limitations to consider. this study included a wide comparison of six different lfas, an elisa and nats, but more tests are available on the market. nevertheless, both lfas and the elisa were limited in sensitivity, suggesting that antibody production is not always detectable or at least not yet detectable during the early phase of infection. second, nat as reference standard remains suboptimal, and it remains possible that in some cases actual infections were missed. in some patients nats were only positive in sputum and negative in nasopharynx, whereas the majority of patients were only tested by nasopharyngeal swabs. third, the subgroup of patients admitted to the icu was limited, precluding definite conclusions in this group. in conclusion, the high specificity of lfas may contribute to rapidly confirm covid-19, accelerate decision-making in emergency rooms and routing to appropriate hospital wards. yet, negative lfa results are unreliable to exclude covid-19 due to the limited sensitivity of these tests. therefore, these lfa tests cannot replace molecular diagnostics in acute care settings, but should only be used as an additional triage tool when improvement of hospital logistics is expected and their limitations are carefully considered. the authors declare no conflicts of interest. clinical characteristics of coronavirus disease 2019 in china rapidly increasing cumulative incidence of coronavirus disease (covid-19) in the european union/european economic area and the united kingdom current information about the novel coronavirus (covid-19) bilthoven: rivm better tests, better care: improved diagnostics for infectious diseases sars-cov-2 viral load in upper respiratory specimens of infected patients detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr performance of vivadiag covid-19 igm/igg rapid test is inadequate for diagnosis of covid-19 in acute patients referring to emergency room department evaluation of a covid-19 igm and igg rapid test; an efficient tool for assessment of past exposure to sars-cov-2 development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis antibody responses to sars-cov-2 in patients of novel coronavirus disease antibody detection and dynamic characteristics in patients with covid-19 boson biotech rapid 2019-ncov igg/igm combo test card 10 / 20 (50% cellex qsars-cov-2 igg/igm cassette dynamiker biotechnology 2019-ncov igg/igm orient gene biotech covid-19 igg/igm rapid test cassette 11 / 20 (55% prometheus bio 2019-ncov igg/igm in the subgroup of patients with time from symptom onset to sample collection >= 14 days, sensitivity and specificity of the lfa were 9/14 (64%; 95%ci 39-89) and 30/32 (94%; 95%ci 85-100), respectively, whereas sensitivity and specificity of the elisa of note, sensitivity was 17/24 (71%) in patients with >=7 days from symptom onset to sample collection and crp >=100 mg/l we would like to thank our laboratory technicians and team managers for their assistance in performing the serological tests.contribution: dsyo and jghk contributed to the conception and design of the study. dsyo, sjm and fal acquired the data. dsyo and sjm analysed the data. all authors contributed to the interpretation of the data. dsyo drafted the first manuscript and all other authors revised it critically for important intellectual content. all authors approved this manuscript version to be submitted. key: cord-325529-pid58g2r authors: ben-ami, roni; klochendler, agnes; seidel, matan; sido, tal; gurel-gurevich, ori; yassour, moran; meshorer, eran; benedek, gil; fogel, irit; oiknine-djian, esther; gertler, asaf; rotstein, zeev; lavi, bruno; dor, yuval; wolf, dana g.; salton, maayan; drier, yotam title: large-scale implementation of pooled rna extraction and rt-pcr for sars-cov-2 detection date: 2020-06-23 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.06.009 sha: doc_id: 325529 cord_uid: pid58g2r objectives: testing for active sars-cov-2 infection is a fundamental tool in the public health measures taken to control the covid-19 pandemic. due to the overwhelming use of sars-cov-2 rt-pcr tests worldwide, availability of test kits has become a major bottleneck, while the need to increase testing throughput only rises. we aim to overcome these challenges by pooling samples together, performing rna extraction and rt-pcr in pools. methods: we tested the efficiency and sensitivity of pooling strategies for rna extraction and rt-pcr detection of sars-cov-2. we tested 184 samples both individually and in pools to estimate the effects of pooling. we further implemented dorfman pooling with a pool size of 8 samples in large-scale clinical tests. results: we demonstrated pooling strategies that increase testing throughput while maintaining high sensitivity. a comparison of 184 samples tested individually and in pools of 8 samples, showed that test results were not significantly affected. implementing the 8-sample dorfman pooling to test 26,576 samples from asymptomatic individuals, we identified 31 (0.12%) sars-cov-2 positive samples, achieving a 7.3-fold increase in throughput. conclusions: pooling approaches for sars-cov-2 testing allow a drastic increase in throughput while maintaining clinical sensitivity. we report the successful large-scale pooled screening of asymptomatic populations. an emerging novel severe acute respiratory syndrome-related coronavirus, sars-cov-2, is the virus behind the global covid-19 pandemic. among the foremost priorities to facilitate efficient public health interventions is a reliable and accessible diagnosis of an active sars-cov-2 infection. the standard laboratory diagnosis of covid-19 involves three main steps, namely, viral inactivation and lysis of the nasopharyngeal swab sample, extraction (or purification) of viral rna, and reverse transcription (rt)-pcr. due to the rapid spread of the virus and the increasing demand for tests, the limited availability of test reagents, mainly rna extraction kits, has become (and will likely continue to be) a major bottleneck as the pandemic expands [1, 2] . of particular importance is the ability to survey large asymptomatic populations-(1) to trace asymptomatic covid-19 carriers which are otherwise difficult to identify and isolate; (2) to assure key personnel (e.g. healthcare personnel) are not contagious; (3) to screen high risk populations (such as nursing homes) to help protect them; (4) to accurately estimate the spread of infection and the effectiveness of community measures and social distancing; and (5) to allow and monitor a safe return to work. efficient and higher-throughput diagnostic approaches are needed to support such efforts. while some of these applications (e.g. (4)) may be achieved with less sensitive detection approaches, most applications do require adhering to the current high standards of rt-pcr. several attempts to address this challenge were recently reported, and can be categorized into three major approaches. the first approach is to replace pcr-based methods by other direct diagnostic methods such as loop-mediated isothermal amplification (lamp) [3] [4] [5] [6] and crispr based diagnostic tools [7] [8] [9] . the second approach involves serological surveys [10] [11] [12] [13] , and the third approach involves the improvement of the pcr methods capacity by optimization and automation [1, 14, 15] or by reducing the required number of tests via pooling samples together, known as group testing. group testing is a field of research in the intersection of mathematics, computer science and information theory, with applications in biology, communication and more. a group testing algorithm is a testing scheme directed towards minimizing the number of tests conducted on a set of samples by using the ability to test pooled subsets of samples. if a pool of n samples tests negative, all samples must be negative, and therefore their status has been determined in only one test instead of n individual tests. various group testing algorithms exist, with different assumptions and constraints [16, 17] . while many such algorithms, most notably binary splitting, may be very efficient in theory, they might be unsuitable because of practical limitations. some key constrains are (1) a limit on the number of stages due to the importance of delivering a test result quickly, exemplified by the urgent clinical context of covid-19 diagnosis; (2) a limit on the ability to dilute samples and still safely identify a single positive sample in a pool; and (3) favorability of simple algorithms which may minimize human error in a laboratory setting. while several pooling approaches for sars-cov-2 detection were recently suggested [2, [18] [19] [20] [21] [22] , these studies mostly discussed theoretical considerations. here we describe and demonstrate practical pooling solutions that save time and reagents by performing rna extraction and rt-pcr on pooled samples. we offer two such pooling approaches, based either on simple (dorfman) pooling or matrix pooling [23, 24] , and demonstrate their efficiency and sensitivity in the daily reality of covid-19. at the hadassah medical center (hmc), two distinct populations are tested for sars-cov-2 at present. first, we receive samples from symptomatic patients, from the hospital and from the community. in these samples, about 10% of sars-cov-2 tests are positive. second, we receive samples from prospectively screened asymptomatic populations such as hospital employees and workers in essential industries. according to the israeli ministry of health guidelines all samples were collected using a single swab for combined deep nasal and oropharyngeal collection from the same patient. nasopharyngeal swab samples were collected in 2ml viral transport medium (vtm) or collected directly to 2ml zymo lysis buffer. the first pooling strategy is a simple two-stage testing algorithm known as dorfman pooling [25] . in the first stage, the samples are divided into disjoint pools of n samples each, and each such pool is tested. a negative result implies that all samples in the pool are negative, while a positive result implies that at least one sample in the pool is positive. in the second stage, the samples of each pool that tested positive are individually tested. to reduce the need to retest positive pools we have also tested a two-stage matrix pooling strategy [23, 24] , where n 2 samples are ordered in an n x n matrix. each row and each column are pooled, resulting in 2n tests, qiasymphony dsp virus/pathogen kit on a qiasymphony platform. we pooled equal volumes of sample lysate to a final volume of 400µl. positive pools were validated by individual tests as described above. both qiagen kits were used with zymo lysis buffers, and therefore we skipped the lysis and proteinase k step. rna was eluted into 60µl; 10µl of rna was used for a 30µl reaction using real-time fluorescent rt-pcr kit (bgi). to reduce the risk of contamination, daily ultraviolet irradiation of rna extraction robots was performed, and different rooms for processing before and after pcr were set up, without mixing personnel or machines between the two compartments. note that analysis of pool results requires close attention to indeterminate-result pools, as these may contain individual positive samples. therefore, all pools detected with ct ≤ 39 were retested (see table 2 , batch 3), while maintaining standard criteria for the individual tests when retesting. we define the efficiency of a pooling algorithm as the total number of samples divided by the expected number of tests conducted on them. we assume all samples are independent and identically distributed, and denote the probability of a sample to be positive by p (prevalence of detectable covid-19 patients in the relevant population) and the pool size by n. the efficiency of the algorithms described above depends on both p and n. the best theoretical efficiency is ݈݃−( ଶ )( − (1 − ݈݃) ଶ (1 − )) ିଵ [26] . the efficiency of dorfman [25] . we chose a pool size of n=8 samples as it allows low false negative rate ( figure 1) and high efficiency for a wide range of covid-19 prevalence ( table 1 and supplementary table s1 ). the prevalence of detectable covid-19 in an asymptomatic population is estimated to be considerably below 1% [27] , and indeed of the 26,576 samples tested in the present study only 0.12% were found positive. therefore, efficiency is likely to be 5-7.5. for higher prevalence the efficiency of matrix pooling is somewhat higher (see table 1 and supplementary note). we provide a tool (https://github.com/matanseidel/pooling_optimization) to help choose the approach and pool size based on the prevalence. these studies were part of the approved diagnosis optimization and validation procedures at the hmc, and therefore no additional institutional review board approvals were required. a key requirement of pooled rna extraction and rt-pcr tests is to retain sufficient sensitivity. theoretically, this approach yielded highly accurate results, with no loss of diagnostic assay sensitivity: each of the pools that contained one or more positive samples was found to be positive, and all the pools that contained only negative samples were found to be negative (figure 1) . of the 5 pools which contained one individual sample with an "indeterminate" result (in each pool), one was found to be negative per definitions of individual tests, but still with ct < 39 that allows pool retesting. in addition, we tested matrix pooling (see methods) by pooling 75 samples into three 5 x 5 matrices, and identified all positive samples accurately (figure 2) . importantly, the positive samples were detected in both the row and the column pools at a similar cycle in all three tested matrices, suggesting the pooling scheme is robust. given the successful validation of both pooling strategies, and the low prevalence in asymptomatic population, we have adopted a dorfman pooling protocol of 1:8 and employed it for the routine testing of nasopharyngeal swab samples from screened asymptomatic healthcare personnel, employees of essential industries, and residents and employees of nursing homes. in the first three batches run at the hmc ( table 2) we demonstrate in a real-life situation the usefulness of pooled sampling starting at the early lysate stage. the simplicity of the method, similarity to currently approved procedures, and the fact that we do not require special sample handling or additional information make it easily adoptable on a large scale. this saves time, work and reagents, allowing a considerable throughput increase of clinical diagnostic labs and opening the door for efficient screening of large asymptomatic populations for the presence of sars-cov-2 infection. an important consideration before implementing group testing is the expected rate of false positive and false negative results. based on our experience with over 26,500 samples from asymptomatic individuals, we did not encounter any false positives in the pools (see figure 1 and table 2 ). false negatives are in principle more worrisome when testing in pools, because samples that failed at the rna extraction step will be missed (while our individual testing includes amplification of a human transcript serving as an internal control for proper rna extraction and rt-pcr of each sample). to define the magnitude of this potential problem, we examined a set of 13,781 individual tests done at our center, which were all expected to show a signal for a human gene serving as internal assay control. amplification of the human gene failed in 52 samples (0.38%). thus, we estimate that our current protocol of pooled sampling carries a risk of missing 0.38% of the positive samples. in a population of 1,000,000 individuals tested, of which 1,000 are positive (rate of 0.1%), this predicts that 4 positive individuals will be missed when using pools. we posit that this is a tolerable situation, particularly given the potentially much higher rates of false negative results due to swab sampling and other errors upstream. largescale implementation of the pooling scheme should be carefully done to assure pre-analytical influences (e.g. inadequate sampling, transport time, temperature influences) do not lead to significant loss of signal, which may further increase the risk of false negatives. increase in throughput applies to rna extraction and rt-pcr, but not to viral inactivation and lysis or reporting of results. reporting at the hmc is automated and was adapted to the pooling scheme and therefore does not require additional work. viral inactivation and lysis typically take 30min while rna extraction and rt-pcr 4-5 hours, and therefore 7.3-fold increase in efficiency translates to ~4.5 increase in efficiency of the entire workflow, or more if efficiency of viral inactivation is increased by other means (e.g. automation). the increase in efficiency allowed the hmc to survey healthcare personnel and multiple nursing homes, and identify a nursing home with 16 positive individuals, helping to stop the spread at that center. specifically, we have demonstrated that pooling lysates from 5 or 8 nasopharyngeal swab samples retains sufficient sensitivity of viral rna detection, allowing identification of sars-cov-2-positive individuals, while increasing throughput 5-fold to 7.5-fold. the prevalence of covid-19 in the tested population is not always known, which could affect the optimal pool size. this could be addressed either by external estimates, such as a previous run of individual samples, rate of symptomatic patients, or alternative methods such as serological screening or wastewater titers monitoring [28, 29] . alternatively, it is possible to dynamically adapt pooling sizes, when the measured rate of positive samples is different than expected. finally, some group testing algorithms (reviewed in [17] ) estimate the number of positive samples while using a relatively small (logarithmic) number of tests, and may be adapted to clinical constraints and parameters. if samples are not independent, and we have information regarding their dependency, we can further improve efficiency by grouping together dependent samples, that is, samples that are likely all positives or all negatives, such as members of the same family, or samples that are likely to be all negative since they have a low risk profile. this will increase the number of negative pools, and therefore decrease the overall number of tests conducted. future improvement of the sensitivity of the test, such as better sets of primers and improved sample collection will allow retaining sensitivity even when pooling a large number of sample lysates together. this will enable further improving efficiency, especially when prevalence is low, by increasing the pool size. all authors disclose no conflicts of interest. agnes klochendler and matan seidel contributed equally to this work. yuval dor, dana wolf, maayan salton and yotam drier contributed equally to this work maayan salton and yotam drier. acquisition, analysis, or interpretation of data writing original draft, review and editing: yuval dor, dana wolf, maayan salton and yotam drier. supervision: yuval dor, dana wolf, maayan salton and yotam drier an alternative workflow for molecular detection of sars-cov-2 -escape from the na extraction kit-shortage evaluation of covid-19 rt-qpcr test in multi-sample pools rapid molecular detection of sars-cov-2 (covid-19) virus rna using colorimetric lamp. infectious diseases (except hiv/aids) 2020 rapid and visual detection of 2019 novel coronavirus (sars-cov-2) by a reverse transcription loop-mediated isothermal amplification 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covid-19 occurrence in a low prevalence area early sars-cov-2 outbreak detection by sewage-based epidemiology key: cord-333334-90q1xkld authors: shengchen, d.; gu, x.; fan, g.; sun, r.; wang, y.; yu, d.; li, h.; zhou, f.; xiong, z.; lu, b.; zhu, g.; cao, b. title: evaluation of a molecular point-of-care testing for viral and atypical pathogens on intravenous antibiotic duration in hospitalized adults with lower respiratory tract infection: a randomized clinical trial date: 2019-06-20 journal: clin microbiol infect doi: 10.1016/j.cmi.2019.06.012 sha: doc_id: 333334 cord_uid: 90q1xkld objectives: the primary objective was to evaluate whether a molecular point-of-care test (poct) for viral and atypical pathogens added to routine real-time pcr could reduce duration of intravenous antibiotics in hospitalized patients with lower respiratory tract infection (lrti) compared with routine real-time pcr. methods: in this single-centre, open-label, randomized controlled study, we enrolled hospitalized adults diagnosed with lrti. patients were randomized to an intervention group (poct filmarray panel for 20 viruses, atypical pathogens and bacteria plus routine real-time pcr) or a control group (routine real-time pcr for ten pathogens). the primary outcome was duration of intravenous antibiotics during hospitalization. the secondary outcomes included length of stay, cost of hospitalization and de-escalation within 72 hours and between 72 hours and 7 days. intention-to-treat analysis was used. results: between october 2017 and july 2018, we enrolled 800 eligible patients (398 in the intervention group and 402 in the control group). duration of intravenous antibiotics in the intervention group was shorter than in the control (7.0 days (interquartile range (iqr) 5.0–9.0) versus 8.0 days (iqr 6.0–11.0); p <0.001). length of hospital stay in the intervention group was significantly shorter (8.0 days (iqr 7.0–11.0) versus 9.0 days (iqr 7.0–12.0; p <0.001) and the cost of hospitalization in the intervention group was significantly lower ($1804.7 (iqr 1298.4–2633.8) versus $2042.5 (iqr 1427.4–2926.2); p 0.002) than control group. more patients in the intervention group achieved de-escalation within 72 hours (7.9%, 29/367 versus 3.2%, 12/377; p 0.005) and between 72 hours and 7 days (29.7%, 109/367 versus 22.0%, 83/377; p 0.024). conclusions: use of molecular poct testing for respiratory viruses and atypical pathogens might help to reduce intravenous antibiotic use in hospitalized lrti patients. clinical trial registration: clinicaltrials.gov identifier: nct03391076. lower respiratory tract infection (lrti) is the leading infectious disease in the world [1] . it is also the fourth commonest cause of death globally, accounting for about 3.0 million deaths worldwide in 2016 [2] . viral infection is one of the most important causes of lrti [3] . because of large overlap in symptoms and clinical presentation between bacterial and viral lrti, antibiotics are inappropriately prescribed to patients with viral infection. this may result in potential risks of antimicrobial resistance with a corresponding financial burden and environmental pollution [4] . furthermore, inappropriate prescription of antibiotics is even more critical in china, which ranks as the world's most frequent user of antibiotics [5, 6] . overuse of intravenous antibiotics in patients hospitalized with lrti constitutes an important part of the inappropriate prescription of antibiotics [7] . in one retrospective study in a teaching hospital in beijing [8] , the median duration of intravenous antibiotics was 10 days (interquartile range 8e14 days) among hospitalized individuals with mild to moderate communityacquired pneumonia (cap). diagnostic uncertainty regarding the lack of microbiological evidence may be one of the most important reasons. laboratory-developed pcr testing is highly accurate for the diagnosis of microbial aetiology, with the turnaround time generally being 1e2 days [9e11]. however, experienced specialists are required for this test and the instruments have to be installed in a central laboratory. filmarray respiratory panel (biofire; salt lake city, ut, usa) is a new molecular point-of-care test (poct) platform, which can simultaneously detect 20 viruses and atypical pathogens and provide results in about 1 hour [12, 13] . the sensitivity and specificity of this new molecular poct for detecting pathogens were high, with the sensitivity and specificity ranging from 92.3% to 97.9% and 96.1% to 99.1%, respectively [12, 13] . a recently published randomized controlled trial showed that use of the filmarray respiratory panel was associated with reduction in proportion of antibiotic use among patients with acute exacerbation of chronic obstructive pulmonary disease (aecopd) and asthma [9] . we speculated that molecular poct for the detection of viruses might reduce the duration of antibiotic use in adult lrti patients [14, 15] . considering predominant overuse of intravenous antibiotics in china, the aim of this study was to evaluate whether combination of poct and routine real-time pcr for pathogen detection could reduce duration and improve deescalation of intravenous antibiotics in individuals with lrti compared with routine real-time pcr only. this was a single-centre, open-label, parallel randomized controlled study that took place between october 2017 and july 2018 in the chinaejapan friendship hospital (cjfh), beijing, china (clinicaltrials.gov identifier: nct03391076). cjfh is a large teaching hospital with 1600 beds. patients were recruited from the general ward of the department of pulmonary and critical care medicine, department of traditional chinese medicine lung disease and department of infectious disease in cjfh. hospitalized patients aged !18 years who were preliminarily diagnosed as radiographically confirmed cap, aecopd or acute exacerbation of bronchiectasis were recruited on the day of hospitalization. patients were excluded if they were <18 years old, pregnant, had hospitalacquired pneumonia, or lung tuberculosis. we also excluded patients with human immunodeficiency virus infection, haematological cancer or solid tumour treated with chemotherapy or radiotherapy in the previous 3 months, organ or bone marrow transplantation, splenectomy, or autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, rheumatic polymyalgia and interstitial lung disease treated with immunosuppressive therapy for >3 weeks. in addition, patients with any other condition that may have increased serum procalcitonin levels, including severe burns, major surgical procedures, major trauma, long-term or severe cardiogenic shock, invasive fungal infection, or an acute attack of plasmodium falciparum, were also excluded. this study was conducted in accordance with the declaration of helsinki. the study was approved by the ethics committee of cjfh (2017-29). written informed consent was obtained from each participant after meeting inclusion criteria and before randomization. random allocation sequence was generated using spss 22.0 software (statistical product and service solutions, ibm co. ltd, armonk, ny, usa) with a fixed random seed. simple randomization was conducted subsequently by sealing the group allocation cards into envelopes according to the sequence number. each envelope was opened only when patients met inclusion criteria and signed informed consent, with allocation of patients to intervention or control group accordingly. study participants, research staff and clinical care providers were not blinded to the group allocations. allocation of patients was blinded to data analysts. demographics and clinical characteristics were collected on enrolment. before the study commenced, research personnel were trained on how to take nasopharyngeal swabs and how to operate the filmarray respiratory panel instrument. routine diagnosis, treatment and microorganism detection of cap, aecopd and acute exacerbation of bronchiectasis followed chinese guidelines and consensus for these diseases [16e18]. in the intervention group, research staff took nasopharyngeal swabs from patients according to standard protocols within 4 hours of admission. the samples were analysed immediately using the filmarray respiratory panel. the panel can detect 17 viruses (influenza a (h1 and h3) virus, influenza b virus, respiratory syncytial virus, rhinovirus or enterovirus, human metapneumovirus, parainfluenza virus types 1 4, coronaviruses (oc43, 229e, hku1 and nl63) and adenovirus), two atypical pathogens (chlamydia pneumoniae and mycoplasma pneumoniae) and one bacterium (bordetella pertussis). the results were reported and explained to physicians via telephone, sending text messages (with mandatory feedback) or face-to-face communication on the day of admission. in both the intervention and control groups, routine real-time pcr assays for the detection of viral pathogens (including influenza a (h1n1, h7n9) virus, influenza b virus, respiratory syncytial virus, parainfluenza virus, adenovirus, epsteinebarr virus, herpes simplex virus and human cytomegalovirus) were performed in the cjfh microbiology laboratory with sputum or nasopharyngeal swab samples (see supplementary material, table s1 ). the results were reported and explained to physicians once obtained. other diagnostic tests such as blood gas analysis, c reactive protein, erythrocyte sedimentation rate, procalcitonin and routine microbiological testing were prescribed by physicians in both groups. the responsible attending physicians decided on antibiotic administration (including moxifloxacin, levofloxacin, types i/ii/iii/ iv generation cephalosporin, carbapenem, b-lactamase/b-lactamase-inhibitors, macrolide, penicillins, tetracycline), de-escalation or cessation of use in both groups without intervention of the research staff. management system and technical support framework for antimicrobial stewardship have been established in china. the medical department and pharmacy department regularly assess of the use of antibiotics in cjfh. all data were collected on a standard case report form and then input into an electronic medical database by an authorized assistant. the primary outcome was the duration of intravenous antibiotics during hospitalization. duration of intravenous antibiotics was defined as the total number of calendar days when one or more than one dose of intravenous antibiotics was used. the secondary outcomes included the proportion of patients who received intravenous antibiotics, the proportion of patients with antibiotics deescalation within the first 72 hours and between 72 hours and 7 days, length of hospital stay, cost of intravenous antibiotics and cost of hospitalization. de-escalation of antibiotics was defined as reduction of antibiotic types, change from intravenous antibiotics to oral antibiotics, or from broad-spectrum antibiotics to narrowerspectrum antibiotics (see supplementary material, table s2 ). cost of hospitalization from the perspective of the hospital before deduction of benefits consist of six parts, including laboratory test (radiation, pathology and blood biochemistry test), medical care (oxygen therapy and doctor visit), surgery, blood storage or processing, drug and other (such as medical material) costs (see supplementary material, table s3 ). all outcomes were measured until discharge from hospital. participants were followed up in person at day 30 by trained research staff if the length of hospitalization was <30 days, with six participants in the intervention group and eight participants in the control group lost to follow up. adverse outcomes included admission to intensive care unit (icu), death during hospitalization, readmission within 30 days, and death within 30 days. according to findings of branche et al. [14] , we assumed that a 1day reduction of antibiotics use in the intervention group would be clinically significant. we estimated that 340 patients would be required in each group to yield a statistical power of 80% to detect a 1-day reduction in antibiotic use in the intervention group at a significance level of p 0.05. we further assumed that 10%e15% of the study participants would be non-adherent or lost to follow up and set a total target recruitment number of 400 patients in each group. data analyses were performed according to intention-to-treat analysis. per-protocol analysis by excluding participants whose diagnosis was ascertained not to be lrti after randomization or who were withdrawn for refusing nasopharyngeal swab was also performed. baseline characteristics were expressed as numbers (proportion), median (interquartile range) or mean ± standard deviation and comparisons were made using the c 2 test, wilcoxon rank-sum test or student's t-test where appropriate. the median and interquartile range of the primary outcome (duration of intravenous antibiotics) and secondary outcomes, including length of hospital stay, cost of intravenous antibiotics and cost of hospitalization, were calculated and the difference between intervention and control group was compared using the wilcoxon rank-sum test. for other secondary outcomes (proportion of intravenous antibiotic use, de-escalation within the first 72 hours and de-escalation between 72 hours and 7 days), any significant differences in the proportions calculated with the c 2 test and unadjusted odds ratios calculated with a logistic regression model were used to look for differences between the intervention and control groups. differences and 95% cis involved in this study were absolute differences expressed as means or proportions. data analyses were performed using sas version 9.4 (sas institute inc.,). between 16 october 2017 and 13 july 2018, we assessed 919 patients for eligibility, and 800 of them were eligible to participate in the study (fig. 1) . a total of 398 patients were randomly assigned ae, acute exacerbation; aecopd, acute exacerbation of chronic obstructive pulmonary disease; cap, community-acquired pneumonia; curb-65, a pneumonia severity score calculator (measured by 5 risk factors in total, with 1 point for each criterion satisfied: confusion defined as an abbreviated mental test score 8; blood urea nitrogen ! 7mmol/l; respiratory rate !30 bpm; systolic blood pressure < 90 mmhg or diastolic blood pressure 60 mmhg; age !65 years). data are presented as mean ± sd (standard deviation) or as median (interquartile range) for continuous variables and as percent for categorical variables. categorical variables were compared using c 2 tests, and continuous variables were compared using wilcoxon rank-sum test or student's t-test. a the denominator is the number of community-acquired pneumonia. b the denominator is the number of study participants that received the procalcitonin test. to the intervention group and 402 to the control group and included in the intention-to-treat analysis, with 15 patients in the intervention group and 24 patients in the control group who were ascertained not to have lrti after randomization or who were withdrawn for refusing nasopharyngeal swab. baseline characteristics of study participants in intervention and the control group included in intention-to-treat analysis and perprotocol are shown in table 1 and the supplementary material (table s4) , respectively. all patients in the intervention group were tested using the filmarray respiratory panel and 46.0% (183/398) of patients were also tested using routine real-time pcr, whereas 47.8% (192/402) of patients in the control group were tested using routine real-time pcr for viral pathogens (see supplementary material, tables s1 and s5). the median duration of intravenous antibiotic treatment in the intervention group was significantly shorter than in the control group (7.0 days (5.0e9.0 days) versus 8.0 days (6.0e11.0 days); difference e1.5 days, 95% ci e2.1 to e0.8 days; p <0.001) ( table 2 ). the proportion of participants who were given intravenous antibiotics was high in both the intervention and control groups, but with no significant differences observed between the two groups (92.1%, 367/398 versus 93.8%, 377/402; difference e1.6%, 95% ci e5.1% to 2.0%; p 0.38). patients in the intervention group stopped having intravenous antibiotics earlier compared with the control group (p < 0.001 for log-rank test) (fig. 2) . eight patients in the intervention group stopped intravenous antibiotic use on the same day as receiving the poct test result (data not shown). more patients in the intervention group achieved de-escalation within 72 hours (7.9%, 29/367 versus 3.2%, 12/377; difference 4.7%, 95% ci 1.4%e8.0%; p 0.005) and between 72 hours and 7 days (29.7%, 109/367 versus 22.0%, 83/377; difference 7.7%, 95% ci 1.4% to 14.0%; p 0.024) than in the control group. the per-protocol analysis also showed shorter intravenous antibiotic treatment and earlier de-escalation of intravenous antibiotics in the intervention group compared with the control group. the median length of hospital stay in the intervention group was significantly shorter than in the control group (8.0 days (7.0e11.0 days) versus 9.0 days (7.0e12.0 days)]; difference e1.0 days, 95% ci e1.6 to e0.4 days; p <0.001). the median cost of intravenous antibiotics and hospitalization were also significantly less in the intervention group ($189.9 (103.5e316.5) versus $245.8 (138.1e397.8); p <0.001 and $1804.7 (1298.4e2633.8) versus $2042.5 (1427.4e2926.2); p 0.002). the per-protocol analysis also showed shorter length of hospital stay and lower cost of intravenous antibiotics and hospitalization in the intervention group compared with the control group. the proportions of adverse outcomes, including icu admission, death during hospitalization, readmission within 30 days, and death within 30 days were not found to be significantly different between intervention and control groups (all p !0.05) ( table 3 ). in our study adding poct to routine real-time pcr testing shortened the duration of intravenous antibiotic treatment, reduced the length of stay and cost of hospitalization, and improved early de-escalation of intravenous antibiotics compared with routine real-time pcr assays in hospitalized lrti patients. admission to the icu and fatality rates were similar between groups. the efficacy of this new poct has been evaluated in retrospective studies [15,19e21] , but most of them were conducted among paediatric patients [19e21] . a few randomized controlled trials have explored the impact of poct among adults, but with small samples [10, 22] . one recent randomized controlled trial with a relatively large sample size evaluated the effect of the filmarray respiratory panel among adult patients with acute respiratory illness [9] . however, only half of them were diagnosed with lrti, including pneumonia and aecopd. the heterogeneity of the study population, including asthma and upper respiratory infections, limited extrapolation to lrti. the advantage of our study is that only individuals with lrti were enrolled, excluding acute upper respiratory infection and non-infectious respiratory illness. another advantage of our study is that we enrolled patients throughout four consecutive seasons. in this study, we focused on duration of antibiotics, but not on withdrawal of antibiotics once the poct result was available. it is easy to understand that physicians can safely stop antibiotics as soon as they know the positive viral results for patients with upper respiratory infections or asthma, as demonstrated in the study by brendish et al. [9] . however, for patients with pneumonia and other kinds of lrti, most chinese physicians refer to local clinical guidelines for duration of antibiotics, such as 5e7 days for cap [16] , 10e14 days for cap with atypical pathogen [16] , 5e10 days for aecopd [17] and 14 days for acute bronchiectasis [18] . although it is difficult to exclude bacterial co-infection, the knowledge of viral aetiology will help physicians to decide whether to stop intravenous antibiotics earlier [10] . considering that length of hospital stay for lrti patients was directly related to duration of intravenous antibiotics, we finally chose duration of intravenous antibiotics as the primary outcome. with the median cost around $225 per hospital-day for patients with lrti, 1 day less of intravenous antibiotics and 1 day less of hospitalization could save billions of dollars in china. there are a number of limitations in our study. first, it was a single-centre study, the results of which need to be verified by future multicentre studies. second, the cost of the filmarray respiratory panel was not considered in the total costs of hospitalization since the filmarray respiratory panel is not commercially available in china. our post-hoc analysis indicated that the cost during hospitalization in the intervention group would be lower than, or at least equal to, that in the control group if the filmarray respiratory panel test cost less than $360. third, because the proportion of patients who received intravenous antibiotic therapy was high and duration of intravenous antibiotics was relatively long in both groups, extrapolation of our results should be carefully interpreted. fourth, the study was conducted in general wards, data are presented as per cent for categorical variables. difference between intervention and control group was compared using c 2 tests and logistic regression model was used to calculate unadjusted odds ratios. a the denominator is the number of study participants that were followed up at day 30. without including patients from icus. the effect of poct needs further rigorous evaluation in patients who are more severely ill, including icu patients. in conclusion, this study found the addition of molecular poct testing to routine real-time pcr testing for respiratory viruses and atypical pathogens might help to reduce intravenous antibiotic use in lrti patients without resulting in adverse outcomes. more multicentre studies will be required to verify these findings. we declare that we have no competing interests. the content of the manuscript has not been published, or submitted for publication elsewhere. the abstract has been accepted for presentation at the 2019 ats international conference. this work was supported by the national science grant for distinguished young scholars (grant number 81425001/h0104) and chinese academy of medical science innovation fund for medical sciences (2018-i2m-1-003). biom erieux provided, free-ofcharge, filmarray panel punches and filmarray instruments. they did not participate in the design, conduction and analysis of the study. bc conceived and designed the trial, supervised the trial and allocated staff, had full access to all of the data in the study and takes responsibility for the content of the manuscript. sd and dy participated in the recruitment of patients and data acquisition. the randomization of participants into intervention or control group according to random sequence number was done by rs, and allocation of patients was finished by hl and yw. xg and gf performed the data analysis. sd, bc and xg drafted and revised the manuscript, and zx and bl ensured the quality control and running environment of the filmarray instrument. all authors reviewed the manuscript and contributed to the study report during the whole progress. all authors approved the final version of the manuscript. world health report: the ten most common infections fact sheet 310: the top 10 causes of death available at: www.who.int/influenza/patient_care/clinical/brave/en impact of antibacterials on subsequent resistance and clinical outcomes in adult patients with viral pneumonia: an opportunity for stewardship bacterial resistance: challenge and strategies deadly sins of antibiotic abuse in china comprehensive evaluation of antibiotics emission and fate in the river basins of china: source analysis, multimedia modeling, and linkage to bacterial resistance potential for cost-savings in the care of hospitalized lowrisk community-acquired pneumonia patients in china routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial the clinical impact of the detection of potential etiologic pathogens of community-acquired pneumonia multiplex pcr point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-randomised study assessing impact on length of stay and antimicrobial use fil-marray, an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection comparison of the biofire filmarray respiratory panel, seegene anyplexii rv16, and argene for the detection of respiratory viruses serum procalcitonin measurement and viral testing to guide antibiotic use for respiratory infections in hospitalized adults: a randomized controlled trial impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients diagnosis and treatment of community-acquired pneumonia in adults: 2016 clinical practice guidelines by the chinese thoracic society, chinese medical association expert consensus on acute exacerbation of chronic obstructive pulmonary disease in the people's republic of china impact of multiplex polymerase chain reaction testing for respiratory pathogens on healthcare resource utilization for pediatric inpatients impact of a rapid respiratory panel test on patient outcomes the rapid diagnosis of viral respiratory tract infections and its impact on antimicrobial stewardship programs the potential of molecular diagnostics and serum procalcitonin levels to change the antibiotic management of community-acquired pneumonia we thank all the patients and clinical staff in the general wards of the respiratory critical medical department, traditional chinese medicine lung disease department, infectious disease department and laboratory of clinical microbiology and infectious diseases in the chinaejapan friendship hospital, including physicians, nurses and laboratory technicians. supplementary data to this article can be found online at https://doi.org/10.1016/j.cmi.2019.06.012. key: cord-252569-9rv1p3qh authors: zanella, m.-c.; lenggenhager, l.; schrenzel, j.; cordey, s.; kaiser, l. title: high-throughput sequencing for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis. a narrative review and clinical appraisal date: 2019-01-11 journal: clin microbiol infect doi: 10.1016/j.cmi.2018.12.022 sha: doc_id: 252569 cord_uid: 9rv1p3qh background: viral aetiologies are the most common cause of central nervous system (cns) infections. approximately one-half of cns infections remain of undetermined origin. high-throughput sequencing (hts) brought new perspectives to cns infection investigations, allowing investigation of viral aetiologies with an unbiased approach. hts use is still limited to specific clinical situations. objectives: the aim of this review was to evaluate the contribution and pitfalls of hts for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis in cns patient samples. sources: pubmed was searched from 1 january 2008 to 2 august 2018 to retrieve available studies on the topic. additional publications were included from a review of full-text sources. content: among 366 studies retrieved, 29 used hts as a diagnostic technique. hts was performed in cerebrospinal fluid and brain biopsy samples of 307 patients, including immunocompromised, immunocompetent paediatric, and adult cases. hts was performed retrospectively in 18 studies and prospectively in 11. hts led to the identification of a potential causal virus in 41 patients, with 11 viruses known and ten not expected to cause cns infections. various hts protocols were used. implications: the additional value of hts is difficult to quantify because of various biases. nevertheless, hts led to the identification of a viral cause in 13% of encephalitis, meningoencephalitis, and meningitis cases in which various assays failed to identify the cause. hts should be considered early in clinical management as a complement to routine assays. standardized strategies and systematic studies are needed for the integration of hts in clinical management. meningitis, encephalitis, and meningoencephalitis are caused by various pathogens, but viral aetiologies are the most common cause [1e4] . among these, enterovirus (ev), herpes simplex type 1 and 2 (hsv-1 and hsv-2), and varicella zoster virus (vzv) are the most frequent viruses associated with encephalitis, meningoencephalitis, and meningitis in paediatric and adult populations [1,2,4e7] . the prevalence of other viruses varies according to the geographical location and immune status of the patient. during the last two decades, the implementation of molecular assays as a complement to serological assays, immunohistochemistry, and culture have improved the diagnosis of viral central nervous system (cns) infections. nevertheless, these assays have limitations because of their targeted approach. apart from technical limitations, the diagnosis of viral cns infections is subject to several issues, such as the type of sample (cerebrospinal fluid (csf) or brain biopsy), the timeline of sample collection, and the different pathogenic mechanisms of viruses. despite technical progress, approximately one-half of encephalitis, meningoencephalitis, and meningitis cases remain of unknown origin [1, 2, 5, 7] . high-throughput sequencing (hts) has brought new perspectives to cns infection investigations. although hts has been recently integrated in encephalitis management guidelines [8] , its use is still limited to specific clinical situations or research. the contribution of hts warrants a better appraisal for further implementation in cns infection management. this narrative review aims to evaluate the contribution and pitfalls of hts for the aetiologic identification of viral encephalitis, meningoencephalitis, and encephalitis in paediatric and adult patients. a comprehensive pubmed search was conducted from 1 january 2008 to 2 august 2018 to identify human studies using the following mesh and keywords research algorithm: '((central nervous system infection or cerebrospinal fluid or central nervous system) and sequencing) and virus'. additional publications were identified from a review of full-text sources. the title and abstract of each citation were screened by two reviewers and assessed for eligibility by detailed analysis. inclusion criteria were studies including patients with encephalitis, meningoencephalitis, or meningitis of unknown origin and reporting the use of hts for the aetiologic identification of a viral origin in cns samples. exclusion criteria were reviews, animal studies, other cns diseases, and studies addressing only technical aspects. a total of 366 references were retrieved (fig. 1 twenty-nine studies were selected for qualitative analysis (19 case reports, ten case series) ( table 1) . fourteen and 13 studies concerned paediatric and adult cases, respectively, and two studies concerned both populations (table 1) . hts was performed in 307 cases (52 adults; 123 paediatric (<18 years); 132 cases with no information on age). twenty-five studies reported patient age (median age, 14 years; range, 3 months to 68 years). diagnostic criteria for encephalitis, meningoencephalitis, and meningitis were inconsistently reported. immune status was reported for 60 patients and comprised 20 immunocompromised (nine paediatric, 11 adults) and 40 immunocompetent patients (22 paediatric, eight adults). studies came from a wide range of regions: europe (ten), north america (ten), asia (six), and oceania (three) ( table 1) . when brain specimens were available, pathological examination provided proof of diagnosis of encephalitis. csf analysis results were reported in 25 patients of 20 studies, with the white blood cell count ranging from 1 to 494 cell/mm 3 in encephalitis and meningoencephalitis cases and 915 cell/mm 3 in the only meningitis case [22] ; three publications reported normal csf analysis without any description [23e25]. hts was performed on individual csf and brain specimens in 129 and 21 patients, respectively. csf samples of 162 patients were pooled for hts analysis [26e28]. hts was performed on csf and brain specimens in five patients [26,29e32] . positive results on both samples were obtained in a cache valley virus chronic meningoencephalitis case [30] and positive results on brain biopsies only were reported in two human astrovirus (hastv)-va1 and tick-borne encephalitis (tbev) cases [29, 33] . in one patient, hts analysis did not identify a viral cause, but balamuthia mandrillaris was identified in both csf and brain biopsy [32] . despite limitations because of publication bias, the overall diagnostic yield for a viral aetiology according to sample type was estimated to be higher for brain specimens (16/21 (76.2%) positive samples) than for csf samples (26/291 (8.9%) positive samples). detailed microbiological investigations performed prior to hts varied according to local practice and were not reported in seven studies [24,34e39] . in most studies, viruses identified with hts were not part of the microbiological work-up, except in three cases where hts identified a virus for which diagnostic assays were negative during routine investigation. these included a west nile virus (wnv) identified in the csf sample of a renal transplant recipient with meningoencephalitis and a negative serological assay [40] . hsv-1 was identified in an encephalitis case [35] . a vaccine strain of mumps virus was identified in a brain specimen of an immunosuppressed child with chronic encephalitis in whom (rt)-pcr for mumps on a csf sample was negative as the assay did not target vaccine strains [41] . hts was performed retrospectively in 18 studies and prospectively as part of the initial work-up in 11 case reports with an impact on the clinical management of three immunocompromised patients: a child with encephalitis associated with hastv-va1 [23] ; an adult with encephalitis associated with hastv-va1 [33] ; and an adult with chronic meningoencephalitis associated with cache valley virus [30] . turnaround times were reported in ten studies [23,31,33,36,39,40,42e45 ]. among publications in which hts was used prospectively, turnaround times ranged from 48 hours to 7 days [23, 33, 39, 40, 42, 44, 45] . hts performed on pooled or individual samples and/or subsequent confirmatory assays allowed the identification of a potential causal virus in 41 of 307 patients (13.4%), comprising 15 paediatric cases (eight immunocompromised cases), 17 adult cases (nine immunocompromised cases) and nine cases for whom age was not specified; median age was 21 years (range 3 months to 68 years). fig. 2 shows the distribution of viruses identified according to patient immune status and clinical manifestations. hts allowed the identification of viruses previously unknown or unexpected as a cause of cns infection (n ¼ 10) and thus not screened during diagnostic investigations (table 1) . these included parvovirus 4 (two) [46] , human coronavirus oc43 (one) [25] , and novel hastv-mlb2 (one) [22] identified in immunocompetent patients. a mumps virus vaccine strain (one) [41] , hastv (undetermined specie; one) [47] , hastv-va1 (four) [23, 31, 33, 43] , and hastv-mlb1 (one) [24] were identified in immunocompromised patients. a gemycircularvirus was also identified, but its causal role in encephalitis is under debate [27] . three novel viral species or strains were identified in csf samples of patients with encephalitis (table 1) : human csf-associated densovirus 1 (hucsfdv1) [37] ; cyclovirus viet-nam (cycv-vn) [28] ; and lymphocytic choriomeningitis virus (lcmv)-related arenavirus [26] . hts analysis also identified viruses known to be responsible for cns infections (n ¼ 11) and not screened or detected by routine assays (hsv-1, hsv-2, vzv, epsteinebarr virus (ebv), tbev, wnv, cache valley, saint louis encephalitis, toscana, mumps, measles, and coxsackie a9 virus) [29, 30, 34, 35, 39, 40, 42, 48, 49] (table 1) . most studies performed nucleic acid extraction protocols dedicated to rna, or rna and dna. thirteen rna and seven dna viral species were identified (table 1) . one study reported the identification of hsv-1 in a csf sample after rna extraction protocol [35] . six studies where hts analysis was not restricted to the detection of viruses resulted in the identification of bacterial (brucella melitensis and leptospira santarosai) [44, 50] , mycobacterial (mycobacterium tuberculosis) [39] , and parasitic (balamuthia mandrillaris) [32, 45] or fungal (candida tropicalis and fusarium solani and oxysporum) [38] pathogens. the use of controls was not systematically reported. nine studies reported various negative control samples, such as brain specimens without encephalitis [34] , csf samples from patients without infection [30, 32, 35, 38, 40, 45] , serum samples, and water or elution buffer [30, 44, 45, 50] . viral sequences of negative controls were not consistently described. positive controls, such as csf or serum samples positive for dna or rna viruses, were rarely reported or used [33, 35, 38, 48, 50] . to address the specificity of hts results, other techniques were performed to confirm hts results in all studies, except one [49] . (rt)-pcr assays were performed in samples from 37 patients and were positive in at least one sample in 36 patients. hts results were also confirmed with serological assays [26, 40, 41, 46] , immunohistochemistry, and in situ hybridization [25, 30, 31, 33, 39, 41, 47] . viral culture confirmed the presence of a replicative saint louis encephalitis virus in a csf sample [42] , but was unsuccessful concerning a cache valley virus [30] . hts offers the possibility of investigating viral aetiologies of cns infections by an unbiased approach when work-up according to guidelines fails to identify a causal pathogen. based on the studies retrieved, its diagnostic yield for a viral aetiology is difficult to estimate, particularly because of publication bias (high number of case reports), methodological heterogeneity, and a lack of systematic prospective studies. when focusing specifically on case series, the diagnostic yield for the identification of a viral cause was approximately 10%, but this result should be interpreted with caution in the light of the evolution of the technique from 2008 to 2017. among the studies reviewed, the hts contribution is evident not only for the identification of a potential causal virus in cns infections of unknown origin, but also in the detection of novel or divergent viruses [26, 28, 37] . similar to other techniques, the type of sample used for analysis is of particular importance. despite diverse hts protocols and publication bias, hts seemed to have a higher diagnostic yield in brain specimens than in csf samples. the diagnostic yield was particularly low in two studies where hts was performed on csf supernatant [27, 28] . hts also shows its clinical value in situations where the viral pathogenic mechanisms and specific clinical situations impairs the results of conventional assays [40] . among immunocompetent patients, hts led to the identification of viruses not previously associated with cns infections (parvovirus 4, cycv-vn, gemycircularvirus, and novel hastv-mlb2) [22, 27, 28, 46] . hts clinical impact was mainly demonstrated among immunocompromised patients, with most studies dedicated to this population. it was performed prospectively in 11 cases and led to a change in clinical management in three [23, 30, 33] . the rapid decision to perform hts, short hts turnaround times, and the efficient interpretation of results were determinant for the management of these latter patients. among immunocompromised patients, hts contributed to the detection of viruses for which no assay was performed during conventional work-up: viruses known to cause cns infections (tbev, wnv, cache valley virus, saint-louis encephalitis virus, ebv) [29, 30, 39, 40, 42] , viruses not known to be responsible for cns infections (novel hastv-va1, hastv-mlb1, human coronavirus oc43, mumps virus vaccine strain) [23e25, 31, 33, 41, 43, 47] , and novel viruses (lcmv-related arenavirus) [26] . focusing on novel hastv, hts brought new insights in our understanding of their association with cns infections [22e24, 31, 33, 43] . furthermore, all (rt)-pcr assays performed retrospectively confirmed hts results, thus highlighting the specificity of hts. in the absence of standardization, the methodological heterogeneity of studies is striking, not only concerning pre-analytic steps, but also hts per se, with the use of diverse hts platforms, single or paired protocols, as well as diverse bioinformatic pipelines and databases. the use of positive controls as quality controls was only reported in seven studies [31,33e35,38,48,50] . addressing the issue of contamination, only a few studies reported the use of negative controls [30, 32, 34, 35, 38, 40, 44, 45, 50] . viral sequences assigned to viruses not considered as the cause of cns infection were not consistently performed: 12 studies provided a description for one cns sample or more [25,27,29,30,32e35,38,40,44,50] . for most of these viral sequences, no interpretation of results was explicitly provided. among reads of viruses known to cause infections in humans, anelloviridae [51] and herpesviridae were the most described in four and seven samples, respectively; human pegivirus reads were identified in one sample. the genome of the torque teno virus, a member of the anelloviridae family, and human pegivirus have been identified in cns samples, but without any association with a cns disease so far [52e57] . other viral sequences were mostly assigned to viruses infecting plants or non-vertebrates and were considered to be reagent contaminants. the minimal description of these hts 'background' results impairs the comprehension of the composition of the cns virome. furthermore, the integration of hts results in the clinical context is of particular importance and the absence of standardization of any reporting methods precludes an objective interpretation of these results. finally, hts-negative results could be interpreted in the context of several clinical and technical aspects that could impact on the sensitivity of the method. first, from a clinical point of view, differences in diagnostic yield from a biopsy compared with csf samples could be explained by several factors: patient selection (cases of encephalitis); the type of sample (e.g. multiple pooled post-mortem brain samples); and the timeline of sampling in the context of encephalitis (biopsy positivity could possibly be less affected by time than csf). from a technical point of view, it should be considered that this narrative review includes studies from 2008 to 2018 and thus takes into account the tremendous evolution of the hts technique over this last decade. several technical issues need to be considered for the interpretation of negative results: pre-analytic steps (e.g. the use of fresh, frozen, or paraffinembedded samples for analysis, extraction protocols, fragmentation methods, library preparation, paired-end versus single-end protocols); sequencing depth; sequencing platforms (table 1) ; and the analysis of hts raw data (e.g. mapping software, viral databases, and pipeline precision). this review highlights that the use of hts in investigations concerning a viral cause of encephalitis, meningoencephalitis, and meningitis could extend not only to immunocompromised, but also to immunocompetent patients. considering the selection and publication bias of the literature reviewed here, the negative predictive value for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis is difficult to quantify and further studies are needed. hts needs to be integrated in clinical management as a second-line technique or in parallel to first-line investigations when a standard work-up according to guidelines [8, 58] and additional investigations considering local epidemiology and specific clinical situations fail to identify a causal agent. brain biopsy should also be considered. furthermore, hts is of particular interest for the screening of a large panel of viruses, particularly to avoid a restricted screening of low-volume clinical samples, such as in paediatric patients. hts brought new perspectives to the investigations of infectious diseases. notably, its unbiased approach is of particular interest in samples that would not usually be tested in specific syndromes. its use may not only be restricted to cns samples, but also extended to other clinical samples. this is illustrated by the positive results of (rt)-pcr assays performed on blood or plasma samples collected at the time of neurological manifestations, which allowed the identification of the same virus detected by hts in cns samples (parvovirus 4, lcmv-related arenavirus, novel hastv-va1, novel hastv-mlb2) [22, 26, 31, 46] . this could be of particular interest when a cerebral biopsy cannot be performed and a disseminated infection occurs or is suspected, particularly in immunocompromised patients. an early decision to perform hts, short hts turnaround times, and an efficient interpretation of results are major issues for allowing hts to contribute to clinical management. for prospective hts use in clinical routine, this timeframe should be as short as possible for clinical decision-making. among publications in which hts was used prospectively, reported turnaround times ranged from 48 hours to 7 days [23, 33, 39, 40, 42, 44, 45] . hts use is still restricted to a limited number of diagnostic laboratories considering the cost of analysis and informatics infrastructures needed (e.g. costs of sequencing platforms, computing resources, data storage). despite the expanding use of hts in clinical microbiology, the surprisingly low number of studies retrieved for this review might be explained for several reasons, including financial limitations when considering the costs of the analysis, the need for shorter turnaround times, and the limitations cited above. addressing the question of the proof of causality, particularly in the context of pathogen discovery, lipkin proposed several criteria for pathogen causality with grading certainty according to confirmation with serological assays or culture for instance [59] . most studies confirmed hts results with (rt)-pcr assays and a few with cell culture, serological assays, and immunohistochemistry. thus, hts should be implemented in clinical routine in association with other diagnostic tests. in most studies, the approach to establish causality was not explicitly described, but was reported as the temporal association of clinical manifestations and the identification of viral sequences of a specific virus using hts on cns samples at the time of manifestations. this process was only described in few studies. similar to other molecular tests such as (rt)-pcr, the detection of viral sequences or genome in a clinical sample should be interpreted with caution in the clinical context. in a near future, the process for the establishment of causality in hts analysis should be more transparent and should comprise multidisciplinary sessions involving infectious disease specialists and bioinformatics experts, not only for results concerning viruses unexpected to cause cns infections. finally, for hts implementation in clinical routine, the question of standardization has to be addressed concerning hts protocols, data analysis algorithms, reference databases and quality controls, and further prospective studies are needed [60] . this review shows that hts contributed to the identification of potential viral aetiologies of encephalitis, meningoencephalitis, and meningitis of unknown origin in approximately 13% of cases and is of particular interest in immunocompromised patients. this unbiased or semi-unbiased approach led to the identification of novel viruses, viruses known or not expected to cause cns infections. standardized strategies are needed for the further implementation of hts in clinical management. in centres where available, the decision 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management of encephalitis: clinical practice guidelines by the infectious diseases society of america the changing face of pathogen discovery and surveillance validation of metagenomic next-generation sequencing tests for universal pathogen detection the authors would like to acknowledge rosemary sudan (geneva university hospitals, switzerland) for editorial assistance. key: cord-300023-2dg7njki authors: pillet, s.; berthelot, p.; gagneux-brunon, a.; mory, o.; gay, c.; viallon, a.; lucht, f.; pozzetto, b.; botelho-nevers, e. title: contamination of healthcare workers' mobile phones by epidemic viruses() date: 2015-12-20 journal: clin microbiol infect doi: 10.1016/j.cmi.2015.12.008 sha: doc_id: 300023 cord_uid: 2dg7njki mobile phones (mps) are potential reservoirs of nosocomial bacteria, but few data are available concerning viruses. we aimed to evaluate the presence of virus rna from epidemic viruses including metapneumovirus, respiratory syncytial virus, influenza viruses, rotavirus (rv) and norovirus on the mps used by healthcare workers (hcws) and to relate it to hygiene measures. an anonymous behavioural questionnaire about mp use at hospital was administered to the hcws of four adult and paediatric departments of a university hospital. after sampling personal (pmp) and/or professional mps (digital enhanced cordless telephone, dect), virus rnas were extracted and amplified by one-step real-time reverse transcription–quantitative pcr. the molecular results were analysed in a masked manner in relation to the behavioural survey. questionnaires from 114 hcws (35 senior physicians, 30 residents, 32 nurses, 27 nurses' assistants) working either in adult (n = 58) or paediatric (n = 56) departments were analysed. medical personnel used their pmp more frequently than paramedical hcws (33/65 vs. 10/59, p <0.001). mps were used during care more frequently in adult wards than in paediatric ones (46/58 vs. 27/56, p <0.001). virus rna was detected on 42/109 (38.5%) collected mps, with rv found on 39, respiratory syncytial virus on three and metapneumovirus on one. the presence of virus rna was significantly associated with mps from the paediatric hcws (p <0.001). mps routinely used in hospital, even during care, can host virus rna, especially rv. promotion of frequent hand hygiene before and after mp use, along with frequent cleaning of mps, should be encouraged. introduction and disinfection could decrease this risk. unfortunately, as reported in reviews about this topic [4, 5] , hcws do not regularly apply hygiene procedures such as regular cleaning of their mobile devices and do not perform hand hygiene before or after their use, even though most physicians are aware that these devices could carry pathogenic microorganisms [9] . in contrast to bacterial contamination, evidence of viral contamination of mps such as digital enhanced cordless telephones (dects) or personal mobile phones (pmps) are, to our knowledge, lacking. however, epidemic viruses such as influenza viruses, rotavirus (rv) and norovirus (nv) have been shown to be able to adhere and contaminate inert surfaces as well as medical devices close to the patients' environment [10] [11] [12] . nv and rv were shown to be particularly resistant; they can survive for weeks, even months, on surfaces and in the hospital environment [11, [13] [14] [15] [16] . contamination of hospital surfaces by these viruses may therefore play a role in nosocomial epidemics [11, 13] . respiratory viruses have been shown to persist on surfaces for a few days [14, 17] , with a potential role in nosocomial transmission, as emphasized during the severe acute respiratory syndrome epidemic of 2003 [18] . epidemic viruses have already been retrieved from electronicdevice surfaces such as keyboards, computers and telephone handsets [10, 12, 17, [19] [20] [21] . however, the viral contamination of hcw mps using up-to-date methods has not been studied. the aims of this study were (a) to evaluate the contamination of mps by epidemic viruses including rv, nv, influenza a and b viruses, syncytial respiratory virus (rsv) and metapneumovirus (hmpv) in clinical settings, (b) to evaluate the behaviour of hcws using their mps in our center by using a blindly recorded questionnaire and (c) to correlate viral contamination of mps with the behaviour of hcws. the study took place at the university hospital of saint-étienne, france, from january to march 2013, i.e. the period of circulation of epidemic viruses (influenza viruses, rsv, gastroenteritis-associated viruses) in our setting [22] (personal data). hcws of the paediatric and adult emergency rooms, as well of those of the general paediatric and the infectious diseases departments, were involved. the term 'mobile phones' was used to indicate both pmps and dects. physicians and residents were considered to be medical staff (n = 55); nurses and nurses' assistants were considered to be paramedical hcws (n = 59). the design of the study is summarized in fig. 1 . medical students were excluded from the survey, but because the sampling of mps was performed in their unit during the study, we also sampled their pmps. none of the hcws declared or presented signs of epidemic viral infection at the time of mp sampling. behavioural patterns in the use of mps by hcws each department was visited twice by sp and ebn during the study period. a questionnaire was administered to all hcws in the visited departments, without previous information about the study being provided. participants were volunteers and answered anonymously; they all agreed that the mps they used could be sampled. general data about the use of pmps or dects during work were recorded, such as using the device close to patients, using an alcohol-based hand rub before and after use and cleaning the mps. the mps were wiped with a 480ce e-swab (copan, brescia, italy), and the swabs, placed in transport medium, were frozen at −80°c before virologic analysis. when hcws used pmps and dects at hospital, both mps were sampled. in some cases, several hcws shared one dect; in this situation, the dect was sampled once. pmps were sampled only if used at hospital. a volume of 200 μl of transport medium was extracted by using the specific b protocol on the nuclisens easymag instrument (biomérieux, marcy l'etoile, france). the elution volume was 50 μl. the amplification step was performed immediately after extraction, without freezing of nucleic acids. ten microlitres of extract was mixed with ready-to-use commercial mastermix, and one-step reverse transcription and quantitative pcr (rt-qpcr) reactions were performed on an abi7500fast real-time cycler (applied biosystems, foster city, ca, usa), according to the manufacturers' recommendations. the enteric viruses (rv and nv) were detected by using khrv and khpnov kits from ceeram (la chapelle-sur-erdre, france); the respiratory viruses (influenza a and b viruses, rsv and hmpv) were detected by using the mws kits from biomérieux [22] . the molecular results were analysed in a masked manner to the results of the behavioural survey. the ethics committee of the university hospital of saint-étienne approved the study. the software used for the collection of data was excel (microsoft, redmond, wa, usa). statistical analyses were performed by spss 20.0 software (ibm, armonk, ny, usa). for the univariate and bivariate analyses, fisher's exact test and t tests were used (p <0.05 was considered significant). to adjust for confounding factors, variables with p <0.2 in univariate analysis were entered into a multiple logistic regression model. during the study period, 114 hcws (all of those interviewed) responded to the questionnaire. the partition of hcws by category and department is presented in fig. 1 . the majority of participants were women (74.6%); the mean age was 33.5 ± 10.0 years. behaviour of hcws in use of mps during work at hospital all hcws owned a pmp, and 99 of them (86.8%) used a dect daily at work. all the hcws declared that they knew that mps could host infectious agents. the participants received more than ten calls per workday in 65.6% of cases (75/114 hcws); no statistical difference was found among categories. table 1 lists the results of the questionnaire analysis. hcws used their pmps in hospital in 37.7% (43/114) of cases, with medical hcws using their pmp more frequently than paramedical hcws (respectively, 33/65 vs. 10/53, p <0.001). seventy-three hcws (64%) used mps during care. among them, 28.8% (21/ 73) never performed hand hygiene before using their mp, whereas 37.0% (27/73) of hcws never performed it after using their mp. overall, 15 hcws (20.6%) never performed hand hygiene both before and after using a mp. as shown in table 1 we also sampled 22 pmps from medical students who were present in the wards during the study; 11 (50%) were found to be positive for virus rna (rv n = 7, rsv n = 2, influenza virus a and influenza virus b n = 1 each). because they did not respond to the questionnaire, they were not included in the multivariate analysis. correlation of viral contamination of mps to the behaviour of hcws table 1 shows the detection of viruses on at least one mp by hcw category. by multivariate analysis, the presence of virus rna was significantly associated with mps from paediatric hcws compared to adult hcws (p < 0.001; 32/59 vs. 10/50; odds ratio increased by 2.76). other recorded behaviours in using mps in hospital were not associated with viral contamination. notably, there were no differences in viral contamination regarding staff categories or hygiene habits related to mp use. we report here for the first time contamination of mps used by hcws with epidemic viruses including rv, rsv and hmpv. this finding raises the possible role of mps in cross-transmission of epidemic viruses in hospitals, with the transfer from nonporous fomites to fingers as described recently [23] and from fingers to fomites including mps. pmps may also play a role in the spread of pathogens from community to hospital as well as from hospital to community. more than one third of sampled mps were found to be contaminated with virus rna in the clinical settings we studied. rv rna was largely recovered from mps, notably in those from the paediatric staff. this finding was concordant with epidemiologic data showing that rv is the prevalent virus during winter epidemics in the paediatric population, including during the time of this study (data not shown), because rv vaccination coverage is low in france [24] . rv has been frequently found on hospital surfaces for several months after the epidemic period and after the surfaces had been cleaned [11, 14, 15] . the high prevalence of rv in patients on the paediatric ward during the study, together with the capacity of rv to persist in the environment, are probably the main factors explaining the high frequency of rv rna detection on mps. it would be interesting to look at the presence of rv rna on mps outside the epidemic winter period. despite nv rna screening in our samples, it was not detected on mps in this study. nvs are, however, largely known to be able to survive on several hospital surfaces [10, 13, 19] . the absence of nv rna was probably due to the reduced circulation of nvs usually associated with benign diarrhoea in adults, and few patients infected with this pathogen may have been hospitalized in adult wards in the course of the study. failure to detect nvs seems to be less probable, as we used internal controls in both rv and nv rt-qpcr assays in order to avoid false-negative results due to inhibitors [15] . the second most frequent virus rna detected on samples was rsv, which may also be found on environmental surfaces and can lead to inoculation after touching contaminated surfaces [25] . finally, other respiratory viruses transmitted by droplets, including hmpv and influenza viruses, were recovered from the pmps of one hcw and two medical students, respectively. these respiratory viruses could also survive on hands and on environmental surfaces [12, 17, 26] , leading to the risk of cross-transmission in hospital settings. the yield of contamination could be high because the same mp could be contaminated by several pathogens, as described in our study, and dects could be used by several hcws. although most hcws are aware that mps may carry pathogenic bacteria [4] , most of them do not perform hand hygiene before or after using mps, and they do not regularly clean their mps [8] . in the present study, all participating hcws said they knew that mps could be contaminated by viruses. however, most of them said they used pmps and dects during their work, notably when they were in contact with patients. this was particularly true among adult staff. a large proportion also did not perform hand hygiene when using a mp, even during physical contact with patients, without difference among categories or departments. hand hygiene should be performed just before patient contact, as highly recommended by world health organization guidelines (http://apps.who.int/iris/ bitstream/10665/44102/1/9789241597906_eng.pdf). beyond possible cross-transmission of pathogenic bacteria [2] [3] [4] [5] 8] , we can hypothesize that cross-transmission may also occur with epidemic viruses, as shown in our study. alcohol-based hand rubs and antiseptic wipes are largely available for all hcws in our hospital; the lack of hand hygiene before and after using mps and the lack of cleaning mps is mostly related to poor adherence to hygiene recommendations by hcws. actually, hand washing with soap and the use of alcohol-based disinfectants have been proven to be efficient at removing viruses, especially respiratory ones, from artificially contaminated hands [27] . gastroenteritis-associated nonenveloped viruses are, however, known to be more resistant to disinfection procedures [28] . hydroalcoholic hand rub that passes the en 14476 viral norm or specific tests is needed to kill nvs. however, rvs are sensitive to alcohol disinfection, meaning that the contamination of mps by these viruses is certainly related to the fact that hcws did not use alcohol-based hand rubs before using their mps, rather than more intensive use. indeed, paediatric mps were found to be more frequently contaminated; paediatric hcws used their mps less frequently than adult hcws. these results suggest that promotion of the cleaning of mps should be performed more actively. indeed, the use of isopropyl alcohol has been shown to be efficient in reducing contamination of fomites [29] ; disinfectant wipe intervention on fomites has also been demonstrated to reduce the load of infectious agents as well as the risk of fomite-to-finger microbial transfer [30] . these recommendations should be promoted in paediatric wards, where rvs circulate intensively during epidemics. our study has several limitations. firstly, only virus rna was detected on mps, without presumption of the possible infectious potential of the different viruses. the samples were not inoculated onto cell cultures for isolation of respiratory viruses, rv and nv being not cultivable [15] . however, this first demonstration of contamination of mps by virus rna should be considered as an effective way for the nosocomial transmission of these viruses, and notably of rvs in paediatric wards. another limit concerns the absence of virus load determination on mps by rt-qpcr, as has been done for food contamination [31] . however, to our knowledge, the correlation between virus load on inanimate surfaces and the risk of nosocomial transmission has not been clearly demonstrated. finally, we were not able to show a correlation between the contamination of the mps and the frequency of hand hygiene; the sample size of 30 workers, chosen to ensure the validity of the estimation per hcw category, was probably too low to permit the evaluation of this interaction. after the demonstration by others of contamination by bacteria [4, 5] , we show here that contamination by virus rna also exists on the mps used by different categories of hcws from several hospital wards. our study indicates that more attention must be paid to disinfecting mps that are largely used in clinical settings and that constitute a reservoir for viral agents. in addition, hand hygiene before and/or after their use must be recommended more strongly, especially in paediatric wards, where viruses may circulate intensively. because restriction on the use of these devices is not feasible [5] , raising awareness in hcws about the risk of pathogen transmission is urgent. regular cleaning of mps should be promoted. beyond use of mps, hand hygiene should be performed just before contact with patients. increasing clinical presence of mobile communication technology: avoiding the pitfalls use of cellular telephones and transmission of pathogens by medical staff in new york and israel use of mobile phones by medical staff at queen elizabeth hospital, barbados: evidence for both benefit and harm ipads, droids, and bugs: infection prevention for mobile handheld devices at the point of care review of mobile communication devices as potential reservoirs of nosocomial pathogens health care workers' mobile phones: a potential cause of microbial cross-contamination between hospitals and community mobile phones as a potential vector of infection in a paediatric ward bacterial contamination of anaesthetists' hands by personal mobile phone and fixed phone use in the operating theatre bacterial contamination of mobile communication devices in the operative environment norovirus in the hospital setting: virus introduction and spread within the hospital environment group a rotavirus detection on environmental surfaces in a hospital intensive care unit survival of influenza virus on hands and fomites in community and laboratory settings role of hospital surfaces in the transmission of emerging health careassociated pathogens: norovirus, clostridium difficile, and acinetobacter species how long do nosocomial pathogens persist on inanimate surfaces? a systematic review virus hazards from food, water and other contaminated environments norovirus gii.4 detection in environmental samples from patient rooms during nosocomial outbreaks survival of influenza a(h1n1) on materials found in households: implications for infection control a novel coronavirus associated with severe acute respiratory syndrome effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces significance of fomites in the spread of respiratory and enteric viral disease viability of human adenovirus from hospital fomites epidemiology and microbiological investigations of communityacquired pneumonia in children admitted at the emergency department of a university hospital transfer efficiency of bacteria and viruses from porous and nonporous fomites to fingers under different relative humidity conditions rotavirus vaccination in europe: drivers and barriers respiratory syncytial virus: its transmission in the hospital environment the survival of influenza a(h1n1)pdm09 virus on 4 household surfaces efficacy of soap and water and alcohol-based hand-rub preparations against live h1n1 influenza virus on the hands of human volunteers reducing viral contamination from finger pads: handwashing is more effective than alcohol-based hand disinfectants nhs connecting for health: healthcare professionals, mobile technology, and infection control evaluation of a disinfectant wipe intervention on fomite-to-finger microbial transfer viral genes everywhere: public health implications of pcr-based testing of foods we thank all the hcws who accepted to participate to this study and e. delorme for technical assistance. c. zintilini, c. janis and p. bourgeois from biomérieux and f. chatigny and f. loisy from ceeram are acknowledged for providing the virologic reagents used in this study. key: cord-023592-w96h4rir authors: nan title: abstracts cont. date: 2015-12-28 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2004.0902c.x sha: doc_id: 23592 cord_uid: w96h4rir nan objectives: in this study we wanted to examine the prevalence of caga, vaca and baba2 status in helicobacter pylori (hp) isolates from patients with gastritis or peptic ulcer; to compare them and to know if there were any relationships between those virulence factors in each group. methods: gastric biopsy specimens from 44 hp positive patients with peptic ulcer (25 cases) and gastritis (19 cases) were studied. dna was extracted and pcr performed to detect caga, vaca s1/ s2 alleles and baba2 gene. results: gastritis: in 74% of strains, the expected caga fragment was amplified by pcr; 68% carried the s1-allele and 32% the s2allele; baba2 gene was detected in 37% of strains. peptic ulcer: 84% of strains were caga+; 72% were vaca s1-allele and 28% were vaca s2-allele; baba2 gene was detected in 36% of strains. no significant differences in the prevalence of caga, vaca or baba2 were found in both groups. neither of them showed relationship between the presence of baba2 gene and caga gene or vaca s1/s2-alleles. conclusions: although the risk of developing more serious gastric lesions increased as the number of virulence factor genes are accumulated in a given hp strain, we did not find any significant differences or relationship in the caga, vaca or baba2 status between the hp isolates from patients with gastritis or peptic ulcer in this study. a low percentage of baba2 gene was found in both groups. helicobacter pylori cells in clinical and wastewater samples p. piqueres, y. moreno, a. jimenez, j. hernández, m. ferrú s valencia, e introduction: the presence of viable but non-cultivable helicobacter pylori cells in environmental samples may underestimate the importance of this way for its transmission. the determination of resistance to antibiotics in these strains is important to a better understanding of the epidemiology of the infection. objectives: we have evaluated the use of a fluorescent in situ hybridisation (fish) assay directly from biopsies and wastewater to detect h. pylori and simultaneously its macrolide resistance genotype. methods: a total of 26 gastric biopsies samples from ulcerpatients were homogenised in 2 ml of selective broth, and a 500 ll aliquot was used for fish detection. twenty-nine wastewater samples collected from different treatment plants were centrifuged and subsequently fixed with 4% paraformaldehyde solution for 4 h at 4 c and then washed with 1% pbs buffer. hpy probe, a 16s rrna targeted fitc-labelled oligonucleotide sequence was used for the detection of all h. pylori strains. in addition to cla1-3, a set of three cy3-labelled probes was used for the detection of 23s rrna mutations associated with resistance to clarithromycin. hybridisation was performed with 35% formamide at 46 c for 2 h. results: fish allowed the detection of h. pylori in 20 out of 26 clinical samples and 12 samples were positive in wastewater. the 35% of the positive biopsies showed the presence of clarithromycin resistant strains and 16.6% of the positive wastewater samples yielded resistance genotype to this macrolide. by using a double filter set we could observe directly the clarithromicyn resistant h. pylori organisms in the samples and its morphology in the different types of environments. the predominant cells' morphology in both clinical and wastewater samples was of helicoidal form. conclusions: the fish is a specific and rapid culture-independent method to determine directly the presence of clarithromycinresistant h. pylori cells in clinical and environmental samples. results showed the presence of macrolide resistant cells in water and, therefore, water must be considered a potential route of h. pylori transmission. acknowledgement: this work was supported by ministerio españ ol de ciencia y tecnología, project agl2002-04480-c03-03. objectives: helicobacter pylori is a leading cause of various gastrointestinal diseases such as atrophic gastritis and gastroduodenal ulcer. the caga gene product caga is directly injected into the bacteria-attached host cells and deregulates intracellular signalling pathways and thereby initiates pathogenesis. caga gene is located on pathoginicty island but the function of other genes on the island is unknown. the goal of the work was to evaluate the impact of cag island genotype on the outcome of the therapy. materials and methods: three groups of 25 patients each with total number of 75 patients were investigated. first group was taking a typical antibiotic therapy (amoxicillin, claritromycin, rabeprasol), patients in the second group were treated with the same antibiotics together with probiotic laminolact (e. faecium strain l-3 in the form of bon-bons together with pectin, soy bean amino acids and sea weed), and the third group was taking only laminolact without any antibiotic. the genotype was determined by pcr with dna primers against three h. pylori genes ureb, caga and cagh. cagh was used as a marker cag island integrity and ureb was a marker of h. pylori presence. five different genotypes were determined: ureb+,cagaàcaghà, ure+,cagaàcagh+, ureb+,caga+cagh+, ureb+,cagaàcagh+ and ureb+,caga+caghà . treatment with antibiotics alone was leading to 68% of eradication. the best eradication percentage (84%) took place in the second group where classical antibiotic treatment was taken together with probiotics. interestingly, probiotic treatment alone was giving 48% of eradication. results of the therapy were highly consistent with cag genotype. patients were found to be statistically less susceptible (p < 0.05) to the therapy in case when the entire cag regulon was present regardless of the therapy used. this fact suggests immunosuppressant function of caga or other proteins encoded by the genes on cag pathogenicity island. conclusion: the effect of h. pylori eradication depends on cag pathogenicity island genotype. probiotics including e. faecium l-3 might significantly improve the anti-h. pylori treatment. screening of p. aeruginosa isolates. this study was done to determine if the p. aeruginosa strains isolated from the cultures of patients hospitalised in the infectious diseases unit were from an individual strain. this technique was preferred because it is cheap and provides a rapid detection opportunity. methods: 45 samples obtained from the clinical specimens of the patients and from the hands of the staff of the infectious diseases unit were cultured. of the 45 samples, 15 were isolated from blood, six from sputum, 10 from drainage and 14 from the hands of the medical staff. p. aeruginosa identification was made by api 20ne system. dna was extracted from the culture material by phenol-chloroform extraction method. ap-pcr was performed by using the primer 5¢-gtt gcg atcc-3¢, and subjected 8% page. band patterns were visualised by silver staining. results: in none of the isolates of the hospital staff p. aeruginosa was cultured. out of the 31 clinical samples of the patients, 15 different genotypes were determined. conclusions: the p. aeruginosa strains of the patients were individual strains, neither related to the staff of the department nor to a specific patient. objectives: pseudomonas aeruginosa is a leading cause of nosocomial infections, particularly pneumonia or sepsis, on intensive care units. its high intrinsic antibiotic resistance and the ability to develop multidrug resistance pose, especially for critically ill patients, serious therapeutic problems. since, culture based techniques for pathogen identification and resistance determination requires at least 2 days, a calculated antibiotic therapy may harbour the risk of an increase in antibiotic resistance and therapy failure. therefore, the development of a fast and reliable identification and antimicrobial susceptibility test is essential for the improvement of the therapy. the aim of the present study was to develop an oligonucleotide-array for a quick, genotypic test of antibiotic susceptibility combined with the determination of relevant virulence factors. methods: dna from different clinical specimen was isolated with a modified qiamp dna blood mini kit. template dna was amplified and simultaneously labelled with cy3 during multiplex pcr. 146 oligonucleotide capture probes (17-24mer), containing a poly-t(15)-spacer at the 5¢-end, were spotted on epoxy-slides to build an array covering regulatory genes of multidrug efflux pumps (mexr, mext, nfxb), alginate synthesis (muca), metallo-beta-lactamases (bla-vim, bla-imp), aminoglycoside modifying enzymes (aac, aad, aph) and virulence factors (exou, exos, exot) . results: 12 of 15 clinical p. aeruginosa isolates could be correctly genotyped. three isolates displayed a hybridisation pattern that could be assigned neither to wild-type nor to known mutations. a sequence analysis of these isolates revealed an unknown mutation in mexr and nfxb. hybridisation with dna from other non-fermenter or enterobacteriacea showed no crossreactivity. genotypic resistance profile of p. aeruginosa deduced from the array data correlated fully with the susceptibility pattern obtained by standard tests. the sensitivity of the array was 100 genome equivalent even with an 10exp7-fold excess of non-pseudomonas dna. the whole analysis, including dna processing, array hybridisation and data evaluation could be performed in less than 5 h. conclusions: due to the good correlation with standard procedures, the pseudomonas-array may be used for a rapid susceptibility test even directly from clinical samples. combined analysis of antibiotic resistance and virulence factors may improve the outcome of an antimicrobial therapy. objectives: because of a high prevalence of pseudomonas aeruginosa infections in cystic fibrosis (cf) patients, we conducted a study to assess 60 p. aeruginosa isolates collected over 10 years from the sputa of 38 cf adult patients attending an italian cf centre. some phenotypic characters of bacteria (o-serotype, motility, production of enzymes and resistance to antibiotics) and their pfge genotypic patterns were evaluated to analyse for the presence of epidemic strains. moreover some sequential isolates collected from 15 cf patients were investigated to look for the chronicisation of the infection. the strains were identified biochemically. the o-serotype was determined by slide agglutination; the production of enzymes (protease, elastase, gelatinase, haemolysin, betalactamase) and motility were detected using specific techniques. the antibiotic susceptibility was analysed by the vitek ams system and disc diffusion method. pfge was used to discriminate the genotypes of p. aeruginosa. results: in our hands, o serotyping failed to identify 26.3% of isolates, considering the bacteria collected at the onset of colonisation; the most frequent serotypes were o: 10, o: 6 and o: 3. moreover, the percentages of protease, haemolysin, gelatinase and elastase production were respectively 78.9, 52.6, 55.3 and 39.5, whereas 42.1% of the microorganisms were non-motile. pfge allowed the typing of all strains except one. the heterogeneity of isolates indicated that cross-infection is unusual; we also observed in several strains isolated in the last years a predominant pattern. some cf patients were harbouring the same p. aeruginosa genotype in sequential isolates and the susceptibility of bacteria to antibiotics tested varies greatly, also in strains belonging to the same pfge profile. conclusion: our results indicate no relationship between genotype and phenotype suggesting that the phenotypic variability is due to an adaptation of the microorganism to the host. moreover, the presence of several strains with the same genotypic profile suggests a possible cross-colonisation in cf patients due to the circulation of a transmissible strain. for ser/thr protein kinases and phosphoprotein phosphatase of pseudomonas aeruginosa and analysis of their properties j. nedvedova, p. lnenicka, k. hercik, p. branny prague, cz objectives: pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. three genetic loci of p. aeruginosa which encodes ser/thr protein kinases were identified. two of them, ppka and stk1, were also characterised but little is known about their function in cell signalling. gene stp1 localised upstream of stk1 encodes stk1 cognate phosphoprotein phosphatase. a possible relationship between quorum sensing and protein phosphorylation in gram-negative bacteria has already been described. the aim of this work was to prepare unmarked deletion mutants in ppka, stk1 and stp1 genes and to find out if the linkage between quorum sensing and protein phosphorylation in p. aeruginosa exists. to prepare the unmarked deletion mutants an improved method for gene replacement in p. aeruginosa which employs a broad-host-range flp-frt recombination system for site-specific excision of chromosomally located dna sequences was used. the phosphorprotein pattern and biochemical properties of the mutants were examined. double mutant in homoserine lactone synthase genes (lasi and rhli) was also subjected to phosphoprotein pattern analysis. results and conclusion: stk1, stp1 and a double mutant stk1/stp1 were prepared. no differences were found in either biochemical properties or phosphoprotein pattern. deletion of ppka gene failed due to the integration of vector into the unknown, but specific site of p. aeruginosa genome. the comparison of phosphoprotein patterns of lasi, rhli double mutant and wild type strain showed important differences. this result suggested that phosphorylation circuit operating in p. aeruginosa is related to quorum sensing system(s). objectives: botulism is a rare but potentially fatal disease generally caused by the neurotoxin produced by clostridium botulinum. symptoms of the disease include paralysis which is due to bont inhibiting neuro-transmitter release. laboratory diagnosis of botulism relies on detecting bont in clinical or food specimens using in vivo tests. diagnosis also includes isolation and identification of the bacterium which again relies on in vivo tests for detection of toxin production from the bacterium growing in vitro. we previously described the development of real-time pcr assays for bonta, b and e gene fragments, and here presented further evaluation data. methods: dna was extracted from faeces, enrichment cultures of naturally contaminated food and clinical samples and from colonies growing on agar plates. taqman-based assays for bonta, b and e gene fragments were performed using a 7700 sequence detector (applied biosystems). the assays were performed as a duplex reaction for bonta and b using fam and vic labelled probes, respectively, and as a monoplex for bonte using a single fam labelled probe. all samples were tested by using the conventional bio-assay and results were compared with real-time pcr assay results. results: pcr and bio-assay were found to be consistent in all samples except those that contained 'silent b' neurotoxin genes in addition to bonta genes. the samples tested comprised: direct examination of six faecal samples, 18 enrichment cultures for six clinical and 12 foods and 34 pure culture growing in vitro. conclusion: this study is the first to report the successful identification of different c. botulinum toxin types for wild type bonta, b and e by using taq-man real-time pcr assay. this assay has already provided a useful adjunct to in vivo tests for the rapid identification of bacteria containing bont genes in wild type c. botulinum. (vntrs) polymorphisms for genotyping 'rickettsia conorii complex' strains l. vitorino, r. de sousa, l. zé-zé, f. bacellar, r. tenreiro lisbon, p introduction: mediterranean spotted fever (msf) is an acute, febrile tick transmitted rickettsiosis caused by strains of rickettsia conorii complex. msf is endemic in portugal and is an obligatory notifiable disease. during 1989-2000 the annual incidence rate of the disease was 9.8/105 inhabitants. in portugal, msf is caused by two strains: r. conorii malish and israeli tick typhus (itt). this strain was isolated, for the first time in 1997, from a patient. moreover, data from the national institute of health points out that half of msf cases occurring in portugal are caused by itt, showing a similar prevalence of infection as r. conorii malish. objective: in this work we present a pcr-based method to detect vntr sequences that enables amplicon size differentiation between r. conorii malish and itt human isolate. vntrs have a high discriminatory capacity not only because they contain greater diversity but also because they often vary in copy, therefore they are being used for molecular typing of many bacteria species. methods: human strains were isolated by shell-vial technique from patient's total blood. dna from tick isolates and reference strains were also used for comparative purposes. vntr loci were identified by the tandem repeats finder software within the r. conorii genome. the vntr65 locus was selected based on the following criteria: repeat units 50 nucleotides in length, 95% nucleotide sequence identity between individual repeat units, and two or more copies of the repeat unit. primers flanking this sequence were designed to enable vntr-pcr amplification. the clinical isolates' identification was also confirmed by ompa gene sequencing. results: the vntr sequence chosen was highly informative since it possesses different repeats of the consensus pattern among the strains tested, namely r. conorii malish and itt. the former contains five tandem repeats and the later only have three repeats of the 65 bp motif unit, which can be easily detected by agarose gel electrophoresis. therefore, the polymorphism observed enabled discrimination between these two strains. these results are in agreement with ompa gene sequence. conclusion: this pcr-based method provides a useful and rapid way for genotyping r. conorii malish and itt isolates (r. conorii complex) which are responsible for msf in portugal. ompa and glta gene amplification are currently the most widely rickettsiae detection method used. however, strain identification is only accomplished by sequencing. p733 monitoring the ability of the human intestinal microflora to become re-established after antibiotic treatment using t-rflp c. jernberg, å . sullivan, c. edlund, j. jansson huddinge, stockholm, s objectives: to study the composition of the human normal faecal microflora during administration of clindamycin and a probiotic or placebo product. since only a small portion of the faecal flora is cultivable the samples were primarily analysed using a culture independent molecular fingerprinting technique, terminal-restriction fragment length polymorphism (t-rflp). methods: the study included eight healthy volunteers. all subjects received clindamycin orally for 7 days. four subjects received a probiotic yoghurt concomitantly containing 10 exp 8 cfu/ml of the strains lactobacillus f19, lactobacillus acidophilus ncfb 1748 and bifidobacterium lactis bb12. the placebo group received ordinary yoghurt. faecal samples were taken before the administration (day 0), the last day of administration (day 7) and 14 days after the administration (day 21). the samples were analysed both by conventional cultivation and by t-rflp. both universal bacterial primers and lactobacillus specific primers were used when analysing the samples using t-rflp. the areas of the different terminal restriction fragments (trfs), each of which theoretically corresponds to one or a group of closely related species, were used to calculate the relative abundance values for the trfs. these values were used for principal components analyses (pca) and upgma analyses to compare the microbial flora at the three different time points. results and conclusions: in the group ingesting the probiotic, the microflora in three out of four subjects became re-established close to their original compositions 2 weeks after antibiotic treatment ceased. by contrast, only one subject out of four in the placebo group had an intestinal microflora that showed tendencies towards normalisation during the same time period. these findings were in accordance with the results from the culture-based analysis. t-rflp was also used to monitor specific bacterial populations that were either positively or negatively impacted by clindamycin. for example, one of the dominating populations, belonging to the clostridium coccoides subgroup, was highly negatively impacted by clindamycin administration in all subjects. when using lactobacilli specific primers, l. acidophilus and lactobacillus f19 were the two dominating populations in the group receiving the probiotic. t-rflp was shown to be a reproducible technique for analyses of antibiotic and probiotic induced alterations in the normal intestinal microflora. campylobacter species using maldi-tof mass spectrometry d. dare, h. sutton, c. keys, h. shah, m. lunt, g. wells manchester, london, uk laser interrogation of bacteria by matrix assisted laser desorption/ ionisation time of flight (maldi-tof) mass spectrometry (ms) reveals unique fingerprint patterns of biomarkers. these patterns are reproducible for a given set of conditions and can be used as the basis for bacterial identification against a database of known bacterial spectra. manchester metropolitan university in collaboration with the health protection agency (uk) and waters corporation have created a maldi-tof mass spectral database of clinical, environmental and food borne pathogens. these pathogens are all supplied from the uk national collection of type cultures (nctc). the database spectra are therefore representative of organisms from a world-renowned collection. this database has continued to grow over the last 3 years from the initial 300 to currently over 3000 spectral entries covering $100 different genera. bacterial identification using this database is often conclusive with the top five matches suggesting the same genera/species. however for identification to be robust, the strains within the database must be well characterised and their identity well established. for campylobacter the number of representative strains in the database has increased significantly from around 10 to 170 over 3 years. the species covered within this taxa are: c. coli, c. consicus, c. curvus, c. fetus, c. gracilis, c. helveticus, c. hyolei, c. hyointestinalis, c. jejuni, c. lari, c. rectus, c. sputorum and c. upsaliensis. this study presents the results of analysing the same datasets for 21 campylobacter strains against the expanding databases containing 300, 1099, 2159 and >3000 mass spectral entries respectively. the results demonstrate a significant improvement (i.e. 29-100%) in the number of campylobacter sp. correctly identified as the number of representative strains increase. therefore maldi-tof ms provides a potential rapid identification system for campylobacter sp. p735 application of 23s-5s intergenic spacer sequencing for the detection and molecular differentiation of legionella species f. grattard, c. ginevra, s. riffard, a. ros, j. etienne, b. pozzetto saint-etienne, f objectives: among the more than 40 species of legionella that have been identified so far, 21 have been reported to be pathogenic for humans. by now, the precise identification of clinical isolates in reference laboratories needs the use of monoclonal antibodies or of molecular markers such as 16s rrna-, mip-, rpob-or dota-gene sequencing. we developed a rapid and convenient technique based on the sequencing of the 23s-5s intergenic spacer using nondegenerated primers specific for legionella spp. methods: we tested 37 legionella species (reference and clinical isolates), including 15 serogroups of l. pneumophila subsp. pneumophila. the amplification step was performed by using a real-time pcr (lightcycler, roche diagnostics) and sequencing was performed on the ceq8000 sequencer (beckman). the comparative analysis of the sequences was done with the computer program mega and the dendrograms obtained by the neighbour-joining method. results: the phylogenic tree of the 23s-5s intergenic spacer sequences was found able to clearly differentiate all legionella species at the subspecies level. actually three subspecies of l. pneumophila (subsp. pneumophila, subsp. fraseri and subsp. pascullei) were clearly distinguished. species sharing the same autofluorescence properties and ubiquinone and fatty acid composition were shown to be phylogenetically related. in addition to rpob sequen-cing that was shown previously to exhibit similar results, our technique was found able to detect and identify strains present in clinical or environmental specimens that could not be cultured on agar medium. although this tool was not discriminatory enough to differentiate all strains of l. pneumophila subsp. pneumophila at the serogroup level, it was used in two different outbreaks to demonstrate rapidly the identity of the sequences between strains responsible for severe human infection and those isolated in the hot water reservoir, suggesting a common origin. conclusion: the 23s-5s intergenic spacer sequencing was found to be suitable for rapid detection and powerful identification of legionella species in clinical settings. whipple's disease (wd) is a rare multisystemic bacterial infection, with variable clinical manifestations occasionally involving the central nervous system. as the cultivation of the aetiologic agent, tropheryma whippelii, is difficult, the laboratory diagnosis is usually based on histological methods. in the last few years, molecular detection of the bacterial 16srrns genes by the polymerase chain reaction (pcr) with two primer sets, has greatly contributed to the diagnosis. we present a cerebral case of wd in a 48-year-old male, successfully diagnosed by pcr of t. whippelii in the blood and the faeces. as far as we know this is the first case reported from greece. for the diagnosis of wd histological examination of duodenum biopsy for diastase resistant, non-acid fast, periodic acid schiff (pas)-positive inclusions in macrophages, and molecular detection of the 16srrna genes of by pcr in csf, blood and faeces were performed. the histological detection was negative. pcr was positive in the blood and the faeces of the patient and negative in the csf. seven months after the onset of antimicrobial therapy, pcr was negative in all three clinical specimens. in conclusion, the application of pcr proved to be an invaluable tool for the recognition, the differential diagnosis and the early start of the antimicrobial therapy of wd, a generally fatal disease, if it remains untreated. results: only one clinical isolate had the same api code profile as the reference strain. fifteen per cent of clinical strains were tested urease positive, as was the reference strain. by fatty acid analysis, clinical isolates could be separated in four different groups (i-iv), containing 77, 98, 5 and 1 isolates, respectively. atcc 49368 was grouped to group ii. sequences were obtained from three strains of groups i and ii, respectively, and from one strain of groups iii and iv, respectively. comparison of the determined eight sequences with public databases showed the greatest similarity score with c. asperum (x82050.1) with values between 98.7 and 100%. c. asperum and c. amycolatum are considered as synonyms, because they exhibit a level of dna-dna relatedness greater than 90% (ruimy r et al. int j syst bacteriol 1995; 45: 740) . homology with c. amycolatum atcc 49368 (x82057.1) was only between 97.1 and 98.3%. sequencing of the c. amycolatum reference strain yielded 100% homology with the published sequence (x82057.1). conclusions: our data confirm the hypothesis that atcc 49368 is atypical for clinical c. amycolatum strains. furthermore, our data are in concordance with the observation, that by pyrolysis-gasliquid chromatography c. amycolatum isolates can be separated in two different groups (voisin s et al. res microbiol 2002; 153: 307) . p738 detection of mutations associated with resistance to tetracycline and clarithromycin in helicobacter pylori using the pyrosequencer a. lawson, c. arnold, r. owen london, uk objectives: clarithromycin and tetracycline are key components of h. pylori eradication therapy. resistance to clarithromycin occurs due to single nucleotide mutations in 16s rdna and an assay to detect these was amongst the first to be developed for the pyrosequencer. recently it has been shown that resistance and reduced susceptibility to tetracycline occur due to single, double or triple mutations in 23s rdna. the aim of this study was to develop a single multiplex assay using the pyrosequencer to determine susceptibility to clarithromycin and tetracycline from h. pylori isolates and direct from gastric biopsy samples. methods: pyrosequencer assays to detect mutations conferring tetracycline and clarithromycin resistance were designed to work singly and in multiplex. the assays were evaluated using 20 isolates with fully characterised 16s and 23s rdna sequences. subsequently, dna extracts from 30 clinical isolates and 20 h. pyloripositive human gastric biopsies -all of unknown antibiotic susceptibility -were examined and the results compared with those achieved by conventional culture-based techniques, namely antibiotic disc diffusion and etest. results: the pyrosquencer multiplex assay correctly determined the 16s and 23s rdna sequences of the 20 characterised control isolates. when applied to dna extracted from clinical isolates and gastric biopsy samples, the pyrosequencer assay was in agreement with the clarithromycin and tetracycline susceptibilities determined by culture-based analysis. conclusion: the pyrosequencer assay allowed rapid determination of clarithromycin and tetracycline susceptibility from both h. pylori isolates and gastric biopsy samples. the sequence data obtained for the mutations occurring in each strain may provide useful epidemiological information and guide patient management. diseases. the aim of this prospective pilot study was to detect iga, igg and anti-caga antibody status and to evaluate the correlation with anti-h. pylori iga, igg western blot and elisa tests in adult dyspeptic patients. methods: upper gastrointestinal endoscopy, two from gastric antrum and two from corpus, was performed in 56 patients (mean age ae46.41) with dyspeptic symptoms. h. pylori was assessed by rapid urease test and by histopathologic examination in these biopsy specimens. patients' sera were tested by anti-h. pylori iga, igg western blot, iga, igg elisa and anti-caga-iga, igg elisa (euroimmun medizinische labordiagnostika, lü beck) tests. results: a total of 56 patients were evaluated and h. pylori infection was diagnosed in 48 (85.71%) patients by rapid urease test and/or histopathology. serological anti-h. pylori test results were shown as below (table 1) . twenty-eight (50%) of 56 adult dyspeptic patients sera were positive for anti-caga-igg elisa and 17 (30.35%) were positive for anti-caga-iga elisa. conclusion: infection with h. pylori results in the production of local and systemic antibodies. cag a is the important pathologic marker with high immunogenic power. a set of serological tests may give more accurate determination of h. pylori infection than one test detecting specific antibody or bacterial antigen. it seems that there is a good correlation with western blot and elisa test results and gold standards. acknowledgement: this work was supported by euroimmun medizinische labordiagnostika, lü beck, germany. p740 susceptibility of helicobacter pylori isolates to the anti-adhesion activity of a high-molecular-weight constituent of cranberry h. shmuely, o. burger, i. neeman, j. yahav, z. samra, y. niv, n. sharon, e. weiss, m. tabak, a. athamna, i. ofek petach tiqva, haifa, rehovot, jerusalem, kfar qaraa, tel aviv, il background: previous studies have shown that a high molecular mass non-dialysable constituent derived from cranberry juice inhibited the adhesion of helicobacter pylori to human gastric mucus and to human erythrocytes. the aim of the present study was to determine the sensitivity of a large number of both antibiotic-resistant and susceptible clinical isolates of h. pylori to the anti-adhesion effect of the cranberry constituent. material and methods: confluent monolayer of gastric cell line in wells of a microtitre plate was exposed to bacterial suspensions prepared from 83 h. pylori clinical isolates, including 17 from patients after treatment failure. adhesion was estimated by the urease assay to calculate the percent inhibition of adhesion by the non-dialysable material. antibiotic susceptibility of h. pylori isolates to metronidazole, tetracycline and amoxicillin were tested by the etest. results: in two-thirds of the isolates, adhesion to the gastric cells was inhibited by 0.2 mg/ml of the non-dialysable material. all isolates were susceptible to amoxicillin and tetracycline and 35 isolates (42%) were resistant to metronidazole. there was no relationship between the anti-adhesion effect of the cranberry material and the resistance to metronidazole in isolates from either the antibiotic-treated or untreated patients. most important, only 13 isolates (16%) were resistant to both non-dialysable material and metronidazole and 30 isolates (36%) were resistant to the non-dialysable material alone. no cross-resistance of the isolates to cranberry constituent and metronidazole was found. conclusions: the data suggest that a combination of antibiotics and a cranberry preparation may improve the eradication of h. pylori. methods: for the seroprevalence study a total of 1041 people of different states from the country were evaluated: 370 symptomatic and 406 asymptomatic adults; 27 symptomatic and 238 asymptomatic children. the determination of specific igg antibodies was made by commercial elisa. the presence of the gene caga was evaluated in 133 patients of the metropolitan area and the center of gastric cancer control of san cristó bal (endemic zone of gastric cancer). the detection of vaca was determined in 29 biopsy from patients of san cristó bal and 36 biopsy from patients of the metropolitan area. the biopsies were analysed by different methods for diagnosis of h. pylori: culture, urease test, polymerase chain reaction and rapds for genotyping the h. pylori isolates. results: the percentage of asymptomatic children with values of specific igg antibodies anti-h. pylori (over 300 u) varies from 30 to 60% (metropolitan area vs. san cristó bal). in symptomatic adults groups, the seroprevalence was between 68 and 93% according to the studied geographic area. a decreased title of igg antibodies anti-h. pylori was observed in patients with diffuse antral gastritis associated with metaplasia type ii. in the group of endemic cancer area the titles of igg anti-hp were elevated in patients with antral diffuse gastritis. the caga gene was detected in 46% of patients of the metropolitan area unlike the group of patients of san cristó bal a smaller frequency was observed (26.41%) (p < 0.001). a high incidence of s1a and m2 genotype was observed in the h. pylori isolated from the patients of endemic gastric cancer area (40%), unlike what we observed in the metropolitan h. pylori isolates where an elevated prevalence of s1b and m1 genotypes was found. samples from gastric antro. no current resident in our area and patients treated previously with eradicated therapy have been excluded. samples were homogenised and cultured in blood-agar, chocolate-agar, pylori-agar and tioglicolate broth. it was incubated to 37 c in microaerofile atmosphere during 5-7 days. we studied the susceptibility to: amoxicillin (am), claritromicin (ch), metronidazole (mz), tetracycline (te) and ciprofloxacin (cp) by detection of imc by e-test (biodiskâ). we have followed nccls criteria for antibiogram lecture. results: from 148 samples, 60 were males and 88 females. we found the follow primary resistance; ch 16 (10.8%), mz 52 (35.1%), te 8 (5.4%), am 2 (1.4%), cp 23 (15.5%) and 10 samples (6.8%) with a mix resistance to ch and mz. ch and mz resistance are more common in females, but the difference is only statistically significant for mz (p: 0.037). conclusions: there is a progressive increased antibiotic resistance in h. pylori in our area. this may be related with a raised used of antibiotics for other indications. ch resistance data agree with other spanish and multicentre european studies, which show a foremost rate in the mediterranean area. the mz resistance is higher than other spanish works. our high prevalence of resistance supports the idea of avoiding imidazol therapy as primary choice treatment. p743 the prevalence and consequences of antibiotic resistance in danish h. pylori strains isolated with an interval of 10 years objectives and background: the treatment of h. pylori (hp) infections is complex and the use of combination therapy is imperative. the choice of the antibiotics is often made exclusively on empirical basis although resistance to many therapeutically relevant antibiotics has been described. the mainstay of hp treatment in denmark is various combinations of normally two of the following antibiotics: metronidazole, amoxicillin, tetracycline and clarithromycin. to clarify whether these compounds were to remain the drugs of choice we decided to determine the susceptibilities of metronidazole, clarithromycin, tetracycline, and amoxicillin against 180 hp strains recently isolated from patients with duodenal ulcer. the results were compared with results previously obtained by us in 1993 using a similar methodology. over a period of 10 years only the development of resistance to metronidazole appears to constitute a problem. otherwise hp has remained remarkably susceptible to these therapeutically relevant antibiotics. on the basis of our results we recommend that surveillance of especially metronidazole resistance in denmark is markedly intensified, e.g. by increasing the use of diagnostic methods of hp infections that allow susceptibility testing. in cases where treatment with metronidazole is considered, susceptibility testing is of course of major importance, if not downright necessary. objectives: helicobacter pylori is the main causative agent of peptic ulcer disease. clarithromycin resistance of h. pylori is the common reason of failure of the eradication therapy, which includes amoxicillin-clarithromycin and proton pomp inhibitor. the aim of this study was to determine the prevalence of clarithromycin resistance among h. pylori strains isolated from gastric biopsies obtained during routine endoscopies at the baskent university medical faculty in ankara, turkey. methods: h. pylori strains were isolated from antral biopsy specimens taken from dyspeptic patients. antibiotic susceptibilities of the isolates to clarithromycin were performed using the nccls approved agar dilution and the e test methods. results: 78 h. pylori isolates were included in the study. clarithromycin resistance was found in 16 (20.5%) of the isolates. the resistance rates were similar by the e test and agar dilution methods. conclusion: the percentage of the clarithromycin resistance among h. pylori strains in our population is significantly high. this information is important to monitoring the eradication therapy and defines regional treatment policies. introduction: helicobacter pylori has been the subject of many studies that contributed to a better understanding of its epidemiology and its clinical importance in the pathology of the upper gastrointestinal tract, being an important cause of duodenal, gastric ulcers and a definite cause of gastric adenocarcinoma in human. objectives: to determine the seroprevalence of h. pylori among population living in rural community, in relation to the epidemiological aspect, and to study the seroprevalence of anti-caga as a virulence factor in a step that might be helpful in studying the magnitude of h. pylori infection. also, to determine a cut-off value among the population in this community. subjects and methods: this is a community based, field study which was performed on 605 randomly chosen subjects representing villagers of eight villages in giza governorate egypt. serological testing for anti-h. pylori and anti-cag a were performed by elisa. results: the overall seroprevalence of anti-h. pylori igg was 91.7% with different degrees of positivity: 40.8% mild, 39.2% moderate and 11.7% high. anti-cag a was present in 10.6%. there was a significant agreement between the presence of the two antibodies; however, on studying the relation of anti-h. pylori igg level with anti-caga no statistically significant relation was found denoting that the level of infection even if mild does not rule out the possible association of virulent strain of h. pylori. no age or sex difference was noted as regards anti-h. pylori seropositivity but subjects seropositive for anti-cag a had a statistically significant higher mean age. when relating the seroprevalence of anti-h. pylori to type of community, it was found to be the same in semi-rural communities and rural ones and when investigating the respective conditions in both communities it was found that the prevalence is rather related to pattern of life, socioeconomic status and to other possible vehicle of transmission as animals or flies than faecaly contaminated water which is not considered the only vehicle for h. pylori transmission in our study. conclusion: h. pylori is holoendemic in egypt; however, infection by virulent strains is not common. objectives: extended-spectrum beta lactamases (esbls) are an increasing cause of resistance in enterobacteriaceae. unfortunately, the laboratory detection of esbls can be complex and, at times, misleading. the aim of this study was to determine whether routine methods performed in a clinical microbiology laboratory of a tertiary care hospital, are adequate for detecting emerging esbl producing clinical isolates. methods: to evaluate the esbl confirmation protocol, we collected 29 enterobacteriacae strains, isolated in our laboratory. each isolate met the nccls screening criteria for potential esbl producers (ceftazidime or cefotaxime mics were !2 for all isolates). we tested 13 kl. pneumoniae, five ent. cloacae, two ent. aerogenes, five e. coli and four pr. mirabilis strains, by methods routinely used in our laboratory. initially, the isolates were tested for clavulanic acid effect by disk diffusion method and all were analysed by the vitek2 automated system (biomerieux, france), which performs a susceptibility testing, by determining the mic breakpoints. the advanced expert system (aes) of vitek2 was set on the phenotypic resistance knowledge-based system and the panel gn020 was used. in parallel, the isolates were tested by the esbl e-test with ceftazidime and cefotaxime plus beta lactamase inhibitor (ab, biodisk, sweden). in order to confirm the esbl production, all strains were tested by isoelectric focusing (ief) followed by pcr for blatem, blashv, blaoxa, blaibc and blactx genes. results: twenty-one out of 29 isolates proved to produce esbls by molecular methods. all enterobacter strains and one proteus mirabilis were not esbl producers. no blaoxa or blaibc genes were detected. the pcr detection of esbl genes results were compared with the double disk diffusion, vitek2 and esbl e-test to estimate the sensitivity, specificity and the predictive value of the methods tested. the sensitivity of the methods was 84.6, 85.7 and 70.5%, respectively, the specificity 62.5, 87.5 and 66.6%, respectively, and the predictive value 64.7, 93.3 and 75%, respectively. discussion: given the increasing incidence of esbl producing clinical isolates, it is important that esbl screening is incorporated into routine diagnostic testing. the backup of the simple disk diffusion method by the automated vitek2 system increases the possibility of identifying esbl activity of clinical strains in the hospital microbiology laboratory setting. objectives: extended-spectrum beta-lactamases (esbl) are plasmid-mediated beta-lactamases and most of them are mutant of tem or shv beta-lactamases. esbls have been associated with clinical failures due to serious interpretive problems of standard laboratory tests. detection of esbls remains a challenge for the laboratory, since routine tests for monitoring a susceptibility to oxyimino-cehalosporins and aztreonam have not been sensitive enough to detect esbl strains and require up to 2 days. we describe an oligonucleotide array for rapid identification of single nucleotide polymorphisms (snps) of the esbl tem beta-lactamases. methods: plasmid dna was amplified and cy5 labelled during pcr with consensus primer pair flanking the blatem gene. oligonucleotide arrays were constructed with 168 oligonucleotide capture probes. the probes were designed with the snp at the central base of the probe sequence for maximum perfect match/ mismatch discrimination. results: 40 of 41 snp positions were correctly identified. the signal intensity values ranged up to 20 000 for the perfect match probes. the discriminatory power of the array expressed as relative intensity of mismatches (rimm) remained for 99% of the mismatches below 0.4. a perfect match was considered as correctly identified, if rimm did not exceed 0.7. analysis of the array reproducibility revealed that in analysed blatem-1 samples all 41 snp positions could be identified. the mean rimm values varied, but 95% remained below 0.4. in dna isolated from clinical samples all mismatches in blatem were identified without ambiguity, and 91% of them remained below the rimm limit. since the reduction of the array-hybridisation time to 30 min had no influence on rimm (rimm limit less than 0.6 for 95% mismatch positions), the assay may be performed within 3.5 h while keeping its discriminatory power. conclusion: the blatem gene variants could be amplified by the use of a single consensus primer pair. using dna-array we were able to discriminate snps in 102 of the 106 tem variants. snp mismatches could be analysed by array within 3.5 h enabling the identification of the corresponding esbls or inhibitor resistant tems. the nccls recommendations. the production of extended-spectrum beta-lactamases (esbl) was detected by double diffusion test. the presence of blatem gene was determined by pcr method. transferability of resistance determinants was studied by bacterial conjugation. results: 70.5% of the clinical isolates were resistant to ampicillin (ampi); 76.5% to cefoxitine (cfox); 55.8% to cefotaxime (ctax); 55.8% to ceftazidime (ctaz); 41.2% to ceftriaxone (ciax); 14.7% to cefepime (cepi); 58.8% to azthreonam (aztr); 5.8% to meropenem (merp); 53.0% to gentamicin (gen); 41.0% to tobramycin (tob); 8.8% to netilmicin (net); 5.8% to amikacin (ami); 8.8% to isepamicin (ise); 55.8% to ciprofloxacin (cip). a total of 61.8% of clinical isolates were identified as esbl producers. the presence of blatem gene coding for tem-type beta-lactamases was detected in 82.4% of clinical isolates tested. resistance determinants to all antibiotics tested, with only one exception of merp, were transferable by bacterial conjugation to the recipient strain escherichia coli k-12 3110. frequency of transfer ranged from 7.1 â 10 à9 to 1.2 â 10 à1 . conclusions: the occurrence of resistance to beta-lactam antibiotics was very high. the most efficient beta-lactams were the carbapenem meropenem and the fourth-generation cephalosporin cefepime. aminoglycoside antibiotics netilmicin, amikacin and isepamicin had high efficiency, too. on the other hand, more than one half of the clinical isolates tested were resistant to the fluoroquinolone ciprofloxacin. beta-lactam resistance was due to the production of esbl and to the presence of the bla-tem gene in the majority of clinical isolates. transferability of beta-lactam and aminoglycoside resistance determinants by bacterial conjugation is important from the epidemiological point of view. objectives: today there are very few significant data on the effectiveness of cephalosporin antibiotics in nosocomial infections caused by the microorganisms producing esbl. methods: the cases of nosocomial infections caused by enterobacteriaceae with a proved esbl production were analysed. esbl producing enterobacteriaceae strains were assayed for susceptibility to different antimicrobials and mics were determined by a broth microdilution method. to determine molecular typing of esbl genes polymerase chain reactions and sequencing reactions were used. patients received initial empiric intravenous antibacterial therapy with third-generation cephalosporin (cefotaxime). in case of failure cefepime 4 g a day was prescribed. results of those infections treatment with third-and fourth-generation cephalosporins were assessed depending on mic. results: esbl production with specific shv and ctx oligonucleotids was proved for six strains of enterobacteriaceae in four patients with nosocomial pneumonia (in two cases mixed infection took place), among them four strains were klebsiella spp. and two strains e. coli. the analysis of the dependence of mic on the results of the treatment gave the following results (table 1) . it may be stated that with the proved enterobacteriaceae esbl production mic values for third-generation cephalosporins of the majority of strains were within the resistance range (more than 32 lg/ml), and these antibiotics were not effective in all cases. as for cefepime, mic showed intermediate sensitivity (32 lg/ml) to the drug only in 33.3% cases; the rest of the strains (mic ¼ 1-8 lg/ml) were sensitive. the therapy with cefepime was effective in three of four patients. , à9/9a (n ¼ 4), à1 (n ¼ 3), à3/à22 (n ¼ 2) and à2/ 20 (n ¼ 1). conclusions: (i) using mic breakpoint >1 mg/l for reduced susceptibility to third-generation cephalosporins we detected esblproducing e. coli and k. pneumoniae with low mic-values (0.5-2 mg/l). (ii) cefotaxime-hydrolysis was the dominating profile in esbl-positive e. coli strains whereas ceftazidime was the most sensitive substrate for detection of esbl-production in k. pneumoniae. (iii) the different methods showed almost the same sensitivity in detecting esbl production assuming that more than one substrate was used, i.e. both cefotaxime and ceftazidime. (iv) ctx-m was the dominating esbl-type. objectives: during 2003, the srmd received isolates of escherichia coli for confirmation of esbl production with a phenotype implying a ctx-m-type beta-lactamase, i.e. cefotaxime (ctx) mics fourfold greater than ceftazidime (ctz) mics. isolates were from hospital patients and, in some instances, from community patients with little or no recent hospital contact. tem-and shv-type esbls are largely confined to nosocomial isolates, so the apparent spread of ctx-m enzymes in the community is cause for concern. we compared the isolates and investigated the genetic basis of their ctx-m phenotype. methods: isolates were compared by pfge of xbai-digested genomic dna and data were analysed using bionumerics software. mics were determined by etest or agar dilution, and interpreted using bsac breakpoints. isolates with a ctx-m phenotype were tested for blactx-m alleles by pcr, initially with universal primers, and then with primers specific for various blactx-m groups. selected amplicons were sequenced, either directly or after cloning into pcr2.1. transfer of ctx-m to e. coli j62 was attempted in broth and on agar plates. results: over 100 ctx-m-producing e. coli were obtained from more than 20 uk centres. these isolates represented multiple strains, although clusters of related isolates (>80% similarity) were observed, some including isolates from more than one centre. sequencing confirmed that 12 e. coli from 11 different centres all produced ctx-m-15. most isolates had substantial resistance to ctx (mics >128 mg/l) and ctz (mics >16 mg/l), consistent with ctx-m-15. isolates (n ¼ 25) associated with a large community cluster produced atypically large amplicons with group i ctx-m primers, as did two related isolates from another centre. these isolates were less resistant to ctx (mics 16-64 mg/l) and ctz (mics 1-4 mg/l), and susceptible to gentamicin; sequencing of a representative isolate identified is26 within the terminal inverted repeat of the isecpi element upstream of blactx-m-15, separating the allele from its usual promoter, and the spacer between isecpi and blactx-m-15 had a t/c polymorphism not seen in other sequenced isolates. we studied also antimicrobial sensitivity of the strains, co-resistance to non-beta-lactam antimicrobials and relationship with antibiotic use. methods: data of monthly non-duplicate ebsl-ec and antibiotic use (hospital: ddd/1000 pat-day and community: ddd/ 1000 inhabitants-day) were collected for january 1999 to october 2003. time series dynamic regression models were adjusted to evaluate the relationship between the use of antimicrobials and the emergence of the bacteria. sensitivity testing was determined by microdilution with gram-negative and urine panels (microscanâ). esbl producing strains were initially selected by screening with microscanâgram-negative and urine panels (mic >1 lg/ml for cefotaxime, ceftazidime or aztreonam, and/or a difference of three or more dilutions between ceftazidime and ceftazidime with 2 lg/ml of clavulanic acid . on univariate analysis only connective tissue disease (p < 0.003), genitourinary pathology (p < 0.008), infections in the past year (p < 0.007) and previous exposure to second-generation cephalosporins (p < 0.001) were factors associated with ca infection due to esbl ec. in our regression model, only previous exposure to secondgeneration cephalosporins was strongly associated (or 18.25, . conclusions: in the last 3 years there has been a marked increase in infections due to esbl ec, especially from the community. only previous exposure to second-generation cephalosporins (not to ciprofloxacin, third-generation cephalosporins or aminoglycosides) was predictive of an esbl ec ca infection. strikingly, neither comorbidity nor previous contact with the healthcare system was risk factors for esbl ec. enzymes of the ctx-m family are currently classified as extended-spectrum beta-lactamases (esbls). over the last decade, ctx-m-type enzymes have been increasingly reported from several countries in europe. the aim of this study was to search for ctx-m-type enzymes in escherichia coli isolates obtained at our institution (varese, northern italy). methods: we studied consecutive e. coli isolates recovered over a 2-year period (2000) (2001) (2002) . stains suspected of producing esbls (according to nccls criteria) were further investigated. the double-disk synergy test and etest esbl strips (ab biodisk, solna, sweden) were used to confirm esbl production. the etest method was also used to evaluate mics of amikacin, gentamicin, ciprofloxacin, and beta-lactams (including last-generation cephalosporins, carbapenems, and aztreonam). esbl-positive isolates were evaluated for the presence of ctx-m-type genes using specific dna probes. patient records were examined to assess risk factors for infections and underlying clinical conditions. results: a total of 12 386 consecutive e. coli isolates were studied. overall, 26 out of 124 esbl-positive strains were found to carry a ctx-m-type gene and to produce a ctx-m-type enzyme. most isolates (21/26) showed high mic values for cefotaxime (>32 mg/l) and borderline values for ceftazidime (1-2 mg/l). the remaining five isolates had also high mics for ceftazidime. ctx-m-positive isolates were obtained both from inpatients (n ¼ 17) and outpatients (n ¼ 9). epidemiological analysis showed that most strains were isolated from urinary tract infections, even though some isolates were recovered from the lower respiratory tract, wounds and blood. most patients (20/26) were treated with immunosuppressive therapy. recurrent urinary infections occurred in five outpatients. conclusions: ctx-m-type enzymes appear to be emerging among e. coli isolates in both the hospital and community environments. the analysis of clinical records demonstrated that these microorganisms can cause severe and persistent infections. therefore, despite the currently low prevalence of ctx-m phenotype, we suggest that a monitoring of this resistance phenotype should be established to avoid the spreading of resistance traits. background and objectives: class c beta-lactamases (cbls) are enzymes that confer broad-spectrum beta-lactam resistance (including penicillin, expanded-spectrum cephalosporins, and cephamycins) and are poorly or not susceptible to commercially available beta-lactamase inhibitors. in strains with reduced outer membrane permeability, they can also provide resistance to carbapenems. a number of these enzymes are chromosomally encoded, but plasmid-mediated cbls are also known as a cause of acquired resistance to expanded-spectrum cephalosporins and cephamycins in clinical isolates of enterobacteriaceae. in italy, only the fox-3 acquired cbl has previously been reported, in klebsiella spp. in this work we report the first detection of acquired cbls of the cmy-lat lineage in escherichia coli and klebsiella pneumoniae clinical isolates from an italian hospital. methods: ten consecutive non-replicate clinical isolates of e. coli (eight) and k. pneumoniae (two) resistant to expanded-spectrum cephalosporins and cephamycins were collected, during 2002, at the laboratory of microbiology of the s. matteo hospital of pavia (northern italy). in vitro susceptibility testing was determined by a microdilution method according to nccls. beta-lactamase production was investigated by analytical isoelectric focusing (ief) coupled with a bio-assay. molecular characterisation of beta-lactamase genes was carried out by a multiplex pcr approach designed for detection of all major lineages of acquired cbls genes, and by sequencing. transferability of resistance genes was tested by mating assays in liquid medium. results: two isolates, one of e. coli and one of k. pneumoniae, were found to be resistant to expanded-spectrum cephalosporins, except for cefepime, and cephamycins (cefoxitin mics >128 mg/l). both isolates produced a beta-lactamase of pi >8.4 that showed hydrolytic activity against cefoxitin, cefotaxime and ceftazidime. molecular characterisation revealed, in both cases, the presence of an acquired cbl gene of the cmy-lat lineage, which was compatible with blacmy-2/lat-3 (the leader peptide-encoding region was not sequenced). the cbl determinant was transferable by conjugation from the e. coli isolate, while conjugal transfer was not detected from the k. pneumoniae isolate. conclusions: these findings reveal that acquired cbls of the cmy-lat lineage, which are the most common acquired cbls, can also be encountered in nosocomial settings from northern italy. enteropathogens p764 dissemination of sulphonamide resistance genes: first sul3 found in salmonella from portugal p. antunes, j. machado, j.c. sousa, l. peixe porto, lisbon, p objectives: the purpose of this study was to determine the distribution of sulphonamide resistance genes sul1, sul2 and sul3 and class 1 integrons in portuguese salmonella isolates collected during 2002-2003, from human and nonhuman sources. methods: eight hundred and seventy-five isolates were tested for resistance to 10 antimicrobial agents by the agar dilution method. sulphonamide resistant isolates were screened for resistance genes sul1, sul2, and sul3 and class 1 integrons by pcr assays. results: resistance was found in 54% and multiresistance in 21% of the isolates. in 151 (17%) sulphonamide-resistant isolates (mics 512 mg/l), 118 (78%) sul1 genes, 57 (38%) sul2 genes and nine (6%) sul3 genes were detected. in 29 isolates, more than one gene encoding sulphonamide resistance was present: sul1 and sul2 in 22, sul1 and sul3 in three and sul1, sul2 and sul3 in four. class 1 integrons were found in 77% of those isolates. among the 116 isolates carrying class 1 integrons, 114 presented sul1 gene, found alone (87 isolates) or simultaneously with sul2 (20) or sul3 (3) and with sul2 and sul3 (4). the two strains with class 1 integrons, which lacked the qaced1 and sul1 genes, carried a sul3 gene. of the 118 sul1-positive isolates, 114 harboured class 1 integrons. conclusion: class 1 integrons and sulphonamide resistance genes are widespread among salmonella. the newly described sul3 gene has now been identified in nine salmonella isolates collected from human and nonhuman sources in portugal. salmonella from portugal p. antunes, j. machado, j.c. sousa, l. peixe porto, lisbon, p objectives: the aim of this study was the characterisation of betalactamase production in portuguese salmonella isolates collected during 2002-2003, from human and non-human sources. methods: eight hundred and seventy-five isolates were tested for resistance to 10 antimicrobial agents by the agar dilution method. a double-disk synergy test for the detection of extended-spectrum beta-lactamase production was performed by disk diffusion method. the identification of beta-lactamases was done in ampicillin resistant isolates by ief and pcr assays, with primers, which detects genes encoding tem, pse-1 and oxa group iii enzymes. to evaluate the association of beta-lactamase genes to class 1 integrons, 50cs-30cs primers were used in a pcr assay. pcr products were purified and both strands sequenced. results: in total, 17% of the isolates exhibited resistance to ampicillin, with mics 64 mg/l. resistance to ampicillin was conferred by a tem-1 beta-lactamase in 99 (68%) of the isolates, pse-1 in 37 (25%) and oxa-30 in nine isolates. it is to be noted that there is the detection of the extended-spectrum beta-lactamase (esbl) tem-52 in one isolate. the tem-type beta-lactamases was not associated with class 1 integrons. in contrast, all the blapse-1 and blaoxa-30 genes were inserted in 1200 and 2000 bp class 1 integrons, respectively. conclusion: a considerable percentage of portuguese salmonella were resistant to beta-lactams, mostly due to the production of tem-1 like beta-lactamase and pse-1 inserted in integrons. the detection of an isolate that produce an esbl, such as tem-52, and nine isolates carrying a class 1 integron with oxa-30, are causes of concern due to the possible therapeutic failures with broad-spectrum beta-lactams. p766 increasing incidence of salmonella typhii with reduced susceptibility to ciprofloxacin in kuwait a.a. dashti, p.w.j. west, d. panigrahi suleibikhat, kwt objectives: to determine the current incidence of reduced ciprofloxacin susceptibility in salmonella typhi, to compare with previous data and to investigate the mechanism responsible. methods: 48 isolates of s. typhi collected in 2002-2003 were tested for susceptibility to ciprofloxacin and other antibiotics using the vitek 2 and e-test. isolates showing reduced ciprofloxacin susceptibility were subjected to pcr to determine if a mutation of the gyra gene was responsible. pcr was carried out using two primers (atgagcgaccttgcgagagaaattacaccg) and (ttcc-atcagcccttcaatgctgatgtcttc). results were compared with those for isolates collected from 1995-1997. results: 18 out of 48 (42%) of the isolates were resistant to multiple antibiotics, including ampicillin, chloramphenicol tetracycline and trimethoprim. of these 12 (67%) showed resistance to nalidixic acid and reduced susceptibility to ciprofloxacin (mic 0.125-0.38 mg/l). of the 30 susceptible isolates, seven (23%) showed reduced ciprofloxacin susceptibility. isolates from 1995 to 1997 showed 11% of 53 multi-resistant strains, but none of 100 susceptible isolates with reduced ciprofloxacin susceptibility. pcr results showed mutations of the gyra gene. conclusion: reduced susceptibility to ciprofloxacin in multi-resistant s. typhi has increased from 11% in 1995-1996 to 67% in 2002-2003 and from 0 to 23% in susceptible strains. mutation of gyra is the mechanism responsible. p767 comparison of antimicrobial resistance in diarrhoeagenic escherichia coli isolates causing traveller's diarrhoea between two periods, 1994-1997 and 2001-2003 e. mendez arancibia, j. ruiz, r. cabrera, j. gascon, j. vila barcelona, e objectives: to compare the antimicrobial resistance levels in escherichia coli clinical isolates causing traveller's diarrhoea in two periods, 1994-1997 and 2001-2003. material and methods: presence of enteroaggregative (eaec) and enterotoxigenic e. coli (etec) was established by pcr among those isolated from travellers with diarrhoea during the periods 1994-1997 and 2001-2003 . susceptibility to ampicillin (amp), amoxicillin plus clavulanic acid (amc), tetracycline (tet), chloramphenicol (chl), cotrimoxazole (sxt), nalidixic acid (nal) and ciprofloxacin (cip) was determined by disk diffusion. results: one hundred thirty-two (50 eaec, 82 etec) and 113 (49 eaec, 64 etec) diarrhoeagenic e. coli were recovered during two periods, 1994-1997 and 2001-2003, respectively . the levels of resistance of eaec to all tested antibacterial agents increased in the second period: amp from 52 to 73%, amc from 0 to 10%, tet from 64 to 86%, sxt from 48 to 69%, nal from 6 to 31% and cip from 2 to 16% (p < 0.0001), whereas the leaves of resistance to chl showed a slight decrease (28-22%) but not statistically significant. in etec strains resistance to amp, nal, cip and amc increased from 43 to 50%; 6 to 17%; 1 to 6%; 0 to 6%, respectively, while resistance to chl decreased from 20 to 14%. the levels of resistance to tet and sxt did not present greater differences, but suggested a slight increase in the resistance (57-61% and 50-53% respectively). conclusions: a trend to an increase in the resistance of eaec and etec to amp, amc, nal, and cip has been detected, and the decrease of resistance to cip is worthy of note due to the fact that this antimocrobial agent is considered a first choice treatment for traveller's diarrhoea. a.j. hakanen, s. pitkänen, a. siitonen, p. kotilainen, j. jalava, p. huovinen turku, helsinki, fin objectives: quinolone-resistant salmonella isolates emerged in finland in the mid-1990s. the main origin of these strains is travellers returning from southeast asia. this study was performed to evaluate the incidence and changes of fluoroquinolone resistance in salmonella isolates between 2000 and 2003 in finland. methods: we collected a total of 805 salmonella enterica isolates which were considered to be epidemiologically unrelated. the isolates were divided into two groups (finnish and foreign isolates) on the basis of travel history. the collection was performed in four phases: each year in 2000, 2001, 2002 and 2003 starting in january, we consecutively collected 100 finnish and 100 foreign isolates. mics for nalidixic acid, ciprofloxacin and 10 additional fluoroquinolones were determined by the standard agar dilution method (nccls). results: during the study period, the number of isolates with decreased ciprofloxacin susceptibility (mic of ciprofloxacin >0.125 lg/ml) increased from 16 to 34% of all isolates (p < 0.01). a similar trend could be seen both among the isolates of foreign and finnish origin. in addition, within the non-susceptible population the mic values were increasing. mic50 of ciprofloxacin increased from 0.25 to 0.5 lg/ml among the isolates with decreased ciprofloxacin susceptibility between 2000 and 2003. the respective figures for mic90 were 0.5 and 1 lg/ml. all isolates with decreased ciprofloxacin susceptibility had also increased mics to additional fluoroquinolones. conclusion: the number of salmonella isolates with decreased ciprofloxacin susceptibility continues to grow in finland. moreover, the mic levels of these isolates have increased. this phenomenon might have serious clinical implications. p769 bacteraemia caused by esbl-producing salmonella enterica serovar. virchow 6.7:r:1,2 -a cause for concern a. guleri, g.d. corcoran, s.r. alcock, d.j. brown glasgow, uk background: antibiotic resistance in salmonellae is now common. in developed countries such strains are largely zoonotic and acquire resistance in the animal host before transmission to humans in food. we present our first case of bacteraemic illness with multi-resistant, extended spectrum beta lactamase (esbl) producing non-typhoidal salmonella. case summary: a 34-year-old male, with a history of recent foreign travel was admitted to hospital with a 7-10 day history of gastrointestinal symptoms/fever. on admission he was febrile and splenomegaly was detected. physical examination was otherwise normal. biochemistry revealed mildly deranged liver function. salmonella enterica serovar. virchow 6.7:r:1,2 was isolated from blood culture. it was sensitive in vitro (nccls disk test) to ciprofloxacin and gentamicin but resistant to ampicillin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, co-trimoxazole, nalidixic acid and streptomycin. mic of ciprofloxacin was 0.094 mg/l. antibiotic treatment was with ciprofloxacin, to which he responded well. esbl detection: the isolate was identified as salmonella enterica serovar. virchow 6.7:r:1,2 [api 20 identification system (bio-mérieux), serogrouping, serotyping and phagetyping]. the isolate, resistant in vitro (nccls) to cefotaxime and ceftazidime, was tested for extended-spectrum beta lactamase/ampc production by phenotypic methods. ab biodisk esbl e-tests (cefepime, ceftazidime and cefotaxime, each ae clavulanic acid) and oxoid esbl combination disks (cefpodoxime, ceftazidime, cefotaxime and cefpirome, each ae clavulanic acid) and cefoxitin alone were used based on modified nccls/manufacturer's guidelines. the isolate tested positive for esbl production by both esbl e-tests and combination disks. molecular typing of the esbl is awaited. conclusion: invasive infection with salmonella virchow is uncommon. the source of infection in this case appears to have been undercooked chicken. the emergence of resistance to antimicrobial agents within the salmonellae is a worldwide problem that has been associated with the use of antibiotics in livestock. invasive infection with s. virchow, resistant to broad-spectrum beta-lactams, is a cause for concern. if antimicrobial therapy is indicated for travellers with a history of recent foreign travel, physicians should be aware of the possibility of treatment failures and in such cases mics of third-generation cephalosporins and ciprofloxacin should be determined. the aim of the present study was to assess the distribution and the antibiotic resistance rates (arr) of the various nontyphoidal salmonella serotypes originated from non-human sources in greece, during a 11-year period (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) . material and methods: a total of 753 isolates, belonging to 27 different serotypes, were selected from the collection of national reference center for salmonella and shigella (nrcss), in order to reflect the prevalence of these serotypes during the mentioned period. the sample consisted of 382 isolates from animals, 295 isolates from foods, and 76 environmental isolates. susceptibilities to 10 antibiotics of various classes were determined using mics broth micro-dilution method. results and conclusions: the highest arr and also the higher incidence of multiresistance have been observed for s. virhow, followed by s. hadar and s. typhimurium. the vast part of s. typhimurium isolates was resistant at least to ampicillin, tetracycline and chloramphenicol, while the main resistance phenotype of s. enteritidis isolates was the monoresistance to ampicillin (table) . the arr and the phenotypes of resistance for the isolates of the above four serotypes were similar with the corresponding ones of human isolates as resulted from a recent greek study derived also from nrcss (eur j epidemiol 2001; 17: 751-755) a fact consistent with possible transfer of antibiotic resistant strains from animals to humans through the food chain. the incidence of resistance for the rest of serotypes was very low. all the examined isolates were susceptible to ceftriaxone and ciprofloxacin. interestingly, almost all the examined isolates belonged to animals bred at a non-industrial scale (e.g. pigeons) and the environmental isolates were sensitive to all tested antimicrobials, possibly because of the reduced antibiotic pressure in these isolates. 1996-1998 and 1999-2001 . the isolation rates of s. enteritidis, s. typhimurium and the others were 74.2, 18.2 and 7.6%, respectively, in the first period. in the second period isolation rates were found 51.6, 29 and 18.4%, respectively. antimicrobial resistance for (amp and tmp-sxt) in s. enteritidis, s. typhimurium and others were found (33.0/11.6%), (31. 6 the laboratory data were analysed in 978 shigella spp. isolated from stool materials of adult patients over a 9-year period (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) retrospectively. shigella isolates were identified by standard biochemical reactions and using specific antisera. antimicrobial susceptibility for ampicillin (amp), trimethoprim-sulfamethoxazole (tmp-sxt) and ciprofloxacin (cip) were determined by the disk diffusion method according to national committee for clinical laboratory standards. results: in order to show the differences in epidemiology and antimicrobial resistance, the study was divided into two periods. the first period was from 1992 to1996, and the second period was from 1997 to 2000. a total of 475 shigella spp. isolates were obtained in the first period and 503 isolates in the second period. isolation rates of the strains in the first and second periods were, respectively, for shigella flexneri 55.7 and 21.5%; for shigella sonnei 30.5 and 60.4%; for shigella dysenteria 8.9 and 11.9%; and for shigella boydii 4.8 and 6.2%. the rates of resistance to (amp) in the first and second periods were respectively in s. flexneri 64.9 and 66.7%; s. sonnei 54.5 and 30.5%, s. dysenteria 32.4 and 62.0%; s. boydii 46.0 and 65.0%. the rates of resistance to tmp-sxt in s. flexneri 22.6 and 40.8%; s. sonnei 38.0 and 41.3%; s. dysenteria 23.2 and 33.7%; s. boydii 40.0 and 56.5%. all strains were susceptible to ciprofloksasin. conclusions: s. flexneri was the most common species isolated in the first period and s. sonnei was the common species in the second period. in ankara, the data showed an increase in the resistance to the commonly used antimicrobial agents are ampicillin and trimethoprim-sulfamethoxazole. ciprofloxacin seemed to be the best choice for the treatment of shigelliosis. to determine the antimicrobial resistance in salmonella and shigella strains isolated from stool specimens during a 2-year period, from patients admitted to our clinics with a diagnosis of diarrhoea. methods: the identification and susceptibility testing was done by vitec 2 (biomerieux, fr) automated system. the antibiotics tested for the study were ampicillin, ampicillin-sulbactam, cefotaxime, cefepim, ciprofloxacin, ofloxacin, and trimethoprim-sulfametaxazole. results: nineteen salmonella and seven shigella isolates obtained between 1 january 2002 and 30 november 2003 were tested for their susceptibilities to seven antimicrobial agents. the total numbers of isolates during 1999-2001 (including the year of big marmara earthquake) were 39. five of six shigella isolates were s. sonnei, one was s. flexneri. thirteen of 19 salmonella isolates were s. typhimurium, three were s. enteritidis, two were identified as salmonella spp., one was s. arizonae. although all of the isolates were found susceptible to the therapeutic agents, ampicillin susceptibility was decreased to 78% from 100% and trimethoprimsulfametaxazole susceptibility was decreased to 89% from 100% in salmonella strains during a 2-year period. only one strain was resistant to cefotaxime. no resistance was found against ofloxacin and ciprofloxacin. all of the shigella isolates were susceptible to all tested antibiotics. conclusions: (1) the incidence of salmonella and shigella infections seemed to decrease significantly over a 5-year period. (2) s. typhimurium and shigella sonnei are the most commonly identified serotypes. (3) there is no significant change in resistance to 'old' and 'new' antibiotics. (4) all of the isolates showed a very good sensitivity all the antimicrobials tested. (5) a careful rotational use of antibiotics might be the best policy to make old drugs again active, and abuse of new agents. objectives: since 1996 the incidence of human campylobacteriosis has shown a significant increase in austria. consumption of contaminated poultry products is a well known risk factor for human infections. during the slaughter process meat products can become contaminated with intestinal organisms. furthermore antibiotic resistance is increasing in humans and animals. the aim of the study was to determine the resistance patterns and the transmission routes of campylobacter sp. on the chicken-carcasses along the slaughter line. to compare the frequency of isolation and occurrence of antimicrobial resistance among c. jejuni and c. coli isolated in humans, retail poultry meat and farm broilers in 2002. methods: fifty-three human, 51 retail poultry meat and 15 campylobacter spp. isolates from broiler cloacal swab were investigated for antibiotic susceptibility to 12 antimicrobials by disk-diffusion method. mics were further determined for erythromycin-and ciprofloxacin-resistant isolates by etest. to confirm ciprofloxacinresistance we used a mismatch amplification mutation assay (mama) pcr to detect the gyra mutation. species were determined by multiplex pcr and genetic diversity by pfge typing. results: c. coli isolated in significant proportion in all three sources, 27.8, 56.9 and 53.3%, respectively. resistance to one or more antibiotic tested was 71.7, 89.5, and 53.8% and multiresistance 30.2, 41.7 and 30.85% in human, retail poultry and farm isolates, respectively. no significant difference was found in the overall resistance rates, and for each antibiotic tested between c. jejuni and c. coli isolates from all three sources, which is unusual finding. moreover, they were higher in c. jejuni. given that after the war population in this region were mostly the muslim, c. coli in humans originated from other sources than pigs. thus, it may suggest that c. coli resistance is origin-related. erythromycin-and ciprofloxacin-resistance was high and almost equal in all three sources (30.2, 30.6, 38.5%, and 32.1, 26.5, 30.8%, respectively) .imported retail poultry meat (from five countries) had higher resistance rates for erythromycin than domestic one, 38.7 vs. 26.7%, but ciprofloxacin resistance was higher in domestic one, 38.7 vs. 22.2%. conclusion: the distribution of c. jejuni and c. coli species and drug resistance in isolates from chicken and farm animals were similar to that seen in humans, even in the absence of antibiotic pressure (57% of patients were under 6 years of age, and growth promoters ban in bosnia and herzegovina), suggesting that poultry may play a role in human infections. when pfge patterns were considered, they were remarkably diverse, suggesting considerable genetic heterogeneity. it may support the hypothesis that campylobacter spp. from food animals and humans may not be represented by discrete populations but rather, form part of a common population shared by food animals and humans, suggesting related sources of infection. objectives: this investigation was designed to study the potential usefulness and economic benefits of oral linezolid as an alternative to outpatient parenteral antibiotic therapy (opat) in the treatment of primary cellulitis. methods: patients with moderately severe cellulitis referred to an infusion centre for antibiotic treatment were enrolled into an open, non-randomised, pilot study. after informed written consent, patients were treated with oral linezolid, 600 mg q12 h, in place of their prescribed parenteral antibiotic. patients were followed with clinic visits and lab monitoring. results: a total of 10 patients, five males and five females (mean age, 49 years), were enrolled. seven were obese (mean weight, 146 kg; range, 101-196 kg), six had lower extremity cellulitis, one had lymphedema, and two were smokers. the average length of linezolid therapy was 12 days (range, 5-27 days). all were compliant with the treatment regimen and had a clinical cure of their infection. mild side-effects (nausea, loose stools, headache, metallic taste) were reported by four patients. none developed thrombocytopenia or prematurely discontinued therapy. a 12-day course of linezolid therapy (drug costs, clinic visits, and lab monitoring) was found to be less expensive than 4 days of vancomycin treatment (1 g q12 h) in the infusion centre. conclusions: in this study, we found that oral linezolid was safe and effective in the treatment of moderately severe cellulitis and could be a suitable replacement for opat. furthermore, oral linezolid has the potential to improve patient satisfaction as well as lower overall treatment costs when compared with opat. objective: vancomycin (v) in combination with rifampin (r) and gentamicin (g) has been the recommended regimen for the treatment of pve caused by mrsa but intolerance to these agents and emergence of mrsa strains with reduced susceptibility to the glycopeptides create the need for alternative agents. we describe the case of a patient with mrsa tricuspid pve who was successfully treated with linezolid (l) after failure of glycopeptides. case: a 67-year-old man was admitted for persistent mrsa bacteraemia. he had been treated with v, g, r, and trimethoprim/ sulfamethoxazole (t/s) for mrsa pve of the tricuspid valve which had recurred after a 45-day course of v and a 60-day course of t/s. due to acute renal failure g was discontinued and v was changed to teicoplanin (t). he was transferred to our department because of persistent bacteraemia of 20 days duration despite adequate blood levels of t. blood cultures revealed a mrsa strain with mics of v, t, and l of 1, 2, and 0.75 mg/l, respectively. he was started on l (600 mg bid) and r (300 mg tid) and bacteraemia cleared the seventh day of treatment. he completed a 6-week course of l and a 3-week course of r. during his treatment he developed anaemia which was managed with blood transfusions and erythropoetin, mild leucopenia and mild thrombocytopenia. he was discharged afebrile with sterile blood cultures and a tee showing reduction in the size of the vegetation. the patient remained well and blood cultures were sterile one month later while pancytopenia fully recovered. the mrsa isolate was investigated for heteroresistance to glycopeptides with (1) a simplified and (2) a detailed population analysis profile method. (1) 0.01 ml of a 10 8 cfu/ml bacterial suspension was plated on bhi agar with v (4 mg/l). subclones that grew after 48 h were submitted to mic determination. (2) tenfold serial diluents of an inoculum of 10 8 cfu/ml were plated on bhi agar plates with increasing concentrations of v or t alone or with 4% nacl. viable colonies were counted at 48 h and plotted against the antibiotic concentration. subclones with v mic 4-6 mg/l were identified, suggesting that the isolate had heterogeneously reduced susceptibility to v which probably explained the failure of v treatment. the population curve showed that 50% of the original inoculum survived on t concentration !8 mg/l suggesting heteroresistance to t. in all three studies, patients treated with lnz had shorter intravenous antibiotic treatment (ivat) duration than patients in comparator groups, which results in increased rates of early patient discharges and reduced use of resources. in two of the studies, patients had significantly shorter mean los and greater odds of early discharge from hospital. indeed, compared with tei, treatment with lnz had 66% greater odds of early discharge (p ¼ 0.049); a similar early discharge potential was also seen when lnz was compared with van (p ¼ 0.005). in select patient populations, such as those with cssti due to suspected/confirmed mrsa, reduction in los may be even more pronounced in lnz-vs. van-treated patients. for the cost comparison in the third study, total mean adjusted cost was also reduced by us $335 (p > 0.05) in the lnz group compared with the tei group in patients from south america and mexico. conclusions: across multiple studies, there is consistent evidence of significant reductions in los and ivat associated with lnz treatment, with significant differences in the rate of early patient discharge. therapy with lnz shows pharmacoeconomic advantages that have the potential to reduce total costs of treatment. in vitro activity against mrsa and its oral administration represents an excellent alternative to iv vancomycin, which is currently recommended for cf patients colonised with mrsa. material and methods: oral lnz (600 mg/12 bid) was administrated in two male cf patients (25 and 29 years) during 14 and 10 days, respectively. s. aureus isolates were cultured from different sputum samples recovered before, during, and after lnz treatment. antibiotic susceptibility was performed by nccls microdilution method using the wider system (fco. soria melguizo, s.a., madrid, spain). pfge-smai was applied to analyse genetic relatedness of s. aureus isolates. results: in the first patient, a total of 10 isolates were analysed during the studied period; six of them recovered in the previous year to lnz administration, two isolates during lnz administration period, and two isolates 4 and 6 months after the end of treatment. with the exception of one isolate that was methicillinsusceptible recovered during lnz treatment period, all isolates were mrsa and presented homogeneous antibiotic susceptibility pattern. a single clone, with a subtype variant that included two isolates, was identified in all isolates, except in the meticillin-susceptible one. in the second patient, two mrsa and one meticillinsusceptible isolates were recovered during 5 months before lnz therapy. another methicillin-susceptible isolate was recovered after the lnz therapy and no s. aureus were identified in the following cf controls during 8 months. mrsa isolates shared the same pfge and antibiotic susceptibility pattern, whereas meticillin-susceptible isolates corresponded to two different clones unrelated with the mrsa clone. independently of microbiology results, patients' pulmonary function remains unchanged after lnz administration. conclusion: oral lnz treatment in cf may affect population dynamics of s. aureus colonisation, being effective in mrsa eradication. despite this fact and assuming the brief follow-up period, maintenance or eradication of mrsa colonisation after lnz treatment seems not to affect pulmonary function, which may be related to the uncertain role of this pathogen in cf patients. p788 in vitro spectrum of linezolid and other agents against clinical isolates of anaerobes k. aldridge, c. manders, s. broyles new orleans, usa objectives: linezolid is an oxazolidinone antimicrobial with established in vitro and in vivo activity against aerobic gram-positive cocci. in infections such as wounds these gram-positive pathogens may be mixed with other pathogens including anaerobes. the role of linezolid as an anti-anaerobe agent has yet to be determined. this study was performed to establish the in vitro activity of linezolid and comparative agents against recently isolated anaerobes. methods: approximately 700 anaerobes were tested for susceptibility to linezolid (lzd), ceftriaxone (axo), cefoxitin (fox), clindamycin (cl), and metronidazole (mrd) using twofold dilutions (0.06-256 mg/l) of each agent using the nccls-recommended broth microdilution method. the sources of test isolates included wounds, abscesses, body fluids, and tissues. results: against all test isolates lzd had an mic range of 0.06-128 mg/l, a mode mic of 4 mg/l, and mic50 and mic90 values of 2 and 4 mg/l, respectively. lzd activity was judged by percentage of isolates inhibited at 2 and 4 mg/l. overall lzd inhibited 51 and 96% of isolates at 2 and 4 mg/l, respectively; at 2 and 4 mg/l, respectively, lzd inhibited 35 and 95% of bacteroides fragilis group; 63 and 92% of clostridium isolates; 71 and 97% of prevotella isolates; 100% of fusobacterium isolates; and 100% of peptostreptococcus isolates. by comparison of mic90 values lzd was 2-to 64-fold more active than axo, 0-to 16-fold more active than fox, and 2-to 32-fold more active than cl against these same groups of isolates. lzd and mrd had virtually equal in vitro activity. interestingly, all isolates with mics of 8 mg/l or higher to lzd had mics of 2 mg/l or less to mrd, while isolates with mics of 8 mg/l or higher to mrd had mics of 4 mg/l or less to lzd. conclusions: based on these results and arbitrary use of nccls breakpoints for gram-positive isolates, we conclude that lzd is highly active against anaerobe pathogens, but this needs to be verified by pharmacokinetic and clinical studies. results: on comparing the mics from the current study with the results from 7 years ago, an unmistakable and very alarming decline in susceptibility was noted for all the antimicrobial agents tested. the greatest difference in susceptibility was noted for cefoxitin (from 91 to 62%), metronidazole (from 98 to 78%), piperacillin (from 84 to 68%) and amoxicillin (from 74 to 60%). the antimicrobial agents for which <5% decrease in susceptibility was found, included meropenem (from 96 to 93%), clindamycin (85 to 81%) and ciprofloxacin (from 74 to 69%). a great concern, however, was an 8% decrease found in the susceptibility for imipenem (from 96 to 88%). conclusions: a decade ago, most anaerobic bacteria were susceptible to antimicrobial agents usually used for infections caused by these bacteria. the results from this study, however, indicate a situation that has undergone some dramatic changes in a relatively short period. it is of concern that the agents most frequently used in the empirical treatment of anaerobic infections, such as metronidazole and the b-lactams such as cefoxitin and piperacillin have shown the most alarming decrease in susceptibility. there is now, more than ever before, a definite need for continuous susceptibility testing of anaerobes and a serious restructuring of the treatment regimes for anaerobic infections. a. camarda, d. pennelli, p. battista, m. corrente, i. alloggio valenzano, i objectives: escherichia coli isolated from fattening-rabbits dead for enteritis were biotypised, tested with pcr for the presence of virulence genes eae and afr2 coding for intimin and the fimbrial adhesin af/r2 and investigated for antimicrobial resistance. methods: fifty-six strains of e. coli isolated in 28 farms were biotypised using the fermentation of sorbose, dulcitol, raffinose, sucrose and l l-rhamnose. detection of drug resistance was determined using the method of kirby-bauer on mueller-hinton agar with antibiotic disks containing gentamicin (gm10), amikacin (an30), tetracycline (te30), erythromicin (e15), spiramicin (sp100), enrofloxacin (enr5), flumequine (ar30), trimethoprim/ sulphametoxazole (sxt), amoxicilline (amx 25), apramycin (apr30), difloxacine (dfx10), marbofloxacine (mar5), nalidixic acid (na30), neomycin (n30), colistin (cl50), streptomycin (s10). results: 10 biotypes (b0, b1, b8, b9, b12, b16, b17, b24, b25, b28) were detected: biotypes 8 and 24 were predominant in rabbitries. eae and afr2 genes were almost observed in e. coli strains belonging to them. results of antibiograms have shown that all the isolates (100%) were e15 resistant. high rate of resistance were also found towards sp100 (98.2%), sxt (92.8%), te30 (87.5%), s10 (73.2%), gm10 (71.4%), n30 (69.3%). about 91% of e. coli tested showed the same susceptibility rate (91.5%) to mar5 and cl50. susceptibility to dfx10, enr5, ar30, na30, was exhibited by the 80.3, 78.5, 75, 71.4%, of the strains, respectively. sensitivity against amx25 was quite high (76.7%). multiple antibiotic resistance was expressed by all e. coli tested. the most prevalent resistotypes were resistant to te30-e15 sp100-sxt, detected in 47 strains (83.9%), te30-e15-sp100-sxt-s10, which accounted for over 60% and te30-e15-sp100-sxt-gm10 detected in 67.8% of isolates. conclusions: no significant correlation was observed between enteropathogenic e. coli (eae+ and af/r2+) and pattern of antibiotic resistance. quinolones have shown very good activity; in particular mar5, which has been recently adopted in veterinary medicine seems to possess high efficacy. on the other hand, e. coli strains exhibited high-level of resistance to antimicrobials. like human e. coli, rabbit strains revealed different patterns of multiresistance, which could make disease control difficult in rabbits and also promote dissemination and increasing of antimicrobial resistance in human strains. objective: to determine the frequency and susceptibility patterns of bacterial pathogens isolated from bloodstream (bsi) of haematology-oncology patients hospitalised at latin american medical centres. material and methods: as part of the sentry antimicrobial surveillance program, a total of 1587 bsi isolates were recovered from haematology-oncology patients from 1997 to 2002. the isolates were susceptibility tested to >20 antimicrobial agents in a central laboratory using nccls broth microdilution method. results: the most frequent isolated pathogen was coagulase-negative staphylococci (cons; 17.7%), followed by escherichia coli (17.5%), staphylococcus aureus (15.8%), klebsiella pneumoniae (10.3%), pseudomonas aeruginosa (8.9%), enterobacter spp. (6.7%), acinetobacter spp. (4.5%), and enterococcus spp. (3.1%). oxacillinresistance rates were 33.9 and 74.4% among s. aureus and cons, respectively, isolates. the prevalence of esbl-producing strains ranged from 9.4% for e. coli to 41.1% for k. pneumoniae. for enterobacter spp., susceptibility rates were 54.7 and 86.9% to ceftazi-dime and cefepime, respectively. all enterobacteriaceae isolates tested were susceptible to carbapenems. the susceptibility of p. aeruginosa to imipenem and meropenem was 83.7 and 86.5%, respectively; 82.2 and 92.8% of the gram-negative bacilli were susceptible to cefepime and meropenem, respectively. only 4.1% of the enterococcus spp. isolates were resistant to vancomycin. conclusions: in contrast to american and european reports, gram-negative bacilli represented the major cause of bsi among haematology-oncology patients in the latin american hospitals evaluated. the antimicrobial agents with the best covered against such pathogens were the carbapenems and cefepime. however, none of the evaluated antimicrobial agents inhibited the growth of 100.0% of the gram-negative bacilli. thus, continued monitoring by surveillance programs is necessary to determine if the observed trends would continue to be recorded. objective: we attempted to verify if the frequency of occurrence (fo) and antimicrobial susceptibility profile (asp) of bacterial isolates responsible for causing bloodstream infections (bsi) in paediatric patients varied along the years and age categories. methods: a total of 2450 bloodstream isolates were collected from paediatric patients hospitalised in latin american hospitals through the sentry program between 1997 and 2002. the asp to various antimicrobials was determined by the nccls broth microdilution method. the fo and asp were studied according to age categories (ac): 1 year, 2-5 years, and 6-12 years. results: overall, s. aureus (sa) was the most frequently isolated pathogen among children 1 year (18.3%) and 6-12 years (26.8%) followed by coagulase negative staphylococci (cons). among children 1 year and 2-5 years, s. pneumoniae (spn) ranked among the top five pathogens. in contrast, it has caused less than 5.0% of bsi among children 6-12 years. curiously, in this age group, acinetobacter spp. and p. aeruginosa (5.8%) assumed the fifth position in the rank order of frequency. in general, among sa, the oxacillin resistance (or) rates were lower in the 6-12-year-old ac (17.2%; p 0.05) than in children 1 year (27.3%) and 2-5 years (25.0%). in contrast, among the cons, elevated rates of or were noticed in all acs ( 1 year, 82.1%; 2-5 years, 81.2%; 6-12 years, 78.3%; p > 0.05). esbl-producing k. pneumoniae were more frequently detected in the ac 1 year (66.8% of the k. pneumoniae isolates) and 2-5 years (64.1%) than 6-12 years (52.0%). on the contrary, esbl-producing e. coli isolates were less frequently encountered among children 1 year (12.1%) than children !6 years (21.9%). however, these differences did not reach statistical significance (p > 0.05). spn isolates showing reduced susceptibility to penicillin were detected more frequently in the ac of 1 year (39.2%) and 2-5 years (32.7%) than in 6-12 years (15.0%; p 0.05). conclusions: although only slight differences in the fo of bsi pathogens was noticed along the years and ac, important differences were observed on the asp of the bsi pathogens according to the age categories, especially for spn and sa isolates. objectives: the aim of this prospective, multicentric study was to assess incidence of gram-positive bacteria in bloodstream infections (bsi) and characteristics of their resistance to antibiotics in the czech republic. methods: the study was done in 15 sites in the czech republic from january to april 2003. consecutive gram-positive strains isolated from blood were assessed and their clinical significance was evaluated. results: the strains of staphylococcus aureus (39%), coagulase-negative staphylococci (34%), streptococcus pneumoniae (11%) and enterococcus spp. (9%) were identified as the etiologic agent of gram-positive bsi. the frequency of oxacillin-resistant strains was in staphylococcus aureus and in coagulase-negative staphylococci 10 and 41%, respectively. all streptococcus pneumoniae strains were susceptible to penicillin and chloramphenicol. no strains resistant to glycopeptides were found in enterococci. clinical significance of isolated gram-positive bacteria was significantly conditioned by bacterial species (p ¼ 0.001) and reached 100% in streptococcus pneumoniae, 87% in staphylococcus aureus, 82% in enterococcus spp. strains and 10% in coagulase-negative staphylococci. production of bacterial biofilm was shown in 56% staphylococcus aureus strains and in 42% coagulase-negative staphylococci. bsi was the immediate cause of death of the patient in 5%. conclusion: we could confirm that presence of artificial material means significant risk factor for bsi. catheter-related infections were present in 32% of cases. forty-six per cent of bsi can be characterised as secondary and pneumonias, git infections and urinary tract infections were the most common sources. the frequency of staphylococcus spp. with positive finding of biofilm was 49% in this study; this finding supports its clinical significance. methods: a total of 6670 s. aureus positive sample isolates between 1 january 1997 and 31 december 2002 from the laboratory were analysed. the susceptibility to antibiotics was assessed by antibiogram based on the api system (biomérieux ò ) according to the french guidelines (ca-sfm). the sa strains were classified as methicillin susceptible and gentamicin susceptible (gentas-mssa), methicillin resistant and gentamicin susceptible (gentas-mrsa), methicillin susceptible and gentamicin resistant (gentar-mssa) or methicillin resistant and gentamicin resistant (gentar-mrsa). the number of isolates was calculated for 1000 admissions. means per year were compared using kruskal wallis test. the spearman coefficient (r) was used to calculate the correlation between the proportion of isolates (for each antibiotic resistance profile) and months. results: the overall proportion of sa positive samples for 1000 admissions during the study period was: 11.2, 12.0, 12.7, 11.9, 11.4 and 11.8 for 1997, 1998, 1999, 2000, 2001 and 2002 , respectively (r ¼ à0.2; p ¼ 0.9). the percentage of mssa was 76.2 (75.7 for gentas, 0.5 for gentar) and the percentage of mssa was 23.8 (20.1 for gentas, 3.7 for gentar) for the total period. patients with mrsa were older (57.3 years) compared with patients with mssa (mean age 41.5, p < 0.0001) but patients with gentar-mrsa were younger (53.2 years) compared with patients with gentas-mrsa (mean age 58.1, p ¼ 0.006). the proportion of gentas-mssa for 1000 admissions was similar by time (9.4, 9.0, 9.9, 8.8, 8.2, 9 .0, p ¼ 0.5) (r ¼ à0.1; p ¼ 0.5). however, the proportion of gentas-mrsa strains increased significantly (1.7, 2.4, 2.4, 2.5, 2.9, 2.5, p ¼ 0.001) (r ¼ 0.4; p < 0.0001) while the proportion of gentar-mrsa strains decreased significantly during the period (0.6, 0.6, conclusion: although the proportion of sa positive samples for 1000 admissions remains constant during the last 6 years, there is a continuous increasing trend of isolates with gentas-mrsa and a decreasing trend of isolates with gentar-mrsa. the age difference between these two sub-groups should be explored. greek region -corfu island e. gatsouli, m. ovrenovits, a. pasxali, a. tzanavari corfu, gr background: in order to assess the regional trends of microbiological resistance pattern, all cultured bacteria isolated in 2003 in our laboratory were reviewed as to specimen source and susceptibility profile. materials and methods: in 2003, 7220 samples were cultured, 75% (5415) of hospitalised patients and 25% (1805) from ambulatory patients. the samples were: 3760 urine, 1276 blood cultures, 580 lesions and 1604 samples of other secretions. classic culture methods, vitek system and nccls breakpoints were used. results: cultivations were positive in 23% (1661, 1381 adults and 280 children samples). the distribution of bacteria differed according to the types of specimens. the distribution of 1147 gram(à) was 1003 enterobacteriaceae and 145 nonfermentative bacilli. there were 460 gram(+) cocci and 54 yeasts, too. e. coli predominated in enterobacteriaceae (65%), followed by klebsiella sp., p. aeruginosa in non-fermentative bacilli (70%) and a. baumanii (29%). among the gram(+) s. aureus was the most frequent (42.5%), followed by cns. ampicillin inhibited growth of 35% for e. coli. ttime/sulfa combination could inhibit less than 16% and the second-generation cephalosporins less than 25%, while fluoroquinolons were very effective against enterobacteriaceae strains (more than 95%). piperacillin inhibited growth of 8% of p. aeruginosa and quinolons less than 17%. enterococcus strains were highly sensitive to teicoplanin (100%) and nitrofurantoin (97.9%). mrsa were 31% but gisa were 1%. a. baumanii and gisa were in icu. conclusion: a permanent surveillance of frequency and sensitivity levels of the most common pathogens responsible for infectious enables to identify local antimicrobial activity and plays a key role in starting empiric therapy pending bacterial identification and in vitro assay. objectives: the biochemistry and genetics of antibiotic resistance are well documented; however, information regarding the medical and social factors that influence its occurrence remains lacking. the aim of this study was to elucidate these latter relationships and to examine the dynamics of their effects. methods: antibiotic resistance data for bacterial isolates obtained from the community was collected from all microbiology laboratories in wales from 1996 to 2003. antibiotic prescribing data, practice demographics, deprivation indices, general practitioner demographics, and details of sampling behaviour was also obtained for the same period for all general practices in wales. initial analyses exploring the nature of these data and the relationships of the various components were undertaken using excel and spss. results: preliminary analyses indicate that both antibiotic resistance and prescribing varied between practices. for coliform utis, there was a clear association between high prescribing and higher levels of resistance, with prescribing accounting for 10-20% of variation in resistance. the correlation between prescribing and resistance was not confined to the urinary coliforms but seen throughout a range of pathogens including those responsible for respiratory and skin infections. there was an association between resistance and social deprivation exceeding that expected from high prescribing in deprived areas and an apparent association between resistance and the number of practitioners in a practice and the practice list size. resistance was more common in infections in the young (<10 years), the aged (>60 years) and for some pathogens resistance was significantly greater in males. multilevel modelling, regression analysis and time series analysis of this complex data set is in progress. conclusions: antibiotic usage appears to affect resistance at practice level and the dynamics of this selection process are currently being investigated. it is hoped that these studies will assist in the design of interventions to limit the future impact of resistance and contribute to our ability to predict their outcomes. objectives: data about the prevalence of antimicrobial resistance in indonesia are limited. the amrin study measured the prevalence of antimicrobial resistance in the indonesian population inside and outside hospitals. methods: 4000 individuals were targeted to be screened constituting four different populations in each of two cities: patients admitted to hospital, patients discharged from hospital, patients visiting primary health centres, and relatives of patients admitted to hospital. nasal swabs and rectal swabs were taken and cultured using phenol red mannitol agar for the isolation of staphylococcus aureus, and chrom agar orientation medium for escherichia coli. susceptibility testing was performed by disk diffusion method recommended by nccls. results: 3996 individuals were included in the study between july and october 2001 in surabaya and between january and may 2002 in semarang equally distributed over the four groups and two cities. s. aureus isolates (n ¼ 298) were frequently resistant to tetracycline (23%) and oxacillin (7%) without obvious differences between the four populations. none of the oxacillin resistant strains of s. aureus harboured mec a gene. e. coli isolates (n ¼ 3284) showed considerable levels of resistance against a number of commonly used antibiotics. the highest levels of resistance to ampicillin (72.5%), chloramphenicol (42.5%), gentamicin (17.5%), cefotaxim (12.5%), ciprofloxacin (22.5%), and cotrimoxazole (55.5%) were among e. coli isolated from patients on the day of discharge from hospitals. resistance rates were consistently lowest among e. coli from relatives of patients on admission to hospital and among patients visiting primary health care centres. conclusions: the results show that antimicrobial resistance among common bacterial pathogens has emerged in indonesia. among e. coli the prevalence of resistance to ciprofloxacin and other antibiotics is remarkable high, especially in individuals after hospitalisation. although the prevalence of mrsa is low, tetracycline resistance is common among s. aureus and not associated with hospital stay. methods: isolates from patients with invasive diseases caused by haemophilus influenzae (hi), neisseria meningitidis (nm), group a streptococcus (gas), and group b streptococcus (gbs) were forwarded to reference laboratories in alaska (2000 alaska ( -2003 , canada (2000 canada ( -2003 , and greenland (2001 greenland ( -2003 for confirmation and serotyping. chart reviews were conducted on confirmed cases to verify illness episode information. data reported for 2003 are preliminary. results: the total numbers of reported cases were 86 hi, 34 nm, 126 gas, and 92 gbs. crude annual rates of invasive disease per 100 000 population varied by country and organism [hi conclusion: native peoples of ak and n can have high rates of invasive bacterial disease caused by hi, nm, gas and gbs. overall rates of nm disease are higher in gn than ak and n can. cases of invasive hib disease continue to occur in children <2 years of age. rates of hia appear to be elevated in n can and increasing in ak, however, caution needs to be used when interpreting rates due to the small number of cases. this trend merits further surveillance. elevated case fatality rates in ak for hi and nm also warrant further investigation. objectives: for tertiary care hospitals, knowing the local patterns of spectrum and susceptibility at the referring institutes can add significantly to the selection of appropriate antimicrobial therapy. our objective was to get information regarding the region specificity, frequency of occurrence and pattern of antimicrobial susceptibility of common bacterial infections in central illinois. methods: we used hospital antibiogram data to assess predominant pathogens and pattern of in vitro antimicrobial susceptibility of bacterial infections in the four regions (west, southwest, central and south) of central illinois from january 2001 to june 2002. results: gram-negative bacteria were predominant in four regions (57, 48, 63 and 52% respectively). in all regions, e. coli was the most common organism (20, 22, 32, and 25%) followed by s. aureus (17, 21, 14, and 24%). e. faecalis, p. aeruginosa, and k. pneumoniae were also among the five most frequently reported species. on the other hand, the frequency of occurrence of s. pneumoniae was 1-3% in the four regions. the pattern of methicillin-resistant s. aureus was different in the four regions (27, 53, 34, and 42%) with only 0.1% of the total number of s. aureus showing intermediate resistance to vancomycin. e. faecalis, 99, 91, 96 and 96%, respectively, were susceptible to vancomycin. susceptibility of s. pneumoniae to penicillin was almost the same in the four regions (67, 74, 64, and 75%). it was not surprising that p. aeruginosa was the least susceptible species among gram-negative bacteria, and this species showed decreased susceptibility to gentamicin (78, 80, 77, and 55%) and to ciprofloxacin (73, 81, 67, and 67%). conclusions: our data show that different communities in central illinois have variable occurrence and pattern of antimicrobial susceptibility of common bacterial infections. we plan to formulate a regional antibiogram, distribute it to all hospitals in the area, and follow the patterns prospectively with renewal of the antibiogram once a year. p800 antimicrobial resistance surveillance of gramnegative anaerobic bacteria isolated in six greek hospitals j. papaparaskevas, n.j. legakis, a. katsandri, a. avlamis -the hellenic study group for gram-negative anaerobic bacteria objectives: the antimicrobial resistance surveillance of gram-negative anaerobic bacteria isolated in six greek hospitals. methods: a total of 122 gram-negative anaerobic clinical strains (72 bacteroides fragilis group, 17 other bacteroides spp. non-fragilis, 25 prevotella spp., 4 fusobacterium spp. and 4 miscellaneous) isolated during the period november 2002 to november 2003 were tested using the etest method on brucella blood agar plates. incubation in a chellab 1.5 anaerobic chamber was performed for 48 h and interpretation was according to nccls guidelines. results: overall gram-negative non-susceptible (intermediate and fully resistant) rates to penicillin, ticarcillin + clavulanic acid, cefoxitin, tetracycline, clindamycin, metronidazole, imipenem and ertapenem were 83, 2, 31, 55, 32, 6, 1 and 5%, respectively. bacteroides fragilis group rates were 93, 4, 38, 65, 31, 1, 1 and 7%, respectively. prevotella spp. rates were 68, 0, 8, 32, 36, 16, 0 and 0%, respectively. overall gram-negative mic90s were 256, 2, 64, 128, 256, 2, 0.5 and 1 mg/l, respectively. bacteroides fragilis group mic90s were 256, 4, 128, 128, 256, 1, 1 and 4, respectively. prevotella spp. mic90s were 256, 1, 16, 64, 256, 256, 0.125 and 0.5, respectively. metronidazole resistance was detected among four prevotella spp., one bacteroides spp., one porphyromonas spp. and one fusobacterium spp. isolates. additionally, a b. fragilis strain was found highly resistant (mic > 32 mg/l) both to imipenem and ertapenem and resistant to all other antimicrobials tested except metronidazole. conclusions: carbapenems, beta-lactam + inhibitor combinations and metronidazole remain the antimicrobial agents of choice against most gram-negative anaerobes. however, metronidazole resistance seems to be an emerging problem in greece, especially among prevotella spp. isolates. in that respect species identification and periodic susceptibility surveillance is mandatory. imipenem and ertapenem activity was comparable, though ertapenem mics were slightly higher. acknowledgements: members of the hellenic study group for gram negative anaerobic bacteria are drs a. avlamis, c. koutsia-karouzou, c. kontou-kastelanou, a. pangalis, e. papafrangas and e. trika-grafakos. the value of quality control strains in susceptibility tests k. huppertz, i. noll, b. wiedemann and the genars group objectives: the goals of a quality control programme are to assist in monitoring the precision and accuracy of the susceptibility test procedure, the performance of reagents used in the test and the performance of persons who carry out the tests and read the results. they are best accomplished by the testing of quality control (qc) strains with known susceptibility to the antimicrobial agents to be tested (nccls). therefore, qc strain measurements done by laboratories taking part in the genars-project (german network for antimicrobial resistance surveillance) were used for a comparison of the performance of three different methods for mic determination. methods: in the genars-project two commercial mic test systems and one manual microdilution system according to nccls are used for the determination of antimicrobial susceptibility. the commercial systems are the vitek 2 (biomérieux) and the micronaut system (merlin diagnostics) with 384-well microtitre-plates. qc strains measured by all test systems were evaluated for those antibiotics where a range of ae1 dilution step of the modal value of the respective qc strain is included in the range of concentrations tested. for reliable assessment of the test quality the distance of the modal value from the lowest and highest concentration tested has to be two or more dilution steps. results: from a multitude of antibiotics tested only few drugs are tested with a range of concentrations which meets the above mentioned requirements. table 1 indicates the number of test combinations available for evaluation. the vitek 2 system offers the shortest ranges of concentrations. however, from the range of concentrations only few are tested, while the others are calculated, e.g. for gentamicin the range includes six concentrations while only three are measured (ast-p526). conclusions: an evaluation of qc strain measurements should be possible for all antibiotics tested. however, due to the concentrations chosen and the short ranges of concentrations available in the different test-systems only few antimicrobial agents can be used for a comparison of the performance of the test methods. therefore, either the range of concentrations has to be extended, or more suitable qc strains have to be implemented in a way that their mics fall into the range of concentrations which are sufficient in clinical terms. p802 lack of evidence for dna in antibiotic preparations as a source of antibiotic resistance genes s.k.p. lau, p.c.y. woo, a.p.c. to, a.t.k. lau, k.y. yuen hong kong, hk objective: to investigate the significance of dna encoding antibiotic resistance genes present in antibiotic preparations in the rapid development of antibiotic-induced antimicrobial resistance. methods: a comprehensive study using sequence alignments and phylogenetic analysis of genes encoding antibiotic resistance in antibiotic-producing bacteria and the corresponding ones in nonantibiotic-producing human or animal bacterial isolates [erythromycin resistant methylase (erm), aminoglycoside 3 0 -phosphotransferase (aph3), aminoglycoside 6 0 -phosphotransferase (aph6), aminoglycoside acetyltransferase (aac), class a beta-lactamase, tetracycline resistance efflux protein, tetracycline resistance ribosomal protection protein and vancomycin resistance proteins (vana, vanh, vanx) and bacitracin transport proteins (bcra, bcrb, bcrc)] was carried out. if dna encoding antibiotic resistance genes present in antibiotic preparations has been important in the development of antibiotic resistance, genes of almost identical amino acid sequences would be expected to be present in antibiotic-producing organisms and other human or animal bacteria, inferring that horizontal transfer of antibiotic-resistance genes had occurred from the former to the latter. results: the maximum amino acid identities of genes among different non-antibiotic-producing bacterial isolates were close to 100% for most genes, but those between antibiotic-producing and human or animal bacteria ranged from <28 to <77%. therefore, recent horizontal transfer of antibiotic resistance genes has not occurred from antibiotic-producing organisms to human or animal bacteria. on the other hand, frequent horizontal transfer of antibiotic resistance genes was observed among the human or animal bacteria, even if they were phylogenetically distantly related. moreover, such transfer was particularly common among gastrointestinal tract flora or pathogens. conclusion: dna encoding antibiotic resistance genes in antibiotic preparations has not been an important source of antibiotic resist-ance genes. dna decontamination during the process of antibiotic synthesis is probably not necessary. the human gastrointestinal tract has been an important place for bacterial gene exchange. the role of the human gut in the dissemination of antibiotic resistance should be further investigated. enterococci and other gram-positive bacteria p803 glycopeptide-resistant enterococci (gre) have emerged as important pathogens since the late 1980s. an important factor associated with the appearance of gre in the community in europe has been avoparcin, a glycopeptide antimicrobial drug used for years in many european countries as a growth promoter in food-producing animals. in europe, evidence suggests that food-borne gre may cause human colonisation or infection. objectives: the objective of this study was to investigate the prevalence and to determine the genotypes of gre from different human and animal sources in styria, austria. methods: stool specimens from each 100 patients with precedent antibiotic therapy and 100 non-hospitalised humans without precedent antibiotic therapy, 166 faecal cattle specimens, 117 faecal pig specimens and 40 faecal poultry specimens were collected in 2003. one millilitre of diluted faeces was added to 9 ml of enterococcosel bouillon (bd) for enrichment. after incubation, 100 ml was subcultured on vre screen agar (bd). species identification was performed with the api strep systems and vitek2 (bio-mérieux). resistance to vancomycin and teicoplanin was determined by the e-test method (ab biodisk). determination of glycopeptide resistance genotypes (vana, vanb, vanc1, vanc2/3) was performed by pcr. results: 4% of the patients with precedent antibiotic therapy harboured vre. among these, two were identified as e. faecium vana, two as e. gallinarum vanc1 and e. casseliflavus vanc2, respectively. eight per cent of the non-hospitalised human specimens contained vre (six e. gallinarum, two e. casseliflavus). a total of 90 vre strains were isolated out of the animal samples, 25.6% e. faecium, 35.6% e. gallinarum, and 38.8% e. casseliflavus strains. no resistant e. faecalis strains were detected. pcrs confirmed that all e. gallinarum were of the vanc1, all the e. casseliflavus of the vanc2 and all the e. faecium strains of the vana genotype. about 95.6% of all e. faecium vana strains were isolated out of the poultry samples. one strain was isolated from a cattle sample, no specimen from pigs yielded glycopeptide-resistant e. faecium. conclusion: the present study indicates that the prevalence of gre in humans and in pig and cattle husbandry appears to be low, but it reveals a high prevalence of gre (e. faecium) in styrian poultry 6 years after the use of avoparcin was banned. glycopeptide resistant enterococci (gre) have become an increasing problem in the us and in europe. enterococci are intrinsically resistant against cephalosporins, aminoglycosides (low-level), polymixins, lincomycin and clindamycin. furthermore, enterococci are able to acquire resistance to a wide range of antibiotics. there remain concerns that antibiotic use for growth promotion, prophylaxis and therapy in animal husbandry may lead to increased resistance to antibiotics used in human medicine. objectives: the aim of this study was to evaluate the species distribution and the antibiotic resistance of gre isolated from styrian food-producing animals. methods: a total of 90 gre strains isolated from cattle, pig and poultry faecal specimens in 2003 were collected. the strains were identified using the vitek2 automated methods (gpc) and the api strep systems (biomérieux). antimicrobial susceptibilities were determined by vitek 2 p524 card and by disk diffusion (linezolid). the strains were studied for susceptibility to 13 antibiotics: ampicillin (am), amoxicillin/sulbactame (amc), ciprofloxacin (cip), erythromycin (ery), gentamicin high level (ge), linezolid (li), norfloxacin (nor), penicillin (p), quinupristin/ dalfopristin (syn), streptomycin high level (str), teicoplanin (tp), tetracycline (te) and vancomycin (va). results: e. casseliflavus was the most common gre species isolated (38.8%), followed by e. gallinarum (35.6%) and e. faecium (25.6%). all e. gallinarum and e. casseliflavus were of the vanc, all e. faecium of the vana phenotype. all investigated strains were sensitive against linezolid and gentamicin high level. p and am resistance (82.6%) and reduced susceptibility to cip (17.4%) was seen in e. faecium only. ery resistance for e. faecium revealed 17.4%, for e. casseliflavus 85.7% and for e. gallinarum 31.3%. resistance against te for e. faecium was 91.3%, for e. casseliflavus 11.4% and for e. gallinarum 68.8%. about 17.4% of e. faecium strains were not susceptible to quinupristin/dalfopristin. conclusions: resistance phenotypes to p, am, cip, ery and te differed among enterococcus species. resistances found against tetracyclines, quinupristin/dalfopristin and erythromycin are causes of concern. high levels of antibiotic and multidrug resistance were observed among the e. faecium strains. the identification was carried out by the vitek system (biomerieux). the susceptibility test was performed either by the breakpoint system:mini api or the vitek system (biomerieux). results: 146 (49%) and 152 (51%) streptococci strains were isolated from outpatients' and inpatients' urine cultures, respectively. the distribution by sex was 65% women/35% men in outpatients and 58% women/42% men in inpatients. a total of 157 (53%) of streptococci strains were enterococcus faecalis, 54 (18%) were enterococcus faecium, 10 (3%) were enterococcus gallinarum and 77 (26%) were streptococci group b. the in vitro antibiotic resistance of enterococci spp. was: penicillin 38.5% (85/221), ampicillin 23% (51/221), gentamicin 39% (86/221), nitrofurantoin 9.5% (21/221), ciprofloxacin 58% (128/221), tetracyclines 56% (123/221), vancomycin 6% (13/221), linezolid 0%. eight vre strains were enterococcus faecium, three were enterococcus gallinarum and two were enterococcus faecalis. the in vitro antibiotic resistance of group b streptococci was: vancomycin 0%, nitrofurantoin 5.2% (4/77), ampicillin 6.5% (5/77), penicillin 7.8% (6/77), erythromycin 14.3% (11/77), tetracyclines 61% (47/77). conclusions: streptococci are responsible only for the 9.4% of urinary tract infections. enterococcus faecalis was the most frequent pathogen (53%). enterococci spp. showed high resistance in ciprofloxacin, tetracyclines, gentamicin, penicillin, and ampicillin. objectives: enterococcal infections are becoming an increasing concern, particularly due to the emergence and spread of resistance to animicrobial agents. we have investigated the phenotypic and genotypic properties of 66 enterococcus faecium clinical isolates, expressing resistance to the combination of quinupristin/ dalfopristin, recovered during a 3-year period in the university hospital of patras. methods: all isolates were characterised at species level by gram stain, catalase production and by the crystal id gram positive system (bbl). minimal inhibitory concentrations (mics) to ampicillin (amp), erythromycin (em), chloramphenicol (chl), gentamicin (gm), ciprofloxacin (cip), vancomycin (va), teicoplanin (tp), quinupristin/dalfopristin (rp) and linezolid (lin) were performed by the e-test (ab biodisk) according to nccls recommendations. the presence of vana and vanb genes was investigated by the evigene commercial kit (statens serum institut), while the presence of vga, vgb and sat1 genes by pcr with specific primers. clonal types were characterised by pfge of smai dna digests. results: in a collection of 88 e. faecium, 66 (75%) expressed mic of rp ! 1 mg/l, and among them 10 isolates (15%) showed mic > 4 mg/l. high-level resistance to gm was detected in 27 (41%) isolates, 53 (80%) to cip, 5 (7.6%) to chl, 58 (88%) to amp and 62 (94%) to em. forty-three (65%) isolates were vancomycinresistant, carrying the vana gene. no isolate was found to carry vga, vgb and sat1 genes. pfge classified 36 isolates to clonal type a, 11 to type b, 6 to type c and the remaining 13 isolates belonged to 10 more types. conclusions: high prevalence of low-level resistance to quinupristin/dalfopristin (mic: 1-4 mg/l) was detected in this collection of e. faecium, with 10 strains expressing higher mic levels. this was mainly due to the dissemination of certain clones in the hospital. in a previous work, we described the dissemination of renc1 and renc4 e. faecalis multiresistant clones colonising patients of four different icus. the renc4 clone was frequently found in bacteraemias, suggesting a blood invasion from an intestinal origin. the aim of this study was to analyse the dynamic population evolution of enterococcal intestinal isolates and if the acquisition of epidemic hospital clones may occurs during icu admittance. material and methods: a close follow-up of four patients from the neurosurgery icu who were admitted after acute traumatism was performed. rectal swabs were collected at the admittance and daily until they were discharged from the icu. stool samples were seeded in m-enterococcus agar, eventually supplemented with selective antibiotics, and multiple colonies were analysed in each sample. pfge-smai and the phoretrix 5.0 software were applied to analyse the genetic relatedness among these isolates and the previously described hospital endemic clones. results: patient 1 and 2 stayed in the icu for 12 days, and patient 3 and 4 for 7 days. patient 1 carried along the 12 days the original e. faecalis and e. faecium clones. moreover, five e. faecalis clones, one identical to the epidemic clone renc1, and one e. faecium clone were acquired during the icu stay, all of them persisting over the rest of the studied period. patient 2 presented at admission three e. faecalis and two e. faecium clones; two e. faecalis were lost in 5 days, and e. faecium were lost at the second day. four new e. faecalis and one e. faecium clones were found during all stays, whereas five more clones were occasionally isolated without persistence. in patient 3 an e. faecium clone was identified along all the studied period, and two new e. faecium clones were later acquired. patient 4 had two e. faecium clones at admission, one of them being lost after the first day; the second persisted during all 7 days; a new e. faecium clone was acquired during the icu stay. patient and methods: the patient was a 62-year-old woman hysterectomised 15 years ago. she reported four surgical interventions due to a cystocele. the last operation took place 11 years ago and she reported no further admittances at any hospital. in the last years the patient also suffered from repeated urinary tract infections. in the present episode she consulted because of typical uti symptoms (dysuria, bladder tenesmus) and a urine sample was collected. after 24 h of incubation, a gram positive coccus was isolated (more than 100 000 ufc/ml). the identification and susceptibility were preliminarily achieved by a commercially available method following manufacturer's recommendations (microscan, dade). identification was confirmed by api rapid strep system (biomerieux). to discard enterococcus species intrinsically resistant to vancomycin the absence of motility was observed with direct microscopic detection and the absence of pigmentation was determined by culture on tsa agar. susceptibility to vancomycin, teicoplanin and ampicillin were assessed by disk diffusion, e-test and broth mcrodilution. results: the isolated microorganism was identified as enterococcus faecalis and showed high mics to vancomycin (>128 mg/l by broth microdilution and 6 mm by disk diffusion) and teicoplanin (8 mg/l by broth microdilution and 10 mm by disk diffusion) but was susceptible to ampicillin (0.5 mg/l by broth microdilution). to characterise the resistance mechanism involved in a series of 11 vancomycin resistant enterococcus faecium (vref) strains recovered in two spanish hospitals of the same city, and to determine their clonal relationship. methods: a surveillance programme was carried out during a 1-year period in ms-hospital in order to detect vref intestinal colonisation. seven vref strains were recovered from seven faecal samples which represents <1% of vref intestinal colonisation. in the same period, four clinical vref strains, implicated in infectious processes were recovered in ms-hospital (n ¼ 3) and rv-hospital (n ¼ 1). all vref strains (n ¼ 11) were recovered from 11 unrelated patients, most of them previously treated with glycopeptides or broad spectrum antibiotics and diagnosed with severe diseases. antibiotic susceptibility testing was performed by agar dilution method and vancomycin resistance genes (vana, vanb, vanc1, vanc-2/3, and vand) were studied by pcr. vanb amplicons were sequenced to determine the subtype and the vanb cluster of genes were also characterised. other resistance genes were studied by pcr: aph(3¢)-iiia, ant(6¢)-ia and erm(b). pfge assays were performed with smai digestion. results: nine of the 11 vref strains (eight of ms-hospital and one of rv-hospital) showed a vanb phenotype [mic (mg/l): vancomycin (16-32) and teicoplanin (0.5)]. the vanb2 gene was detected in these nine strains and in addition, the intergenic vansb-yb region showed the characteristic mutations of the vanb2 subtype. the vanb2 gene cluster was integrated into the tn5382like element in all of them, as it was demonstrated by specific pcrs and sequencing. these strains were resistant to streptomycin, kanamycin and erythromycin and ant(6¢)-ia, aph(3¢)-iiia and erm(b) genes were detected by pcr. all of them were included in the same pfge clonal type a and two closely related subtypes were distinguished: a1 (seven strains from both hospitals) and a2 (two strains from ms-hospital). both subtypes were found in clinical strains as well as in strains recovered from faecal samples. only three of them were from serious infections, the rest was isolated from carriers. all but one present vana phenotype and harbour vana gene. the only vanb harbouring strain was resistant to teicoplanin. the outbreak at haematology was at the beginning polyclonal (10 pfge clones) but eventually three of them became predominant in both wards. the outbreak in cracov centre was spread to two other wards of this hospital (surgery and geriatry) with 53, 27 and 7 vrem isolated up to now, respectively. five were from serious infections, 15 were from wounds in the surgery, the rest represents for carriers detected during infection-control measurements. all but two of them were vana phenotype/genotype (2 vanb phenotype/genotype isolates). one predominant pfge clone was observed, differentiated into 14 pfge sub-types ('hospital clone'). five other pfge clones detected seemed to be unique (one to five isolates). in both outbreaks two basic mechanisms of vre spread were detected, clonal spread of vre strains and the vana-elements horizontal transfer. conclusion: after time of vrem presenting vanb phenotype caused sporadic outbreaks two of haematology centres in poland become the stages of multi-drug-resistant vana vrem outbreaks, eventually turning into endemic. the colonisation rate was 10-15 times higher than infection in both cases. the danger of transmission to other centres and non-haematological hospitals in the country appears very high in these circumstances. material and methods: thirty-three selected vrefls from different patients at three hospitals (huc, hsa and hst) in the north and centre of portugal (1996 portugal ( -2002 were studied. susceptibility to 12 antibiotics was performed by the agar dilution method (nccls). isolates were searched for genes coding for resistance to glycopeptides, macrolides, and aminoglycosides. tn1546 characterisation was done by an overlapping pcr strategy and sequencing when necessary. clonal relatedness was performed by smai-pfge. virulence traits (cyl, agg, gele, esp) were investigated by a multiplex pcr assay. results: all vrefls showed vana phenotype and were mostly resistant to ery, cipro, hlrgm, and hlrkm (91, 88, 82, and 82%, respectively). resistance genes found were vana, erm(b), aac6-aph2, and aph30-iiia. nine pfge types were isolated: eight from eight patients and one (clone b) from 25 patients. clone b was disseminated among the three hospitals for 7 years giving eight pfge subtypes, each one characteristic of a specific hospital. vsefls showing pfge patterns identical to two clone b subtypes were found in hst. six variants of tn1546 were found, five of them among isolates of clone b. tn1546-pp4 was found in all hospitals for 7 years and predominates in huc and hst. it contains an isef1 insertion in the intergenic vanx-vany region. tn1546-pp15, only found in hsa, lacks genes involved in transposition. pp5 and pp2 were variants of pp4 and were recovered at huc. pp16 was a pp-15 variant found in hsa. all vre but one isolate of clone b were agg+ and gel+. cyl and esp were present in 43% of the vre. conclusion: our findings indicate that the dissemination and establishment of successful e. faecalis clones in the hospital setting amplify particular genetic determinants in local metagenomes resistant to vancomycin, and therefore influences future evolutionary events. we also report the first tn1546-variant containing an isef1 insertion. staphylococcus spp. is widely distributed in medical and veterinary pathology and represents one of the most important causes of infection. many strains are antibiotic-resistant even for the presence of an eso-polysaccharide matrix. the aim of this work was to individuate, among 396 different staphylococci of human and animal origin, the slime producing strains and to correlate the presence of biofilm to the resistance to eight antibiotics. a total of 185 coagulase negative staphylococci (cns) and 211 s. aureus isolated from different sources and identified with sceptor system, were tested for antibiotic susceptibility (kirby bauer method) and for slime production (polystyrene plates -stained with alcian blue -spectrophotometric reading at 450 nm). the strains were classified as weak, strong and no slime-producing on the basis of od results. the results were submitted to statistical analysis using student's t-test and chi-square tests. evaluating the differences of slime production among medical and veterinary strains, we found different statistical frequencies (p > 0.001). no statistical differences were obtained between s. aureus and the other cns. instead, the statistical analysis on s. epidermidis vs. the other staphylococci has shown no statistical differences among average values using student's ttest (p < 0.052) and significant frequency differences using chi square tests (p < 0.02). finally in the cns, between s. epidermidis and the other strains, no statistical differences were found. the relation between slime production and the origin of strains was evaluated and no correlation was found. about the correlation between antibiotic-resistance and slime production a resistance increment of about 30% was obtained in strongly slime producing strains. staphylococcus spp. is often involved in nosocomial infections as complication of post-surgery wounds, catheters and orthopaedic devices. the presence of antibiotic-resistant strains interferes in the therapy successes and seems to be strictly related to biofilm production beyond that genetically acquired. human and veterinary strains have shown a similar behaviour towards biofilm production and antibiotic-resistance. the results confirm that s. epidermidis is one of the most slime-producer and introduce s. aureus as a new high slime-producer. (2000) recommendations. the macrolide resistance phenotypes were determined using the erythromycin-clindamycin double disk test. results: all the s. agalactiae isolates tested were found susceptible to penicillin g and vancomycin while the resistance rate to erythromycin was 8.1% (seven strains). the expression (%) of the macrolide resistance phenotypes among the resistant strains as they were evaluated by the double disk test were: constitutive (cmlsb) phenotype 57% (four isolates) and inducible (imlsb) phenotype 43% (three isolates). no s. agalactiae strain was assigned to the m resistance phenotype. the overall resistance rate to clindamycin was 8.1%. conclusions: our findings demonstrate that s. agalactiae remains fully susceptible to penicillin and vancomycin while there are relatively low resistance values to macrolides and lincosamides. the mlsb phenotype predominated among the macrolide-resistant strains, a finding that raises concern about the use of clindamycin instead of erythromycin in prophylaxis or treatment of s. agalactiae infection in patients allergic to beta lactams. however, continuing surveillance is needed to detect any change in susceptibility patterns. effect of enterovirus infection on the risk of type 1 diabetes mellitus has been studied mainly using indirect serological evidence of past infections, or using rt-pcr detection of the virus in plasma. with respect to enterovirus biology, we decided to assess the exposure to enterovirus using real-time rt-pcr detection and quantification from stool samples. this exposure is studied in relation to signs of autoimmune process ultimately leading to type 1 diabetes. methods: the study population comes from the norwegian 'midia' study which screens newborns from the general population for the highest hla-encoded risk of type 1 diabetes mellitus. the high-risk babies are followed-up by questionnaires, serum samples for markers of beta-cell autoimmunity, and stool samples collected in monthly intervals from month 3 to month 24. the stool samples are collected by parents and mailed to the laboratory where rna and dna is co-purified on qiagen columns together with a low quantity of exogenous control rna. enterovirus is quantified by real-time rt-pcr using armored rna as a standard. control rna is detected in late cycles in the same reaction using a differently coloured probe reporter. adenovirus quantity is simultaneously investigated as a viral exposure which has not been implicated in triggering type 1 diabetes. here we present the results of the pilot study. objective: there are conflicting reports regarding cmv-dna positivity among healthy cmv-seropositive individuals. we aimed to determine the frequency of cmv-dna positivity among healthy subjects and to evaluate its association with physical and mental stress in a longitudinal study. subjects and methods: weekly peripheral blood samples were drawn into from 17 healthy cmv seropositive subjects aged between 24 and 53 years during a 8-week study period. each subject rated their physical and mental stress and they also recorded their alcohol consumption and any change in their health status. cmv dna was screened in plasma and peripheral blood leukocyte samples with a nested pcr using primers targeting mie gene of cmv. results: in total, 272 samples (136 plasma and peripheral blood leukocytes, each) were screened and only one peripheral blood sample obtained during the second week of the study gave positive result. this sample belonged to the oldest subject of the study. according to our results, cmv-dna positivity among healthy cmv seropositive individuals seems to be a rare event. results: all 16 centres reported qualitative results; four centres also reported quantitative results. all samples were correctly identified by all centres. various extraction and amplification methods were used. fourteen centres reported results of internal controls. most of the centres controlled only the amplification step and did not adjust the detection sensitivity of the internal control to the detection limit of the target. three centres failed to detect one internal control in two positive samples and one negative sample. for quantification of hcmv dna all centres used real-time quantitative pcr. cv of hcmv dna load between centres were low (3.6-7%) except for one sample (13%), but this could be attributed to a heterogeneous preparation of this sample by the organisers. using student's t-test, no statistically significant difference was observed between hcmv load whatever the medium or the number of added cells. conclusion: results of this external quality assessment for molecular detection and quantification of hcmv dna were excellent. almost all centres used internal control of pcr inhibition; however, control of the whole pcr process, including extraction and better adjustment of the detection sensitivity of the internal control to the sensitivity limit of the pcr target is desirable. the most accurate way to identify false negative results, e.g. those caused by pcr inhibitors, in real-time pcr assays is to spike samples with an internal control that will be co-amplified with the target (pathogen) dna. however, current internal control procedures, which usually involve the introduction of a dna fragment, are complex, time consuming and expensive. we present a novel technique for simple internal control of real-time amplification assays. methods: single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time pcr assays. mismatches were included in the probe-binding region of the internal control oligonucleotide (ico) to prevent probe-control hybridisation during the fluorescence acquisition step of the pcr. icos could be added directly to the sample material prior to dna extraction. results: to demonstrate the feasibility of the new approach, we designed icos for the following lightcycler hybridisation probe assays: mycobacterium tuberculosis complex, hepatitis b virus, herpes simplex virus and varicella zoster virus. in each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis of the pcr products. conclusion: a single-stranded oligonucleotide, which mimics the target region of the pathogen yet is clearly distinguishable from the target during analysis, can serve as a simple, cost-effective internal control for real-time amplification assays. such control oligonucleotides are easy to design and cheap. a costly second probe system is not necessary. moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications. objectives: biomérieux has developed a new nucleic acid isolation method (nuclisens magnetic extraction reagents) that uses boom chemistry in combination with magnetic silica particles. the nuclisens mini mag instrument is facilitating the washing and collection of the silica particles in a user friendly and efficient way. in principle the extraction method is generic and can be applied to a broad range of different sample types. the objective of this study was to measure the performance of this new extraction platform in terms of rna and dna recovery, purity and integrity. in addition, user aspects were also addressed in the study. methods: rna recovery was measured by spiking e. coli rna to human normal edta plasma, extracted rna was quantified by using a fluorescence dye for rna detection (sybr green ii). dna recovery was measured by spiking plasmid dna (pbr322); extracted dna was determined by a260 measurement. an indication of rna and dna purity was obtained by measuring a260/ a280 ratios. the integrity/intactness of the extracted nucleic acid was determined by gel analysis or by using the bioanalyzer (agilent technologies) for rna and dna, respectively. the extraction method was tested on three external test sites in order to score relevant user aspects. results: the average recovery rates were 83 and 85% for rna and dna, respectively. for rna extracts an average a260/a280 ratio of 2.13 was measured, whereas for dna this value was 1.82. these values indicate that the purity of both preparations is high since for pure preparation the expected values are 2.0 and 1.8 for rna and dna, respectively. in addition, it was found that both rna and dna were intact recovered since no degradation products were detected. in addition, all users scored the method as labour friendly. the total amount of time needed to process 12 samples was <60 min, the throughput time was improved further by using two instruments in parallel, in this way 24 samples can be completed within 90 min. in addition the method was also verified for a broad range of different sample types including plasma, serum, csf, sputum and stool. dna sequencing is the gold standard method for accurate genotyping of human papillomaviruses (hpv) and provides nucleic acid sequence information, which is the core of every organism. pyrosequencing method has been successfully used for hpv genotyping with sequencing of only 14-21 bases. multiple hpv infections are a common phenomenon in clinical samples with a varying rate depending on the group investigated. dna sequencing techniques cannot differentiate between different genotypes as uninterruptible sequence results are obtained when multiples infections and unspecific amplification products are present in the amplicon. to address these problems, a type-specific multiplesequencing primer dna sequencing strategy, suitable for genotyping and detection of hpv-6, -11, -16, -18, -31, -33 and -45 has been developed. in the new method seven type-specific sequencing primers, combined in a pool, are added to the dna sample. the oligonucleotide hybridising to the dna sample will function as a primer during the subsequent dna sequencing procedure. the new method is especially suited for detection and typing of samples harbouring different hpv genotypes (multiple infections) and unspecific amplifications, which eliminates the need for nested pcr, stringent pcr conditions and cloning. furthermore, the method has proved to be useful for samples containing subdominant types/species, and samples with low pcr yield, which avoids re-performing 'failed' pcrs. we also introduce the sequence pattern recognition when there is a plurality of genotypes in the sample, which facilitates typing of more than one target dna in the sample. moreover, target specific sequencing primers could be easily tailored and adapted according to the desired applications or clinical settings based on regional prevalence of hpv as well as other microorganisms and viruses. as the cost for dna sequencing is dropping, a sample could be sequenced in parallel with two or three different target specific primer pools covering a broader range of genotypes. the pyrosequencing hpv detection assay is fully automated and could be used for detection and identification of different microorganisms and viruses. reagents to reference extraction methods for the isolation of rna and dna from various sample types results: by performing several extractions (up to 12) of a dilution series of strain coxacksie b5 in csf, it was shown that the analytical sensitivity of the enterovirus rt-pcr was found to be independent of the extraction method used, whereas in very low frequency higher sensitivities were obtained in combination with magnetic extraction. as expected the higher input samples gave better reproducible results than lower input samples. after evaluation of the enterovirus pcr using csf and stool samples a 100% correlation between the two extraction methods was found. in addition, using a broad panel of clinical specimens for m. tuberculosis pcr, the same samples were identified as positive using the boom extraction method and magnetic extraction. however, the latter method resulted in less samples having inhibition in pcr, but this needs to be confirmed in a larger study group. methods: 23 conjunctival scrapings were sent to our laboratory in 2 ml of viral transport medium and were inoculated to monolayers of a-549 and mrc-5 cells in tubes, incubated at 37 c in stationary phase, and scored daily for cytophathic effect (cpe) for 7 days or until cpe developed. when a characteristic adenovirus cpe was observed (usually after 5 days of culture), a passage was done to two homologous monolayers in shell vials that were incubated 24 h at 37 c and stained with specific fluorescent reagents to adenovirus. dna from 0.2 ml of the remaining transport medium was purified by a commercial procedure and resuspended to a final volume of 50 ll. five microlitres of this purified dna was used for real-time amplification in a final 20 ll reaction volume, using 1â fast start sybr green i master mix (roche), mgcl 2 (3 mm m), and 0.5 lm m of each primer. the region amplified belongs to the hexon gene. total processing time was less than 3 h. results: adenovirus was isolated in 10 of 23 samples processed by conventional cell culture and all theses culture-positive samples were positive by real-time pcr; of 13 samples testing negative with conventional cell culture, real-time pcr detected eight as negative and five as positive. gel electrophoresis analysis showed amplification bands of the expected molecular weight in all these real-time pcr positive, cell culture negative samples. a control group of 20 samples from patients with bacterial conjunctivitis was tested and all of them were negative by pcr. a plasmid containing the hrv-5 sequence spiked into a negative synovial tissue or blood specimen was used as a positive control. extracted dna from a negative synovial tissue or blood specimen was included between every two specimens as a negative control. suitability of dna for pcr was verified using a pcr assay for beta-globin. positive specimens were subjected to bidirectional sequencing. fisher exact test (two-tailed) was used for statistical analysis. results: all 600 specimens were positive for beta-globin (extraction control). cloned hrv-5 proviral dna spiked into tissue, mononuclear cells and granulocytes followed by extraction yielded an amplified product in all cases. the limit of detection of the assay was 166.6 copies/ml blood and 6.6 copies/mg tissue. two hundred tissue specimens, 200 mononuclear cells, and 196 of 200 granulocyte specimens tested negative for hrv-5 proviral dna. two ra and two oa granulocyte specimens, however, yielded a positive signal for hrv-5 proviral dna. all were detected at a low copy number (quantitated by comparison to a known quantity of cloned hrv-5 proviral dna spiked into blood), range 83-1365 copies of hrv-5 proviral dna/ml blood. all four showed 95-98% identity to genbank sequence af480924 by ncbi blast search. conclusion: we did not find an association between hrv-5 and ra or oa ( p ¼ 0.516) using a real time pcr assay. recently it has been shown that hrv-5 is actually rabbit endogenous retrovirus h (j virol 2002: 76; 7094-7102). we hypothesise that experimental rabbit studies ongoing in our laboratory while the granulocyte specimens were being prepared account for the low level of 'hrv-5' proviral dna detected in 0.7% of the specimens tested. results: csf virus load ranged from 10 2 to 5 â 10 4 copies per millilitre. in comparison the virus load in vesicular fluid was 3 â 10 6 copies per millilitre. the highest virus loads (5 â 10 4 and 2 â 10 4 ) were detected in a patient with paresis of facial nerve and a young patient with relatively mild disease. the lowest virus load (10 â 10 2 copies per millilitre) had a child with varicella meningitis and an old patient with severe herpes zoster of the trunk. quantitative pcr has good reproducibility and is useful for assessment of viral load in csf samples. however, the correlation between virus load and severity of illness remains uncertain. the purpose of this study was to develop enzyme immunoassay (eia) for the detection of igg anti-hsv-2 activity using two new recombinant proteins as antigenic targets, and to evaluate these eia with the aid of statistical methods. methods: fragments of glycoprotein g (gg-2), comprising residues 525 to 578aa of herpes simplex virus type 2 (hsv-2) and glycoprotein d of hsv-2 (gd-2(266-394aa)), were expressed in the e. coli as gst fusion proteins to develop an assay for the detection and hsv-2 type-specific antibodies. results: a new enzyme immunoassay for the detection of igg anti-hsv-2 (igg-eia) in sera was developed using two new recombinant proteins. the igg-eia was evaluated using serum specimens obtained from patients with culture-proven hsv-2 infection (cp) (n ¼ 13) and from normal blood donors (bd) (n ¼ 629). all specimens were additionally tested for igg anti-hsv-2 activity by two commercially available eias. this new igg-eia detected anti-hsv2 activity in all specimens from hsv2 infected patients. when bd were tested the overall concordance between these three assays varied between 39 and 63.6%, concordance between positive samples ranged from 18.4 to 46.7%. in the absence of a gold standard the accuracy of these eias was assessed by the computer program based on a maximum likelihood approach using a 'latent class' model. this analysis estimated the igg-eia sensitivity and specificity to be within the range 98-100% and 95-100%, respectively. dna was detected in one csf specimen from a patient with aseptic meningitis, in three aqueous humor specimens from patients with uveitis, in one swab from a patient with herpetic vesicular skin lesions and in three conjunctival swabs from patients with conjunctivitis, and (b) vzv dna was detected in two aqueous humor specimens from patients with iridocyclitis, in two swabs from patients with vesicular skin lesions, and in the vesicle aspirate and bronchoalveolar lavage from the patient with varicella pneumonitis. the precise diagnosis of herpetic infection was available within 24-48 h, which allowed for an early initiation of adapted antiviral therapy. conclusion: the detection of the six commonest human herpesviruses in clinical specimens by the herpesvirus consensus pcr methodology allowed rapid, sensitive and specific results. objectives: bovine herpesvirus-1 (bhv-1) is the aetiological agent of many infections and may predispose infected animals, possibly through immunosupression, to secondary bacterial infections. immunosupression may directly be associated with the induction of programmed cell death (pcd) in some virus infected cells. nitric oxide (no) has an important mediating role against fungal, bacterial, protozoal, viral pathogens and tumours. in this study, role of no was questioned in the pcd process. methods: this study was planned in two consecutive stages. in the first stage, the morphological (with and without staurosporin) and biochemical changes caused by virus-induced pcd in mdbk cells were investigated. morphological assessment of pcd was performed using hoechst 33342 nuclear staining and fluorescence microscopy technique. in the second phase of the study, the induction of pcd with staurosporin (ss) (alone or with bhv-1 addition) and apoptotic route of bhv-1 infections (with/without staurosporin) were analysed by applying 1, 3, 8, 9 caspase inhibitors (r&d, germany). results: it was interesting to see that bhv-1 inhibited pcd following 1 h of poi instead of being induced by staurosporin and induced apoptosis alone between 0.5 and 3 h of poi in mdbk cells, however, between 3 and 6 h of poi, pcd response has found to be decreased. these results showed similarities with those obtained from herpes simplex type-1 infections in human epithelial cells. following caspase 1, 3, 8, and 9 inhibitors applications pcd responses decreased after 1 h whereas no responses increased following 3 h of infections with caspase 1, 3, 8, and 9 inhibitory peptides. conclusion: in conclusion, bhv-1 inhibited the apoptotic response in a caspase-independent way and bhv-1 may modulate the no response through the apoptotic pathways. objectives: the aim of this study is the questioning the programmed cell death (pcd) process in acute phase of bhv-1 infection in cultured epithelial like cells' microenvironments and to investigate its relation with possible nitric oxide responses in hep-2 cells infected with bhv-1 with and without staurosporin induction. methods: this study was planned in two consecutive stages. in the first stage, the morphological (with and without staurosporin) and biochemical changes caused by virus-induced pcd in hep-2 cells were investigated. morphological assessment of pcd was performed using hoechst 33342 nuclear staining and fluorescence microscopy technique. in the second phase of the study, the induction of pcd with staurosporin (ss) (alone or with bhv-1 addition) and apoptotic route of bhv-1 infections (with/without staurosporin) were analysed by applying 1, 3, 8, 9, and total caspase inhibitors (r&d, germany). results: it is known that following hsv-1 infection of 3-6 h of poi anti-apoptotic activity is triggered in human cells. and this activity is through caspase 3. it is interesting to see that in these experiments following 1 h of bhv-1 infection the number of apoptotic cells reduced whereas no response continuously increased following 1-h poi. conclusion: anti-apoptotic activity of bhv-1 seems to be activated through caspase 3 like hsv-1, and this inversely proportional relation between no and pcd responses seem to be related with the triggering effect of no on pcd response. this effect was explained as non-specific stimulation of the host immune system. however, direct anti-viral effect cannot be excluded. the goal of present study was to evaluate the effect of probi-otics strains derivative metabolites on the reproduction of herpes simplex virus type 1 (hsv-1). materials and methods: probiotic strains used were: lactobacillus plantarum 8a-p3, enterococcus faecium-l3, escherichia coli m17. one hundred and six vero cells were infected with 103-104 id of hsv-1 and then incubated with supernatants from bacteria or bacteriocin preparations applied in serial dilutions. acyclovir 20% (lek, slovenia) was used as anti-viral drug control. cytopathic effect of the virus was determined by light or immunoflourescence microscopy after 72 h. results: hsv-1 alone or in the presence of the e. coli m17 extracts caused the most profound cytopathic effect. addition of acyclovir completely inactivated the effect of the virus that was taken for 100%. supernatants obtained from l. plantarum, and e. faecium generated dose dependant effect from 90 to 35% of viral inhibi-tion. e. faecium strain l-3 extract was 10-20% more active than l. plantarum. extract from the strain l-3 was analysed for the presence of bacteriocins. two types of peptides were determined -enterocin a and enterocin b (5.5-5.6 kd). bacteriocin preparation demonstrated similar anti-viral effect (65-85% of inhibition) which allows to consider enterococcal bacteriocins as major antiviral agents in present model. conclusions: extracts of several probiotic bacterial strains express a specific activity against reproduction of hsv-1 in vitro. antiviral effect of e. faecium strain l-3 was the strongest due to the presence of enterocins a and b in the supernatant. acknowledgement: work was supported by public health service grants ai19304 and tw00188 from nih, grant 03-04-49760 from rffi and regional foundation of support to new technologies in medicine. we detected 13 ribotypes, among toxin b producing strains (tcdaàtcdb+) only one ribotype was detected. among non-toxigenic strains four ribotypes were detected. it seemed to be interesting to observe the dominating ribotypes. between toxigenic (tcda+tcdb+) five belonged to ribotype 014 and four to 046. all strains (n ¼ 20) (tcdaàtcdb+) belonged to one ribotype -017. in summary, pcr-ribotyping is a good method to discriminate c. difficile strains. we decided to continue further epidemiological study in poland. objectives: the aim of the study was to identify risk factors of c. difficile-associated diarrhoea due to adp-ribosyl transferase producing strains. materials and method: a retrospective case control study was performed. each case (patient with a diarrhoea due to an actin-specific adp-ribosyl transferase producing strain) was compared with two controls (patient with diarrhoea due to a c. difficile strain which does not produce an actin-specific adp-ribosyl transferase) matched on ward and on date of hospitalisation. cdta and cdtb genes were screened by pcr (stubbs et al., fems microbiol letters 2000; 186; 307-312) . production of cdt was studied by western blot using an antiserum anti ia and ib from c. perfringens and the activity of the toxin was assessed using an adp-ribosyl transferase assay. results: twenty-six cases (14 males and 12 females) were identified in 1999 and 2000. they were hospitalised in six different hospitals of paris and its surrounding area. all the cdt positive strains were also positive for toxins a and b. cases were compared with 42 controls. cases and controls did not differ significantly for sex, age, previous administration of antibiotics, of chemotherapy or immunosuppressive treatment. endoscopic examination was performed in 30.5% of cases and in 23.8% of controls (p ¼ 0.52) and frequency of mucosal abnormalities was similar. diarrhoea was more often community-acquired in cases than in controls (65.4 vs. 35.7%, p ¼ 0.017) and represented more often the cause of hospitalisation (61.5 vs. 26.2%, p ¼ 0.003). moreover, diarrhoea from cases was more frequently associated to abdominal pain (63.6 vs. 39.4%, p ¼ 0.007) and to liquid stools (76.9 vs. 59.5%, p ¼ 0.14). conclusions: these results suggest that there could be a correlation between the production of binary toxin and the severity of diarrhoea. the binary toxin could induce intestinal lesion independently of toxins a and b or it may act in synergy with these toxins. methods: outbreak was detected by the c. difficile surveillance programme survey of the infection control unit. c. difficile infection was diagnosed by stool culture and by detection of toxin a with a qualitative rapid immunoassay. isolates of c. difficile were genotyped using pulsed-field gel electrophoresis. results: incidence of c. difficile-associated diarrhoea increased from 16 cases per 100 000 patient-days before to 90 cases per 100 000 patient-days during the outbreak. this outbreak involved 21 patients of four geriatrics wards, located on two geographically distinct sites (with the same medical team). mean age was 83 (range 71-100) years; sex-ratio (f/m) ¼ 1.1; 90% (19/21) of cases had received one or more antibiotics before onset of diarrhoea. about 24% (5/21) of cases were long-term care facilities-acquired diarrhoea, and secondary hospital transmission resulted in three clusters involving 16 cases. serotyping and genotyping were performed on isolates from 19 different stools; 16 of these strains belonged to the same type a1 whereas three displayed profiles different from the outbreak strain. management of this outbreak consisted in reinforcement of contact isolation precautions for patients with diarrhoea, cohortage of infected patients in the same ward and in promotion of hand disinfection with an alcoholic solution. environmental disinfection with hypochlorite was introduced during the outbreak. the ward where most transmission occurred was closed during 10 days for a completed disinfection after last patient discharge. after resolution of the outbreak, incidence for acquisition was 12 cases per 100 000 patient-days. ninety per cent (19/21) of patients were treated by metronidazole or vancomycin. relapses occurred in 29% (6/21) of patients. two patients died with severe colitis. mean hospital stay was 39 (range 11-97) days (annual mean of length of stay in the department ¼ 21 days). conclusion: rapid control of this nosocomial outbreak of c. difficile among geriatric patients was obtained with early implementation of cohortage and ward closure associated to reinforcement of environmental disinfection, hand hygiene and enteric isolation. introduction: toxigenic clostridium difficile is the main cause of nosocomial diarrhoea and has recently been described as involved in community acquired infections. two main toxins have been classically described as the main virulence factors although strains that lack one of them are emerging with increasing frequency. objective: we aimed to characterise toxigenic phenotypes in an institution with high prevalence of c. difficile-associated diarrhoea (cdad). materials and methods: c. difficile isolates were obtained and collected over a 6-month period from diarrheic stools submitted to our laboratory. specimens were cultured in ccfa plates with blood and presumptive colonies identified by standard procedures. toxin b was detected with a standard cytotoxicity assay on human fibroblast culture using both diluted samples and pure broth cultures of the microorganism. toxin a was detected by a commercial enzyme-immunoassay (cdtox a oia, biostar, finland) using colony suspensions in order to increase the sensitivity of the test. all negative results for any of both toxins were checked by pcr using previously published primers and conditions. results: a total of 220 c. difficile isolates were obtained during the study period. one hundred and ninety-nine isolates (90.5%) produced both toxins (a+b+); 10 isolates (4.5%) were classified as non-toxigenic (aàbà) by phenotypic procedures; in 11 isolates (5%) only toxin b was detected (aàb+), while no isolates were classified as producers of toxin a exclusively (a+bà). all non-toxigenic strains showed pcr positive results for gene b and four of them also for gene a (six isolates were aàb+ and four were a+b+). from all aàb+ isolates, only five were confirmed by pcr, while in six of them, toxin a gene was also detected. conclusion: the vast majority of c. difficile isolates obtained in our laboratory were toxigenic (a+b+) by traditional approaches. we have detected, using classical methods and confirmation by pcr, the presence of aàb+ isolates in our collection. all isolates considered as non-toxigenic by phenotypical methods were pcr-positive for one or both toxins. disagreements between results of phenotypical and genetic methods can be justified as the presence of incomplete or unexpressed genes or a lack of sensitivity of the former methods. background: it has been estimated that the extra cost to the nhs for every patient that contracts c. difficile in hospital is £4000. in light of this it seemed imperative that the possible components involved in the mode of transmission of this nosocomial infection be investigated with a view to control the spread. objective: to look at the level of contamination of health care workers' hands with c. difficile after dealing with a known positive patient. methods: hands were sampled using the finger streak method on a c. difficile moxalactam norfloxacin (cdmn) agar plate. plates were incubated for 48 h under anaerobic conditions and then examined for any possible colonies of c. difficile. these were identified using the gram stain and rapid ana ii system. hands were sampled directly after patient contact and the type of contact was also noted. hands were also sampled after the removal of gloves and after hand washing. in all, 54 duplicate samples were taken after various contacts with 14 colonised patients. results: 21% of samples taken immediately after patient contact were positive. nine per cent of samples taken after the removal of gloves were positive. no samples taken after hand washing were positive. conclusion: this study showed that hands do readily and regularly become contaminated after contact with a known positive patient and that this contamination can follow fairly minimal contact with the patient. objectives: during the conduct of a phase 2 clinical trial on the efficacy of tolevamer 1 or 2 g tid for 14 days compared with vancomycin 125 mg qid for 10 days, we collected serial faecal samples on study entry, days 4, 7, 10, 14, 21, 28 and 42 to determine if non-antibiotic therapy can neutralise c. difficile toxin b in faecal filtrates, promote restoration of the normal microbiota and achieve clinical response. methods: 33 patients were randomised into the study at calgary study sites (out of 289 patients/58 centres). faecal filtrate concentrations of c. difficile cytotoxin b, quantitative counts of c. difficile vegetative organisms, c. difficile spore counts were determined. quantitative aerobic/anaerobic cultures using serial dilutions of faeces 10e-3,5,7,9,11 g à1 wet weight were performed using criteria as outlined in the wadsworth anaerobe laboratory manual. stools from healthy donors served as normal microflora controls. results: thirty of 33 patients provided one or more samples, and 22/30 provided serial samples beyond 7 days and up to 42 days. normal flora controls showed an average of four different bacteroides species in counts of 10 10à12 g à1 faeces wet weight, plus other anaerobic genera in a more inconsistent manner. using bacteroides species as a marker genus for the anaerobic microflora, 15, 8, and 7 patients had bacteroides counts below the limit of detection, between 10 3à8 , or >10 8 cfu/g faeces, respectively, at study entry. vancomycin treatment eliminated vegetative c. difficile with variable spore persistence, and the bacteroides genera remained suppressed in the majority of patients during and after the course of therapy. on the other hand, response to tolemaver therapy appears to be accounted for by the inter-relationship between toxin neutralisation, c. difficile growth/persistence, and the pattern of recovery of the microflora. in general, patients who responded to toxin binding therapy exhibited non-emergence of toxin combined with increase in the numbers of anaerobic organisms. recovery of the anaerobic microflora appeared not to be complete at 14 days in the majority of patients. objectives: the treatment of choice for a. baumannii bacteraemia has not been established. there are few data to guide the selection of agents for treating these infections. carbapenems are generally considered the drugs of choice, but an increasing of the resistant strains has been described. several alternatives guide lines have been proposed: ampicillin-sulbactam (sam) alone or associated with an aminoglycoside, piperacillin-tazobactam (tzp) or tetracyclines. the aim of this study is to know the best alternative in the empirical treatment of these infections according to the temporal evolution of the nosocomial outbreaks or endemic infections in our hospital. methods: from june 1995 to december 2001 we collected all a. baumannii strains from bacteraemia infections and their related focus. all the isolates were characterised by molecular methods in order to obtain different clones using pfge and rep-pcr. susceptibility study was performed by disk diffusion to 23 antibiotics and mic-e-test in the mainly treatment alternatives (imipenem, meropenem, sam, tzp, tobramicine (tm), amikacine (an), and ceftazidime) and interpreted according to nccls criteria. results: in 1995-1996 the empirical antimicrobial treatment (eat) of choice was imipenem because all 64 isolates were carbapenem sensible (s), with two mainly molecular clones (34 isolates c1-aminoglycosides resistant (r), and 30 c2-gentamicin-r, but an, netilmicin and tm-s). according to detection of an outbreak carbapenem-r in 1997 (153 of the 163 isolates) clone c3 multiresistant (only some strains sam or an-tn-sensible) the eat changed to sam and an or tm. this clone was persistent until 1999 and replaced with another multiresistant outbreak (c3b -94% sam-r and 25-75% aminoglycosides-r). then the eat was chosen as monotherapy with an or tn (the only ones sensible of the 23 antimicrobial tested). in the last period (2000-2001) emerges a new clone (c5-carbapenems, sam, aminoglycosides and doxycycline-s) and imipenem returns like the actual eat in our hospital to control bloodstream a. baumannii infections. conclusions: empirical antimicrobial treatment on patients with bloodstream a. baumannii infections in a hospital, with changes in the temporal evolution of the clones associated to outbreaks or endemic infections must be established according to the susceptibility test and molecular characterisation of the strains in different clones. gentamicin-resistant enterococci in paediatric blood-stream infections in a tertiary hospital in tanzania b. blomberg, s.c. mohn, k.p. manji, n. langeland on behalf of the joint study group on antimicrobial resistance at muhimbili national hospital, dar es salaam, tanzania and the university of bergen, norway objectives: enterococci have emerged as major pathogens causing urinary tract, wound and blood stream infections (bsi). nosocomial spread of enterococci resistant to multiple antimicrobials is a great therapeutic challenge. little is known about the role of these pathogens in bsi in east africa. the objective of the study was to assess the prevalence and resistance patterns of enterococcal isolates causing bsi in children at muhimbili national hospital, dar es salaam, tanzania. methods: blood cultures were obtained from 1789 children (age 0-7 years) with fever or signs of serious infection admitted to the hospital during the period august 2001 to august 2002. isolates were identified by standard methods. the identities of enterococcus fecalis and e. faecium isolates were confirmed by polymerase chain reaction (pcr), the isolates were susceptibility tested by e-test and assessed for genetic relatedness by pulsed field gel electrophoresis (pfge). twelve e. faecium isolates were also investigated by mlst. results: thirty-two of 1789 children (1.8%) had growth of enterococcal isolates in blood culture. nine of 17 e. faecium isolates showed combined resistance to ampicillin (are), ciprofloxacin and high-level gentamicin resistance (hlgre). six of 15 e. fecalis isolates were hlgre, but none of these were resistant to ampicillin or ciprofloxacin. all except one of the hlgre were also resistant to chloramphenicol. the resistant strains were recovered from several geographically separated wards, including the neonatal ward. the majority of the e. faecium and e. fecalis were closely related when investigated by pfge. mlst conducted on 12 e. faecium strains also confirmed this result. conclusion: this is the first study to identify outbreaks of bloodstream infections caused by combined are/hlgre e. faecium and hlgre e. fecalis in tanzania. e. faecium was more frequent than e. fecalis. the commonly used treatment regimens at the hospital (ampicillin and gentamicin or penicillin and chloramphenicol) are insufficient for infections caused by are/hlgre enterococci. nonrepetitive (one per patient) resistant to fox k. pneumoniae strains were isolated from clinical specimens (10 from blood, two from bronchial secretions, four from urine, two from wound and five from catheter tips). patients were cared in different wards including intensive care unit (icu) and neonatal intensive care unit (nicu). species identification was done by using the vitek system (biomerieux, france). mics were determined with vitek automated microdilution system and by disk diffusion method. the criteria of the nccls were used to define susceptibility or resistance to antimicrobial agents. expanded spectrum a-lactamase (esbl) production was assessed by the double disk synergy test. the isolates were typed by enterobacterial repetitive intergenic consensus (eric) pcr with the eric-2 primer. isoelectric focusing (ief) of blactamases was performed to representative group isolates. results: antimicrobial profiles demonstrated that all isolates were resistant to third-generation cephalosporins, to aztreonam, cefoxitin, amoxicillin/clavulanate, ticarcillin/clavulanate and piperacillin/tazobactam. four isolates were also resistant to cefepime and cefpirome. all isolates were susceptible to imipenem. ief showed that all isolates expressed two b-lactamases, one with pi of 8.2 which correlated with the shv-5 and one with pi of 9.1 which corresponded to lat-2. eric-pcr analysis demonstrated three strain types. type i, consisting of two subtypes, was common to 15 strains, indicating that the clonal spread was mainly responsible for the outbreak. type ii comprised two isolates and type iii was unique. five isolates were not identified with eric-pcr. conclusions: k. pneumoniae strains, harbouring plasmid-coding for ampc-type b-lactamase, have been established in our hospital. nosocomial infection surveillance, such as restriction of particular antibiotics and adjustment of the infection control measures, has been recommended. acinetobacter baumannii producing the per-1 extendedspectrum b-lactamase objectives: recently per-1 extended-spectrum b-lactamase (esbl) was discovered in a peudomonas aeruginosa strain in france and was subsequently detected in acinetobacter spp. and pseudomonas aeruginosa in other countries including turkey. the purpose of this study was to clarify the molecular epidemiology of infection caused by a strain of cefepime-resistant a. baumannii and also to determine the mechanism of drug resistance. methods: cefepime-resistant a. baumannii strains were isolated from clinical specimens of nine patients hospitalised in an intensive care unit in busan, korea. antimicrobial susceptibilities were determined by the disk diffusion and agar dilution methods. the double disk synergy (dds) test was performed for screening of esbl production. isoelectric focusing and conjugation experiments were performed. blaper-1 and blaper-2 alleles were detected by pcr, and sequences of amplified products were determined by using the dideoxy-chain termination method. pulsed-field gel electrophoresis (pfge) was performed for molecular typing of isolates. results: the isolates showed same antimicrobial susceptibility pattern, positive dds results and pfge patterns. the isolates contained three b-lactamase bands: pi 5.3, 7.9, and 9.4. pcr-based experiments detected blaper-1 genes. mics of ampicillin, piperacillin, cephalothin, cefoxitin, cefoperazone, ceftazidime, cefotaxime, cefepime, and aztreonam to these isolates were !256 mg/l, respectively, and those of imipenem were 8-16 mg/l. despite repeated attempts, the resistance to cefepime of a. baumannii isolates was not transferred to the recipient. conclusions: a. baumannii isolates from clinical specimens of nine patients hospitalised in a same intensive care unit were shown to be of the same clone. all these isolates contained blaper-1 gene which caused resistance to cefepime. to the best of our knowledge, outbreaks caused by per-1 esbl-producing a. baumannii have not previously been described. objectives: hospital outbreaks of salmonella spp. infections are not uncommon not only in europe but also in the united states, but in neonatal units it is rarer. the maternity unit has approximately 4000 deliveries each year. we described the results as well as infection control that stopped the outbreak. methods: from october 2001 to january 2002 six neonates were infected in the neonatal unit of our hospital. the index case corresponding to a newborn delivered in our hospital, she was born by normal vaginal delivery. the 15-day-old patient was admitted again by neurological deficits. seven days at the hospital she developed diarrhoea. the group included five prematures, only case index was not premature. all stool specimens from family case index were negative. stool samples were request for culture from asymtomatic staff and all babies from the neonatal unit (n ¼ 66). the isolates were identified by standard methods and serotyped by agglutination with monospecific antisera. the antibiotics (ab) taken in the study were: ampicillin (a), ticarcillin (t), amoxicillin/clavulanate (a/c), cefalothin (ce), ciprofloxacin (cp), co-trimoxazole (co), nalidixic acid (na), gentamicin (g), thirdgeneration cephalosporins (3gc). its was evaluated by a microdilution method and confirmation by e-test. results: eight strains of salmonella enteritidis serotype o 9,12:h g,m were identified. the phage type (pt) involved was same in all cases, pt 6a. seven were isolated from faeces and one from blood culture. all isolates demonstrated same antibiotic susceptibility pattern with resistance to ampicillin and ticarcillin. fortunately no babies died. no salmonella from stools of nurses, staff personnel and mothers was isolated. conclusions: a hand washing was not sufficiently frequent the infection was probably transmitted by hand contact to prepared milk, infusion and other equipment from the index case. this hypothesis was subsequently confirmed as the outbreak was terminated after eradication of the presumed contamination sources by changing the mattresses, disinfecting the unit and ensuring strict observance of hand washing before and after every manipulation. salmonella enteritidis pt6 is third commonest phage type in spain. objectives: to investigate the cause of an outbreak of pseudomonas aeruginosa isolations following bronchoscopic procedures. methods: from 16 to 31 january 2003, we detected a cluster of p. aeruginosa isolates associated with bronchoscopy (nine samples from eight patients). laboratory culture, bronchoscope and medical records of all cases were reviewed. all of them were related with one bronchoscope and environmental samples were obtained from it. microbiological identification and susceptibility testing were performed with the microscan walkaway 40 system (dade behring). random amplified polymorphic dna analysis (rapd) and pulsed field gel electrophoresis (pfge) were performed on all available isolates of p. aeruginosa (eight clinical isolates from seven patients and six bronchoscope related isolates). results: two of the eight patients showed clinical evidence of infection and required specific antimicrobial therapy (the index case and other patient with two isolates separated by 8 days). all isolates were ceftazidime, aminoglycosides and ciprofloxacin susceptible and imipenem resistant. rapd and pfge patterns revealed that all the clinical and bronchoscope isolates (eight and six, respectively) were indistinguishable. the bronchoscope was replaced and no further cases appeared. conclusions: we documented contamination of a bronchoscope with p. aeruginosa and possible secondary infection of at least one patient. microbiologists have an essential role in the detection of medical devices contamination, especially by surveillance of the emergence of infrequent bacterial recovery. in all three cases klebsiella pneumoniae was isolated from the blood cultures. the aim of this study was to investigate the epidemiological relation among the isolates and to try to find a common source for the infection. methods: environmental samples, including different i.v. fluids and drugs, and skin samples were obtained in order to detect the source of the infection. microbial identification and in vitro susceptibility tests were carried out automatically with the microscan system (dade). clinical isolates were molecular typed by random amplification of polymorphic dna (rapd) using one 10mer oligonucleotide, pcr-ribotyping with oligonucleotide of the intergenic 16s/23s region and pfge using xbai as a restriction enzyme. an unrelated strain was also included in all the experiments, as a control, in order to check the discrimination power of the techniques. results: all three clinical isolates of k. pneumoniae obtained from blood cultures shared the same biotype and antibiotype, and were all resistant to ampicillin, gentamycin and tobramycin. molecular typing methods proved clonal identities among the clinical strains. patterns generated were different from those of the control strain. the source of the infection could not be demonstrated in any of the environmental or newborn skin samples. conclusions: a single k. pneumoniae strain was the cause of the fulminant sepsis in the three newborns. all three molecular typing methods, rapd, pcr-ribotyping and pfge accurately demonstrated clonal identities of the isolates. the common source of the infection could not be detected due, probably, to the logical delay in culture growth and identification. the objectives of this presentation are to describe the outbreak and the infection control measures implemented, and to constitute an example for handling possible future outbreaks with limited resources. this is the first ekc outbreak reported from turkey. methods: eye clinic of kou hospital is equipped with modern devices; however it has limited physical conditions (e.g. insufficient hand-washing facilities) because of temporary settlement of the hospital after the earthquake of 1999.on 12 december 2001, the infection control team (ict) was alerted to ekc cases. an investigation began and infection control measures (icm) were implemented. conjunctival swabs of patients with ekc and environmental swabs were obtained and studied in gata and hacettepe university microbiology laboratories. infection control protocol (icp) was implemented as recommended by apic guidelines with some modifications. in addition, terminal disinfection (td) was applied two times and after tds clinic was closed for first 2 and than 4 days (figure 1) . results: a total of 116 ekc cases were diagnosed among the 1033 patients who visited the eye clinic during the outbreak (general attack rate: 11.2%). seventy-five of the ekc cases were male and average age was 41.39 ae 21.98 (range: 6 months to 85 years). primary and secondary attack rates were found to be 26 and 119%, respectively. adenovirus type d was isolated from patient samples, biomicroscope and device solution. with the implementation of icp and td, ekc cases decreased by time and outbreak disappeared about 14 days after the second td and closing the clinic for four days (figure 1) . conclusion: this is the first outbreak reported from turkey. isolation of virus from biomicroscope and device solutions which are used for more than one patient is an evidence of transmission from environment. although several reports have described icm that terminated outbreaks of nosocomial ekc, this study demonstrates that implementing td and/or closing the clinic for four days in addition to icm, may control nosocomial ekc outbreaks. background: because of severity of underlying disease, multiple venous accesses, parenteral nutrition and often increased length of stay, intensive care unit (icu) patients are at increased risk for catheter-related candidemia (crc). we investigated health and economic outcomes in icu patients with crc. methods: in a retrospective matched cohort study (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) attributable mortality and excess length of stay for crc was investigated. matching was (1:2 ratio) based on severity of underlying disease and acute illness (apache ii score and admission diagnosis) and length of icu stay prior to the onset of the candidemia. as expected mortality can be derived from apache ii; this matching procedure results in an equal prognosis for cases and control subjects. attributable mortality is determined by subtracting the hospital mortality rate of the controls from this of the candidemic cases. excesses in length of icu stay and hospitalisation were determined by subtracting the median length of stay of the controls from this of the cases. results: during the study period 21 icu patients developed a microbiologically documented crc (out of a total of 83 candidemic patients). nineteen catheters were removed within 24 h. cases (n ¼ 21) and controls (n ¼ 42) had an equal age (resp. 49 ae 20 vs. 53 ae 19 year; p ¼ 0.470), apache ii score (resp. 23 ae 8 vs. 23 ae 8; p ¼ 0.754) and incidence of respiratory failure (95 vs. 86%; p ¼ 0.479), acute renal failure (33 vs. 14%; p ¼ 0.153) and haemodynamic instability (76 vs. 69%; p ¼ 0.767). the excess length of icu stay was 11 days (median 31 vs. 20 days; p ¼ 0.002). although patients with crc had a longer length of hospital stay this difference was not significant (52 vs. 30 days; p ¼ 0.508). the attributable mortality of crc was 19.1% (95% ci: à6 to 44%) as hospital mortality rates in cases and controls were 42.9 and 23.8%, respectively (p ¼ 0.207). conclusion: our data revealed that, after careful adjustment for severity of underlying disease and acute illness, crc is not associated with a significantly higher mortality in icu patients. it is, however, associated with a significant excess in length of icu stay, thereby representing an important economic burden. patients were hospitalised in two internal medicine departments, two surgical departments, the nephrology department and the intensive care unit, during the period july 2001 to november 2003. the catheter tips were cultured using the following methods: (a) semi-quantitative maki's and co. and (b) quantitative cleri's and co. samples for culture were taken also from the site of catheter insertion into the skin and from the hub. blood culture samples were taken from a peripheral vein in cases of clinical suspicion of bacteraemia or sepsis and they were incubated using the bactalert (organon teknika) automated system for 6 days. results: 33 cases of ccbri were recorded. the incidence of ccbri was 9.3 per 1000 catheter days. in 29 of the 33 cases of ccbri the origin of colonisation was determined. in 10 cases which all had positive catheter tip and hub cultures with the same strain, the gram-negative bacteria prevailed (6/10, analytically e. aerogenes three, k. pneumoniae two and p. aeruginosa one) while in four cases candida spp. (three cases) and coagulase negative staphylococcus (cons) (one case) were isolated. in contrast, in 19 cases of ccbri with positive catheter tip and skin point entry cultures with the same strain, the gram-positive bacteria prevailed (15/19, analytically s. aureus eight, cons six and corynobacterium spp. one). conclusions: (1) the incidence of ccbri was 9.3 per 1000 catheter days. (2) ccbri caused from gram-positive bacteria was mainly derived from the catheter site entry, whilst colonisation of hub caused mainly gram-negative ccbri. (3) the preventive measures should be focused on better aseptic techniques and hand hygiene, care of the catheter's entry site and better training of the medical staff. we studied 104 patients, suspect for catheter-related infections (cri) -52 from icu and 52 from hdu with central venous catheters used for parenteral nutrition, drug administration or haemodialysis. the preferred vein in icu was v.subclavia, 48, and in hdu, v. femoralis, 50 catheters. mean duration of catheterisation -10 days in icu and 22.8 days in hdu. signs for colonisation of the catheters were found in 57 cases -32 in icu and 25 in hdu. the most common microorganisms in icu were gram-negative rods (kesgroup, b. cepacia, pseudomonas spp.) -21 (65.6%) followed by coagulase-negative staphylococci (cns) -10 (31.2%). in hdu in most of cases were isolated cns -19 (73.0%) and s. aureus -4 (15.3%) (p < 0.01). as catheter-related bacteraemias (crb) were considered in 16 cases, 11 of them in icu and 5 in hdu. causative microorganisms of crb in icu were most gram-negative rods -9(81.8%) and syaphylococcus spp. 3 (60.0%) in hdu (p < 0.01). conclusions: the frequency of crb in icu is significantly higher, 21.1-9.6%, in hdu (p < 0.01).they developed earlier and were caused by gram-negative rods. more probable way to development of crb in icu is the catheter hub and hdu is the skin of the patients. catheter-related infection (cri) is considered as a cause of increased hospital morbidity but its influence on hospital mortality remains a matter of debate. in critically ill patients, baseline severity, underlying conditions and various confounding factors may explain the observed increased mortality rather than cri itself. in order to determine the influence of cri on hospital mortality in icu, all episodes of nosocomial septicaemia were reviewed. material and methods: retrospective analysis of all nosocomial septicaemia occurring over a 7-year period in a teaching hospital. septicaemia episodes were separated in secondary, primary and proven catheter-related bloodstream infections. baseline severity (saps score), delay between admission and infection, and hospital mortality were determined. results: over this 7-year period, 195 853 patients were admitted to the hospital and 2720 episodes of cri were recorded (1.38%, 1.5/1000 catheter-day (ktd)). hospital mortality for all septicaemia was 21.7% while mortality related to secondary septicaemia was 28.5% (p < 0.05). during the same period, 22 313 patients were admitted to the icu, corresponding to 81 740 ktd. four hundred twenty-four episodes of septicaemia occurred in these patients (5/1000 ktd), of which 166 were primary septicaemia and 87 were proven cri (1.06/1000 ktd). mean saps score for all icu patients was 30 and hospital mortality 6.9%. icu patients developing infection had a mean baseline saps score >40. cri occurred more than 2 weeks after icu admission (median : 14 days, mean 20.5 days). pathogen-associated cri were scn 31%, s. aureus 18%, e. faecalis 12%, candida spp. 10%, other 29%. hospital mortality in patients developing cri was 42/87 (48.2%). conclusions: in this study, hospital mortality in critically ill patients developing cri was high but seemed to be primarily determined by baseline severity and underlying conditions as reflected by saps score and prolonged delay between icu admission and septicaemia. staphylococcus epidermidis is the most important pathogen of these systemic infections. objectives: to study the genomic dna profiles of s. epidermidis isolated from catheter-related infections and bloodstream infections comparing with the strains isolated from skin and nasal swab in patients hospitalised in a tertiary care university hospital. methods: catheter-related infections were defined according to the cdc definitions. patients with a culture for s. epidermidis from blood and catheter tip (>15 cfu) were selected to have swabs from skin and nasal for s. epidermidis. the s. epidermidis were typed using pfge, antibiotic susceptibility testing and biofilm detection, by congo red method, were performed. results: twelve patients with 17 episodes of catheter-related infections were included in this study and 255 strains were analysed. in 10 episodes, the same dna profile was detected in cvc/blood and in the skin/nasal and in seven episodes the clone causing cvc/blood infections were not found in skin/nasal. the mean time of isolation of s. epidermidis with clonal relation between cvc/blood and skin/ nasal colonisation from the first day of hospitalisation until the detection in cvc/blood was 25.3 days. in episodes without s. epidermidis clonal relation, the mean time was 13.7 days. pfge identified three hospital endemic profiles that were present in 46.6% (119/225) of all strains from 10 episodes, including the strains from cvc/blood infections and in skin/nasal colonisation. in the strains from skin/nasal colonisation, the endemic profiles were present in 47.9% (93/194) of the strains. the endemic dna profiles were biofilm producers and were resistant to penicillin g, oxacillin and ciprofloxacin, variable susceptibility to aminoglycosides and were susceptible to vancomycin. conclusion: patients with long term hospitalisation were previously colonised by hospital endemic s. epidermidis strains that were responsible for catheter-related infections. , and requiring a cvc were included in this retrospective cohort. the following data were analysed: patient and cvc characteristics, risk factors and microbiological results. the diagnosis of cvc-ri was based on brun-buisson methodology (1987) . the comparisons were done using the chi-square and student's t-tests. a multiple logistic-regression model was used to identify risk factors of cvc-ri according to their adjusted odds ratio (aor; 95% ci) and completed by a survival analysis adjusted on duration of hospitalisation and cvc, the unit and the timing of cvc implementation (before or after admission in the unit). results: a total of 89 patients were included who required 102 cvc (60 cvc were implanted as per the hospitalisation in this study units [gr#1] and 42 before the patient admission in this same units [gr#2]). the total of catheter days was 1086 (respectively 581 for gr#1 and 505 for gr#2). the number of cvc-ri was 21 that is 20.6% of cvc and the incidence rate per patient was 21%. the part number of cvc-ri were respectively 9 (15%) for gr#1and 12 (28.6%) for gr#2. the incidence density of cvc-ri was 1.93/100 catheter days for the totally cohort, 1.55/100 for gr#1 and 2.38/100 for gr#2. in the totally cohort cvc-ri was monomicrobial in 16 cases (76.2%). in that case the most prevalent bacteria were: coagulase-negative staphylococcus spp. (43.8%) and staphylococcus aureus (37.5%) and in case of plurimicrobial infection the most prevalent agents were: staphylococcus aureus (60%), coagulase-negative staphylococcus spp. and enterococcus faecalis (40% for each). intestinum somatoplasty was not was a risk factor for cvc-ri in this study. the crude mortality rate was 5.3% (1/19) and 10% (7/70) for cvc-ri and non-cvc-ri, respectively (p ¼ 0.5). conclusion: in these surgical units, the incidence of cvc-ri is high and was related to the frequency of manipulations of the line such as infusion, parenteral nutrition, injections and dressing even after adjustment on the duration of cvc and timing of cvc implantation. an intervention focused on these risk factors is planned to reduced cvc-ri and improve the quality of care. case 1: a schizophrenic 41-year-old man was admitted to the hospital because of fever of 2 weeks duration; he was affected by diabetes type ii and nh lymphoma diagnosed 6 months earlier and treated with chemotherapy through a groshong cvc and, subsequently, with chronic steroid. multiple blood cultures, performed from cvc and peripheric veins, were positive for e. faecalis and e. coli; the patient was treated with ceftriaxone 2 g ev qid â2w + lock-in therapy with teicoplanin 60 mg (in 3 ml) and ciprofloxacin 6 mg (in 3 ml) for 6 h a day for 10 days. it was obtained a clinical and microbiological resolution without removal of cvc. case 2: a 49-year-old man was admitted to the hospital for septic fever; 3 months earlier a groshong cvc had been placed to treat with chemotherapy a rhinopharyngeal carcinoma. multiple blood cultures (from cvc and peripheric veins) were positive for a multi-drug-resistant stenotrophomonas maltophilia (s only to chloramphenicol, trimethoprim-sulfamethoxazole and levofloxacin). the patient was successfully treated, without removal cvc, with systemic trimethoprim-sulfamethoxazole + levofloxacin combined to antibiotic lock (ciprofloxacin 8 mg in 4 ml for 12 h a day for 7 days). conclusion: the cases reported by the aa confirm that many catheter infections can be maintained in place and sterilised with lock-in therapy avoiding to replace expensive intravascular lines with unnecessary and risky insertions. one of the questions to resolve will be whether or not concomitant systemic antibiotic therapy is necessary. background: nosocomial infections influence upon the mortality, quality of patients' life, costs and length of hospitalisation. the source of those infections might be staff members, contaminated water system, air-conditioning or pests. disinfectants are helpful in reducing or eradicating harmful pathogens existing in hospital environment. some bacteria are able to grow on a surface as a biofilm. this form is more resistant to external harmful conditions such as antibiotics, disinfectants or host defence. bacterial adhesion was recognised as the important virulence factor for colonisation of patient or biofilm formation. in our study the susceptibility of 65 bacterial strains isolated in hospital environment (colonising or infecting patients or carried by german cockroaches) to antibiotics and chemical disinfectants was determined. moreover the efficacy of the disinfectant working solution (active ingredients: sodium dichloroisocyanorate 1795.2 mg/l; glucoprotamine 5200 mg/l; potassium persulphate 4300 mg/l) on selected bacterial strains adherent to catheter (after growing for 5 days on it) by treating then for 15 min was determined. results: susceptibility profile to antibiotics varied; among grampositive bacteria the mlsb, mrcns strains were found; among gram-negative bacteria the esbl, ampc phenotype were described. determined mic values or disinfectants were in range: sodium dichloroisocyanorate 7.8125-2000 mg/l; glucoprotamine 1.453-500 mg/l; potassium persulphate 7.8125-1000 mg/l. the results indicate that the working solution of the disinfectant might be ineffective to some strains of well-known pathogens: serratia marcescens, citrobacter freundii, enterobacter cloacae and staphylococcus epidermidis. the examination of disinfectants efficacy on selected strains showed that some bacterial strains were more resistant when they were grown on catheter for 5 days. the mic value was lower than working solution of that chemical even more than 300 times. moreover it was found that all tested disinfectants were ineffective to some strains adherent to catheter ex. s. marcescens and e. cloacae strains isolated from the body surface of german cockroaches. conclusions: the possibility of biofilm formation could explain the increase of resistance to disinfectants of some strains. german cockroaches carrying them in hospital should be considered not only as nuisance insects, but also as a real source of resistant to antibiotics and disinfectants bacteria. background: indwelling catheters are commonly colonised by skin flora. propionibacterium spp. are among the commonest bacteria of normal human skin but currently recommended catheter-culture procedures would not detect its presence. furthermore, propionibacterium is nearly always regarded as a blood culture contaminant and automated blood culture methods may not detect a proportion of them. our objective was to determine the rate of catheter colonisation by propionibacterium spp. in unselected intravascular catheters submitted for culture. methods: 368 intravascular catheters were processed by the rollplate technique and incubated in air at 37 c for at least 2 days. organisms that were present in significant counts were subcultured for identification and susceptibility testing. when the conventional aerobic processing was finished, all primary culture plates were reincubated in an anaerobic jar. after 7 days of anaerobic incubation the plates were read looking for bacterial colonies that were not initially present. control plates were inoculated with a suspension of p. acnes to assess the influence of aerobic preincubation on the final number of colony forming units (cfu). conventional processing detected significant growth of bacteria in 24.8% of all catheters and no significant number of colonies (<15) in an additional 17.6% samples. anaerobic reincubation yielded p. acnes in significant counts in 3.5% of all catheters (15% of all positive catheters) and no significant number of colonies in an additional 14.1% of samples. three samples yielded significant growth of both aerobic and anaerobic bacteria. of all the organisms recovered in significant counts, coagulase-negative staphylococci represented 55.5%, p. acnes 11.1%, s. aureus 7.7% and corynebacterium spp. 6%, enterococcus spp. 5.1% and other bacteria and yeast 14.5%. anaerobic bacteria other than p. acnes were rarely recovered in non-significant counts. aerobic preincubation for 5 days did not substantially affect the final number of cfu. conclusion: p. acnes is the second most frequent coloniser of intravascular catheters. anaerobic reincubation of plates used in standard routine is a simple method that could be useful for catheterrelated research projects. the potential of p. acnes as a cause of catheter-related bacteraemia merits further studies. results: nineteen patients included 10 males and nine females, whose ages ranged from 15 to 67 years (mean 41 years). all of the patients were hospitalised in the neurosurgical department. the most common underlying conditions were intracranial haemorrhage (8/19 cases), followed by hydrocephalus (4/19 cases) and cranial injury secondary to trauma (3/19). all patients underwent surgical procedures prior to infection, which included 15 craniotomies and four ventriculostomies. all patients were receiving antibiotic therapy at the onset of infection. mean time between surgical procedure and diagnosis of meningitis was 15 days (6-27 days) . fever and neck stiffness was found in eight and seven patients, respectively. in 12 patients serum leukocyte count was higher than 10 â 1000/cu mm. mean leukocyte count in serum and cerebrospinal fluid was 16 â 1000/cu mm (min 10 â 1000/cu mm, max; 35 â 1000/cu mm) and 19 â 1000/ cu mm (min 10/cu mm, max; 5620/cu mm) respectively. mean csf protein concentration was 210 mg/dl and mean csf glucose concentration was 44 mg/dl. only in 14 of 19 cases the microorganism was isolated from cerebrospinal fluid. acinetobacter spp. (11 cases), k. pneumoniae (two cases) and e. cloacae were the isolated microorganisms. most of the acinetobacter isolates were susceptible to carbapenems but all of them were resistant to thirdgeneration cephalosporins. a combination of carbapenem plus an aminoglycoside and/or vancomycin therapy was applied most of the patients. an additional intrathecal aminoglycoside dosage was needed for seven patients who responded poorly. the overall mortality rate in these patients was 43%. conclusion: there has been an increase of post neurosurgery meningitis cases. in addition, the emergence of strains resistant to third-generation cephalosporins in this group has also been noted in recent years, and has become a great therapeutic challenge. early diagnosis and initiation of appropriate antibiotic therapy is needed in this potentially fatal disease. objectives: pulmonary resection is associated with considerable risk of infection, so antimicrobial prophylaxis has become routine practice in thoracic surgery. the aim of this study was to assess changes in microflora of upper respiratory tract in hospitalised patients with non-small cell lung cancer (nsclc) before and after preoperative antimicrobial prophylaxis. methods: 51 patients with nsclc aged 37-73 years were subdivided into two groups: (a) control group (21 patients without antimicrobial prophylaxis and surgery), (b) 'prophylaxis' group (30 patients undergoing pulmonary operation with preoperative antimicrobial prophylaxis, including piperacillin, cefuroxime or ceftriaxone alone or in combination with amikacin). throat and nasal specimens were taken up two times: examination i -on the day of hospital admission and examination ii -on the third or fourth day of hospitalisation in group a and on the third or fourth day after the surgery in group b. the routine microbiological methods were used for isolation and identification of bacteria and fungi. statistical analyses were performed by nonparametric tests. results: the colonisation of nasal mucous membranes by pathogenic microflora did not differ significantly during hospitalisation between group a and b. similar situation was observed in the case of pathogenic microflora on throat mucous membranes in group a. different results were obtained in group b. the increased prevalence of pathogenic microflora on throat mucous membranes was observed -from 46.67% in examination i to 80% in examination ii. this difference was statistically significant (p ¼ 0.007). in group b colonisation of throat mucous membranes by enterobacteriaceae family and candida spp. was increased significantly during hospitalisation (from 10 to 26.67% and from 36.67 to 70%, respectively). conclusion: our results indicate that antimicrobial prophylaxis can be regarded as an important predisposing factor for changes of upper respiratory tract microflora and for colonisation of mucous membrane of throat with enteric gram-negative rods and yeast-like fungi -candida spp. these microorganisms are potential causative agents of endogenous infections in immunocompromised patients with lung cancer. objectives: the purpose of this study was to determine aerobic and anaerobic bacteria colonising pleural drains in patients with non-small cell lung cancer (nsclc) undergoing thoracic surgery and to define antimicrobial agents susceptibility of isolated strains. routine antimicrobial prophylaxis included piperacillin or cefuroxime. in some cases beta-lactam was used in combination with amikacin. methods: material for research was fluid from pleural drains collected from 34 patients aged 38-72 years two times -on the day of pulmonary resection and on the fourth day after operation. samples were routinely cultured under aerobic and anaerobic conditions and determined using api system (biomerieux). antimicrobial resistance was estimated by the disc diffusion method according nccls recommendations. results: aerobic (49 strains) and anaerobic (23 strains) bacteria were found in 30 (44%) and 15 (22%) samples, respectively. among aerobic bacteria, gram-negative rods (22 strains; 18 -belonging to non-fermenting rods) and coagulase negative staphylococci (cns; 14 strains) were most often cultured. fifteen strains of non-fermenting rods and 11 isolates of cns were classified as multidrug resistant (mdr) organisms. two isolates of s. marcescens were producers of extended spectrum beta-lactamases (esbls) and inducible beta-lactamases (ibls). all staphylococci were susceptible to vancomycin and teicoplanin. cns strains resistant to penicillin and oxacillin but sensitive to amoxicillin/clavulanate were most frequently isolated. only two methicillin-resistant strains, belonging to s. haemolyticus were found. the most common anaerobic bacteria were from the genera eubacterium (nine strains) and actinomyces (six strains). all of them were highly susceptible to antimicrobial agents except metronidazol (69.6% resistant strains) and chloramphenicol (52.2% resistant isolates). conclusion: colonisation of pleural drains does not mean infection, however knowledge about bacterial species found in drain fluid in a local population and antimicrobial resistance (especially mdr strains) has a major impact on the success of prophylaxis and therapy of potential postoperative infections. a. artero, j.j. camarena, r. zaragoza, s. sancho, j. tamarit, r. gonzález, j. nogueira valencia, e objectives: to know the clinical and microbiological characteristics of diabetic patients with severe bacteraemia. to identify the differential features of severe bacteraemia between patients with and without diabetes mellitus (dm). materials and methods: during a 7-year period (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) we have evaluated all bacteraemias with severe sepsis or septic shock in an intensive care unit of a teaching hospital. clinical and microbiological features were recorded from clinical charts. the spss package (9.0) was used to identify significant differences between dm and no-dm cases, and to determine if the presence of dm was associated with mortality by a multivariate analysis. results: the prevalence of dm in patients with severe bacteremic infections was 23.8% (n ¼ 60). in the group of dm the mean age of patients was 69.2 ae 7.9 years, the relation between men/women was 1.06, the origin of the bacteraemias was nosocomial in 88.3%, severe sepsis was present in 61.6% and septic shock in 31.6%. the focus of infection in diabetic patients was: unknown (n ¼ 29), catheter (n ¼ 11), respiratory (n ¼ 9), urinary (n ¼ 5), abdominal (n ¼ 3), vascular (n ¼ 2) and cutaneous (n ¼ 1). the main microorganisms causing of bacteraemias in patients with dm were: cns (21.6%), acinetobacter baumannii (15%), staphylococcus aureus (11.6%), escherichia coli (8.3%) and enterococcus spp. (8.3%). a higher proportion of nosocomial cases in dm was the only differential feature between patients with and without dm (p ¼ 0.041). the global mortality in patients with and without dm were 43.3 and 56.7% (p ¼ 0.068), respectively, and the related mortality were 21.6 and 27.6% (p ¼ 0.361), respectively. dm was related neither to global (or ¼ 0.523, 95% ic 0.269-1.015) nor related mortality (or ¼ 0.730, 95% ic 0.337-1.583) by multivariate analysis. conclusion: dm is prevalent between critically ill patients with severe bacteraemic sepsis and bacteraemic septic shock. diabetic patients had a higher proportion of nosocomial origin of bacteraemia. we did not find that dm was related to mortality in severe bacteraemic infections. a. poulou, f. markou, x. efthimiou, f. mountaki objectives: brucellosis is a zoonotic disease whose prevalence in northern greece is high and constitutes a significant problem for the local health authorities. the aim of this study is to report a rare case of transmission of brucella melitensis. patients: a female infant showed signs of respiratory distress during delivery. the obstetrician in charge tried to clear the respiratory tract of saliva and amniotic fluid. in his attempt he swallowed some secretions. a blood culture from the infant was incubated in the bactec 9120. after 3 days b. melitansis was isolated. the case was proved to be a rare case of congenital brucellosis. the family of the infant was checked and the mother was found to be positive at 1/80 titre by brucella agglutination test though her blood culture was negative. neither her husband nor her other two children were positive on the wright agglutination test. both parents were involved in animal husbandry. two months after the delivery of the infected infant the obstetrician reported pains in the back of his neck and low fever. a blood test revealed leucopenia and neutropenia (white cell count 2800/mm 3 ). the wright agglutination test was positive at titre 1/80. a blood culture was taken and b. melitensis was isolated. transaminases were normal. the obstetrician reported that he had not consumed unpasteurised milk or dairy products. he was treated with vibramycin and rifadin for 40 days. two months later the wright agglutination test was found negative and the white cell count was normal. conclusions: b. melitansis is usually transmitted through consumption of unpasteurised diary products. in these cases we had transplacental transmission and transmission through infectious secretions via the gastro-intestinal tract. therefore it is essential that detailed medical case histories should be taken from pregnant women in order to avoid congenital infections and that medical personnel should be aware of the possibility of such transmission. objectives: coryneforme bacteria have gradually acquired greater importance in infectious pathologies, especially as opportunistic nosocomial pathogens, some of them displaying resistance to various antibiotics. the aim of this report is to describe some of these bacteria with significant implication in different clinical pictures. methods: over a 3-year period we characterised the coryneforme isolates with presumable clinical significance. clinical significance of the isolates was evaluated according to clinical information received (fever, intravascular devices, underlying disease, prolonged antibiotic therapy, etc.) as well as microbiological criteria (more than one isolation from habitually sterile anatomical areas and/or repeated isolations as predominant flora in sites contaminated with comensal flora). results: in 35 patients the isolations were clinically significant. the most frequent isolations (18) were found in blood culture: seven corynebacterium amycolatum, five corynebacterium jeikeium, two corynebacterium minutissimum, two dermabacter hominis, one corynebacterium group g, one brevibacterium sp. in another eight cases bacteraemia was accompanied by isolation of the same species in intravenous catheters (two c. amycolatum, one c. striatum, one c. jeikeium, one c. group g), a pace-maker cable (c. minutissimum) or soft tissue wound (one c. urealyticum, one brevibacterium sp.). in addition, four c. striatum were isolated (three in respiratory secretions and one in a lower limb abcess), two c. amycolatum in mammary abscesses, one c. jeikeium in articular fluid and two c. urealyticum in urine. all the isolates were sensitive to vancomycin (mics <0.5 mg/l), while sensitivity to beta-lactamics, macrolides and fluorquinolones was variable. conclusions: (1) c. amycolatum and c. jeikeium were the most frequently found corynebacteria with presumable clinical significance. dermabacter and brevibacterium were the genera identified among the non-corynebacterias. (2) outbreaks of nosocomial ssss and impetigo bullosa in infants have been well-described to be associated with the well baby nursery. the source of infection has been traced to health care workers in the delivery room or the newborn nursery. the initial site of s.a. colonisation/infection may be the anterior nares, nasopharynx, conjuctiva, umbilicus and/or the blood rather than the skin. often the personnel are asymptomatic carriers of the epidemic strain of s.a. objectives: the aim was determine the genetic relatedness of s.a. isolated from patients and staff and investigation of the potential source of the infection. material and methods: in november 2001 27 strains of s.a. were isolated from various materials from newborns hospitalised at neonatological and obstetrician departments as well as from the staff. biochemical test api staph was used for the species identification. to molecular typing of isolates was used pulsed field gel electrophoresis (pfge). interpretation criteria for the gels followed manufacturer's guidelines: isolates with identical restriction profiles were assigned the same type, isolates that differed by one genetic event (one to three bands) were considered closely related, isolates with a four-to five-band difference were considered possibly related, and isolates that differed by more than six bands were different strains. results: comparative analysis of the banding pattern for the isolates can be divided into several categories: genetic: type asix strains from newborns with impetigo bullosa and one from staff (baby nursery); type b -two strains from the staff; type c -seven strains from the staff; type d -two strains from the staff; nine other types -each one from one person from the staff. conclusions: all the cases of impetigo bullosa were caused by one genetic type of s. aureus which allows to characterise the infection as a hospital infection. strains isolated from the staff (except one person) belonged to different genetic types (unrelated strains). isolation of the same genetic type from infected newborns and a person from the stuff may suggest that this person was the source of the infection, but we can not exclude that she was accidentally colonised during the hospital outbreaks. to define whether bacterial translocation is a process involved in the series of events following multiple trauma. methods: crushing fracture of the middle of the right femor was performed in 12 new zealand rabbits. blood sampling was performed before and 4 h after fracture for the determination of tumour necrosis factor-alpha (tnf-alpha) and of nitric oxide (no). tnf-alpha was estimated by a bioassay on l929 fibrosarcoma cell line and no by a colorimetric assay. survival was recorded and after death segments of liver, spleen and lower lobe of the right lung were cut for quantitative culture. < 11.5. conclusion: in this in vivo model of pp (1) gat was strongly effective on the fully susceptible strain and its efflux derivative, despite the emergence of rm for this later one. (2) gat was ineffective, as expected on resistant gyra strain, but more surprisingly on parc mutated strains, mainly due to the presence of rm. (3) these mutants were selected in vivo in a msw more precisely defined by pkpd parameters using mpc. (4) low levels of resistance to fq should be detected by simple tests to guide the therapeutic options. objective: boost of systemic neutrophil count by g-csf prior to infection leads to diminished growth of pneumococci in experimental meninigitis and improves survival. whether this protective effect also includes attenuation of hearing loss is reported here. materials and methods: rats -infected intracisternally with $1 â 105 s. pneumoniae serotype 3 -were randomly allocated to receive g-csf (10 lg/kg s.c. td) 48 h prior to infection (n ¼ 16), late treatment (28 h postinfection, n ¼ 16) or no g-csf (n ¼ 22). all animals also received ceftriaxone started 28 h postinfection. infection was documented by blood and csf tap 24 h post infection. just before, 24 h and 7 days after infection, assessments of hearing was made by measurements of distortion product otoacoustic emissions (dpoae) at f 2 ¼ 4-70 khz and by assessment of hearing thresholds by auditory brain stem responses (abr) at 52 khz in levels from 20 to 100 db spl. results: 24-h postinfection hearing loss was significantly increased in g-csf treated animals compared with untreated (hearing loss in 37.5 vs. 14.3% of animals from f 2 ¼ 10-50 000 hz and 75 vs. 42.9% f 2 > 50 000 hz, respectively, mann-whitney, p ¼ 0.026). on day 8 postinfection among surviving animals, severity of hearing loss in g-csf pretreated animals was furthermore increased compared with the control group (severe hearing loss in 92.3 vs. 50% from f 2 ¼ 10-65 000 hz, respectively, mann-whitney, p ¼ 0.013). late g-csf treatment did not affect hearing loss significantly compared with the control group. objective: bacterial meningitis is characterised by an intense inflammatory host response that contributes to the high mortality and morbidity of the disease. doxycycline is a clinically used antibiotic which has anti-inflammatory effects that are separate and distinct from its antimicrobial action, including the reduction of cytokine release and the inhibition of matrix metalloproteases. the present study assessed the effect of doxycycline, when given as adjuvant therapy in experimental pneumococcal meningitis. methods: eleven-day-old rats were infected intracisternally with 10 ll of saline containing 2.5-1.5 â 10 6 cfu/ml streptococcus pneumoniae. at 18 h after infection all animals received ceftriaxone (100 mg/kg i.p., q12 h) and were randomised for administration of a single dose of doxycycline (30 mg/kg s.c.; n ¼ 67) or an equal volume of saline (500 ll; n ¼ 65). at 40 h after infection, surviving animals were sacrificed. albumin concentration in the brain was assessed as an index for blood-brain barrier (bbb) leakage. brain damage was quantified by histomorphometry. results: a single dose of doxycycline (30 mg/kg) vs. saline improved survival (survival rate: 80 vs. 52%, p < 0.001), protected the bbb (cortical albumin/total protein: 7.9 vs. 12.7 lg/mg, p < 0.04) and reduced injury in the cerebral cortex (damage in percent of cortex; median [range] 0 [0-2.8] vs. 0 [0-26.9], p < 0.05). conclusion: adjuvant treatment with doxycycline may be a promising approach to prevent death or neuronal injury as a consequence bacterial meningitis. establishing conditions resulting in null survival by antibodies protection or antibiotic treatment. methods: a fully amoxicillin-resistant (mic of 8 mg/l) serotype 6b streptococcus pneumoniae was used as infecting strain. amoxicillin was administered at a dose (3.12 mg/kg) producing serum concentration lower than the mic of the infecting strain all over the treatment period (c max : 6.1 mg/l). passive immunisation was performed with hyperimmune serum (hs; obtained from mice weekly inoculated with whole cell heat-inactivated inoculum for 5 weeks) diluted in pbs up to dilution 1/6 that had shown null protection (0% survival) in preliminary experiments. groups of 10 balb/c mice weighing 19-22 g were passively immunised with one-single intraperitoneal (ip) injection of the 1/6 dilution of hs, 1 h prior to infection with the 6b pneumococcus. amoxicillin treatment was started 1 h after inoculation and continued t.i.d for 48 h. groups of animals receiving placebo (pbs), non-immune serum, non-diluted hs, 1/6 dilution of hs or amoxicillin 3.12 mg/kg alone were included as control groups. mortality was recorded over the 7-day follow-up period. results: survival rates in all control groups were lower than 10% except in the non-diluted hs that was 100%. antibiotic treatment in passively immunised animals produced survival rates of 100%, with significant differences vs. controls (except the non-diluted hs). conclusion: since amoxicillin concentrations were below the mic (8 mg/l) of the infecting organisms all over the treatment period (c max of 6.1 mg/l), the presence of specific antibodies produced in vivo efficacy of sub-inhibitory concentrations. the in vivo combined effect antibodies/amoxicillin is synergistic and not only additive considering the survival rates obtained by the antibodies (0% survival) and amoxicillin sub-inhibitory concentrations (10% survival) alone and those obtained when acting together (100% survival). (15 mg/kg) and cro (100 mg/kg) were injected at hour 0 and v (20 mg/kg) were injected at hours 0 and 4. cro and v were standard doses. d corresponded to high doses in humans. csf samples were repeatedly collected during therapy in order to determine antibiotic levels and killing rates. d serum levels peaked at 200 mg/l decreasing slowly to 36 mg/l 8 h later. d csf levels ranges between 5 and 3 mg/l. d penetration into inflamed meninges was 5%. results of bactericidal activity of the different regimens are expressed in delta log 10 cfu/ml h and delta log 10 cfu/ml over 8 h. results are presented in table 1 . conclusions: (1) d is highly efficacious against penr and penr+ qurr pneumococci in experimental meningitis, sterilising the csf of rabbits within 4 h (9 out of 10 in both d treatment groups). (2) d as monotherapy is significantly superior to the standard regimen based on a combination of cro with v against both strains. (3) the efficacy of d was also confirmed in time-killing assays over 8 h. objectives: skin-temperature is an effective measure of the severity of pneumococcal pneumonia in mice and can be used to predict lung bacterial counts and imminent death. skin-temperatures vary considerably in groups of infected mice and thus, drug intervention at a particular skin-temperature more closely resembles that which is used in humans. in this study, we compared the efficacy of moxifloxacin (mfx) with levofloxacin (lvx) in the treatment of pneumococcal pneumonia using our novel skin-temperature model. methods: swiss webster mice were inoculated endotracheally with 5-log 10 cfu of the streptococcus pneumoniae a66 strain (mics: mfx, 0.12 lg/ml; lvx, 0.5 lg/ml). skin temperature at 35 h was used to assess disease severity prior to drug treatment. a skin temperature of !32 c is indicative of a moderate infection with a pulmonary bacterial count of 6-log 10 cfu whereas temperatures <32 c but >30 c are suggestive of a severe infection with a count of 7-log 10 cfu. all mice with a temperature of 30 c were excluded from the study, as death is imminent within 24 h. a 50 mg/kg subcutaneous dose of mfx or lvx was given twice daily for 5 days. skin temperature was measured daily to monitor clinical improvement or failure ( 30 c for at least 48 h). all mice deemed to have failed therapy were euthanised immediately. viable counts in the lungs were determined for all mice. results: of the mice classed as moderate, 24/29 (83%) mice treated with mfx and 10/20 (50%) mice treated with lvx survived. complete eradication was obtained in 93 and 20% of mice treated with mfx and lvx, respectively, in this group. of the mice classed as severe, 24/31 (77%) and 5/20 (20%) mice treated with mfx and lvx, respectively, survived. complete eradication was obtained in 84 and 15% of mice treated with mfx and lvx in this group. conclusions: mfx showed significantly enhanced activity over lvx at both an early and late stage pneumococcal lung infection. . a partial knee replacement was performed with a silicone implant fitting into the intramedullary canal of the tibia, and 107 cfu of mrsa, were injected into the knees. rx was started 7 days after inoculation and continued for 7 days intramuscularly. results: mics (mg/l) of lzd, van and rif were 1.5, 1.5 and 0.008, respectively. in vivo, lzd reduced significantly the mean log 10 cfu/g of bone (2.58 ae 0.85, n ¼ 9) vs. controls and van (6.22 ae 0.43, n ¼ 7; 4.87 ae 0.61, n ¼ 8), respectively (p < 0.01). both rx were not sufficient to sterilise animals (1/9 and 0/6 respectively). the combination of rif with lzd (1.6 ae 0.01, cfu/ g of bone, 6/6 sterile animals) or with van (1.6 ae 0.08 cfu/g of bone, 6/6 sterile animals), was significantly more effective than monotherapy (p < 0.01). emergence of resistance to rif was not detected in vivo. conclusion: in this mrsa joint prosthesis infection, lzd combined with rif was highly effective in vivo and prevented the selection of mutant resistant of rifampin. lzd should be of interest for treating mrsa joint prothesis infection. staphylococcus aureus nasal decolonisation model to study the role of the multidrug efflux system acrab-tolc in resistance of salmonella typhimurium dt104 to detergents and bile salts. to evaluate the importance of the components acrb and tolc of this efflux system in the colonisation of a multidrug-resistant s. typhimurium dt104 strain in chicks. methods: acrb and tolc mutants of a multidrug-resistant s. typhimurium dt104 strain were constructed by deletion or insertional inactivation of the genes. mics of detergents and bile salts were determined for the acrb and the tolc mutants, comparatively to the wild type mutidrug-resistant strain. the effect of sodium choleate on the in vitro growth of these three strains was evaluated. the ld50s of the strains were measured in a one day old chicken model, inoculated with several doses (3-9 log cfu) by the oral route, during 7 days post-inoculation. the colonisation levels were assessed at the subletal dose 7 days post-inoculation by determining the number of cfu of salmonella in the faeces, caeca, spleen, and liver. results: the decrease of resistance to detergents and bile salts was much more important for the tolc mutant than for the acrb mutant. for example, mics of sds decreased of 1024 and 128 times, mics of sodium deoxycholate decreased of 64 and 8 times, for the tolc and acrb mutants, respectively. addition of choleate in culture medium had no effect on the growth of the wild type strains and of the acrb mutant but inhibited the growth of the tolc mutant. the ld50s in the 1-day old chicken model, were 6 log cfu and 7 log cfu for the wild type strain and the acrb mutant, respectively, and not calculable for the tolc mutant because of a too small number of dead chicks. furthermore, in contrast to the acrb mutant, the tolc mutant was unable to colonise the caeca, spleen, and liver after 1 week of infection. moreover, in most chicks no intestinal excretion was detected for the tolc mutant. the colonisation levels of the acrb mutant were the same as those of the parental strain. conclusion: tolc but not acrb appears to be essential in multidrug-resistant s. typhimurium dt104 colonisation of chicks, which is in accordance with their respective roles in resistance to detergents and bile salts. therefore, tolc could be a better target than acrb for the development of efflux system inhibitors. (kp17) and its derivative producing the plasmid-mediated ampc-type b-lactamase cmy-2 (kp27). in vitro studies: mic/mbc: microdilution method (nccls), inoculum: 105, 106 and 107 cfu/ml. the in vitro postantibiotic effect (pae) was investigated by exposing the bacteria to imp and cep at concentration equal to two and six times the mics for 1.5 h. the pae was quantitated calculating the difference between the times required for the numbers of drug-exposed and untreated organism to increase 10-fold above the numbers present immediately after removal of the antibiotic. pk/pd parameters (c max and time above the mic) were determined after a single dose of antimicrobials. in vivo studies: experimental pneumonia in c57bl/6 mice, with intratracheal inoculum of 108 cfu/ml. the animals were grouped in: con (no treatment), cfp (360 mg/kg/day) and imp (240 mg/kg/day), during 72 h. variables: mortality rates and bacterial clearance from lungs. statistical analysis: chi-squared and fisher tests, anova, and posthoc tests. results imp (16.9, 1.32, 0.23) . in vivo: for kp17, cfp and imp decreased the mortality respect to con (0 vs. 73%, p < 0.003) and (33.3 vs. 60%, p < 0.05); for kp27, imp was the only therapy that decreased the mortality compared with con and cfp (13 vs. 60% and 60%, p < 0.01). bacterial clearance from lungs: for kp17, cfp and imp cleared the lungs respect to con (1.74 and 3.38 vs. 9.16 log cfu/ml, p < 0.01), cfp being better than imp (p < 0.001); for kp27, cfp and imp cleared the lung respect to con (4.33 and 4.06 vs. 9.07 log cfu/ ml, p < 0.000). conclusions: the presence of plasmid-mediated ampc-type b-lactamase cmy-2 in k. pneumoniae diminished the in vivo efficacy of cefepime and not that of imipenem. the inoculum effect for cefepime and the pae of imipenem partially explain these results. m. abscessus is a rapidly growing mycobacterium (rgm) that is emerging as a significant pathogen in humans, both as a respiratory pathogen in patients with or without recognised comorbidities, and as the agent of inoculation infections. the histopathologic features of the human infection suggest that m. abscessus causes a tuberculosis-like infection. we investigated the systemic challenge of c57bl/6 mice with the type strain of m. abscessus through intravenous and intraperitoneal routes. with both high (107 cfu) and low (105 cfu) doses, the initial bacterial load remained stable for 5 days in liver and spleen until the establishment of a granulomatous response. the differentiation of the granuloma (central f4/80+ epithelioid cells with a peripheral cd4+ and cd8+ lymphocytic crown) was contemporary to a drastic decrease of the bacterial load in the organs studied. however, 90 days following the challenge some mice still harboured bacteria capable of in vitro growth in their livers and spleens despite an overall effective control of the infection, and all mice infected presented granulomas of various differentiation stages in their livers. this response is highly reminiscent of the ifnc dependent response to m. tuberculosis. mice deleted for the gene encoding ifnc were challenged intraperitoneally with m. abscessus and significantly failed to reduce the bacterial load by day 14. we show for the first time that the rapidly growing m. abscessus can cause a long lasting, tuberculosis-like, ifnc dependent infection in c57bl/6 mice. these results show promise for the elucidation of m. abscessus disease since data from m. tuberculosis might be relevant. reciprocally, m. abscessus faithfully models key features of mycobacterial infection. campylobacter jejuni infection is the most common antecedent in the axonal variant of guillain-barré syndrome (gbs). antibodies against nerve gangliosides found in gbs patients recognise cross reactive epitopes in the lipopolysaccharide (lps) of c. jejuni. this led to the molecular mimicry hypothesis of gbs. to investigate the connection among c. jejuni, antibodies anti gangliosides and gbs we designed an animal model employing a lps isolated from a gbs patient. methods: we immunised eleven rabbits with a lps extracted from penner serotype 0:19 c. jejuni strain isolated from patient with gbs and freund's adjuvant (cfa) (group i). in a second experiment we immunised seven rabbits with lps, cfa and keyhole limpet hemocyanin (klh) (group ii). results: all rabbits of groups i and ii developed a strong humoral response to lps. elevated igm and igg antibodies to lps could be detected as early as 2 weeks after the first immunisation. igg raised during the immunisation period up to 25 600 in group i and 6400 in group ii. anti-gm1 igm antibodies were detectable at low titres 2 weeks after the first immunisation in both groups and raised up to 3200 in group i and to 6400 in group ii. igg anti-gm1 could already be detected at low titres in both groups 2 weeks after the first immunisation and increased up to 51 200 in group i and up to 25 600 in group ii. titre of anti-gm1 igg showed a steep rise during the 6 weeks following the first immunisation. in western immunoblotting of c. jejuni lps, the serum of immunised rabbits reacted strongly with a band that co-migrated at 10 kd at the same level of ct, pna and serum of the patient with anti-gm1 antibodies. the kinetics of igm and igg anti-gd1b was similar to that of antibody anti-gm1 but the maximal titres were lower as igg raised up to 3200 in group i and 12 800 in group ii. igm anti-gd1a were at low titre in both groups throughout the experiment whereas igg anti-gd1a raised up to 3200 in group i and to 800 in group ii. igm and igg anti-gq1b were not detectable in group i and ii sera. conclusion: c. jejuni lps is a potent b-cell stimulator capable to induce a strong antiganglioside response in rabbits. however to induce the neuropathy is crucial to employ klh a glycoprotein known to stimulate both humoral and cellular responses. this is the first animal model reproducing the pathogenetic process hypothesised in axonal gbs with antiganglioside antibodies post-c. jejuni infection. methods: three separate experiments were conducted in order to screen the ability of five clinical c. concisus isolates and the atcc 33237 type strain of oral origin to infect balb/ca mice. all mice were pre-treated with vancomycin, and half of the animals received cyclophosphamide to disturb immune functions, prior to c. concisus challenge by direct intragastrical inoculation with 0.3 ml 10 9 cfu, controls received 0.3 ml of pbs. measured parameters were bacterial isolation from stool and internal organs, loss of body weight and histological examinations of tissue samples. mice were sacrificed on days 7, 21 and 56 of the studies. isolation of c. concisus was performed by the selective filter method and pcr. results: isolation and identification: c. concisus was isolated on day 7 from the cyclophosphamide treated group infected with the clinical isolate 10776 (study 1). liver (3/3), ileum (3/3) and jejunum (1/3) were culture positive. pcr results from tissue samples were only positive in one mouse from the same group (liver, ileum and jejunum). faecal pellets were consistently negative. during the two following studies, no isolation of c. concisus was possible. histological examination: microabscesses (1/3) were found in the liver in two untreated groups. oedema of villi in the ileum was occasionally noted in infected groups, but not in controls (study 2). two mice in the untreated group infected with the atcc 33237 type strain, presented leukocyte infiltration of colon. loss of body weight: compared with controls, the c. concisus infected mice had a significant weight loss (p < 0.05) (study 3). loose stools: on days 2 and 3, c. concisus inoculated groups had loose and slimy stools compared with control groups (study 3). one mouse inoculated with the clinical isolate 10776 died on day 5 (study 3). discussion: the present model mimics a relevant intragastrical exposure to c. concisus infection of imunocompetent balb/ca mice upon cyclophosphamide treatment and results indicate a possible transient colonisation of liver and ileum, with clinical signs of illness as loss of bodyweight and loose stools. histological examination was inconclusive. isolation of c. concisus was not reproducible in two subsequent studies, which severely hampers the present model. future studies should concentrate on the first days of infection, as the organism is rapidly cleared from the gi tract. . they were co-adminis-tered with antimicrobials in an experimental model of sepsis by an mdr isolate. methods: sepsis was induced in 30 rabbits after the iv infusion of an 8 log 10 inoculum of a p. aeruginosa isolate resistant to ceftazidime (cz), imipenem, ciprofloxacin and amikacin (am) by a catheter inserted into the right jugular vein. animals were assigned into five groups of treatment of six animals each: a controls; b iv cz and am; c iv cz, am and alcohol 99%; d iv cz, am and an alcoholic solution of gla; and e iv cz, am and an alcoholic solution of aa. therapy was administered 30 min after bacterial challenge. cz was given at a 50 mg/kg dose, am at 15 mg/kg and both n à 6 pufas at 25 mg/kg. n à 6 pufas were infused within 10 min. all agents were administered by a catheter inserted into the left jugular vein. survival was recorded; after death segments of various organs were cut for quantitative cultures. results this synergy is tested in an experimental model. methods: thirty-five wistar rats became neutropenic by the intraperitoneal injection of 100 mg/kg of cyclophosphamide on day 1 and 150 mg/kg on day 3. on day 5 an 8 log 10 inoculum of one mdr isolate was intramuscularly injected into the right femor of animals. rats were assigned into four groups of treatment: a (n ¼ 6) controls; b (n ¼ 8) rf treated; c (n ¼ 11) cl treated; and d (n ¼ 10) treated with both agents. therapy was given four hours after bacterial challenge. cl was administered im 3 mg/kg into the left femor and rf iv from a catheter inserted into the right jugular vein at 5 mg/kg. survival was recorded. results: mean ae se survival of animals of groups a, b, c and d were 1.75 ae 0.17 days, 3.13 ae 0.52 days (p: 0.031 compared with a), 3.86 ae 0.50 (p: 0.004 compared with a) and 5.50 ae 0.11 (p: 0.011 compared with a) respectively. conclusions: co-administration of cl and rf is beneficiary accompanied by prolonged survival in an experimental model of sepsis by mdr a. baumannii. infections by acinetobacter baumannii (ab) with high-degree resistance (hdr) to carbapenems have recently increased. only colistin seems to keep its in vitro efficacy, but clinical practice is scarce. to our knowledge, no clinical data are currently available to evaluate the systematic use of the beta-lactam (bl)-aminoglycoside (ag) combination to treat serious ab infections in a way similar to that in other infections by gram-negative bacteria. objective: to analyse the efficacy of the combination of two bl (imipenem [i] methods: we used immunocompetent c57bl/6 mice and three strains of ab with susceptibility, moderate-degree resistance and hdr to carbapenems (a, d and e respectively). mics (mg/l) were (strains a, d, e): i: 1, 8, 512; s: 2, 4, 128; and t: 128, 8, 8 . the in vivo activity was examined by quantitative evaluation of the lung homogenate cultures after 48 h of induction of pneumonia. results: in control (con) animals (n ¼ 45), the bacterial counts in lungs at 48 h were (mean ae sd): 10.66 ae 0.37, 10.83 ae 0.32 and 10.77 ae 0.35 log 10 cfu/g of tissue for strains a, d and e, respectively (p ¼ ns between strains). results of antibiotic activity were expressed as differences between treated (n ¼ 4 in each therapy) and con groups (delta log 10 cfu/g) (see table) . conclusions: in this mice pneumonia model, i or s kept his efficacy for ab with moderate resistance to carbapenems. in infections caused by this strain d, t in combination conferred a possible greater efficacy on these bls. in infections by ab with hdr to carbapenems, t alone was also effective. interestingly the combination bl + ag also showed a higher effect on the infection by this hdr strain e, against which monotherapy with i or s were totally ineffective. although the pharmacodynamics of t in this model may have been overestimated, because of the peak levels achieved are not usually found in humans at the recommended doses (c max 32.87 ae 5.45 mg/l), these results are promising to treat multiresistant ab infections. objectives: to investigate the effect of orally administered cranberry juice and its organic acids on escherichia coli in an experimental mouse model of ascending urinary tract infection. methods: e. coli c175-94, a clinical isolate from a patient with uti was used. it expresses type 1 fimbriae but not p or s fimbriae. the transurethrally infected mice were at all times were allowed free access to chow and water (control group) or treatments. the control group and the treated groups all consisted of six mice in every trial; after 1 week, the mice were sacrificed and urine, bladders and kidneys collected for determination of bacterial counts. most of the treatments were repeated two or more times in independent trials and these data were pooled. treatments were commercially available cranberry juice cocktail, freshly prepared cranberry juice, the hydrophilic fraction of cranberry juice (contains sugars and organic acids) and organic acids (quinic, malic, shikimic and citric acid in concentrations corresponding to cranberry juice). results: a reduced number of organisms could be recovered from the bladder (p < 0.01) and urine (p < 0.05) of mice orally treated with unsweetened cranberry juice. commercially available cran-berry juice cocktail also reduced the cfu in the bladder (p < 0.01), as did the hydrophilic fraction of cranberry juice (p < 0.05). quinic, malic, shikimic and citric acid were administered in combination and one by one. the four organic acids decreased the cfu in the bladder when administered together (p < 0.001), and so did the combination of malic plus citric acid (p < 0.01) and malic plus quinic acid (p < 0.05). these data indicate that the beneficial effect of the organic acids from cranberry juice during urinary tract infection is obtained when the acids are administered together. conclusion: for the fist time the effect of cranberry juice and its dominating organic acids has been tested in an experimental mouse model of long-term ascending urinary tract infection under controlled conditions. cranberry juice inhibited e. coli colonisation of the bladder, and the organic acids were the active component involved. the active treatments reduced the bacterial load in the bladder to sub therapeutic concentrations, which indicates that cranberry juice is no final treatment but a remedy that could help the patient to clear the infection, before it eventually becomes a final cystitis. (mellado et al., mol microbiol 1996; 20: 667-679) . we describe a kinetic microbroth method of measuring the growth rates of aspergillus fumigatus spectrophotometrically. using this method, growth rates (as defined by v max values) were determined for nine aspergillus fumigatus isolates for which an ld90 value in temporarily neutropenic cd-1 mice, infected intravenously, had previously been obtained. methods: an inoculum of 10 4 spores in 50 ll sab medium gave us uniformly shaped growth curves and allowed the measurement of v max values with greater sensitivity. soft max pro software was used to determine the v max value for each growth curve by performing linear regression on as many five data point line segments as possible, calculating the slope for each line segment and reporting the steepest slope as the v max (mod/min). growth rate was determined in quadruplicate in three separate experiments and the average v max measurement across these experiments calculated. results: mean growth rate varied from 1.558 (af10) to 2.411 (af71). ld90 varied from 3 â 10 5 to 5 â 10 6 . comparison of the growth rates and ld90 values of these isolates suggests a correlation exists between the two parameters, omitting the one significant outlier (af65, which is amphotericin b resistant), r 2 ¼ 0.6687. conclusion: these data are important in describing a simple method for measuring the growth rate of the common filamentous fungus a. fumigatus, and proving a direct link between pathogenicity in vivo and growth rate in vitro. objective: to compare the histological changes, viral persistence and localisation of the virus in the pancreas and the small intestines of mice, experimentally infected by oral or intraperitoneal route. method: mice were infected with cvb 3 (nancy) by the oral or intraperitoneal route. doses ranged from 5 â 10 3 to 5 â 10 9 tcid50. selected organs from each mouse were embedded into paraffin and sections were attached on silanised slides. for histological observation the sections were stained by mayer's haematoxylin eosin method. for localisation of the antigen by immunohistochemical staining, the vp1 protein served as an indicator for the presence of the virus. the method was standardised. the tissue sections were processed and stained by the avidinbiotin method, using the monoclonal mouse anti-enterovirus antibody against vp1 protein. results: the histological observations reveal that the tissue of exocrine pancreas showed inflammatory changes on the 3rd, 7th, 10th, 14th and 21st day post-infection in exocrine pancreas of the intraperitoneally infected mice. after oral infection no destruction of the exocrine pancreas was observed, but on day 35th post-peroral infection liposis was seen. vp1 was detected mainly on the third and seventh days after infection in the small intestine. we found differences in vp1 localisation between oral and intraperitoneal infection. in small intestine of orally infected mice positive staining was localised in smooth intestinal muscles whereas after intraperitoneal infection. vp1 was detected within the villi. there was no correlation between the virus concentration and tissue damage. conclusions: the pathogenesis of cvb3 infection is influenced by the route of virus administration, which has direct implications for the use of mouse models to study the pathogenesis of coxsackieviruses. objectives: the portal of entry of coxsackieviruses may influence the pathogenesis of infections caused by these viruses. in this study an outbred murine model (swiss albino mice) was used for experimental infection with coxsackie b3 virus (cvb3), strain nancy to follow-up the virus shedding in the stool and the presence of replicating virus in the small intestine of mice after oral and intraperitoneal route of infection. methods: for infection of mice different concentrations of the virus (10 4 , 10 6 , 10 8 and 10 10 ) were used. the stool and small intestine specimens of dissected mice were collected on days 3, 7, 10 post-infection (p.i.) and from day 14 in weekly intervals up to a day 147 p.i. the suspensions made from the collected specimens were studied for presence of replicating virus in hep-2 cell cultures. the virus titre was determined in hep-2 monolayers on microtitre plates and calculated by reed and muench method. results: the replicating virus in the stool pellets was detectable from day 3 p.i. to day 14 p.i. in both orally and intraperitoneally infected mice with a virus titre reaching the level 10 1:5 tcid50/ ml. in the small intestine of orally infected mice the presence of replicating virus was detected up to day 35 p.i. in the small intestine of intraperitoneally infected mice the replicating virus was present for a shorter time, up to day 21 p.i., irrespective of the dose of infection. conclusion: there was no difference in the length of virus shedding in stool specimens of mice infected by oral or intraperitoneal route. however a longer presence of replicating virus in the small intestine of orally infected mice in contrast to intraperitoneally infected mice was observed. this was confirmed by the immunohistopathological studies, these observations support the suggestion that the pathogenesis of coxsackieviral infections is influenced by the route of virus administration. object: in xenotransplantation with porcine neonatal pancreatic cell clusters (npccs), the risk of cross-species porcine endogenous retrovirus (perv) infection remained as problem. we used the severe combined immunodeficient (scid) mouse and the lewis rat model to identify the perv transmission with the time course and the differences between the models. methods: npccs were transplanted to scid mice and lewis rats and left for 1-70 days before being sacrificed. dna and rna were extracted from the liver, spleen, pancreas, lung, kidney and testis. to examine the perv transmission, nested-pcr and rt-pcr were used upon pol/env/gag regions of perv. the pig mitochondrial cytochrome oxidase ii subunit gene (coii) was amplified simultaneously to monitor the microchimerism. results: total 264 samples from seven mice and five rats were tested. ten weeks after xenotransplantation, two mice and four rats were identified to have permissive perv infection. in the scid mice, 92.9% of tested organs were positive for perv-pol gene and 85.7% were positive for coii gene with dna examination. in the lewis rats, 86.7% of organs were positive for perv-pol gene and 14.1% for coii gene with dna examination. examinations of organs of mice showed that 35 (83.3%) organs were positive for the perv-pol gene and coii gene simultaneously that presumed as microchimerism, but 15 (50%) organs of rats were presumed as microchimerism. results of perv-pol positive and coii negative that presumed as permissive perv infection were observed in 9.5% of organs in the scid mice and 36.7% in the lewis rats. organs presumed as permissive perv infection were the spleen (day 3), liver (day 70), lung (day 70), and testis (day 70) in the scid mice by dna examinations. in the lewis rats, the spleen and testis of day 1; the liver, spleen, and kidney of day 5; the testis and kidney of day 7; the liver, spleen, lung, and testis of day 14 were identified to have permissive perv infection. conclusion: the cross-species perv infection was identified from these animal models. expression of perv depends on the immunity of the recipients, because the xenotransplanted scid mice had more perv microchimerism but less permissive infection than that of the lewis rats. detection rate was increased with the time course, accordingly in the early period after transplantation, perv considered to exist as an inactive form. the therapy of numerous antimicrobial classes including the recently introduced quinupristin/dalfopristin, telithromycin and the oxazolidinones. clearly the need for antimicrobial discovery persists, and this should be a continued priority for the pharmaceutical industry. this report addresses the spectrum of activity for pdf7 tested against a collection of recent (2002) clinical isolates cultured from patients infected with pathogens within the spectrum for peptide deformylase inhibitors. methods: pdf7 was acquired from novartis. the compound was dispensed into reference broth microdilution trays in appropriate media over the range of 0.06-8 mg/l. mueller-hinton broth was supplemented with 2-5% lysed horse blood when testing fastidious streptococci, and the corynebacteria. nccls qc strains were used concurrently and all pdf7 mic results were within proposed ranges. results: 1837 gram-positive strains were tested with a species rank order of s. aureus (875 strains) > cons (381) objective: nvp-pdf713 is a new peptide deformylase inhibitor active against a wide variety of gram-positive and -negative bacteria. the current study examines the activity of nvp-pdf713 compared with those of ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, vancomycin, teicoplanin, linezolid, ranbezolid, dap-tomycin, oritavancin and quinupristin/dalfopristin against 131 s. aureus (62 methicillin resistant) and 127 coagulase-negative staphylococci (60 methicillin resistant). microdilution using frozen trays containing cation-adjusted mueller-hinton broth and inocula of 1 â 105 cfu/ ml with trays incubated in air. results: mic 50 and mic 90 values (lg/ml) were as seen in the following table. nvp-pdf713 was equally active against all staphylococcal strains (mics <0.06-4 lg/ml), irrespective of susceptibility to other agents. quinolone resistance was mainly seen in methicillin r strains. vancomycin, linezolid, ranbezolid, daptomycin, oritavancin and quinupristin/dalfopristin were all active at mics <4.0 lg/ml and teicoplanin was less active against coagulase-negative strains. conclusions: nvp-pdf713, a new peptide deformylase inhibitor, was active in vitro against staphylococci. p916 antipneumococcal activity of nvp-pdf713 compared with 18 other agents p. appelbaum, l. ednie, m. jacobs hershey, cleveland, usa background: drug resistance in pneumococci is found worldwide. objective: nvp-pdf713 is a new peptide deformylase inhibitor active against gram-positive and -negative bacteria. this study tests activities of nvp-pdf713, amoxicillin ae clavulanate, imipenem, meropenem, ceftriaxone, cefuroxime, cefpodoxime, cefdinir, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, azithromycin, clarithromycin, linezolid, quinupristin/dalfopristin, vancomycin and teicoplanin against 80 pen s, 88 pen i, 132 pen r pneumococci (154 macrolide r and 30 quinolone r strains with defined r genotypes). methods: agar dilution using cation-adjusted mueller-hinton agar + 5% sheep blood and inocula of 1 â 104 cfu/spot; plates incubated in air. results: mic50 and mic90 values (lg/ml) are shown in table 1 . nvp-pdf713 was equally active against all pneumococci, irrespective of activity of other drugs. beta-lactam mics rose with those of pen g. moxi was the most potent quinolone followed by gati, levo cipro. vanco, teico, linez, quin/dalf were all active at mics <4.0 lg/ml. conclusions: nvp-pdf713 was active in vitro against beta-lactam, macrolide and quinolone s and r pneumococci. objective: nvp pdf-713 is a new peptide deformylase inhibitor active against gram-positive and gram-negative strains. this study tested activity of nvp pdf-713, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, vancomycin, teicoplanin, linezolid, ranbezolid, daptomycin, tigecycline, oritavancin and quinupristin/ dalfopristin against six s. aureus (3 methi r) and six cons (3 methi r). methods: nccls macrodilution mic was used. for time-kills, 5 â 10 5 -5 â 10 6 cfu/ml inocula in cation-adjusted mueller-hinton broth were incubated aerobically in a shaking water bath at 1â, 2â, 4â mic. viabilities were done after 3, 6, 12, 24 h. ca 2þ was added for dapto. results: mic ranges (lg/ml) were: nvp pdf-713, 0.25-2; cipro, 0.25 to >32; levo, 0.25-32; gati, 0.125-16; moxi, 0.03-8; vanco, 1-4; teico, 0.5-16; linez, 1-4; ranbez, 0.125-4; dapto, 0.125-2; tige, nvp 0/0 0/0 0/0 1/1 1/1 0/0 1/2 1/1 0/0 0/0 0/0 0/0 cipro 3/5 1/2 0/0 6/7 2/3 2/2 6/7 2/5 2/3 6/7 2/7 2/4 levo 4/5 1/2 0/0 8/8 3/5 1/1 9/9 4/8 1/5 9/9 7/8 3/7 gati 6/10 2/2 0/0 10/12 5/9 3/4 11/12 9/12 2/7 10/12 7/12 3/8 moxi 8/10 2/5 0/0 9/11 4/9 1/3 10/12 7/9 2/7 8/12 3/9 2/7 vanco 33 0/0 0/0 9/10 1/2 0/0 11/12 4/9 3/3 8/12 5/10 3/9 teico 02 0/0 0/0 4/8 0/0 0/0 10/10 4/6 0/0 9/12 6/11 3/6 linezolid 00 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 ranbez 00 0/0 0/0 1/1 0/0 0/0 1/3 0/0 0/0 2/10 0/1 0/0 depto 911 6/9 0/5 12/12 9/11 6/9 12/12 11/12 5/10 11/12 10/12 8/11 tigecyt 00 0/0 0/0 0/0 0/0 0/0 0/2 0/1 0/0 0/6 0/0 0/0 oritavan 1212 10/12 2/7 12/12 12/12 8/12 11/12 9/11 9/10 8/12 6/12 6/12 quin/dalf 00 0/0 0/0 4/5 0/0 0/0 5/7 1/2 0/0 5/8 1/3 0/1 0.25-1; orita, 0.25-1; quinu/dalfo, 0.125-1. no. of strains at mic/ 2 â mic with delta-1 log 10 cfu/ml (90%), delta-2 log 10 cfu/ ml (99%) and delta-3 log 10 cfu/ml (99.9%) killing at the various time periods are shown in table 1 . nvp pdf-713 was not cidal at 1 â mic and 2 â mic, but was static against all 12 strains at mic after 24 h. cipro and moxi were cidal against four to seven strains at 2 â mic after 24 h. vanco was cidal at 2 â mic for nine strains after 24 h. oxazolidinones, tigec and quinu/dalfo were mainly bacteriostatic and dapto and orita rapidly cidal. conclusions: nvp pdf-713 gave low mics and static activity against all strains, irrespective of methicillin susceptibility status. p920 time-kill study of the antipneumococcal activity of nvp pdf-713, a new peptide deformylase inhibitor, compared with 13 other agents p. appelbaum, g. pankuch, m. jacobs hershey, cleveland, usa background: drug-resistant pneumococci are an increasing worldwide problem. objective: nvp pdf-713 is a new peptide deformylase inhibitor. this study used time-kill analysis to examine the antipneumococcal activity of nvp pdf-713 compared with imipenem, meropenem, ceftriaxone, moxifloxacin, levofloxacin, gatifloxacin, azithromycin, clarithromycin, vancomycin, teicoplanin, linezolid, daptomycin, and quinupristin/dalfopristin. twelve strains were tested: three penicillin sensitive, two intermediate, and seven resistant pneumococci. of the 12 strains tested; 10 were macrolide resistant [4 erm (b), 4 mef, 2 l4], and two quinolone resistant. methodology: nccls macrodilution mic methodology was used. time-kill analyses were in cation-adjusted mueller-hinton broth with 5% lysed horse blood, and final inocula of 5 â 10 5 -5 â 10 6 cfu/ml. mueller-hinton broth was supplemented to a final concentration of 50 mg ca 2þ /l for testing daptomycin. viability counts were done after 0, 3, 6, 12, and 24 h. results: mics (lg/ml) were as follows: nvp pdf-713, 0.125-2.0; imipen, 0.004-0.25; meropen, 0.008-1.0; ceftriax, 0.016-4.0; moxi, 0.125-4.0; levo, 1.0-16; gati, 0.25-8.0; azithro, 0.06 to >64; clarithro, 0.016 to >64; vanco 0.25-0.5; teico, 0.06-0.125; linez, 0.5-2.0; dapto, 0.125-0.5; quin/dal, 0.25-1.0. the number of strains at mic/ 2 â mic with log 10 cfu/ml values of à1 (90% killing), à2 (99% killing) and à3 (99.9% killing) at the various time periods are shown in table 1 . conclusions: nvp pdf-713 had kill kinetics similar to those of linezolid. nvp pdf-713 at 2 â mic was bactericidal (99.9% killing) against six strains after 24 h. linezolid at 2 â mic was bactericidal against seven strains at the same period. daptomycin and quinupristin/dalfopristin showed rapid killing. imipenem, meropenem, vancomycin, and quinupristin/dalfopristin were bactericidal against all 12 strains at 2 â mic after 24 h. objectives: nvp-pdf-713 is a new peptide deformylase inhibitor antimicrobial with excellent activity against gram-positive cocci, including methicillin-resistant staphylococcus aureus (mrsa) and penicillin-resistant streptococcus pneumoniae (prsp). we used the neutropenic murine thigh-infection model to measure in vivo postantibiotic effects (paes) and determine which pk/pd parameter best correlated with in vivo efficacy. methods: mice had 106.6-7.4 cfu/thigh of staphylococcus aureus atcc 29213 and streptococcus pneumoniae atcc 10813 when treated for 24 h with 40-1280 mg/kg/day of nvp-pdf-713 fractionated for 3-, 6-, 12-, and 24-h dosing. mice were sacrificed at the end of therapy. ten per cent thigh homogenates were prepared, and serial dilutions were plated for cfu determinations. serum levels after both oral and subcutaneous injection of doses of 20, 80 and 320 mg/kg were measured by microbiologic assay. non-linear regression analysis was used to determine which pk/pd parameter (24-h auc/mic, peak/mic or time above mic) best correlated with cfu/thigh at 24 h. in vivo paes were measured from serial 2-6 h cfu/thigh values after doses of 80 and 320 mg/kg. results: pharmacokinetic studies exhibited linear kinetics with doses from 20 to 320 mg/kg, with peak/dose values of 0.10-0.12, auc/dose values of 0.09-0.11 and half-lives of 26-30 min. oral bioavailability was 67-88%. protein binding in mouse serum was low at 25%. nav-pdf-713 produced in vivo paes of 3-5 h with s. aureus and 10-13.5 h with s. pneumoniae. the 24-h auc/mic was highly correlated with efficacy (r 2 ¼ 84-87% for 24-h auc/mic compared with 34-76% for peak/mic and 40-60% for time above mic for s. pneumoniae and s. aureus, respectively). because of the rapid half-life in mice, oncedaily dosing was slightly less effective than the more frequent dosing regimens. conclusions: the 24-h auc/mic is the parameter that best correlates with in vivo activity of nvp-pdf-713. the prolonged in vivo paes would support at least twice daily dosing. p922 in vivo pharmacodynamic activity of nvp-pdf-713 against multiple bacterial pathogens w. craig, d. andes madison, wisconsin, usa objectives: the 24-h auc/mic is the pk/pd parameter that best correlates with in vivo activity of nvp-pdf-713, a new peptide deformylase inhibitor. we used the murine thigh-infection model nvp 0/0 0/0 0/0 2/3 0/1 0/0 8/9 1/2 1/2 7/10 4/8 2/6 imipen 11/12 4/5 0/0 11/12 6/10 3/3 12/12 11/12 8/11 12/12 11/12 10/12 meropen 8/9 2/3 1/1 11/12 7/7 3/3 11/12 7/11 4/9 8/12 7/12 5/12 ceftriax 4/6 1/2 0/0 8/12 5/7 3/3 9/12 9/12 6/10 11/12 8/12 4/10 moxi 1 5/8 0/1 0/0 8/10 4/6 1/2 9/10 8/10 5/9 10/10 8/10 7/10 levo 1 5/5 0/0 0/0 8/10 4/6 1/1 10/10 8/10 3/6 9/10 9/10 5/10 gati 1 4/6 0/1 0/0 7/10 2/7 1/1 10/10 6/10 3/7 9/10 8/10 4/9 azithro 2 1/1 0/0 0/0 1/1 1/1 1/1 2/2 1/2 1/2 2/2 2/2 2/2 clarithro 2 0/0 0/0 0/0 2/2 1/2 0/0 2/2 2/2 2/2 2/2 2/2 2/2 vanco 4/4 0/1 0/0 12/12 5/6 2/3 12/12 11/11 9/9 11/12 10/12 9/12 teico 0/0 0/0 0/0 5/6 1/1 0/0 9/10 6/7 3/4 9/12 8/12 7/10 linez 0/0 0/0 0/0 2/4 0/1 0/1 6/11 1/4 0/1 8/12 4/11 2/7 depto 7/10 2/4 2/2 12/12 11/11 4/8 11/12 10/11 7/11 8/12 5/11 3/11 quin/dal 10/11 7/9 4/5 12/12 11/11 6/9 11/12 10/11 7/11 7/12 5/12 3/12 in normal and neutropenic mice to determine (1) the magnitude of the 24-h auc/mic needed for efficacy of nvp-pdf-713 with various pathogens (including mrsa and penicillin-, macrolideand tetracycline-resistant strains of s. pneumoniae) and (2) the impact of neutrophils on the drug's in vivo activity. methods: mice had 106.7-7.9 cfu/thigh of five isolates of staphylococcus aureus (two mrsa) and six isolates of streptococcus pneumoniae (five penicillin-resistant, four macrolide-resistant, three tetracycline-resistant strains) when treated for 24 h with 20-320 mg/kg of nvp-pdf-713 subcutantously every 6 h. streptococcus pneumoniae atcc10813 and staphylococcus aureus atcc 29213 were studied simultaneously in normal and neutropenic mice. mice were sacrificed at the start and end of therapy. ten per cent thigh homogenates were prepared and serial dilutions were plated for cfu determinations. serum levels were determined by microbiologic assay after subcutaneous doses of 20, 80 and 320 mg/kg. a sigmoid dose-response model was used to estimate the dose (mg/kg/24 h) required to achieve a net bacteriostatic effect over 24 h. results: pk studies exhibited linear kinetics with auc/dose values 0.09-0.11 and half-lives of 26-30 min. protein binding was 25%. mics ranged from 0.5 to 2.0 mg/l. static doses for the various organisms ranged from 48 to 124 mg/kg/day. mean 24-h auc(free)/mic values (aesd) were 33.3 ae 8.9 for s. aureus and 34.9 ae 10.0 for s. pneumoniae. the differences were not significant. methicillin and penicillin resistance did not alter the magnitude of the auc/mic required for efficacy. the presence of neutrophils reduced the 24-auc(free)/mic required for efficacy by about fourfold. conclusion: the 24-h auc/mic of nvp-pdf-713 required for in vivo efficacy was relatively similar among various pathogens, was not altered by drug resistance, and was reduced fourfold by the presence of neutrophils. p923 determination of quality control guidelines for mic dilution and disk diffusion methods when testing nvp-pdf713, a novel peptide deformylase inhibitor t. fritsche, t. anderegg, r. jones north liberty, usa background: quality control (qc) guidelines remain necessary for accurate determination of antimicrobial susceptibility testing and should be established early in the development of new antimicrobial classes. nvp-pdf713 is a pdf inhibitor rapidly progressing into phase ii and iii human clinical trials, thus qc guidelines appear necessary for nccls methods. methods: multi-laboratory (seven or eight sites) trials were initiated using the nccls m23-a2 guideline for qc determinations. key technical details were: mic phase -four mueller-hinton (mh) broth lots, eight participant sites and 10 replicates of four appropriate qc strains; and disk diffusion phase -three mh agar lots, seven sites and 10 replicates of three qc strains. results were analysed by statistical methods found in m23-a2. control drugs included vancomycin, clarithromycin, linezolid and levofloxacin; 99.9-100.0% of control results were within published nccls ranges (640 and 1050 results for mic and zone tests, respectively). inoculum concentration controls averaged 3.5 â 10 5 (mic trial only). results: seven or eight participants provided qualifying results in the two separate qc studies, and the calculated (proposed) ranges were (range; % results in range): e. faecalis atcc 29212 (2-8 mg/ l; 95.6), s. aureus atcc 29213 (0.5-2 mg/l; 99.4), s. pneumoniae atcc 49619 (0.25-1 mg/l; 97.5 and 30-37 mm; 97.6), h. influenzae atcc 49247 (1-4 mg/l; 97.5 and 24-32 mm; 99.8), and s. aureus atcc 25923 (25-35 mm; 97.8). all qc ranges were maximised to contain %95% of reported results and zone sise variation was elevated due to the bacteriostatic character of this pdf inhibitor, creating non-discreet zone edges. conclusions: qc ranges for nccls methods when testing nvp-pdf713 have been established. results from these nccls m23-a2-conforming trials can be utilised to control the accuracy of the susceptibility testing of this pdf inhibitor projected to be among the 'first' to reach human clinical studies. p924 determination of dry-form commercial reagent reproducibility and mic validations for nvp-pdf713, a novel peptide deformylase inhibitor g. moet, r. jones, p. rhomberg, t. fritsche north liberty, usa background: nvp-pdf713 is a new pdf inhibitor rapidly being advanced to human clinical trials. commercial reagent broth microdilution mic panels will be required for investigator laboratory use, especially those products with extended shelf-lives (dry-form). this study reports the results of reagent qualifying tests. methods: the experiment was performed by nccls m23-a2 guidelines to assess dry-form mic reproducibility (10 organisms â 3 tests/day â 3 days ¼ 90 tests) and comparative mic accuracy to the reference mic (ref; m7-a6, 2003) using %100 strains representing the following organism groups: staphylococci, enterococci, s. pneumoniae, other streptococci, h. influenzae, and selected species refractory to pdf inhibitor action. all trays were manufactured by sensititre (trek diagnostics, cleveland, oh). results: reproducibility results showed 80% of mics were identical and 97.8% of mics were within one log 2 dilution step. validation test results comparing dry-form to ref mics were (% identical/twofold/fourfold): for staphylococci (71/99/100%), for enterococci (55/99/100%), for s. pneumoniae (33/91/97%), for other streptococci (69/100/100%) and for h. influenzae (36/97/ 100%). consistent variations were detected with spn (49% of dry-form panel results being one dilution higher than ref) and hi (60% of results being one dilution lower than ref). nvp-pdf713 mics were off-scale (mic values, >32 mg/l) for enterobacteriaceae and non-fermentative gram-negative bacilli (40 strains). overall, 97% of sensititre mic results for were within one log 2 dilution of ref mic values. conclusions: nvp-pdf713 dry-form diagnostic mic panels have been validated for accuracy and reproducibility using 520 recent clinical isolates from five major pathogen groups. the spectrum of activity for this pdf inhibitor compound appears focused toward gram-positive cocci and specific fastidious respiratory tract pathogens. objectives: the emergence of antibiotic resistance among grampositive pathogens has impacted the clinical management of these infections. paratek pharmaceuticals initiated a programme to apply medicinal chemistry to the core structure of tetracycline (tet) with the goal of creating novel classes of proprietary antibiotics that would (a) be unaffected by the known tet resistance mechanisms and (b) retain the safety and tolerability profile of the tet family. since there is no cross-resistance between the tets and other antibiotics, such new agents would be expected to be active against isolates resistant to all other currently available classes. the aim of the programme was to synthesise new agents active against gram-positive, common gram-negative, atypical and anaerobic bacteria. methods: a series of 7-position and 7,9-position derivatives of sancycline were synthesised and tested for activity in vitro against mrsa, vre, enterococcus faecalis and streptococcus pneumoniae by microdilution. the presence of tet-resistance determinants was assessed by pcr and confirmed by resistance to currently available tets. results: a number of 7-dimethylamino-9-aminomethylcyclines (amc) and 7-aryl or heteroaryl sancyclines with potent activity in vitro (mic range less than or equal to 0.06-2.0 mg/l) were identified. both novel series were more potent against one or more of the resistant strains than currently available antibiotics tested (mic range 16-64 mg/l). the amc derivatives were active against bacteria resistant to tet by both efflux and ribosome-protection mechanisms. conclusions: this study identified the amcs as a novel class of antibiotics evolved from tet that exhibit potent activity in vitro against tet-resistant bacteria, including gram-positive bacteria resistant to currently available antibiotics. one agent of this class, bay 73-7388 (discovered by paratek pharmaceuticals, inc., boston, ma, and designated ptk 0796) has been chosen for development. bay 73-7388 is a novel antibiotic compound being developed for the treatment of severe bacterial infections. it is the first compound selected from the novel class of aminomethylcyclines and was designed to meet an increasingly significant need for additional therapies for treatment of infections, including those resistant to currently available antibiotics. the efficacy of bay 73-7388 in different mouse models of skin and soft tissue infection (ssti) was compared with that of vancomycin (van) and linezolid (lin). methods: two mouse models were employed to determine the efficacy of bay 73-7388: (1) infected abscess model (induced by implantation and subsequent infection of gelfoam (tm)) and (2) infected thigh muscle model in neutropenic mice. staphylococcus aureus strain dsm11823 (mssa) was used to infect the respective structures in the skin and soft tissues. infected abscess bearing mice were treated i.v. bid for 2 days, while thigh muscle infection model mice were treated s.c. 30 min post-infection. cfu reduction of infected tissues and bacterial load in different organs (spread from the infection site) were used as read-out for therapeutic efficacy. results: as measured by reduction of bacterial load, therapy of infected abscesses with bay 73-7388 (cfu reduction >4 log units at 10 mg/kg) was superior to van and lin (no reduction in bacterial load) . furthermore, bay 73-7388 reduced the overall bacterial load in spleen, liver, lung and heart. in the reduction of organ load, bay 73-7388 was as efficacious as van conclusions: this dal activity survey indicates that this new glycopeptide has significant gram-positive activity (96.9-100.0% inhibited at 1 mg/l), superior to available agents in the class, and the potency was similar for european isolates when compared with prior experience in other geographic areas. background: tigecycline (tig) is a novel glycylcycline with broad spectrum activity. increasing reports of resistance (r) among commonly occurring gram-positive cocci (gpc) that produce respiratory tract and skin and soft tissue infections has created a need for development of new antimicrobial agents. in this study the activity and potency of tig, tetracycline (tc) and other comparator agents was evaluated using contemporary isolates of commonly occurring species of gpc, including the presence of r organism subsets. table. 1 organism ( methods: the activity of tig and nine comparators was challenged with a collection of gpc including oxacillin (oxa)-susceptible (s; 3196 strains) and -r (1881 strains) s. aureus (sa); oxa-s (321 strains) and -r (1111 strains) coagulase-negative staphylococci (cons); penicillin (pen)-s (1126 strains) and non-susceptible (ns; 459 strains) s. pneumoniae (spn); penicillin-s (161 strains) and -ns (51 strains) viridans-group streptococci (vgs); beta-haemolytic streptococci (bhs; 405 strains); and vancomycin-s (1294 strains) and -r (122 strains) enterococci (ent). broth microdilution susceptibility tests were performed and analysed using nccls reference methods and interpretive criteria. results: whereas oxa-r subsets of both sa and cons displayed cross-resistance to tc, macrolides, clindamycin and quinolones, no differences were seen with tig (mic50/90 being 0.25 and 0.5 mg/l, respectively). among streptococci, all spn and vgs (regardless of pen-s), and bhs demonstrated tig mic50/90s of 0.12 mg/l (one exception being pen-intermediate vgs with the mic90 at 0.25 mg/l). tig was also uniformly active against enterococcal isolates, with mic50/90s of vancomycin-s and -r subsets being 0.25 and 0.5 mg/l, and 0.12 and 0.25 mg/l, respectively. when using the nccls tc s breakpoint of 4 mg/l, all 10 127 staphylococci, streptococci and enterococci tested would be classified as s to tig. conclusions: tig displays a remarkable spectrum of activity and potency against s and r subsets of gpc with the highest mic90 being 0.5 mg/l. in addition to for use in treating communityacquired respiratory tract infections, tig may also be a candidate for treatment of complicated skin and soft tissue infections and, possibly, urinary tract infections caused by gpc. p938 endemic, highly resistant acinetobacter in the intensive care unit -is tigecycline the answer? objective: to find satisfactory antibiotic treatment against an organism, acinetobacter baumanii, that became endemic on the intensive care unit of a busy district general hospital. this organism is resistant to many antibiotics and in one case was ultimately resistant to all currently marketed antibiotics. methods: (1) surveillance of patients in the intensive care unit for the presence of acinetobacter baumanii. (2) clinical assessment of patients with the organism to establish those needing antibiotic therapy. (3) patients requiring treatment were given an antibiotic combination using colistin (usually combined with oral minocycline) or tigecycline monotherapy, a first-in-class glycylcycline agent. (4) treatment and outcome were monitored. the study was observational. allocation to treatment categories was not randomised or blinded. the tigecycline was used on a compassionate basis. results: the intensive care unit was free of acinetobacter until the beginning of 2001. by the end of 2001, 5-10 new isolates of acinetobacter baumanii were isolated per quarter. initially these pathogens were sensitive to imipenem, meropenem, tobramycin, amikacin, colistin, and minocycline. this sensitivity began to wane and, by the end of 2002, one patient had died with acinetobacter baumanii in his bloodstream that was resistant to everything available. after this death, we tested further isolates of acinetobacter against tigecycline, a new broad spectrum agent currently in phase 3 development, and found it to be active against the endemic strain. two patients with ventilator-associated pneumonia caused by this organism were treated with tigecycline and made a full recovery. there were no adverse effects related to tigecycline treatment. conversely, five patients with ventilator-associated pneumonias caused by the same organism were treated with colistin and failed to respond. acinetobacter finds the respiratory system a favourable environment, and this, combined with the fact that the vast majority of the patients were ventilated resulted in ventilator-associated pneumonia being the commonest infection. conclusion: tigecycline is likely to be a useful agent in clinical practice on intensive care units when dealing with this difficult organism. further evaluation is warranted. it may well be the antibiotic of choice. p939 antimicrobial activity of tigecycline (gar-936) tested against enterobacteriaceae, and selected non-fermentative gram-negative bacilli, a worldwide sample r. jones, t. fritsche, h. sader, m. beach north liberty, usa background: as resistances (r) among gram-negative bacilli (gnbs) expand, few antimicrobial agents have been developed to address this clinical problem. tigecycline (tig), a novel glycylcycline, has an expanded spectrum of activity and potency, tigecycline covers many routine gram-negative resistant strains and additionally possesses activity versus some uncommonly isolated non-fermentative gnbs. this study compares tig with contemporary broad-spectrum agents using recent clinical isolates from europe and other continents. methods: all strains (2420) were centrally processed by reference, broth microdilution methods against more than 20 antimicrobials. all concurrent qc results were within nccls published ranges, with identifications performed by traditional methods and/or the vitek system. over 2400 isolates were tested from the enterobacteriaceae (ent) and non-fermentative gnbs categories. susceptibility (s) for tig was defined as 4 mg/l, that breakpoint used for all tetracyclines by the nccls. results: the ent were divided into three groups for analysis: esbl-producing isolates (154 strains), proteae group (131 strains; includes p. mirabilis and indole-positive species) and all enteric bacilli. tig was very active against all esbl-producing isolates (mic90, 0.25-2 mg/l; highest among tc-r subsets), and all ent (mic50/90, 0.25/1 mg/l). proteae had a mic90 at 4 mg/l and all but one of tig-r or intermediate strains (mics, 8 and 16 mg/l) were m. morganii or p. mirabilis. p. aeruginosa was marginally inhibited by tig (mic90, 32 mg/l). in contrast, acinetobacter spp. (mic90, 2 mg/l; 96.1% s) and s. maltophilia (mic90, 2 mg/l; 100.0% s) were readily inhibited by tig. among all ent studied, 31.0% were tc-r, but only one strain (p. mirabilis) was tig-r (mic at 16 mg/l). conclusions: remarkable potency and breadth of spectrum was observed for tig against ent (99.4% at 4 mg/l vs. 66.8% for tc), s. maltophilia and acinetobacter spp. limited activity was noted versus p. aeruginosa (16.0% at 4 mg/l) and some proteae (mic90, 4 mg/l). tig should be of value for the treatment of infections caused by several commonly r gnb groups. background: tigecycline (tig, formerly gar-936), is a novel glycylcycline which is currently in phase 3 clinical trials. the in vitro activity of the tig was evaluated in comparison with tetracycline (tet) and other antimicrobial agents against recent (2000) (2001) (2002) clinical isolates collected worldwide from patients with respiratory infections and meningitis. methods: a total of 1727 isolates were tested against tig and more than 20 comparator agents by broth microdilution according to the nccls reference methods and interpretative criteria. the collection included, h. influenzae (hi; 1215 strains, 20% betalactamase-producing), m. catarrhalis (mcat; 495 strains, 96% beta-lactamase-producing), and n. meningitidis (nm; 17 strains). results: tig demonstrated excellent activity against these organisms with all isolates being inhibited at 4 mg/l (tet susceptibility breakpoint). tig was highly active against hi (mic90, 1 mg/l) and mcat (mic90, 0.25 mg/l), and its potency against these pathogens was not affected by beta-lactamase production. tig was fourfold more potent than tet against hi and tetresistant isolates showed low ( 1 mg/l) tig mics. nm isolates were highly susceptible to tig (mic90, 0.12 mg/l) and to the vast majority of antimicrobial agents evaluated. conclusions: these results indicate that tigecycline has potent in vitro activity against clinically important gram-negative bacteria that cause community-acquired respiratory infections and meningitis, including tet-r isolates. further evaluations of tig activity, as well as, clinical studies are necessary to assess the role of this compound in the treatment of both community-and hospitalacquired infections. background and objectives: beta-lactamase production is the major mechanism of bacterial resistance to beta-lactam antibiotics in gram-negative pathogens, and surveillance of beta-lactamase determinants is an important issue of microbial drug resistance. given the great diversity of beta-lactamases and their overlapping substrate specificities, molecular analysis is necessary to identify the nature of beta-lactamase genes in clinical isolates. in this work we investigated the potential of the dna microarray technology for a rapid and comprehensive detection of beta-lactamase genes in drug-resistant bacteria. methods: a total of 104 oligonucleotide probes were designed for specific recognition of beta-lactamase genes of 65 different lineages (18 of molecular class a, 19 of class b, 13 of class c and 15 of class d). a dna chip was designed including a triplicate set of probes, as well as positive hybridisation controls. the microarray was printed on epoxy-modified glass slides using an affymetrix gms 417 robotic spotter. genomic dna was labelled with cy3 or cy5 by random priming. hybridisation signals were then detected using an affymetrix 418 laser scanner and images were analysed by the genepix pro (version 5.0) software. results: the dna chip was tested with 23 gram-negative strains (including both reference strains and clinical isolates) in which the repertoire of beta-lactamase genes was partially known or unknown. all the predicted beta-lactamase genes (among which there were members of the blatem, blashv, blactx-m, blaper, blavim, blaimp, blacmy-lat groups of acquired genes) were correctly detected by microarray hybridisation. in clinical isolates of unknown beta-lactamase content, the microarray detected genes whose presence was subsequently confirmed by conventional pcr assays. false-positives were observed with a subset of probes, which had to be redesigned to overcome the problem. conclusions: successful detection of several different beta-lactamase genes of clinical importance was achieved by using a dna microchip. the dna microarray technology appears to be a sensitive and specific tool for rapid detection and characterisation of beta-lactamase genes in clinical isolates. objectives: some members of the genus citrobacter are potential pathogens of debilitated hospital patients. they can become resistant to beta-lactamases, including third generation cephalosporins due to over-expression of a chromosomal beta-lactamase. eleven species are currently known, but speciation is often difficult using biochemical tests. isolates previously typed as citrobacter diversus are now known as citrobacter koseri. here we measured sequence variation at the beta-lactamase structural gene amongst a group of clinical isolates, originally identified as c. diversus by api 20e profiling. methods: nine c. diversus isolates were collected from faecal samples of children being treated in the oncology department of bristol children's hospital in the early 1980s. beta-lactamase and 16s rrna genes were amplified by pcr and sequenced by standard methods. beta-lactamase induction was attempted in liquid-grown cultures using cefoxitin (10 mg/l for 2 h). nitrocefin hydrolysis assays were performed using a spectrophotometer. results: analysis of 16s rrna gene sequences confirm that, of the nine clinical isolates, five, which all have an inducible betalactamase gene whose sequence is closely related to c. diversus nf85 and ula27, are actually citrobacter amalonaticus. given that c. diversus isolates have all been renamed c. koseri, this error in nomenclature must be addressed. the reason for the error is that c. diversus was known to have variability in its ability to utilise malonate, the only differentiation between c. koseri and c. diversus. four of the test isolates do type as c. koseri using 16s rrna sequencing. these true c. koseri isolates produce a novel, acidic, class a beta-lactamase, named ckoa, constitutively. the sequence of this beta lactamase gene was determined, and is only 40% identical to the c. diversus (now c. amalonaticus cdia). conclusions: we present a new beta-lactamase sequence, from c. koseri and shows that c. koseri nf85 and ula27 should be retyped as c. amalonaticus. beta-lactamase-specific pcr may provide a valuable tool for typing citrobacter spp. isolates, and is very suitable for separating c. amalonaticus and c. koseri, which are very closely related biochemically. the knowledge that clinical c. koseri isolates produce a beta-lactamase constitutively at low levels may be useful clinically. p944 a single-tube pcr with mgb eclipse probes for detection of shv-type extended-spectrum beta-lactamases (esbls) a. ekimov, m. edelstein, e. belousov smolensk, rus; bothell, usa objectives: esbls of the shv-type are one of the most common and clinically significant beta-lactamases. the number of shv variants is continuously growing; however esbl activity of shv enzymes has been associated with mutations at relatively few amino acid positions (aa-s) as compared with the tem enzymes. here we propose a simple and rapid method that allows detection of all the known shv esbls in a single real-time pcr reaction. methods: the proposed method is based on amplification of blashv genes in the presence of short (13-14 nt) fluorogenic probes capable of hybridisation-triggered fluorescence. these probes commercially known as mgb eclipse probes contain a dark quencher with a conjugated minor groove binder at the 5¢-end and a fluorescent dye at the 30-end. this structure allows detection and differentiation of nucleotide polymorphisms at targeted sites by post-pcr melting curve analysis. four probes were designed to perfectly match the wild-type (wt) sequences at mutation sites corresponding to aa-s 146, 149, 156, 179 and 238. thus, mutations conferring esbl activity were expected to specifically lower the melting temperatures (tm-s) of the probe-template duplexes. each probe was labelled with a unique dye permitting analysis of mutations at multiple sites in a single reaction. results: the method was validated using laboratory strains producing the shv-1 (wt, non-esbl control), shv-2, 3, 4, 5 (g238s), shv-18 (g238a), shv-6 (d179a), shv-8 (d179n) and strains carrying cloned blashv fragments to which the naturally occurring mutations d179g, g156d, t149s and a146v were introduced by site-directed mutagenesis. following careful design of the probes and optimisation of pcr conditions, all the above mutations were successfully detected and discriminated from the wt sequence and each other according to specific tm-s. the detection was precise and highly reproducible in repeated experiments. furthermore, when applied to the analysis of 10 clinical isolates of klebsiella pneumoniae expressing esbl phenotype, the method was able to detect multiple shv alleles (wt and g238s or d179a) in the same isolates. this observation is particularly important considering the high frequency of co-production of the shv-1 and esbls in klebsiellae. conclusions: a pcr with mgb eclipse probes has a great potential for studying the epidemiology of shv esbls and possibly for analysis of other antimicrobial resistance mechanisms associated with mutations at defined loci. methods: a total of 356 non-repeat enterococcal blood isolates (250 e. faecalis and 106 e. faecium) were collected during 1994 to 2001 from hospitals located in south east of sweden. the bacterial isolates were identified by standard microbiological methods and susceptibility testing was performed with a 30-lg gentamicin disk on pdm-agar (ab biodisk) to detect hlgr isolates. all isolates were tested for the presence of the aac(6¢)ie-aph(2¢¢)ia gene using the polymerase chain reaction (pcr) technique. results: there was complete correlation between the gentamicin disk diffusion test and the pcr results. all 40 hlgr isolates, as defined by disk diffusion, and the positive control (e. faecalis atcc 51299) carried the aac(6¢)ie-aph(2¢¢)ia gene as judged from the pcr results. the resistant gene was not found in the negative control atcc 29212 or any of the 316 non-hlgr enterococci. conclusion: this study shows that in our setting the sensitivity and specificity of the disk diffusion method for the detection of hlgr enterococci is very high and there is a total agreement with the results obtained by using a pcr technique for detection of the aac(6¢)ie-aph(2¢¢)ia aminoglycoside modifying gene. objectives: the main objective was to develop a pyrosequencing method for identification of enterococcus spp. species with pyrosequencing method. also, development of antibiotic resistance with special reference to macrolide resistance will be studied by susceptibility testing in samples isolated serially from subject exposed to clindamycin. methods: biochemical identification of the enterococcal strains from faecal samples was done by growth at 45 c, catalase and hydrolyse of 1-pyrridonyl-beta-naphtylamide (pyr). species identification was done with pyrosequencing method. psq 96ma pyrosequencing technique enabled identification of different enterococcus species based on their 16s rrna v2-regions signature-sequences. antibiotic susceptibility testing was done by agar dilution method on mü ller-hinton ii medium, according to nccls. mic values were tested against erythromycin, clindamycin, ciprofloxacin, ampicillin, gentamicin, vancomycin and tetracycline. macrolide resistance genes; erm(b), erm(tr) and mef(a) was studied by multiplex-pcr. results: with pyrosequencing method, we identified 46 enterococcus faecium, 22 e. faecalis, 11 e. avium and 33 e. casseliflavus species, and 54 non-enterococci species. the antibiotic susceptibility testing showed that 26.5% of the enterococcus strains were resistant to erythromycin, 14.8% to ciprofloxacin and 17.4% to tetracycline. about 31.6% of the enterococcae had erm(b)-gene. conclusion: pyrosequencing was rapid and easy method for identification of bacterial strains even to the species level. antibiotic resistance varied a lot between different bacterial strains, as e. faecium and e. casseliflavus species being the most resistant ones. pyrosequencing results correlated well with species phenotype and antibiotic resistance. objectives: to determine the species distribution of vancomycin resistant enterococci (vre) isolated from hospitalised patients and detect genes encoding resistance to vancomycin and teicoplanin, by sandwich hybridisation method. and cpha genes by pcr, but not actual enzyme production, may be attributed to so-called 'silent' genes. susceptible strains are known to be able to convert to high-level beta-lactam/carbapenem resistance by increasing the expression of 'nearly' silent metallobeta-lactamase genes. metallo-beta-lactamases have been found to be carried on a small plasmid (13.6 kb) that appears to be selftransmissible, posing a potential threat of rapid spread of resistance. therefore early recognition of metallo-beta-lactamase producing strains is imperative. to describe the distribution of species in our nocardia isolates and to evaluate the usefulness of an easy and rapid method based on a short battery of susceptibility tests to identify clinical nocardia isolates compared with pcr and restriction analysis of hsp65 routinely used in our laboratory. methods: nocardia sp. isolated from 1995 to 2003 were selected to study. molecular identification was performed by hsp65 pcr-rflp. identification by susceptibility testing was by disk diffusion with gentamicin (cn), tobramycin (tob), amikacin (ak) and erythromycin (e) and by broth microdilution and e-test with ampicillin (amp), ciprofloxacin (c), cefotaxime (ctx) and amoxicillinclavulanate (aug). results: 34 isolates of nocardia sp. were studied. distribution of species according to results from pcr-rflp was: n. asteroides i (4), n. asteroides vi (15), n. farcinica (10), n. nova (3), n. otitidis-caviarum (2). n. asteroides i isolates had two different susceptibility patterns, two isolates were cn-s, tob-s, ak-s, e-r and the other two were cn-r, tob-s, ak-s, e-r. all n. asteroides i isolates were amp > 8 lg/ml and c > 4 lg/ml and ctx < 2 lg/ml. eightyseven per cent of n. asteroides vi were cn-s, tob-s, ak-s, e-r, amp > 8 lg/ml, ctx < 8 lg/ml whereas c was variable. hundred per cent of isolates of n. farcinica were cn-r, tob-r, ak-s, e-r, amp > 8 lg/ml, c < 4 lg/ml and ctx > 32 lg/ml. n. nova isolates were cn-s, tob-s, ak-s, e-s, amp < 4 lg/ml, ctx < 2 lg/ml and c < 4 lg/ml. n. otitidis-caviarum isolates were cn-s, tob-s, ak-s, e-r, amp > 8 lg/ml, ctx > 32 lg/ml and c < 4 lg/ml. medium time to obtain results by both methods was 48 h. conclusions: 94% of isolates belonged to the former n. asteroides complex. n. farcinica and n. nova were easily distinguished from other nocardia species by its susceptibility patterns. the main group n. asteroides vi was more difficult to distinguish from n. asteroides i and n. otitidis-caviarum. a short battery of susceptibility tests permits rapid differentiation of our most frequent nocardia isolates, although genotypic tests are more discriminatory. the ixodes ricinus tick, common ectoparasite of animals and humans, is the main vector of lyme disease in the czech republic. detection of borrelia under microscope, isolation in bsk-h medium and pcr identification was the aim of this work. methods: a tick was crushed in drop of sterile phosphate buffer saline and admired under microscope in dark-field. samples, in which spirochetes had been detected, were incubated in liquid bsk-h medium (sigma) at 33 c and admired weekly for 6 weeks. each strais was passaged twice and was frozen in 1.8-ml aliquots at à70 c. direct fluorescence assay (dfa) with fluorescein labelled polyclonal antibody to borrelia burgdorferi was used for screening. deoxyribonucleic acid of borrelial strains was isolated with invisorb genomic dna kit iii (invitec). three sets of primers (for b. burgdorferi sensu lato, b. garinii and b. afzelii) derived from 16srrna gene (rosa and schwan) were used for elementary identification of strains. detailed analysis of strains was made by light cycler real-time pcr (rt-pcr). primers and probe derived from reca gene were used in this method. results: there was a collection of 6283 ticks in urban and suburban localities of the czech republic from 1998 to 2002 years. incidence of spirochetes in tick population differed from 1 to 23.5% in different localities. spirochetes were cultured from at least one of six ticks (27 out of 156) that were tested positive by dark-field microscopy. all strains reacted positively by dfa and gave positive response with primers specific for b. burgdorferi sensu lato complex. nineteen strains belonged to b. garinii, four to b. afzelii and two to b. burgdorferi sensu stricto genospecies. one strain did not react with 16srrna primers for b. garinii but had melting temperature of reca gene product identical with b. garinii type strain. we identified the genotype of two strains determined as b. burgdorferi sensu lato neither by pcr, nor by rt-pcr. uors, blood and tissue were subjected to sequencing with the dideoxy chain termination technique using ceq 2000cx sequencer. cultivation, immunocytochemistry and western blots were used for confirmation. results: we cultured four blood, six skin, six csf isolates, numerous tick and two animal isolates. real-time pcr targeted reca, 16s and ospa genes showed that involvement of the nervous system, joints and skin in czech patients was predominantly caused by b. garinii, serotypes 3,5,6 (54%), then b. burgdorferi ss (26%) and b. afzelii (10-16%). the remaining 4-10% comprise coinfection with anaplasma phagocytophila or mixed borrelial infections. similar results were found in 424 animals. among game animals 22% tested positive with b. garinii and b. burgdorferi. wild boars and murids hosted borrelia sp. in 7 and 12% with prevalence of b. afzelii. no significant differences were noticed between the infection of adult and nymphal ticks, both reaching 20 and 12% in june and september, respectively. diferences were also between regions, in east bohemia with b. garinii prevailing and in moravia with prevalence of b. afzelii and human cases of erythema migrans and acrodermatitis atrophicans. infection prevalence data for patients were in agreement with data for the tick and animals. objectives: the aim of our study was to identify the strains of borrelia isolated from ticks and lyme disease patients in the russian far east and to analyse their taxonomic positions based on ospa gene phylogeny. methods: we have analysed 30 strains of borrelia burgdorferi sensu lato isolated from ixodes persulcatus ticks (25) and skin biopsies of erythema migrans from lyme disease patients (5) isolated by standard methods during last 6 years in the russian far east. after amplification with newly designed primers, we obtained full-length ospa gene sequence of each of the 30 strains. results: we identified four strains as b. afzelii completely identical to the strain xj23, isolated in japan. all of them were isolated from ticks. the other 26 strains were found to be genetically variable, but the closest homology found was with b. garinii. after phylogenetic analysis of ospa gene we found that these strains form three distinct and well-defined clades at the phylogenetic tree. genogroups 1 and 2 represent only species isolated in the far easter regions of the russian federation and in japan only, whereas genogroup 3 represents mostly european isolates, including seroand genogroups defined in the works of b. wilsske et al. and g. will et al. and four isolates from the russian far east. european serogroups 3 and 7 form the clade localised between genogroups 2 and 3. human strains were found within genogroups 1 and 2. conclusion: b. garinii was found to dominate among other b. burgdorferi sensu lato strains isolated from ticks and lyme disease patients form the russian far east. phylogenetic analysis showed that the species identified as b. garinii have significant variability in the ospa gene and form three major groups. two groups consisting only of strains isolated in the far east are significantly remote from all other b. burgdorferi sensu lato species. bootstrap values and distances among these groups suggest their solidity, especially genogroup 1. this, probably, indicates the distinct origin of defined genogroups 1 and 2 of b. garinii and may suggest another taxonomic status. objectives: for diagnosis of lyme borreliosis (lb) a two-step approach is recommended by cdc and dghm (screening elisa followed by immunoblot (ib) in case of reactive elisa). though borrelia ibs are widely used, they are still poorly defined regarding sensitivity, specificity and standardisation. a recently described recombinant western immunoblot (wib) complemented with borrelia antigens produced in vivo but not in culture (i.e. vlse) could improve previous tests (1). here a recombinant borrelia line ib (lib) was developed where each recombinant antigen is separately detectable, even those antigens with identical molecular weight. methods: the following recombinant igg and igm ibs were compared: (a) the wib described in (1) with p83/p100 (strain pko, b. afzelii), p58 (strain pbi, b. garinii ospa-type 4), bmpa (strains pka2, b. burgdorferi sensu stricto, pko, and pbi), vlse (strain pka2), ospc (strains pka2, pko, pbi, and b. garinii strain 20047) , and dbpa (strains pko and pbr, b. garinii ospa-type 3). (b) the lib with all antigens of the wib and in addition vlse (strains pko and pbi), ospc (strain ple, b. afzelii) and dbpa (strains b31 and pbi). to verify sensitivity and specificity, 65 sera of patients with early lb (50 early neuroborreliosis, 15 erythema migrans) and 110 control sera (60 blood donors, 10 rheumatoid factor positive, 10 syphilis patients and 30 patients with fever of unknown origin) were studied. results: ib interpretation criteria defining a serum as positive with at least two reactive bands or in case of igm at least one strong ospc band were used (2). sensitivity significantly increased from 63% (wib) to 80% (lib) for igg and from 46% (wib) to 69% (lib) for igm while specificity remained unchanged (99% for igg tests and 98% for igm tests). the increase of sensitivity was mainly due to the line blot technique, which allows detection and identification of antibodies differently reactive with homologues of the same protein. conclusion: the lib is more sensitive than the wib for both igg and igm antibody detection in acute lb while specificity remains unchanged. the lib is better to standardise and results are easier to interpret. background: tick of the ixodes ricinus group are well known as major vectors of the causative agents of lyme borreliosis, granulocytic anaplasmosis, ehrlichiosis and babesiosis in european countries. the humans infected with these agents can experience a wide range of clinical manifestations. i. ricinus is a widely distributed tick in lithuania and may transmit pathogens to mammalian hosts, including human beings. a single tick may contain several different pathogens so double-infection with borreliosis and ehrlichiosis may be seen. objectives: the aim of this study was to determine whether i. ricinus ticks collected in different regions of lithuania were infected with the causative agents of lyme borreliosis, anaplasmosis, ehrlichiosis and babesiosis agents and to estimate the prevalence of mixed infections in them by pcr. no investigations have been carried out to assess the prevalence of borrelia, anaplasma, ehrlichia and babesia infection in i. ricinus in lithuania using the pcr method before. methods: altogether, 243 i. ricinus ticks collected from 10 different regions of lithuania, were included in this study. all ticks were analysed individually. the presence ehrlichia/anaplasma group pathogen was determined by using pcr with ehrlichia/anaplasma-specific primers hr521/ehr747, multiplex pcrs using species-specific borrelia primers gi-r/gi-l (borrelia burgdorferi s.s.), gii-r/gii-l (b. garinii), giii-r/giii-l (b. afzelii). real-time pcr method with the abi prism 7000 system was used to detect babesia divergens. ehrlichia/anaplasma species were determined using the reverse line blot hybridisation. results: of the 243 individually processed ticks, 12 (5%) were positive for ehrlichia/anaplasma (hge -3, hge variant -1, e schotii -2 and 6 were not identified), 38 (16%) for borrelia (b. burgdorferi s.s -one (0.4%), b. garinii -12 (5%), b. afzelii -25 (10%) and 5 (2%) were positive for babesia divergens. one tick contained both ehrlichia/anaplasma and babesia, two contained both babesia and b. afzelii and one ehrlichia/anaplasma and b. garinii. conclusions: our results represent the first study in lithuania in which borrelia, ehrlichia, anaplasma and babesia parasites were directly identified in i. ricinus ticks by pcr, multiplex pcr, reverse line blot hybridisation and real-time pcr. it was detected that b. afzelii was the dominant genospecies in lithuanian ticks (10%) and ehrlichia/anaplasma and babesia were found in ticks too and might cause human diseases. molecular bacteriology: characterisation of agents p957 improved automated ribotyping using hindiii to discriminate previously uniform listeria monocytogenes serotype 4b strains i. heller, k. grif, m. dierich, r. wü rzner innsbruck, a objectives: to develop improved automated subtyping approaches for listeria monocytogenes, we characterised the discriminatory power of different restriction enzymes for ribotyping. pvuii and hindiii were evaluated for their ability to differentiate among isolates representing one of the two major serotype 4b epidemic clones, having ribotype reference pattern dup-1038 (which differs from the other clone dup-1042 in the ecori pattern only). this is of utmost importance, as the presence of only two major patterns within the serotype 4b does not allow sufficient epidemiology of listeria infections. methods and results: the eight selected l. monocytogenes isolates (serotype 4b) with the ribotype reference pattern dup-1038 were responsible for human listeriosis outbreaks in france, canada, switzerland and turkey from 1978 to 2002, and for sporadic foodborne cases in austria (2002), england (1987 and 1989) and the usa. ribotyping was performed using the riboprinter microbial characterisation system according to the manufacturer's instructions using ecori, pvuii and hindiii as restriction enzymes. we found that the eight isolates belonging to dup-1038 (i.e. indistinguishable by ecori) were also indistinguishable by pvuii but yielded two clearly different patterns when using hindiii. conclusions: we conclude that automated ribotyping using hin-diii allows discriminating previously uniform l. monocytogenes 4b isolates. this discrimination may facilitate the tracing of outbreaks and may also improve epidemiological surveys. p958 detection of bft, the isoforms of the enterotoxin gene and cfia gene in bacteroides fragilis isolates of different origins g. terhes, j. soki, k. ago, e. urban, e. nagy szeged, hun objectives: bacteroides fragilis is an obligate anaerobic, gram-negative rod constituting 1% of the normal intestinal flora of humans, and is the gram-negative anaerobic rod most frequently isolated from human clinical samples. some of the b. fragilis isolates produce a zinc-dependent metallo-protease, enterotoxin coded by the bft gene. this protein has enterotoxic activity; it causes fluid accumulation in a lamb ligated ileal loop model. to date, three different isoforms, designated bft-1, bft-2 and bft-3, have been identified. the literature regards the enterotoxin-producing property of b. fragilis as a virulence factor since these strains can be isolated more often from severe infections such as sepsis, or abdominal and deep soft-tissue abscesses. it is also thought to be involved in diarrhoea in 1-5-year-old children. aims and methods: the aim of the present study was to examine the prevalence of enterotoxin production among b. fragilis strains isolated between 2001 and 2003 from specimens originating in clinical wards of our university or in other hospitals by ht-29 cytotoxicity testing or pcr detection of the bft gene. the results obtained with the two methods were compared. the frequencies of three alleles of bft genes in enterotoxigenic strains from different sources were determined by using pcr-restriction fragment length polymorphism analysis. the b. fragilis strains can be divided into two major groups by molecular typing methods and most importantly according to the carriage of the cfia gene. we therefore also examined the occurrence of the cfia gene by pcr and the co-incidence of bft and cfia among the above collection of strains. results: the average occurrence of toxigenic b. fragilis strains in the different groups of clinical samples was 10% and in deep-tissue infections was 15% by both the pcr method and the cytotoxicity assay. bft genes were found only in the cfia-negative group. the prevalence of the cfia gene corresponded to our earlier findings and data from the literature and we did not observe co-incidence of the bft and cfia genes in this study. introduction: in addition to the two large clostridial cytotoxins (lct -toxins a and b) some strains of clostridium difficile also produce an actin-specific adp-ribosyltransferase (binary toxin cdt). cdt may serve as an additional virulence factor. methods: we used pcr and southern blotting methods for detection of genes encoding the enzymatic (cdta) and binding (cdtb) components of binary toxin in 369 strains isolated from patients with suspected c. difficile-associated diarrhoea or colitis. binary toxin production was assessed by western blotting using antisera against the iota toxin of c. perfringens (anti-ia and ib). toxin activity was detected with an adp-ribosyltransferase assay. pcr amplification was performed to detect the gene encoding for toxin b. binary positive strains were subjected to toxinotyping and were characterised by phenotypic (serogrouping) and genotypic markers (pcr-ribotyping, arbitrarily primed pcr (ap-pcr) and pulsed-field gel electrophoresis (pfge)). results: twenty-two strains (prevalence 6%) harboured both genes cdta and cdtb; 19 out of the 22 strains reacted with antisera against the iota toxin of c. perfringens; the binary toxin activity was positive in only 17 of the 22 strains. all strains also produced toxins b. however, they had significant changes in tcda and tcdb genes and belonged to variant toxinotypes iii, iv, v, vii, ix and xiii. with typing methods used we could differentiate 16 profiles, indicating that most of binary toxin positive strains were unrelated. conclusion: binary toxin-producing isolates of c. difficile are widespread but prevalence varies from one country to another. more studies are needed to define the role of binary toxin in pathogenesis. clostridium difficile in singapore w.y. leong, r. das ramadas, t.h. koh, k.p. song singapore, sgp objective: occurrence of nosocomial clostridium difficile-associated diarrhoea and pseudomembranous colitis is related to the production of toxins a and b (encoded by tcda and tcdb, respectively) from the pathogen. tcda and tcdb, together with their accessory genes, tcdc-e are arranged within a well-defined chromosomal region termed pathogenicity locus (paloc). another virulence factor, adp-ribosyltransferase binary toxin (encoded by cdt genes) was reported to be found in approximately 12% of pathogenic strains of c. difficile. despite the availability of a number of detection methods, the identification methods commonly used are not designed to detect all the virulence factors known. we present here an alternative characterisation of the toxigenic and the related genes of c. difficile based on genotyping. the correlation between paloc and cdt genes was also examined. methods: all 110 clinical isolates from singapore general hospital (sgh) were screened with pcr and multiplex pcr for the presence of tcda-e and cdta-b in the paloc region and cdt operon, respectively. the production and activity of toxins a and b were analysed by commercial kit and cytotoxicity testing. results: the isolates could be classified into 16 groups based on the genotypic analysis of the paloc and cdt genes. approximately 21% of them shared a common profile with the reference strain vpi 10463, and about 36% were completely devoid of the genes tested. variations demonstrated in tcdc-e were complicated and no specific profile could be attributed to a particular genotype. an atypical toxigenic variant was discovered which contains only tcdb. in contrast to data reported elsewhere, none of the pathogenic strains was found to contain complete cdt genes. when tested for tcda and tcdb production, six strains were identified to be toxins a-negative, b-positive. conclusion: the great genetic polymorphisms displayed by the c. difficile isolates here confirm that these strains were highly heterogeneous and could originate from endogenous source. there is no significant correlation between presence of the structural genes (tcda-b), accessory genes (tcdc-e) and cdt genes. pathogenic strains do not necessarily contain all the genes in the paloc. in conclusion, our results using this toxino-genotyping method for the studies of genetic distribution of toxinogenic genes correlates well with the phenotype of the bacteria i.e. toxin expression. p961 characterisation of clostridium difficile strains isolated in different time periods and belonging to different ribotypes p. spigaglia, v. carucci, p. mastrantonio rome, i objectives: seventy-four clostridium difficile clinical isolates, collected in different time periods, were typed by pcr-ribotyping. strains belonging to the two main pcr-ribotypes were characterised for virulence determinants and for antibiotics resistance. methods: paloc genes analysis, detection of binary toxin gene and antibiotic resistance determinants (ermb, tetm and catd) were performed by pcr assays. erm(b) sequence type was identified by a rflp-pcr. mics for erythromycin, clindamycin, tetracycline and chloramphenicol were determined by e-test. results: two main pcr-ribotypes named a and r, respectively, were identified. pcr-ribotype a collected 20 strains whereas 15 strains belonged to pcr-ribotype r. old strains (from 1985 to 1990) belonged to pcr-ribotype a, whereas recent strains (from 2000 to 2001) belonged to pcr-ribotype r. all strains with pcr-ribotype a had classical paloc genes and did not have the binary toxin gene. ninety percent of these strains were multiresistant and the sequence type of the ermb genes was similar to that of c. difficile 630. all strains belonging to pcr-ribotype r had the binary toxin gene, four of them showed major variations in the toxin a gene and 87% had a mutated toxin negative regulator. none of these strains was multi-resistant although one showed all three antibiotic resistance determinants. fifty three percent had a tetm gene, 13% tetm and ermb genes and 7% only an ermb gene with a sequence similar to that of c. perfringens cp592. interestingly, as far as resistance is concerned, there was no correspondence between phenotype and genotype in 75% of these strains. in particular, all strains with a tetm or a catd gene were susceptible to tetracycline and chloramphenicol in vitro, whereas five strains, resistant to erythromycin but not to clindamycin, did not have an ermb gene. all these strains showed, after induction with erythromycin, some clindamycin resistant colonies. conclusions: the results seem to indicate a recent spread of c. difficile clones that add together a potential increase of virulence by acquisition of the binary toxin, variations in genes belonging to the paloc and acquisition of different mechanisms of antibiotic resistance. enterococci are natural inhabitants of the gastrointestinal flora of humans and animals and are widely distributed in the environment. members of this genus are recognised as important opportunistic pathogens responsible for serious infections but the molecular mechanisms of enterococcal virulence are not yet completely understood. in this study 42 enterococci from different sources, including clinical isolates (from human and veterinarian origin), non-clinical isolates and reference strains from 19 enterococcal species, were typified and their virulence potential characterised. the relationships among these enterococci were first analysed using smai pulsed-field gel electrophoresis and m13 pcr-fingerprinting, in order to evaluate the genomic heterogeneity of the isolates. enterococci were also screened for several virulence traits such as cytolysin (cyl genes), adhesins (agg, esp, efaafs and efaafm genes) and gelatinase (gele), revealing distinct virulence potentials. in enterococcus faecalis, it was recently described that some virulence determinants can be clustered on large pathogenicity islands and not only in pheromone-responsive plasmids. dot-blot dna-dna hybridisation was used to locate virulence determinants in the bacterial genome of the enterococci under study. no conclusive results were obtained for esp and gele, whereas efaafs and efaafm were found on the chromosome as expected. although cyl genes and agg are plasmidic, in most isolates, they were detected on the chromosome of five strains, suggesting that these enterococci may harbour a pathogenicity island. beyond the widespread nature of virulence traits, chromosomal integration of virulence genes seems to occur in different enterococcal species and isolates from non-clinical sources. p963 identification of salmonella serotypes in sheep by pcr t. zahraei-salehi tehran, ir introduction: salmonella abortusovis, s. dublin, s. montevideo and s. typhimurium are more common serotypes in sheep. one way of transferring of contamination is from visceral organs specially gallbladder, intestine and liver, which can be transferred from meat to human. because of this, this research was essential to consider about it. objectives: (1) isolation of salmonella serotypes from visceral organs of sheep and goats. (2) detection of inva gene in isolated serotypes by pcr. materials and methods: for these goals, samples from 96 livers, 86 gallbladders, 110 mesenteric lymph nodes and 10 faeces (totally 302 samples) were taken, and then cultured in enrichment and selective media. doubtful colonies were selected and transferred to tsi agar, urea agar, sim, mr-vp broth and nitrate broth. pcr reaction was carried out in master cycle (eppendorf). for dna extraction isolated salmonella serotypes was cultured in lb broth for 24 h at 37 c. lb broth (200 ll) was boiled for 10 min and centrifuged at 6000âg for 3 min. a total of 1.5 ll of the supernatant was used for amplification by pcr with salmonella-specific (139 and 141) primers. results: three salmonella serotypes were isolated from mesenteric lymph nodes (two cases) and gallbladder (one case). serotyping test showed that two of them belong to group b and one of them to group d of salmonella. when subjected to salmonellaspecific primer inva, all isolates, including positive control, generated a single 284-bp amplified dna fragment, on 1.5% agarose gel. conclusion: salmonella-specific pcr with primer set inva is rapid, sensitive, and reliable for detection of salmonella in many clinical samples. the present research supports the ability of this specific primer set to confirm the isolates as salmonella. all isolates, including positive controls (s. typhimurium and s. dublin), screened by pcr resulted in 284-bp amplified product. no amplified products were obtained from negative controls (water and o2k12 escherichia coli serotype). objectives: the variability of salmonella typhimurium strains was studied by pcr-based methods. methods: 28 strains of s. typhimurium were isolated from food or animal sources in the course of surveillance programmes. strains were phagotyped and their antibiotic resistance was determined by disk diffusion method. fluorescent aflp was done using eco-ri and msei enzymes and aflp products were separated by capillary electrophoresis. results: the presence of integrons was analysed in all 28 strains of s. typhimurium and three different integron profiles (ips) were detected by amplification of variable region of the integrons. the ip-1 profile, characterised by two pcr products of 1.0 and 1.2 kb, was present in six strains. all these strains were multiresistant with resistance acssut or acssutna. the ip-2 profile contained single 1.6 kb pcr product and was present in six strains resistant to asutmp or assutmp. the dhfra1 gene was confirmed to be an integral part of ip-2 integron. a total of 0.2 kb pcr product (ip-3) was amplified in two strains sensitive to all antimicrobials. as lysogenic bacteriopahges could frequently transfer their dna into the bacterial cell and thus change chromosomal composition, phage-related sequences were probed in s. typhimurium strains by pcr with primers complementary to four genes of phage p22 (g8, g13, eae, eac). three different types of pcr products were detected in multiplex reaction: the presence of g8 sequence only, the simultaneous occurrence of g8 and eac or presence of g8 and g13. nine strains did not contain any from the tested phagerelated genes. the relatedness between strains was further monitored by aflp. we observed high strain-to-strain similarity as dice coefficients fell in the range of 94-100%. according to the presence of several dna fragments, strains were separated into eight aflp clusters. conclusions: by comparison of all methods we obtained corresponding results in strain clustering. all methods can be used for subtyping of s. typhimurium strains. producing klebsiella strains isolated from nosocomial infections k. matusiewicz, b. maczynska, d. olejniczak, a. przondo-mordarska, r. franiczek wroclaw, pl objectives: klebsiella bacilli present many pathogenic properties, which determine their ability to survive and rapid spreading in hospital environment. the adhesive properties of klebsiella bacilli associated with the presence of fimbrial and non-fimbrial adhesins play a very important role in pathogenicity of these bacteria. rapid spread of patogenical factors is often connected with presence of their plasmid-mediated genes. the aim of our study was to detect plasmid and chromosomally born fimh and mrkd genes encoding main adhesins: ms and mr, respectively. methods: a total of 55 klebsiella clinical isolates obtained from patients hospitalised in different hospital wards were studied. the phenotypic activity of fimbriae was characterised by haemagglutination method. the genomic and plasmid dna were isolated using manual method as well as qiagen dna kits. the presence of genes encoding main adhesins were detected using pcr-method with primers detected fimh and mrkd genes results: 40% of strains displayed phenotypic activity of both type 1 and type 3 fimbriae, 25.5% showed only activity of type 1 fimbriae, 30.9% only of 3 type fimbriae and 3.6% strains showed the lack of hemagglutination activity. the percentage of detected genes using pcr, was higher then showed results of phenotypic activity. the presence of mrkd genes was detected in 100% investigated strains in chromosomal dna and 85.4% showed both mrkd and fimh genes. a total of 5.5% strains demonstrated only fimh genes in chromosomal dna and 3.6% strains showed no genes. in plasmid dna, the presence of main adhesin genes confirmed in 33% klebsiella strains (mrkd genes in 20% strains, both fimh and mrkd in 9% and only fimh in 4% strains). conclusions: the presence of fimh and mrkd genes in genomic and plasmid dna not always leads to phenotypic expression of fimbrial adhesins. the activity of type 3 fimbriae is connected with chromosomal variant of mrkd gene. in case of fimh genes, the plasmid variant is enough for haemagglutination activity of type 1 fimbriae. the percentage of detected fimh and mrkd plasmid genes depended on hospital units from which these strains were isolated. this suggests the spread of plasmid-encoded adhesins among klebsiella strains. objectives: bacteria of the genus klebsiella are opportunistic pathogens responsible for an increasing number of multiresistant infections in hospitals. the two clinically and epidemiologically most important species, klebsiella pneumoniae and k. oxytoca, have recently been shown to be subdivided into three and two respective phylogenetic groups. the aim of this study was in-depth evaluation of the amplified fragment length polymorphism (aflp) genetic characterisation method. methods: first, we investigated the variability of aflp patterns for klebsiella strains within and between different outbreaks. second, by use of carefully characterised, phylogenetically representative strains, we examined whether different klebsiella species and phylogenetic groups can be discriminated using aflp. twenty-four strains originating from seven presumed outbreaks and 31 non-associated strains were investigated. results: the aflp fingerprints of all epidemiologically associated strains showed three or fewer fragment differences, whereas unrelated strains differed by at least four fragments. cluster analysis of the aflp data revealed a very high concordance with the phylogenetic assignation of strains based on gyra sequence and ribotyping data. the species k. pneumoniae, k. oxytoca, k. terrigena and the possibly synonymous pair k. planticola/k. ornithinolytica each formed a separate cluster. similarly, strains of the phylogenetic groups of k. pneumoniae and k. oxytoca fell into their corresponding cluster, with only two exceptions. conclusion: this study provides a preliminary cut-off value for distinguishing epidemiologically non-related klebsiella isolates based on aflp data, confirms the sharp delineation of the recently identified phylogenetic groups and demonstrates that aflp is suitable for identification of klebsiella species and phylogenetic groups. objectives: k-serotyping, i.e. determination of the capsular antigen, has been the preferred typing method for klebsiella isolates, as it is highly discriminatory (77-types are known) and as k-types are known to differ in their pathogenic potential. unfortunately, k-serotyping requires a large collection of sera and is restricted to a few reference centres. moreover, k-serotyping suffers from cross-reactions and is not applicable to non-capsulated strains. the objective of this work was to develop a molecular method that would enable to determine the k-serotype without using antiserum. methods: we amplified by pcr the capsular antigen gene cluster (cps) and the pcr product (10-18 kb long) was digested with hincii, followed by agarose gel electrophoresis (cps pcr-rflp). results: the profiles (called c-patterns) obtained for 228 strains representing the 77 known k-serotypes showed four to 13 bands in the size range 0.2-4.361 kb. a total of 128 distinct c-patterns were obtained. the following important observations were made: (i) the c-patterns obtained for strains of any k-serotype were distinct from the c-pattern of all other k-serotypes, with the only exception of serotypes k22 and k37, which are known to cross-react. (ii) for 12 k-types, c-pattern variation was found among strains with the same k-serotype; in most cases, the strains with variant c-patterns belonged to other klebsiella species than the reference strain. thus, cps pcr-rflp has a higher discriminatory power than classical k-serotyping. (iii) within k. pneumoniae, we observed c-pattern identity among strains of a given k-type, for example k1 or k3, that were collected many years apart and from distinct sources. this stability of the c-pattern indicates that cps pcr-rflp is suitable for long-term epidemiology of capsular types. (iv) only 2.8% (compared with 8-23% for classical k-serotyping) of the strains analysed by cps pcr-rflp were non-typable, because pcr amplification failed. (v) the value of cps pcr-rflp for k-serotype determination was tested on 21 recent k. pneumoniae clinical isolates. the k-serotype of 18 (86%) of them could be deduced from the comparison of their c-pattern with the database. (vi) four of five non-capsulated strains analysed showed a recognisable c-pattern. conclusions: cps pcr-rflp allows determination of the k-serotype, while being easier to perform and more discriminatory than classical serotyping, and allowing the characterisation of non-capsulated strains. (1) the composition of the vaginal microbial community of eight of these vaginal swabs (three grade i, two grade ii and three grade iii), were studied by culture and by cloning of the 16s rrna genes obtained after direct amplification. (2) species-specific pcr for atopobium vaginae and gardnerella vaginalis was carried out for all 150 vaginal swab samples. (3) forty-six cultured isolates were identified by tdna-pcr and 854 cloned 16s rrna gene fragments were sequenced, yielding a total of 38 species. results: cloning revealed that a. vaginae was abundant in four out of the five non-grade i specimens and that lactobacillus iners was the only lactobacillus species that was present in non-grade i specimens, while it was absent from grade i samples. respectively 1.8% (grade i), 15.7% (grade ii) and 77.7% (grade iii) of the vaginal swab samples were positive for both a. vaginae and g. vaginalis species-specific pcr (p < 0á001, chi square). discussion: culture independent, molecular analysis revealed a higher microbial diversity in non-grade i specimens than did culture. together, culture, 16s rrna gene cloning and species-specific pcr point to the presence of nine presumptively novel bacterial species and to a strong association between a. vaginae, g. vaginalis and bacterial vaginosis and to an ambiguous role for l. iners. it appears as if a. vaginae may be a constituent -in low numbers -of the human vagina, possibly attaining replicative dominance in association with decreasing lactobacillary grading. the presence of a. vaginae in bacterial vaginosis(-like) microflora may shed new light on the aetiology of this condition. using multilocus pcr tests with various primers in the genomes of 186 strains isolated in the territories of russia and turkmenistan we were able to detect three housekeeping genes (hapa, toxr, rtxa) and nine virulence genes located in prophages and 'pathogenicity' and 'persistence' islands: ctxphi (ctxa, zot, ace), rs1phi (rstc), vpi (tcpa, alda, toxt), vpi-2 (nanh), epi (mshq). besides, we used the methods of ribotyping and pcr typing which involved the 'random' primer, 1281, to elucidate genetical relationship between the strains of varying epidemic significance. the genome of clinical isolates obtained from patients during several epidemic outbreaks, was shown to be stable and to contain all the genes tested. c. vibrios isolated during the interepidemic period from natural ecosystems, formed a heterogenous population represented by single virulent clones that had retained the complete set of the genes under study, by non-toxinogenic strains, that had lost only individual genes and (or) pathogenicity blocks of genes, i.e. either ctxphi and rs1phi, or ctxphi, vpi and rs1phi, or by those carrying deficient prophages ctxphi (ctxaà zot+ ace+) and vpi (ctpaà alda+ toxt+), as well as by clones containing only housekeeping chromosomal genes and sometimes a gene from the 'persistence island'. as soon as virulent clones get into water environment, they lose their virulence blocks in the following order: ctxphi and rs1phi, then vpi, gene vpi-2 being the last one to be lost. in conformity with the results of the above three genotyping methods, epidemically hazardous strains, represented a homogenous group, suggesting a single clonal origin. close genetical relationship between these strains and non-toxinogenic vibrios, that partly retained their virulence genes, was also established. at the same time, as shown by ribotyping and pcr typing studies, avirulent 'water' vibrios formed an independent group, because their genotypes manifested quite distinct features, in contrast to the first two vibrio groups. thus, the observed genotype heterogeneity of el tor cholera vibrios living in water ecosystems was likely to be a result of the loss of dna fragments varying in their length and functions. the genotyping procedures used in the work made it possible to discover evolutional relationships among the bacterial strains under study. bacteroides fragilis gram-negative anaerobic rods, 132 strains isolated in poland and 53 in france (from intestinal and extraintestinal sources) were compared in this study. the identification of bacterial strains was done on the basis of gram staining, growth on selective bbe (bacteroides bile esculine) medium, and biochemical characteristics determined by the api 20 a test (biomérieux, france). for assessment of the presence of enterotoxin (fragilysin) gene in analysed strains, the pcr method was used. dna for pcr was isolated using genomic dna prep plus (a&a biotechnology, poland) and amplification was performed in a techne thermocycler with primers 404 (5¢-gag ccg aag acg gtg tat gtg att tgt-3¢-tgc tca gcg ccc agt ata tga cct agt-3¢). the pcr program consisted of the following steps: 94°c for 4 min, 40 cycles of 94 c (1 min), 52 c (1 min) and 74 c (1 min). among the polish 132 strains, 16 contained the fragilysin gene. of the 53 french strains 10 contained the fragilysin gene. for all these strains, pulsed field gel electrophoresis (pfge) was performed. bacteria were suspended in se buffer (75 mm nacl, 2.5 edta ph 8.0), embedded in 0.5% agarose plugs and lysed overnight at 55 c. plugs were washed five times in se at room temperature afterwards. dna in the plugs was digested using not i (boehringer mannheim, germany). electrophoresis was performed in a chef mapper (biorad, venendaal, the netherlands). the voltage was 10 v/cm for 18 h with linear ramping from 5 to 35 s at ae60 angles. in conclusion, 19% strains isolated in france and 15% of those isolated in poland contained the fragilysin gene. the pfge analysis revealed that strains isolated in poland and in france show genetically differentiation (these strains are genetically not homogenous). objectives: different molecular mechanisms of resistance to azole antifungal agents, that can exist simultaneously, have been described in candida albicans strains. one of these mechanisms includes alterations in the gene encoding the target enzyme erg11. in the present study we used pyrosequencing method to conduct an epidemiologic survey in ketoconazole-susceptible and -resistant strains of clinical c. albicans strains isolated in our region, to determine differences in the gene encoding lanosteroldemethylase (erg11). methods: the strains of c. albicans were obtained by swabbing the oral mucosa of subjects with oropharyngeal candidiasis. susceptibility to ketoconazole was tested using the broth microdilution method recommended by the nccls document m27-a. concentrations of ketoconazole tested were in the range 0.03-16 mg/ml. the mic endpoint was defined as the lowest concentration at which 80% of growth was inhibited, compared with the drug-free control. yeasts were grown in sabouraud agar and dna was extracted by using qiaamp dna mini kit (quiagen). pcr primers matched an erg11 gene region of 178 bp. one of the primers of pcr fragment was biotinylated, a single strand of pcr products was obtained with streptavidin-coated beads method. samples were analysed using a psq 96 system with sqa software and sqa reagent. results: a total of 31.2% of strains exhibited dds or resistance to ketoconazole (mic >0.25 lg/ml). the sequence analysis was designed to cover a region of the erg11 gene including codons 464-483. previous studies showed that in this region, the mutations g464s, g465s, r467k and i471t are associated with azole resistance in c. albicans. in our study the sensitive strains have shown no mutations. among dds and resistant strains, only the mutation g464s was found in two strains, while no mutations were demonstrated in the remaining isolates. conclusion: this study is the first to use the pyrosequencing system to characterise changes in nucleotide sequence of the erg11 gene fragment involved in azole resistance of c. albicans strains. the observation of one point mutation in only two resistant strains tested suggests a limited role of the region of the erg11 gene analysed in the azole resistance among c. albicans strains present in our region. however, the pyrosequencing system has shown to be a fast and specific technique for detection of point mutations in the region of erg11 gene of c. albicans strains. escherichia coli verocytotoxin 2 variants. correlations to the clinical manifestations s. persson, f. scheutz, k.e.p. olsen copenhagen, dk background: verocytotoxin 2 (vt2) of verocytotoxin producing escherichia coli (vtec) is a potent toxin, capable of producing serious complication, when excreted from the bacteria colonising the intestinal tracts. the mature toxin is composed of one a-subunit and five identical b-subunits, and is encoded by the approximately 1240 bp vtx2ab operon. based on the variable nucleic acid sequence of both subunits, several toxin variants have been identified. objectives: the subtype designation, important sequence motifs and clinical significance of the vtx2 variants, are not consistent throughout the literature. to shed more light on these features, a novel typing method was developed for the investigation of subtype-specific correlations to the clinical outcome. methods: the subtyping method relies on pcr and sequencing. by use of vtx2 universal primers, a 630-bp fragment covering the most variable regions of subunit a and b was amplified by pcr, and subsequently sequenced. results and conclusion: the present method was used for the analysis of vtx2-positive strains from our strain collection, counting 274 strains, isolated from patients with known clinical manifestations (hus, hc, bloody diarrhoea, diarrhoea, fever, etc.). compared with traditional subtyping, our preliminary results indicate that most strains in our strain collection harbour the vtx2 or vtx2c subtype, in addition to a few strains containing the activatable carboxy-terminus of subunit a, referred to as vtx2d. correlations between these subtypes and the clinical complications will be presented. additionally, the novel sequences from our strain collection will be investigated for other sequence motifs connected to the clinical outcome. as sequencing has become more accessible and less expensive, we believe that this method, offers a good and reliable alternative for diagnostic subtyping of vtec strains from these infections. p976 shiga toxin-producing escherichia coli o157 in slovenia p. zabukovnik, a. andlovic, a. zore ljubljana, si objectives: in the institute of microbiology and immunology (department for bacterial diagnostics of diarrhoeal infections), medical faculty in ljubljana, we wanted to introduce multiplex pcr test for detection of shiga toxin-producing escherichia coli (stec). until recently we used only enzyme immunoassay (eia) to detect production of shiga toxin (stx) in specimens. institute of microbiology and immunology has extensive collection of e. coli isolates from human faeces (mostly from hospitals in ljubljana). we decided to test isolates in our collection from 1993 to 2002, with serogroup o157. we used multiplex pcr assay that amplified sequences in four virulence genes (shiga toxin 1 (stx1), shiga toxin 2 (stx2), intimin (eaea), enterohemolysin (ehxa)). methods: all isolates were serotyped with rabbit o antisera. we used multiplex pcr to detect presence of shiga toxin 1, shiga toxin 2 (and sub variants, but did not discriminate between them), intimin and enterohemolysin genes. we also tested those strains for production of stx with eia. results: we tested 20 e. coli isolates with serogroup o157 and found 10 stec. stx2 and ehxa genes were present in almost all stec o157 isolates. the most common pcr profile (five of 20) of o157 isolates had stx2, eaea and ehxa genes. one isolate had stx2 gene but did not produce shiga toxin (or possibly eia did not detect produced shiga toxin). most of those 10 stec o157 were isolated in summer months of july and august. two o157 stec were isolated in the year 1997 shortly one after another. they had identical multiplex pcr profile. the same happened in the year 2002. conclusion: we notice increase in the number of stec o157 isolates per year in years after 1997. this may be because of use of better diagnostic methods. in last years stec o157 with pcr profile stx2, eaea and ehxa is dominant. in years 1993 to 1998 the dominant pcr profile had stx1, stx2, eaea and ehxa genes. background: chronic prostatitis is recognised to be caused by infectious and non-infectious prostatic inflammation as well as non-inflammatory diseases, but the separation of various prostatitis syndromes is difficult to perform. bacterial prostatitis is a common diagnosis and a frequent indication for antimicrobial therapy. however, confirmation of aetiology of inflammation is exceedingly uncommon. objectives: the aim of this study was to determine the prevalence and aetiology of chronic bacterial prostatitis among the patients with clinically confirmed diagnosis. methods: between october 2002 and october 2003 the patients with suspected prostatitis were examined. the clinical diagnosis was confirmed in patients within 3 months or greater duration of the following signs and symptoms: perineal discomfort, pain following ejaculation, urinary frequency, urgency, dysuria, low back pain, suprapubic pain, palpation of a tender prostate on physical examination. the bacteriological diagnosis was determined in patients, who had not been taking antibiotics in the previous month, by meares and stamey technique. prostatitis was categorised according to nih classification. results: a total of 129 patients were examined. chronic bacterial prostatitis (nih category ii) was found in nine patients (7.0%), inflammatory chronic pelvic pain syndrome (nih category iiia) -in 59 (45.7%), non-inflammatory chronic pelvic pain syndrome (nih category iiib) -in 61 (47.3%). the following pathogens were isolated in nih category ii: staphylococcus spp. -in three (33.3%), anaerobic bacteria (prevotella spp., prevotella spp. and peptostreptococcus spp.) -in three (33.3%), escherichia coli -in two patients (22.2%), acinetobacter lwoffii -in one (11.1%). conclusions: chronic bacterial prostatitis is an important but rare clinical entity. careful examination using quantitative segmented bacteriologic cultures leads to proper categorisation into the recognised forms of the prostatic syndrome. the most common pathogens of chronic bacterial prostatitis were staphylococcus spp., anaerobic bacteria (prevotella spp. and peptostreptococcus spp.) and e. coli. objectives: a prospective multicenter urology outpatient survey, undertaken to examine prostatitis in italy, is used to compare the prevalence, characterisation, diagnosis and treatment of prostatitis patient with the north american (na) prostatitis patient. methods and materials: seventy urologists, representing a crosssection of urologic centres in italy, counted and recorded the overall total male patients reported in the clinic and the overall total patients diagnosed with prostatitis over a 5-week period. results were compared with published practice prevalence and cohort data (in particular the nih chronic prostatitis cohort study -cpc and seattle prostatitis cohorts) examining similar data in na. results: a total of 1148 patients were identified with prostatitis (12.8%). the mean age of the prostatitis patients was 47.1 (range 16-83). the most common urinary diseases were benign prostatic hyperplasia (17.4%), recurrent urinary tract infections (11.2%) and urinary calculogenesis (11.1%), while the most common concurrent diseases were diabetes (7.2%) and depression (6.8%). the most frequently reported and most severe symptoms at time of evaluation were irritative voiding symptoms, perineal and suprapubic pain and discomfort. over three quarters of the patients were dissatisfied with their quality of life. bacteria were cultured in 15.6, 17.7 and 14.0% of eps, vb3 and semen specimens, respectively. comparison to na data suggests that the european prostatitis patient and the european urologists' approach to the diagnosis and treatment of prostatitis are not that dissimilar to prevalence and management of prostatitis in na. conclusion: prostatitis is a common worldwide outpatient diagnosis, comprising a significant percentage of male outpatient visits to urologists in both europe and na. the similarities in prevalence, characterisation and management of the typical prostatitis suggests that an international collaborative research effort is indicated in this important urological condition. observation unit for <24 h. before discharge (7.0%) or admitted (38.3%). the two factors that significantly correlate to hospital admission were the severity of uti (53% of complicated ac, 34% of aup, 80% of complicated ap and 57.3% of acute prostatitis) and patients' age (76.2 ae 17.9 years in those admitted with complicated ac vs. 48.8 ae 24.9 years in the non-admitted, 44. 2002) . demographic factors, underlying conditions, symptoms and signs, laboratory, radiological and microbiological data, antimicrobial therapy, outcome and final diagnosis were evaluated. results are expressed by percentages or median as appropriate. results: median age was 86 years, 50% were female and 56% were nursing home residents. seventy-four per cent were dependent for activities of daily living, 22% had a permanent urinary catheter and 58% had cognitive impairment. the most frequent symptoms were fever (72%), decline in function (54%) and dyspnoea (50%); only 7% referred dysuria. stupor (46%), crackles (40%) and ronchi (34%) were the commonest signs. leucocytosis (14065/ul), elevated urea (64 mg/dl), respiratory failure (59%) and high c-reactive protein (142 mg/l) were the main laboratory abnormalities. pyuria was observed in 71%, chest x-ray showed a pulmonary infiltrate in 48%, and 52% of cases fulfilled criteria of severe sepsis. blood and urine cultures were positive in 18 and 52% of patients, respectively; gramnegative bacilli (gnb) were found in 82% of positive cultures, escherichia coli being the most common agent. no pneumococci were isolated either in blood or sputum. amoxicillin-clavulanate was the antimicrobial therapy most frequently administered (51%). median hospital stay and mortality were 6 days and 27% respectively. urinary tract infection was the commonest final diagnosis (61%). conclusion: respiratory manifestations predominate in disabled old patients with gnb severe urinary sepsis initially diagnosed as suari. respiratory distress may underlie this presentation. further studies are required to support this contention. enterococcus in patients hospitalised through the emergency department d. raveh, i. rosenzweig, b. rudensky, a.m. yinnon jerusalem, il objectives: to determine the incidence of, and risk factors for, isolation of pseudomonas aeruginosa or enterococcus from urine cultures obtained from patients in the emergency department (ed). methods: one year prospective, non-interventional study of all urine specimens collected in the ed, out of which one organism was isolated at a concentration of >100 000 cfu/ml. in this study were included all patients with p. aeruginosa or enterococcus bacteriuria (study patients), and control patients with escherichia coli bacteriuria subsequently hospitalised, at a ratio of two controls for each study case. patients were interviewed with a structured questionnaire and charts were reviewed for demographic, clinical and laboratory indicators of enterococcus or pseudomonas bacteriuria as compared with e. coli bacteriuria. results: over the 1-year study period, 744 positive urine samples were obtained from ed patients: 610 (82%) enterobacteriaceae (including 476 isolates of e. coli) and 134 (18%) other organisms, of which 39 (5%) were p. aeruginosa and 28 enterococcus (4%). comparison with a randomly chosen control cohort of 80 patients with e. coli bacteriuria revealed several indicators for pseudomonas bacteriuria, including male gender (odds ratio 3.3, 95%ci 1.5-7.2, p < 0.005), presence of a permanent urinary catheter (or 15.4, 95%ci 2.2-140, p < 0.005), past prostatectomy (or 13.4, 95% ci 1.5-315, p < 0.01), hospitalisation in the previous 2 months (or 4.2, 95% ci 1.5-4.9, p < 0.005), and pregnancy (or 2.8, 95%ci 2.1-3.6, p < 0.05). in addition, both enterococcus and pseudomonas, as compared with e. coli, significantly more often indicated asymptomatic bacteriuria in patients with other diagnoses, as opposed to clinically manifest bacteriuria, than isolation of e. coli (or 4.2, 95%ci 1.2-14.4, p < 0.05). conclusions: pseudomonas (5%) and enterococcus (4%) are isolated from a significant minority of urine samples obtained from ed patients with clinically suspected bacterial infection. isolation of these organisms, as compared with e. coli, more often indicates asymptomatic bacteriuria in patients with other infectious disease diagnoses. in addition, several independent clinical indicators for pseudomonas bacteriuria were identified. these data may assist in selecting optimal antibiotic treatment for patients admitted with suspected urinary tract infection. objectives: certain virulence factors (vf), particularly pap fimbriae, are able to trigger production of cytokines, especially through activation of toll-like receptor 4 (tlr-4), and therefore produce inflammation. the aim of this study was to assess the influence of certain vf in the degree of inflammation in febrile urinary tract infections (futi). methods: from 2002 to 2003 adult patients with febrile community acquired futi (81 female with acute pyelonephritis (mean age 50 (sd ¼ 22)) and 36 acute prostatitis (mean age 60 (sd ¼ 16)) caused by escherichia coli were prospectively included. levels of c reactive proteins (crp), white blood cell count (wbcc) and days until apirexia after beginning antibiotic treatment were recorded in all patients and considered as indirect markers of inflammation. genes encoding haemolysin, type 1 fimbriae, pap g fimbriae, cytotoxic necrotising factor, aerobactin and autotransporter toxin were detected by a pcr. additionally expression of type 1 fimbriae and haemolysin were detected by agglutination and growth on blood agar. results: strains carrying pap g fimbriae were involved in futi with higher crp levels than pap g fimbriae negative strains (14.95 vs. 10.51; p ¼ 0.028). the relation between the rest of vf and crp levels did not reach statistical significance. no differences were found regarding the wbcc and the duration of the fever. conclusions: these data indirectly suggest that the degree of inflammation in futi caused by e. coli is associated with the presence of pap g fimbriae, which is coherent with the fact that pap g fimbriae are coreceptors of tlr4. mycology: candida and aspergillosis p986 initiation of an active surveillance programme on yeast-related bloodstream infections in france (aspyrif) an active surveillance program has been implemented in france to prospectively analyse yeast-related blood stream infections. a pilot study was conducted from 1 october 2002 through 30 september 2003 in 23 medical centres in paris and suburbs. for each patient, one isolate of each identified species was sent to the nrcm together with clinical data filled on a standard form. identification was confirmed using phenotyping tests and a pcr assay was performed on all candida albicans isolates to identify c. dubliniensis. antifungal susceptibility testing to amphotericin b, flucytosine, fluconazole, itraconazole, voriconazole and caspofungin was performed according to eu-cast recommendations. the median age of the 282 patients was 57 years [0-94 years], with a male predominance (59%). underlying factors for yeast-related blood stream infections were often multiple for a given patient dominated by recent surgery (69%), central venous catheter (66%), hospitalisation in intensive care unit (53%), malignancy (42%), immunosuppressive therapy (28%), hiv infection (10%), solid organ (6%) or bone marrow (3%) transplantation and prosthetic devices (6%). overall, the mortality rate was high with 43% of deaths within 30 days after the first positive blood culture. candida spp. was the most frequent genus (96%) with c. albicans (49%), c. glabrata (14%), c. parapsilosis (13%) and c. tropicalis (10%) being the most frequent species isolated. other candida were recovered below 3% (c. krusei, c. kefyr, c. lusitaniae). non-candida spp. were trichosporon asahii, t. mucoides, geotrichum capitatum and cryptococcus neoformans. our data show that the percentage of nonalbicans species equal that of c. albicans among the yeasts recovered during fungaemia. the proportion of the four major species differed significantly according to the presence of central venous catheter (p ¼ 0.02). analysis of the antifungal susceptibility testing results revealed that most of the isolates had usual antifungal susceptibility profiles. in conclusion, aspyrif is a powerful tool that should allow us to accurately describe the epidemiology of yeast-related blood stream infec-tions in france without restriction to any underlying disease or species. background: nosocomial candidaemia is associated with significant morbidity and mortality in the critically ill. emergence of fluconazole resistance raises further problems, but the newer antifungal drugs [voriconazole, caspofungin, ambisome and abelcet] offer alternative therapeutic options. they also raise the issue of treatment-associated costs. an 8-year [1996 to november 2003 clinical audit was conducted across two tertiary care hospitals [western infirmary and gartnavel general hospital, glasgow] . the distribution of candida species and fluconazole/itraconazole resistance, with emphasis on high-risk areas was studied. it also addresses the newer antifungal options, cost implications and patient risk-stratification approach. objectives: to evaluate the outcome and complications in patients with candidaemia treated with antifungals. to identify the most common candida species isolated in the vamc patients with candida and evaluate the risk factors and epidemiological data of the patients. methods: all patients admitted in the vamc from august 1995 to august 2002 with blood cultures positive for candida were included in this study. epidemiological data, medical history, risk factors, co-morbid diseases and laboratory results were evaluated in record review. candida species were identified to determine the prevalence of candida species in the vamc. the patients were assigned to three different groups according to the therapeutic regime provided to the patient by the primary physician. outcome and complications including nephrotoxicity, electrolytes disturbances and hepatotoxicity were evaluated in each therapeutic group. statistical analysis was performed using the spss (statistical package or social science). a regression model was used for the analysis of risk factors associated with mortality in patients with candidaemia. results: one hundred and seven patients were randomised in the study. c. tropicalis was the most commonly isolated candida species 45%, followed by c. albicans 31%. mortality rate is high 73%, especially in those patients infected with c. tropicalis and c. glabrata 88% (p ¼ 0.01). the mortality rate increased to 81.4% if no treatment was given (p < 0.0001) and was worse if c. tropicalis was isolated and not treated 92%. the patients treated had a similar mortality rate irrespective of the administered agent, amphotericin (64%), abelcet (60%) and diflucan (60%), but was worse in those patients admitted to an icu, amphotericin (86.6%), abelcet (60%) and diflucan 71.4% (p < 0.0001). response rate in the patients infected with c. albicans was 38.7 vs. 9% in patients with c. tropicalis. nephrotoxicity developed in 54% of patients and no difference was found in those patients treated with amphotericin b vs. abelcet. conclusion: candidaemia has been increasing in frequency. c. tropicalis is the most commonly isolated candida species in our institution. candidaemia has a high mortality rate and is worse if c. tropicalis is isolated and the patient is admitted to an icu and no treatment is given. there is no difference in response rate within the different therapeutic options. nephrotoxicity is higher in patients treated with amphotericin irrespective of the formulations administered. background: invasive candidaemia is a life-threatening complication occurring especially in hospitalised cancer patients due to surgical operation and application of aggravating chemotherapy. candida colonisation, dysfunction of humoral and cellular immune system and prolonged periods of hospitalisation are considered to be the risk factors of invasive candidaemia development. early diagnosis and evaluation of the risk factors are still a major challenge. objectives: the aim of our study was to evaluate the relationship between the rate of candida colonisation, disorders in immune responses (associated with adverse changes in concentration of tnf-alpha, il-12, and myeloperoxidase) and development of invasive candidaemia in hospitalised cancer patients. methods: study group included 78 patients with lung cancer admitted for surgical operation and 31 women with carcinoma ovariorum after the third course of treatment with taxol and cisplatin. patients were examined for fungal colonisation of mucosal membranes with culture methods. presence of candida antigens and dna of the pathogen in the bloodstream was determined with elisa and pcr assay, respectively. cytokine and myeloperoxidase concentration in serum of the patients was specified with elisa commercial kits. results: the study revealed that 43 (39%) lung cancer patients were colonised with candida in nosepharynx before the operation. pneumonia and wound infections were observed in 15 patients of this group, candida albicans was isolated as the only pathogen from three patients colonised previously with candida. in case of patient group with ovariorum carcinoma, colonisation with candida of two or three sites was demonstrated in five (15%) of 31 women. the candida antigen was present in blood in four of them; positive pcr result was found in blood sample collected from one of them. significant relationships between candida colonisation or infection and myeloperoxidase concentration were found (5.9-13.1 vs. 170 ng/ml in healthy persons). conclusions: high rate of candida colonisation and drastic decrease in myeloperoxidase serum concentration in patients with lung and ovariorum cancer are predisposing risk factors for invasive candida infection. detection of candida antigens and dna of the pathogen may improve early diagnosis of the candidosis. p990 evaluation of bact/alert 3d system to diagnose bloodstream infections due to yeasts p992 effect of voriconazole on ergosterol content of s. costa-de-oliveira, c. pina-vaz, e. pinto, a. oliveira, c. tavares, a. gonc¸alves rodrigues porto, p voriconazole (vor) is a new azole antifungal agent with a similar structure to fluconazole (flu). as with other azoles, its primary mechanism of action is through disrupting the normal sterol biosynthetic pathway, leading to a reduction in ergosterol content (1). nevertheless vor is more potent against most candida spp., and shows a wide spectrum of activity. thus candida krusei, which is intrinsically resistant to fluconazole (by unknown mechanism), shows low mic values to vor. this lack of cross-resistance and the fact of being fungicidal to some fungi suggest a distinct mechanism of action. objective: to study the effect of vor on the amount of ergosterol of c. krusei strains, in comparison with flu. methods: the mic to vor was determined according to the nccls protocol m27-a on 24 strains of c. krusei, all resistant to flu (mic ! 64 lg/ml). ergosterol was isolated from c. krusei cells by saponification and the non-saponifiable lipids were extrac-ted with heptane. ergosterol was identified by its spectrophotometric absorbance profile (240-300 nm) (2). a quantification of ergosterol was determined after incubation with and without both azoles at mic and sub-inhibitory concentrations. results: in all the strains, mic to vor ranged between 0.125 and 0.5 ug/ml. all c. krusei have a significant amount of ergosterol, with no significant differences among the strains. after incubation with mic concentrations of vor an 80-100% reduction of the ergosterol content was observed. a similar effect was obtained with fluconazole but only with highest concentrations (64 ug/ ml). conclusion: the vor induces a considerable impairment on the biosynthesis of ergosterol by c. krusei strains. it is much more potent inhibitor of ergosterol biosynthesis than flu. background: mycotic infections of hospitalised patients are emerging as a significant public health issue. numerous studies have shown that candidaemia is associated with a significant attributable mortality and prolonged hospital stay, but only a few reports analyse the incidence of candida spp. in wounds. objective: to analyse the species distribution and antifungal susceptibility of candida infection in wounds in our hospital during a 6-year period (1997-2002) . methods: the in vitro activities of amphotericin b (ab), fluconazole (fz), itraconazole (iz), ketoconazole (kz) and flucytosine were determined by the broth microdilution method following nccls criteria. mics were visually determined after 24 and 48 h incubation at 35 c results: from 1997 to 2002 we processed 18 573 wound samples in our laboratory. of these, 14 060 (75.7%) were positive, 13 413 (95.4%) showed bacterial growth without candida and 647 (4.6%) with candida. the rate of isolation of candida in wounds/year was as follows: 1997 (2.7%), 1998 (3.7%), 1999 (4.8%), 2000 (4.3%), 2001 (6.3%) and 2002 (5.7%). globally, candida albicans was the most frequently isolated species per patient (296; 57.9%), followed by c. parapsilosis (71; 13.9%), c. glabrata (40; 7.8%) and c. tropicalis (37; 7.2%). the trends in species distribution were similar in both the adult and paediatric population. the evolution in the successive years of wounds with more than one species of candida was as follows: 2/59, 8/94, 8/97, 4/92, 16/163 and 16/ 142 . overall, the percentages of resistance of candida spp. isolated were: ab (4.6%), fz (10.2%), iz (17%), kz (12.5%) and fc (2.3%). conclusion: our study shows an increasing presence of candida spp. among the wound isolates in the microbiology laboratory. a high proportion is due to species other than c. albicans and it can be probably attributed to the increase in antibiotic burden in our hospital. infants were performed to estimate disease burden, short-term outcome and microbiological characteristics of causative organisms. methods: prospective enhanced surveillance of invasive fungal infections in vlbw (<1500 g) infants began in february 2003, with cases defined as meeting of one or more of the following diagnostic criteria: (1) culture from a sterile site -csf, blood (peripheral sample), urine (supra-pubic aspirate or in-out catheter sample), bone/joint, peritoneal or pleural space; (2) pathognomonic findings on ophthalmological examination; (3) pathognomonic findings on renal ultrasound examination; and (4) autopsy diagnosis of invasive fungal infection. cases were identified through three separate surveillance schemes: monthly notifications from paediatricians to the british paediatric surveillance unit; continuous reports from microbiology laboratories to the communicable disease surveillance centre (england) and scottish centre for infection and environmental health (scotland). reports from the three systems were reconciled and analysed. rates were calculated using office for national statistics total live birth estimates. results: between february and july, 38 confirmed cases of invasive fungal infection in vlbw infants were reported, 10.02/1000 births of vlbw. median age at diagnosis was 11 days (range 1-126) and birth weight 800 (520-1200) g. thirty-four of the 38 infants were of extremely low birth weight (<1000 g). candida albicans was the most common pathogen, found in 55% of cases, and c. parapsilosis in 23%. organisms were most commonly isolated from blood (73%), followed by urine (23%), csf (8%) and central line tips (53%). just over a third of cases (36%) had received prophylactic antifungal therapy. one case of drug resistance was identified during this period (fluconazole resistance in a non-albicans candida spp.). of the 32 infants for whom outcome data were available, 22 were alive at 37 weeks post-conceptional age. conclusion: preliminary findings from enhanced surveillance suggest an incidence of invasive mycoses in vlbw infants of one in 100. as per adult cases, c. albicans was the most common fungal pathogen involved, although c. parapsilosis was relatively more common than in adults. the majority of cases occurred in extremely low birth weight infants, and mortality was found to be high. methods: surveillance swabs of throat and rectum were taken on admission and twice weekly afterwards. diagnostic samples were obtained on clinical indication. all samples were processed using standard mycological techniques. overgrowth was defined as !3+ or !1 000 000 yeast cells/ml of saliva and/or gram of faeces. carriage index is the ratio of the sum of all semi-quantitative growth densities of positive surveillance swabs divided by the total number of swabs; on a particular sampling day. oral polyenes were started following the identification of the carrier state. results: a total of 1241 children requiring minimally 4 days of ventilation were enrolled in this 4-year observational, prospective study [01/03/99 to 28/02/03]. the median paediatric index of mortality was 0.06 [iqr 0.02-0.13], and the actual mortality was 9.6%. enteral polyenes as part of selective digestive decontamination [sdd] were administered to half of the study population [53%] . the median length of stay was 8 days objective: candida dubliniensis is a newly described pathogenic species, first isolated from hiv-infected patients with oropharyngeal candidiasis. it shares many phenotypic features with c. albicans, including the ability to form germ tubes and chlamydospores. these similarities have caused significant problems in its differentiation from c. albicans in routine clinical microbiology laboratories. this study reports isolation and identification of c. dubliniensis for the first time from kuwait and presents data on antifungal susceptibility profile. methods: over a period of 21 months, 800 germ-tube positive yeasts identified as c. albicans and recovered from different clinical specimens were screened for their ability to grow at 45 c on sabouraud dextrose agar. isolates which failed to grow at 45 c were presumptively identified as c. dubliniensis. the identity of c. dubliniensis isolates was further confirmed by formation of rough colonies and chlamydospores on sunflower seed agar, by vitek 2 system, and by semi-nested pcr using species-specific primers corresponding to unique sequences within the internally transcribed spacer 2 (its2) of c. dubliniensis and by direct sequencing of its2. the antifungal susceptibility testing was performed on rpmi 1640 medium as recommended in nccls, m27a document. results: of the 800 germ tube positive yeast isolates, 27 (3.3%) were identified as c. dubliniensis. they were isolated from sputum (n ¼ 12), vaginal swabs (n ¼ 5), endotracheal secretion (n ¼ 3), throat swabs (n ¼ 2), urine (n ¼ 2) and one each from bronchoalveolar lavage, catheter tip and peritoneal fluid. none of the isolates originated from hiv-positive patients. all the c. dubliniensis isolates were susceptible to amphotericin b, fluconazole, itraconazole and voriconazole. however, 19% of the isolates were resistant to 5-flucytosine (>32 lg/ml) without any known previous exposure. conclusion: identification of c. dubliniensis from 3.3% of the yeast isolates in our study suggests that this species is not uncommon in kuwait. there is a need to carry out a systematic study in high-risk patient groups to know its epidemiologic significance. acknowledgement: the work is supported by kuwait university research grant mpi-118. background: fungaemia remains a severe nosocomial complication and the emergence of non-albicans species is posing new challenges both to clinicians and to microbiologists. objective: to assess the incidence and clinical presentation of c. glabrata fungaemia, its susceptibility and its clinical outcome. methods: from 1987 to 2001, we had 479 episodes of fungaemias and 37 cases corresponded to c. glabrata (7.7%). thirty cases were six cirrhosis patients and 20 miscellaneous). following the eortc/msg criteria, these patients were classified as proven ia (n ¼ 30), probable ia (n ¼ 37), possible ia (n ¼ 2) and 'colonisation' (n ¼ 20). mean saps ii score was 52 with a predicted mortality of 48.6%. overall mortality was 80% (n ¼ 71). mortality of the proven and probable group was 96.7 and 86.5%, respectively. among the 18 patients who survived, 10 just had 'colonisation' with aspergillus. post-mortem examination in the non-haematooncological group was done in 46 out of the 71 patients who died (70%) and 29/46 autopsies (63%) showed hyphael invasion with aspergillus (mainly the lung as target organ). there were five proven cases in patients without compromising host factors according to the eortc/msg definitions (three liver cirrhosis, one pneumonia in a 95-year-old man, one klebsiella sepsis with mof). conclusion: ia is an emerging infectious disease in non-haematooncological icu patients. there seems to be a broad group of patients at risk of ia. ia was diagnosed in patients without characteristics described in the eortc/msg definitions. it seems worthwhile to investigate the validity of the available diagnostic tools in non-haemato-oncological patients at risk for ia in a prospective manner. p1001 epidemiology of invasive aspergillosis in a teaching hospital, france: a 6-year survey (1998-2003) a. cornillet, c. camus, s. nimubona, v. gandemer, p. tattevin, c. belleguic, s. chevrier, c. meunier, c. lebert, m. aupée, b. lelong, c. guiguen, j.-p. gangneux for the aspergillosis study group objectives and methods: the aim of this survey was to characterise a file of patients who developed an invasive aspergillosis (ia) in our institution, their risk factors and management. we analysed retrospectively the cases of ia, which occurred between 1998 and 2001, then prospectively all new cases until the end of 2003. the overall survey covered a 6-year period. cases were classified as suspected, probable or proven ia, using criteria derived from the eortc/msg classification. results and discussion: until 11/2003, out of the 80 cases of ia analysed, nine were histologically proven, 47 were probable ia and 24 were suspected ia. the sex ratio was 1.5 male:one female with a mean age of 53 years (ranging from 4 to 89 years). fifty percent of cases were diagnosed in the intensive care units, and 36% in haematology units (28% in adults and 8% in paediatrics). neutropenia was the major risk factor in 55% of the patients (during haematological malignancies and solid cancers). however, we also noted an increasing number of ia in patients under corticosteroid therapy for cobp, asthma, rheumatoid arthritis, horton and microvascular diseases, in comparison to available data in the literature. other cases occurred in solid organ transplant recipients and only one out of the 80 patients was infected by hiv. prognosis factors will be discussed. regarding biological diagnosis, good sensitivities of the mycologic examination (microscopy + culture) and the galactomannan antigen detection by enzyme immunoassay (platelia aspergillus, biorad) were noted: 75 and 71%, respectively. the sensitivity reached 80% when both tests were combined. pulmonary imagery was less efficient, probably due to the fact that, in our institution, ct scans are performed later than proposed in the literature. during this survey, we observed great modifications in therapeutic approaches. first line treatment progressively switched from deoxycholate amphotericin b (amb) to voriconazole and second line treatments now include lipid formulations of amb and caspofungin acetate. amb deoxycholate and voriconazole were the two drugs used for empirical therapy. the overall mortality was >70%. conclusion: ia remains a major life-threatening infection among immunosuppressed patients, although protective measures such as air filtration significantly reduced its incidence in neutropenic patients. however, this 6-year-survey points out the increasing number of cases in non-neutropenic patients hospitalised in wards without air filtration. this emerging population of patients must be taken into account and imposes to reinforce surveillance for high-risk groups and to rethink our preventive measures. priate chest ct appearance) was the sole basis of the diagnosis in 13 (65%) pts. in the remaining seven (35%) pts, positive elisa accompanied either histopathological or microbiological evidence of ia. five (38%) of these 13 pts were later upgraded to definite ia. nineteen of the 20 pts were assessed for efficacy at the end of cas rx. the favourable response rate was 26% (5/19). pts whose only evidence of ia at cas onset was elisa (and characteristic chest ct findings) had a 38% (5/13) success rate. follow-up elisa data was available in 17 pts. four of five pts with a favourable response to cas had negative elisa by the end of rx. the one other pt with a favourable response had quantitative elisa improvement that was temporally associated with clinical and radiographic response. of the 12 pts with unfavourable responses and follow-up elisa data, 10 had no elisa improvement and two had normalisation of elisa while on cas. conclusions: in this study, the use of elisa did not result in an exaggerated favourable response rate. in general, the elisa was associated with clinical/radiographic response. paradoxical elisa increases in pts clinically/radiographically responding to cas were not noted. best rapd patterns with respect to number, spreading and intensity of the bands, but the highest level of discrimination was achieved by a combination of data generated by both of them. therefore, we emphasise the convenience of using at least two primers for rapd typing. objectives: to gain insight into the molecular epidemiology of staphylococcus aureus at a tertiary hospital. methods: all s. aureus isolates recovered from blood samples over a 1-year period were analysed. demographic, clinical and microbiological data from these patients were collected. antimicrobial susceptibility tests were performed by the wider system and the disk diffusion method; all methicillin-resistant s. aureus (mrsa) isolates underwent confirmatory pcr analysis for the meca gene. molecular characterisation was performed by pulsed-field gel electrophoresis (pfge) following dna extraction and smai digestion. patterns differing by less than seven dna fragments and with a dice coefficient of correlation >80% were considered a common bacterial type while subtypes included isolates with indistinguishable pfge patterns. univariate and multivariate analyses were performed with epi-info 2002 and spss 10.0 softwares. results: one hundred and sixty-two episodes of s. aureus bacteraemia, whether methicillin-resistant or methicillin-susceptible (mssa), were nosocomial in origin (77.2%) or were cases associated with the healthcare system (15.4%). only a total of 12 cases of bacteraemia (7.4%), one mrsa and 11 mssa, were strictly considered to be community-acquired. thirty-five unique s. aureus pfge types were identified among 154 dna macrorestriction patterns. within the isolates of mrsa, four major genotypes were identified, with 36 isolates (85.7%) represented by a single pfge type. in contrast, the 112 isolates of mssa comprised 31 different pfge types, of which 16 represented more than one isolate. three pfge types were found to represent 42% of all mssa isolates. these common strains were found with equal frequency among adults and paediatric patients, and were evenly distributed between nosocomial and community-acquired cases. conclusion: our results provide indirect evidence of ongoing transmission of mrsa and mssa in our hospital. in the case of mrsa, the spread is predominantly due to a single clone, with transmission favoured by increased length of stay in hospital and the administration of beta-lactam antibiotics. in contrast, the spread of mssa bacteraemia in this population is associated with multiple, genetically distinct strains. (2) to use a real-time pcr to detect the presence of meca gene in s. aureus clinical isolates. methods: seventy-three strains obtained from clinical specimens were identified by microscan (dade behring) and coagulase test (28 s. aureus, 15 s. epidermidis and 30 other coagulase negative staphylococci). in vitro susceptibility was determined by microscan and disc diffusion. a total of 39 s. aureus strains were classified according to methicillin susceptibility: 21 resistant and 18 susceptible to methicillin. dna was obtained by incubation at 100 c in lysis buffer. real-time pcr was performed in a lightcycler instrument (roche diagnostics, spain) using two commercially available kits: (1) lightcycler staphylococcus kit mgrade: pcr was positive in all staphylococci and they were differentiated according to melting temperature (62.1 ae 2 c for s. aureus, and 43.4-59 c for cns) and (2) lightcycler mrsa detection kit: pcr was positive in meca positive s. aureus. an internal control excludes the presence of inhibition. once the dna was extracted the whole process takes 1 h. results: twenty-seven out of 28 s. aureus strains were clearly identified by real-time pcr due to the melting temperature (range from 60.2 to 63.4 c). one s. aureus showed melting temperature of 59.8 c. all s. epidermidis strains showed melting temperature from 50.1 to 53.4 c. s. lugdunensis showed melting temperature of 58.6, 54.6 and 53.1 c. other cns showed melting temperature from 51 to 57.1 c. twenty-five out of 39 (64.1%) strains tested were meca positive by using this lightcycler mrsa kit and realtime pcr. among the 25 meca positive, 21 were fenotipically methicillin resistant (84%) whilst four were methicillin susceptible (16%). all 14 meca negative strains were susceptible to methicillin by phenotypic methods. conclusions: real-time pcr (lightcycler) seems to be an accurate method to identify s. aureus and differentiate it from different cns and to detect resistance to methicillin in s. aureus. both reactions could be done simultaneously and the whole process takes less than 2 h (dna extraction plus real-time pcr). objectives: surveillance of methicillin resistant staphylococcus aureus (mrsa) in canada began in 1995. from this surveillance, six epidemic strains of mrsa have been identified and named cmrsa1-6. in order to better understand the relatedness of these strains, as well as their genetic content, we have used microarrays to compare their genomes to that of the fully characterised genome of the mrsa strain col. methods: genomic dna from representatives of the six epidemic strains, as well as col was fragmented and labelled using random primers with cy5 or cy3 labelled dctp. col and each of the epidemic strains (labelled with different dyes) were hybridised to arrays containing pcr products or 70 bp oligomers representing 2740 of the open reading frames (orfs) in the col genome. data were processed with the arraypro software package, positive/ negative cut-off values were determined using genomotyping analysis by charles kim and then analysed using the genemaths program. macrorestriction digest patters were generated using smai. results: results indicate that all canadian epidemic strains have six common regions of deletion, a portion of the type i sccmec region, bacteriophage l54a, and four smaller areas composed of two to four orfs. the only gene of known function in these smaller areas was the staphylococcal enterotoxin b. apart from these major deletions, many sporadic, single deletions are seen throughout the strains. larger regions of deletion that are not present in all strains also occur. the only obvious orf duplication is of is431 in cmrsa3, 5 and 6, which is found in multiple copies in the typeiii sccmec region in these strains. macrorestriction digest data were used to approximate the sizes of the cmrsa genomes. cmrsa4 shows the smallest genome ($1862 kb), and the least genetic content in common with col (79%). though cmrsa1 and 2 appear to have larger genomes ($2378 and $2706 kb, respectively), they show fewer orfs in common with col than other strains (81 and 86%, respectively), suggesting a substantial portion of the genome may be novel. conclusions: this is the first study of epidemic mrsa using the comparative genomic hybridisation approach. while the cmrsa strains show a high degree of relatedness to col, there are considerable differences in genetic content. this study also indicates that there may be genetic content which is unaccounted for in the col genome. other studies are being devised to identify and characterise the novel genetic content. objectives: in finland, the annual number of mrsa isolates notified to the national infectious disease register (nidr) has constantly increased, especially outside helsinki metropolitan area. molecular typing has revealed numerous outbreak strains of mrsa, and some of them have been associated with community acquisition. we analysed strain types identified by pulsed-field gel electrophoresis (pfge) of mrsa isolates sent to the national reference laboratory (nrl) during 1997-2003. methods: all isolates of mrsa notified by the finnish clinical microbiology laboratories were sent to nrl for further verification and characterisation, including pfge analysis. pfge profiles differing by fewer than six bands were interpreted as identical or closely related. one isolate per person were included in the analysis. strain types were categorised as sporadic (strain type only found from one person), domestic outbreak or international epidemic (strain type found from more than one person) as well as community-acquired (strain type associated with community acquisition in our previous study). the proportions of mrsa isolates included in each category were assessed. results: a total of 2496 mrsa isolates were studied. the number of mrsa isolates increased from 138 in 1997 to 804 in 2003. pfge identified more than 200 different strain types. of the mrsa isolates, 9% were sporadic, 66% domestic outbreak and 25% international epidemic. one strain type disappeared compared with years before 1997, and 11 new strain types appeared during 1997-2003. the proportion of sporadic strains varied between 3 and 18% during the study period. of the international epidemic strains, bel ec-3 increased from <1% in 1997 to 27% in 2003, mainly outside helsinki metropolitan area. uk emrsa-16 decreased from 13% in 1997 to <1% in 2003, and helsinki i (a representative of mlst st-5 strains) from 18 to 6%, respectively. uk emrsa-15 varied between 1 and 6%. the three main strains with community-acquisition fluctuated during the study period (range, 10-30%). conclusions: intensive national surveillance with molecular typing revealed that the predominant mrsa strains change over time. the internationally spread epidemic strains of mrsa have also been found in finland. however, most of them show a decreasing trend or have disappeared. these results encourage us to continue aggressive interventions with each new mrsa case. introduction: according to recent studies, community-acquired (ca)-methicillin-resistant staphylococcus aureus (mrsa) strains often contain a type iv sccmec cassette and panton-valentine leukocidin (pvl) locus. it has also been shown that certain multilocus sequence types (st) seem to be connected to ca-mrsa strains from different continents. materials and methods: we studied 108 finnish ca-mrsa strains for their genotype by pulsed-field gel electrophoresis (pfge) and multilocus sequence typing (mlst), the methicillin resistance genes by sccmec pcr and the presence of pvl gene locus by pcr. mrsa was defined as community acquired if the mrsa specimen was obtained outside hospital settings or within 2 days of hospital admission from a person who had not been hospitalised within 2 years before the date of mrsa isolation. to confirm the functionality of the pvl-pcr reaction and the quality of the dna, nuc gene was amplified at the same time. results: the majority of ca-mrsa strains studied (91/108, 84%) possessed sccmec cassette type iv but only 13 (12%) were pvl positive. all but two pvl positive strains contained sccmec type iv. one strain had sccmec cassette iii subtype, and for one strain the type was not determined. the pvl positive strains were mostly (9/13, 69%) of multilocus st80. the four remaining pvl-positive strains were of st1 (two strains) and st8 and 96. the sequence types correlated well with the pfge results: all strains with st80 were analysed as pfge profile hkiviii and strains with st1 as pfge profile nurmes. st96 (sccmec cassette -iii subtype) and st8 (sccmec not determined) strains were considered as sporadic. the 95 pvl negative ca-mrsa strains belonged to 15 different shared and four sporadic pfge profile types. the mlst analysis of pvl negative strains is currently underway. conclusions: most of the finnish ca-mrsa strains have sccmec cassette type iv but only a minority contain pvl gene locus, which is in contrast to previous reports. majority of the pvl gene positive strains possessed st80. in spite of the strict definition for community-acquisition we used, majority (88%) of finnish ca-mrsa were pvl negative and showed heterogeneous pfge profiles. objectives: methicillin-resistant staphylococcus aureus (mrsa) is among the major pathogens. the most common methods currently used for identifying methicillin (oxacillin) resistance in many clinical laboratories are susceptibility tests. the performance of these tests has been erratic because the expression of resistance is variable and commonly heterogeneous within strains. methods: a retrospective laboratory-based study was carried out with clinical isolates of s. aureus in a tertiary care providing uni-versity hospital in thrace, greece. methicillin (oxacillin) susceptibility of 118 s. aureus isolates, which were recovered from various clinical specimens (blood cultures, tracheal aspirates, wound swabs and central venous catheters) were studied by four different methods: (1) agar screening test [mh-oxacillin (6 lg/ml) agar supplemented with 4% nacl], (2) susceptibility determination by the vitek 2 (biomerieux), (3) mic was determined by e-test (ab biodisk), (4) mec-a gene detection by pcr, using specific primers. the strains were evaluated by using the presence of meca gene detected by pcr, as definitive criteria for mrsa and non-mrsa. the susceptibility tests were carried out as recommended by the nccls. results: among all the isolates, 44 were identified as meca-positive and the remaining 74 as meca-negative. the percentages of correct results (% sensitivity/% specificity) were: oxacillin agar screen, 98/100; e-test, 93/100; and vitek-2, 100/89. ten isolates, negative for the mec-a gene by pcr, were recognised by at least one phenotyping method as oxacillin resistant. only one strain meca-positive was incorrectly identified as oxacillin-negative by the oxacillin agar screen. conclusions: as shown in this and other studies, no phenotypic method is completely reliable for the detection of oxacillin resistance in s. aureus. the specificity was generally high, especially with the agar screening and e-test methods, while the sensitivity varied between the different methods. in particular, the oxacillin screen test is the most accurate test and approaches the accuracy of pcr. although, the presence of the meca gene, as detected by pcr, still remains the 'gold standard', agar-screening test should be considered in association with other susceptibility methods to maximise the ability to correctly detect oxacillin-susceptibility in s. aureus. p1016 amplification of dna fragments surrounding rare restriction sites (adsrrs-fingerprinting) for typing staphylococcus aureus isolated from patients with recurrent furunculosis w. baranska-rybak, r. nowicki, e. scieburako, j. kur, e. arlukowicz, a. samet gdansk, pl introduction: in the present study we report data on phenotypic and genotypic characteristics of staphylococcus aureus strains. the aim of our research was to identify sa genotypes in the patients suffering from recurrent furunculosis. materials: we obtained 70 isolates from 45 patients with recurrent furunculosis. purulent discharge from furuncle, nasal and throat swabs were taken for culture. methods: the identity strain of sa was confirmed by novel dna-typing technique. amplification of dna fragments surrounding rare restriction sites (adsrrs-fingerprinting ) is an effective and rapid method for molecular typing of isolates of bacteria. this method is based on suppression of pcr (polymerase chain reaction) reaction. sa dna was digested with two restriction enzymes: bamhi (10 u/ml) (sigma) and xbai (10 u/ml) (sigma). cohesive ends of dna were ligated with adapters (xbai short adapter and bamhi long adapter) and amplified. pcr products were electrophoresed on polyacrylamide gels, stained by ethidium bromide and photographed under uv. results: adsrrs-fingerprinting of 70 sa isolates revealed 10 unique patterns. in most cases the strains isolated from the same patient (nose, throat and furuncle) gave identical pattern. the reverse situation was found in five patients. conclusions: (1) in most cases we confirmed the identity between nasal/throat and furuncle sa isolates. (2) we found no specific genotype, which is responsible for recurrent furunculosis. (3) adsrrs-fingerprinting seems to be a very useful method for epidemiological studies of sa. objectives: rapid and efficient epidemiologic typing systems may be useful to investigate dissemination of the lineages of staphylococcus aureus. we have compared the usefulness of well-established methods to those of newly developed rapid typing methods as epidemiological tools. methods: a total of 59 s. aureus isolates were analysed by pulsedfield gel electrophoresis (pfge), multilocus sequence typing (mlst), repetitive-element pcr technique (rep-pcr) based on the presence of dna sequence that are homologous to mp3 repeat in mycoplasma pneumoniae, multiple-locus variable-number tandem repeat analysis (mlva), and multiplex pcr-based method with primer mix of the spa gene, the coa gene, and the hypervariable region adjacent to meca gene. results: fifty-nine s. aureus isolates clustered by pfge in 50 different genotypes. mlva, which had the highest compatibility with pfge of all testing methods in this study, clustered into 38 different genotypes, multiplex pcr-based method clustered into 23, and rep-pcr clustered into 16 different genotypes. rep-pcr differentiated s. aureus isolates in a way similar to mlst that clustered these isolates in 19 groups. conclusion: although pfge is still the gold standard, owing to its high discriminatory power amongst molecular typing methods, genotyping methods based on pcr may be useful in respect of speed and ease of performance. mlva, multiplex pcr-based methodology and rep-pcr are rapid, reproducible, and easy to perform. however, mlva and multiplex pcr-based method generate more unambiguous results than those of rep-pcr. objectives: to determine whether the variable visual outcome in endophthalmitis secondary to coagulase-negative staphylococci spp. are due to different strains causing intraocular infection, with a possible difference in virulence of each strain or resistance to the antibiotics given. methods: twenty-eight intraocular samples infected with coagulase-negative staphylococci spp. were analysed using both biotyping and pulsed-field gel electrophoresis for strain identification. the results were correlated with the visual outcome after 6 months post-treatment. results: four different strains of coagulase-negative staphylococci spp. were found to cause endophthalmitis; s. epidermidis, s. haemolyticus, s. equorum and s. warneri. twenty-one out of the 28 isolates were identified as s. epidermidis and the others were grouped as non-s. epidermidis for correlation with the clinical data. comparing the s. epidermidis with the non-s. epidermidis infected cases, it was found that the mean visual gain was significantly better for the non-s. epidermidis infected cases [(mean visual gain of 38.1 vs. 83.3 logmar letters, respectively) (p ¼ 0.029)].the visual outcome was significantly worse for patients infected with s. epidermidis and antibiotic resistance was more common among these isolates although all were sensitive to at least one of the three/four antibiotics given. comparing the non-s. epidermidis infected cases to the s. epidermidis infected cases that were sensitive to all four antibiotics used, the visual outcome was still significantly better in the non-s. epidermidis group [mean visual gain 83.3 vs. 25.75 logmar letters, respectively) (p ¼ 0.022)]. use of arbitrarily primed pcr to study salmonella ecology in turkey production environment detection of salmonella serovars from clinical samples by enrichment broth cultivation -pcr procedure p979 aetiology and resistance of community urinary tract infections in são paulo, brazil: a three-year survey with 27 437 positive cultures 8%) positive cultures were analysed in this survey. chi-square test for trend (altman, 1999) was performed to evaluate the resistance prevalence ordering in the years surveyed (p < 0.05 was considered significant). results: among the 27 437 positive cultures, 88.8% were from female and 11.2% from male patients. among the positive cultures 89.3% presented growth of enterobacteriaceae followed by 6.7% of gram-positive cocci conclusions: an important difference in the resistance pattern was observed among pathogens and age groups. the difference in age groups suggests the possibility of selective pressure due to previous antimicrobial use in the community setting. ciprofloxacin could be used for empiric therapy in community uti. however, its apparent ascending resistance should raise awareness as to possible usage restriction in this setting. surveillance studies are useful for guiding therapy and helping curbing resistance. p980 resistance of escherichia coli isolates from pregnant and non-pregnant women with community-acquired urinary tract methods: one hundred and forty-four non-pregnant and 117 pregnant women with signs of upper or lower communityacquired uncomplicated utis were enrolled in two multicentre prospective epidemiological studies (eight medical centres), utiap-2002 and arimb, respectively. the strains isolated from the patients who had significant bacteriuria (>105 cfu/ml) were included in the microbiological analysis. the mics of antibiotics (ampicillin -amp, amoxicillin-clavulanate -amx-clv, cefuroxime -cfr, cefotaxime -cft, gentamicin -gnt, co-trimoxazole -ctz, nitrofurantoin -ntf, fosfomycin -fsf) were determined by the agar dilution, as described in the nccls (2003) guidelines. quality control was performed using reference strains including e. coli atcc 25922, e. coli atcc 35218. results: resistance rates of e. coli from pregnant and non-pregnant women with ca-uti in russia are shown in figure. there are some statistically significant differences in antimicrobial resistance between studied groups. ampicillin resistance was higher among uti isolates of e. coli in non-pregnant women (45.8%) than in pregnant women (31.6%), p < 0.05 (chi-square statistic) methods: consecutive patients with presumed uti were included during 14 days if they were older than 15 years and had a positive urine dipstick. subsequently, urine culture (uc) was prescribed and patients classified according to nine uti categories. centres were also required to notify all visits motivated by infectious diseases (id) during the study period. results: of 109 potential participants, 78 included 1054 uti period, prevalence of id is estimated at 13.4% of nontrauma visits and prevalence of uti at 15.9% of all id. the main uti categories were acute cystitis (ac ¼ 43.3%), acute pyelonephritis (ap ¼ 38.0%), bacterial prostatitis (bp ¼ 9.0%). mean age of patients was 46.0 ae 23.8 years and sex ratio f in 71%. however, both differ significantly according to uti category all bc received in the microbiology service were included in our study. all the specimens were performed with bact/alert 3d (biomerieux) initially during 5 days or 30 days in special cases related to the detection time in aerobic bottles, 36.62% gave a positive result in the first 12 h of incubation (average 6.8 h) cumulative percentage of 50% at 24 h. in the second day, 80.28% were positive. in anaerobic bottles 50.68% gave a positive result in the first 12 h of incubation (average 7.8 h) cumulative percentage of 67.11% at 24 h. in the second day 93.14% were positive. candida albicans was isolated in 47.37% cases methods: from 1/03/2003 to 31/11/2003 we studied all yeasts considered pathogens from all body sites, from paediatric pts in all in-hospital locations. isolation and yeasts species identification were carried out by conventional methods. on isolates, flu and vor susceptibilities were assessed by the nccls m44-p method, with disks tested in mueller-hinton medium with glucose and methylen blue, 0.5 macfarland inoculum. all susceptibility test results were read by biomic plate reader system (giles scientific). c. albicans (ca) atcc 90028 was included. nccls flu breakpoints (mcg/ml) were s < 8, s-dd 16-32, r > 64 with corresponding zone interpretative criteria (mm) s > 19, s-dd 15-18, r < 14. breakpoints for vor have not yet been established. results: in the study period we recovered 65 ca, 21 c. parapsilosis (cp), 16 c. tropicalis (ct), two c. krusei, two c. glabrata (cg), two c. lusitaniae (cl) and one tricosporon beigelii. species were isolated: 20% from urinary tract, 25%, upper respiratory tract, 19% miscellaneous fluids, 13% lower respiratory tract, 10% blood, 9% cvc, 4% various. patients with yeasts infections were hospitalised: 49% in picu/nicu, 14% haematology-oncology, 11% surgery, 6% infectious diseases, 6% nephrology, 5% pneumology, 5% medicine, 2% orthopedics, 2% dermathology. distribution of bloodstream isolates were: four cp, three ct, one ca and one cl. seventy percent of cp strains were recovered from picu/nicu pts. the average zone diameter (mm) -mic50/mic90 (mcg/ml) (agar disk gradients) were: ca flu 34 flu with vor mics >48 mcg/ml, one ct was flu sdd, one gg was r to flu and inhibited with 1.1 mcg/ml of vor. conclusions: our results show that ca is still the predominant species recovered from paediatric pts; cp and ct appear to be recovered with increased frequency in serious infections of critically ill pts p993 trends in species distribution and antifungal susceptibility in candida wound infections: an overview of a 6-year period when compared with c. albicans, patients with c. glabrata fungaemia were older (58 vs. 42), had received more previous antifungals (37 vs. 10%, p ¼ 0.01) and antimicrobial agents (97 vs. 73%, p ¼ 0.01), had more indwelling bladder catheters (90 vs. 50%, p < 0.001) and had more septic metastasis (23 vs. 6%, p ¼ 0.07). iv catheters were more commonly withdrawn in patients with c. glabrata fungaemia (60 vs. 33%, p ¼ 0.03), whereas these patients received fewer antifungals (57 vs. 70%, ns). mic90 of c. glabrata were fluconazole (flu) 16 mg/l, itraconazole 1 mg/l, amphotericin b (amb) 1 mg/l and voriconazole 0.5 mg/l. surprisingly, flu was more frequently selected to treat patients with c. glabrata (57 vs. 24%). mortality was similar (50 vs. 53%). six of the 10 patients treated with flu died, as well as four of the seven treated with amb. two patients had persistent fungaemia despite catheter withdrawal and flu therapy cs p1002 invasive aspergillosis in patients with copd of patients with copd and aspergillus spp. in respiratory samples to determine risk factors and outcome. results: we identified 219 patients with copd and aspergillus spp. in respiratory samples. median age was 72 ae 9.3 years. eighty-three percent were men. forty-one patients had criteria for 'probable' ifi none of 107 cases had criteria to suspect an ifi, however, nine were treated and all but one died. the remainder were colonisations. conclusions: a progressive increase of copd patients with aspergillus spp. has been observed but frequently, this is a colonisation. however, we observed that patients in 'probable' category have a high rate (17%) of 'proven' ifi, similar to other known risk groups. we think that these categories could help in clinical practice and to identify homogeneous groups for clinical research in diagnostic methods and therapeutic interventions p1003 evaluation of serum galactomannan elisa during caspofungin therapy: results from the caspofungin salvage invasive aspergillosis study a sandwich elisa assay, which detects circulating aspergillus galactomannan antigen using a rat monoclonal antibody has recently been licensed (plateliaâ, biorad). yet, animal models of ia have shown that treatment (rx) with an echinocandin may result in a paradoxical increase in antigenemia despite clinical/radiographic improvement. concern also remains that using elisa as the sole means of ia diagnosis may result in exaggerated favourable outcomes. to address these concerns, we reviewed the elisa experience from the caspofungin (cas) salvage invasive aspergillosis (ia) study. methods: patients (pts) with proven/probable ia were eligible for enrolment. probable ia was limited to pulmonary sites. probable pulmonary ia could be diagnosed serologically provided the pt had an appropriate chest ct appearance (halo sign, air-crescent sign) and positive elisa on more than two consecutive tests. all pts were refractory (>7 days) or intolerant of prior antifungal rx. cas, with doses ranging from 50-100 mg/day, was administered as monorx. efficacy was assessed at the end of cas rx. favourable responses were limited to complete or partial responses. results: of the 127 pts enrolled, 20 (16%) had consecutively positive serum elisa at the onset of cas underlying diseases were: lymphoid: 55%; myeloid: 40%; non-malignant: 5%. clinical efficiency of the test was tested at three different cut-off values 1.5, 1.0 and 0.7. results: results are summarised in the table. the overall incidence of invasive aspergillosis (ia) was 6.4% (43/667 admissions). following eortc definition criteria, the repartition was: two definite ia, 16 probable ia and 25 possible ia. the definition of 'probable' ia was substantiated by positive gm antigen tests (eight cases); both by microbiological (positive cultures) and positive gm antigen tests (four cases) or only by microbiological criteria (four cases). gm antigen was detected at all different cut-off values in 30 cases corresponding to: 1/2 definite ia, 12/16 probable ia. results were considered as false-positives in 17 patients: four cases without clinical context; 15 cases with a negative chest ct-scan conclusion: detection of circulating gm antigen may be helpful for the diagnosis of ia, particularly in the absence of microbiological data, but a substantiated number of false-positive results do occur among patients undergoing antibiotic therapy with pipera/ tazobactam or amoxi/clavulanate. considering different cut-off values did not improve the sensitivity or the specificity of the assay a results were reported as the number of isolates of each species per plate of the pair. results: a total of 332 pairs of samples were evaluated. of these, 42 showed growth of mucor spp. (40 in sd and two in czapeck) and could not be studied for aspergillus. of the remaining 290 pairs, 157 pairs (54.1%) were positive for aspergillus spp a. fumigatus was the most frequently isolated species, 98 pairs (33.8%) were positive [28 (28.6%) on both plates, 39 (39.8%) only in sd and 31 (31.6%) only in czapeck conclusions: our data supports the recommendation that both media (czapeck and sd) should be used for correct air sampling antifungal combination of caspofungin with flucytosine has been shown to be additive to synergistic in vitro against aspergillus fumigatus. the aim of the present study was to evaluate the interaction between these two drugs in vivo in an animal model of disseminated aspergillosis. methods: for in vivo experiments survival rates of mice treated with the combination of caspofungin at 0.25 mg/kg/day with flucytosine at 500 and 1000 mg/ kg/day were 79 and 86%, respectively. mice treated with caspofungin at 0.5 mg/kg/day combined with flucytosine at 500 and 1000 mg/kg/day had a 92 and 79% survival the study was performed on 57 strains of enterococci all from patients with severe underlying diseases. strains were isolated from urine (37.9%), blood cultures (18.9%), pus (5.4%), peritoneal fluid (5.4%), intravenous catheter (21.6%), infection of the drainage site (10.8%). identification to the species level was performed by vitek 2 (bio-merieux, france). antibiotic susceptibility testing was done by kirby-bauer and mic by e-test and vitek 2. the sandwich hybridisation method was performed in all strains using the commercially available evigenetm vre detection kit (statens serum institute), for the presence of vana and vanb genes.results: from the stains tested, 37 were vancomycin and teicoplanin resistant (vana phenotype) and 20 susceptible to these antibiotics, as determined by kirby-bauer and mics by vitek 2 and e-test methods. of them, 10 were e. faecalis, 22 e. faecium, three e. casseliflavus and two e. hirae. all the vres strains, which were suggesting the presence of vana phenotype by kirby-bauer and mic, were identified to be vana positive by the sandwich hybridisation method. the 20 susceptible strains were negative for the detection of the genes vana and vanb. conclusions: identification of vre to the species level and knowledge of the type and the profile of resistance is critical for infection control purposes in the hospital environment. the sandwich hybridisation is a rapid (3.5 h) and easy to use commercially available molecular method to detect the vana and vanb genes, while the phenotypic resistance determination requires incubation for at least 24 h and other molecular methods require specific instruments and experienced technicians. the sensitivity and specificity of the method is 100%.p948 evaluation of the evigene tm vre detection kit for detecting of enterococci including vancomycin resistance genes a. kilic, m. baysallar, g. bahar, a. kucukkaraaslan, l. doganci ankara, tr objectives: evaluating the correlation of the evigenetm vre detection kit using pcr, which is the golden standard for gene detection and correlating the minimum inhibitory concentration (mic) for vancomycin and teicoplanin are the aim of this study. methods: the vancomycin-resistant enterococci (vre) detection kit is based on microwell plates where to dna probes specific for the bacterial targets dna are bound. test wells include: a positive (16s rrna) and a negative control, a vana microwell and a vanb microwell. the pcr detects the vana, vanb, and vanc-2 genes. the mic determination was performed by e-test according to the nccls guidelines. results: we tested a total of 64 diverse vancomycin resistant enterococci: enterococcus casseliflavus (n ¼ 50) and enterococcus faecium (n ¼ 14). all strains were vana positive (od: all strains >1.192). all results obtained with the vre kit were confirmed by the pcr. the mic determination correlated with the pcr and kit results for all vana positive strains with high mic for vancomycin. conclusion: as a result, the evigene vre detection kit can clearly distinguish vre with the vana and vanb genotypes among a large collection of enterococci and with the same specificity as pcr.p949 development of antibiotic resistance in enterobacteria s.d. nyberg, a. hakanen, m. ö sterblad, p. huovinen, c. edlund, j. jalava turku, fin; stockholm, s objectives: the main objective is to get a better knowledge of the human microflora in gastro-intestinal organ by following variations among intestinal enterobacteria in four healthy subjects receiving oral clindamycin. the microflora in the chosen subjects will be monitored for a 2-year period. the presence and stability of specific resistance genes will be studied in samples collected serially from selected antibiotic exposed subjects. blatem and blashv that code for an extended spectrum beta-lactamase in enterobacteriaceae will be studied. the study will be done by using identification, susceptibility testing, pcr and molecular fingerprinting methods. methods: serially collected faecal samples from four healthy subjects who had received clindamycin perorally for 7 days were cultured and screened for enterobacteriaceae. sampling was performed pretreatment, day 7, 3 weeks, 3, 6, 9, 12 and 18 months after clindamycin administration. between 20 and 50 colonies of suspected enterobacteriaceae were picked from each sample. biochemical identification of the bacterial isolates was done by oxidase, indole production and activity of beta-glucoronidase. mics were determined according to nccls by standard agar dilution method on mü ller-hinton ii medium. the following antimicrobials were tested: ampicillin, cephalothin, cefuroxime, piperacillin/ tazobactam, amoxicillin-clavulanic acid, ceftazidime, cefotaxime, imipenem, aztreonam, gentamicin, streptomycin, chloramphenicol, tetracycline, nalidixic acid, trimethoprim, sulfamethoxazole and ciprofloxacin. results: a total of 521 isolates were identified as oxidase negative, gram-negative rods and thus belonged to the enterobacteriaceae. the isolates were then screened for indole and betaglucoronidase activity. these results showed that 81% of all strains were e. coli. of all strains, 60% were resistant to ampicillin, 46% against sulfamethoxazole, 11.9% against cephalothin and 7.7% against nalidixic acid. the variation of antibiotic resistance between subjects is broad. conclusion: enterobacteriaceae are naturally resistant to clindamycin. however, after clindamycin treatment alterations in the susceptibility to other antimicrobial agents still occur in the microflora. additional research needs to be done to clarify if these alterations in antibiotic resistance are caused by variation of strains/species or exchange of resistant elements.p950 prevalence and implication of the cfia and cpha genes in imipenem resistance among bacteroides spp.m. theron, m.n. janse van rensburg, c. roussouw bloemfontein, za objectives: bacteroides is a major cause of intra-abdominal and female genital tract infections as well as subcutaneous abscesses. beta-lactam agents and carbapenems are currently used in monotherapy against anaerobic infections. the study was done to: (1) investigate the susceptibility of bacteroides strains isolated from bloemfontein academic hospitals; (2) compare results with a previous study; (3) determine the prevalence of carbapenemases/ metallo-beta-lactamases in bacteroides spp. methods: fifty-one bacteroides spp. strains were isolated from patients in the universitas and pelonomi hospitals in bloemfontein. mics of 12 antimicrobial agents were determined by the nccls agar dilution method. a bioassay was used to screen for carbapenemase or metallo-beta-lactamase production. pcr amplification was performed for the detection of cfia and cpha genes. plasmids were extracted using a high pure plasmid isolation kit. results: susceptibility levels were relatively high for imipenem (95%), meropenem (90%) and metronidazole (88%). comparing the results with a previous study (isolates from 1996/1997), showed a reduction in susceptibility to imipenem (100-95%), meropenem (100-90%) and metronidazole (100-88%). the bioassay results gave no indication of the presence of significant concentrations of a carbapenemase or metallo-beta-lactamase. pcr amplification showed the cfia gene (747 bp) in 4/18 strains (imipenem mic 1 to >128 lg/ml) and the cpha gene (769 bp) in 3/18 of the isolates (imipenem mic 1-4 lg/ml). no plasmids were detected. conclusions: although >90% of the isolates were susceptible to the carbapenems, it is evident that resistance has increased over the last decade. fortunately the production of metallo-beta-lactamases has been found to give rise to mics that only range from 2 to 4 lg/ml. this study supports these findings with the exception of one isolate with a mic >128 lg/ml. demonstration of the cfia p973 application of molecular biological techniques to the study of alterations in hamster gut microflora and assessment of treatment with saccharomyces boulardii l. coroler, g. philippe-taine, e. bayart, t. cécile, j.-m. gillardin, h. goïot compie`gne, f objectives: studies of the intestinal microbial ecosystem by classical culture techniques suggest that only 30% of the microflora can be cultured. pcr procedures based on 16s rrna gene specific for bacteria were developed to detect bacterial populations in hamster faeces. methods: a total of 30 populations of bacteria were characterised by their genomic dna sequences and targeted by pcr probes: actinomyces group, bacteroides distasonis, bacteroides fragilis, bifidobacterium group, b. adolescentis, b. angulatum, b. catenulatum, b. infantis, b. longum, clostridium group, c. clostridiiforme, c. coccoides, c. difficile, c. leptum, c. perfringens, fusobacterium prausnitzii, lactobacillus group, peptosteptococcus productus, propionibacterium group, pseudomonas aeruginosa, ruminococcus obeum, citrobacter group, c. freundii, escherichia group, enterobacteria group, enterobacter cloacae, morganella morganii, proteus mirabilis, staphylococcus group, salmonella group. results: sensitivity was measured by extraction of total genomic dna and pcr amplification and a significant detection level of 10 3 bacteria/faecal sample was obtained. qualitative variations of bacteria population were observed during the first 2 weeks of acclimatisation, suggesting a stabilisation period for hamster microflora in new environmental conditions. after oral antibiotherapy, with one dose of 30 mg/kg amoxicillin-clavulanic acid, some groups were eradicated from hamster faeces: propionibacterium, staphylococcus and c. leptum, c. clostridiiforme. as reported in the literature, no antibiotic effect was observed on levels of dominant faecal groups: bifidobacterium, peptostreptococcus. antibioticassociated perturbations are linked with the disruption of the normal intestinal flora leading to a colonisation of pathogen bacteria species. in order to understand the role of saccharomyces boulardii (s.b.) in prevention of antibiotic-associated diarrhoea, 4 â 10 10 cfu/kg/day of s.b. were administered to hamsters during oral antibiotic treatment. the results showed that populations that were eradicated by antibiotic administration remained expressed and stabilised with concomitant s.b. treatment, suggesting an effective protection by s.b. on the intestinal flora. conclusions: these pcr results should be used to quantify the intestinal microflora by dna microarray analysis. objectives: the acinetobacter calcoaceticus-acinetobacter baumannii complex (acb complex) includes a. calcoaceticus (genospecies 1), a. baumannii (genospecies 2), unnamed genospecies 3 and 13tu. these species are difficult to differentiate by phenotype. in this study, the feasibility of using sequences of the 16s-23s rdna spacer region (its) for identification of the acb complex was evaluated. methods: the bacteria-specific universal primers 13 bf (gtgaa tacgt tcccg ggcct) and 6r (gggtt ycccc rttcr gaaat) (y ¼ c or t, and r ¼ a or g) were used to amplify a dna fragment that encompassed a small portion of the 16s rdna region, the its, and a small portion of the 23s rdna region. the its regions from 108 reference strains (42 species) of nonfermenters including strains of acb complex were amplified by pcr and sequenced; the sequence data in combination with those available in genbank were used to construct an its sequence database for the identification of acb complex. for reference strains of each species of the acb complex, the sequence similarities of the its regions were obtained by comparing their its sequences with that of the type strain of the same species. the database was used to test 82 clinical isolates of acb complex, including 63 isolates of a. baumannii and 19 isolates of a. calcoaceticus, as identified by api 20ne. results: a. baumannii had the shortest its fragment (607-609 bp) followed by genospecies 13tu (608-615 bp), genospecies 3 (619-621 bp) and a. calcoaceticus (627-638 bp). the intraspecies its similarity of the acb complex was very high, ranging from 0.98 to 1.0, whereas the interspecies its similarity was relatively low (range: 0.86-0.91). among the 63 clinical isolates of a. baumannii, two isolates were genospecies 3 and 14 isolates were ungroupable, as revealed by its sequence analysis. therefore, about 25% of clinical isolates of a. baumannii was misidentified. furthermore, among the 19 clinical isolates of a. calcoaceticus, 16 isolates were genospecies 3 and three isolates were ungroupable. therefore, the designation of a. calcoaceticus to clinical isolates is, under most conditions, not correct. these results were confirmed by amplified rdna restriction analysis (ardra). conclusions: its sequence analysis provides a simple and useful alternative for species delineation of the acb complex. objectives: enteroaggregative escherichia coli (eaec) are increasingly implicated in acute and persistent diarrhoea around the world. phenotypically, eaec have a defining 'stacked brick' pattern of aggregative adherence (aa) to epithelial cell lines in vitro. genotypically, they are diverse, and while a range of eaec pathogenicity factors are known, their distribution amongst strains varies. the most widely used dna probe for eaec is cvd432, which has been reported to have limited sensitivity in some studies, but it is presumed to be specific for eaec. the aim of this study was to determine whether the cvd432 probe is a specific tool for identifying eaec strains imported into the uk. methods: a total of 520 e. coli isolates (four per patient) were obtained from consecutive stool samples of 130 diarrhoeal patients with a recent history of foreign travel (33 different countries). all were screened for hybridisation with the cvd432 probe, as well as eaec plasmid encoded virulence factors aggr (aggregative adherence regulator), aap (dispersin) and the chromosomal pathogenicity-island-encoded mucinase pic. other pathogenic e. coli were identified using standard probes. cvd432 probe positive strains were then examined for adherence to hep 2 cells after coincubation for 3 h. results: the prevalence of eaec-associated genes amongst the 520 isolates was: cvd432 8.3%; aggr 6.3%; aap 11%; pic 13.3%. adherence assays on the 43 isolates that were cvd432 positive revealed a mixture of aggregative (24 isolates) and non-adherent strains (nine isolates) plus 10 isolates that gave an unusual pattern of loose, highly localised aggregation, present on <5% of the hep-2 cells. of the cvd432 positive strains, 65% were aap, aggr, and pic positive as well, but this group also contained strains of all three adherence types. none of the other eaec-associated probes was unequivocally predictive of actual aa among cvd432 positive strains. unexpectedly, cvd432 positive isolates that hybridised with the enteropathogenic e. coli probe eae were isolated from one patient (returning from turkey). conclusions: this study suggests that the cvd432 probe may not be specific for true eaec, even when combined with the other probes used here. the significance of the newly described adherence pattern in relation to diarrhoeal disease remains to be elucidated, as does the finding of e. coli with both eaec and epec properties. objectives: otomycosis represents a significant percentage of clinical external otitis and is usually caused by candida, aspergillus, penicillium and malassezia. clinical symptoms such as otorrea, erythema and stenosis of the external auditory canal are commonly present and create appropriate conditions for fungal growth. the objectives of this study were to determine the prevalence of candida otomycoses and to evaluate the relationship between albicans and non-albicans species. methods: from april 2002 to november 2003, a total number of 67 patients were found to be suffering from symptoms indicating otitis externa. the specimens were taken by cotton swab from bony portion of external ear. all specimens were inoculated on sabouraud dextrose agar, incubated at 26 and 37 c for 7 days and examined macroscopically every day. suspected cultures were examined microscopically in order to confirm finding of candida spp. the identification of isolated candida strains was carried out by germ tube test and api 20c aux assimilation test (biomerieux, france). results: in a base of microbiological findings 12 (17.9%) patients considered to be negative, 39 (58.2%) confirm bacterial or mould results and in 16 (23.8%) patients candida spp. was found. out of 16 patients with diagnosed candida otomycosis, in 11 patients only candida spp. was isolated and in five patients otitis externa was caused by candida associated with bacterial or mould infection. c. albicans was identified in three (3/16) cases, while all other was non-albicans strains as three cases of c. guilliermondii (3/16), four of c. famata (4/16) and six of c. parapsilosis (6/16) . conclusion: in clinical finding of otitis externa mycological examination could be very important in setting the accurate diagnosis and appropriate therapy. these results suggest that c. albicans is not the predominant causative agent of otitis externa. isolation of non-albicans species has particular interest in therapy of otitis externa because of their reduced susceptibility to antifungal agents. the study was focused on the species involved and their in vitro antifungal susceptibility. molecular typing of the isolates involved in subsequent episodes of rvvc allowed establishing if the strains showed the same dna type. methods: isolates were identified by standard morphological and biochemical methods. mics of amphotericin-b, itraconazole, fluconazole, ketoconazole, 5-fluorocytosine, voriconazole were determined by sensititre yeastone colorimetric antifungal panel plates according to nccls document m27-a. the strains were typed using pulsed-field gel electrophoresis (pfge) and repetitive extragenic palindromic-pcr dna fingerprints. results: c. glabrata was isolated in 53.8%, c. albicans in 30.7%, c. krusei in 15.5% of cases. the yeasts involved in each recurrence were characterised by identical biochemical profiles and drug resistance phenotypes. c. albicans strains isolated from one rvvc resulted in in vitro resistant to azoles. the genotyping by pfge revealed that c. albicans and c. glabrata obtained from different patients were clinically unrelated to each other while an identical profile, indicating clonal relatedness, was observed with yeasts recovered from the same patient. conclusion: our data underline the persistence of strains, with the same antifungal susceptibility profile and clinically related genotypes in patient with recurrent infections, suggesting a colonisation with the same strain over different periods of time despite therapy. these results stress the need for molecular tools for strain typing in order to clarify the epidemiology of the rvvc and to control drug-resistant fungal agent spread. objectives: using criteria designed for invasive aspergillosis (ia) in neutropenic patients, the present study aimed to determine the impact of invasive aspergillosis in different groups of non-haematooncological icu patients. methods: this study is a retrospective analysis of all patients that were hospitalised in the 17-bed medical intensive care unit (micu) between 1 january 2000 and 1 january 2003. any admitted patient fulfilling one or more of the following criteria was included in the study: (a) histopathological evidence of aspergillosis (including autopsy) or (b) microbiological evidence of aspergillosis during stay in the micu (positive culture or positive circulating galactomannan). ia was classified as proven, probable or possible, according to the eortc/msg definitions. aspergillus isolation from a non-sterile site in patients without appropriate clinical setting was considered as 'colonisation'. results: between 2000 and 2003, 127 of 1850 patients (6.9%) fulfilled the inclusion criteria. thirty-eight patients (29%) had haematological malignancies and were not further analysed. eightynine (71%) were non-haemato-oncological patients (37 copd, nine solid organ transplant recipients, 17 autoimmune diseases, objectives: we evaluated the value of aspergillus pcr as a tool for diagnosing invasive aspergillosis during antifungal therapy from whole blood samples. methods: in a 3-year study, 36 patients receiving antifungal therapy due to chest radiographic findings highly suggestive for fungal pneumonia were evaluated. the pcr results of whole blood samples were compared with those obtained from bronchoalveolar lavage fluids and/or tissue specimens. results: a total of 205 whole blood samples, 15 fine needle aspirations or tissue biopsy specimens, 21 bronchoalveolar lavage fluids and tracheal secrets were analysed using pcr. fifteen patients had proven, nine probable and 12 possible invasive aspergillus infections according to european organization for research and treatment of cancer/mycosis study group definitions. in patients with proven infections, the sensitivities of pcr of lung and blood samples were 100 and 40%, respectively. the specificities were 100%. the negative predictive value of blood monitoring under antifungal treatment was 44%. in patients with probable infections, the sensitivities of pcr of lung fluids and blood were 66 and 44%, respectively. the specificities were 100%. the negative predictive value of blood monitoring under antifungal therapy was 58%. conclusions: the benefits of pcr diagnosing of whole blood are limited if sampling takes place once treatment has started. the performance of aspergillus pcr should be recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection. objective: air is considered the main vehicle of aspergillus spores causing community or nosocomially-acquired invasive aspergillosis (ia). air surveillance is nowadays performed in protected air environments in many institutions. sabouraud dextrose agar irradiated (sd) is used for the control of air in our institution but czapeck agar is also recommended for this purpose. the aim of our study was to compare the efficiency of both media for aspergillus isolation in air samples. methods: we collected 332 samples using the merck air sampler mas 100 ò with a volume of air per culture of 200 l. every sample was cultured in both media (pair of samples), and agar plates were incubated at 35 c for 5 days. aspergillus spp. was identified by conventional methods. the pairs were checked daily to observe the growth of fungi and after the incubation period the objectives: the spreading of aspergillus hyphae into the brain of immunocompromised patients is a complication of invasive asper-gillosis that leads to death in nearly 100% of the cases. the most frequent species for induction of cerebral aspergillosis is aspergillus fumigatus. our aim was to study the interaction of a. fumigatus with the complement system to determine the reason for the failure of the cerebral immune system. furthermore, these experiments might give first approaches for a putative immune therapy to support current antimycotical treatment. methods: different pools of cerebrospinal fluid (csf) were tested for their ability to opsonise fungal hyphae with different complement factors. germinated conidia were fixated, incubated in csf, and the deposition of complement was shown via indirect immunofluorescense (if) by suitable specific antibodies. the extent of surface labelling on aspergillus was compared with pseudallescheria boydii, another neurotropic fungus. immunohistochemical (ihc) staining of paraffin-embedded tissue sections derived from patients with cerebral aspergillosis allowed the comparison with the complement deposition in vivo.results: the levels of the complement factors c1q, c4, c3, c5, c6 and c7 in the csf of normal persons were sufficient for opsonisation of the fungal hyphae, although the deposition was much weaker than in human serum. however, the recognition of aspergillus surface was not optimal in comparison to p. boydii that showed a clearly stronger deposition. concentrations of different complement proteins and complement activation products were highly elevated in csf derived from a patient with cerebral aspergillosis. this csf showed a significantly stronger complement deposition on the fungal surface than the non-inflammatory csf. however, ihc-analyses in tissue sections of patients with cerebral aspergillosis showed only limited opsonisation on the fungus. conclusion: csf harbours the ability of complement deposition on the surface of neurotropic fungi. frequent pathogens like aspergillus fumigatus have adopted their surface to minimise recognition by the complement cascade. cerebral complement production is upregulated as a consequence of fungal infection, which might contribute to antifungal immune defence but also to inflammation and tissue damage. the amount of deposited factors on the fungal hyphae in vivo is low, indicating the expression of complement inhibitory factor(s) by a. fumigatus. objectives: staphylococcus epidermidis is a major pathogen in nosocomial infections, and infectious isolates display a high prevalence of oxacillin resistance (oxar). tn917 mutagenesis of rsbu, encoding a positive regulator of the alternative sigma factor sigma b lead to a reduced oxar in s. epidermidis 1057. however, the mechanism of this regulatory pathway is still unknown. the role of sigma b in the regulation of oxar in s. epidermidis was investigated in this study. methods: two mutants with inactivation of the entire sigma b operon (1057rsbuvwsigb) or the regulatory cascade rsbuvw (1057rsbuvw) were generated by allelic gene replacement in s. epidermidis 1057, which displays a heterogeneous oxacillin resist-ance phenotype. rna was extracted at 9 and 17 h from cultures in mueller hinton + 2% nacl (mhnacl) and mhnacl supplemented with 1 lg/ml oxacillin (mhoxa). quantitative transcriptional analysis of meca, femabcdf, fmta, mrp (fmtb), and mprf (fmtc) were performed by real-time rt-pcr. at least a 2.5-fold difference compared with the wild type in the average of three independent experiments was defined as cut-off for differentially expressed genes. results: population analysis of the mutants and the wild type strain revealed that mutant 1057rsbuvwsigb displayed a more heterogeneous phenotype with a smaller subpopulation expressing methicillin resistance compared with the wild type. mutant 1057rsbuvw with constitutive expression of sigma b displayed a strong increase of methicillin resistance and a homogeneous resistance phenotype compared with the wild type. transcriptional analysis revealed that the homogeneously resistant mutant 1057rsbuvw displayed no differences compared with the wild type under all conditions investigated, except of the gene fmta, which was downregulated in mhoxa at 9 h. interestingly, in the less resistant mutant 1057rsbuvwsigb the genes meca, femb, femd, fmta, and mprf were upregulated in mhnacl compared with the wild type at both time points, whereas in mhoxa only the genes femd, fmta, and mprf were upregulated at 9 or 17 h. conclusions: none of the investigated genes including meca is responsible for the homogeneous expression of oxar in mutant 1057rsbuvw. mutant 1057rsbuvwsigb displayed a less resistant phenotype compared with the wild type strain, despite the upregulation of several genes required for oxar. therefore, an additional sigma b dependent factor must be required for homogeneous expression of oxar in s. epidermidis. objectives: to develop methods to measure the initial response of s. aureus after exposure to antimicrobial agents. such an approach has the potential to allow both the sensitivity and mechanism of resistance to be rapidly determined from isolated bacterial strains. methods: mrna was extracted from a selection of s. aureus isolates either with or without 30 min exposure to antimicrobial agents (including oxacillin and mupirocin). the mrna extracted was then used to produce labelled nucleic acid suitable for hybridisation to a low-density flow through oligonucleotide array targeting specific genes. these arrays are suitable for high throughput screening and provide very rapid hybridisation kinetics.results: distinctive changes in mrna levels were detected for each agent tested and for isolates with different phenotypic susceptibilities. oxacillin resulted in a significant increase in the levels of penicillin binding protein 2 (pbp2) mrna in both sensitive and resistant isolates and an increase in the levels of pbp2prime mrna in resistant isolates only. in contrast mupirocin resulted in very high levels of ile-trna synthetase in both strains with high-or low-level mupirocin resistance but not in sensitive strains. conclusion: future developments in rna extraction and labelling as well as the increased availability of dna array technology will allow this approach to be more widely used. this and similar methods have the potential to provide information on both the resistance phenotype of the isolate and the mechanism of resistance, in contrast to 'classical' molecular tests for drug resistance which generally target known genotypes. key: cord-009664-kb9fnbgy authors: nan title: oral presentations date: 2014-12-24 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2009.02857.x sha: doc_id: 9664 cord_uid: kb9fnbgy nan [ primary immunodeficiency diseases are a heterogeneous group of disorders, caused by inherited defects in the immune system, and characterised by wide spectrum of clinical manifestations, particularly an increased susceptibility to infections and a predisposition to autoimmune diseases and malignancies. recurrent infections or infection with unusual organisms are the most commonly presentation of primary immunodeficiency diseases. although recurrent respiratory tract infections and gastrointestinal manifestations are the most common features of these diseases, especially in predominantly antibody deficiencies and combined immunodeficiencies, other organs can be involved as well. recurrent cutaneous abscesses with unusual organisms or deep abscesses may represent infections with an association with immunodeficiencies, particularly in phagocytes defects. meningococcal infections could have an association with complement deficiencies. meanwhile other bacterial infections, mainly streptococcus pneumoniae and staphylococcus aureus, as well as infections with viruses, fungi and parasites are also common in several primary immunodeficiency diseases. autoimmune diseases such as idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia, systemic lupus erythematosus, juvenile arthritis, sclerosing cholangitis, and vasculitis are common in primary immunodeficiency diseases. whilst some syndromic immunodeficiencies (e.g., wiskott aldrich syndrome, di george syndrome) have a strong association with autoimmunity, there are a group of disorders (e.g., alps, apeced, ipex) that the autoimmune manifestations are typically the first and most significant findings. malignancies are also common in some primary immunodeficiency diseases (e.g., cvid, alps, xlp, and dna repair defects). other manifestations such as dysmorphic features, associated anomalies, skeletal dysplasia, and oculocutaneous hypopigmentation can be unique characteristics of some cases with primary immunodeficiency diseases. the clinical manifestations of these diseases are often helpful in guiding the appropriate evaluation of the patients. prompt and precise diagnostic laboratory evaluation should be performed in the patients with such features, whereas early diagnosis and successful management of these patients prevent irreparable organ system damage and improve the prognosis. immunodeficiency specialists from all over europe have composed a multistage diagnostic protocol that is based on their expert opinion, in order to increase the awareness of pid among doctors working in different fields. the protocol starts from the clinical presentation of the patient; immunological skills are not needed for its use. a list of relevant symptoms and signs from the history and physical examination that should alert any physician to potential pid is given. these are grouped together to form eight typical clinical presentations of pid: recurrent ent and airway infections; failure to thrive from early infancy; recurrent pyogenic infections; unusual infections or unusually severe course of infections; recurrent infections with the same type of pathogen; autoimmune or chronic inflammatory disease, or lymphoproliferation; characteristic combinations of clinical features in eponymous syndromes; and angioneurotic edema. these presentations lead the user towards different algorithms, which in fact represent the traditional division into antibody, complement, lymphocyte, and phagocyte deficiencies, respectively. the algorithms each are comprised of several steps. this multistage design allows cost-effective screening for pid within the large pool of potential cases in all hospitals in the early phases, while more expensive tests are reserved for definitive classification in collaboration with an immunologist at a later stage. g. schmid°(geneva, ch) in 1986, articles suggesting that male circumcision (mc) decreased the risk of hiv infection appeared. over the next 15 years, studies of two epidemiologic types − ecologic and observational − increasingly supported this contention. ecologic studies showed strong correlations between prevalences of mc and hiv, e.g., tribes with low prevalences of mc had high prevalences of hiv infection. observational cross-sectional studies showed that uncircumcised men had higher rates of hiv than circumcised men. observational cohort studies confirmed these weaker study design findings. a systematic review of observational studies in 2000 found a relative risk (rr) of 0.42 (95% ci, 0.34−0.54), a 58% protective effect. in 2005 and 2007, results from three randomised controlled trials, all from sub-saharan africa, were reported. results were consistent, and the pooled rr of 0.42 (95% ci, 0.31−0.57) was identical to that of the observational studies. the protective effect in the three trials, found at about 21−24 months' follow-up, has been extended in one trial to a protective effect of 64% at 42 months of follow-up. who and unaids have strongly endorsed mc as an effective hiv prevention strategy in generalised hiv epidemics where mc is uncommon. what about europe? mc is uncommon with an adult male prevalence of <20%. hiv incidence is low enough that mc for hiv prevention purposes is unlikely to have much impact. no public health authority recommends routine neonatal circumcision. increasingly, however, data are showing benefits of mc in addition to hiv prevention. lessened risk of urinary tract infection in infants (rr 0.13, 95% ci 0.08−0.20) and lifetime avoidance of phimosis and associated conditions occur when mc is performed neonatally. other benefits occur in males circumcised at any age. mc protects against acquiring sexually transmitted infections characterised by genital ulcers-syphilis, chancroid and herpes-and possibly trichomoniasis. circumcised men may be less likely to acquire hpv and are more likely to clear the infection. through the protective effect against hpv, mc halves risk of penile cancer (rr 0.52, 95% ci 0.33−0.82) and partners of circumcised men are at lessened risk of cervical cancer. other issues must be considered in making public health decisions about mc. cultural objections may occur, but mc in the developing world is readily accepted in non-circumcising societies. studies of sexual pleasure and function have found no relationship to circumcision status. mc may be advised for subgroups, even if not for the entire population. and, surgical risk and cost must be considered. while many sub-saharan african countries are scaling up mc services to prevent hiv infection, public health agencies in many industrialised countries are reconsidering mc policies-the outcomes of both efforts are being followed with interest. acute otitis media (aom) is generally considered a bacterial infection that is treated with antibiotics. however, despite extensive use of broadspectrum antibiotics for this condition, the clinical response to the treatment is often poor. this fact, together with vast clinical experience connecting aom with viral respiratory infections, has prompted research into the role of viruses in aom. to date, ample evidence from studies ranging from animal experiments to large clinical trials supports a crucial role for respiratory viruses in the aetiology and pathogenesis of aom. in most cases, viral infection of the upper respiratory mucosa initiates the whole cascade of events that finally leads to the development of aom as a complication. the pathogenesis of aom involves a complex interplay between viruses, bacteria, and the host's inflammatory response. recent studies indicate that with sensitive techniques viruses can be found in the middle-ear fluid in most children with aom, either alone or together with bacteria. viruses appear to enhance the inflammatory process in the middle ear, and they may profoundly impair the resolution of otitis media. it is important to understand, however, that our increasing knowledge of the importance of viruses in the etiopathogenesis of aom does not diminish the central role of bacteria in aom. therefore, while viruses may explain many of the problems encountered in treating aom, the ultimate decision on whether or not to treat aom with antibiotics cannot be based solely on the degree of viral involvement in aom. the non-judicious use of antibiotics has lead to an epidemic in antimicrobial resistance. acute otitis media (aom) is the most common indication for use of antibiotics in children in the united states (us). despite available evidence that supports a wait and see approach, most us physicians immediately prescribe antibiotics for the treatment of aom. the american academy of pediatrics published a guideline in 2004 that addressed the diagnosis and treatment of aom. this guideline recommends the use of observation as a potential strategy for the treatment of aom. the key components of this published guideline will be discussed, as well as the evidence and rationale that supports the use of observation as an initial strategy to treat aom. otitis media (om) is the most common bacterial infection in children aged <5 years for which antibiotic treatment is prescribed worldwide. although most of the time this entity resolves spontaneously it is associated with morbidity, family dysfunction, antibiotic use and burden on the medical system. efforts to reduce the burden of om by vaccination have not been extremely rewarding, but some progress has been made. the first obvious step would be to reduce viral infections leading secondarily to om. in the modern era, the only viral vaccine with proven effect on aom is the influenza virus vaccine. both the inactivated and the live virus showed some effect, but since influenza virus has only a limited season yearly the effect on the overall om rate is far from being remarkable. haemophilus influenzae (hi) b vaccine did not reduce om since most hi causing om are nontypable (nthi) and not hib. the newly developed pneumococcal conjugate vaccines (pcvs) have all been shown to reduce >50% of the om caused by the serotypes included in the vaccines, but some replacement with serotypes not included in the vaccines and non pneumococcal organisms was demonstrated to reduce the overall effect of pneumococcal vaccines. the effect of pcv on the reduction of recurrent om, om with effusion, the need for ventilation tubes and frequent visits for aom has been suggested, and the real impact is still being studied. aiming with pcv at those with established recurrent om has proved disappointing. pcvs can reduce om caused by antibiotic-resistant s. pneumoniae but the continued overuse of antibiotics is responsible for the increase in antibiotic resistance in non-vaccine serotypes. a newly developed pcv with an outer membrane protein for hi (pnpd) is suggested to reduce also om caused by hi, but confirmation studies are needed. the expansion of the 7 serotypes included in the current licensed pcv to 10 or more serotypes may add to the prevention of om in the near future. in the next decade, om will continue to be an important disease in children. however, we can expect it to be modified in terms of bacteriologic aetiologies, antibiotic resistance and hopefully short and long term consequences. v. korten°(istanbul, tr) infectious consequences of an earthquake mainly involve several types of communicable diseases and crush related infections. water-borne and food-borne illnesses often result from the disruption of the public water and sewage systems and contamination of water supply. overcrowding, poor hygiene and sanitation in temporary shelters also may be factors. the type of infectious diseases are associated with the epidemiology of communicable diseases in the area where the earthquake occurred. the most common outbreaks associated with earthquakes are gastroenteritis, infectious hepatitis and pulmonary infections. in unvaccinated populations, there are reports of increased measles. tetanus can be seen in populations where vaccination coverage levels are low. the risk for diarrhoeal disease outbreaks following earthquakes is higher in developing countries than in industrialised countries. an outbreak of acute watery diarrhoea involved >750 cases occurred in a camp after the 2005 earthquake in pakistan. acute respiratory infections, hepatitis e clusters and measles (>400 clinical cases in the 6 months) also occurred among the displaced victims after the same earthquake. contamination of drinking water led to an outbreak of rotavirus after the 2005 earthquake in kashmir, india. an unusual outbreak of coccidiomycosis associated with exposure to increased levels of airborne dust occurred after the 1994 southern california earthquake. persons who have been trapped by rubble for several hours or days may develop compartment syndromes requiring fasciotomy or amputation. infectious complications were common in renal victims of the1999 marmara earthquake in turkey and were associated with increased mortality when complicated by sepsis. of 639 renal victims, 223 (34.9%) had infectious complications, mainly sepsis and wound infections. most of the infections were nosocomial in origin and caused by gram-negative aerobic bacteria and staphylococcus spp. multivariate analysis of the risk-factors for nosocomial infections revealed a significant association with fasciotomy and length of hospital stay in a back up university hospital. the most frequent pathogens isolated from pus and/or wounds culture in 2008 wenchuan earthquake survivors were s. aureus, e. coli, a. baumannii, e. cloacae, and p. aeruginosa. disaster-preparedness plans, focused on trauma and mass casualty management and also on health needs of the surviving affected populations may decrease the health impact of earthquakes. s16 infections in the disaster setting: famine. experience from darfour, sudan clinic malnutrition is a known risk factor for id worldwide. subsaharan africa and india is at higher risk due to vegetarian habits on absolute absence of animal meat proteins, resulting to depletion of micronutritients (zinc, iron, selenium), responsible for recovery of postmalarial anaemia. in addition, depletion of proteins results to immunoglobulinaemia and to delayed response to many bacterial pathogens causing id in topics (pneumococci, salmonella, etc.) . third problem is absence of vitamins dissolved in oil and fat, resulting to delayed phagocytic activity. therefore proteinocaloric malnutrition results to significant adverse outcome in hiv, tb (diarrhoea, pneumonia), the major killers of children under five. st. elizabeth university tropical programme runs 4 antimalnutrition centres: 1 in sudan, darfour and 2 in kenya amaong upcountry refugees from major conflict areas (sudan − turrana border) and 1 in uganda trying to rehabilitate malnourished children under 5 and helping them to combat disease, responsible for 12.5 million deaths in children mean 5 a year − malaria (1.2 mil), tb (1.1 mil), hiv (2.0 mil), pneumonia (7.5 mil) and diarrhoea (0.5 mil. children deaths approximately a year). h. giamarellou°(athens, gr) for the last six years greece has faced a large number of infections, mainly in the intensive care units (icu), due to carbapenemsresistant klebsiella pneumoniae. the proportion of imipenem-resistant k. pneumoniae has increased from less than 1% in 2001, to 23% in isolates from hospital wards and to 53% in isolates from icus in 2008. likewise, in 2002, these strains were identified in only three hospitals, whereas now they are isolated in at least 32 of the 40 hospitals participating in the greek surveillance system. until 2007 this situation was due to the spread of the blavim-1 cassette among the rapidly evolving multiresistant plasmids and multiresistant or even panresistant strains of mainly k. pneumoniae and also other enterobacterial species. however, the fact that most strains display mic values below or near the clsi resistance breakpoint create diagnostic and therapeutic problems, and possibly obstruct the assessment of the real incidence of these strains. as of 2007, the emergence of kpc-producing k. pneumoniae has been noted in icus of some greek hospitals and has now spread to most hospitals throughout the country creating a countywide outbreak in 2008. in attikon university hospital we recently described the icu outbreak of kpc-producing k. pneumoniae. twenty-nine patients (admitted from february to december 2008) were colonised mainly in gi tract. fifteen patients were male (52%) and the median apache ii was 19. patients had already long hospital stays preceding icu admission with a median of 25 (17−40) days. in twenty-two of these patients (76%) kpc-producing k. pneumoniae colonisation was definitely icuacquired while in 7 (24%) acquisition in other wards or other hospitals was hypothesized. five of these patients are still hospitalised in the icu and, of the remaining 24, 11 died (icu mortality 46%). ten of the 29 colonised patients were clinically infected. fifteen infections were documented, mostly bsi (11/15), followed by vap (2/15) and ssi (2/15). only 1 patient died from this infection (1/15, 6.7%). an evidence-based consensus on the therapeutic strategy for these infections has been reached by keelpno and the greek ministry of health which proposed the use of high dose meropenem (6−8 g/day) combined with an active aminoglycoside or colistin for strains with an mic 4 mg/ml whereas for strains with a higher mic the use of carbapenems is contraindicated and active alternatives (monotherapy with tigecycline, colistin, or an aminoglycoside or aztreonam-based combinations) could be used. antibiotic stewardship is of great importance in such a dismal situation but stringent adherence to infection control measures is probably of even greater importance for the effective containment of these pandrugresistant strains. the presentation of clostridium difficile infection (cdi) varies from mild diarrhoea to a potentially fatal pseudomembranous colitis. the recent emergence of types 027 and 078 of c. difficile has been associated with increased virulence. c. difficile takes advantage of disruption of the normal intestinal flora as caused by antibiotic therapy. the antibiotical class and the antimicrobial resistance pattern of c. difficile influence the development of disease. in the netherlands, significantly more patients with cdi due to type 027 used fluoroquinolones (or, 2.88; 95% ci, 1.01−8.20) compared with those who were infected with other pcr ribotypes. similar as type 027 cdi, patients infected with type 078 also more frequently received fluoroquinolones therapy (or, 2.17; . the risk to develop cdi due to type 027 was particularly high in persons receiving a combination of cephalosporin and fluoroquinolone (or 57.5, ). this association was also strongly dependent on the duration of therapy. the use of clindamycin was found as a protective factor. however, the recent detection of clindamycin-resistant c. difficile type 027 strains in other european countries is an important and worrying development. since the association of cdi with fluoroquinolones has only been investigated at patient level, a study was performed to investigate the relationship between cdi incidence and the preceding use of different antibiotic classes at hospital level in the netherlands. comparisons were made between hospitals where type 027 caused an epidemic, hospitals where only isolated cases of type 027 were observed and hospitals where no outbreak of cdi or type 027 were encountered. in the pre-epidemic period, the total use antibiotics was comparable between affected and unaffected hospitals. higher use of secondgeneration cephalosporins, macrolides and all other studied antibiotics were independently associated with a small increase in cdi incidence, but the effect was too small to predict which hospitals might be more prone to 027-associated outbreaks. despite the fact that the netherlands is known by its restrictive and conservative use of antibiotics, outbreaks of cdi due to new emerging types have been recognized. this is probably associated with the use of antibiotics at patient level and hospital department level rather than the use of antibiotics at the level of the healthcare institute. m. peiffer, j. bulitta, h.a. haeberle, m. kinzig-schippers, m. rodamer, v. jakob, b. nohé, f. sörgel, w.a. krueger°(trier, de; albany, us; tubingen, nuremberg, constance, de) piperacillin-tazobactam (pip-tazo) is a broad spectrum antibiotic, used for treatment of severe infections such as ventilator-associated pneumonia (vap). the effectiveness of betalactams is best predicted by the duration of free drug concentrations above the minimal inhibitory concentration (t > mic) of infecting pathogens [1] . animal experiments suggest that more than 50% of t > mic should be reached. continuous infusion (ci) of pip-tazo may enhance the therapeutic performance, but there is little data on pharmacokinetic/-dynamic (pk/pd) parameters, when ci is used in critically ill patients. objectives: the aim of our study was to determine concentrations of pip-tazo in plasma and broncho-alveolar epithelial lining fluid (elf) at steady state during ci. based on these results, the penetration ratio (plasma/elf) and pk/pd parameters for pip-tazo are derived. methods: after approval by the ethics committee, 16 mechanically ventilated critically ill patients were enrolled during treatment in 3 intensive care units. each patient received a loading dose of 4 g/0.5 g of pip-tazo, followed by ci of 12 g/1.5 g over 24 h. at steady state (67.8 + 39.5 h after loading dose), a total of 30 blood samples were drawn and bronchoalveolar lavage (bal) was simultaneously performed in 8 cases (1 sample discarded for technical reasons). samples were stored at −80ºc until analysis by liquid chromatography coupled with mass-spectrometry (lc-ms). elf-concentrations were calculated from bal-samples using the relation of ureaplasma:ureabal as dilution factor. results: plasma concentrations of pip and tazo (n = 30 in 16 pts.) amounted to 15.38+8.89 mg/ml, and 1.31+0.95 mg/ml, respectively. elflevels (n = 7) were 56.63+27.24 mg/ml, and 5.95+3.74 mg/ml. elf-levels were 368+236%, and 587+584% of corresponding plasma levels (n = 7) for pip and tazo, respectively. the ratio pip:tazo was 11.74:1 in plasma, and 9.52:1 in elf. conclusions: using advanced analytical techniques, elf concentrations were higher compared to traditional bolus administration [2] . ci yielded steady state plasma concentrations in excess of mics of susceptible bacteria (<8 mg/ml, according to eucast) in 76.6% of measurements, respectively, but elf levels exceeded 8 mg/ml in all cases. taken together, our data provide further arguments for ci being the preferred mode of administration for pip-tazo in critically ill patients with suspected vap. [ objectives: staphylococcus aureus is a potential pathogenic microorganism and a causative agent of~25% of infections in intensive care patients. an optimal empiric choice for the treatment of these infections will result in a reduction in morbidity and mortality. therefore, it is essential to provide the clinician with resistance data of the bacterial population to be treated. to optimise the empiric choice and to monitor the emergence of microbial resistance, a national surveillance program of the swab was started in the netherlands in 1996.this study describes the results of the resistance development of s. aureus from icu's of 14 hospitals all over the netherlands over a ten year period. methods: in the first 6 months of each year, the participating hospitals collected clinical isolates from among others blood and respiratory samples. in total 943 isolates were collected: 250 from 3 hospitals in the north, 187 from 2 in the east, 229 from five in the west and 280 from four in the south. the antimicrobial susceptibility was determined as a micro broth dilution method according to the clsi guidelines. results: an increase in resistance to ciprofloxacin was observed from 4% until 2002 to 14% from in 2005, which dropped again to 7% in 2006. the resistance to moxifloxacin was rather constant over time, i.e. 2%, only in 2003 8% resistance was found. resistance to clarithromycin increased to 10% in 2003, but decreased in 2006 to 6% the level before 2003. resistance to penicillin, clindamycin and tetracycline fluctuated over time at~75%, 4−8% and 2−10% respectively. during the study period seven methicillin resistant s. aureus were isolated, no resistance to vancomycin, teicoplanin and linezolid was observed. resistance to gentamicin and rifampicin was sporadicly found. regional differences were observed for ciprofloxacin, being the highest in the western and southern part and tetracycline being the lowest in the northern part. conclusion: during the 10 year study period only an increase in resistance to ciprofloxacin was observed. the data presented justify the empiric choice of flucloxacillin, (with rifampicin or gentamicin depending on the indication) in case of an infection in icu patients probably caused by s. aureus. j.j. lu°, p.r. hsueh, s.y. lee (taichung, taipei, tw) objectives: to investigate the prevalence of visa in hospitalised patients with mrsa infections or colonisations at a teaching hospital in taiwan and to evaluate the possible clonal spread of visa in the hospital. methods: from september 2001 to august 2002, 1500 consecutive mrsa isolates were collected from various clinical specimens of 637 patients hospitalised at a teaching hospital in taiwan. minimum inhibitory concentrations (mics) of vancomycin for all mrsa isolates were determined by the broth microdilution method in accordance with clsi guidelines. molecular characteristics and antimicrobial susceptibilities of visa isolates were investigated and pulsed-field gel electrophoresis was used to evaluate the clonality of the isolates. results: among the 1500 mrsa isolates, 43 (2.9%) were visa. of the 43 visa isolates, 35 had vancomycin mics of 4 microgram/ml and 8 had vancomycin mics of 8 microgram/ml. all isolates were inhibited by tigecycline at 0.5 microgram/ml, linezolid at 1 microgram/ml, and ceftobiprole at 2 microgram/ml. five (11.6%) isolates had reduced susceptibility to daptomycin (mics of 1−2 microgram/ml). six of the 43 visa isolates had decreased susceptibility to autolysis in 0.05% triton x-100. the 43 visa isolates were recovered from 21 patients; 13 of these patients had received glycopeptide treatment prior to the isolation of visa. five (23.8%) patients died despite vancomycin therapy. all 43 visa isolates carried sccmec type iii and agr group i but were negative for pvl gene (luks-lukf). none of the enterococcal van genes were detected in the 43 visa isolates. results of pfge analysis revealed that one major clone of visa isolates (90.5%, clone a exhibiting sccmec type iii, agr group i, and absence of pvl gene) had disseminated in the hospital. conclusion: this retrospective study demonstrated that clonal dissemination of visa had occurred in the hospital. rapid and correct detection of visa and proper use of antibiotics are the most effective approaches for preventing its emergence and spread. x. zheng°, c. qi, a. o'leary, m. arrieta, s. shulman (chicago, us) objectives: vancomycin remains one of the major options for treating methicillin-resistant s. aureus (mrsa) related infections. some but not all studies have shown an increase in prevalence of mrsa isolates with elevated vancomycin mic values among recent clinical isolates, so called "mic creep". although still within the susceptible range, higher mics may be associated with increased chance of treatment failure. because of the conflicting reports and lack of published data from paediatric patients, we sought to assess possible mic change over time and to compare results generated by using different methodologies including etest, agar dilution, and broth microdilution (microscan) methods. methods: we studied 318 mrsa isolates predominantly community acquired including all blood and normally sterile site isolates collected in our large children's hospital in 2000/2001, 2003, 2005, and 2007 molecular bacteriology o41 genome sequence of a virulent, methicillin-sensitive staphylococcus aureus clinical isolate that encodes the panton-valentine leukocidin toxin l. faraj, l.a.s. snyder, n.j. loman, d.p. turner, m.j. pallen, d. ala'aldeen, r. james°(nottingham, birmingham, uk) objective: to determine the genome sequence of a virulent meticillinsensitive staphylococcus aureus (mssa) clinical isolate sanot01. methods: roche 454 sequencing determined the genome sequence of the clinical isolate at 12 times coverage. newbler sequence assembly (roche) generated 10 scaffolds that were annotated using gendb and compared with other s. aureus genome sequences. results: an 11-year-old asian girl presented with fever and a 1-week history of knee pain following a trivial fall. an mr scan revealed a large subperiosteal abscess around the upper tibia secondary to metaphyseal osteomyelitis. a pvl-positive, mssa was isolated from blood cultures and pus. the child deteriorated, required repeated debridement and developed septic shock. further investigation revealed aortic valve endocarditis with an aortic root abscess. whole genome sequencing revealed that sanot01 is the first sequence of an st30 s. aureus isolate to be determined. sanot01 is agr type iii and carries three coding regions that are not found in any other s. aureus genome sequences. amongst the unique genes present in these regions is a dihydrofolate reductase gene (dfrg) which is present in addition to the usual dfrb gene. downstream of the orfx gene, a 6.5 kb remnant of sccmec type ivc was found. this sequence has only previously been found in the mrsa252 genome sequence where it is located between the orfx and sccmec type ii sequences. mrsa252 is unique in sharing 14 genome regions with s. aureus strain rf122, a causative agent of contagious bovine mastitis. all but one of these 14 genome regions are also present in sanot01. conclusions: comparison of the genome sequence of sanot01 and the closely related mrsa252 ha-mrsa (emrsa-16) isolate reveals new insights in the evolution of both ca-mrsa and ha-mrsa isolates and the link to s. aureus rf122. pvl-encoding mssa strains can be significant pathogens but are not currently under mandatory surveillance in uk. as the cost of whole genome sequencing falls further it will become feasible to use this technology to monitor the evolution of both mssa and mrsa in healthcare settings and reveal clinically relevant information that will help to improve patient outcomes. objectives: ca-mrsa often produce panton-valentine leukocidin (pvl), a leukocidin encoded by two co-transcribed genes located on lysogenised phages. five pvl-encoding phages have been described in s. aureus: phipvl, phi108pvl, phislt, phisa2mw and phisa2958. single nucleotide polymorphisms (snps) in the pvl genes tend to vary with lineage and may have structural and functional implications. we examined a selection of pvl-positive ca-mrsa reported in our hospital to determine whether sequence variation and the pvl-encoding phage vary with lineage. methods: twenty-two pvl-positive isolates were chosen to reflect mlst clonal complexes identified in our hospital: cc1, 5, 8, 59, 80, 88 and 154 . isolates were characterised by antimicrobial resistance profile, sccmec and spa type, pulsed-field gel electrophoresis (pfge) profile and multilocus sequence typing (mlst); an oligonuleotide array (clondiag arraytube) was used to detect a range of toxin and antimicrobial resistance genes. primers were designed to amplify and sequence the luksf-pv genes. the pvl-encoding phage was characterised using a recently described pcr-based assay (ma et al. j clin microbiol 2008; 40:3246−58) . results: snps were identified at seven positions in the luksf-pv genes and the snp profile varied with lineage. three of the snps were coding mutations, which may have structural and functional implications. cc1 and cc80 isolates were both found to carry phisa2mw. the pvlencoding phage was not definitively identified in the other lineages, although the cc59 isolates carried a phisa2958-like phage and the cc8, cc80 and cc154 isolates carried elongated head-type phages. one of the cc1 isolates had an unexpected snp pattern compared with other cc1 isolates; this isolate also carried a novel or variant phage. conclusion: pvl gene sequence and the pvl-encoding phage vary with lineage in pvl-positive ca-mrsa isolates. this suggests that certain lineages are susceptible to infection or lysogeny with certain phage types. although ca-mrsa commonly carry pvl genes, some strains do not; it is possible that some pvl-negative types are resistant to infection with pvl-encoding phage, perhaps via restriction modification systems. crucially, our findings suggest the pvl genes have co-evolved with their phage and are not freely transmitted between different phages. further work is required to characterise the pvl-encoding phage in other isolates and to investigate whether the pvl sequence variants result in biological differences. objectives: community-associated mrsa (ca-mrsa) of many different mlst clonal complexes (ccs) can harbour lysogenised bacteriophage dna (prophage) encoding panton-valentine leukocidin (pvl). five pvl phages (phipvl, phislt, phisa2mw, phi108pvl, and phisa2958) have been reported to date. we sought to determine the distribution of chromosomally integrated copies of these lysogenised pvl-phages amongst dominant clones of pvl mrsa in england and wales. methods: seventy isolates of previously characterised pvl-mrsa were analysed by pcrs developed by ma et. al, (jcm, 2008) , to identify and discriminate between the five known pvl phages. to maximise any underlying diversity, representatives of each cc were selected based upon their spa, staphylococcal cassette chromosome mec (sccmec), toxin gene and pulsed-field gel electrophoresis (pfge) profiles. these included isolates of internationally disseminated pvl-mrsa lineages ccs 8, 30 and 80 which resemble the usa300, south west pacific (swp) and european clones, respectively. in addition we analysed pvl-mrsa from ccs 1, 5, 22, 59, 88 and st93. results: all seven cc80 isolates, which included representatives of the european clone, possessed an elongated-head-type phage and were positive by the pcr specific for the phisa2mw phage. one of the cc30 isolates possessed a phi108pvl phage, four swp representatives had elongated head type phages, whilst the remaining four cc30 isolates harboured an icosahedral-head-type phage. one cc30 was positive for both head shapes. the 12 cc8 (including representatives of usa300), eight cc1, six cc88 isolates and the st93 isolate were all positive for elongated-head-type phage. nine cc5 isolates were non-typeable for phage head shape and specific phage pcrs. three of four cc59 isolates, harboured a phisa2958-like phage of an unknown head type and the other cc59 isolate was non-typeable. all 14 cc22 isolates possessed an icosahedral-head-type phage, 13 were positive for the phipvl phage type and one possessed phi108pvl type. we have determined the pvl phages present in a diverse panel of distinct pvl-mrsa clones and found considerable inter-lineage variation in the pvl prophage present. there was also evidence of intra lineage variation in some major ccs such as ccs 22, 30 and 59. together with variation in mlst cc and sccmec, these data suggest pvl-mrsa have evolved on multiple occasions, sometimes within the same lineage. o44 transcriptional profiling of klebsiella pneumoniae genes controlled by the transcription factor, rama objectives: rama is an arac/xyls family transcriptional activator where over expression is associated with a multidrug resistance phenotype. in both multidrug resistant klebsiella and salmonella isolates, the rama gene has been associated with increase in expression of the acrab efflux pump. in salmonella it has been shown that a deletion of the rama locus prevents the emergence of multidrug resistant mutants. therefore in order to understand the role of this key regulator in the emergence and development of antibiotic resistance, transcriptomic analyses of its regulon were undertaken in k. pneumoniae. methods: rna was extracted from a combination of isogenic mutants and clinical isolates using the qiagen or ribopure kits. rna integrity was assessed using nanodrop and agilent nanochip systems. the rna was transcribed into double stranded cdna prior to labelling with cy3. the cdna was hybridised to the nimblegen expression array platform designed from the k. pneumoniae mgh 78578 genome. results: approximately 50 genes were found to be affected by rama expression, of which twenty (involved in metabolism, physiology, transcription, drug efflux, protection responses and the cell envelope) were confirmed by rt-pcr. the rama protein appears to affect drug efflux operons not previously shown to be associated with multidrug resistance and or affected by similar proteins such as mara. comparative transcriptome analyses of different k. pneumoniae clinical isolates overexpressing rama showed that variations exist in the levels of expression of the drug efflux genes. of note genes shown to be directly regulated by rama have a marbox-like sequence within the promoter sequences. conclusion: in this study, the transcriptome of the regulatory protein, rama, was determined in the pathogen k. pneumoniae. drug efflux proteins not previously associated with rama overexpression were found to be directly affected. the rama regulon overlaps with the mara and soxs regulons in e. coli and salmonella but is directly associated with regulating the expression of a subset of genes via a marbox sequence. interestingly, variations in the levels of the expression of the regulon genes were found in the different rama overexpressing strains. m. eshoo°, c. crowder, h. li, h. matthews, s. meng, s. sefers, r. sampath, c. stratton, d. ecker, y.w. tang (carlsbad, nashville, us) objectives: the potential for fatal outcome from tick-borne human infections such as ehrlichiosis emphasizes the need for rapid diagnosis. we developed and validated an ibis t5000 assay (ibis biosciences, inc., carlsbad, ca) that can detect and identify a wide range of tick-borne pathogens from clinical samples. methods: a multi-locus assay was used that employs 16 broadrange pcr primer pairs targeting all known bacterial tick-borne pathogen families. electrospray ionisation mass spectrometry of the pcr amplicons was used to determine their base composition. these base composition signatures were subsequently used to identify the organisms found in the samples. the assay was developed using field collected ticks and a wide range of clinical sample types and has been shown to be sensitive to the stochastic limits of pcr. results: whole blood (198) , cerebrospinal fluid (20) and plasma (1) samples, which were originally submitted for ehrlichia species detection by a colorimetric microtiter plate pcr (pcr-eia), were collected consecutively from january 5 to august 1, 2008 at vanderbilt university hospital. among the total 219 specimens, pcr-eia detected 40 ehrlichia species with a positive rate of 18.3%. the ibis system detected ehrlichia in 38 of the 40 pcr-eia-positive samples and 1 in 179 of the pcr-eia-negative specimens, giving sensitivity and specificity of 95.0% and 99.4%, respectively. the ibis system further characterised the 38 ehrlichia-dual positive specimens to the species level (e. cheffeensis, 35; e. ewingii, 3) with a 100% agreement to that identified by pcr-eia using additional species-specific probes. in addition we demonstrated the detection of borrelia burgdorferi from the blood and skin of a patient with lyme disease. conclusions: we demonstrate broad-range detection of tick-borne pathogens in a single assay using skin, whole blood, plasma, skin and csf. in addition to ehrlichia, the ibis system detected 4 rickettsia rickettsii positive specimens, which were confirmed by serology and clinical findings. the ibis t5000 system, which can be completed within five hours from specimen processing to result reporting, provides rapid and accurate detection and identification of a broad range of pathogens causing tick-borne human infections. r. sampath°, l. blyn, r. ranken, c. massire, t. hall, m. eshoo, r. lovari, h. matthews, d. toleno, r. housley, s. hofstadler, d. ecker (carlsbad, us) objective: to investigate the use of a novel platform-based approach for rapid characterisation of hai organisms. pathogens that cause healthcare-associated infections (hais) pose an ongoing and increasing challenge to hospitals, both in the clinical treatment and in the prevention of the cross-transmission of these problematic pathogens. here we describe the utility of a pcr electrospray ionization mass spectrometry (pcr/esi-ms) detection platform as an innovative, rapid approach for detection and complete characterisation of important hai pathogens. methods: we have developed pcr/esi-ms based methods to rapidly identify and characterise mrsa, vre, c. difficile (nap-1 strain), p. aeruginosa and a. baumannii. each target organism can be analyzed using an independent 8-well assay that can be run on the same platform and can provide species and strain id, virulence factors, antibiotic resistance and genotyping as appropriate. validation studies were performed using 100-300 retrospective, well-characterised clinical isolates for each organism. this was followed by a prospective study for one of the 5 organisms, mrsa, that included screening of 557 clinical specimens (nares swab) from patients who were admitted to a medical unit with a high prevalence of mrsa clinical infections. results: for each of the five hai organisms, pcr/esi-ms species identifications were compared to gold standard testing results from the clinical microbiology laboratory and showed 100% concordance. for s. aureus, p. aeruginosa and a. baumannii, molecular genotyping by pcr/esi-ms was compared to pulse field gel electrophoresis (pfge) clusters and showed >95% concordance. characterisation of virulence and/or drug resistance was performed for mrsa, vre and c. difficile and showed 90−95% correct detection compared to existing testing methods. analysis of clinical specimens for mrsa showed that of the 557 swabs, 95 (15%) contained mrsa, either singly or as a dual infection with cons, 33 (5%) were mssa and 358 (58%) contained meca+ coagulase negative staphylococcus (mr-cons). comparison to gold standard analysis showed 100% sensitivity for mrsa detection with 96.8% specificity, 84% ppv and 100%npv. the pcr/esi-ms technology is a high throughput assay system useful for infection control and for epidemiological studies. it is capable of simultaneous identification of hai organisms while detecting presence of key phenotypic markers and genotypic strain characterisation. m. reijans°, j. ossel, j. keijdener, g. simons (maastricht, nl) objective: molecular diagnostics play an increasingly important role in the detection of infectious agents in cerebrospinal fluids. however, the growing list of targets and the relatively small sample volumes are challenges that demand an improved molecular diagnostic approach. the meningofinder is a multifinder assay allowing the simultaneous detection of 7 viruses and 1 internal control in 1 reaction. until now, the analysis of multifinder assays was based on size-fractionation, identifying each multifinder probe due to its specific length. here we present an alternative approach allowing realtime detection of eight meningofinder probes in a single tube. the realtime detection enables a faster analysis, less handling and lowers the risk of contamination. method: the meningofinder assay is a multifinder assay which detects herpes simplex virus 1 and 2 (hsv1−2), human parechovirus (hpev), cytomegalovirus (cmv), epstein-barr virus (ebv), enterovirus (ev) and varicella-zoster virus (vzv) plus an internal control in a single reaction. each meningofinder probe can be distinguished based upon the specific length of each probe by size-fractionation using gel or capillary electrophoresis. we developed an alternative detection method using fluorescently labelled probes which allow specific identification of 8 multifinder probes in a realtime pcr machine. results: a large number of qcmd samples (n = 44), several enterovirus types (n = 27) and characterised clinical samples (n = 66) were analyzed using the meningofinder. all meningofinder reactions were analyzed by capillary electrophoresis and by fluorescently labelled probes in a realtime pcr machine. the results of the meningofinder showed a very good correlation with the expected results (>95%). furthermore, the results of both meningofinder analyses showed a high degree of correlation. the realtime detection of the meningofinder probes decreases the analysis time and post pcr handling dramatically. we developed a new assay for the realtime detection of 8 meningofinder probes. the realtime analysis showed a very good correlation with the conventional capillary electrophoresis analysis. in addition, the realtime detection reduced contamination risk and patient results became available more quickly. the combination of multifinder technology combined with realtime detection shows great potential in fast and easy multiparameter screening of clinical samples for infectious pathogens. in-house naats were applied to nucleic acid extracts obtained by own in-house methodology in each centre. results: sensitivities for the detection of the respiratory viruses were 40% for commercial mx naat, 86% for in-house mw naat, and 90% for mono in-house naat. the viral load was low each time false-negative results were obtained. false positive results were obtained by all methods used, resulting in specificities ranging from 88%-97%. for the atypical bacteria, the 2 multiplex naats failed to detect low l. pneumophila positive samples and low m. pneumoniae positive sample resulting in sensitivities of 25% and 75% compared to 100% in the inhouse mono naats. the commercial mx naat also failed to detect strong positive samples. no false positive results were obtained for the atypical bacteria. revisiting phage therapy against problematic pathogens s61 how the past feeds the future: from d'herelle to modern phagotherapy the increasing antibiotic resistance problem boosts the interest in alternative treatments for infections. a prominent example for this is the so-called phagotherapy. it makes use of bacterial viruses − bacteriophages − as drugs against bacterial agents. these bacteriophages are isolated from nature, characterised and then tested against the bacterial strains that are targeted. in theory, this approach has several advantages. for instance, bacteriophages infect, as a rule, their bacterial prey very specifically. therefore, they do not harm the commensal bacteria of the patient. additionally, if a bacterial strain becomes resistant against a certain bacteriophage strain, evolution will provide for new and active bacteriophage strains. in practice, phagotherapy has been used for a long time. already one of the two discoverers of bacteriophages, félix d'herelle, was an ardent advocate of this method. in fact, he was the first to use bacteriophages against infections − 1919 against bacterial diarrhoea (shigella spp.). after that, phagotherapy has been used to quite some extent in europe, the us and other parts of the world until penicillin entered the market in the 1940 s. in some parts of the former soviet union and the eastern bloc, the method has been utilised until today. now, several companies and university researchers are developing bacteriophages for therapeutical purposes again. historical documents related to phagotherapy and oral history reveal a fascinating past. bacteriophages have been employed against a wide variety of bacterial diseases in a time in which there were virtually no other anti-infectives. for example, in india, millions of cholera patients were treated with bacteriophages in the 1930 s. anti-cholera phages were also poured into drinking wells as prophylactics. bacterial viruses have also been utilised by the german and soviet armies in the second world war. the history of phagotherapy makes for more than an exciting story, however. analysis of the old literature helps identify important factors for success and failure. this is especially relevant for a field which holds promise but which has had limited funds at its disposal in the past few years − and which, therefore, has been making rather slow progress. additionally, examination of the strategy used for phagotherapy in the soviet union and poland also contributes to a better application of this method today. the discovery of bacteriophages, particularly their ability to replicate and lyse pathogenic bacteria may have been among the most important milestones in the history of biomedical sciences. in the pre-antibiotic era of the early 20th century, phage therapy was becoming a powerful weapon against infectious diseases of bacterial aetiology. unfortunately, phage treatment and research was largely forgotten in the western world as antibiotics became widely available. nowadays, the rapid propagation of multi-drug resistant bacterial strains is leading to renewed interest in phage therapy. in contrast to its decline in the west, phage therapy remained a standard part of the healthcare systems in eastern europe and the ussr during the second half of the 20th century. phage preparations were used for diagnostic, therapeutic and prophylactic purposes to combat various bacterial infections. the eliava institute of bacteriophages, microbiology and virology (tbilisi, georgia) is perhaps the most famous institution in the world focused on the study of bacteriophages, particularly the isolation and selection of phages active against various bacterial pathogens. phages have been isolated against bacterial strains received from all over the former ussr and socialist east european countries; consequently, a huge collection of phages and pathogenic bacterial strains has been constructed at the institute. thousands of people were treated with individual phages and phage mixtures during the soviet era. the preparations developed in tbilisi have been studied through extensive preclinical and clinical trials. however, little of this information has ever been published and even when details are available, the trial reports do not meet internationally approved regulations and standards. bacteriophages have a number of advantages in comparison to antibiotics. phage therapy as an alternative approach for treatment of infections has become an evident and promising remedy. today, many people from various parts of the world express their willingness to take phage treatment against different infections, including those that are caused by antibiotic-resistant bacterial pathogens. the eliava institute has elaborated new, phage-based products and technological schemes for their production. strong collaboration with the medical community in the design of clinical trials according to international standards is absolutely critical to supporting the broader implementation of phage therapy. an australian male aged 57 years died from an intracerebral haemorrhage ten days after he returned from a trip to rural yugoslavia. his kidneys and liver were donated to three female recipients aged 44 years (kidney), 63 years (kidney), and 64 years (liver). four to five weeks after the organ donation, all three recipients died. all had febrile illnesses with altered mental status. subsequent testing of post-mortem tissues from the recipients identified a novel arenavirus, which was related to lymphocyctic choriomeningitis virus (lcmv). this viral detection process involved the use of high-throughput sequencing techniques to identify novel microbial rna sequences. confirmatory testing was performed using the techniques of reverse transcriptasepolymerase chain reaction, immunohistochemical analysis for arenavirus antigens, and immunofluorescent testing for igg and igm antibodies. the clinical features in these four patients as well as other similar problems with transplant-related illness from classic lcmv will be discussed, as well as details of the laboratory identification of this new virus, and implications for organ transplantation protocols in future. successful management of invasive fungal infections depends on timely and correct treatment. over the last decades a number of new tests have become available which have improved the diagnostic options. in contrast to the scenario for bacterial infections, acquired resistance in fungi is rare and thus species identification is a valuable tool guiding choice of treatment. therefore, microscopy & culture is still a corner stone in diagnosis, but culture and identification are time consuming (app. 1−5 and 1−3 days, respectively). the sensitivity and speed of microscopy have been improved by the use of fluorescent brighteners such as calcofluor white or blankophor. but only with the recent development of pna probes specific for a number of the candida spp. has species identification become possible directly from a positive blood culture before subculture on agar media. chromogenic agars allow a presumptive identification of several candida spp. and facilitate the recognition of yeast isolates in samples containing several yeasts or yeast and bacteria in combination. the use of such plates has been shown to lead to a better identification of mixed cultures in a recent nordic eqa scheme including more than 50 laboratories. rapid species identification of the most important candida spp. is possible in the routine laboratory using easy commercially available kits. thus, a species identification of c. albicans, c. dubliniensis and c. krusei can be obtained within minutes using latex agglutination kits (bichro-dubli, krusei-color; fumouze diagnostics) and c. glabrata can be rapidly identified due to its high amounts of preformed intracellular trehalase enzyme (glabrata rtt; fumouze diagnostics). finally, pna probes and fluorescence microscopy can also be used for a same day identification of a range of the clinically relevant candida spp. (advandx). susceptibility testing is possible using etest and the results are comparable with those obtained by reference methodologies in head to head comparisons. however, recent data from eqa distributions suggest that detection of isolates with acquired resistance causes many laboratories difficulties. this illustrates that a critical number of isolates should be tested per technician per week and quality control strains should be included on a regular basis. in conclusion, a number of new diagnostic tests have become available over the last decade and the diagnostic laboratories are encouraged to take advantage of these new options. 19th eccmid, oral presentations since the introduction of newer antifungals with different in vitro spectra, the aetiology of invasive fungal infections (ifi) has become a major diagnostic issue as a prerequisite for a guided antifungal therapy. while molecular methods, such as pcr and sequencing for the diagnosis of ifi have been evaluated from specimens such as blood and bronchoalveolar lavage fluid for some years, they have been less studied for biopsies. characteristics inherent to these molecular methods, e.g. sensitivity, specificity and short turnaround time makes them promising as adjuncts to conventional diagnostic tests, e.g. culture and histopathology from organ biopsies. studies using tissue from animal models of mould infections suggest that pcr might be more sensitive than culture and allows for a better species identification than histopathology. however, most of these studies used assays detecting only a small range of agents or even single organisms. while this may increase the sensitivity of the assays and reduces the likelihood of contaminations it limits the usefulness in the clinical setting, given the broad range of potential fungal pathogens. studies using fresh clinical samples suggest that the detection and identification of a wide range of fungi is possible using broad range assays in combination with sequencing or by combining more specific pcr assays. further studies are needed to optimise dna extraction, define the best molecular targets and the best method for amplicon detection. the prevention of contaminations due to ubiquitous fungi and unspecific amplifications are a major problem, especially when using broad range assays. in contrast, fish probes may potentially be more specific than pcr due to the visualisation of fungal elements in tissue. in contrast to pcr, they appear to work well with formalin fixed specimens. species identification might be more challenging than by pcr and sequencing. direct comparisons between fish and pcr are needed to characterise the pros and cons of each method in determining the aetiology of ifi. molecular tissue diagnosis has the potential to evolve into a useful method to describe the aetiology of ifi even in culture negative samples. results might be obtained fast enough to guide the antifungal therapy in patients with ifi progressive to empiric antifungal therapy. in these patients, the risk associated with invasive tissue sampling might be outweighed by potential benefits of a guided antifungal therapy. the two groups of carbapenemases (serine carbapenemases and metallobeta-lactamases (mbls)) can be encoded by genes that can be carried on plasmids. the serine carbapenemases are distinctly either class a or oxa (class d); the latter being mainly associated with acinetobacter spp. the dominant mbl subgroups, vim and imp have genes that are reportedly carried on plasmids and chromosomes. recent evidence has shown that the majority of blavim-2, even those initially reported, are indeed plasmid mediated and probably accounts for their rapid dissemination. blavim-1 genes have been recently shown to be carried on incn and incw plasmids. the "brazilian" mbl gene, blaspm-1, is exclusively chromosomally encoded. the mbls sim-1 and aim-1 are both chromosomally encoded whereas gim-1 is encoded from a plasmid of approx. 48 kb. the recently described blakmh-1 gene is also carried on a plasmid (200 kb). hitherto, only two mbl-positive plasmid sequences are available thus far -those carrying blaimp-8 and blavim-7. the former carries other resistance genes and are approx. 302 kb (inchi2), whereas the latter is a small plasmid (24 kb) and shows similarities with incp plasmids. oxa carbapenemase genes have been shown to be both plasmid and chromosomally mediated. thus far, the blaoxa-23 and blaoxa-24/40 clusters can be both plasmid and chromosomal and have mainly been found in acinetobacter spp. the blaoxa-48 and blaoxa-58 clusters have been found in k. pneumoniae and acinetobacter spp., respectively, and both are plasmid mediated. blaoxa-48 and blaoxa-58 have been shown to be carried on 70 kb and 28-100 kb plasmids, respectively. a blaoxa-58 plasmid has been recently sequenced and shown to carry two different replicases. the class a carbapenemase genes, blakpc, blaimi-2 and blages are all carried on plasmids. blakpc is found mainly in k. pneumoniae and carried on plasmids that vary in size 12−95 kb and mostly possessing the origin of replication incn. however, kpc-2 has recently described in a pseudomonas as being chromosomally mediated. blaimi-2 is exclusive to the usa and carried on a 66 kb plasmid although blaimi-1 is chromosomal. the blages genes have been found in p. aeruginosa and enterobacteriaceae of which ges-2, 4, 5 and 6 have been shown to be plasmid mediated although little else in known. this lecture will provide a synopsis, discuss the evolution of resistance due to plasmids and briefly predict what we may face in the 21c with respect to carbapenemase resistance. nosocomial infections caused by multidrug-resistant pathogens, especially gram-negative bacilli, have become a serious clinical concern in every healthcare setting worldwide. as well as carpapenemhydrolysing metallo-b-lactamases, ctx-m-type b-lactamases, and qunolone-resistance genetic determinants such as qnr, aac(6 )-ib-cr, and qepa, plasmid-mediated novel molecular mechanisms such as rmta, rmtb, rmtc, rmtd, arma, and npma responsible for pan-resistance to aminoglycosides have recently been identified in pseudomonas aeruginosa, acinetobacter spp., serratia marcescens, esherichia coli, klebsiella pneumoniae, proteus mirabilis etc. since 2003, and these enzymes have indeed methylation activity of 1405g or 1408a at the a-site of the bacterial 16s rrna as found in aminoglycoside-producing actinomycetes. these plasmid-mediated 16s rrna methylases are speculated to be originated from some nonpathogenic environmental microbes that produce aminoglycosides or some similar compounds, so it is quite natural that several new enzymes would be further identified hereafter in both clinical and livestock farming environments. rmtb and arma have widely spread in asia, europe, america and australia via various pathogenic gram-negative bacilli, we should pay special attention to the further spread of such hazardous microbes. in my talk, i would like to give an outline of newly identified molecular mechanisms that confer pan-resistance to aminoglycosides in pathogenic microbes isolated from both human and veterinary environments. [ acquired resistance to quinolones mainly results from chromosomal mutations responsible for modification(s) of dna gyrase and topoisomerase iv, and for a decrease of drug accumulation into bacteria due to decreased permeability and/or overexpression of efflux systems. plasmid-mediated quinolone resistance (pmqr) was first reported in 1998 from the usa, and two other mechanisms have been identified to date. the first pmqr determinants, qnr proteins, belong to the family of pentapeptide repeat proteins. five determinants have been identified: qnra, qnrb, qnrc, qnrd, and qnrs with 6, 20, 1, 1, and 3 different variants, respectively. they may act by binding directly to both dna gyrase and topoisomerase iv leading to protect them from quinolone inhibition. they confer resistance to nalidixic acid and reduced susceptibility to fluoroquinolones (fqs), but may facilitate recovery of mutants with higher level of resistance. the overall prevalence of qnra, qnrb, and qnrs determinants generally ranges from 1 to 5%, and they have been identified worldwide mostly in esbl-producing enterobacterial isolates. the origin of the qnra and qnrs genes were identified as shewanella algae and vibrio splendidus, respectively. the second type of pmqr determinant, aac(6 )-ib-cr, is a variant of the aminoglycoside acetyltransferase aac(6 )-ib which confers resistance to kanamycin, tobramycin and amikacin. this variant possesses two substitutions (trp102arg and asp179tyr) that are sufficient to acetylation of ciprofloxacin and norfloxacin with a 2-to-4-fold mic increase. the overall prevalence of aac(6 )-ib-cr may range from 0.4 to up to 34%, and it has been reported mainly in escherichia coli and klebsiella pneumoniae. the third type of pmqr determinant, qepa, has been identified in two e. coli clinical isolates from japan and belgium. the qepa gene encodes a 14-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily. this protein confers decreased susceptibility to hydrophilic fqs (e.g. norfloxacin, ciprofloxacin and enrofloxacin) with an 8-to-32-fold mic increase. the two epidemiological surveys for qepa may indicate its low prevalence (<1%). the natural reservoir of qepa remains unknown but might be an actinomycetal species. discovering of three main mechanisms of pmqr within the last ten years is peculiar. it may reflect the emergence of novel mechanisms of resistance but also a deeper investigation of resistance mechanisms in clinical isolates. emerging infections: can we cope with them? a. kühn°, c. schulze, h. ranisch, p. kutzer, h. nattermann, r. grunow (berlin, frankfurt-oder, de) objective: little is known about the prevalence of francisella tularensis in humans and animals in germany. interestingly, the pathogen emerged recently when several marmosets (callithrix jacchus) died from tularaemia and a group of hunters became infected in the areas of western germany. to find out more about the distribution of the pathogen also in eastern germany we investigated the seroprevalence of tularaemia under foxes (vulpes vulpes) and raccoon dogs (nyctereutes procyonoides) in the area of brandenburg (around berlin). methods: sera of animals (n = 351 and n = 32, respectively) from the years 2007 and 2008 were tested for f. tularensis − lps antibodies in an indirect elisa and suspicious samples were confirmed by western blot for lps ladder recognition using protein g − pod conjugate. furthermore we investigated the serum samples by a competitive elisa using a peroxidase-conjugated anti − lps monoclonal antibody. results: from the serum collection, we tested 31 (8.8%) foxes and 3 raccoon dogs (9.4%) positive for specific f. tularensis antibodies. the geographical distribution showed hot spots in the area of the investigated region. our results indicate for a higher seroprevalence in wildlife for tularaemia in eastern regions of germany than assumed. since the reported human cases for the last decade seem to be underestimated, the real prevalence of the pathogen is unknown. the high number of tularaemia antibody positive foxes and raccoon dogs indicates that this zoonose is present in wildlife in eastern germany. however, the impact of transmission of zoonotic pathogens from wildlife to domestic animals and humans is not yet well studied. in conclusion, the obtained data will contribute for creating of up-to-date strategy for more efficient control of the two rickettsial zoonoses. objective: helicobacter pylori is established as the primary cause of gastritis and peptic ulceration in humans. in a minority of patients with upper gastrointestinal symptoms long tightly coiled spiral bacteria, clearly distinct from h. pylori, and provisionally named as "h. heilmannii", can be observed in gastric biopsies. our objective was to isolate and identify the spiral organism, resembling "h. heilmannii" from the gastric mucosa of a finnish patient presenting with severe dyspeptic symptoms. methods: we used two different selective media for the isolation of the bacteria from gastric biopsy samples before and after treatment of the patient with a 7-day course with lansoprazole, tetracycline and metronidazole. the isolates were characterised by testing for urease and catalase activity, light and electron microscopy, and sequencing the partial 16s rrna and ureab genes. single enzyme aflp was used to analyse the genetic diversity among the isolates. results: growth of long spiral organisms was obtained from 7 out of 8 antrum and all 8 corpus biopsies before and all three antrum biopsies after treatment of the patient. the partial 16s rrna gene sequence showed high sequence similarities with other gastric helicobacter species. the partial ureab gene showed high sequence similarity with h. bizzozeronii and was clearly distinct from other gastric helicobacter species. aflp indicated that the isolates belonged to the same clone however some minor genetic diversity was observed among the isolates. results: b. pseudomallei was primarily found in close proximity to streams and in grass-rich areas but was also correlated with environmentally disturbed soil such as caused by the presence of animals, farming or irrigation. prediction maps are currently being verified by sampling predicted b. pseudomallei "hot-" and "cold-spots". see in figure a prediction map for rural darwin with red areas indicating high probability for presence of b. pseudomallei. this study contributes to the elucidation of the environmental distribution of b. pseudomallei in endemic tropical australia and to the clarification of environmental factors influencing its occurrence. it also raises concerns that b. pseudomallei are spreading due to changes in land management. o82 concurrent multi-serotypic dengue infections in various body fluids w. kulwichit°, s. krajiw, d. chansinghakul, g. suwanpimolkul, o. prommalikit, p. suandork, j. pupaibool, k. arunyingmongkol, c. pancharoen, u. thisyakorn (bangkok, th) objectives: dengue virus infection is one of the rapidly-spreading emerging diseases worldwide. the virus is divided into 4 distinct serotypes with limited cross-protective immunity; therefore, one can be reinfected with different serotypes. while each episode is usually caused by a single serotype, an individual can occasionally be infected by concurrent multiple ones. our group has previously detected dengue virus from urine and oral specimens of some patients. in this study, we sought to determine the characteristics of multi-serotype infections when analysing beyond the patients' blood compartments. methods: during 2003 during -2007 and adult patients suspected of dengue infections were enrolled. plasma, peripheral blood mononuclear cells (pbmc), urine pellets, buccal brushes, and saliva were collected during and after the febrile episode. only specimens from patients with both positive dengue serology and pan-dengue-specific rt-pcr were included. serotype-specific rt-pcr was then performed on the aforementioned various specimens of each patient. results: 95 patients met the above criteria. serotyping was successful in 85 patients. den-4 was the most common serotype, accounting for half of the cases. 20 of these 85 (23.5%) demonstrated multiserotypic infections when combining data from all specimen types in each individual. serotyping using single, conventional serum/plasma specimens, however, would detect only half of the cases. the phenomenon of concurrent multi-serotypic infections was present in all examined specimen types, including urine pellets, buccal brushes, and saliva. the most frequent combinations were den-1 + den-4 and den-2 + den-4 (5 cases each). two patients were simultaneously infected by serotypes 1, 2, and 4 and one by serotypes 1, 3, and 4. there was no demonstrable significant difference in clinical severity between single-and multi-serotypic infections. conclusion: in a dengue-hyperendemic country with simultaneous circulation of all four serotypes, the phenomenon of concurrent multiserotypic infections are more common than previously demonstrated by traditional serotyping on single serum/plasma specimens. this may be explained by the sensitivity limitation of the detection method or by biological behaviour of the virus. our findings have an implication for potentially more accurate epidemiologic studies in the future, and for further exploratory investigations regarding dengue virus in various secretions and excretions. o83 emerging concepts about the evolutionary history of hantaviruses h.j. kang, s.n. bennett, l. sumibcay, s. arai, a.g. hope, j.a. cook, j.w. song, r. yanagihara°(honolulu, albuquerque, us; tokyo, jp; seoul, kr) objective: recent discovery of genetically distinct hantaviruses in shrews (family soricidae), captured in widely separated geographic regions, challenges the conventional view that rodents are the principal and progenitor reservoir hosts of hantaviruses, and raises the possibility that other soricomorphs, notably moles (family talpidae), harbour hantaviruses. methods: using oligonucleotide primers based on conserved genomic regions of rodent-and soricid-borne hantaviruses, rna extracts from tissues of the japanese shrew mole (urotrichus talpoides), american shrew mole (neurotrichus gibbsii) and european common mole (talpa europaea) were analyzed for hantavirus sequences by rt-pcr. newfound s-, m-and l-segment sequences were aligned using clustal w and were analyzed phylogenetically by the maximum-likelihood and markov chain monte carlo tree-sampling methods, with the gtr+i+g model of evolution. results: novel hantavirus genomes, designated asama virus (asav), oxbow virus (oxbv) and nova virus (nvav), were detected in tissues of urotrichus talpoides, neurotrichus gibbsii and talpa europaea, respectively. sequence and phylogenetic analyses indicated that asav and oxbv were related to hantaviruses harboured by soricine shrews in eurasia and north america, respectively. by contrast, phylogenetic analyses of full-length s-and l-segment sequences showed that nvav formed a unique clade, clearly distinct and evolutionarily distant from all other hantaviruses. despite the high degree of sequence divergence at the nucleotide and amino acid levels, the secondary structures of the nucleocapsid proteins, as well as the l-segment motifs, of the moleassociated hantaviruses were well conserved. conclusions: while cross-species transmission has influenced the course of hantavirus evolution, such host-switching events alone do not satisfactorily explain the co-existence and distribution of genetically distinct hantaviruses among species in two taxonomic orders of small mammals spanning four continents. when viewed within the context of molecular phylogeny and zoogeography, the close association between distinct hantavirus clades and specific subfamilies of rodents, shrews and moles is likely the result of alternating and variable periodic codivergence at certain taxonomic levels through evolutionary time. thus, the primeval hantavirus might have arisen from an insect-borne virus, with ancestral soricomorphs, rather than rodents, serving as the original mammalian hosts. from south-eastern france m. kaba, b. davoust, j.l. marié, m. barthet, m. henry, c. tamalet, j.m. rolain, d. raoult, p. colson°(marseille, toulon, fr) objectives: autochthonous hepatitis e is currently considered as an emerging disease in industrialised countries and several studies suggest that hepatitis e is a zoonosis, especially in pigs, boars and deer. we aimed to study whether hepatitis e virus (hev) is commonly present in domestic pigs in southern france, and to determine the relationship between hev sequences detected from pigs and those described in human hepatitis e cases. methods: serum and stools samples were collected from 207 three or six-month-old pigs from different regions of southern france. 107 sixmonth-old pigs were from a slaughterhouse, and 100 three-month-old pigs were from a pig farm. swine igg anti-hev antibodies testing was performed using a commercial elisa kit for clinical diagnosis with minor modifications. swine hev rna detection was conducted by realtime pcr and amplification/sequencing assays using in house protocols targeting the 5 orf2 region of the hev genome. results: 40% of pigs were seropositive, and 65% of three-monthold pigs were hev rna-positive, whereas none of the six-monthold pigs were hev rna-positive. hev rna was significantly more frequently detected from stools than from serum (65% versus 22%; p < 0.001). phylogenetic analysis showed that swine hev sequences belong to genotype 3f or 3e and formed two clusters within which sequences showed high nucleotide homology (>97%). these clusters were correlated with the geographical origin of pigs as well as with their repartition into pens and buildings in the pig farm where samples were collected. swine hev sequences from the present study were genetically close to hev sequences found from humans or swine in europe, although no strong phylogenetic link could be observed neither with these latter sequences nor with those from human hepatitis e cases diagnosed in the laboratory. conclusion: our data indicate that three-month-old farm pigs from southern france might represent a potential source of contamination to humans, and they underscore the great potential of hev to cause epizootic infections in populations of farm pigs. o85 clostridium difficile: changing epidemiology trends, 2000 -2007 objectives: clostridium difficile infection (cdi) has become a growing concern world-wide with an increased reported incidence and an increase in the associated financial burden. our aim therefore was to review trends in cdi occurring from 2000-2007 inclusive. methods: all patients admitted to lothian university hospitals division (luhd) tested for c. difficile toxins a+b by eia were included. retrospective analysis of prospectively collected data was performed. the number of occupied bed days was provided by nhs-lothian statistics department. the most recent published costs associated with cdi were used to estimate potential costs to lothian nhs trust. results: 50,590 faecal samples were tested for c. difficile toxins from 2000-2007 inclusive; of these 7301 samples were positive. overall cdi was identified in 15.2 cases/10000 patient days and 5.8 cases/1000 inpatient hospital admissions. the incidence of identified cdi rose from 3.6cases/10000 patient days in 2000 to 14.8cases/10000 patient days in 2007. incidence also increased with age from 3.3cases/10000 patient days in the 0−20 years age group to 18.1cases/10000 patient days in the 61−80 years age group. renal medicine and intensive care had the highest incidences of identified cdi with greater than 57cases/10000 patient days each followed by infectious diseases and gastrointestinal medicine whose rates were 47.5 and 42.6 cases/10000 patient days respectively. medicine of the elderly in comparison had an incidence of 19.5cases/10000 patient days. of note 10% of all patients were transferred through a minimum of two specialties during the period in which they remained positive for c. difficile toxins. estimated costs over the study period for toxin testing alone were in the region of £126,500 and the minimal potential hospitalisation costs of patients with cdi was in the region of £20,000,000. conclusion: the incidence of patients identified with cdi has risen markedly and not surprisingly the incidence has also been noted to increase with age. medicine of the elderly however had a much lower incidence than several other specialties and therefore risk assessment of cdi development and containment should now also be targeted within other specialties. with 10% of identified cdi patients transferred through different specialties and the significant financial burden cdi imposes on healthcare institutions judicious application of infection control measures remains an important factor to prevent cdi spread. isolates of this strain were pvl negative, but positive for enterotoxin a (sea) and, in most cases, also for seb, sek and seq. a fifth strain was the "taiwan clone", st59/952-mrsa-v (wa mrsa-9 and -52) which also comprised two closely related sequence types. this strain carried a sccmec element of type v(t) or vii as well as pvl and, usually, seb, sek and seq. it was the most common cc59 strain in wa. the sixth strain differed from the "taiwan clone" in the presence of a sccmec type v element and in the absence of pvl. the differentiation of this clonal complex into various different strains indicates a rapid evolution and spread of sccmec elements, and the diagnostic microarray technology allows one to distinguish beyond mlst level and hence to accurately trace outbreaks and spread of these strains. a sample taker 12 has daily contact with poultry and is excluded from analysis. b sample taker 5 reported no contact with livestock elsewhere than in this study at that moment (spa-types of sample taker 5 and farm are not corresponding). c sample taker 6 tested mrsa-negative in following tests. d sample taker 9 was not tested again. complete data sets (samples taken before, directly after and 24 hours after a visit) were collected on 141 visits by 29 sample takers visiting 50 farms. on 28 farms mrsa was collected from pigs or stabledust (56%). these farms were visited 78 times by 23 different sample takers. one sample taker (#12) was positive for mrsa before visiting a farm, he was removed from the following analysis. fifteen of the 78 (19%) visits to mrsa-positive farms resulted in acquisition of mrsa and 11/23 (48%) sample takers acquired mrsa at least once after visiting a positive farm. of these 11 positive sample takers 2 acquired mrsa twice and 1 sample taker acquired mrsa three times after separate visits. of the 15 acquisitions of mrsa, 13 were negative after 24 hours. the spa-types of mrsa isolates found on the farms and sample takers were grossly comparable. on the 32 negative farms, none of the 60 visits resulted in mrsa acquisition. for further information see the table. discussion: mrsa-cc398 was acquired by 48% of the sample takers after occupational exposure in this study. however, in 11 of the 13 cases the strain was not recovered the next day, therefore acquisition was of short duration, posing a limited treat to human health. some persons seemed to be more vulnerable to acquire mrsa during their work. the sample size of this study was too small to draw final conclusions concerning this inter-personal variation. this requires a more extensive study. [ objectives: community-associated mrsa is an increasing problem and an association with food animal contact has been made in some regions. this has led to concerns about the potential role of food in mrsa transmission. the objective of this study was to evaluate the prevalence of mrsa colonisation of retail pork in canada. methods: pork chops, ground pork and pork shoulders were purchased at retail outlets in four canadian provinces in conjunction with the canadian integrated program for antimicrobial resistance surveillance. both direct inoculation of meat into enrichment broth and rinsing of meat in broth were performed for pork chops and shoulders, followed by inoculation onto chromogenic agar. ground pork was tested only using the direct method. mrsa isolates were typed by pfge and spa typing. real time pcr was used to detect panton-valentine leukocidin genes. results: mrsa was isolated from 31/402 (7.7%, 95% ci 5.5−10.7%) of samples. there was a significant difference between provinces (p < 0.001) but no difference between different products, with mrsa isolated from 23/296 (7.7%) pork chops, 7/94 (7.4%) ground pork and 1/12 (8.3%) pork shoulders (p = 0.99). 21/403 (5.2%) samples were positive using direct culture while mrsa was isolated from 15/355 (4.2%) of samples testing using the rinse method. nine samples were positive on direct culture but negative using the rinse method, while 10 others were positive only with the rinse method and only 5 were positive with both methods. seven samples (ground pork) that were positive on direct culture were not tested using the rinse method. 3 main clones were present. the most common (40% of isolates) was a group of 3 related spa types (t064, t008 and new related type) were classified as canadian epidemic mrsa-5 by pfge, an st8 human epidemic clone that has been associated with horses. pfge-non-typable spa t034 were not surprisingly common, accounting for 30% of isolates. the 3rd main group was 3 related spa types (t002, t045 and new type) that were cmrsa-2 (usa100), an st5 clone that is common in humans in canada, that also accounted for 30% of isolates. the clinical relevance of mrsa contamination of pork is currently unclear. it is possible that contact with contaminated food could be a mode of mrsa transmission in the community, although further study of the prevalence of contamination, amount of mrsa in contaminated samples, sources of contamination and implications on human health are required. o95 prevalence of the novel trimethoprim resistance gene dfrk among german staphylococcal isolates of the bft-germvet monitoring study k. kadlec°, s. schwarz (neustadt-mariensee, de) objectives: very recently a novel trimethoprim resistance gene, dfrk, was identified on a tet(l)-harbouring plasmid in a porcine mrsa isolate from the bft-germvet monitoring study. this study included in total 248 independent coagulase-positive and coagulase-variable staphylococci collected between 2004 and 2006 all over germany: 46 isolates from infections of the urinary-genital tract of pigs, 44 isolates from skin infections of pigs, 57 isolates from respiratory tract infections of dogs/cats, and 101 isolates from infections of skin/ear/mouth of dogs/cats. in this study, we investigated the prevalence and the plasmid location of the dfrk gene among these isolates. methods: pcr primers were designed and a pcr with subsequent restriction analysis of the pcr product was established to detect dfrk. isolates with positive results were tested for a plasmid location of dfrk by transfer experiments and dfrk-carrying plasmids were further analysed. the trimethoprim resistance gene dfrk was detected in another 10 isolates. all isolates were from pigs: 9 from skin infections and the remaining 1 from a urinary-genital tract infection. six staphylococcus hyicus subsp. hyicus isolates, 3 s. aureus isolates (2 mrsa and 1 mssa) and 1 s. pseudintermedius. all these isolates harboured plasmids. in 7 isolates (4 s. hyicus, 2 mrsa and the single s. pseudintermedius), the plasmid location of dfrk was confirmed by protoplast transformation with subsequent susceptibility testing and pcr analysis of the transformants. in all 7 cases, the plasmids harbouring dfrk also carried a tet(l) tetracycline resistance gene. the results of a combined pcr assay with primers from tet(l) and dfrk confirmed that the dfrk gene was always located immediately downstream of the tet(l) gene. further analysis of these dfrk-and tet(l)-harbouring plasmids showed that they varied in size between 6 and 40 kb and that similar sized plasmids differed in their ecorv and hindiii restriction patterns. the novel trimethoprim resistance gene dfrk occurred in 11 (12.2%) of the 90 porcine staphylococcal isolates from the bft-germvet study. in 8 (72.7%) of the 11 isolates, it was located on structurally diverse plasmids, however, always in close proximity to a tet(l) gene. the linkage of the dfrk and tet(l) genes allows the maintenance and coselection of such plasmids under selective pressure by either tetracyclines or trimethoprim, both of which are widely used in veterinary medicine. (table) . the isolates were resistant to ciprofloxacin, clindamycin, erythromycin, gentamicin but susceptible to vancomycin. only one se was methicillin-susceptible and two isolates were quinupristin/dalfopristin non-susceptible. all strains were clonally related and clustered into three subtypes (a, a1 and a2). cfr gene was detected in a linezolid non-susceptible strain (mic, 64 mg/l), which was recovered from a 57 y/o male who underwent liver transplantation. plasmid analysis identified six plasmid bands ranging from c.a. 1.5-to 154-kb in the cfrcarrying strain. hybridisation signals were observed from the 154-kb plasmid band as well as from a chromosomal band after i-ceui digestion. mutations at the 23s rrna, l4 or l22 were not detected. the cfr increased the linezolid mic value between 8-and 16-fold. this report highlights the ability of se to acquire linezolid resistances. the potential mobility of cfr combined with the clonal tendency for dissemination among staphylococcus spp., represent a serious threat to several potent gram-positive-active agents, including oxazolidinones. active surveillance combined with effective infection control and molecular studies seem prudent to minimise the spread of these resistance mechanisms. the objective is to get a glimpse of the potential impact of infectious diseases on music, as regards to the composer's or performing musician's own disease, living conditions or other relevant elements which might have affected the end result, the music we enjoy today. as music is an art of senses, full of drama, despair, realities of life − or just the opposite, blissful ignorance of those realities, full of romance, beauty, and delicacy − various forms of music was researched paying special attention to infections which potentially have played a significant role in the birth of that particular piece or performance. the entire research process was subjective, biased, and emotional, but done wholeheartedly. it aimed at to taking into account, not only the personal life of a composer or performing musician, but also the historical context in which the music was born. musical examples, served to the audience along with the essential background data, will show the extent to which infections have impacted music. regarding the aetiology of those infections, bacterial, viral and parasitic agents are well represented. in addition, many epochs in history have played their role. sometimes, the connections are surprising, even dramatic. if listened to with a tender ear, music quite often turns out to be affected also by infectious diseases. as physicians we should realise the strength with which some people are driven by this demonic, divine − but altogether beautiful force: music. the prevalence of antibiotic resistance has been increasing in asian countries in recent years. this problem has most likely arisen due to a combination of inadequate infection control practices particularly in hospital settings and the widespread misuse of antibiotics in hospital and community settings. factors that lead to antibiotic misuse include inappropriate antibiotic prescription due to a lack of clinical, microbiological and/or imaging data in many clinical settings in the asian region. a lack of separation of prescribing and dispensing by medical practitioners as practised in many countries in asia as well as the easy availability of over the counter medications also contribute to antibiotic misuse. optimal control of antibiotic use can only be achieved through a multipronged approach that includes better education of the public and medical practitioners on rational use of antibiotic, a review of the health system structure, as well as better control of over the counter sales of antibiotics. upgrading of microbiology and other laboratories and radiological facilities that will enhance the accuracy of clinical diagnosis is also urgently needed in most developing countries to keep pace with the complexities of managing patients in this new era to minimise the widespread practise of inappropriate antibiotic use. examination of the csf for microorganisms, wbc and differential counts, and concentrations of glucose and protein is the primary investigation to diagnose meningitis. however, this csf examination may not always be conclusive, and it can be difficult to distinguish bacterial from viral meningitis. therefore, improvement in diagnostic sensitivity and specificity of bacterial meningitis and development of rapid test for a bacterial aetiology are still needed. this presentation gives a review of the strength and weakness of several analyses and methods to reveal the microbiological agent (i.e. csf microscopy and culture, antigen or antibody detection, molecular methods to detect dna or rna) and the use of several mediators of the host immune response for diagnostic and prognostic purposes. bacterial meningitis is a medical emergency that requires a multidisciplinary approach. a diagnosis of bacterial meningitis is often considered, but the disease can be difficult to recognize. recommendations for antimicrobial therapy are changing as a result of the emergence of antimicrobial resistance. in this lecture, current concepts of the initial approach to the treatment of adults with bacterial meningitis will be summarised. the management of the critically ill patient with bacterial meningitis poses important dilemmas. controversial areas (i.e., prehospital admission antibiotics) will be reviewed and relevant literature will be discussed in the framework of current treatment guidelines, highlighting new developments in adjunctive dexamethasone therapy. acute bacterial meningitis (abm), especifically when caused by infection with streptococcus pneumoniae, still has an unacceptably poor prognosis with a mortality of 10−30%. bacterial infection of the meninges causes one of the most powerful inflammatory reactions known in medicine. yet 50 years ago, this inflammatory reaction was suggested to contribute substantially to brain damage. this concept underlies the use of anti-inflammatory agents as adjunctive therapy in abm. of all adjunctive treatments in abm, only corticosteroids have been properly evaluated in clinical trials. these trials recommend corticosteroids in patients with haemophilus influenzae type b and pneumococcal meningitis (pm). however, adjunctive corticosteroid therapy has several weaknesses such as a narrow treatment window and borderline effects on neurologic sequelae. thus, there is still the need for additional or alternate adjuvants in the therapy of abm. experimental studies using animal models (predominantly of pm) have provided insight into the pathogenic mechanisms underlying brain injury in abm. it is now clear that the autodestructive inflammatory reaction is initiated by the interaction of bacterial components with host pattern recognition receptors (prr) like toll-like receptors (tlr). prr signaling results in the activation of transcription factors like nf-kb which up-regulate the production of proinflammatory cytokines. cytokines like il-1b are also potent triggers of nf-kb activation and therefore can exaggerate the inflammatory reaction (via positive feedback loops). as a consequence, great numbers of neutrophils are recruited to the meninges. activated neutrophils release many potentially cytotoxic agents including oxidants and matrix metalloproteinases that can cause collateral damage to brain tissue. additionally to the inflammatory response, direct bacterial cytotoxicty has been identified as a contributor to tissue damage in abm. thus, experimental studies point at four different targets of adjunctive therapy, namely interference with (i) the induction of inflammation (e.g., tlr blockade), (ii) the exaggeration of inflammation (e.g., il-1 antagonism), and (iii+iv) the generation of cytotoxic factors (either of host or bacterial origin, e.g., scavenging of oxidants). this presentation will give an overview of the pathophysiology of abm (with special emphasis on pm) and highlight promising targets for adjunctive therapy in abm, as deduced from experimental studies. a clinician's approach to managing difficult infections s120 acute post-surgical prosthetic joint infection optimal management of prosthetic joint infections (pji) remains undefined. important issues such us when the implant can be retained (conservative strategy), optimal duration of antimicrobial therapy (at) or the role of rifampin are yet matter of controversy. in spite of a number of reports, literature appears confusing. among the limitations of the literature we must emphasize: 1) different criteria to classify pji; 2) different criteria to select for conservative strategy (cs); 3) no description of the initial population from which patients were selected for cs; 4) very different at (from 4 weeks to chronic suppressive therapy); 5) low numbers of patients or short follow-up; 6) absence of clinical trials. it is not so surprising that the rates of cs success have varied from 0 to almost 100%. the most useful classification to approach pji was proposed by tsukayama (1996) . in his series 25 out of 35 patients with early pji managed by a cs (debridement, exchange of polyethylen and implant retention) were cured after 4 weeks of at. the spanish group for the study of pji was constituted in 2003 within the spanish network for the study of infectious pathology (reipi), a public funded initiative. data from 139 consecutive cases of early pji attended in 10 hospitals were recorded in an online database. 117 cases managed with cs could be analysed (mean followup of 2 years). sixty-seven patients (57.3%) were cured after a mean of 81 days of at. in 35 (29.9%) the infection was not controlled (or relapsed) after a mean of 84 days of at, and the implant had to be removed. in other 15 patients (12.8%) the implant was not removed, but suppressive at was given because of suspected ongoing infection. results were significantly worse in one hospital. no other factors resulted statistically significant, but there was a trend of worse results for mrsa produced infections (p = 0.06). time from the symptoms appearance to debridement was shorter in successfully treated cases (median, 7 days) than in failures (median, 10 days); p = 0.08. good functional results were obtained in patients with successfully cs. in summary, a substantial proportion of early pji can be managed with cs strategy and a definite (non suppressive) at. it is difficult to identify patients at higher risk for failure, although mrsa aetiology and longer time until debridement seem to predict failures. different outcomes in some centres suggest that surgical technique could be an important factor for failure. more than 3 million cardiac pacing systems are implanted worldwide and the estimated rate of infections after implantation of permanent endocardial leads is 1% to 2%, but varies between 0.1 to 20%. pacemaker infections correspond to different clinical situations including localised infection in the device pocket, pacemaker leads to systemic infection associated with bacteraemia and lead-associated endocarditis. this latter represents 10 to 25% of all cases of pacemaker infections. the severity of pacemaker related infective endocarditis is sustained by a mortality range between 10 to 20%. risk factors related to infections of implanted pacemakers are correlated with fever before 24 h before implantation, temporary pacing before implantation and early re-interventions (haematoma, lead dislodgment). in contrast, an inverse correlation is observed between development of infection and antibiotic prophylaxis and implantation of a new system. data to guide therapy in patients with pacemaker infection are limited and the most appropriate management remains to be determined. according to different series, staphylococci accounted for 60 to >90% of the responsible organisms. coagulase-negative staphylococci (cns) are reported as predominant pathogens following by staphylocococcus aureus. the biofilm production, responsible for bacterial survival, and the emergence of methicillin-resistant in s. aureus and cns have complicated the management of pacemaker infections. this implies that empiric treatment of suspected pacemaker infection should coverage for staphylococci including methicillin-resistant strains. streptococci, corynebacterium spp, propionibacterium acnes, gram-negative bacilli and candida spp can cause occasional infections. the optimal therapy combines complete device extraction (percutaneous ablation or surgical removal during extracorporeal circulation) and prolonged course of antibiotics, in particular in case of multiresistant bacteria. leaving the device intact is associated with increased mortality and risk of relapsing or persistent infections. in absence of prospective studies, the duration of antibiotic treatment remains to be determined but 1 month has been shown not to be associated with an increased incidence of relapse. shortest course of treatment (2 weeks) has been proposed in case of vegetations strictly localised to leads without affecting cardiac valves. antibiotic therapy working alone should be reserved for highly selected patients. infection remains the most critical complication of ventriculoperitoneal shunt placement with an incidence of 2.2−39%. factors as the age of patient, aetiology of hydrocephalus, the type of shunt implanted, and the surgeon's experience are determined to be associated with increased risk of infection. children are more likely than adults to acquire shunt infection. the possible reasons are longer hospital stay, higher skin bacterial concentrations, immature immune systems, or more adherent strains of bacteria. staphylococci, as skin commensals, are the main causative organisms. nevertheless, in recent years a change in the epidemiology of microorganisms was observed with an increase of gram-negative bacteria. appropriate systemic antibiotics according to the antimicrobial susceptibility testing and surgical removal of the shunt with temporary external cerebrospinal fluid drainage and shunt replacement following the eradication of the infection are the cornerstone of the treatment of cerebrospinal fluid shunt infections. good compliance with infection control practices, inserion of the catheter under aseptic techniques and short-term perioperative antimicrobial prophylaxis in order to prevent the emergence of drug-resistant subpopulations are important steps in the prevention of shunt infections. o125 influenza in adults admitted to canadian hospitals: data from two seasons a. mcgeer, d. gravel, g. taylor°, c. weir, c. frenette, j. vayalumkal, a. wong, d. moore, s. michaud, b. amihod (toronto, ottawa, edmonton, montreal, saskatoon, sherbrooke, ca) objective: seasonal influenza (flu) remains a cause of substantial morbidity and mortality. antiviral treatment should be considered for all hospitalised patients with influenza. to better understand the epidemiology and burden of illness within the hospital sector in canada and the current use of antiviral therapy, we carried out a multihospital survey of virologically confirmed flu in hospitalised adults. methods: cnisp is a network of largely teaching hospitals across canada that collaborates to collect data on infections in hospitalised patients. during two consecutive years (2006/2007 and 2007/2008) hospitals within cnisp identified inpatients >16 years who had virologically confirmed flu. case patient charts were reviewed to capture demographic and clinical data and to determine whether flu was community (ca) or hospital acquired (ha). cases were reviewed at 30 days to determine outcomes. deaths at 30 days were reviewed to determine whether flu was a main or contributing cause. results: fifteen (06/07) and 11 (07/08) hospitals were recruited from the cnisp network. 532 virologically confirmed cases of flu were found, 182 in 06/07 (95% flu a) and 358 in 07/08 (56% flu a). mean patient age was 67 years, 52% were male. there was documentation of patient vaccination that season in 29%. incidence of ca flu was 11/10,000 admissions in 06/07 (range by hospital 2 − 23) and 27 in 07/08 (1 − 47). admitting diagnoses in ca cases were: pneumonia or influenza 48%, exacerbation of copd 20%, sepsis or fever not otherwise specified 9%, cardiac diagnoses 7%, other diagnoses 16%. 24% of cases were ha, range by hospital 3.9 − 5.4/100,000 patient days. 68% of patients were managed with droplet and contact isolation practices, an n-95 mask was used in 19%. 29% of ca cases but 75% of ha cases received antiviral therapy p < 0.01, almost entirely oseltamivir. 9% of cases were admitted to an icu; 30-day mortality was 8% with 2.6% attributed to influenza. conclusion: there is considerable season-season and hospital-hospital variation in flu in patients in canadian hospitals. hospitalised patients ca flu present with a wide spectrum of clinical diagnoses; nearly a quarter of all cases were ha. few ca cases but most ha cases were treated with antiviral drugs. attributable 30 day mortality was 2.6%. v. papastamopoulos, e. kakalou°, t. panagiotopoulos, j. baraboutis, m. samarkos, a. skoutelis (athens, gr) objectives: our study sought to describe influenza vaccination coverage among adults in greece for the season 2007/08. methods: we conducted a random-sampling, telephone based household survey among adult individuals in greece. for this purpose a sample of 1104 adults representative of the basic demographic, social and geographical characteristics of the overall greek population according to the latest national survey, was used. two target groups were determined for analysis: persons >65 years of age and persons with chronic conditions such as respiratory and heart conditions (other than hypertension), diabetes mellitus and other conditions. results: the influenza vaccination rate for the season 2007/08 among the adult population in greece was: 16% for the overall adult population (19.5% for men, 12.7% for women), 48.1% for people >65 years of age, 31% for persons with chronic illness (32.5% for persons with respiratory illness, 50.2 for persons with heart conditions, 35% for persons with diabetes mellitus). a high rate of 81% of the overall population reaching 88% among persons with chronic conditions report having had any type of contact with the national health system or a private physician within the last three years. among them only 20.1% had been recommended to get vaccinated. among the ones recommended any vaccination, 80.5% of persons with respiratory illness, 100% of persons with diabetes mellitus and 89.1% of persons with heart conditions had been recommended to get the influenza vaccine. conclusions: available data show unacceptably low levels of influenza vaccination coverage among vulnerable groups such as the population over 65 years of age and people living with chronic illness. influenza vaccination is the only preventive measure reducing influenza morbidity and mortality and its use has proven cost-effective among high risk groups. it is also the main vaccine recommended by physicians. however the overall rate of physicians recommendation of vaccination is very low. dynamic efforts are thus needed to design and implement strategies and policies that have demonstrated their rigorous effectiveness in enhancing influenza vaccination coverage rates. conclusions: nasopharyngeal sampling with flocked swabs is well tolerated and suitable to be used in an outpatient setting. implementation of real-time mono and multiplex naats results in a significant improvement of the rate in diagnosing lrti. hrv account for the majority of viral lrti in primary care followed by influenza and coronaviruses but also rsv and hmpv are prevalent in an adult population. in this study, 19 polyomaviruses were detected of which 10 were involved in a double infection. methods: observational analysis of a prospective cohort of 1041 nonseverely immunosuppressed adults with pp requiring hospitalisation (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) . of them, 556 were diagnosed by urinary antigen and/or 650 were diagnosed by culture. overall, 86% of pneumococcal strains were available for serotyping (quellung) and 58% for pfge (smal) and or mlst. the diagnosis of septic shock was based on a systolic blood pressure <90 mmhg and peripheral hypoperfusion with clinical or bacteriologic evidence of uncontrolled infection. results: a total of 114 (11%) patients with pp had septic shock at presentation. patients with shock were younger (61 vs 66 yrs; p = 0.003), were more frequently current smokers (45% vs 28%; p = 0.002), had received more commonly corticosteroid therapy (13% vs 6%; p = 0.015), and were more frequently classified into high-risk psi classes (81% vs 60%; p < 0.001) than those who did not have this complication. they were also less likely to have received prior influenza vaccine (31% vs 48%; p = 0.007) and had more frequently bacteraemia (41% vs 30%; p = 0.014). no significant differences were found in rates of penicillin-(2% vs 2%) and erythromycin-resistance (16% vs 12%). serotype 3 was more commonly associated with shock (40% vs 24%; p = 0.007), whereas serotype 1 was rarely associated with this complication (2% vs 9%; p = 0.041). no significant differences were found regarding genotypes: st2603 (26% vs 16%), netherlands-ser8-st53 (10% vs 3%), netherlands-ser3-st180 (10% vs 8%), spain-ser9v-st156 (10% vs 12%). patients with shock required more frequently mechanical ventilation (38% vs 4%; p < 0.001), and had longer los (19 vs 10 days; p < 0.001). early (10% vs 1%; p < 0.001) and overall case-fatality rates (25% vs 5%; p < 0.001) were higher in patients with shock. conclusions: pp presenting with septic shock is still associated with a poor outcome. it occurs mainly in current smokers, patients receiving corticosteroids, and in those infections caused by serotype 3. prior influenza vaccination and pp caused by serotype 1 are associated with a lower risk of shock. o131 high long-term mortality rate after initial recovery from severe community-acquired pneumonia background: despite the presence of antibiotics and vaccination strategies against pneumocci, community-acquired pneumonia (cap) is still a major cause for mortality in developed countries. however, it is unclear how an episode of cap influences long-term survival after initial recovery. therefore, we determined mortality up to 5 years after discharge in patients hospitalised because of an episode of severe cap in a non-intensive care setting. methods: in 5 hospitals in the netherlands, patients (pts) with severe cap (psi class iv and v without need for treatment in icu) were prospectively followed for 28 days and mortality up to 5 years after discharge was determined using the dutch municipal public records database. we used cox regression analysis to examine predictors for mortality. results: compared to strategy 2, strategy 1 resulted in slightly higher costs (chf 8,748 vs. 8,981) but fewer infections (.008 vs. 0.006) during patients' mean length-of-stay, producing an incremental costeffectiveness ratio (icer) of chf 83,303 per mrsa infection avoided. strategy 3 was dominated by strategies 1 and 2 (both more costly and less effective). sensitivity analyses suggest that prevalence of colonisation on admission is a stronger predictor of cost-effectiveness than the costs of infection or rapid screening, the probability of cross-transmission, or the incremental costs of isolation and contact precautions. increasing the relatively low on-admission prevalence at our centre by 20% lowers the icer to chf 60,973 per infection avoided. in contrast, increasing the cost of each infection, the cost of rapid screening, or the risk of cross-transmission by 20% only marginally affects the icer. conclusion: this analysis suggests that compared to risk factor identification and pre-emptive isolation, universal rapid screening upon surgical admission is not strongly cost-effective at our centre. however, local epidemiology plays an important role. in particular, settings with higher prevalence of colonisation on admission may find universal rapid screening more cost-effective. of note, no screening is undesirable, as costs and infections would be higher. results: admission and weekly screening coupled with patient isolation was found to dramatically reduce the number of mrsa acquisitions. the largest reductions were obtained with pcr technology, followed by chromogenic agar. the differences, however, were surprisingly small, and all screening technologies achieved reductions in mrsa acquisition of close to 80% compared with the no-intervention scenario. nonetheless, chromogenic and pcr-based systems were able to decrease the number of unisolated mrsa-bed-days by approximately 15 and 35% respectively. conclusions: the small differences in the ability of the screening technologies to reduce mrsa acquisition reflect both a relatively low estimated isolation efficacy and the observed highly skewed distribution of icu-stays, and may provide some important insights into the reasons for recent disappointing trial results. in particular, the skewed length of stay distribution means that most mrsa-bed days are accounted for by relatively long-stay patients for whom rapid detection will make the least difference. key sources of uncertainty were found to be isolation effectiveness and attributable mortality due to mrsa infections, both of which are difficult to accurately estimate with currently available data. the model results allow us to quantify the expected value of reducing these key uncertainties, and help to provide a rational basis for setting future research priorities. objectives: we have shown that there is substantial colonisation of mrsa among nursing home residents and staff with our recently conducted point prevalence study in 45 nursing homes which revealed an overall prevalence rate of 24% in residents and 7.6% in staff.1 the aim of this study was, therefore, to test the effectiveness of an intervention in nursing homes which sought to improve standards of infection control as a means of reducing mrsa prevalence. methods: a cluster randomised controlled trial (crct) involving 32 nursing homes, with each home representing the unit of analysis, was performed. the study ran for 12 months with data collected at baseline, 3, 6 and 12 months. nasal swabs were taken at baseline from consenting residents and staff in all homes prior to randomisation with an audit of infection control procedures also undertaken. following collection of these baseline data, nursing homes were allocated to the intervention or control arm (1:1). intervention home staff were trained in infection control, specifically hand hygiene, catheter care, barrier approaches such as use of gloves, aprons and masks, and decontamination of equipment and the environment with usual practice continuing in control homes. after each data collection timepoint, feedback was given to the intervention homes in terms of their performance and further education and training provided as required. the primary outcome was the prevalence of mrsa in intervention homes compared to control sites. results: preliminary analysis of the data has revealed no significant change in the prevalence of mrsa in the intervention and control homes, taking account of the clustering, over the one-year intervention period [risk ratio 0.83; 95% confidence intervals (ci) 0.53−1.29]. however, there was an improvement in infection control audit scores in the intervention homes, with a mean score in control homes at 12 months of 64.4% compared with 81.7% in the intervention sites; these scores were significantly different (paired t-test, p < 0.0001). the results suggest that infection control education and training as implemented in this study was not sufficient to affect mrsa prevalence. therefore, a more detailed education and training package either alone or in combination with mrsa decolonisation of staff and residents, may be required to reduce mrsa prevalence within this unique environment. [ objectives: in a response to the rapid global increase in the nosocomial prevalence of multi-resistant micro-organisms, infection control measures, such as patient isolation, are increasingly used. it is unknown how these measures influence the quality of life (qol) of patients during short-term isolation, and this was determined in a prospective matched cohort study. methods: all adult patients needing isolation in a single-patient room between 11/06 and 03/07 in the umc utrecht were eligible and included 24−48 hours after start of isolation (after giving informed consent and being able to fulfil study requirements). for each index patient we identified two control patients, admitted to the same wards at the same time, yet not subjected to any isolation measure. anxiety and depression and qol were assessed using the hospital anxiety and depression scale (hads) and visual analogue scale (eq-5d-vas) in all patients. opinions on and experiences with isolation were measured in isolated patients by means of a self-developed 'isolation evaluation questionnaire'. results: 42 isolated patients and 84 controls were included, with comparable baseline characteristics (age, sex, nationality, level of education, length of hospital stay and severity of underlying disease and co-morbidity (using the cumulative illness rating scale)). reasons for isolation were clostridium difficile-associated disease (n = 17, 40%), high risk for mrsa carriage (n = 12, 29%), or resistant gram-negative bacteria (n = 7, 17%). mean scores of questionnaires are presented in table 1. isin univariate analysis only duration of isolation of 48 hours (compared to 24 hours) was associated with a reduced quality of life (vas 57.7 compared to 68.7, p 0.02). on a visual analogue score of opposite terms isolation measures were rated with means of 87.5, 83.3 and 70.8 for safety, usefulness and quietness, respectively. conclusion: short-term isolation (up to 48 hours) is not associated with anxiousness or depression, but with positive feelings about safety, usefulness and quietness. index patients (n = 42), mean (sd) 4.7 (3.5) 5.3 (3.5) 9.9 (6.0) 62.3 (15.5) control patients (n = 84), mean (sd) 5.4 (3.7) 5.2 (3.6) 10.6 (6. objectives: there is a lack of data about the impact of healthcare associated infection (hai) on the experience of individual patients. this information is essential to empower health organisations to understand, prioritise, develop and implement solutions that will minimise risks to patients. this study explored comparable narratives from patients who had experienced a staphylococcus aureus blood stream infection with patients who had not. we conducted qualitative semi-structured interviews with eighteen adults who had previously been an in-patient in an acute teaching hospital in scotland. nine patients had had a laboratory diagnosed staphylococcus aureus blood stream infection and nine had no blood stream infection. all patients were interviewed for 20−40 minutes. the interviewer asked patients about their thoughts around hai, what concerns they had or still do, what measures they took to safeguard themselves from hai and how their experience impacted on their confidence of the nhs. probing questions were then asked depending on the responses given to the initial questions. all interviews were recorded, transcribed and analysed thematically. results: analysis of transcribed interviews is ongoing. preliminary analysis showed that all patients had positive and negative comments about infection prevention and control practice in the hospital. specific concerns included poor communication, poor cleanliness, awareness of patient boarding, lack of facilities, staff shortages and multi-tasking. some patients who had experienced bacteraemia said they had not been informed about the infection. those who had been informed were not given clear information about treatment or subsequent results. most patients were not specifically told what they or their family should do to safeguard them from infection and little or no written information about hai was provided. most patients are worried about hai on future admissions. the concerns of patients were not fundamentally different if they did or did not experience blood stream infection. the patient's reported experiences show that they have a broad awareness of systems issues that may increase risk of infection. consequently we need to involve patients in the design and evaluation of systems change and information that will improve patient experience. improving the safety and reliability of the system will have direct benefits for all patients in the hospital, not just the ones at risk of hai. analysis of surgical specialties separately revealed a significant reduction of mortality in cardiothoracic surgery who had been treated with mup-chx (2.3% (5/218) vs. 6.5% (11/170), p = 0.040, figure) . in other surgical specialties no significant difference was found. conclusion: peri-operative application of mup-chx in nasal carriers of s. aureus undergoing cardiothoracic surgery results in a threefold reduction of mortality after one year. o142 a lot done, more to do − a survey of teaching about healthcare-associated infections in uk and irish medical schools h. humphreys°, d. o'brien, j. richards, k. walton, g. phillips (dublin, ie; norwich, newcastle-upon-tyne, dundee, uk) objectives: patient safety and the prevention of healthcare-associated infections (hcai) are increasingly important health issues. medical doctors have traditionally been poor in complying with preventative measures to minimise hcai such as hand hygiene compliance. we surveyed medical schools in the uk and ireland to assess what is being taught and assessed in this area. methods: a questionnaire was drafted, piloted and then subsequently forwarded to the heads of medical schools as well as to known contact professionals with an interest in hcai in 38 medical schools. the questionnaire surveyed topics covered in the curricula, the modalities used to assess knowledge and practice, the usefulness of various teaching methods and materials, e.g. lectures, and what education resources were available. results: replies were received from 31 (82%) medical schools; two supplied data on their undergraduate and postgraduate courses. only 18 (60%) covered hcai as a quality and safety issue but over 90% covered prevalence, recognised risk factors, transmission, and preventative measures. 24 (80%) medical schools assessed competence in undertaking aseptic techniques and the disposal of sharps and mcqs were the most common (87%) means of assessment. case scenarios, resource materials and clinical skills stations were used in educating students in 26 (87%), 22 (73%) and 22 (73%) medicals schools respectively. 25 (83%) medical schools would be willing to share educational resources on hcai with other medical schools. conclusions: medical schools in the uk and ireland include hcai in their curricula but its importance as a safety and quality issue needs to be further emphasized. there is potential for agreeing a core curriculum on hcai and for sharing teaching resources such as videos and e-learning material. objectives: noroviruses are most common cause of outbreaks of gastroenteritis in uk national health service hospitals, leading to ward closure costing as much as £115 million per annum. using a detailed data set on norovirus outbreaks from three hospital systems in the south west of england, we estimated (1) the relative importance of introduction of norovirus from the community and within the hospital and (2) the cost effectiveness of ward closure at different time points during an outbreak. methods: using regression models we examined the association between number of new outbreaks in a hospital and community levels of activity and number of outbreaks currently occurring in other wards within the hospital. we examined the effect of different ward types (admission, general and long stay units) and whether the ward was open or closed to new admissions on a given day. we then undertook as analysis of cost (-effectiveness) of unit closure by developing a dynamic transmission model taking into account that ward closure may reduce norovirus transmission within and between wards. the stochastic simulation model was based on the actual characteristics of an acute hospital and the norovirus transmission parameters quantified in the statistical analysis. we measured the costs and benefits of closing affected wards at 1, 3 and 5 days after the onset of symptoms in the first case. results: community level of norovirus infection had a significant effect on the occurrence of new outbreaks as did outbreaks in admission and general medical units. the cost of closing wards to new admissions varied between £0.5 million to £0.9 million depending on the assumed effectiveness of closure in curtailing transmission. cost of bed day loss − compared with staff illness -accounted for around 90% of the total cost of closure. although the total number of cases tends to fall with rapid ward closure (by around 50% compared with no closure), the actual cost of control is similar regardless of when the closure is performed. we have developed a modelling framework to assess the effectiveness and cost-effectiveness of strategies to control norovirus outbreaks in hospital settings. ward closure is effective at preventing cases but since closure itself is an expensive intervention, it may not always be cost-effective. . other prevalent ribotypes were 001 (25%) and 106 (36%). 76% of the 027 isolates originated from 5 hospitals located in 2 healthboard areas. the remaining 18 isolates of ribotype 027 originated from 11 hospitals across scotland. in vitro 96% of 027 isolates were resistant to clindamycin with a mic range of 8−24 mg/l, mic50 of 12 mg/l and mic90 of 16 mg/l. furthermore 100% of the 027isolates were highly resistant to erythromycin (mic50 256 mg/l, mic90 256 mg/l), and to levofloxacin and moxifloxacin (mic50 32 mg/l, mic90 32 mg/l for both), while 65% of these isolates were resistant to cefotaxime (mic50=64 mg/l, mic90=96 mg/l). all 027isolates were susceptible to metronidazole, vancomycin, meropenem and piperacillin-tazobactam. high frequencies of clindamycin, erythromycin, levofloxacin, moxifloxacin and cefotaxime resistance were also found among isolates of ribotype 001 (90−99%) and 106 (94-100%). conclusion: until 2008 c. difficile ribotype 027 was only reported infrequently in scotland. in 2008, reports of ribotype 027 became more frequent and clusters were detected in 5 hospitals. the majority (96%) of ribotype 027 isolates were resistant to clindamycin. three other european countries have previously reported clindamycin resistance in pcr ribotype 027, albeit with a higher mic90 of >256 mg/l. objectives: to analyze trends in mortality due to clostridium difficile enterocolitis and to describe the most affected groups in order to better understand current clostridium difficile changing epidemiology. methods: we reviewed mortality data from the flanders and brussels regions in belgium (about 7 million inhabitants). we selected those records in which icd-10 code a04.7 (enterocolitis due to clostridium difficile) appeared as underlying cause of death within the death certificate. age-and sex-specific mortality rates were calculated for the period 1998-2006. direct standardisation was performed using the european standard population and 95% confidence intervals were calculated. stata 10 ® and excel ® were used as statistical software. objectives: toxigenic clostridium difficile is an enteric pathogen typical in the hospital environment but also community-acquired cases have been reported. however, relatively few attempts have been made to clarify the role of soil or water as a source of c. difficile infection. in november-december 2007, the drinking water distribution system in the town of nokia, finland was massively contaminated with treated sewage effluent resulting in a large gastroenteritis outbreak. the aim of the present study was to evaluate if contaminated water in this outbreak was also a potential source of c. difficile infection. a sample from the contaminated tap water and a treated sewage effluent sample were collected as soon as possible after the massive faecal contamination of the drinking water distribution system had occurred. c. difficile was isolated from heat-treated water samples by filtrating of 100 ml, 10 ml and 1 ml volumes of water and placing the membranes on selective ccey agar plates, which were anaerobically incubated for 3 d. stool samples from the patients fallen ill during the epidemic were examined for enteric pathogens, including c. difficile. all potential c. difficile colonies were subcultured on ccfa agar plates and toxin-positive isolates were identified by pcr. pcr ribotyping was performed according to the protocol of the anaerobe reference unit in cardiff, uk, using the cardiff-ecdc culture collection as a set of reference strains. after gel electrophoresis, the band patterns were analyzed using the bionumerics software. results: altogether 22 c. difficile isolates were found in water samples. twelve isolates were toxin-positive; 5 isolates were from contaminated tap water and 7 isolates from treated sewage effluent, the latter being the contamination source. among the tap water and sewage effluent isolates, 4 and 5 distinct pcr ribotype profiles were identified, respectively. the 9 human faecal c. difficile isolates detected were divided into 4 distinct pcr ribotype profiles. none of the profiles were identical with that of the hypervirulent pcr ribotype 027. two isolates, one from tap water and another from a patient, had an indistinguishable pcr ribotype profile. conclusion: our observation implies that c. difficile contamination of a tap water distribution system had occurred. waterborne transmission of toxigenic c. difficile and subsequent c. difficile infection seems possible. objectives: an accurate and rapid method is needed for typing of toxigenic clostridium difficile. a commercial automated repetitive pcr system (rep-pcr; diversilab ® , biomérieux inc., st louis, usa) utilises amplification and subsequent automated electrophoretic separation of the repetitive extragenic palindromic sequences of c. difficile. our aim was to evaluate the performance of this rep-pcr method for genotyping of c. difficile isolates and to compare it to pcr ribotyping. in addition, the correlation between the rep-pcr and the virulence gene profiles of c. difficile strains was studied. methods: a total of 195 toxin-positive c. difficile isolates were studied. we included consecutive isolates from two laboratories in finland, containing also strains of the hypervirulent c. difficile ribotype 027. in addition, selected c. difficile strains with >18 bp deletions in their tcdc genes were analyzed. the dna was extracted and the rep-pcr performed according to the manufacturer's instructions. the amplification products of rep-pcr were detected and analyzed using the diversilab system. further analysis was performed with the web-based software accompanying the system. the usefulness of the library construction option of the diverslab system for isolate comparison was tested. the virulence genes (tcda, tcdb, cdta, cdtb and tcdc) were analyzed by conventional pcr and the whole gene sequencing of tcdc was performed from isolates with deletions >18 bp. pcr ribotyping was performed using the protocol of the anaerobe reference unit in cardiff, uk. the correlation between the rep-pcr profile and the ribotype was excellent. all major ribotype groups were clustered in their own rep-pcr groups. interestingly, subgroups could be found with rep-pcr within two most prevalent ribotypes 001 and 027. the automated rep-pcr proved to be reproducible; the results from separate dna isolations and pcr-runs/microfluid electrophoresis as well as the results performed by different individuals of laboratory personnel were comparable. the rep-pcr profiles and pcr ribotypes correlated also with the virulence gene profiles. conclusion: this automated rep-pcr represents an effective and reproducible method for the genetic characterisation of c. difficile strains in clinical laboratories with molecular biology facilities. the constructed c. difficile library allows comparing the relatedness of c. difficile strains and their fingerprints over time. objectives: clostridium difficile infection (cdi) is a serious diarrhoeal illness associated with high morbidity and mortality. currently available treatments (oral vancomycin or metronidazole) usually produce good resolution of diarrhoea but are associated with a 20% to 30% incidence of recurrence. opt-80, the first in a new class of macrocyclic antibiotics, is bactericidal via unique inhibition of rna polymerase. this phase 3, non-inferiority clinical trial was conducted in more than 100 sites in north america and compared the efficacy and safety of opt-80 and vancomycin in treating cdi. methods: eligible patients were adults with acute cdi symptoms and a positive stool toxin test. patients received oral opt-80 (200 mg twice daily) or oral vancomycin (125 mg 4 times daily) for 10 days. primary end point was clinical cure (resolution of symptoms and no further need for cdi therapy 2 days after stopping study drug). secondary end point was cdi recurrence (diarrhoea and positive stool toxin test within 4 weeks after treatment). global cure was defined as a clinical cure with no recurrence. results: 629 patients were enrolled and 87% were evaluable. in the per protocol (pp) population (n = 548), mean age was 61.3±17.1 years and 44.0% of patients were male. equivalent rates of clinical cure were observed with opt-80 (92%) and vancomycin (90%) in the pp analysis; similar outcomes were observed in a modified intent-to-treat (mitt) analysis. significantly fewer patients treated with opt-80 (13%) than vancomycin (24%) experienced recurrence in the pp analysis (p = 0.004) and in the mitt analysis (15% vs 25%; p = 0.005). significantly more opt-80-treated patients achieved global cure (78%) than vancomycintreated patients in the pp analysis (67%; p = 0.006) and in the mitt analysis (75% vs 64%; p = 0.006). opt-80 was well tolerated with an adverse event profile similar to that of vancomycin. in this study -the largest comparative trial of a new antimicrobial agent versus vancomycin for the treatment of cdi -clinical cure rates after treatment with opt-80 or vancomycin were equivalent. however, opt-80 was associated with a significantly lower recurrence rate and a higher global cure rate than vancomycin. opt-80 is an oral, non-absorbed agent that has a convenient (twice daily) dosing schedule and low risk of adverse events. opt-80 represents a potential new treatment option for cdi that is associated with a lower recurrence rate than currently available treatments. results: sequence analysis (sa) revealed that locus a is absent in type 078 and that some mismatches are present in the primer annealing sites for loci b, c and g. lowering the annealing temperature and increasing the magnesium chloride concentration for loci b, c and g resolved the low yield of pcr products. applying the mlva on 54 type 078 strains revealed that 42 (80%) strains, encompassing isolates from human (n = 42) and porcine (n = 11) origin, are genetically related with a summed tandem repeat differences (strd) 10). three clonal complexes (cc, defined by strd 2) were recognized; one cc contained both human (n = 4) and porcine (n = 3) strains. the optimised mlva identified 3 genetically related clusters and 6 cc among the 67 isolates from e and ni. ccs contain isolates from more than one hospital and indeed for several clusters isolates from both e and ni. 2 isolates obtained from ni 8 years earlier were part of one large cc. the optimised mlva can distinguish and/or group type 078 strains from distinct settings. type 078 strains from human and animal origin are genetically related. the clustering of some isolates from distinct settings is consistent with community sources for type 078. the last 2 observations suggest zoonotic transmission. objectives: this paper updates our assessment of the contribution that community-associated clostridium difficile infection (cdi), as reported to the english mandatory surveillance scheme since 2007, makes to both the acute and community sectors of the national health service (nhs) in england. methods: nhs acute trusts (hospital groups) in england are required to report all c. difficile toxin positive diarrhoeal specimens processed by their laboratories whether the patients were in hospital or the community at the time of onset of the illness or when the specimen was taken via a web enabled reporting system. positive specimens from the same patient within 28 days are not reported. reported cases in patients under 2 years of age were omitted from this analysis. enhanced surveillance data (including information on date of admission, patient location prior to testing, sex, age and patient category) on cdi have been collected through a web-enabled reporting system since april 2007. risk factor information is completed on a voluntary basis. results: more than 75,000 cases of cdi in patients aged >2 years were reported, 23% of these cases were taken in non-acute settings of which 74% were taken by a general practitioner. a further 17% of specimens were taken on presentation or <2 days of admission into an acute trust. approximately 32% of all cases had at least one risk factor field completed, >19,000 cases reported risk factor information on episode category; 23% of these cases were community associated and 77% were hospital acquired. the information reported suggests that only 3% of the community associated cases were from patients with continued infection or relapsed episodes of cdi, this is compared to 8% of the hospital acquired cases who had continued infection or relapsed episodes of cdi. conclusions: 23% of the c. difficile specimens reported by acute trusts were diagnosed in a community setting. published studies suggest that 12−15% of these might be expected to have been acquired during a hospital stay within the previous month (i.e. were community onset hospital acquired cases). future work is required to investigate whether there are differences in the epidemiology, risk factors e.g. antibiotic exposure and outcome of patients with community onset disease. o152 clostridium difficile-associated disease: a newly notifiable disease in ireland m. skally, f. roche, d. o'flanagan, p. mckeown, f. fitzpatrick°( dublin, ie) new cases of clostridium difficile-associated disease (cdad) became notifiable in ireland on 4th may 2008. the main objective of this new notification process was to provide a national overview of the epidemiology and burden of cdad. this paper review the first six months of preliminary data notified. methods: the interim case definitions for new and recurrent cdad cases proposed by the european society for clinical microbiology and infectious diseases (escmid) study group for c. difficile were employed. this report reviews the weekly events of cdad extracted from the computerised infectious disease reporting (cidr) system in january 2009. census of population 2006 figures were used as denominator data in the calculation of incidence rates. results presented represent 34 weeks of data submitted. results: there were 1581 new cdad cases notified on cidr between the 4th may 2008 and 27th december 2008, representing a crude incidence rate (cir) of 37.3 cases/100000 population (estimated annual cir is 57.0 cases/100,000). all cases were laboratory confirmed. there was a higher occurrence of cases in females. the male:female ratio for the period was 1:1.6. in 0.4% of cases the sex was unknown. 71.4% of cases were in the greater than 65 years age category. the preliminary data submitted on cidr indicate that 63.0% of cases were hospital inpatients and 8.9% of cases were either gp patients or outpatients. the origin of 28.1% of samples is unknown. there was large variation between the 8 public health regions (table 1) . the incidence of cdad in ireland is prominent in older age groups and in healthcare settings. what is more remarkable is the regional variation of cases reported. this varies from 9.1 per 100,000 in the north east to 52.4 per 100,000 in the west. the seasonal trend is indistinguishable at present due to late and batch notifications from institutions. o153 clostridium difficile-associated diarrhoea in immunosuppressed patients with cancer objective: to assess the epidemiology, clinical features and outcome of clostridium difficile (cd) associated diarrhoea in immunosuppressed patients with cancer. methods: review of all episodes of cd associated diarrhoea documented in adults with cancer and haematopoietic stem cell recipients (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) . microbiologic diagnosis included cd isolation from stool samples, direct detection of cd toxin, and testing for cytotoxin production by the isolated strain. we documented a significant increase of cd associated diarrhoea, from 0.34/1000 admissions in 2000 to 4.05/1000 admissions in 2008 (p < 0.01). there were 56 episodes in 54 patients. thirty-one patients were male (55%) with a mean age of 52 years (± 16). forty three (77%) patients had an haematological underlying disease and 13 had solid tumour; 41 (73%) had received previous chemotherapy, 14 (25%) were stem cell transplant recipients (3 presenting with gvhd) and 17 (30%) were neutropenic (<500). in the previous month 52 patients (93%) had received one or more antibiotics (cephalosporins 63.5%, glycopeptides 40%, carbapenems 38.5%, betalactam + betalactam inhibitors 29%, quinolones 19%). fever >38ºc (71%) and abdominal pain (44%) were the most frequent manifestations, and the diarrhoea was hemorrhagic in 8% of the cases. most patients (77%) were treated with metronidazole (median 11 days), and the antibiotic therapy was discontinued in 56%. in 5 patients who had recovered from neutropenia, the diarrhoea resolved just by discontinuing the antibiotic therapy. no patient developed toxic megacolon or needed surgery. three patients (5.5%) had relapses. overall mortality (<30 days) was 22% (12 patients). the incidence of cd associated diarrhoea in cancer patients has increased significantly in recent years. it is related with important morbidity and mortality. better strategies to improve its prevention and treatment are needed. s154 linking research to the clinic: how laboratory findings relate to management of invasive candida infections the role of the research laboratory in the management of invasive candida infections goes beyond routinely available tests for identification of candida species and susceptibility testing of antifungal agents. cutting-edge molecular epidemiology technologies have been used to type isolates of candida species based on their dna sequences. multilocus sequence typing schemes have been designed for c. albicans, c. dubliniensis, c. glabrata, c. krusei and c. tropicalis. multi-locus sequence typing can be used to investigate possible hospital outbreaks of infection (finding widely different strain types within a unit indicates no outbreak, although the converse is not true). for c. albicans, typing multiple isolates from the same patient has shown that people tend to harbour as commensals a mixture of closely related but different strain types, which may provide for selection of the most appropriate type for invasion of a particular tissue or in response to antifungal treatment. strains in c. albicans clade 1, the largest group of related strain types, have a higher proportion of isolates resistant to flucytosine than other clades, and they all share a common resistance mechanism. research on mechanisms of resistance of candida species to many types of antifungal has progressed to the point that some investigators are looking to design dna chips that could be used both for identification and for susceptibility testing of a candida isolate. much research effort goes into detailed study of host-fungus crosstalk in experimental candida infections. animal models of infection have been greatly refined and the latest research shows how early release of chemokines that attract neutrophils into infected tissues contributes to the immunopathology of candida infection. this rapid, innate immune response also emphasizes the need for antifungal intervention at the earliest possible stage to provide the best chance for successful treatment of a disseminated candida infection − a finding now supported by clinical data as well as experimental models. translation of the latest research advances into practical diagnostic tests and new therapeutic approaches for candida infections always takes a long time − typically years − and not all research results find clinical applications. however, the level of effort invested in basic candida research ensures support for steady progress in diagnosis and management. the echinocandins are semi-synthetic lipopeptides that are increasingly used for the prevention and treatment of invasive fungal infections. understanding the pharmacokinetic and pharmacodynamic (pk/pd) characteristics of these compounds is critical for their optimal clinical use. the echinocandins have potent in vitro activity against candida spp., although c. parapsilosis is less susceptible than other candida species. the molecular mechanisms of resistance in candida species, which relate to amino acid substitutions in 'hot spots' within the fks1 gene, are becoming well characterised. susceptibility breakpoints for all three clinically available compounds have been determined recently by the clinical laboratory standards institute, with a 'susceptible-only' breakpoint of >2 mg/l suggested. the pk/pd of the echinocandins have been determined in experimental models of disseminated candidiasis, and of both disseminated and pulmonary invasive aspergillosis. these studies suggest that the echinocandins: (1) display concentration-dependent antifungal killing (or effect); (2) are extensively distributed into peripheral tissues, where they exhibit prolonged mean residence times at the site of infection; (3) are fungicidal against candida spp. and induce dose-dependent morphological changes in aspergillus spp.; and (4) result in a diminished propensity for angioinvasion by aspergillus spp. recent evidence also suggests that the echinocandins have important immunomodulatory properties, which may contribute significantly to their observed antifungal effect. pk/pd modelling and laboratory animal-to-human bridging techniques have been used to identify safe and effective dosages for the echinocandins for relatively uncommon clinical syndromes such as neonatal haematogenous candida meningoencephalitis. these techniques are an efficient method of identifying effective regimens for humans that can be expedited for study in clinical trials. pk/pd modelling techniques can and should be used to address outstanding clinical queries in relation to these compounds, including optimal dosages, decision-support analysis for the setting of in vitro antifungal susceptibility breakpoints and the clinical relevance of inherent or acquired reduced antifungal susceptibility. s156 invasive candidiasis: which antifungal treatment for which patient? management of patients with invasive candidiasis represents a complex issue owing to the heterogeneity of patients in whom these infections occur. established risk factors for invasive candidiasis, which include total parenteral nutrition, multiple organ failure and candida colonisation, are common to many types of patients that are treated within the critical care setting. furthermore, the severity of the underlying condition in these patients necessitates swift antifungal treatment to ensure optimal outcomes. an additional factor for consideration when treating candida infections is the changing epidemiology of candida species; potentially fluconazole-resistant species such as c. glabrata and c. krusei are becoming more common, particularly in patients with prior fluconazole exposure. a range of antifungal agents is available with in vitro activity against candida species. however, not all of these agents are suitable options for the clinical management of invasive candidiasis because of the overall complexity of both infection and underlying condition. for example, the position of the polyenes, particularly amphotericin b deoxycholate, is becoming less tenable as the risk of renal complications is increasingly regarded as unacceptable in patients that are likely to have or be at risk of multiple organ failure. furthermore, because of the increasing prevalence of fluconazole-resistant species, recent guidelines no longer recommend the use of azoles as first-line treatment for invasive candidiasis except in special cases, focusing instead on the echinocandin agents. there is now a wealth of clinical data available for the echinocandins. micafungin, for example, has been assessed in invasive candidiasis in clinical trials that included a wide variety of underlying conditions and patterns of infection, including neutropenic patients and those with deep infections such as peritonitis. furthermore, micafungin is the most extensively evaluated of the echinocandins in paediatric patients, having been tested both in children up to the age of 16 years and in premature infants and neonates. optimal management of patients with invasive candidiasis depends on a strategy that takes into account the complex nature of the disease. judicious selection of antifungal treatment should be accompanied by consideration of non-drug-related factors that improve survival, such as careful assessment of intravenous catheters and their potential involvement in candida infections. patients with invasive candidiasis often have underlying conditions that are severe illnesses in themselves. these range from neutropenia during cancer chemotherapy to the multi-organ failure of intensive care unit patients. against this background of severe underlying illness, it can be difficult to appreciate the success or otherwise of treatment strategies for candida infections. in the last decade, major advances have been made in antifungal therapy with the introduction of 1. echinocandins; 2. extended-spectrum azoles; and 3. lipid formulations of amphotericin b. robust clinical studies for their successful use in candidaemia have been published. however, it is important to translate these studies into practical strategies for the care of individual patients. in this presentation, individual cases will be used to provide insights into the successes and failures of these antifungal classes for the management of invasive candidiasis. specific interest will be focused on the use of fluconazole versus the echinocandins. these micafungin-based cases will be supported by insights from the evidence-based literature combined with practical experiences at the bedside. the factors to be considered are: 1. spectrum of activity; 2. drug toxicity; 3. drug interactions; 4. drug resistance; 5. pharmacology; 6. diagnosis; 7. site of infection; 8. use of biomarkers/cultures in treatment strategies; and 9. costs. it is important to realise that large clinical trials exclude many patients with invasive candidiasis. therefore, with the use of individual cases, it is possible to provide further insights into the clinical use of these outstanding antifungal agents. patient management: the era of rapid diagnostic results (symposium organised by cepheid) s161 will community mrsa and clostridium difficile change infection control in hospitals? infections caused by methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococci, and clostridium difficile are inter-related in healthcare institutions. the emergence of epidemic mrsa and c. difficile strains has placed a greater burden on infection control systems in healthcare facilities, which often must increase surveillance and change disinfection strategies to halt the transmission of these pathogens in hospitals. ironically, the usa300 mrsa strain arose in the community but now is being transmitted frequently in healthcare settings, while the epidemic nap1/bi/027 c. difficile strain was originally a healthcare-associated pathogen, which now is causing considerable morbidity in community settings. to successfully slow the spread of these pathogens, infection control must work closely with both the laboratory and pharmacy services to ensure that these organisms are detected rapidly and that the selective pressure to maintain the organisms in the institution are reduced. clearly, bundles of interventions, rather than single approaches, are necessary to contain the spread of these organisms in hospitals. the continued influx of patients with communityacquired mrsa and c. difficile infections into healthcare institutions is a challenge for infection control practitioners that will clearly increase in the future. the food borne pathogen l. monocytogenes discovered by murray in 1926 is responsible for a severe infection with various clinical features (gastroenteritis, meningitis, meningoencephalitis and materno foetal infections) and a high mortality rate (30%). the disease is due to the ability of listeria to cross three host barriers during infection: the intestinal barrier, the placental barrier and the blood brain barrier. it is also due to listeria capacity to survive in macrophages and to enter into non phagocytic cells, such epithelial cells. recovery from infection and protection against reinfection are due to a t-cell response, explaining why listeria has since many years has become a model in immunology. nearly three decades of molecular biology and cell biology approaches coupled to genetic and post-genomic studies have promoted listeria among the best models in infection biology. in depth studies of the mechanism of entry into cells has help unraveling how listeria crosses the intestinal and placental barrier. unsuspected concepts in cell biology were discovered. post-genomic studies have recently allowed to unveil the listeria transcriptional landscape during switch from saprophytism to virulence. the talk will give an overview highlighting recent results in the frame work of well established data. the last several decades of research in medical mycology have offered great insights into fungal cell biology, epidemiology, phylogenetics and the cells and molecules involved in the pathogenesis of fungal disease. a legitimate question is to ask to what extent our extensive advances in comprehension of the biology of fungal pathogens have contributed to improvements in diagnosis and treatment. to what extent do patients benefit from translation of basic research into tools for clinical management? and the equally valid question: to what extent does biological science benefit from study of fungi that are opportunistic pathogens? the speaker will examine some of these questions from the perspective of long experience in the field and the curmudgeonly attitude that develops with age. objectives: the incidence of invasive meningococcal disease (imd) has been reported in the czech republic since 1943. in response to the emergence of a new hypervirulent clonal complex, cc11, nationwide enhanced surveillance of invasive meningococcal disease was implemented by the national reference laboratory for meningococcal infections (nrl) in 1993. the case definition is consistent with the ecdc guidelines. culture and pcr are used for confirmation of cases. notification is compulsory and is performed by local epidemiologists. strains of neisseria meningitidis isolated from imd cases are referred by the field laboratories to the nrl to be characterised by serogrouping, pora and feta sequencing (http://neisseria.org/nm/typing/) and multilocus sequence typing (mlst) (http://pubmlst.org/neisseria/). in the nrl, the epidemiological database is matched against that of strains to avoid duplicate reporting in the final enhanced surveillance database. results: despite the stable trend in imd incidence (0.8/100 000) since 2005, the case fatality rate was high (11.8%) in 2007. the disease was caused mainly by serogroup b meningococci (67.4%) in 2007, followed by serogroups c (20.9%) and y (9.3%). the most frequent clonal complexes were cc18, cc41/44 and cc32 (typical for serogroup b) and cc11 (typical for serogroup c). the highest age-specific morbidity rates were observed in the lowest age groups, i.e. 0−11 months and 1−4 years (11.4/100 000 and 4.5/100 000, respectively), and were associated with high prevalence of serogroup b. the case fatality rate was the highest in infants under 1 year of age (38.5%). the incidence of imd caused by serogroup c is currently low and there is no indication for mass vaccination with menc conjugate vaccine. menb vaccine is needed for infants, but the sero/subtype coverage by the currently developed porin-based vaccines is low for czech meningococcal isolates (maximum 56.8% for nine-valent meningococcal pora vaccine). methods:the vaccination programme incorporates dedicated vaccine clinic with a multi-disciplinary team including a nurse, data manager, a pharmacist specifically appointed to the unit. additional interventions to improve vaccine uptake and outcome have included use of sms texting to announce availability of influenza annually and improve adherence to completion of hepatitis b vaccination, educational programmes changes in guidelines e.g. varicella vaccination and creation of a vaccine passport. we reviewed vaccination clinic activity in the cohort of 1,700 hiv positive patients since introduction of a dedicated vaccine service. results:there has been a large increase in the uptake of vaccinations since introduction of this service. the varicella vaccination uptake increased from 8 (2007) to 43 (2008) due to targeted vaccine programme.(see graphic, legend reads left to right) conclusion: strategies implemented increased the uptake of recommended vaccinations in our hiv population. these included appointment of a dedicated health professional team, use of it supports, education of staff and patients and development of a vaccine passport. we developed the vaccine passport to help with patient education and awareness and it will serve as a record of vaccine administration for physicians off site. in the latter year, post guideline change, we have targeted our varicella non immune population. the next intervention planned is to assess all late entrants to our healthcare system to determine need for catch up vaccines, including mmr. results: column purified recombinant protein sspb1 was found to be a good antigen for both groups of animals used for immunisation. antibodies against the recombinant sspb1 tested by opsonophagocytosis were found to enhance phagocytosis of 4 gbs strains belonging to different serotypes at the average 5.5 times relatively to control. affect against gas strains was less pronounced (2.5 times) but still statistically significant. antibodies were also capable to interfere with adherence of gbs strains carrying sspb1 relatively to the strain without the protein. adherence of the strain with sspb1 towards different cell lines was dramatically higher which proves the function of the protein as adhesin. in passive protection test carried out with mice challenged with virulent gbs or gas strains introduced intranasaly were eliminated from the lungs of the animals 20 times faster in case of the usage of anti sspb1 serum relatively the control. in the experiments with active protection sspb1 immunised animals were found be significantly better protected against gbs and gas infection. (table 1) . similar results were obtained in the analysis of factors associated with 90-day mortality. conclusion: these data suggest that outcomes of both community-onset and nosocomial bloodstream infections due to s. aureus may be improved by an expert consultation service. the factors most critical for better outcomes and modifiable in time by id specialist consultation remain to be determined and may be explored as process of care quality indicators. objective: worldwide, the present tuberculosis epidemic is characterised by an alarming emergence in drug resistance. given the limited therapeutic options in mdr (and especially xdr) tuberculosis, there is a need to define the resistance levels and mechanisms present in clinical isolates categorised as drug resistant on the basis of critical concentration testing, so as to facilitate rapid therapeutic decisions. methods: we determined quantitative resistance levels of drug resistant isolates of mycobacterium tuberculosis sampled in switzerland over the past 3 years. resistance-conferring genetic alterations were identified by probe assays and pcr-mediated gene sequencing. results: rifampicin resistant isolates unanimously showed a high-level resistant phenotype (>50 mg/l) associated with mutations in rpob. in contrast, a significant fraction of clinical tb isolates categorised as isoniazid resistant on the basis of critical concentration testing showed a low-level resistant phenotype (mostly mutations in inha); heterogeneous phenotypic resistance levels were associated with mutations in katg. one third of streptomycin resistant clinical isolates had a low-level resistance phenotype (<10 mg/l). ethambutol resistance occurred mostly in mdr strains and was linked to alterations in embb, but resistance never exceeded 25 mg/l. our data indicate that some first line agents may be considered as therapeutic treatment option despite in vitro resistance at the critical concentration. diagnostic mycobacteriology would benefit from standardised measures of quantitative drug susceptibility testing in particular for those drugs were significant variations in phenotypic resistance levels are found in clinical isolates, e.g. isoniazid, ethambutol and streptomycin. introduction recent advances in the diagnostics of varicella zoster virus (vzv) infections have changed the perception of this virus as a cns pathogen. a real-time pcr method amplifying a 70 nt segment of the vzv gb region gave 0.5 log improved sensitivity over conventional pcr and was employed for routine diagnosis of vzv dna in samples of cerebrospinal fluid (csf). in addition, a new elisa method for detection of antibodies in the csf to glycoprotein e was developed, using a mammalian cell expression system for optimal glycosylation of the antigen. these methods were utilised for studies of vzv-induced cns infections. in a retrospective study, almost all patients had a reactivated vzv infection, but only 60% showed skin lesions. the following diagnoses were made: acute aseptic meningitis (aam), n = 34; encephalitis, n= 22; meningoencephalitis, n = 6; cranial nerve affections, n = 20; encephalopathy, n = 5; and cerebrovascular disease, n = 6. in 66 patients in whom vzv dna levels were determined, significantly higher viral loads were found in those with aam and encephalitis compared to patients with cranial nerve affection (including ramsay hunt syndrome). of the 50% (n = 50) who had a follow-up, 50% (n = 25) had neurological complications after 3 months. sixty-two percent had a ct/mri scan of the brain performed and 46% of these had pathological findings. vzv encephalitis showed a more broad disease spectrum as compared with herpes simplex encephalitis (hse), as will be presented. detection of intrathecal synthesis of vzv ge antibodies was positive in the vzv encephalitis patients, as well as in some of the hse patients, arguing for a previous suggested role for vzv as a co-pathogen at least in some cases of the latter disease. vzv vasculitis was a more common finding (6% of all cases) than expected from the literature of case reports. mr findings showed that middle and posterior cerebral arteries were targeted. surprisingly, despite substantial vzv dna loads in the csf of these patients, investigated serum samples were pcr negative. thus, vzv might be suggested to be neuronally transported to the arterial walls rather than haematogenously spread. conclusions: vzv is a serious and underestimated cause of cns infection. a substantial number of the patients presented with serious neurological symptoms and sequela, and pathological findings on ct/mri of the brain were abundant, especially in patients with encephalitis and vasculitis. pk/pd controversies for the clinician s190 pk/pd and azoles the triazoles have revolutionised the treatment of invasive and allergic fungal diseases. fluconazole, itraconazole, voriconazole and posaconazole are available for clinical use. isavuconazole and ravuconazole are in development. the triazoles have broad spectrum antifungal activity. the pharmacokinetics and pharmacodynamics (pk-pd) of the triazoles have been extensively investigated in murine models of disseminated candidiasis. the pd parameter that optimally links drug exposure with the observed antifungal effect is the ratio of the area under the concentration-time curve (auc) to mic (auc:mic). there is increasing information on the magnitude of the auc:mic that is required for optimal antifungal effect. pk-pd principles have been used to define in vitro susceptibility breakpoints. the triazoles are fungistatic against candida spp. their mode of action against aspergillus spp. is less well defined, although they clearly exhibit dose-dependant decrement in fungal burden in laboratory animal models of invasive pulmonary aspergillosis. the triazoles accumulate in tissues and this is important for an understanding of their antifungal effect. in humans, the triazoles are characterised by complicated pharmacokinetic properties. both itraconazole and voriconazole exhibit nonlinear pharmacokinetics. the triazoles all exhibit clinically relevant exposureresponse relationships. recent work from our laboratory suggests that itraconazole exhibits clinically relevant concentration-toxicity relationships. higher concentrations of voriconazole are associated with a progressively higher probability of hepatotoxicity, photopsia and central nervous system toxicity. because of the significant pharmacokinetic variability and clinically relevant drug exposure-response relationships, therapeutic drug monitoring (tdm) is frequently used. a strong argument can be made for the routine monitoring of itraconazole and voriconazole. there may also be grounds to consider monitoring posaconazole levels. tdm should be considered for all patients receiving triazoles who have refractory disease. furthermore, tdm should be considered when compliance, drug interactions and variable pharmacokinetics result in uncertainty about resultant drug exposures. an understanding of the pk-pd relationships of the triazoles has been instrumental in optimising their clinical efficacy. innate immunity s192 the inflammasomes: danger sensing complexes triggering innate immunity the nod-like receptors (nlr) are a family of intracellular sensors of microbial motifs and 'danger signals' that have emerged as being crucial components of the innate immune responses and inflammation. several nlrs (nalps and ipaf) form a caspase-1-activating multiprotein complex, termed inflammasome, that processes proinflammatory cytokines including il-1beta. amongst the various inflammasomes, the nalp3 inflammasome is particularly qualified to sense a plethora of diverse molecules, ranging from bacterial muramyldipeptide to monosodium urate crystals. the important role of the nalp3 inflammasome is emphasized by the identification of mutations in the nalp3 gene that are associated with a susceptibility to inflammatory disorders. these and other issues related to the inflammasome will be presented. it is now 20 years since charles janeway hypothesized the existence of clonally derived pattern recognition receptors and pointed to the importance of these in initial responses to bacterial and viral infections. janeway's hypothesis has been validated by the discovery of three groups of prrs. first, are the toll-like receptors which detect microbial lipids and non-self nucleic acids at the cell surface an in intracellular compartments. in addition cytoplasmic sensors of bacteria (nods) and of viral nucleic acids (rigs) have also been characterised. as well as being critical for responses to infections, these prrs also underlie a large burden of autoimmune and inflammatory disease in the human population and are thus important targets for therapy. in my talk i will describe the molecular mechanisms by which these conserved pathogen associated moecules are recognized by the tlrs with particular reference to lipo polysaccharide and single stranded viral rnas. i will also present new results which show how receptor activation is coupled to downstream signal transduction and in particular the role played by oligomeric signaling platforms assembled form adaptors and other signaling molecules involved in the pathway. i will discuss the potential for structural analysis to be used in the rational design of new drugs. this session proposes a critical review of the most salient recently published papers in the field with a special focus on control of multi drug-resistant organisms, prevention of infections in the intensive care unit, surgery etc. and highlights the need for validity/scope assessment. it emphasizes also the importance to prioritise information published in the abundant literature available so as to be able to summarise and understand the potential changes in clinical practice, and identify unresolved issues and areas of possible future clinical research. tourism is europe's face to the world. it is also a major source of revenue, employment and productivity. each year over 450 million arrivals are recorded into the continent, and of those, approximately 4 million are from latin america. returning travelers are even more numerous and more often associated with disease transmission into europe. within countries of the european continent, imported cases of environmental and zoonotic illnesses such as cholera, dengue, malaria, viral haemorrhagic fevers and west nile virus infections are a rare but established fact. diseases imported from latin america with the potential for autochthonous transmission (chikungunya, malaria, yellow fever) and or high infectivity (viral haemorrhagic fevers) will be described in detail and the possibility of european outbreaks from latin american countries will be discussed. cutaneous leishmaniasis (cl) is a worldwide disease, endemic in 88 countries, that has shown an increasing incidence over the last two decades. so far, pentavalent antimony compounds have been considered the treatment of choice, with rates of curing close to 85%. however, the high efficacy of these drugs is counteracted by their adverse events. recently, in vitro and in vivo studies have shown that no plays a key role in the eradication of the leishmania parasite objective: to determine whether a no donor patch (developed by electrospinning technique) is as effective as meglumine antimoniate in the treatment of cl while causing less adverse events methods: a double-blind, randomised, placebo-controlled clinical trial was conducted with 178 patients diagnosed with cl in santander, colombia, south-america. the patients were randomly assigned to two groups. during 20 days group 1 received simultaneously meglumine antimoniate and placebo of nitric oxide patches while group 2 received active nitric oxide patches and placebo of meglumine antimoniate. biochemical determinations (aspartate aminotransferase, alanine aminotransferase, creatinine and pancreatic amilase) were measured at he beginning and at the end of the treatment. a follow up was realised 21, 45 and 90 days after the beginning of the treatment results: the study included 69 (38.77%) women and 109 (61.23%) men. the average age in group 1 was 30.80±14.23 years; while in group 2 it was 27.88±13.79 years. clinical and demographic data were similar in the two groups. after the follow up period, the complete clinical healing of group 1 was 94.81% versus 37.14% for group 2 (p= 0.0001). treatment with no patches generated both, a lower frequency of non-serious adverse events (fever, anorexia, myalgia, arthralgia, headache), and a reduced variation in biochemistry determinations (asat 26 the treatment with no patches resulted in a lower percentage of complete clinical response compared with meglumine antimoniate. despite its inferior effectiveness, the safety, the lower frequency of adverse events, the facility of administration (topical) and the low cost of the patches justifies its evaluation in further poblational studies, especially in populations as the colombian ones, where the serious adverse events due to glucantime have increased dramatically. objectives: trichinellosis is a zoonotic disease which has never been reported in taiwan and is rarely linked to consumption of reptiles. we investigated the first documented outbreak of trichinellosis in taiwan consisting of 8 patients who became acutely ill after eating at the same restaurant in may 2008. we conducted a retrospective cohort study by interviewing the patients and persons who ate together with them. a case was defined as illness in an attendee who had fever (>38.0ºc) or myalgia 4 weeks after the festivals and was seropositive to trichinella antigen using an enzymelinked immunoassay and immunohistochemical staining. environmental study of the soft-shelled turtle farm was performed. results: of the 23 attendees, 8 persons met the case definition (attack rate = 35%). the most common presenting symptoms were myalgia (88%), fever (88%), and periorbital swelling (38%). all 8 patients sought medical care; five were hospitalised. of the 7 patients who underwent blood test, all had moderate eosinophilia. all 8 patients' serum samples were strongly reactive to trichinella excretory-secretory antigen. the only food item significantly associated with illness was the raw softshelled turtle meat (relative risk undefined; p = 0.005). traced back to the farm, histological examination of soft-shelled turtles was negative for trichinella species. the most likely cause of this outbreak was consumption of raw soft-shelled turtle served in the festivals. this investigation indicates taiwan is not free of trichinellosis. prevention and control programs of trichinellosis should be established. the public should be aware of the risk of acquiring trichinellosis from consumption of raw soft-shelled turtle. objective: to develop and evaluate a modified, rapid giemsa staining procedure for detection of malaria parasites in blood smears. disadvantage of the rapid commercially available staining methods is that they require highly experienced technicians for interpretation of results because the interpretation can be difficult. for this reason, many laboratories use the giemsa stain. shorter giemsa staining times have been reported previously, however, to our knowledge, the effect of 5 and 10 minute staining in different giemsa dilutions have not been evaluated. the stock solution of giemsa stain (merck, darmstadt, germany) was used in different dilutions (1:10 and 1:5) and incubated for different lengths of time (10 min and 5 min). the staining effect was compared to our standard giemsa stain (1:40, 45 min). sensitivity was determined by examining smears of p. falciparum from fresh and edta blood. the level of parasitaemia was followed in two patients admitted to our hospital with p. falciparum parasitaemia's of 21.5% and 28.8% (see table; patient a and b) by examination of blood smears taken at different time points after initiation of therapy. these samples were used to evaluate the different giemsa dilutions and staining times. smears were read by three independent observers (a clinical microbiologist, a laboratory technician specialised in parasitology, and a resident in clinical microbiology). in the table results of the three staining methods on blood from two patients from ghana with high parasitaemia's on admission and during follow-up are shown. all smears were equally easy to read and yielded parasite counts within internationally accepted ranges of variation (see united kingdom national external quality assessment service). conclusion: staining blood smears for detection of plasmodium falciparum parasites with a 1:5 dilution of giemsa stain for five minutes provides easy to read slides and results comparable to those obtained with the standard giemsa staining. advantage of the rapid method is the shorter turnaround time, disadvantage is the larger amount of stain used. objectives: diarrhoeal diseases are common in developed and developing countries and are major causes of morbidity and mortality worldwide. the need to differentially diagnose protozoan parasites versus other gastrointestinal (gi) aetiologies is well recognized. the most common gi protozoan parasites infecting humans worldwide are considered to be entamoeba histolytica, giardia lamblia, blastocystis hominis, dientamoeba fragilis and cryptosporidium spp. laboratory detection of these parasites is relying on microscopic analysis of stool samples and water concentrates, as well as enzyme immunoassay (eia) tests. utilising the microscopic examination usually results in underdetection of gi parasites, while usage of eia is often not cost-effective. methods: savyon diagnostics is currently engaged with developing an approach aiming to address the unmet needs and the current limitations in this field. this approach includes 3 major aspects: (1) the ability to detect a panel of all the above 5 organisms in one test kit, (2) the possibility to perform the diagnosis in two steps − first, simultaneous detection of these organisms without distinguishing between the different species for screening of large number of specimens, and second, distinctive detection of the specific aetiology in the positively-found specimens, and (3) the ability to apply eia diagnosis in formalin-preserved specimens for all the mentioned parasites. results: polyclonal antibodies were produced in-house based on native antigen extracts, recombinant antigens and synthetic peptides. the resulted inventory of antibodies enabled finding the optimal combination that provided the desired performance parameters for separate detection of each of the parasites in fresh, frozen or formalin preserved faeces specimens. the analytical limit of detection and the performance in characterised clinical specimens were comparable to microscopy or to reference eia, when available. the results show unique detection of e. histolytica in formalin-preserved specimens, which is comparable to detection in fresh specimens. furthermore, we demonstrate simultaneous detection of the parasites without compromising performance characteristics in fresh or preserved specimens. the presented work is a paradigm of an innovative approach, expected to advance the diagnosis of protozoan parasites in gi patients, thus, enabling appropriate and cost-effective diagnosis and treatment. objectives: systemic administration of certain facultative anaerob bacteria to mice bearing solid tumours leads to accumulation in tumours compared to normal target organs, like spleen and liver, and to retardation of tumour growth. salmonella enterica serovar typhimurium (s. typhimurium) as well as escherichia coli 1917 nissle (ecn) are such bacteria. preliminary experiments showed that such bacteria that exhibit the ability to form biofilms in vitro might also do so in tumours. in the present study this was systematically investigated. methods: biofilm formation of bacteria were detected on low-salt biofilm plates. additionally, salmonella-or e. coli-infected ct26tumours of balb/c mice that were left untreated or were treated with anti-gr1 to deplete neutrophilic granulocytes were removed two days post infection, fixed and prepared for electron microscope analysis. the expression of different genes which are probably involved in the biofilm formation were tested via real-time pcr. results: when examined after colonising tumours s. typhimurium sl7207 and sl1344 as well as ecn are almost exclusively found extracellular although they are able to invade the ct26 cells in vitro. interestingly, like in vitro all three bacteria form biofilms to various extend when residing in the tumours. this was followed in more detail for s. typhimurium sl7207. biofilms were not formed by sl7207 when neutrophils had been removed by antibodies. in addition, when arda a central switch for biofilm formation in the salmonellla had been deleted no biofilms could be found. importantly, now bacteria could be found intracellularly most likely in neutrophilic granulocytes. conclusion: the formation of biofilms by facultative anaerobic bacteria when residing in solid tumours is a novel and surprising finding. when neutrophils were removed, no biofilms are formed, while uptake into neutrophils is allowed when the ability of the bacteria to form biofilms was blocked. hence, it appears that the bacteria use biofilm formation as a defence system against the immune system of the host. objectives: rama is an arac/xyls family transcriptional activator found in klebsiella pneumoniae, salmonella spp. and enterobacter spp., the overexpression of which is associated with an mdr phenotype. recently a tetr-like gene that lies upstream of rama, known as ramr, has been identified as a repressor of rama. k. pneumoniae kp342 is a diazotrophic endophyte strain which has been reported to exhibit notable resistance to antibiotics. despite its mdr phenotype kp342 has been shown to exhibit attenuated pathogenicity in mouse models in comparison to clinical k. pneumoniae strains. the aims of this study were to: determine the levels of rama expression and establish its role in kp342's mdr phenotype; determine the effect of ramr complementation on rama expression and antibiotic susceptibility. methods: genome and sequence analysis performed in k. pneumoniae strain kp342 demonstrated a 96 bp deletion within the ramr gene. cloning and complementation with full size wild type ramr was performed in kp342 (hereby known as kp342/ramr). rt-pcr was used to assess levels of gene expression which were subsequently quantified using bio-rad quantity one software. mic testing was performed against chloramphenicol (cm), norfloxacin (nor) and tetracycline (tet) according to bsac guidelines. biofilm formation was measured using a modified protocol of o'toole and kolter. results: kp342 containing the mutated ramr gene (96 bp deletion) was shown to overexpress rama and the putative outer membrane protein roma. complementation of the ramr gene resulted in the repression of both rama and roma transcription by 3−4 fold. interestingly, the ramr complemented strain demonstrated increased biofilm formation (up to 9-fold increase) over a 72 hour period in both lb and m9 medium after static growth at 37ºc. mics of the tested antibiotics were reduced up to 16-fold in kp342/ramr compared to the ramr mutated kp342. conclusions: this result demonstrates that ramr acts as a repressor of both rama and putative outer membrane protein roma thereby increasing its susceptibility to antibiotics. however the restoration of a functional ramr in kp342 also increases biofilm formation significantly, suggesting that ramr plays a role in the regulation of biofilm formation genes and possibly bacterial virulence. rifampicin showed the highest activity on biofilm matrix and bacteria in sa and pa biofilms. results also indicated that biofilm viable mass was more susceptible to treatment than the biofilm matrix, which is mainly responsible for biofilm persistence. further research should specifically focus on compounds destroying matrix and which can be used as an adjunct to antibiotic therapy. [ objectives: staphylococcus epidermidis is a common cause of foreignbody infections (fbi) because of its ability to form biofilms. biofilms are very resistant to antibiotics. active and passive immunisation against biofilm-associated bacterial antigens may be an alternative. we studied the effect of immunisation against the lpxtg protein sesc in s. epidermidis biofilms in vitro and in vivo. we previously reported that sesc is present in all s. epidermidis strains tested. sesc is mainly expressed during the early and late fbi and at a higher level in sessile cells than in planktonic cells. methods: we used rabbit polyclonal anti-sesc-iggs (4 mg/ml) to study biofilm inhibition in vitro and in vivo in our rat model (50 mg igg per rat) on 1-day old biofilms. we also vaccinated rats twice with sesc according to standard protocols. serum samples taken at day 0 and 2 weeks after the 1st and 2nd immunisation were tested by elisa and showed an increase in anti-sesc antibody levels. s. epidermidis strains 10b and 1457 are biofilm forming strains and have been described before. for in vitro experiments, s. epidermidis 10b or 1457 were mixed with anti-sesc-iggs and incubated for 2 hours at 4ºc. subsequently 10 6 cells were added to each well. after 24 h at 37ºc biofilms were washed and stained with crystal violet and od595 was measured. for in vivo experiments, catheter fragments were pre-incubated with s. epidermidis 10b and implanted subcutaneously in each rat. after explantation, the average number of cfu was determined after 24 hrs. results: our data show that rabbit anti-sesc-iggs inhibit in vitro biofilm formation by s. epidermidis strains 10b and 1457 by 74% and 65%, respectively (n = 9). in the in vivo rat model, rabbit anti-sesc-iggs reduced the bacteria in a 1-day old biofilm 60-fold (n = 18). active immunisation with recombinant sesc led to a 10-fold reduction of cfu compared to control rats in 1 day-old biofilms (n = 10). after 3 days, the reduction in biofilm-associated bacteria in the immunised rats was 15-fold (n = 10) (fig 1.) . conclusion: sesc represents a promising target for prevention of s. epidermidis biofilm formation. the higher effect of passive immunisation compared with active immunisation is probably due to the subcutaneous injection of anti-sesc-iggs at the place of catheter insertion. objectives: staphylococcus epidermidis has emerged as a pathogen associated with infections of implanted medical devices impeding their long-term use. characteristics of s. epidermidis that allow persistence of infection are the ability of bacteria to adhere to surfaces in multilayered cell clusters, followed by the production of a mucoid substance more commonly known as slime, encoded by the ica operon. the adherent bacteria and slime are collectively known as biofilm. the coupled effects of specific chemical terminal surface groups and flow conditions on slime production and biofilm formation by s. epidermidis were investigated in correlation to the expression of two genes of the ica operon. methods: reference control strains (atcc35984, slime-positive and atcc12228, slime-negative), and two clinical strains isolated from different hospitalised patients, (one ica-positive/slime-positive and one ica-positive/slime-negative) were tested. bacteria grown in bhi medium were suspended in physiological saline at a concentration of~3×10 9 cells/ml. hydroxyl (oh)-terminated (hydrophilic) and methyl (ch3)terminated (hydrophobic) glass surfaces were used as substrates in a parallel plate flow chamber. bacterial adhesion was examined under two flow rates: 2 ml/min and 20 ml/min for two and four hours. total rna from both planktonic (p) and adherent (a) bacteria, after detachment with trypsin, was isolated by the trizol method. reverse transcription followed by relative real-time pcr (rrt-pcr) towards a 207 bp part of 23s rrna gene, allowed the detection of expression levels of icaa and icad. adherent bacteria were investigated with scanning electron and confocal laser microscopes. results: higher expression levels of both icaa and icad genes onto glass and especially methyl-terminated glass surfaces were calculated by rrt-pcr, under higher flow rate in two hours by the reference and the clinical slime-positive strains. these results correlate well with adherent bacterial cell counts and images taken by both microscopes. the icapositive slime-negative clinical strain showed lower expression levels of ica genes, less adherent ability and pia production on glass surfaces, as observed by microscopes. higher flow rate enhances the expression level of both ica genes, with a peak in two hours. hydrophobic biomaterial surfaces seem to play a crucial role to initial adherence, increasing ica gene expression and pia synthesis. consenting men and women with dfi (predefined by clinical signs and symptoms) caused by mrsa were potentially eligible including those associated with bacteraemia. patients with initial osteomyelitis were excluded. patients could receive l 600 mg bid either iv or po. primary end point were cure or improvement rates (c+i) and microbiologic eradication (me) at 60 days after the beginning of l. secondary end points were c+i on days 5 and 30 after the beginning of treatment and hospital discharge day, need of amputation, duration of therapy and mortality rates. all the adverse events were collected. results: 70 patients were enrolled. relation men:women was 2.1.the age of patients was 63.2±13 years and the average period from the diagnosis of diabetes was 16.5±9.7 years. associated bacteraemia was present in 27.1% of patients included. primary end points: c+i 60 days after the beginning of l was achieved in 91.4% of patients and me was obtained in 84.3% of patients. secondary end points: c+i on day 5, hospital discharge day and day 30 after the beginning of treatment and were; 70%; 84.3% and 88.6% respectively. only 8 patients needed a minor amputation. the primary and secondary end points in the subgroup of bacteraemic episodes were not statistically different of those previously described. the mean duration of therapy was 29.5±18.4 days. global mortality was 4.3%. only one episode of polineuropathy was reported. neither thrombocytopenia nor lactic acidosis was found. conclusions: l achieved excellent c+i even at first evaluation visit in documented dfi caused by mrsa. l also showed high me rates. although patients received prolonged periods of treatment, l was a safe drug. objectives: azithromycin microspheres formulation (azm) was developed to enable a higher dosage of 2 g to be administered as a single oral dose without decreasing the safety profile. this study compared azm with moxifloxacin (mox) aimed at confirming the efficacy and safety of azm in acute exacerbations of chronic bronchitis (aecb). methods: this prospective, multicentre, randomised, double-blind, double dummy study compared azm 2 g single dose with mox 400 mg once daily for 5 days, enrolled aecb patients 50 years old and above, with anthonisen type 1 exacerbations, and with at least 2 exacerbations of aecb in the past 12 months. subjects were to have a history of smoking of at least 20 pack-years and documented forced expiratory volume in 1 second (fev1) less than 80% of predicted. they were followed up for up to 9 months. results: a total of 396 patients were treated (198 in each of the treatment groups) the distribution of the age, and mean fev1 were similar for the 2 treatment groups. pathogens were isolated from 62.9% of the patients (61.1% of patients on azm and 62.9% of patients on mox). the clinical success (signs and symptoms related to the acute infection had returned to the subject's normal baseline level, or clinical improvement was such that no additional antibiotics were deemed necessary) rate for the per protocol population at test of cure (toc) at day 12−19 was 93.0% for azm and 94.2% for mox group (95% ci −5.8, 3.9). bacterial eradication rate (bacteriologic pre protocol population) at toc was 96.0% for azm group and 96.7% for mox group (95% ci −4.5, 3.3). although the study population had history of at least 2 exacerbation in the past 12 months, less than half of the subjects experienced a recurrence during the follow-up, and there was no statistically significant treatment difference in time taken to first occurrence of aecb. both treatments were well tolerated. the incidence of treatment related adverse events was low, being reported by 17% of subjects receiving azm and 12% of subjects receiving mox. most aes were mild or moderate in severity. the most common aes were gastrointestinal disorders, being reported by 14% of subjects receiving azm and 8% of subjects receiving mox. conclusions: a single oral dose of azm was as effective as a 5-day course of mox in the treatment of aecb and was well tolerated. objectives: optimal duration of gentamicin containing regimen for therapy of human brucellosis is not clearly determined. methods: this randomised clinical study was conducted to compare the efficacy of gentamicin 5 mg/day for 5 days plus doxycycline 100 mg twice daily for eight weeks (gd group) versus streptomycin 1gr im for 2 weeks plus the same dose of doxycycline for 45 days (sd group). all cases were followed for one year after cessation of therapy. efficacy of both regimens (failure of therapy or relapse) were compared. results: seventy-nine patients with the mean age of 35±14.5 years and 75 cases with the mean age of 36.7±13.9 years were treated with regimen of gd or sd, respectively. the clinical manifestations in these two treated groups were similar. failure of therapy was seen in one patient in gd group and in 2 cases in sd group ( objectives: to study the efficacy of telavancin (tlv), an investigational bactericidal lipoglycopeptide, for the treatment of complicated skin and soft tissue infections (cssti) caused by presumed or confirmed grampositive organisms. methods: atlas 1 and atlas 2 were methodologically identical, double-blind, randomised, multinational, phase 3 studies. adult men and women presenting with cssti including major abscess were randomised 1:1 to tlv 10 mg/kg intravenous (iv) q24 h or vancomycin (van) 1 g iv q12 h for 7 to 14 days. test-of-cure (toc) visit was conducted 7 to 14 days after end of study treatment. the all-treated population (at) included patients with confirmed diagnosis of cssti who received 1 dose of study medication. this analysis examined the baseline characteristics and cure rates at toc for patients with major abscess in the combined atlas at population. results: in the pooled at population of atlas, 772 patients presented with major abscess. more than 60% of these patients required hospitalisation. the baseline lesion surface area exceeded 5 cm 2 in 98% of the cases, while 65% of the patients presented with lesions exceeding 50 cm 2 (table 1) . elevated white blood cell counts were found in more than 40% of the cases (table 1) . nearly all patients required surgical drainage, with approximately 2/3 performed prior to the first dose of study medication. very few patients required a surgical procedure more than 4 days after the start of study medication. clinical cure rates at toc are presented in table 1 . overall, adverse events in the at population were similar between the treatment groups with regard to type and severity. conclusion: telavancin administered once daily was non-inferior to vancomycin for the treatment of major abscess. objectives: b. fragilis and related species, members of the normal bowel flora, are the most widely isolated anaerobic bacteria from different infections. to follow the development and spread of the resistance among these strains is difficult, as antibiotic susceptibility testing of clinically relevant anaerobes in different routine laboratories in europe is less and less frequently carried out due to the fact, that clinicians treat many presumed anaerobic infections empirically. to follow the changes in the antibiotic resistance of bacteroides strains three europe-wide studies were organised during the past twenty years. the evaluation of the results of these studies may show changes in the resistance to different antianaerobic drugs. only clinical isolates and no normal flora members of bacteroides strains belonging to different species were collected from different countries throughout europe during these studies. agar dilution method was used for the antibiotic susceptibility determination. actual breakpoints accepted by nccls (clsi) and eucast were used. molecular genetic investigations were carried out to detect resistance mechanisms. since the first study the chromosomally mediated beta-lactamase production and tetracyclin resistance is the most prevalent among bacteroides strains in europe. clindamycin resistance in bacteroides is mediated by a macrolide-lincomycin-streptogramin (mls) mechanism and its frequency differs in different countries in europe. resistance to beta-lactam-beta-lactamase inhibitor combinations was studied using amoxicillin-clavulanic acid and/or piperacillintazobactam. increase in resistance was observed to both combinations throughout the years. the same is true for cefoxitine and in the third study several hetero-resistant isolates were found. the occurrence and spread of resistance to imipenem and metronidazole among bacteroides strains merit special clinical importance. the presence of the cfia gene is much more prevalent than the expression of the imipenem resistance; however the spread of the cfia gene among species other than b. fragilis is still very rare. the molecular genetic methods looking for the resistance genes among strains with elevated mics against these antibiotics prove that resistance breakpoints should be reconsidered. the resistance to moxifloxacin shows great differences in different countries. the lowest resistance rate was observed in the case of tigecyclin. many factors may affect the response to treatment such as site of infection, surgical procedures, severity of the illness, patient status, presence of other pathogens (mixed infection), pk/pd parameters of the antibacterial drugs. thus, correlation between treatment failure and antibiotic resistance among anaerobes remains difficult to assess. the main discrepancies came from intra-abdominal infections and a worrisome disjunction between surgeon and microbiologist opinions emerged in the 1990's. but, patients in whom primary therapy failed had more resistant strains compared with patients in whom therapy succeeded. in contrast many failures may be due to the lack of isolation of anaerobes from clinical samples! during anaerobic bacteraemia, salonen et al. demonstrated that mortality increased dramatically from 17% for initially effective treatment to 55% when an ineffective treatment was started. facing new mechanisms of resistance and global increase resistance to many antibiotics among anaerobes may lead nowadays to a different answer. clindamycin vs. penicillin studies for the treatment of lung infections pointed out the failure due to b-lactamase production among gram-negative anaerobes. we found many reports of failure after clindamycin treatment in osteomyelitis, septic arthritis, brain abscess in presence of clindamycin-resistant anaerobes (bacteroides fragilis group and prevotella), probably because when resistance occurs, clindamycin mic's are high. similarly, the lack of coverage of an undetected resistant anaerobe allows the selection of an anaerobic strain resistant to the treatment chosen against the associated aerobes such as imipenemresistant eghertella lenta or metronidazole-resistant strains of prevotella or bacteroides fragilis. the later failures may give opportunity to set up a new metronidazole breakpoint for resistance (mic > 4 mg/l). the main problem is related to the difficulty to detect some heterogeneous resistant strains, that needs prolonged incubation period on agar medium. this kind of situation is probably the most suitable to correlate the bacterial antibiotic resistance with the failure of the antibiotic treatment. methicillin-resistant s. aureus isolates causing community-acquired infections (ca-mrsa) in children is a major problem in several areas around the world. ca-mrsa are associated with both skin and soft tissue infections and invasive infections. recurrent soft tissue infections and infections within the family caused by ca-mrsa isolates are common. ca-mrsa s. aureus isolates containing gene coding for pvl have been associated with serious staphylococcal pneumonia as well as osteomyelitis complicated by subperiosteal abscesses or venous thromboses. in addition to vancomycin, ca-mrsa generally are susceptible to clindamycin and trimethoprimsulfamethoxazole. treatment of superficial skin and soft tissue infections involves surgical drainage of abscesses followed by an oral agent such as tmp-smx or clindamycin. minocycline or doxycycline is a consideration for children >8 years old. empiric vancomycin is typically administered for more serious and invasive infections such as osteomyelitis, septic arthritis, serious head and neck infections or suspected staphylococcal pneumonia. clindamycin is efficacious in treating invasive ca-mrsa infections caused by susceptible organisms. linezolid or daptomycin is another option in selected circumstances. mri is the optimal imaging modality for assessing children with ca-mrsa osteomyelitis. aggressive surgical drainage of subperiosteal abscesses or sites of pyomyositis is recommended. venous thombosis is increasingly recognized as a complication of ca-mrsa osteomyelitis. anti-coagulation until the thrombus has resolved is recommended. the optimal approach to prevention of recurrent ca-mrsa infections is unclear but a strategy that includes emphasizing personal hygiene, plus/minus antimicrobial soaps, mupirocin to the nose or "bleach baths" is frequently suggested. s226 understanding the pathogenesis of group a streptococcal disease: the bedside-to-bench approach invasive group a streptococcal (gas) infection presents itself in a range of guises, most notoriously necrotising fasciitis and the streptococcal toxic shock syndrome. as a human pathogen, gas pathogenesis research should ideally be shaped by clinical questions arising from either epidemiological or case-based investigation of human disease. in the mid 1990 s, large epidemiological studies pointed to a central role for specific t cell-stimulating superantigens in the aetiology of streptococcal toxic shock. this sparked a series of clinical and laboratory investigations that demonstrated production of superantigens during infection which were indeed capable of triggering massive t cell activation in patients but were unlikely, alone, to account for all the features observed in toxic shock. genomic, clinical and laboratory-based investigations have identified novel and highly potent superantigens that appear to directly contribute to sepsis pathogenesis and, together, may constitute targets for adjunctive treatments in invasive disease. epidemiological, clinical, and laboratory studies have highlighted a role for blunt trauma in the aetiology of at least a quarter of cases of gas necrotising fasciitis. one of the most striking findings on examination of tissues from patients suffering with necrotising fasciitis is the failure of neutrophils to migrate to the focus of infection. investigation of patients with invasive gas infection led to the discovery that gas produces an enzyme that can cleave and inactivate human chemokines and study of patients with bacteraemia has highlighted a likely role for the causal enzyme spycep in disease pathogenesis; this bacterial surface enzyme has also shown promise as a potential vaccine antigen. notwithstanding a potential role for individual virulence factors in disease causation, clinical studies have demonstrated that gas bacteria may persist at the site of infection despite high concentrations of bactericidal antibiotics, and this has been borne out by experimental studies; the reasons behind such persistence are unclear but may include internalisation of gas by immune cells, formation of biofilm, and antibiotic penetration of necrotic tissues. the persistence of viable bacteria in such cases is not widely recognized and deserves focused consideration in the research laboratory. genome-wide analysis of microbial pathogens and molecular pathogenesis processes has become an area of considerable activity in the last 10 years. these studies have been made possible by several advances, including completion of the human genome sequence, publication of genome sequences for many human pathogens, development of microarray technology and high-throughput proteomics, and maturation of bioinformatics. despite these advances, relatively little effort has been expended in the bacterial pathogenesis arena to develop and use integrated research platforms in a systems biology approach to enhance our understanding of disease processes. we have exploited an integrated genome-wide research platform to gain new knowledge about how the human bacterial pathogen group a streptococcus causes disease. results of these studies have provided many new avenues for basic pathogenesis research and translational research focused on development of an efficacious human vaccine and novel therapeutics. new data stemming from use of a systems biology approach to provide new data about group a streptococcus pathogenesis will be presented. streptococcal toxic shock syndrome and necrotising fasciitis caused by group a streptococcus are rapidly progressive invasive diseases that are associated with significant morbidity and mortality, ranging from 30−80% despite prompt antibiotic therapy and surgical debridement. s. pyogenes is known to primarily cause disease by activating and modulating host immune responses. the exotoxins with superantigenic activities have been demonstrated to be crucial triggers of excessive inflammatory responses and consequently systemic toxicity, organ dysfunction, tissue necrosis and shock. another important virulence determinant is the m-protein, which is classically known for its antiphagocytic properties, and lately, was shown to trigger pro-inflammatory responses as well as induction of vascular leakage and shock. this likely represents important mechanisms contributing to the rapid development of shock and systemic toxicity in patients with severe invasive group a streptococcal infections. the understanding of these infections as hyperinflammatory diseases highlighted the potential of immunotherapy to improve outcome. one such strategy includes the administration of intravenous polyspecific immunoglobulin (ivig) as adjunctive therapy. the mechanistic actions of ivig in this setting are believed to include opsonisation of the bacteria, neutralisation of the superantigens and suppression of the pro-inflammatory responses. there is growing evidence to support the use of ivig in patients with streptococcal toxic shock syndrome. these studies include one observational cohort study based on canadian patients identified through active surveillance of invasive group a streptococcal infections, and one european multicentre placebo-controlled trial. however, the question remains whether ivig is efficacious also for the severe streptococcal deep tissue infections. an observational study of seven patients with severe streptococcal deep tissue infections suggested that the use of high-dose ivig in patients with severe gas soft tissue infections may allow an initial non-operative or minimally invasive approach, which can limit the need to perform immediate wide debridements and amputations in unstable patients. the fact that seven patients with severe group a streptococcal infections survived with this approach definitely warrants further studies to be conducted on the use of ivig in these severe infections. hepatitis o229 prevalence and outcome of pregnancy in chronic hepatitis c virus infection i. julkunen°, a. sariola, m. sillanpää, k. melen, p. koskela, p. finne, a.l. järvenpää, s. riikonen, h.m. surcel (helsinki, oulu, fi) objectives: in the western countries the incidence of hepatitis c virus (hcv) infection has steadily been increasing especially among young adults. it is thus likely that an increasing prevalence of hcv infection is also found in pregnant women. methods: to assess the frequency of hcv infection in the metropolitan area of helsinki selected anti-hcv antibody testing was carried out for pregnant women during the years 1991-1999. in addition, hcv prevalence was analysed in serum specimens collected from pregnant maters during the years of 1985-2005. results: altogether 145 mothers were identified among 44680 mothers. the frequency of anti-hcv positivity rose from 0.13% in 1991 to 0.43−0.53 in 1997-1999. in early 90's only 20% of mothers knew about their seropositivity, whereas by the end of the follow-up period almost 70% of mothers knew about their hcv infection already before the pregnancy. intravenous drug abuse was the major risk factor (71% of cases) for contracting the disease. in 90% of the mothers chronic hcv infection was well under control and in this population the mean serum alanine aminotransferase (alt) values decreased towards the end of the pregnancy. however, 10% of anti-hcv ab positive mothers developed intrahepatic cholestasis (odds ratio 16.4) as characterised by itching and elevated serum bile acid levels. the correspondig value in the control pregnancies was only 0.7%. anti-hcv ab positive mothers were younger, delivered earlier and gave birth to babies with smaller birth weight as compared to control deliveries. to have a more comprehensive view of the problem of hcv infection during pregnancy randomly selected serum specimens from the finnish maternity cohort were tested. 2000-5000 serum specimens were tested in selected cohorts (1985, 1990, 1995, 2000 and 2005) . in 1985 the nationwide prevalence was 0.19% and it steadily role to 0.50% in 2005. in the metropolitan area of helsinki the prevalence was higher being 0.68% and 0.70 in 1997 and 2002, respectively. conclusion: our study indicates that there is an increasing problem of hcv infection in pregnant women in finland. although most women cope well with their disease during pregnancy there is a subpopulation of mothers who develop cholestasis and their liver status should thus be followed-up carefully. testing of all mothers for serum anti-hcv antibodies is recommended. objectives: the viral genome of hepatitis c virus constitutes a 9.6kb single-stranded positive-sense rna which encodes altogether 11 viral proteins. in order to study the humoral immune responses against different hcv proteins in patients suffering from chronic hcv infection, we produced three structural (c, e1 and e2) and six nonstructural proteins (ns2, ns3, ns4a, ns4b, ns5a and ns5b) in sf9 insect cells by using the baculovirus expression system. the recombinant hcv proteins were purified and used in western blot analysis to determine antibody responses against individual hcv protein in 68 hcv rna and antibody positive human sera that were obtained from patients suffering from genotype 1, 2, 3 or 4 infection. results: these sera were also analysed with inno-lia score test for hcv antibodies against core, ns3, ns4ab and ns5a, and the results were similar to our western blot method. based on our western blot analyses we found that the major viral antigens were the core, ns4b, ns3 and ns5a proteins and they were recognized in 97%, 86%, 68% and 53% of patient sera, respectively. there were no major genotype specific differences in antibody responses to individual hcv proteins. a common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized ns2 protein and only three sera (from genotype 3) recognised ns5b. the data shows significant variation in the specificity in humoral immunity in chronic hcv patients. anti-hcv antibody pattern also remains very stable within one individual. alt and ast levels were tested in all subjects. the presence of hbv-dna was determined quantitatively in plasma samples of hd patients with anti-hbc alone (hbsag negative, anti-hbs negative and anti-hbc positive) by real-time pcr using the artus hbv rg pcr kit on the rotor-gene 3000 real-time thermal cycler. results: of 289 patients enrolled in this study, 18 subjects (6.2%, 95% ci, 3.5%-8.9%) had anti-hbc alone. hbv-dna was detectable in 9 of 18 hd patients (50%, 95% ci, 27%-73%) with anti-hbc alone. plasma hbv-dna load was less than 50 iu/ml in all of these patients. our study showed that detection of anti-hbc alone could reflect unrecognized occult hbv infection in hd patients. the majority of these infections are associated with low viral loads. were included in the study. all the subjects had never been exposed to antiretroviral therapy. genotypic resistance testing was performed at the time of diagnosis with a sequence-based assay (trugene hiv-1 genotyping test) targeted at the protease region (codons 1 to 99) and rt region (codon 40 to 247) of the hiv-l genome. results: 21 of 218 patients (9.63%) harboured a virus with at least one mutation associated with phenotypic resistance; 1/218 with mutations associated with resistance to nucleoside reverse-transcriptase inhibitors (nrtis), 17/218 to non-nucleoside reverse-transcriptase inhibitors (nnrtis) and 3/218 to protease inhibitors (pi). resistance to nrtis was associated with the key mutation m184v, while resistance to nnrtis was associated with y181c and k103n mutations. among mutations to pi, major resistance mutations l90m and d30n were found in three patients, whereas there was a high prevalence of accessory pi resistance mutations at positions 10, 20, 36 and 63. conclusion: our data estimate the prevalence of primary resistance and mutations patterns among naive hiv patients, underlining the importance of genotypic resistance testing in hiv patients before starting treatment, especially when nnrtis would be included in the initial antiretroviral therapy. objectives: few data are available on the genetic mechanisms of protease inhibitor (pi) resistance in non-b hiv-1, and pi resistanceassociated mutations (rams) are commonly observed in pi-naive patients with subtype a/e infection. this study aimed to compare pi-rams between pi-naive and -experienced patients. methods: genotypic resistance testing was conducted among a cohort of hiv-1 infected patients who had virologic failure. patients were categorised into 2 groups: pi-naive and pi-experienced. we focused on pi-rams previously described by ias-usa 2008. results: we studied 137 patients (mean age, 41.8 years; 64% male). median cd4 cell count and hiv-1 rna at virologic failure were 169 cells/cu.mm. and 14100 copies/ml, respectively. 85% of patients were infected with subtype a/e; the others had subtype b (12%), ab (2%), and c (1%). there were 75 patients in pi-naive group and 62 patients in pi-experienced group. the clinical characteristics between 2 groups were similar (p > 0.05) except for the duration of antiretroviral therapy which was shorter in pi-naive group (31.5 vs. 46.8 months, p = 0.028). percentage of patients who had primary pi-rams was 1% in pinaive and 19% in pi-experienced groups (p = 0.001). the most common primary pi-rams in the latter group were v82a (10%) and i54v (7%). percentage of patients with secondary pi-rams in the corresponding groups was 99% and 98%, respectively (p = 1.000). median number of secondary pi-rams was also similar between 2 groups (p = 0.244). the most common secondary pi-rams in both groups were m36i (91%), h69k (34%), l89m (30%), i13v (26%), l63p (25%), l10i we also defined a "silent score" (ss) and a "resistance score" (rs) as the number of synonymous mutations and of resistance mutations (in the second sequence in comparison with the first one) divided by number of days between the two tests, respectively. (12); pts with drms in non-b-st (%) were 7 (23.3), 6 (14), 5 (7) and 3 (4.2). a significant increase of non-b-st (p = 0.021) and a significant decrease in drms (p < 0.001) were observed. crf02_ag was the prevalent non-b st (44%). 35.3% of non-b st pts were italians. among b-st, drms predicted a reduced susceptibility to one drug class in 23, 17, 15 and 14 cases in the different periods; to two drug classes in 4, 6, 5 and 8; to three classes in 3, 2, 0 and 0. in non-b-st, a reduced susceptibility to one drug class was found in 6, 6, 4 and 0 cases; to two drug classes in 1, 0, 0 and 2; to three drug classes in 0, 0, 1 and 1, respectively. among pts with one or two classes of resistance, a decrease of percentage of protease inhibitors related drms, and a persistence of non nucleoside rt inhibitors involving drms, mainly 103n and 190a, were observed. methods: from hiv+ persons with a history of, or an acute episode of opc, oral fungal burden was evaluated bi-weekly and buccal mucosa tissue was collected bimonthly for a period of one year. tissue was evaluated for the presence of cd8+ t cells and e-cadherin by immunohistochemistry or flow cytometry. objectives: to define the secular trends in the epidemiology of candidaemia in queensland, australia (population, 4.1 million) over a 10-year period. methods: all episodes of candidaemia within queensland public hospitals from 1999-2008 were identified from laboratory information systems. data on species identification, antifungal susceptibility, demographics, and hospital ward of diagnosis, and denominator data (hospital admissions, accrued patient-days (pt-days) and fluconazole usage) were collected. results: over the 10-year period, 1137 unique episodes (100% case ascertainment) were identified from 42 healthcare facilities (8 tertiary, 2 paediatric, 11 secondary and 21 smaller hospitals). the median patient age was 56.4 years. the overall incidence-density was 0.45/10000 ptdays, highest in paediatric (1.28/10000 pt-days) and tertiary hospitals (0.62/10000 pt-days). over the 10 years, the incidence-density increased 3.2-fold in tertiary hospitals and 6.6-fold in secondary hospitals (both p < 0.0001 for trend), but not in paediatric or smaller hospitals. the incidence-density in icus (5.2/10000 pt-days) was 10-fold higher than in non-icu wards, but did not significantly increase over the study period. the relative proportion of episodes occurring in adult general medical/surgical (ie non-oncology/non-icu) wards significantly increased (p < 0.001), accounting for 62% of episodes at the end of the 10-year period, whereas that occurring in paediatric and adult oncology wards decreased (p < 0.001 and p = 0.07 respectively). overall, c. albicans accounted for 44%, c. parapsilosis 27% and c. glabrata 13%. although the incidence-density of all species increased over the study period, the relative proportion caused by c. albicans decreased (p = 0.007) and c. parapsilosis increased (p = 0.01). despite significantly increased fluconazole usage (from 19.7 to 30.6 ddd/1000 pt-days, p < 0.0001), the relative proportion caused by c. glabrata/c. krusei did not change (p = 0.5). the overall incidence of candidaemia has increased almost 400% in queensland public hospitals over the last 10 years. the relative proportion of episodes occurring among general medical/surgical patients and caused by c. parapsilosis has increased. candidaemia is an increasing problem the epidemiology of which continues to evolve. it is increasingly affecting patients outside traditional risk groups. conclusions: this surveillance study and pharmaco-economic modelling has proved immensely beneficial in setting up inhouse processing, improved tat, reduced costs of outsourcing and subsequent use of expensive antifungals. reduction in mortality has been noted but is not statistically significant. c. albicans was the commonest isolate; fluconazole resistance is minimal and associated mortality is lower than reported from europe. many pts received systemic prophylaxis (72%); itraconazole and fluconazole were used in 68 and 33 pts respectively. no differences emerged between empirical vs pre-emptive therapy and none of the drugs resulted to significantly influence outcome. in 66% of pts initial empirical/pre-emptive drug remained unchanged after ia diagnosis, while in 16% clinicians shifted to a combined treatment. conclusion: this study allows as to analyzed multiple factors as potentially influencing outcome. we confirmed that aml phase and neutropenia influence ia outcome. present data confirm the perception that during last years the application of a correct and timely diagnostic work-up and the availability of more efficacious and less toxic drugs (i.e. voriconazole, liposomal amphotericin b, caspofungin) have modified the course of ia. however none of the new drugs emerged as the most efficacious in our series. even combined treatment did not confer any advantage in survival analysis. (<3% each). the first line therapy was monotherapy with voriconazole (49%), caspofungin (14%), lipid formulations of amb (9%) or used antifungal drugs combination (20%). the mortality rate at day 90 was 41% when first line therapy included voriconazole compared to 60% when it did not (p < 0.001). conclusion: comprehensive collections of cases based on systematic reporting and description of cases using a dedicated network of hospitals in selected regions and stringent definition criteria applied by trained clinicians and microbiologists are useful to describe ia, to assess its burden and secular trends, and to identify potential changes in diagnostic and therapeutic procedures. this network will expand to other regions in the near future, and data will help assessing the impact of new management strategies such as prophylaxis with posaconazole, the impact of modification of new diagnostic criteria as recently proposed (clin infect dis, 2008), and identifying new populations at risk for ia. nosocomial aspergillosis represents a serious threat for severely immunocompromised patients and outbreaks have been attributed to airborne sources. the role of hospital-independent fungal spread sources e.g. the private homes or business suites are not known. we investigated the relationship between fungal exposure prior hospitalisation and the ensuing onset of invasive mould infections (imi) in patients at risk. patients admitted to the department of haematology and oncology or to the department of transplant surgery of the innsbruck medical university received a structured questionnaire regarding their fungal exposure prior hospitalisation. questions inquired heavy fungal exposures up to five days prior hospitalisation. 234 patients were enrolled in this study and 19% were smokers, 22% suffered from an airborne allergy, 62% lived in old buildings, 73% were ruralists, 82% and 92% were exposed to any outdoor or indoor fungus sources. poor housing conditions and other fungus exposures were associated with the onset of community-acquired imi only in patients with acute myelogenous leukaemia (p < 0.01). aml patients being more at risk for imi when smoking cigarettes (p < 0.05), living on the country site (p < 0.05), having two or more fungus exposures (p < 0.05) and suffering from allergy to dust, pollen and/or moulds (p < 0.05). a similar trend was for lung transplant recipients receiving extensive immunosuppressive agents to treat allograft rejection. overall, 88% of imi were community-acquired cases. hospital-independent fungal sources highlight risk-factors for imi in severe immunocompromised patients and the rate of communityacquired imi does increase. an analysis of an individual patient's risk factors for fungal infection and the type of fungus to which they are most susceptible, indicates the preventative strategies that are likely to be successful. to the icu-mhs with aspergillus spp detected in significant amounts in clinical samples. the underlying conditions of the patients were heart transplantation (n = 5), major heart surgery (n = 4), and other (n = 2). eight (72.7%) patients developed proven/probable ia (4 with lung infection, 2 with mediastinitis, 1 with disseminated ia, and 1 with prostate involvement). the mortality of patients with ia was 87.5%. the icu-mhs is divided into 3 areas, one of which is equipped with hepa filters. only 1 case of ia occurred in the protected area. we measured the fungal conidia levels in the air of each of the 3 areas (508 samples analyzed) monthly. a total of 172 strains of a. fumigatus (110 clinical strains from 10 patients and 62 environmental strains) were genotyped using microsatellites (de valk et al, jcm 2005) . the mean airborne conidia levels (6 months) before and after the outbreak were, respectively, 5.6 (0−15) cfu/m3 and 1.8 (0−10) cfu/m3. no cases of ia occurred during these periods. however, all cases of ia were linked to 4 peaks of abnormally high airborne conidia levels (65, 70, 200 and 500 cfu/m3). a. fumigatus was involved in 7 cases of ia; 1 patient was infected by non-fumigatus aspergillus (not further genotyped). in 4 patients (1 mediastinitis, 2 pulmonary ia and 1 colonisation), we demonstrated similar genotypes in the air and in clinical samples. patient 1 was located in the protected area and had a unique genotype. patient 2 had two different clusters of genotypes: one cluster was similar to that of patient 3 and the other was also found in patient 4 and in the air. the genotype present in patients 2 and 4 was also detected in the air during a 6-month period. conclusions: epidemiologic and molecular typing suggests that there is a causal relationship between aspergillus causing ia and those present in the air. our finding also supports the need for hepa filtration in icu-mhs. j. guinea is contracted by fis (cm05/00171). sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were calculated in reference to proven and probable cases of ia. reasons for performing bronchoscopy on patients were also recorded. the protocol received approval by the local ethic committee. results: from the 117 samples studied, 5 (4.3%) were classified as proven, 6 (5.1%) as probable, and 35 (29.9%) as possible cases of aspergillosis. twelve samples (10.3%) represented colonisation, and 59 bal samples were obtained during routine surveillance. pulmonary aspergillosis was the main clinical presentation of ia (63.6%). using roc analysis, the best cut-off for galactomannan testing in bal was defined as 1.5 (sensitivity 90.9%, specificity 90.6%, ppv 48% and npv 99.1%). median bal gm index for the group of patients with proven/probable aspergillosis and for 'negative cases' were 3.3 and 0.5, respectively (p < 0.001). overall mortality was 20% (n = 12). the odds for death for patients diagnosed with ia were 11.8, in comparison to patients who did not have this infection (95% ci 2.9−48.4). conclusion: gm testing in the bal added to the diagnosis of ia in lung transplant recipients. in order to avoid false-positive results, a higher test cut-off should be applied to bal samples, in comparison to sera. increasing the cut-off to 1.5 resulted in a very high npv, with an associated sensitivity of >90%. objectives: 1) determine the performance characteristics of the galactomannan (gm) assay in broncho-alveolar lavage (bal) in haematology-oncology patients; 2) evaluate the prognostic value of the gm assay in this particular population. methods: the platelia gm eia assay (bio-rad) was performed on all bal specimens obtained from haematology-oncology patients at our institution between march 2005 and april 2008, in addition to routine laboratory stains and cultures. all results were reported to physicians. we conducted chart reviews to classify cases as proven, probable, possible or without invasive pulmonary aspergillosis (ipa) according to the revised definitions of invasive fungal disease from the eortc/msg consensus group. for performance characteristics, proven and probable cases were considered as ipa; possible cases were considered as without ipa. the result of bal gm was not considered as a criterium to classify cases in order to avoid incorporation bias. in patients with >1 positive (gm index >0.5) specimen, only the first one was considered for the analysis. mortality was calculated at 60 days following the first bal procurement. data were analyzed with stata 8.0. results: there were 173 bal samples from 145 patients, including 101 haematopoietic stem cell transplant (hsct) recipients. we found 5 proven, 7 probable and 35 possible cases of ipa (total of 12 ipa cases; 6.9%). gm on bal was positive in 47 (27.2%) specimens. the sensitivity and specificity of the gm assay in bal were 100% and 78.3% respectively. positive predictive and negative predictive values were 25.5% and 100%, respectively. false-positive results were found in 21 patients without ipa and in 14 with possible ipa. an index value 0.5 was significantly associated with a 60-day mortality risk (12/39 patients with a positive gm died within 60 days after bal compared to 13/106 with a negative gm (or = 3.2, 95%ci 1.3−7.8; p = 0.01). this association was even stronger when restricted to hsc recipients (or =4.6, 95%ci 1.5−13.6; p = 0.006). the clinical utility of gm assay in bal mainly lies in its negative predictive value, identifying patients at low risk of ipa. this test also carries a prognostic value in predicting patients at higher risk of mortality. (see table below) . not significant differences have been found among pneumocystis colonisation and copd status evaluated by fev-1%. as well as no significant differences respect to age, sex or lymphocytes and leucocytes blood count were found. background: infliximab, a monoclonal antibody targeting tumour necrosis factor alpha (tnf-a), is indicated for the treatment of rheumatoid arthritis (ra) and other autoimmune diseases. however, its use has been associated with opportunistic infections, including pneumocystis jirovecii pneumonia (pcp). moreover, p. jirovecii has been observed colonising to humans with several disorders. objectives: to obtain information about p. jirovecii colonisation among patients with rheumatologic disease treated with infliximab. this information could be useful for assessing new strategies in the prevention of pcp in patients at risk. methods: 62 consecutive patients treated with infliximab for rheumatic disorders were included in the study. oropharyngeal washes (ow) samples were collected for p. jirovecii detection. clinical and demographic data were collected (sex, age, rheumatologic diagnosis, duration of infliximab use, concomitant use of other drugs for rheumatologic treatment, use of any other anti-tnf-a agent, use of anti-pc drugs in the last six months, smoking, and diagnosis of chronic pulmonary respiratory disease). p. jirovecii colonisation was identify in ow samples by pcr at mtlsu-rrna gene, with primers paz102-x and paz102-y. we adapted a method previously described to a real-time pcr setting, using a lightcycler 1.5 (roche, germany). individuals in whom the presence of p. jirovecii was detected at two independent assay in the absence of respiratory symptoms or radiological findings suggestive of pcp were considered to be colonised. results: clinical and demographic data for 62 patients treated with infliximab are presented in table 1 objectives: most research with human bocavirus, a recently found respiratory pathogen, has been done by molecular biology (polymerase chain reaction, pcr). the results have been ambiguous because the virus has often been found in co-infection with other viruses, and also in clinically healthy subjects. it has been proposed that, for bocavirus, antigen detection could better indicate the aetiology than qualitative nucleic acid detection. we have developed a rapid antigen detection test for the virus. the one-step test for bocavirus vp2 antigen is based on a separation-free two-photon excitation fluorometry (arcdia tpx assay technique). the assay protocol is simple; the swab sample is dissolved in sample buffer, and the solution is dispensed (20 ml) onto a 384-well microtitre plate (containing the reagents in dry form) for incubation and automated quantitative measurement. the immunoassay applies microspheres as solid-phase carriers of purified bocavirus-specific polyclonal antibodies. the virus antigens concentrate onto the solid-phase which is probed in real-time with fluorescently labelled antibody reagents. strong positive samples are reportable in 15 minutes, while low positive and negative samples are reported in 2 hours. the performance of the method was studied with recombinant human bocavirus-like particles (vp2), and purified respiratory pathogens (group a streptococci, streptococcus pneumoniae, and influenza a and b, respiratory syncytial, metapneumo, adeno, and parainfluenza 1−3 viruses). results: analytical detection sensitivity of the method (lowest limit of detection, 0-control + 3sds) was 3 ng/ml, dynamic concentration range was three orders of magnitude, and intra-assay imprecision was 5−10%. cross-reactions with the other respiratory pathogens were not found. the new method enables rapid detection of bocavirus antigens. the new test is very easy to perform in comparison to standard elisas. the analytical sensitivity of the method is expected to allow analysis of clinical samples. the sensitivity of the antigen detection test could be significantly increased by the use of monoclonal antibodies (10-100 fold). our future objectives include increasing the detection sensitivity, and analysis of clinical samples in order to study the correlation of antigen detection and the clinical aetiology. life-year for patients who survived. all analyses were performed using treeage software (2008). results: the overall mortality rates for empiric vancomycin (v) and semi-synthetic-penicillin (ssp) was 30% and 35%, respectively, as apposed to 24% for those receiving the rapid mrsa pcr testing. these mortality rates were similar in both the eu and us subsets. furthermore, the number needed to test in order to save one life was 20 and 11 for empiric v and ssp, respectively. using sensitivity analysis the prevalence of mrsa was varied from 5% to 80% and yielded an absolute mortality difference favouring the pcr testing group of 10% and 2%, respectively as compared to empiric v and 1% and 18% compared to empiric ssp. in eu the c/e for empiric v and ssp treated patients was €873 and €949, respectively as compared to €807 for rapid pcr testing. in the us the c/e for empiric v was $1,049 as compared to $971 for rapid pcr testing. using sensitivity analysis the prevalence of mrsa was varied from 5% to 80% and yielded favourable c/e in both the eu and us for rapid pcr testing regardless of the empiric treatment regimen. conclusion: rapid mrsa pcr testing using the xpert mrsa/sa blood culture pcr assay appears to improve mortality rates and is cost effective in the eu and us across a wide range of mrsa prevalence rates. background: rapid detection of gastro-intestinal carriage of glycopeptide-resistant enterococci (gre) from screening cultures is crucial for an efficient control of their spread. we assessed 4 media − 2 chromogenic, chromid, (biomérieux), and chromagar (chromagar microbiology), and 2 selective, vre selective (oxoid) and eccv (bd) − for their ability to detect gre using well-characterised isolates and stool samples from hospitalised patients at high risk of gre colonisation. methods: twenty-five isolates consisting of 13 gre. faecalis/faecium carrying various van genes and 12 non-vre at concentrations of 10 6 -10 1 cfu/ml and 10 6 cfu/ml, respectively, and 37 stool samples were randomised and spiral plated on all media and scored by 5 blinded investigators for characteristic colonies after 24 hrs incubation. standard confirmatory tests were done on 1 putative gre colony or on 1 characteristically coloured colony each for e. faecalis/faecium from the selective and chromogenic media, respectively. detection of van genes, and ddl or soda based speciation was done on pcr-sequencing. mean sensitivity (sen) and specificity (spec), and confidence intervals (cis) were estimated for each medium by a logistic regression model using a penalised likelihood approach based on the reader response for the stool samples and isolates, and additionally on confirmation test results for the stool samples, both at the aggregated (gre detected) and penalised level (correct species-colony colour correlation). results: chromagar showed the highest sen based on reader response at the aggregated and penalised level for both stool samples and isolates (table) . using confirmation test results at the aggregated level, sen for eccv was highest while the two chromogenic media showed a decrease in sen by at least 11% in comparison to the values obtained based on reader response. sens for the 2 chromogenic media were even lower (<70%) based on confirmation test results at the penalised level. eccv and chromid showed the highest specs with both reader response (stool samples) and confirmation test results at the aggregated level, and chromid also at the penalised level, with narrow cis indicating a high precision of this parameter estimate. for isolates, specs were highest for chromagar at both levels. conclusions: chromagar showed the best overall performance considering both sen and spec estimates. eccv performed well as a selective medium for gre detection from stool samples. objectives: metallo-beta-lactamases (mbls) expressed from pseudomonas are able to confer resistance to all beta-lactams with the exception of aztreonam. however, enterobacteriaceae possessing mbls exhibit moderate cephalosporin and low carbapenem mics and thus are often underestimated. herein, we describe data from new etest prototypes specifically designed to detect this problematic resistance mechanism. methods: 82 mbl-positive (vim or imp derivatives) enterobacteriaceae clinical isolates from 8 countries and 27 randomly selected enterobacteriaceae negative controls (including the atcc type strains) were tested against the 4 different etest mbl prototypes. beta-lactam substrates used were imipenem (ip), meropenem (mp), ceftazidime (tz) and cefotaxime (ct) with or without the inhibitors dipicolinic acid (dpa) and edta. the etest standard procedure for gram negative aerobes was used and a reduction of beta-lactam mic by equal to or greater than 3 dilutions by edta or dpa was interpreted as positive for mbl. presence of esbls was tested using the etest ct/ctl, tz/tzl and cefepime (pm)/pml strips. ampc production was detected using the etest cefoxitin (fx)/fxi and cefotetan (cn)/cni strips. of the 784 select specimens that were negative for gbs, 345 grew turquoise-blue colonies, but the majority that required further work to rule out gbs grew after 48 hours. two strains of gbs that were missed grew as white colonies on select, and even at 48 h, did not exhibit the characteristic turquoise-blue colour. conclusion: ssb enrichment followed by select subculture was extremely sensitive (99.2%) and superior to cna/ssb for detection of gbs from genital specimens. however, non-gbs organisms can produce turquoise-blue colonies on select and further work must be performed to rule out the presence of gbs. objectives: screening for chlamydia trachomatis (ct) specific antibodies is valuable in investigating recurrent cause of miscarriage, pelvic inflammatory disease and tubal damage following repeated episodes of pelvic inflammatory disease. immunofluorescence (if) is considered the gold standard for detection of ct antibodies. the present study aims to compare the performance of 4 other commercial tests for the detection of serum igg antibodies specific for ct: two ct igg pelisa both using major outer membrane protein (momp; ["momp-medac", ct-igg-pelisa; medac, wedel, germany and "momp-ruwag", ct pelisa; ruwag, bettlach, switzerland), one ct hsp-60 igg pelisa ("hsp60-medac", chsp60-igg-pelisa; medac, wedel, germany), and a new automated epifluorescence immunoassay ("inodiag", "must chlamydiae; inodiag, signes, france). methods: a total of 405 patients with (n = 251) and without (n = 154) miscarriages were tested by all 5 serological tests described above. sensitivity and specificity were calculated using if as gold standard. a second standard, defining true positive or negative samples as sera respectively positive and negative in all 4 others tests, was also used (see table) . objectives: participation in diagnostic microbiology internal and external quality control (qc) processes is good laboratory practice, an essential component of a quality management system and compulsory in some european countries. currently, there is no qc scheme for diagnostic oral microbiology. the aim of this study was to collate information on current qc needs, and processes undertaken in diagnostic oral microbiology laboratories. method: an on-line questionnaire was devised to ascertain interest in participating in an oral microbiology qc scheme and sent to oral microbiology diagnostic laboratories. the laboratories were identified from participants attending the european oral microbiology workshop in helsinki, 2008. following this, a pilot round of qc samples was distributed to all interested laboratories. results: we identified 12 individuals that worked in diagnostic oral microbiology laboratories and received 7 (58%) positive responses. of these 7 laboratories (representing 6 european countries) 71% did not participate in either internal or external qc. each laboratory processed on average a total of 4135 samples annually. 86% of participants were in favour of a european-wide oral microbiology qc scheme. the preferred frequency for receiving external qc specimen was once in 3−4 months. the most preferred specimen types were periodontal pocket and oral pus specimens (both 29%), followed by oral mucosal swabs and caries activity tests. all participating laboratories were willing to share and harmonise their specimen processing and interpretation standard operating procedures. the pilot round specimen was a periodontal pocket sample. six laboratories reported their findings in the specified time. the predominant pathogens (aggregatibacter actinomycetemcomitans, porphyromonas gingivalis) were identified by 5 of 6 laboratories. in addition to conventional culture, one laboratory used pcr. 5 laboratories performed antibacterial sensitivity testing primarily by disc diffusion. conclusions: this is the first attempt to a standardised europeanwide approach to diagnostic oral microbiology. the findings from this feasibility study have indicated that a qc scheme for oral microbiology is of interest and have raised a number a pointers for subsequent rounds of specimens. further work to improve the quality, to standardise the methodology and the interpretation of diagnostic oral microbiology at the european level is on-going. objectives: since severe sepsis with acute organ dysfunction can be fatal within hours, it is customary to start empirical broad-spectrum antimicrobial therapy in all patients hospitalised for a suspicion of systemic inflammatory response syndrome. however, increased use of broad-spectrum antimicrobials over the years has contributed to the emergence of drug resistant strains of bacteria. especially, drug resistance among gram-positive bacteria, the leading cause of sepsis, is now a serious problem. the objective of this preliminary study was to develop a method for distinguishing between gram− and gram+ bacterial infection. methods: in this prospective study, leukocyte and neutrophil counts, crp, esr, and quantitative flow cytometric analysis of neutrophil complement receptors 1 (cr1/cd35) and 3 (cr3/cd11b), were obtained from 289 hospitalised febrile patients, of which 89 had bacterial and 38 viral infection. the patient data were compared to 60 healthy controls. results: it was noticed that in gram− infection (n = 21) the average amount of cd11b on neutrophils was significantly higher than in gram+ infection (n = 22). on the contrary, serum crp level was significantly higher in gram+ than in gram− infection. other measured parameters did not differ significantly between gram+ and gram− infections. we derived a crp/cd11b ratio dividing the serum crp value by amount of cd11b on neutrophils. in thirteen (76%) out of 17 patients with gram+ sepsis had crp/cd11b ratio cutoff value of 3.1 (figure 1 ). of these 13 patients, 9 (70%) were diagnosed with streptococcus pneumoniae, 2 with staphylococcus aureus, 1 with enterococcus faecalis, and 1 with both streptococcus intermedius and streptococcus oralis. corresponding percentages in patients with local gram+ infection, gram− infection, clinical pneumonia, other clinical infection, and viral infection were 20%, 14%, 30%, 15%, and 0%, respectively. conclusion: the detection of gram+ sepsis is possible after combination of neutrophil cd11b data and serum crp level. crp/cd11b ratio viral infections of the central nervous system s61 displayed 76% sensitivity and 80% specificity for detection of gram+ sepsis. the proposed crp/cd11b ratio test could, for its part, assist physicians to decide appropriate antibiotic treatment in patients with severe bacterial infection. a bacterial biofilm is a structured consortium of bacteria cells surrounded by a self-produced polymer matrix. biofilms may be monospecies or polyspecies biofilms. biofilm growing bacteria give rise to chronic infections, which persist in spite of therapy and in spite of the host's immune-and inflammatory responses. biofilm infections are characterised by persisting pathology and immune response (in contrast to colonisation). bacterial biofilms use both biofilm specific (b) and conventional (planktonic) resistance mechanisms (p) when they are exposed to antibiotics. the following resistance mechanisms have been described in bacterial biofilms: 1. stationary phase physiology (b), low oxygen tension (b) and slow growth (b) especially inside biofilms whereas the surface of biofilms is more similar to planktonic growth. 2. penetration barriers (b), binding to the polymer matrix (b). 3. mutations, hypermutators (b, p). 4. chromosomal betalactamase is upregulated (b, p). 5. antibiotic tolerance/adaptive resistance (b). 6. efflux pumps (b, p). 7. alginate production (b). 8. high cell density and quorum sensing (b, p). 9. pbp 3 − sos response ? (b). the knowledge of these resistance mechanisms can, however, be used to design new therapeutica approaches especially as regards quorum sensing inhibitors. we consider two factors that contribute to treatment failure in the absence of inherited resistance, the density of the population being treated and the physiological state of the bacteria. we also explore how these factors might contribute to the evolution of inherited resistance during the course of treatment. we conclude with a computer-and chemostat-assisted consideration of the potential clinical implication of these density and physiology effects and make suggestions for treatment protocols to deal with them. using in vitro cultures of staphylococcus aureus atcc25923 or the clinical isolate ps80 and antibiotics of six different classes we determined the functional relationship between the inoculum density and the efficacy of the antibiotics. as measured by the rates and extent of kill and/or the minimum inhibitory concentration (mic), the efficacy of all of these antibiotics declined with increases in the density of bacteria, albeit to different extents. for daptomycin and vancomycin, much of this density effect can be attributed to bacteria-associated declines in the effective concentration of the antibiotic in the medium. for gentamicin, vancomycin, ciprofloxacin and oxacillin, our bioassays failed to reveal significant reductions in their effective concentration in the medium. the effects of the physiological state of s. aureus on the efficacy of these antibiotics were examined for bacteria from cultures in "stationary phase" for different times and from chemostats run at different generation times. these experiments are currently under way but by the time of the symposium we will have the full (and true) story. it is, however, clear that the efficacy of all of these antibiotics declines with the time in stationary phase (its "age"). and, even slowly dividing cultures from chemostats are more susceptible to antibiotic-mediated killing that early stationary phase batch cultures. the efficacy in killing non-growing bacteria varies among the bactericidal antibiotics examined. to ascertain the potential clinical implications of these density and physiological effects, we use both computer and in vitro simulations of antibiotic treatment. the results of these simulations provide compelling support for the proposition that antibiotic treatment regimes, including those designed to prevent the ascent of resistance, should take into account the anticipated density and physiological state of the target population of susceptible bacteria. there have been an increasing number of neurotrophic viral infections playing an important role in the world over the last decade. the list includes west nile virus, nipah and hendra virus (both paramyxoviruses), as well as chikungunya virus which suddenly emerged. furthermore, the relation between jc virus in progressive multifocal leukoencephalopathy (pml) in patients with multiple sclerosis treated with a new immunosuppressive drug, has triggered our attention. the development and implementation of molecular based amplification method has assisted us to detect these viruses more efficiently. these technologies have been used now routinely in a large number of laboratories to enable the detection of more commonly known neurotrophic viruses, like hsv, vzv and the neurotrophic picornaviruses like enterovirus and parechovirus. the pitfalls of these molecular methods have been generally solved by implementing regular quality control testing schemes, like organised by qcmd (quality control of molecular diagnostics) and the introduction of internal controls during the whole diagnostic process. finally, with the ability to quantify the amount of nucleic acid present in csf, more information on the pathogenesis of these viral infections, as well as significant tool to monitor the antiviral effect of treatment options for these viruses, has become available. to as a rare disease in europe restricted to some endemic foci. however, current data suggest that the incidence of ae has significantly increased, and the disease is spreading to the north, west, and east. ae has become an emerging disease in the baltic countries. thus, human infections with e. multilocularis have arrived in the "centre" of europe. ae is a lifethreatening disease, and is characterised by a tumour-like lesion in the liver. the larva can infiltrate the surrounding tissues and metastasize to distant organs. in an attempt to classify the large variety of anatomical findings in ae, the pnm-classification system was developed and serves as a benchmark for standardised evaluation of diagnostic and therapeutic measures. modern imaging techniques, such as ultrasound, ct or mri and pet/ct contributed not only to a much better description of the lesions, but also to a judgment upon the activity of the metacestode. the differential diagnosis of ae varies from haemangioma-like lesion of the liver or cancer. the diagnostic skills are limited, and are the reason for frequent misdiagnosis in geographic areas where ae is rather unknown. continuous treatment with benzimidazoles is the backbone of a lifelong management of ae. however, radical resection is the procedure of choice and should always be strived for. ae is still a rare disease in europe, but where it occurs, it is often diagnosed too late. patients are misdiagnosed for months and years, before receiving the correct treatment. at that late stage the disease has progressed, and radical cure of the liver lesion(s) is not anymore possible. recent reports provided hints for an accelerated larval growth of echinococcus spp. in the immunodeficient host. a careful monitoring of patients receiving immune-modifying drugs is warranted. the modern clinical management and long-term parasitostatic treatment with benzimidazoles are highly effective. thus, a higher alertness for the "tumours from the centre" would increase the prognosis of this hepatic disease resembling liver cancer. the percutaneous treatment of liver hydatid cysts were considered to be contraindicated due to two main potential risks: anaphylactic shock and abdominal dissemination of the disease. since the first case percutaneously treated was published, several series of successful percutaneous treatment of the liver and the other abdominal organs, peritoneum, thorax, soft tissue and orbital cavity hydatid cysts have appeared in the literature. percutaneous treatment of hydatid liver disease is an effective and safe procedure with its unique advantages (e.g., shorter hospital stay, low complication rate). today, the percutaneous approach has an important role in treatment of hydatid cysts not only in the liver but also in the other organs and tissue. therefore it must be first treatment option whenever it is indicated. in europe, dirofilaria immitis and dirofilaria repens are responsible of autochthonous filariases in dogs. adults of d. immitis kills the dogs with an heart location and d. repens is often found in subcutaneous nodules in dogs and cats. the microfilariae are present in the blood of these animals. dirofilariasis is due to the transmission of microfilariae by some mosquito bites (aedes, culex, anopheles, mansonia, psorophora and taeniorhynchus). usually non pathogenic to humans, these parasites are particularly present around the mediterranean basin. d. immitis is very rare in humans in europe, sometimes found in a pulmonary nodule and the heart location is not described. d. repens is more frequent and emerging in humans. usually, only one larva develops, producing an immature adult worm inside a subcutaneous nodule. ultrasound examination may suggest the parasitic origin of the lesion before an extraction and a parasitological diagnosis of the worm. more often, a fortuitous diagnosis is made on histological examination. very rarely, an adult worm may mature and produce systemic diffusion of microfilariae. dirofilariasis due to d. repens can present problems in diagnosis and treatment. an ocular and subconjunctival location of the worm and a subcutaneous nodule enclosing an immature adult are the commonest clinical forms. exceptional pulmonary locations are described. the subcutaneous locations described are: skull, cheek, breast, inguinal area, buttocks, arms and legs. cases of testicular location with painful symptoms have been observed. blood hypereosinophilia was exceptionally observed in human. it is treated surgically, by excision, without chemotherapy. while the majority of esbls, isolated in clinically-relevant gram negative bacteria (gnb) (mostly enterobacteriaceae, p. aeruginosa, a. baumannii) are tem-, shv-or ctx-m-types, a few others have been reported (sfo, bes, bel, tla, ges, bel, per, veb-types, and some oxa-esbls). laboratory detection of esbl-producers is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. selective culture media (macconkey and drigalski agar supplemented with cefotaxime and/or ceftazidime) have been proposed for detection of gnb resistant to expanded-spectrum cephalosporins (esc). media using chromogenic based substrates and selective antibiotics have been developed recently for the detection and presumptive identification of esbl-producing enterobacteriaceae directly from clinical specimens. detection of esbls based only on susceptibility testing is not easy due to the variety of b-lactamases and their variable expression of blactam resistance. commercially available esbl detection methods yield at most 90% accurate esbl identification, since some esbl-producers may appear susceptible to some escs. therefore, any organism showing reduced susceptibility to esc should be investigated using esbl confirmatory tests. these tests should be able to discriminate between esbl-producers and those with other mechanisms conferring esc resistance. these phenotypic tests (double-disk synergy test, esbl etest, and the combination disk method) are based on clavulanate inhibition and esc susceptibility testing. they often need slight changes by either reducing the distance between the disks of esc and clavulanate, the use of cefepime (not hydrolysed by ampcs), the use of cloxacillincontaining plates (that inhibits ampc), or by double inhibition by edta and clavulanate (masking metallo-enzymes). enzymatic tests have also been proposed for identification of esbl-producers. several pcr-based techniques (end-point or real time) have been developed on clinical samples or on colonies. several esbl genes have been detected using pcr coupled to either pyrosequencing, inverse hybridisation, to dhplc, or to fluorescent probes. these techniques even though more specific require technical knowledge, special equipment, are costly and detect only known genes, regardless of their expression. detection of esbl-producer remains a challenge for the microbiology laboratory and one shall be aware that esbl screening media are now available. resistance to antimicrobial agents has become common in many bacterial species, particularly those that cause human infections. the rapid detection of resistant organisms directly in clinical samples by real-time pcr coupled with molecular beacons, or of potentially resistant bacteria and yeast in blood culture bottles by peptide nucleic acid-fluorescence in situ hybridisation (pna-fish) is already having a positive impact on antimicrobial therapy. the direct detection of mycobacterium tuberculosis in sputum in approximately 2 hours with concomitant detection of mutations in rpob indicating rifampin resistance (as a surrogate for multidrug resistance) in the near future will likely improve the outcomes for tuberculosis patients in many developing and developed countries. several molecular technologies, including microarrays, bacterial tag encoded flx amplicon pyrosequencing (btefap), and ultra deep sequencing, have not yet transitioned to clinical laboratories but will likely provide even greater information about antimicrobial resistance not in just a single species, but in a whole community of microorganisms. complex wounds, like diabetic foot ulcers, containing multiple resistance genotypes are amenable to analysis by btefap. the implementation of these technologies in the clinical laboratory will be expensive but the potential to dramatically improve therapeutic outcomes especially for life-threatening diseases is unprecedented. objective: to determine the appropriateness of antimicrobial therapy (amt) in 11 dutch hospitals. method: data were obtained from a prevalence survey performed within the dutch surveillance network for nosocomial infections (prezies). amt administrated on the day of the survey was registered. antiviral and antifungal drugs, tuberculostatics, cements containing amt and prophylaxis administrated in the operation-theatre were excluded. the appropriateness of amt was assessed according to a standardised algorithm based on the local antimicrobial prescription guidelines. per patient a classification in appropriate use, inappropriate use and insufficient information was made. figure: relative risk of ia use of amt against largest hospital (hospital c). results: a total of 3,546 patients were included of which 1,075 (30%, range per centre (rpc): 23−37%) received amt. in the latter group, amt was considered appropriate in 70% (rpc: 57−84%), inappropriate in 17% (rpc: 3−32%) and was not judged because of insufficient information in 13% (rpc: 1−30%). there was considerable variation in inappropriate use among the participating centres (figure). in univariate analysis older age, the use of quinolones, being on the urology ward and presence of a suprapubical catheter were associated significantly with inappropriate use. admission on the icu and presence of an intravascular catheter were associated significantly with appropriate use. in a multivariate analyses the presence of suprapubical catheter, being on the urology ward and the use of quinolones were determinants for inappropriate use. this study showed large differences in overall use and appropriateness of use of amt between hospitals. based on these results it is possible to define targets for intervention to improve the prudent use of amt. the high fraction of patients with insufficient information in several centres may have influenced the analyses and should be addressed in future studies. m. struelens°, s. metz-gercek, r. mechtler, f. buyle, a. lechner, h. mittermayer, f. allerberger, w. kern objectives: the eu-project antibiotic strategy international (abs) qi team developed process qis for auditing the performance of key treatment and prophylactic practices. an international network of pilot hospitals tested these tools for feasibility, reliability and sensitivity to improvement. methods: qis included: 1. surgical prophylaxis (indication, drug choice, timing and duration of administration); 2. management of community-acquired pneumonia (cap) (blood culture and legionella antigen tests and drug choice for empirical treatment); 3. management of s. aureus bacteraemia (echocardiography, iv catheter removal and duration of therapy); and 4. iv-po switch for bio-available antibiotics. a minimum of 40 consecutive cases per centre and qi were retrospectively reviewed from clinical, laboratory and administrative records and assessed for data availability, inter-observer reliability, data collection workload and performance score. results: a total of 1240 patients were evaluated in 11 acute care hospitals from 5 countries, with a range of 80 to 500 cases and 2 to 9 centres per indicator. seven centres had already implemented antibiotic quality improvement and audit programmes. availability of data was >85% of cases and ranged between 87% (catheter removal in s. aureus bacteraemia) and 100% (diagnostic tests for cap). 13/14 indicators were found to be reliable with kappa 0.60 (good to excellent agreement). the workload per case ranged from a median time of 16 (cap) to 35 min (iv-po switch). the intention to treat qi scores showed high levels of adherence to the surgical prophylaxis qi bundle, with median values of 81 to 97% for hip prosthesis and 65 to 92% for colo-rectal surgery. for cap management, diagnostic testing appeared sub-optimal (<56% compliance with idsa guidelines). for s. aureus bacteraemia management, indicator results ranged from 60 to 65%. for use of bio available antibiotics, a median of 45% iv administrations were avoidable. there were marked differences of scores between centres for all qis. conclusions: the abs qis are reliable and broadly applicable tools for auditing antibiotic treatment and prophylactic practices. inter-hospital variation in adherence to recommended practice indicates substantial potential for improvement with different local priorities. these qis can be recommended for assessing the effect of quality of care interventions at either local or multi-centre level. d.j. noimark°, e. charani, s. smith, b. cooper, i. balakrishnan, s.p. stone (london, uk) introduction: reduction of clostridium difficile infection (cdi), which often follows use of third generation cephalosporins, is a national priority. over a three year period, antibiotic policies were reviewed and changed in an elderly medicine department according to local sensitivities of common pathogens and levels of cdi. a laminated pocket-sized card describing antibiotic policies was given to all doctors in the department on induction with instructions not to depart from these without microbiologists' approval. this prospective controlled interrupted time series examines whether this intervention increased compliance with antibiotic policy and decreased cdi incidence. methods: the department's "narrow-spectrum, no cephalosporin" antibiotic policy was changed on 1st august 2006 to replace trimethoprim with cephradine (1st generation cephalopsporin) as empiric treatment for urinary tract infection, reflecting local escheriscia coli sensitivities. in october 2007, all cephalosporins and quinolones were removed from the policy as cdi levels had increased. notional 7 day antibiotic usage was calculated from prospective pharmacy generated data with aspirin, calcium, bisphosphonate & laxative prescription use as a non-antibiotic control, and analysed by segmented regression with a robust variance estimator. cdi rates were prospectively collected separately & analysed by a poisson regression model. results: an immediate response to change in antibiotic guidelines was observed (figure) . from august 06-sep 07 there was a highly significant increase in cephalosporins (85-100% of which was cephradine alone) (p < 0.001), a significant fall in trimethoprim (p < 0.004) and a significant increasing trend in cdi ( no tools existed to assess the readiness of public hospitals to receive this technology, and therefore guide resource allocation to facilitate implementation. aim: to assess the readiness of victorian public hospitals to introduce electronic antimicrobial stewardship. method: literature on readiness for change, organisational culture and information technology acceptance were reviewed. group interviews with project teams at site initiation meetings, one on one interviews with project officers at subsequent meetings, and observation where appropriate were all used to determine potential barriers and enablers. this information was recorded using a 'readiness assessment tool' and analysed to identify a number of key domains. to triangulate the data, questionnaires were distributed to project officers asking them to assess their sites' readiness to implement the system. results: a novel 'readiness assessment tool' was developed. it covered the domains of technical readiness, skills readiness, process readiness, administrative support readiness, resource readiness and hospital organisational characteristics. assessments at several hospitals highlighted a variety of issues at different sites and allowed early efforts to address these. a formative readiness assessment can be used to identify systematic problems that might facilitate or hinder uptake of electronic antimicrobial stewardship and to inform the adopters of potential resources required. [1] buising, k, thursky, k, robertson, m, black, j, street, a, richards, m & brown, g (2008) . electronic antibiotic stewardship-reduced consumption of broad-spectrum antibiotics using a computerised antimicrobial approval system in a hospital setting. j antimicrob chemother. w.v. kern°, m. steib-bauert, a. pritzkow, g. peyerl-hoffmann, h. von baum, u. frank, m. dettenkofer, c. schneider, k. de with, h. bertz (freiburg, ulm, de) objectives: fluoroquinolone prophylaxis (fqpx) may reduce morbidity and mortality in cancer patients (pts) with neutropenia, but the development of fluoroquinolone resistance (fqr) in escherichia coli and other target organisms limits its usefulness. we evaluated changes in the incidence density of gram-negative bloodstream infection (gnb) and in the in vitro fqr rates after the introduction of fqpx (with levofloxacin) as a standard of care for pts with high risk neutropenia in a university hospital. methods: we collected individual data for 357 pts admitted during baseline and during the first months following the intervention to assess clinical outcomes. individual pt data were compared with aggregate data (3-month periods). aggregate data analysis (unit-wide antibiotic consumption, gnb and numbers of in vitro fqr bloodstream isolates) was continued for a total of eight 3-month periods for both the haematology-oncology service and for general internal medicine. the new policy was introduced in the second half of the year 2005 when unit-wide baseline fqr of e. coli and of coagulase-negative staphylococcal (cons) bloodstream isolates had been 15% and 80% in the haematology-oncology unit, and 8% and 60% in general internal medicine, respectively. the individual pt data analysis revealed that pts not given fqpx had a much higher incidence of gnb than those given fqpx ( -2007) . the monthly use of iv and oral quin was calculated based on data from the pharmacy department. statistical analyses were performed using segmented linear regression analysis. bayesian model averaging was used to account for model uncertainty. results: before the interventions the use of quin (both iv and total) was stable. the best fitting models indicated that the first intervention was associated with a stepwise reduction in iv use of 71 prescribed daily doses (pdd) (95% ci: 47, 95 (p < 0.001)). there was also an indication of smaller reduction in iv use associated with intervention 4, but only the intervention 1 effect was robust to model uncertainty. the overall use of quin was also significantly reduced (figure) with a large stepwise reduction of 107 pdd (95% ci: 58, 156) associated with intervention 2. this study showed that the hospital-wide use of quin can be significantly improved (and decreased) by an active policy consisting of multiple interventions. marwick°, j. broomhall, c. mccowan, s. gonzalez-mcquire, k. akhras, s. merchant, p. davey (dundee, high wycombe, uk; raritan, us) aim and objectives: to describe the antibiotic treatment and outcomes stratified by severity in a representative sample of adult patients aged 18 or older who were treated in hospital for skin and soft tissue infections. inadequate. we also judged that 43% of patients received unnecessarily broad spectrum therapy. conclusions: ssti is common and is associated with significant mortality. however, choice of empirical therapy is not evidence based, with significant under treatment of high risk patients. ab were mostly (16/17) prescribed by gps and delivered by public (n = 14) or hospital pharmacies (n = 3). surveillance of ab use in nhs was organised in only 4 ms. in 3 countries a nh specific pharmaceutical formulary was available. prescription profiles by prescriber were available in 5 countries. other quality improvement initiatives in nhs such as regular training of prescribers, promoting microbiological sampling, collection of antimicrobial resistance profiles or pharmacist advice on ab prescription were scarce. guidelines for ab treatment of most frequent infections were available in many countries but were focussing on ambulatory care and did not consider the specific nh situation. only in 1 country the presence of an infection control practitioner was compulsory and partnership with hospital infection control teams was legally imposed in 3 ms. conclusion: important structural, functional and regulatory nh differences exist between eu countries. specific tools to improve infection prevention and ab therapy in nhs should take into account these differences. a european nh network was created in the framework of the esac nh subproject, which will organise point prevalence surveys on ab use in 2009. c. escherichia coli in south-western finland j. jalava°, o. meurman, h. marttila, a. hakanen, m. lindgren, k. rantakokko-jalava (turku, fi) objectives: extended-spectrum betalactamases (esbls), especially enzymes of the ctx-m group, are spreading rapidly in europe. enterobacteriaceae with reduced susceptibility to third generation cephalosporins and a positive esbl confirmatory test are also increasing in southwest finland. the purpose of this work was to study the resistance genetics of these esbl-positive enterobacteriaceae. methods: the study comprises a total of 271 clinical enterobacteriaceae strains isolated from both inpatient and outpatient specimens. all enterobacteriaceae strains that were esbl confirmatory test positive between january 2004 and december 2008 were included in this study (263 escherichia coli, 8 klebsiella pneumoniae, one isolate per patient). of these strains, 225 (83%) were urine isolates. resistance determinations were done using disk diffusion method (clsi) or vitek 2 and esbl confirmations by the double disk method using cefotaxime and ceftatzidime with and without clavulanate. thus far, 219 strains (those collected by end of june 2008) have been analysed for the presence of the most important esbl genes (tem, shv and ctx-m) using pcr and pyrosequencing as described before (haanpera et al. aac, 52:2632; 2008) . results: in 2004 only 10 esbl-positive strains were found. all of them harboured a ctx-m type esbl gene. since then, the number esblproducing enterobacteriaceae strains has increased significantly being tenfold in 2008 compared to year 2004 (figure) . a high majority, 197 (90%) of the 219 strains analysed thus far had a ctx-m-type esbl gene. most of those (79%) belonged to the ctx-m-1 group according to the pyrosequencing results. ctx-m-9 group was the next common, with 20% of the ctx-m genes belonging to this group. only two strains with ctx-m group 2 enzyme were found. conclusions: enterobacteriaceae strains which produce esbl are increasing rapidly in southwest finland. this is especially true with e. coli strains isolated from urine. towards the end of the study period, the esbl enzymes were almost exclusively ctx-m, ctx-m-1 group being the most common. further research is needed to characterise genetic elements that carry these esbl genes. esbl strains and the proportion of ctx-m genes in 2004-2008. (2000) (2001) (2002) (2003) (2004) (2005) (2006) in france (n = 6), spain (n = 4), portugal (n = 6), uk (n = 11), kuwait (n = 2), canada (n = 13) and china (n = 10), including hong kong (n = 3) were studied. clonality was established by pfge and phylogenetic groups of ec and kp were determined as reported. susceptibility testing (clsi), blactx-m-14 transferability and location (i-ceu-i/s1 nuclease) were investigated. plasmid analysis included determination of inc group (pcr-replicon typing, hybridisation, sequencing) and comparison of rflp patterns. association of blactx-m-14 with isecp1, isecp1-is10 or iscr1 was established by pcr and sequencing. we identified 42 pfge types among 52 isolates: 38/47 ec, 3/4 kp and 1/1 cf. distribution among phylogroups were as follows: i) ec: a (n = 7), b1 (n = 3), b2 (n = 5) and d (n = 23), and ii) kp: kpi (n = 2) and kpii (n = 1). resistance to tetracycline (76%), nalidixic (74%), streptomycin (67%), sulfonamides (67%), ciprofloxacin (60%) and trimetroprim (43%) was common. were spreading horizontally in our hospitals and, here, we characterised the plasmids responsible in the major k. pneumoniae strains identified during the survey. methods: plasmids from representative k. pneumoniae strains with ctx-m-15 enzyme were extracted by alkaline lysis and compared by apai, psti and ecori restriction analysis. they were transferred into e. coli dh5a by electroporation. transformants were selected on cefotaxime-containing agar and were screened by pcr for beta-lactamase genes, the aminoglycoside resistance genes aac(6 )-ib and aac3-iib, and the plasmid-mediated quinolone resistance genes qnra/b/s. results: twelve isolates were characterised, representing 5 major strains (a-d, and f) found in the most-affected hospitals. restriction analysis divided their plasmids into several groups. representatives of strain a (n = 4) had essentially the same plasmid (group 1), as did the two representatives of strain d (group 2a). one strain f isolate had a plasmid (group 2b) very similar to plasmid 2a from strain d, indicating possible horizontal transfer. plasmids of group 3 were retrieved from representatives of strains b and c, again indicating probable transfer. plasmids from three other strains differed substantially from each other and from plasmids 1, 2a, 2b and 3. nevertheless, on all plasmids, blactx-m genes were linked to an upstream isecp1 element, known to be involved in their mobilisation. all encoded multi-resistance: all but one group 1 and one ungrouped plasmid carried aac(6 )-ib; blaoxa-1 and aac(3)-iia were detected on all except group 1 plasmids; blatem was found on group 1, 2b, one group 3 and two ungrouped plasmids. blashv and qnra/b/s genes were not detected. the considerable diversity of plasmids encoding ctx-m-15 enzyme in major slovenian k. pneumoniae strains suggested only limited transfer, even when multiple strains were present in the same hospital. evidence of plasmid transfer was between strains b and c, and possibly between strains d and f, although these plasmids were not strictly identical. analysis of resistance genes encoded by the plasmids revealed diversity, with groupings coinciding largely with those based on restriction profiles. a. ingold, g. borthagaray, a.k. merkier, d. centrón, h. bello, c.m. márquez°(montevideo, uy; buenos aires, ar; concepción, cl) objectives: to examine the genetic context of class 1 integron harbouring blactx-m-2 in fifteen nosocomial k. pneumoniae isolates from south america in order to enhance the understanding of the antibiotic resistance spread among the region. methods: dna was extracted with the use of axypreptm bacterial genomic dna miniprep kit. the analysis of the cassette array was carried out with the use of primers hs458/hs459 targeting adjacent conserved regions. the examination of the surroundings were performed using two pcr primer pairs, hs817/hs818 and hs825/hs826, to amplify the initial(iri) and the terminal(irt), inverted repeat boundary, respectively. the primer pair hs825/hs911 was used whenever a negative result was obtained with hs825/hs826. all pcr products were purified and sequenced and the data was analyzed with ncbi blast tool. the sequence obtained with primers hs817/hs818 revealed the presence of three different transposons backbones at the iri end. the tn5036-like module and the tn21-like module were present in 4 isolates, the tn1696-like module was present in 7 isolates. no amplicons were obtained with the use of primers hs825/hs826 that amplify a tn21-like insertion. two uruguayan isolates with a tn5036 boundary at the iri end were tested with hs825/hs911 that target a tn5036-like backbone and one generated a product consistent with a tn5036-like mer region. uruguayan isolates carried a single aada1 cassette (4/5) and the other one contained a dfra17-aada5 array, while the four argentinian isolates carried the combination aaca4-aada1-orfd. chilean isolates arrays are in process. conclusions: among the extended-spectrum beta-lactamases, the cefotaximases constitute a rapidly growing cluster of enzymes that have disseminated geographically. there is a high frequency of isolation of ctx-m-2 producing k. pneumoniae associated with a class 1 integron in the region. despite being common the presence of iscr1 linked to blactx-m-2 in k. pneumoniae isolates, this study provides new and relevant information in the sequence context at the iri. here we report about the cassette array diversity and the diversity of elements in which the class 1 integron are embedded. different integron/transposons carrying the blactx-m-2 gene seem to be circulating and different regional patterns could be emerging, this study highlights the ability of different genetic elements to act cooperatively to spread and rearrange antibiotic resistance. l. vinué, a. garcía-fernández, d. fortini, p. poeta, m.a. moreno, c. torres, a. carattoli°(logroño, es; rome, it; vila real, pt; madrid, es) objectives: ctx-m enzymes are frequently detected in europe. in particular, ctx-m-1 and ctx-m-32-producing strains have been recovered from both humans and farm animals in spain, italy, greece, and portugal, suggesting the existence of community reservoirs for these enzymes. the aim of this study was to compare escherichia coli strains and plasmids harbouring blactx-m-1 and blactx-m-32 genes isolated from human and animals. methods: four e. coli ctx-m-1 and eight ctx-m-32 epidemiologically unrelated producers from sick or healthy animals (pig, dog, cow and chickens) and from humans (urine, blood and faecal samples) were analysed by xbai-pfge, plasmid transferability, pcr-based replicon typing, plasmid restriction analysis and southern blot hybridisation. all isolates were from spain but the dog isolate was from portugal. the genetic context of the blactx-m genes was previously investigated for all the strains. results: three ctx-m-32 strains (one from healthy chicken and two from hospitalised patients) showed the same pfge pattern. a chromosomal localisation of the blactx-m-32 gene was suspected in these strains. the five remaining ctx-m-32 producers showed the blactx-m-32 gene on plasmids belonging to the incn (4 strains) or untypable groups (1 strain). two incn plasmids showed identical pvuiirestriction patterns: one was identified in a strain from a healthy chicken and one was from a hospitalised human patient; these two strains were isolated in 2002 and 2004, respectively and showed different pfge patterns. ctx-m-1 producers (three from animal strains and one a healthy human) did not show clonality by pfge and the blactx-m-1 gene was always located on plasmids, three belonging to the incn and one to the inci1 groups. two of the incn plasmids carrying the blactx-m-1 gene showed highly related restriction patterns: one was from a healthy dog and one from a healthy human. conclusion: this study demonstrated the presence of clonal e. coli ctx-m-32 producers in animal and human sources and also detected epidemic incn plasmids disseminating among unrelated isolates from humans and animals, clearly suggesting a potential animal reservoir for the blactx-m-1/32 genes. o309 characterisation of bladim-1, a novel integron-located metallo-beta-lactamase gene from a pseudomonas stutzeri clinical isolate in the netherlands l. poirel°, j. rodriguez-martinez, n. al naiemi, y. debets-ossenkopp, p. nordmann (k.-bicetre, fr; amsterdam, nl) objectives: characterisation of the mechanism involved in the uncommon resistance to carbapenems observed from a pseudomonas stutzeri isolate recovered from a patient hospitalised in the netherlands with a chronic tibia osteomyelitis. that strain was resistant to ticarcillin, piperacillin-tazobactam, imipenem and meropenem, of intermediate susceptibility to ceftazidime and cefepime, and susceptible to aztreonam. methods: screening for metallo-beta-lactamase (mbl) production was performed using the e-test method with a strip combining imipenem and edta. shotgun cloning was performed with xbai-digested dna of p. stutzeri and pbk-cmv cloning vector. selection was performed on amoxicillin and kanamycin-containing plates. results: e. coli top10 (pdim-1) recombinant strains were obtained, displaying resistance to penicillins and ceftazidime, reduced susceptibility to cefepime, imipenem and meropenem, and full susceptibility to aztreonam. sequence analysis identified a novel ambler class b betalactamase dim-1 for "dutch imipenemase" (pi 6.1) weakly related to all other mbls. dim-1 shared 52% amino acid identity with the most closely related mbl gim-1, and 45 and 30% identity with the imp and vim subgroups, respectively. dim-1 hydrolyzes very efficiently imipenem and meropenem, expanded-spectrum cephalosporins, but spares aztreonam. the bladim-1 gene was as a form of a gene cassette located at the first position in a class 1 integron, but the 59be of that gene cassette was truncated giving rise to a fusion with an aadb gene cassette encoding an aminoglycoside adenylyltransferase. the third and last gene cassette corresponded to the qach cassette encoding resistance to disinfectants. conclusion: a novel mbl gene was identified in p. stutzeri further underlining (i) the diversity of acquired mbl genes, especially among non-fermenters, (ii) that pseudomonas sp. may be a reservoir of these genes and (iii) the possibility of spread of important resistance determinants in northern part of europe. isolates in greece p. giakkoupi, o. pappa, m. polemis, a. bakosi, a. vatopoulos°( athens, gr) objectives: metallo-beta-lactamases of the vim family are the main mechanism of carbapenem resistance in p. aeruginosa in greece. in this preliminary report we attempted to survey the subtypes of vim betalactamase currently prevailing in p. aeruginosa clinical isolates in greek hospitals, the genetic relatedness of the respective isolates, as well as the genetic environment of the blavim gene. methods: fifteen mbl producing and epidemiologically unrelated p. aeruginosa clinical isolates were collected in september 2006 from fifteen different hospitals around greece. mbl production was initially identified by an edta synergy test. identification of blavim gene, as well as mapping of the blavim cassette carrying integrons were performed by pcr and sequencing of the products. the o serotypes of the isolates were determined by a slide agglutination test using p. aeruginosa antisera (biorad). molecular typing was performed by pulse-field gel electrophoresis of spei-restricted genomic dna. results: blavim-2 gene was detected in nine isolates, blavim-4 in five and blavim-1 in only one isolate. the blavim-2 cassette of all nine isolates was located on the 1600 bp variable region of a class i integron, preceded by aaca29 gene cassette. blavim-4 cassette of all five isolates was the first cassette of the 3200 bp variable region of a class i integron, followed by the aaca4 and blapse-1 gene cassettes. blavim-1 was the unique cassette of a class i integron. vim-2 producers belonged to o8, o11 and o12 serotypes, whereas four isolates were non-typeable. vim-4 producers belonged to the same three serotypes, whereas only one was non-typeable. the vim-1 producer belonged to o12 serotype. the nine vim-2 producing p. aeruginosa isolates revealed a great degree of variability in pfge molecular typing, belonging to seven types. contrary, the five vim-4 producing p. aeruginosa isolates displayed higher genetic similarity and fell into one major type with 85% homology, which also included the vim-1 producing isolate. there was no correlation between the results of serotyping and molecular typing. conclusions: mbl production in p. aeruginosa in greece seems to be mainly due to specific class i integrons harbouring either blavim-2 or blavim-4 genes. genetic variability was higher among bacteria carrying vim-2 beta-lactamase, a fact indicating wider intraclonar spread of the respective integron. j.m. rodriguez-martinez, l. poirel°, p. nordmann (k.-bicetre, fr) objectives: extended-spectrum beta-lactamases of ampc-type (esacs) contributing to reduced susceptibility to imipenem have been recently reported from enterobacteriaceae. the aim of the study was to evaluate the putative role of natural ampc-type beta-lactamases of p. aeruginosa in a similar resistance profile. methods: thirty-two non-repetitive p. aeruginosa clinical isolates recovered in our hospital in 2007 were included. they were selected on the basis of criteria of intermediate susceptibility or resistance to ceftazidime and intermediate susceptibility or resistance to imipenem. mics were determined by agar dilution and e-test techniques. the level of expression of the ampc beta-lactamases was evaluated by measuring specific activities. pcr, sequencing, and cloning allowed to characterise the different bla(ampc) genes. identified esacs were purified and their km and kcat values for beta-lactams determined by spectrophotometry. results: using cloxacillin-containing (an ampc beta-lactamase inhibitor) plates, the susceptibility to ceftazidime was restored for 25 out of 32 isolates, suggesting overproduction of the ampc. in addition, in presence of cloxacillin, reduced mic values were also observed with ceftazidime, cefepime and imipenem for 21 out of those 25 isolates. cloning and sequencing identified 10 distinct ampc b-lactamase variants among the 32 isolates. recombinant plasmids expressing the ampcs were transformed into reference p. aeruginosa strain and reduced susceptibility to cefepime and imipenem was observed only with recombinant p. aeruginosa strains expressing ampc beta-lactamases that had an arginine residue at position 105. the catalytic efficiencies (kcat/km) of the ampc variants possessing this arginine residue were increased against oxyiminocephalosporins and imipenem. in addition, in-vitro assays demonstrated that those ampc variants constituted a favourable background for selection of additional degree of carbapenem resistance. conclusions: some ampcs of p. aeruginosa possessing extended activity torward carbapenems may contribute to carbapenem resistance. background: most oxa-type esbls are oxa-10, oxa-2 or oxa-1 derivatives. they display a very low homology, the percentage of which is between 20% and 30%. oxa-type esbls are divided into five groups according to the different homology by frederic bert, etc. group 1 includes oxa-5, oxa-7, oxa-10 and its derivants;group 2 includes oxa-2, oxa-3, oxa-15 and oxa-20;group 3 includes oxa-1, oxa-4, oxa-30 and oxa-31; group 4 is named after oxa-9; group 5 only includes a single enzyme called lcr-1. oxa-type esbls has been reported widespread in the world since the first report in 1987, such as turkey, france, england and so on. but there is few report about it in china. objective: to investigate the prevalence and genotype distribution of oxa-type extended-spectrum beta-lactamases (esbls) in clinical pseudomonas aeruginosa strains isolated from xiangya hospital of central south university in changsha city, hunan province, china. methods: ninety-seven non-repetitive clinical isolates of p. aeruginosa were collected between october 2006 and january 2007 from the hospital. they were screened for oxa-type esbls production by polymerase chain reaction pcr with five pairs of primes specific for blaoxa genes, respectively. then amplification of oxa-type esbls production was performed by pcr with specific primers. the purified and amplified products were sequenced to confirm the genotype of the oxa-type esbls. results: the sequences of the three oxa-type esbls pcr products were then compared in genbank database and there were no the completely same ribonucleotide and amino acid sequence with them. they were two novel oxa-type esbls, named as blaoxa-128 and blaoxa-129, which have been registered in genbank database under accession numbers eu573214 and eu573215, respectively. conclusions: there have occurred infections caused by p. aeruginosa producing oxa-type esbls in xiangya hospital of central south university. two novel oxa-type esbls in p. aeruginosa strains have been discovered in our study, which are named blaoxa-128 and blaoxa-129, respectively. pneumonia is one of the most common nosocomial infections and is associated with high mortality. in the last 15 years, gram-positive bacterial pathogens have risen in prevalence as a cause of hospitalacquired pneumonia (hap), including that occurring during mechanical ventilation (ventilator-associated pneumonia; vap). in particular, staphylococcus aureus is a major cause of hap, including vap. the rise of multidrug-resistant infections is a source of concern, with methicillinresistant s. aureus (mrsa) accounting for >40% of s. aureus isolates in some european hospitals. this symposium will take the format of a question-and-answer roundtable session in which experts will answer questions and initiate discussion surrounding emerging concerns and appropriate therapeutic strategies in nosocomial pneumonia, including that caused by multidrug-resistant gram-positive pathogens. recently, shifts in the susceptibility of s. aureus to established therapeutic agents for nosocomial pneumonia have added to the challenge of selecting appropriate empiric therapy. in patients with suspected multidrug-resistant infections or those who are mechanically ventilated, prompt initiation of therapy, often before the pathogen has been confirmed, is critical. vancomycin is the gold-standard treatment for multidrug-resistant infections and resistance has been remarkably slow to emerge. however, clinical reports in europe of 'mic creep' and the emergence of vancomycin-intermediate s. aureus (visa), hvisa and linezolid-resistant mrsa have presented new clinical dilemmas. elevated vancomycin mics are linked to treatment failure and increased mortality. hence, while vancomycin remains a useful therapeutic tool, treatment decisions present an increasing challenge, especially in groups of patients in whom rapid eradication of infection with appropriate agents is critical. telavancin is a novel lipoglycopeptide under investigation for treatment of nosocomial pneumonia. a number of key features suggest telavancin as a potentially attractive option for nosocomial pneumonia. telavancin has a unique dual mechanism of action that disrupts both bacterial cell wall biosynthesis and cell membrane integrity. the agent is rapidly bactericidal against a broad range of clinically relevant grampositive bacteria, including mrsa. two pivotal phase iii studies have demonstrated telavancin efficacy equivalent to vancomycin in hap, including vap, including in seriously ill patient subgroups and in that caused by mrsa. hantaviruses are enveloped rna viruses, each carried primarily by rodents or insectivores of specific host species. they have coevolved with the hosts in which they cause almost asymptomatic and persistent infections. in humans some hantaviruses cause disease: haemorrhagic fever with renal syndrome (hfrs) in eurasia. in europe puumala (puuv) from bank voles and saaremaa (saav) from field mice cause mild hfrs and dobrava (dobv) from yellow-necked mice severe hfrs. in asia hfrs is caused mainly by hantaan and seoul viruses. in americas some viruses cause hantavirus cardiopulmonary syndrome: sin nombre, andes and other viruses carried by sigmodontine rodents, not found in eurasia. in addition, in europe the common vole carries tula and rats seoul virus. however, they have not been definitely associated with disease in europe, although both can infect humans. we discuss the epidemiology, molecular genetics, detection of infection in carrier hosts and humans (including rt-pcr and 5-min serological tests), functions of hantaviral proteins, risk factors for humans to catch hantavirus infection (including smoking) and disease (including risk and protective hla haplotypes), role and mapping of epitopes of cytotoxic t-cells, mechanisms of hantavirus-induced apoptosis, newly discovered clinical features (including hypophyseal haemorrhages in puuv infection), and long-term consequences and pathogenesis of hfrs (endothelial permeability, thrombocytopenia, tnf-alpha and il-6). puuv occurs widely in europe except in the far north and mediterranean regions, saav in northern, eastern and central europe and dobv mainly in the balkans. the epidemiological patterns differ: in western and central europe hfrs epidemics follow mast years with increased oak and beech seed production promoting rodent breeding. in the north, hantavirus infections and hfrs epidemics occur in 3−4 year cycles, driven by prey-predator interactions. the infections and hfrs are on the increase in europe, partly because of better diagnostics and partly perhaps due to environmental changes. in several european countries hantavirus infections are notifiable and in some countries (e.g. belgium, finland, france, germany, scandinavian countries, slovenia) their epidemiology is relatively well studied. in large areas of europe, however, hantavirus infections and hfrs have not been studied systematically and they are still heavily under-diagnosed. mrsa screening − will we ever agree? s330 mrsa: universal screening! the successful control of any outbreak or epidemic relies on detection of those harbouring the pathogen (infected and colonised persons) combined with eliminating spread to new individuals. the approach to containment and reduction of the global mrsa pandemic is now being discussed. a challenge for this infection is that most persons harbouring mrsa do not exhibit signs of disease and thus in order to detect all potential spreaders of this organism some surveillance must be done. the required level of detection (surveillance through screening) is not known and likely varies with the prevalence of colonisation and disease. for a given mrsa prevalence, the factor that seems most crucial in reducing spread is the percentage of potential isolation days captured. the operational processes that highly influence this are 1) the sensitivity of screening detection (including sites tested and laboratory methods used), 2) the speed at which results of newly detected positive patients are reported from the laboratory (assuming pre-emptive isolation is not employed), and 3) the selection of patient populations who are to undergo screening. laboratory testing has a major impact on detecting mrsa colonised patients with real-time pcr having a sensitivity of 98% and a possible 2 hour reporting time compared to direct chromogenic agar cultures with a sensitivity of 80% and >24 hour reporting and enriched chromogenic agar testing with a sensitivity of 90% and >48 hour reporting (am j clin pathol, 2009); both reduced sensitivity and prolonged reporting time negatively impacting the success of mrsa timely isolation. we have shown that capturing 33% of mrsa isolation days in a modest mrsa prevalence setting (9 infections/10,000 patient days) with a high sensitivity test having a >24 hour result reporting time did not reduce hospital-wide mrsa disease (ann int med 148:209, 2008) . others have demonstrated that surveillance in an icu with similar mrsa prevalence, again with a high sensitivity test having 1 day result reporting, did not reduce icu disease until preemptive isolation was initiated (crit care 10: r25, 2006) . finally, we demonstrated that universal admission surveillance and decolonisation capturing 85% of possible mrsa isolation days had a dramatic impact by reducing 70% of all in-hospital infections from mrsa. future research in this area should focus on better defining those patients that benefit from mrsa screening and the role of decolonisation in these programs. clostridium difficile infection (cdi) is a toxin-mediated intestinal disease and extraintestinal manifestations are exceptional. clinical outcomes can range from asymptomatic colonisation to mild diarrhoea and more severe disease characterised by inflammatory lesions and pseudomembranes in the colon, toxic megacolon or bowel perforation, sepsis, shock, and death. the main clinical symptoms, secretory diarrhoea and inflammation of colonic mucosa, can be in great part explained by the actions of two large protein toxins, toxin a (tcda) and toxin b (tcdb). both toxins are cytotoxic, destroy the intestinal epithelium and decrease colonic barrier function by disruption of the actin cytoskeleton and tight junctions resulting in a decreased transepithelial resistance allowing fluid accumulation. in addition, c. difficile toxins also cause release of various inflammatory mediators which affect enteric nerves, sensory neurons and promote inflammatory cells, adding to the fluid secretion, inflammation and transmigration of neutrophils. some experimental evidence points also to possible extraintestinal action of c. difficile toxin b. in zebrafish embryos tcdb caused damage and edema in cardiac tissue and in hamsters the same toxin caused lung damage. only recently efficient systems have been developed to genetically manipulate c. difficile. comparison of knock-out mutants producing only one of both toxins have shown that tcdb-positive-only mutants retain the ability to kill hamsters, whereas tcda-positive-only mutants were not virulent for hamsters. these results are in concordance with epidemiological findings that naturally occurring a-b+ strains still cause the entire spectrum of cdi, but are not in concordance with effects observed after intragastric challenge of hamsters with purified toxins tcda and tcdb. the role of the third toxin produced by c. difficile, binary toxin cdt in the development of human disease is not well understood. cdt was shown to have enterotoxic effect in rabbit ileal loop assay, but natural strains producing cdt but neither tcda nor tcdb colonised animals but were not lethal in hamsters. comparative genomic analysis will most likely reveal additional factors involved in pathogenesis and in increased virulence (including cell surface layer proteins, sporulation characteristics and antibiotic resistance). additionally, the role of the host immune response in cdi has just started to be better understood. since 2002, there has been an escalation in rates of clostridium difficile infection (cdi) with epidemic c. difficile (pcr ribotype 027/north american pulsed-field type 1 [nap1]) responsible for outbreaks of severe infection in north america and europe. while fluoroqinolone resistance and over-use are thought to be driving the epidemic, the ageing population and improved case ascertainment are contributing to the dramatic increase in cases. other factors may also be important, such as the increase in prescription of proton pump inhibitors. in the netherlands, since 2005, there has been an increase in prevalence of human cdi with ribotype 078 strains usually found in animals. these infections were in a younger population and more frequently community acquired. there was alarm when it was reported that 20% of retail beef samples in canada contained c. difficile. the figure is higher in the usa where more than 40% of packaged meats (beef, pork and turkey) from 3 arizona stores contained c. difficile. most animal isolates of c. difficile produce binary toxin, and both pigs and cattle harbour pcr ribotype 078 a strain that, like ribotype 027, also produces more toxins a and b, and binary toxin. in the eastern part of the netherlands where >90% of pig farms are located, >20% of human isolates are now ribotype 078, and human and pig strains of c. difficile are highly genetically related. it has been suggested that the overlap between the location of pig farms in the netherlands and the occurrence of human ribotype 078 infections involves a common source. that source is likely to be the environment. the upsurge in cdi has prompted diagnostic companies to try to either improve current tests or develop new ones. laboratory diagnostic methods can be divided into 3 groups; traditional faecal cytotoxin detection (with or without culture), enzyme immunoassays (eias) and molecular methods. faecal cytotoxin detection is specific but lacks sensitivity, culture is sensitive but lacks specificity. new eias should find a niche in medium sized laboratories. current in-house pcr methods have the potential for great sensitivity and specificity but have been available only in larger laboratories. new commerciallyavailable platforms will make this methodology more accessible to smaller laboratories. whatever method is chosen, it is necessary for the laboratory to have as fast a turn-around-time as possible, particularly in an outbreak situation. d. lévy-bruhl°(saint-maurice, fr) in 2005, the advisory board on immunisation (abi) has been asked to make recommendations to the ministry of health regarding the inclusion or not in the french immunisation schedule of the soon to be licensed first hpv vaccine. the main elements considered in the establishment of the benefit-risk balance of routine hpv vaccination were: on the benefit side: -the very significant potentially preventable burden of diseases; -the very high efficacy of the vaccine against persistent hpv 16/18 infections in naive subjects; -the expected additional impact on other hpv16/18 related lesions and cancers; -the fact that vaccination, by preventing the pre-cancerous lesions, has the advantage over screening to reduce the cost and anxiety related to their detection and management; -the available data in favour of a satisfactory safety profile; -the benefit of vaccination for the women not covered by the opportunistic screening program. on the "risk" side: -the high cost of vaccination; -the unknown duration of protection; -the need for continuation of screening, even for vaccinated women; -the fact that the majority of residual cervical cancers could be prevented by the organisation of the screening program; -the risk of a decrease in compliance to screening for vaccinated women; -the low benefit if vaccinated and screened women were the same. a cost effectiveness analysis, carried out on a multi cohort markov model, showed that, over a 70 years period, the impact of vaccinating 80% of 14 years old girls or of organising the screening were comparable (reduction of cancer deaths close to 20%). however, the cost-effectiveness ratio of the vaccination was higher than that of the screening organisation, resp. 45,200 and 22,700 € per life year saved (at a 3% discount rate). on the basis of the economical analysis, the screening organisation was therefore the first priority. however, if both interventions were implemented, the overall reduction in cervical cancer deaths was estimated at 32%. the cost-effectiveness of the addition of vaccination on the top of the organisation of the screening appeared acceptable (55,000 € per life year saved). based on those results, the abi issued in march 2007 a recommendation to include the hpv vaccination in the immunisation schedule for 14 years old girls, together with a catch up for 15 to 23 years old women not having started their sexual life more than one year ago. the vaccine cost has been reimbursed since july 2007. clinical microbiology − is outsourcing the way to go? s338 the (r)evolution of clinical microbiology in europe − is it good or bad? laboratory medicine in general and clinical microbiology in particular is presently subject to rapid (r)evolution. are we aware? are we in command? do we know where we are going? should we oppose or cooperate? do we have a choice? do we recognise a driving force other than money? is it good, bad or just plain necessary? and are we gaining or losing? it is not one evolutionary process -it is several parallel processes with varying emphasis in different areas. there are at least four distinctive major trends over the last 15 years; the gradual formation of bigger and bigger units (concentration), the amalgamation of many different laboratory services into one (laboratory medicine), accreditation and an explosion of professional proficiencies and backgrounds of staff in microbiological laboratories. personally i have withstood the first two, with pleasure succumbed to the latter. a recent 5th trend, outsourcing microbiology services to large private consortiums, is splitting clinical microbiology into a purely analytical high-throughput money-saving activity, often leaving the consultative, clinical part of microbiology and health care infection control adrift. what is driving the evolution? not only cost-saving but also our inability to recruit medically trained microbiologists, the need to broaden the knowledge base of microbiology laboratories, automation, the development of new techniques and apparatus common to many laboratory disciplines, computerised medicine, political trendiness, power struggles, and much more. there is much to be gained by both concentration and amalgamation but much to be lost as well and many consider the heart and soul of clinical microbiology at risk. over a period of years, rational high-throughput production has won over consultation and personalised microbiology. that may be fine for the production of negative hiv-antibody/antigen analysis as for the screening of blood-donors but certainly not for the bacteriological cultures taken in conjunction with a hip replacement. or when it comes to understand and advise on the intricacies of antimicrobial resistance development. in other cases "outsourcing" and/or "amalgamation" mean that blood cultures are sent to x-town, cmv-antibodies to y-town and everything else to z-ville. when that happens clinical microbiology is lost. there are several instances where concentration, amalgamation and/or outsourcing of clinical microbiological services, alone or with other services, have meant that the tie between clinical microbiology and infection control has been severed and that many, both small and large hospitals have lost the personalised service so necessary to control outbreaks of multi-resistant bacteria and other health care related infections. a good service requires a strong knowledgeable and enthusiastic champion. a service which encompasses too many branches of laboratory medicine cannot be expected to champion each and every one with equal strength and fervour. and when outsourced to "big companies", there is no "clinical", only "microbiology". in 2008 "medical microbiology" broke out from "laboratory medicine" in uems. we are now striving towards a strong "medical microbiology" service in europe. it will have many facets, much strength, some weakness, great opportunities, but many threats. escmid certainly intends to help shape microbiology in europe. the optimal organisation of microbiology laboratories in european metropolis is an evolutionary task, driven by the evolution in laboratory tasks, laboratory technologies, communication technologies, regulations and financial issues. in the past five-ten years, medical and societal query for a more rapid and refined detection and identification of pathogens and antimicrobial resistance determinants coincided with the expansion of internet-based and remote tools for communication, an unprecedented revolution in laboratory technologies and new financial constraints. the concentration of laboratory workforces into one unique laboratory is one way to address these apparently contradictory issues. the tertiary medical school hospital system in marseille, a 2-million metropolitan area in france, comprises four hospitals for a total of 3,500 beds. the system had once four microbiology laboratories which have been progressively embedded into a unique, 600,000 acts per year, laboratory which deals with bacteriology, virology and environmental microbiology and hygiene. the medical staff comprises of 17, the ingenior staff of 11, technical staff of 88 and support staff of 13 persons for a total of 129 persons. this organisation allowed reducing labour time for routine microbiology, to develop prospective and sophisticated time-consuming diagnostic methods and to develop advanced diagnostic methods such as molecular methods (real-time pcr-based tests, sequencing, and mass spectrometry identification) and new generation serology. new, sophisticated technologies such as automated serology and mass spectrometry were corner-stones on which to base the constant diminution of routine labour time and the development of time-consuming tasks such as fastidious organisms' isolation. these evolutions paralleled the exponential increase in the ratio of ingeniors in the laboratory. this paradigm allowed for the constitution of large collections of biological specimens for retrospective analyses, the specialisation of every medical senior in one particular field of internationally recognized expertise and the increase in knowledge output in terms of peer-reviewed papers, patents and grants. implantation of point-of-care in the emergency department, in permanent internetbased connection with the central laboratory, was the last, but not least, evolution of this system. when tuberculosis epidemiology is seen in a global perspective, and the millennium development goals are considered, it is clear that two regions of the world, africa and europe, are severely behind in the control of the disease. in africa, especially sub-saharan africa, the tb problem is closely related to the endemic hiv/aids situation. in europe, especially the eastern part and in parts of the former soviet union, the main obstacle to an effective tb control is related to drug resistant forms of m. tuberculosis. the prevalence of the most severe forms of resistance, mdr-and xdr-tb, is so high that it makes control efforts both extremely complicated and very expensive. unfortunately, increasing levels of drug resistant tb are today also seen in many african countries, and hiv infection is spreading in eastern europe. during the last ten-year period new tools, based on molecular fingerprinting of m. tuberculosis strains, have been increasingly adapted to study tb transmission. with such molecular methods to characterise clinical isolates of m. tuberculosis it is now possible to study the spread of individual strains of the bacteria in detail. the laboratory tools used, rflp, miru/vntr, spoligotyping and others, will be presented and their use exemplified. how molecular epidemiology contributed to the detection and characterisation of a major outbreak of drug resistant tb in the stockholm area will be discussed. molecular characterisation of clinical isolates from different parts of the world has led to an increased recognition of the differences between different families of m. tuberculosis strains. to further describe and understand the role of these differences in the clinical field as well as for tb epidemiology is an ongoing and interesting field of research. an increased understanding of how tb is transmitted will hopefully help in the efforts to control this global health threat both on the local level and in a global perspective. living in the era of increasing tuberculosis drug resistance, the importance of making an early and accurate diagnosis with drug sensitivities has never been greater. the epidemiology of tuberculosis defines the extent of latent disease and the proportion which becomes active. accurate diagnosis is vital if patients are to be treated in a timely manner and to reduce the amount of time infectious individuals go untreated in the community disseminating disease. in many areas of the world, dots programmes are at the forefront of tuberculosis control. however, as a diagnostic this currently relies on sputum smear microscopy which is known to miss 50% of cases of tuberculosis and provides no data on drug sensitivity. the second major issue around tb is the lack of worldwide diagnostic facilities. there is a need for a simple, low cost, easily implemented diagnostic test. this talk will briefly consider the issues around the diagnosis of latent and active disease which are quite distinct. the focus will be on the diagnosis of active infection. in particular, the use of mods (microscopic observation drug-susceptibility) assay in diagnosis of tuberculosis will be discussed. the potential for using this in resource poor countries will be reviewed as well as the way sophisticated technology maybe harnessed to improve reporting and allow translation to all parts of the world. the important issue of how to distinguish patients with latent and active disease will also be considered. key issues and principles in diagnosis both now and in the future will be reviewed. in terms of treatment, there are 2 main issues. the first is that even short-course therapy is prolonged being a minimum of 6 months leading to issue of compliance. this may result in drug resistance. the massive rise of multi-drug resistant tuberculosis to approximately 500,000 cases world-wide with around 50 countries reporting extensively drug-resistant disease means that the need for new approaches to therapy are urgent. the second part of this talk will review different approaches to using current anti-mycobacterial drugs, the emergence of a small number of new drugs such as the diarylquinolones and entirely novel approaches to control and treat tuberculosis. there has been great success and also many threats in the field of infectious diseases during the previous year. the antimicrobial resistance, especially increasing carbapenem resistance among aerobic gram-negative rods and xdr mycobacterial tuberculosis strains are already big threats in some countries and they will probably spread to many other areas all over the world in the future and we will need new drugs for these indications but unfortunately very few new promising drugs seem to be in the pipeline at the moment for these purposes. the virulent clostridium difficile 027 strain spreads rapidly to many new countries and e.g. in finland it killed many times more people compared with mrsa and esbl strains in 2008. however, it is possible to stop its spreading but it needs new thinking in antibiotic use policy and infection control policy in hospitals. clostridium difficile 027 infection has a high relapse rate after metronidazole or vancomycin therapy, but an experimental "stool exchange treatment" is a promising therapy although controlled studies are needed to prove this assumption. an interesting research area during the last years has been the role of infections in the etiopathogenesis of chronic diseases like cancer, atherosclerosis, cardiovascular diseases and many autoimmune diseases. we can fight against many cancers like liver cancer and cervix cancer with virus vaccines and gastric cancer with antimicrobial drugs. also the high incidence of malignant tumours seems to decrease during haart treatment in hiv patients. the role of infections in the etiopathogenesis of cardiovascular diseases and atherosclerosis is complex. it is obvious that infections play a role in the etiopathogenesis of atherosclerosis, stroke and myocardial infarction but the undirected routine antimicrobial treatment is not recommended for these patients but there seems to be subgroups in patients with various cardiovascular diseases which may benefit from antimicrobial treatment. recent studies seem to suggest that there are hla types which protect or make people susceptible for coronary heart disease. the hla type hla-b*35 seems to be a risk factor for coronary heart disease but it is also a risk factor for chronic chlamydia pneumoniae infection. the feared pandemia due to h5n1 influenza a did not appear during the recent year and the world is now much more prepared to meet the next pandemia which, however, hopefully does not come during the next year. ø. samuelsen°, c. giske, u. naseer, s. tofteland, d.h. skutlaberg, a. onken, r. hjetland, a. sundsfjord (tromsø, no; stockholm, se; kristiansand, bergen, oslo, førde, no) objectives: the worldwide dissemination of kpc-producing multidrugresistant enterobacteriaceae is worrisome. the first kpc-producing klebsiella pneumoniae in norway was isolated late 2007 from a patient after hospitalisation in greece. throughout the following year seven additional kpc-producing k. pneumoniae isolates have been detected in clinical samples from six new patients. the aim of this study was to perform molecular characterisation of the strains and examine their epidemiological relatedness. materials and methods: antimicrobial susceptibility was examined by etest. molecular characterisation was performed by mlst, pfge and sequencing of the blakpc genetic structure. plasmid analysis was carried out by pfge of s1 nuclease-digested total dna and southern blot hybridisation using a blakpc probe. relevant epidemiological data were collected retrospectively. results: eight kpc-producing clinical isolates of k. pneumoniae have been identified from seven patients in two different regions of norway from the following specimens: blood culture (n = 3), urine (n = 2), expectorate (n = 1), perineal swab (n = 1) and wound secretion (n = 1). two blood culture isolates with clonally related but different pfgeprofiles were observed in one patient. the detection of kpc-producing k. pneumoniae isolates in norwegian patients was associated with import in four cases after hospitalisation in greece. two patients had been hospitalised at the same hospital in greece. isolation of a kpc-producing isolate in a fifth patient was epidemiologically linked to one of these imported cases and was a case of nosocomial transmission in norway. for the latter two cases no risk factors were identified with respect to recent hospitalisation or travel abroad. molecular analysis of six isolates has shown genetically related pfge-patterns and a common sequence type (st258). st258 has been associated with dissemination of ctx-m-15 in hungary. the blakpc gene was localised in tn4401 on a~97 kb plasmid. the two most recent isolates are currently undergoing similar analysis. conclusion: the first seven cases of kpc-producing k. pneumoniae in norway are associated with hospitalisation abroad, nosocomial transmission in norway, or urinary tract infections in outpatients without obvious risk factors. the clonal relationship between isolates underlines the existence a biological fit genetic lineage of kpcproducing k. pneumoniae with an epidemic potential. objectives: two recent publications have reported the isolation of kpc producing k. pneumoniae from infections in two patients, one in france and one in sweden, who originally had been hospitalised in greece. since this resistant mechanism had not been identified before in this country, the purpose of this report was to confirm the presence of blakpc producing k. pneumoniae in greece, to assess the extent of its spread and to study the genetic relatedness of the respective bacterial strains and the transferability of the blakpc harbouring plasmids. methods: for a three month period (february to april 2008) 40 hospitals participating in the greek system for surveillance of antibiotic resistance (www.mednet.gr/whonet) were asked to seek for possible kpc producers among k pneumoniae isolates displaying reduced susceptibility to imipenem (equal or higher than 1 mg/l), a positive hodge test for the presence of carbapenemase and a negative edta synergy test for the presence of metalloenzymes. the presence of blakpc gene in these strains was confirmed by pcr and sequencing. mics to carbapenems were determined by etest. conjugation experiments were carried out both in broth and on agar. the possible absence of ompk36 porin was detected by pcr. molecular typing was performed by pulse-field gel electrophoresis of xbai-restricted genomic dna. results: ninety two k. pneumoniae clinical isolates (one per patient) from 13 hospitals all over greece were found to harbour blakpc-2 gene. although colonies present in the inhibition zone made the exact determination of imipenem mic difficult, the absence of ompk36 porin was always associated with mic of imipenem higher than 32 mg/l. all isolates exhibited resistance to all other drug classes except colistin, tetracycline and tigecycline. pfge analysis revealed that 85 isolates from 12 hospitals displayed more than 95% similarity and were classified into one pulsotype, whereas the remaining seven isolates belonged into four different pulsotypes. blakpc-2 gene could not be transferred by conjugation from strains belonging to the main pulsotype. however, it was transferred from strains belonging to three out of the four remaining pulsotypes. conclusion: production of kpc-2 betalactamase seems to be a new emerging resistance mechanism in klebsiella pneumoniae in greece. blakpc-2 gene's possible clonal spread imposes the urgent need of implication of infection control practices in the affected hospitals. i. galani, m. souli, e. papadomichelakis, f. panayea, n. mitchell, a. antoniadou, g. poulakou, f. kontopidou, h. giamarellou°(athens, gr) background: until now, carbapenem resistance among klebsiella pneumoniae (kp) clinical isolates in greek hospitals has been attributed to the dissemination of vim-1 metallo-beta-lactamase. we describe the first outbreak of kpc-producing kp in greece; the first to occur outside the usa or israel. setting: 21-bed icu of attikon university hospital, athens. methods: kp isolates with an imipenem mic > 1 mg/l and a negative edta-imipenem disk synergy test were submitted to boronic acid disk test, to pcr for a kpc gene with specific primers and sequencing. records from patients colonised or infected with a kpc-producing kp were retrospectively reviewed for clinical and epidemiological data. environmental cultures for kpc-producers were performed. clinical isolates were submitted to molecular typing using pfge. results: from february to november 2008, 552 kp were isolated from 95 patients, 132 (23.9%) of which were boronic acid positive and produced kpc-2. most of them (126/132, 95.5%) were isolated since august. a total of 24 patients were identified as colonised or infected by a kpc producer which in 22 of them belonged to the same genetic clone. the source was faeces (73), bronchial secretions (26), blood (7), cvc tip (5), urine (15), pus (4) and throat (2). among patients whose medical records were available, median age was 74, apache ii score; 21, length of preceding hospital stay; 28 days, total length of stay; 50 days, immunosuppresion was identified in one and crude mortality was 71%. the kpc-producing kp was more frequently icu acquired whereas in a minority of patients it was already present on icu admission. seventy percent of the patients had previously received a carbapenem for a median of 15 days. environmental colonisation was not identified. ten (7.6%) of the kpcproducers from 8 (33.3%) patients were identified as the cause of an infection: bacteraemia (7), ventilator-associated pneumonia (2) and surgical site infection (1) and exhibited mic90 (mg/l) for imipenem, >8; meropenem, >8; gentamicin, 4; ciprofloxacin, >2; fosfomycin, >128; colistin, 0.5 and tigecycline, 4. most patients were successfully treated with a colistin-containing combination mostly with a beta-lactam. there was no attributed mortality. isolates from the same bacterial species were typed by pfge or automated ribotyping. kpc-encoding gene was fully sequenced. plasmid preparations and i-ceu digestion of total dna were resolved in agarose gels, blotted and hybridised with a blakpc probe. the blakpc-carrying element (tn4401) was amplified with various primer pairs, digested with eag i and sequenced. results: 30 strains each carried kpc-2 and kpc-3. one e. cloacae carried kpc-4. 13 k. oxytoca were kpc-2-producers and 2 s. marcescens harboured blakpc-3, all from usa. great genetic diversity was observed among the isolates (41 different types). one clone of 10 e. cloacae was detected in new york state (2006) (2007) . small clusters of 2 and 3 strains were detected among e. coli, e. cloacae, k. oxytoca. plasmids were present in all but 3 isolates. persistence of clones throughout the years was not observed. in 35 isolates the kpc-encoding gene was located in high molecular weight plasmids (>54 kb). blakpc was located in the chromosome of 11 strains (e. cloacae, e. coli and k. oxytoca) and the location of this gene could not be determined in 15 strains. small plasmids were present in several strains, but did not harbour blakpc. tn4401 carried blakpc in 46 isolates, and the transposon element was conserved. this structure was not detected in 12 strains. conclusions: kpc-encoding genes were most often located in tn4401 among several enterobacteriaceae species collected in usa and israel. this blakpc-carrying element was located in plasmids and on the chromosome. this study highlights the importance of tn4401 in the dissemination of blakpc genes in several genetically diverse bacterial species. blakpc was not associated with tn4401 in only 12 of 61 strains. these strains are under further investigation. objective: to evaluate the carbapenem resistance mechanism in a raoultella planticola bacteraemia isolate recovered from a patient hospitalised in ohio, usa. methods: species identification was performed by vitek 2 and confirmed by 16s rrna sequencing. susceptibility testing used clsi broth microdilution method. blakpc was amplified and sequenced. the blakpc genetic element (tn4401) was amplified and sequenced. plasmid extractions and conjugation experiments were carried out and the isolate was screened for esbl-encoding genes, qnr and qepa. a 83 year old female patient was admitted to a hospital located in akron with a diagnosis of cap in may/2008. sputum, paracentesis and blood cultures were negative. urine culture grew e. coli and patient received courses of moxifloxacin, ceftriaxone, azithromycin and meropenem. the patient was discharged and returned after three weeks with respiratory problems. tracheal aspirate grew a multidrug resistant a. baumannii and the blood culture grew the enteric-like gramnegative bacillus. the isolate was identified as r. planticola by the vitek 2, which was confirmed by 16s sequencing. r. planticola strain demonstrated resistance against most b-lactams, including carbapenems. screening for kpc-encoding genes was positive and this strain carried blakpc-2. fluoroquinolone and aminoglycoside mic values were elevated. kpc-2-encoding gene was located in tn4401, but conjugation experiments failed. esbl and qnr/qepa genes were not detected. conclusions: kpc serine-carbapenemases have been detected in several gram-negative species commonly isolated from clinical specimens. kpc genes are embedded in transposon-like structure usually harboured in conjugative plasmids carrying multiple antimicrobial resistance mechanisms. this is the first report of kpc-producing r. planticola that is an environmental organism related to klebsiella spp. the similarity between these organisms could facilitate the transfer of genetic material. kpc-producing isolates appear to be prevalent among different enterobacteriaceae species in usa hospitals and was detected in an isolates of apparent environmental origin. objectives: it is long known that not all individuals with a specific disease present with the same clinical manifestations, nor do they have identical prognoses or responses to treatments. it has become clear that variations in the human genome are likely to have an impact on these aspects. tank-binding kinase 1 (tbk1) is a central molecule in the induction of a.o. the type i interferon response to pathogens. our goals for this study were 1) to investigate the frequency of single nucleotide polymorphisms (snps) in the promoter and coding region of tbk1 in a dutch caucasian population and 2) to search for potential associations between these snps and bloodstream infections. methods: whole blood samples or samples of positive blood cultures were collected and after genomic dna was isolated, pcr and sequencing were performed for snp identification. functional studies included promoter activity measurements using a luciferase assay as well as electrophoretic mobility shift assays (emsa) to study binding of the transcription factor usf1 to the wt and mutant promoter. snp incidences were studied in a case control study. results: in samples from dutch caucasian healthy volunteers, 4 snps were found with allele frequencies higher than 5% whereas 6 other known snps had frequencies lower than 5% in our cohort. two snps (rs89208169 and rs89208163) located in the promoter region were studied in a larger cohort of 350 anonymised patients from the maastricht university medical center with either gram-positive or gram-negative blood cultures. we found that the prevalence of rs89208169 was significantly increased in patients with positive blood cultures in comparison with those with negative blood cultures or healthy volunteers. further investigation of this snp showed that it is located just outside a usf1-binding site. measuring the promoter activity using luciferase assays, the mutant promoter exhibited a decreased activity of <35%. this observation was confirmed by emsa which showed that recombinant usf1 protein had a reduced binding affinity to the mutant promoter. conclusions: snp rs89208169 in the promoter region of tbk1 has a significant association with gram-positive infections. our results demonstrate that this is likely due to a decreased expression of tbk1 due to reduced binding of the transcription factor usf1 to the mutant promoter. our results support recent findings that tbk1 plays also an important role in the host response to gram-positive infections. objective: lymphocyte apoptosis has been recognized as an important factor contributing to both the onset of sepsis post infection and to the progression into septic shock. animal data suggest that prevention of lymphocyte apoptosis by caspase inhibition stabilises the immune system, improves bacterial clearance and decreases mortality in experimental sepsis. the present study evaluated the potential of vx-166, a novel broad caspase inhibitor, as a therapy for sepsis. methods and results: initial characterisation of vx-166 in a number of enzymatic and cellular assays clearly demonstrated that the compound is a broad caspase inhibitor with potent anti-apoptotic activity in vitro. in vivo, vx-166 was tested in a murine model of endotoxic shock and a clinically relevant model of peritonitis. in the endotoxic shock model, male cd-1 mice (n = 28 per group) were administered lps (20 mg/kg iv) and survival was monitored for 96 h. vx-166 administered by repeat iv bolus (0, 4, 8 and 12 h post-lps) significantly improved survival in a dose-dependent fashion (p < 0.0001). in the rat peritonitis model, adult male sprague-dawley rats (n = 12 per group) underwent caecal ligation and puncture (clp) and survival was monitored over 10d. continuous administration of vx-166 by mini-osmotic pump (0.9 mg/kg/h) immediately following surgery significantly improved survival (p < 0.01) from 38% in the control group to 88% in the compound-treated group. mode of action studies in the rat clp model confirmed that vx-166 reduced thymic atrophy and lymphocyte apoptosis (p < 0.01), supporting the anti-apoptotic activity of the compound in vivo. in addition, vx-166 reduced plasma endotoxin levels (p < 0.05), strongly suggesting an improved clearance of bacteria from the bloodstream. most importantly, we demonstrated that vx-166 fully retained its efficacy when dosed 3 hours after insult (p < 0.01) by improving survival to 92% versus 42% in control animals, further highlighting the potential of anti-apoptotic therapy in sepsis. overall these data demonstrate that vx-166 inhibits lymphocyte apoptosis, improves the clearance of bacterial endotoxin and improves survival in experimental sepsis. importantly vx-166 improves survival in the clp model when dosed post insult, and therefore represents significant progress in the development of therapeutically viable broad caspase inhibitors for the treatment of this disease. v. vankerckhoven°, s. van voorden, n. hens, h. goossens, g. molenberghs, e. wiertz (wilrijk, be; leiden, nl; hasselt, be) objectives: toll-like receptors function as key regulators of both innate and adaptive immunity. lactobacilli modulate the immune system in different ways. the aim of this study was to examine toll-like receptor (tlr2, tlr2/6 and tlr4) signalling induced by clinical and probiotic lactobacillus strains. methods: a total of 45 lactobacillus strains (19 l. paracasei and 26 l. rhamnosus) of different origin (22 probiotic, 2 faecal, and 21 clinical) were tested for tlr2, tlr2 in combination with tlr6, and tlr4. tlr signalling was measured as relative il-8 promotor activation in transfected human embryonic kidney (hek) 293 cells. il-8 concentrations were measured using an enzyme-linked immunosorbent assay. heat-killed listeria monocytogenes (hklm) was used as positive control in all assays, whereas pam3, pam2, and lps were used as positive controls for, respectively, tlr2, tlr2/6, and tlr4. all assays were performed at least in duplicate. linear mixed model analyses and stepwise model selection were used to identify the statistically significant effects. random effects were used to account for heterogeneity across and homogeneity within isolates. p < 0.05 was considered statistically significant. results: hek-tlr2 and hek-tlr2/6, but not hek-tlr4, cells released il-8 upon stimulation with uv-inactivated lactobacilli, which was enhanced by co-transfection with cd-14. interestingly, the production of il-8 was shown to be variable for the different lactobacillus isolates. although similar results were seen for all isolates for tlr2 and tlr2/6, il-8 production was significantly higher for tlr2 (8.4 log pg/ml) compared to tlr2/6 (6.05 log pg/ml) (p < 0.0001). no significant differences in il-8 production were seen between clinical and probiotic isolates. however, l. rhamnosus isolates induced a significantly higher il-8 production compared to l. paracasei isolates in both cell lines, 7.88 and 6.84 log pg/ml, respectively (p = 0.0004). intra-isolate correlation was found significant (p < 0.0022). conclusions: our study shows that lactobacilli activate both tlr2 and tlr2 in combination with tlr6. our results also indicate that heterodimerisation of tlr2 with tlr6 does not lead to an improved recognition of lactobacilli. furthermore, taking intra-isolate correlation into consideration proved to be important. finally, our results suggest that differences in immunomodulation by lactobacilli may be related to differential signalling through tlrs, including tlr2 and tlr2/6. m.c. gagliardi, v. sargentini, r. teloni, m.e. remoli, g. federico, m. videtta, g. de libero, e. coccia, r. nisini°(rome, it; basel, ch) objective: to gain insights into the mechanisms used by mycobacterium tuberculosis and bacillus calmette guérin to cause human monocytes differentiation into cd1 negative dendritic cells (my-modc), unable to present lipid antigens to specific t cells. methods: human monocytes infected or not with mycobacteria were induced to differentiate into dc with gm-csf and il-4 in the presence or absence of p38 or erk specific inhibitors. kinases activation was detected by western blot using antibodies specific for phosphorylated and non phosphorylated isoforms. differentiation of monocytes into dc and the cd1a, cd1b and cd1c expression was evaluated by flow cytometry and by real time pcr at different time points from infection. functional expression of cd1 molecules was assessed by recognition of lipid antigens by cd1 restricted t cell clones. results: we show that mycobacteria trigger phosphorylation of erk and p38 mitogen-activated protein kinase in human monocytes as well as of activating transcription factor (atf)-2. mycobacteria-infected monocytes treated with a specific p38 inhibitor, but not with a specific erk inhibitor become insensitive to mycobacterial subversion and differentiate into cd1 positive my-modc, which are fully capable of presenting lipid antigens. data indicate that phosphorylation of p38 is directly involved in cd1 inhibition. conclusions: we propose p38 signaling as a pathway exploited by mycobacteria to affect cd1 expression, thus representing a novel target of possible pharmacological intervention in the treatment of mycobacterial infections. s. ebert°, s. ribes, r. nau, u. michel (gottingen, de) objective: activin a (act a) is a multifunctional cytokine with roles in the immune system and the inflammatory response. act a levels are elevated in the cerebrospinal fluid of patients with meningitis. microglial cells, the major constituents of innate immunity within the brain, express toll-like receptors (tlrs) recognising exogenous and endogenous ligands. upon stimulation with tlr agonists, primary mouse microglial cells become activated and release nitric oxide (no), cytokines, and also act a, suggesting that they are a source of elevated conclusions: pre-treatment with act a enhances no release from microglial cells activated by agonists of the principal tlrs involved in the recognition of bacteria. these findings provide further evidence for a role of act a in the innate immune response and suggest that act a acts as an pro-inflammatory modulator during infection and inflammatory processes in the cns. insertion sequences (is) are genetic tools that can mediate expression of previously silent genes or be responsible for the overexpression of certain genes (in each case by providing promoter sequences). in addition to be involved in gene transcription levels, is elements also play a very important role for gene acquisition/mobilisation. an is is usually made of of two inverted-repeat sequences (irs) bracketing a gene encoding the transposase which activity enables this entity to replicate and target another sequence. the is-related mechanisms at the origin of antibiotic resistance gene acquisition are diverse, including composite-transposition, rolling-circle transposition, one-ended transposition. is elements may be also involved in gene acquisition by mediating co-integration processes, or recombination events as hypothetized for is26 in relation with blashv extendedspectrum b-lactamase (esbl) genes originating from the chromosome of klebsiella pneumoniae. the blactx-m esbl genes known to be extremely widespread worldwide are encoded on plasmids, and have been found in association with isecp1 (acting by one-ended transposition) or iscr1 (acting by rolling-circle transposition). in that case, iss have played a role in the mobilisation from the chromosome of kluyvera spp. being the blactx-m progenitors and then in their expression. also, genes encoding acquired ampc b-lactamases, being of the blaacc, bladha, and blacmy-types, are mostly found in association with iscr1 or isecp1. sometimes antibiotic resistance genes are mobilised by composite transposons which are made of two copies of a given is bracketing the mobilised fragment. in acinetobacter baumannii, the worldwide disseminated blaoxa-23 carbapenemase gene is part of a composite transposon structure made of two copies of isaba1, forming transposon tn2006 which had mobilised a chromosomal fragment from acinetobacter radioresistens that actually corresponds to the progenitor of blaoxa-23. another possibility can be the forming of composite transposon structure bracketed by two different is (sharing similar irs) as observed with the blaper-1 esbl gene in pseudomonas aeruginosa. this diversity of iss elements at the origin of mobilisation/acquisition of antibiotic resistance genes is therefore responsible for the very efficient dissemination of many of them. s362 resistance islands − their role in the accumulation and spread of antimicrobial resistance genes historically, multi-antibiotic resistance in many bacterial species was largely attributed to the acquisition of resistance (r)-plasmids encoding one or more resistance determinants. however, over the last decade the r-plasmid paradigm has begun to be challenged. 'resistance islands' comprising large, chromosomally-integrated spans of alien dna harbouring multiple antibiotic resistance genes have been identified in the major hospital pathogens methicillin-resistant staphylococcus aureus (mrsa) and multi-resistant acinetobacter baumannii, and the foodand water-borne diarrhoeal pathogens shigella, salmonella and vibrio cholerae. in addition, comparative genomics analysis of the archetypal haemophilus influenzae conjugative resistance element that had spread worldwide revealed that it belonged to a large syntenic family of integrative islands, members of which could be found in at least 15 other b-and g-proteobacteria. with the exception of the a. baumannii island, these elements can be described as classic self-excising, -circularising and -integrative elements. all three functions are mediated by short island-flanking direct repeats and cognate integrase proteins encoded by the islands. in 2006 fournier et al. described an 86 kb a. baumannii island (abar1) which harboured 45 resistance genes packaged within a highly mosaic, integron-rich element that had almost certainly evolved via recombination, transposition and integron-mediated cassette capture from an 'empty' ancestral prototype. abar1 probably represents a new class of resistance island as it exhibits several features reminiscent of complex nested transposons, suggesting a distinct functional natute. however, despite the widespread distribution of resistance and genomic islands only a minority are known to code for part or all of the conjugative machinery necessary for their dissemination; others have been mobilised by helper plasmids or bacteriophages. regardless, data on the mechanisms of mobilisation of the vast majority of similar nonresistance islands remain sparse. importantly, resistance islands may not consists merely of packages of resistance genes. on the contrary, these diverse and frequently hybrid entities could potentially confer upon their hosts other advantageous traits relating to host-pathogen interaction, virulence, survival in the environment and/or transmissibility, truly justifying the label 'selfish islands' and further explaining their evolutionary success. due to the availability of new techniques, genome sequencing of bacteria has become fast and inexpensive. furthermore, recent methods using paired-end reads located several kb apart in the genome eases the assembling process, even though no reference sequence is available. in a reasonably close future, it should be possible to obtain the fully assembled sequence of a bacterial isolate overnight. the new sequencing techniques generate enormous amounts of genomic data and, thereby, a need for new tools. these should able to quickly analyze genomes and point to zones of interest, prompting further analysis on a reduced number of regions or genes, such as genomic islands. pathogenicity islands, a subset of genomic islands, carry genes such as toxins or resistance genes and have the particularity to be mobile, i.e. they may transfer to other species or strains. thereby, they confer their new hosts a more resistant or infectious phenotype, making this phenomenon particularly important to study. nucleotide composition of genomes is fairly homogeneous inside bacterial genomes. in general, horizontally transferred regions can be spotted due to their particular nucleotide content, because they tend to retain the composition of their original host and don't share the one of their new hosts. to do an analogy with languages, genomes speak dialects, and as one would easily spot a paragraph in finnish in an english text while not knowing finnish, one can spot genomic and pathogenicity islands transfers in a given genome. several techniques relying on various compositional aspects and on different algorithmic methods have been recently developed to detect pathogenicity islands in bacterial genomes. even very simple measures of the genome composition, such as the variation in t vs. a bias (ta skew) can lead to the identification of all known prophages in streptococcus pyogenes. it can even trigger the discovery of a putative ancient genomic island carrying a large number of genes related to pathogenicity in all strains of that species. in conclusion, with the rise of fast and inexpensive genome sequencing, new quick and simple methods are being developed. they take the advantage of the homogeneous nucleotide composition of bacterial genomes to uncover mobile genetic elements carrying genes involved in pathogenicity. in the past 10 years, significant progress has been achieved in the management of chronic hepatitis b with the successive development of six potent antiviral medications (lamivudine, adefovir dipivoxil, pegylated interferon alpha, entecavir, telbivudine and tenofovir). however, the clinical results of antiviral therapy have been limited by the emergence of antiviral drug resistance especially with the first generation of nucleoside analogs (lamivudine, adefovir and telbivudine). furthermore, the unique mechanism of viral genome replication and persistence within infected cells is responsible for viral persistence even after prolonged therapy with the newer antivirals (entecavir and tenofovir). this is the major reason why life-long treatment is envisaged in the majority of patients, which may expose them to long-term risk of developing resistance. the use of in vitro phenotypic assays has been crucial for the characterisation of newly identified resistant mutants and determine their cross-resistance profile. results allowed to understand the different mechanism of viral resistance to lamivudine and adefovir, the mechanism of primary failure to adefovir therapy, the unique mechanism of entecavir resistance, and to characterise the emergence of multi-drug-resistant strains in patients receiving sequential antiviral therapy. the crossresistance profile for the main resistant mutants was determined which allowed to provide recommendation to clinicians for treatment adaptation based on molecular virology data. the understanding of the development of hbv drug resistance has allowed to significantly improve the management of antiviral resistance and to design better treatment strategies to prevent resistance. the current standard of care relies on treatment initiation with antivirals combining a strong antiviral potency and a high barrier to resistance. a precise virologic monitoring is required to measure antiviral efficacy, and to diagnose partial response or viral breathrough at an early stage. this allows to adapt antiviral treatment preferrably using an add-on strategy with a drug having a complementary cross-resistance profile. this strategy has been shown to be efficient in controling viral replication and preventing liver disease progression in the majority of patients. treatment of chronic hepatitis b virus (hbv) infection is aimed at suppressing viral replication to the lowest possible level. in many prospective clinical trials it has been shown that a sustained hbv dna response was correlated with serologic, histologic, or biochemical responses. despite the recent progress in hepatitis b antiviral treatment, it is shown that antiviral drug resistance is inevitable against many of the nucleoside analogs. the emergence of antiviral-resistant strains of hbv leads to viral and subsequently biochemical breakthrough and may lead to disease progression and increased death. most of the data on the clinical impact of antiviral resistant hbv came from the data derived from studies of lamivudine therapy. there is limited data on other hbv antiviral drugs like adefovir. it is shown in several studies that treatment of hbeag-negative chronic hepatitis b with lamivudine effectively suppresses hbv replication and results in biochemical remission and histologic improvement in more than two thirds of patients. however, relapse has occurred in the majority of hbeag-negative patients after the cessation of therapy. there are several studies to support the occurrence of severe hepatic flares, and liver failure after the emergence of lamivudine resistance. several studies, where liver biopsies were taken, demonstrated that histological improvement was reduced in those patients experiencing lamivudine resistance. the clinical outcome for patients with antiviral resistance is related to their age, the severity of the underlying liver disease and the severity of the hepatic flares. on the other hand in a different study it was found that long-term lamuvidine treatment was associated with a reduced chance of developing cirrhosis and hcc in patients without advanced disease but, although resistant mutants reduced the benefits from lamivudine therapy, the outcome of these patients was still better than untreated patients. results of several clinical trials have shown that the addition or substitution of newer antiviral agents can restore suppression of viral replication, normalisation of liver function and reverse histological progression in patients with antiviral resistance. consequently, well-tolerated, potent therapies that offer a strong genetic barrier against the development of resistance are desirable, since antiviral resistance and poor adherence are key risk factors for treatment failure and subsequent reversal of clinical improvement. resistance of enteric fever-causing and non-typhoid salmonella serovars to agents traditionally used to treat these infections in the past shows extensive geographical variation. decreased susceptibility to ciprofloxacin is rapidly increasing all over the world with target alteration and increased efflux being the most important mechanisms behind. infections with such strains often result in extended hospitalisation or even in therapeutic failures. furthermore, it is likely that moderately increased mic values facilitate the development of strains with higher level of resistance, i.e. a pattern described at various locations. screening methods based on quinolone sensitivity testing may fail to indentify decreased fluoroquinolone susceptibility both in typhoid, as well as in non-typhoid salmonella. plasmid mediated quinolone resistance genes are detected increasingly all around the world although neither the frequency nor the variety of genes identified has approached that seen in some other members of enterobacteriaceae. treatment with gatifloxacin or azithromycin are alternative options for invasive and systemic infections caused by strains with decreased susceptibility to ciprofloxacin. at some parts of the world resistance to extended spectrum cephalosporins reached such incidence that may have therapeutic implications particularly when initial, empiric treatment of invasive infections is concerned. resistance is due to plasmid coded ampc type beta lactamases (particularly to cmy-2), and most often to esbls of which usually some of ctx-m types are the frequently encountered ones. carbapenem resistance is still rare, albeit does occur, among salmonella isolates. the recent description of a non-typhoid salmonella strain with the blaimp-4 gene co-located on a class-1 integron with several other resistance determinants on a conjugative plasmid is of particular concern. campylobacters exhibit natural resistance to a variety of antimicrobials. the drugs of choice used to be fluoroqunolones or macrolides. however, the current incidence of ciprofloxacin resistance made the former drugs already obsolete or seriously limited their use at several parts of the world. with the exception of few locations the incidence of macrolide resistance is still relatively low and is seen more frequently in c. coli than in c. jejuni. however, strains exhibiting resistance against both groups of drugs have been emerging, particularly in south-east asia. neisseria meningitidis, the meningococcus, is a major cause of meningitis and septicaemia worldwide while neisseria gonorrhoeae, the gonococcus, is responsible for one of the most widespread sexually trasmitted disease. the behaviour of these two species towards antibiotics is very different: resistance in n. gonorrhoeae is now widespread occuring as both chromosomally and plasmid mediated to a variety of drugs, whereas, besides resistance to sulphonamides, n. meningitidis remains largely susceptible to antibiotics used both for therapy and prophylaxis. however, as in the gonococcus, the resistance to antibiotics of n. meningitidis is also evolving, as documented by the ever higher frequency of strains with intermediate resistance to penicillin in many countries. transformation has apparently provided both species with a mechanism by which they can increase resistance to penicillin by replacing part of their pena gene, which encodes pbp2, with part of the pena gene of related species that fortuitously produces forms of pbp2 less susceptible to the antibiotic. n. meningitidis is still at this step, whereas n. gonorrhoeae has acquired also mutation in the pona gene that encodes pbp1, mutation in porin ib, increased expression of efflux pump and the tem-1 b-lactamase plasmid. the emergence and the spread of gonococci fully resistant to penicillin since the second half of the 1980 s years led to the recommended use of fluoroquinolones as primary therapy. however, this class of antibiotics became rapidly unefficacious, mainly in asia, due to the emergence of mutations in gyra and parc which are able to block the activity of the quinolones on gyrase and topoisomerase iv. since 2006, cdc no longer recommends their use for treatment of gonococcal diseases. fortunately, the occurrence of quinolone resistant meningococci, due to mutations in gyra, is still rare but even if cases are still few they are of great concern for the epidemic potential of this pathogen and the required prophylaxis of contacts. also for the other antibiotic, frequently used to this aim, rifampicin, some meningococci have showed to be resistant, again for the presence of mutations, in this case in the rpob gene coding for the b-subunit of the meningococcal rna polymerase. the molecular epidemiological identification of clonal clusters for both neisseria species with distinct resistance profiles is required to monitor ongoing trends that may pose problems both in therapy and prophylaxis. l. brookes-howell°, c. butler, k. hood, l. cooper, h. goossens (cardiff, uk; antwerp, be) introduction: grace is a european network of excellence established to focus on antibiotic use for community-acquired lower respiratory tract infection (lrti) and antimicrobial resistance across europe. grace-02, the second study to begin within grace, is a large qualitative study that explores the attitudes of clinicians and patients to antibiotic use for lrti and antibiotic resistance. aims: this presentation will focus on clinicians' accounts of the factors that contribute to variation in management of lrti and patient views on when antibiotics are necessary. methods: semi-structured interviews with 81 clinicians and 121 patients were conducted in primary care networks in nine european countries. interviews were audio-recorded, transcribed and, where necessary, translated into english for analysis. themes were identified, organised and compared using a framework approach. results: analysis of clinician interviews shows that, beside clinical findings, factors which influence the management decision for patients can be divided into two main areas. firstly, within each european network there is a group of country specific factors imposed by the system in which consultations take place. these factors include: near patient test usage, self-medication, patients' finances and lack of consistent, local prescribing guidelines. secondly, there is a group of factors, similar across all networks, that relate to personal characteristics of certain groups of clinicians. these include clinicians' professional ethos, self-belief in decision making and attitude towards the doctorpatient relationship. analysis of patient interviews shows that beliefs about antibiotic use tend to draw on clinical factors, namely the severity of specific symptoms (fever and/or coughing). many patients also implied a period of waiting or alternative action required before antibiotics are used − to identify whether the immune system would fight the infection or whether nonantibiotic management was effective before turning to antibiotics. discussion and conclusion: with a greater understanding of the factors that contribute to the decision to prescribe, we discuss ideas to enhance appropriate prescribing. this analysis highlights the need for interventions to be sensitive to factors relating to the systems in which different european networks operate, to target the individual characteristics of specific groups of clinicians and to build on the clinical beliefs already held by patients. o377 pre-treatment with low-dose endotoxin prolongs survival from experimental lethal endotoxic shock k. kopanakis, i. tzepi, e.j. giamarellos-bourboulis°, a. macheras (athens, gr) objective: clinical trials of immunointervention with anti-endotoxin antibodies in patients with severe sepsis have failed to disclose survival benefit. these failures led us to the assumption that the opposite approach with a low endotoxin stimulus may result to low level immunoaralysis and subsequent survival benefit. this approach was tested in an experimental setting. methods: a total of 36 male c57b6 mice were studied divided into two groups: group a stimulated with the ip injection of sodium saline followed after one day by the ip injection of 30 mg/kg of lipopolysaccharide (lps) of escherichia coli o155:h5; and group b stimulated with the ip injection of 3 mg/kg of lps of the same isolate followed after one day by the ip injection of 30 mg/kg lps. lps was diluted in sodium saline and the volume of each injection was 0.2 ml. survival was recorded at six hour time intervals. results: survival of group b was considerable prolonged compared with group a (log-rank: 5.435, p: 0.020) as shown in figure 1 . thirteen mice of group a died (72.2%) compared with seven mice of group b (38.9%, p: 0.044 between group). conclusions: administration of low doses of lps prolongs survival after lethal endotoxic shock. this approach opens a promising novel pathway for immunointervention in sepsis. fragilis isolates with an mxf mic of 2 mg/ml (n = 5), 4 mg/ml (n = 20) and 8 mg/ml (n = 8), which were virulent in the mgp model, were used to determine the efficacy of mxf. for the mgp model, pouches were created by injecting 5 ml of air and 0.5 ml of 0.1% croton oil in olive oil under the skin of the back. on day 3, the air was withdrawn and replaced by 1 ml soft agar. on day 5, a bacterial suspension was injected into the pouch. infected mice (n = 6 mice/group) were treated with mxf 100 mg/kg iv, b.i.d. for 2 days. this dose simulates the auc of the human 400 mg once-daily mxf iv dosage. efficacy was assessed by the reduction in colony forming units (cfus) in pouch exudates 48 hours post-infection compared with the untreated infection control. results: in the mgp model, mxf, 100 mg/kg b.i.d., displayed good efficacy in term of cfu reduction against all used strains in this study. there were no non-responders in terms of cfu reductions. conclusion: the loss of atle had no impact on the mics of cloxacillin and vancomycin. conversely, the mutant atle(−) strain was less susceptible to bactericidal activity of both antibiotics, supporting the implication of atle in the tolerance of s. epidermidis to cell wall active antibiotics. the loss of atle did not alter the virulence of s. epidermidis in the mouse peritonitis model, whereas it decreased virulence in previously published experiments using an intravenous catheter infection model. therefore, the mouse peritonitis model was suited to compare antibiotics efficacy against atle(+) and atle(−) strains. our results showed that the loss of atle did not alter significantly the activity of cloxacillin and vancomycin in the mouse peritonitis model. this study shows that the loss of atle results in decreased susceptibility to bactericidal activity of cell wall active antibiotics, with no apparent impact on the activity of these antibiotics in the mouse peritonitis model. in infant rat pneumococcal meningitis, ceftriaxone plus daptomycin versus ceftriaxone attenuates brain damage and hearing loss while ceftriaxone plus rifampicin versus ceftriaxone does not d. grandgirard, m. burri, k. oberson, a. bühlmann, f. simon, s.l. leib°(berne, ch) objectives: lytic antibiotics for therapy of bacterial meningitis (bm) increase the release of pro-inflammatory bacterial compounds which, in turns, induce inflammation. exacerbation of the inflammatory response in cerebrospinal fluid (csf) contributes to the development of neurological sequelae in survivors of bm. daptomycin, a nonlytic antibiotic acting on gram-positive bacteria has been shown to decrease inflammation and brain injury vs. ceftriaxone in experimental pneumococcal meningitis. with a view on the clinical application for empiric therapy of paediatric bacterial meningitis we investigated, whether therapies combining daptomycin or rifampicin with ceftriaxone are beneficial when compared to ceftriaxone monotherapy in infant rat pneumococcal meningitis. methods: eleven day old wistar rats were infected by intracisternal injection of s. pneumoniae and animals were treated with daptomycin (10 mg/kg, s.c., daily) plus ceftriaxone (100 mg/kg, s.c., bid), rifampicin (20 mg/kg, i.p., bid) plus ceftriaxone or ceftriaxone alone. csf was sampled at 6 h and 22 h after the initiation of therapy and assessed for concentrations of chemo-and cytokines (mcp-1, mip-1a, il-1b, il-6, il-10; il-18 and tnf-a). a subset of animals was sacrificed 40 h post infection (h pi) and brain damage quantified by histomorphometry. the remaining animals were treated for 3 d and were tested for hearing loss, by assessing the auditory brainstem response (abr) at 3 weeks after infection. results: compared to ceftriaxone alone, daptomycin plus ceftriaxone significantly (p < 0.04) lowered csf concentrations of mcp-1, mip-1alpha and il-6 at 6 h and mip-1a and il-1b at 22 h after initiation of therapy, led to significantly (p < 0.01) less apoptosis assessed at 40 h pi, and significantly (p < 0.01) improved hearing capacity. while rifampicin plus ceftriaxone also led to lower csf inflammation (p < 0.02 for il-6 at 6 h), apoptosis and hearing loss were not significantly different from the ceftriaxone group. conclusion: compared to ceftriaxone monotherapy, daptomycin plus ceftriaxone lowers the level of pro-inflammatory mediators in the csf and reduces hippocampal apoptosis and hearing loss in infant rat pneumococcal meningitis. d. croisier-bertin°, l. piroth, p.e. charles, d. biek, y. ge, p. chavanet (dijon, fr; alameda, us) objectives: ceftaroline (cpt) is a novel, parenteral, broad-spectrum cephalosporin exhibiting bactericidal activity against gram-positive organisms, including methicillin-resistant s. aureus (mrsa) and multidrug-resistant s. pneumoniae, as well as common gram-negative pathogens. the efficacy of simulated human dosing with cpt or ceftriaxone (cro) was evaluated in a rabbit model of penicillin-resistant pneumococcal pneumonia. methods: 3 s. pneumoniae strains were used to induce pneumonia in rabbits: pssp, pisp, and prsp. mics (mg/l) were 0.06/0.015, 1/0.125, and 4/0.25 for cro and cpt, respectively. the animals were randomised to no treatment (controls), intravenous (iv) cpt human equivalent (he) dosage (600 mg/12 h), iv cro he dosage (1 g/24 h), or intramuscular (im) cpt (5 or 20 mg/kg) for prsp-infected rabbits. serum levels were measured by microbiological assay and pk data were obtained. evaluation of efficacy was based on bacterial counts in lungs and spleen (per gram tissue). results: 5−7 animals/group were tested. for iv cpt/iv cro, mean auc0−24 was 155/938 mg.h/l, cmax was 20/158 mg/l and cmin was 1.3/6 mg/l, respectively. bacterial counts in target tissues are listed in the iv cpt and iv cro were highly efficacious against pssp and pisp. iv and im cpt were superior to iv cro against prsp with a quasi sterilisation of lungs and spleen. combined results from the iv and im studies indicated that %t > mic for cpt of 30% and 45% were associated with 50% and 100% bacterial count reductions, respectively. in this rabbit model of penicillin-resistant pneumococcal pneumonia, cpt administered iv (with he dosing) or by im administration was more effective against prsp than iv cro. r. endermann°, d. hoepker, k. merfort, m. glenschek-sieberth (wuppertal, de) objective: moxifloxacin (mxf) is approved in the usa and other countries for the treatment of complicated intra-abdominal infections (ciais). we compared the efficacy of mxf with piperacillin/tazobactam (pip/taz), a commonly used treatment for ciais, in three different models: ( . c. clp model: survival over 10 days was significantly higher in the mxf group than in the pip/taz group (p < 0.0001). conclusions: using humanised dosages, mxf had greater antimicrobial activity and provided higher survival rates that pip/taz in three different models for ciai. m. nairz, i. theurl, a. schroll, m. theurl, s. mair, t. sonnweber, g. fritsche, r. bellmann-weiler, g. weiss°(innsbruck, at) mutations in hfe predispose to hereditary haemochromatosis type i, a frequent genetic disorder characterised by progressive parenchymal iron deposition and eventual organ failure. since hfe mutations are associated with reduced iron levels within mononuclear phagocytes, we hypothesized that hfe deficiency may be beneficial in infections with intramacrophage pathogens. using hfe+/+, hfe+/− and hfe−/− mice in a model of typhoid fever, we found that animals lacking one or both hfe alleles are protected from systemic infection with salmonella typhimurium, displaying prolonged survival and improved bacterial control. this increased resistance can be referred to an enhanced production of the siderophore-binding peptide lipocalin 2 and the reduced availability of iron for salmonella engulfed by hfe deficient macrophages. this effect is mediated via stimulation of lipocalin 2-dependent iron export from infected cells since hfe−/− macrophages concurrently knocked out for lipocalin 2 are unable to efficiently control the infection or to withhold iron from intracellular salmonella. correspondingly, infection of hfe+/+ and hfe−/− mice with siderophore deficient salmonella abolishes the protection conferred by the hfe defect. thus, by inducing the formation of the iron-capturing peptide lipocalin 2, the hfe mutation harbours a genetically determined immunological advantage towards infections with intracellular pathogens such as salmonella. i. koutelidakis, a. kotsaki, p.d. carrer, k. louis, a. savva, e.j. giamarellos-bourboulis°(thessaloniki, athens, gr) objective: the majority of clinical trials of immunointervention in severe sepsis have failed to disclose survival benefit. a likely explanation may be administration of therapy when immunoparalysis of the septic host supervenes. in an attempt to reverse immunoparalysis, injection of mononuclear cells was attempted in experimental sepsis by multidrugresistant pseudomonas aeruginosa (mdrpa). methods: peripheral blood mononuclear cells (pbmcs) diluted in rpmi were isolated from five healthy human volunteers after gradient centrifugation over ficoll. 1×10 7 /kg of one mdrpa live or heat-killed isolate from one patient with severe sepsis was injected intraperitoneally for bacterial challenge. a total of 72 male c57b6 mice were studied divided into four groups: group a (n = 26) pre-treated with rpmi and challenged after one hour with live isolate; group b (n = 26) pretreated with 5×10 7 pbmcs/kg and challenged after one hour with live isolate; group c (n = 10) pre-treated with rpmi and challenged after one hour with heat-killed isolate; group d (n = 10) pre-treated with 5×10 7 pbmcs/kg and challenged after one hour with heat-killed isolate. survival was recorded for 20 mice of groups a and b and for all mice of groups c and d. six mice of groups a and b were sacrificed six hours after challenge. blood was collected from the lower vena cava and tnfalpha and il-6 were estimated in serum by an enzyme immunoassay. bacterial growth of liver and lung at the same time was assessed. results: median survival of group a was 24 hours and of group b 88 hours (log-rank: 4.524, p: 0.033). nineteen animals of group a died (95%) compared with eight animals of group b (40%, p: 0.038). four animals of group c died (40%) compared with nil animals of group d (0%, log-rank: 4.274, p: 0.03). median serum tnf-a of groups a and b at sacrifice was 31 and 184 pg/ml respectively (p: 0.048). respective values for il-6 were 2084 and 2231 pg/ml (pns); for liver bacterial cells 3.63 and 4.99 log10 cfu/g (pns); and for lung bacterial cells 2.56 and 4.22 log10 cfu/g (pns). conclusions: allogeneic transplantation with pbmcs prolonged survival in experimental sepsis by mdrpa. its mechanism of action was related with a) blockade of cell wall structures as shown by survival experiments with heat killed isolate; and b) reversal of immunoparalysis as evidenced by increase of serum tnf-a. this approach creates a promising novel perspective for immunointervention in sepsis. a. marangoni°, c. nanni, m. donati, r. aldini, d. di pierro, s. trespidi, s. accardo, s. fanti, r. cevenini (bologna, it) objectives: chlamydia trachomatis is one of the world's major causes of sexually transmitted diseases of the cervix and urethra and it is a major agent of pelvic inflammatory disease. genital tract infection of female mice with chlamydia muridarum closely mimics acute genital tract infection in women. aim of this study was to assess the predictivity of 68ga-chloride small animal positron emission tomography ( o387 inadequate statistical power of published comparative cohort studies on ventilator-associated pneumonia to detect mortality differences between the compared groups m. falagas°, v. kouranos, a. michalopoulos, s. rodopoulou, a. athanasoulia, d. karageorgopoulos (athens, gr) objective: comparative cohort studies are often conducted to identify novel therapeutic strategies or prognostic factors for ventilator-associated pneumonia (vap). we aimed to evaluate the statistical power of such studies to provide statistically and clinically significant conclusions. methods: we searched in pubmed and scopus for comparative cohort studies evaluating the mortality of patients with vap. we calculated for each of the included studies the statistical power to detect the observed difference in mortality between the compared groups (observed power), as well as 3 expected, clinically relevant, effect sizes (expected power). we identified 39 (20 prospective) comparative cohort studies on vap as eligible for inclusion in this analysis. the median observed power of these studies was 17.9% [interquartile range (iqr), 9.8−52.4%]. the median expected power was 10.0% (iqr, 7.2−13.6%) for a risk ratio for mortality of 0.85 between the compared groups; 14.7% (iqr, 10.6−21.8%) for a risk ratio of 0.80; and 7.9% (iqr, 6.3−10.2%) for a reduction in mortality from 30% to 25%. all expected power measures were significantly lower than the observed power. the statistical power of most cohort studies to detect the observed difference in mortality between compared groups of patients with vap is low. the power is even lower when expected, clinically relevant, differences in mortality are considered. for a wiser utilisation of resources allocated to research, we favour the conduction of cohort studies with larger sample size so that potential differences between the compared groups are more likely to be shown. objective: to clarify issues regarding the frequency, prevention, outcome, and treatment of patients with ventilator-associated tracheobronchitis (vat), which is a lower respiratory tract infection involving the tracheobronchial tree, while sparing the lung parenchyma. methods: we performed a systematic review and meta-analysis of relevant available data, gathered though searches of pubmed, scopus, and reference lists, without time restrictions. a conservative random effects model was used to calculate pooled odds ratios (or) and 95% confidence intervals (ci). results: out of the 564 initially retrieved articles, 30 papers were included. frequency of vat was 10.2%. selective digestive decontamination was proved an effective preventive strategy against vat. presence, as opposed to the absence, of vat was not associated with higher mortality (or: 1.18, 95% ci 0.90−1.53). administration of systemic antimicrobials (with or without inhaled ones), as opposed to placebo or no treatment, in patients with vat was not associated with lower mortality (or: 0.56, 95% ci 0.27−1.14). most of the studies providing relevant data noted that administration of antimicrobial agents, as opposed to placebo or no treatment, in patients with vat was associated with more ventilator-free days and lower frequency of subsequent pneumonia, but without shorter length of intensive care unit stay or shorter duration of mechanical ventilation. conclusions: approximately one tenth of mechanically ventilated patients suffer from vat; an infection potentially prevented by the implementation of selective digestive decontamination. antimicrobial treatment of patients with vat may protect against the development of subsequent ventilator-associated pneumonia. degranulation. subsequently, allergen specific ige to chlorhexidine was demonstrated and skin prick/intradermal testing was positive to chlorhexidine, confirming the diagnosis of chlorhexidine-precipitated anaphylaxis in each patient. a detailed review of the case-notes revealed that each patient had manifest evidence of minor cutaneous reactions to pre-operative chlorhexidine use that had not been ascribed to chlorhexidine at the time. discussion: fda issued a public health notice [1998] following 1st description of anaphylaxis to chlorhexidine coated central venous catheter. a recent case cluster has also been reported from another cardiac centre in the uk [3-cases over a 9-month period]. references to be presented. it is interesting that these reports of chlorhexidine anaphylaxis have all occurred in patients undergoing cardiac surgery. these patients receive multiple exposures to chlorhexidine during their pre-operative investigations and preparation. this has increased recently as a result of the drive to reduce the incidence of hospital-acquired infections. we wish to postulate that these patients have been sensitised by repeated topical exposure to chlorhexidine and have exhibited anaphylaxis when this allergen was presented to the patient in the form of the chlorhexidine coated central venous catheter. type i strains of helicobacter pylori possess the cag pathogenicity island to deliver virulence factors. cag is a specialised type iv secretion machinery that is activated during infection and comprises 31 genes originated from a distant event of horizontal transfer. after translocation the effector protein caga is phosphorylated on tyrosine residues restricted to a previously identified repeated sequence called d1. this sequence is located in the c-terminal half of the protein and contains the five amino acid motif epiya, which is amplified by duplications in a large fraction of clinical isolates. tyrosine-phosphorylation of caga is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. in addition, we observed that two members of the src kinases family, c-src and lyn, account for most of the caga-specific kinase activity in ags cell lysates. translocated caga interacts with the zo-1 and jam host-cell proteins causing disruption of the apical junctional complex. transfection of the caga gene into polarised epithelial cells induces disruption of cell-to-cell contacts and altered morphology. strikingly caga-expressing cells become migratory and invasive penetrating into collagen gel. the study of different portions of the molecule revealed the presence of two distinct functional domains and both are necessary to induce abnormal cell differentiation through interactions with host cell morphogens. cell polarity and invasion have been suggested to contribute to both early and late stages of cancer formation. these results suggest a mechanism by which caga may acts at the early stage of tumorigenic progression causing loss of cell polarity, increased cell motility and invasiveness of epithelial cells. after a period of 50 years of silence, a disease with an unpronouncable name, "chikungunya" (chik), has recently become a medical reality and reached the public throughout the world. conclusion: low mhla-dr expression after septic shock independently predicts ni. this promising biomarker may be of major interest in identifying patients at increased ni risk who could benefit from targeted and tailored therapy aimed at restoring immune functions. pneumonia, the leading infectious cause of death in the us, kills more people annually than aids, tuberculosis, meningitis and endocarditis combined. from a wide range of observational studies of communityacquired pneumonia (cap), only half of the cases had an aetiologic agent identified. streptococcus pneumoniae was consistently the predominant bacterial aetiology. this lecture will primarily focus on the innate immune response to pneumococcal pneumonia. toll-like receptors (tlrs) are key molecules that recognize pathogen associated molecular patterns (pamps) and induce an inflammatory response. pneumolysin, an intracellular toxin found in all s. pneumoniae clinical isolates, is an important virulence factor of the pneumococcus that is recognized by tlr4. although tlr2 is considered the most important receptor for gram-positive bacteria, tlr2 does not play a decisive role in host defence against s. pneumoniae pneumonia; likely, pneumolysin-induced tlr4 signalling can compensate for tlr2 deficiency during respiratory tract infection with s. pneumoniae. besides tlr2 and tlr4, tlr9 contributes to an effective host defence against s. pneumoniae in the airways. the importance of tlr signaling for host defence against pneumococcal pneumonia is illustrated by the fact that mice lacking the common tlr adaptor protein myd88 are highly susceptible to this infection. activation of tlrs results in the production of proinflammatory cytokines. there is ample evidence that underlines the importance of tumour necrosis factor (tnf) and interleukin (il)-1 in host defence in bacterial pneumonia: in a murine s. pneumoniae pneumonia model, treatment with a neutralising anti-tnf mab strongly impaired antibacterial defence. in addition, il-1a receptor type i deficient mice infected with s. pneumoniae displayed an increased bacterial outgrowth. of considerable interest, treating il-1 receptor deficient mice with a neutralising anti-tnf antibody made them extremely susceptible to pneumococcal pneumonia. infection of the lower airways by s. pneumoniae is associated with complex interaction between the pathogen (e.g. cell wall components, pneumolysin) and the host (e.g. tlrs, cytokines). these interactions play a crucial role in the outcome of this clinically important infection. severe bacterial pneumonia remains uncommon unless specific conditions exist that tip the balance between the host and pathogen in favour of the microorganism. such conditions include: persons at the extremes of age; exposure to especially virulent organisms; patients with concomitant illness impairing pulmonary clearance mechanisms; and immunocompromised hosts. pathogens overcome an array of innate and acquired host defences to successfully invade the host. the known virulence traits of three common respiratory pathogens (streptococcus pneumoniae, staphylococcus aureus, and pseudomonas aeruginosa) will be briefly reviewed. the capsular polysaccharide of pneumococci is the major anti-phagocytic virulence trait but many other factors contribute to disease pathogenesis including the critically important exotoxin known as pneumolysin, bacteriocins, adherence factors, choline binding proteins, lipoteichoic acid, iron, manganese and magnesium transporters, pili, competence and biofilm capacity, and virulence genes that promote invasion and impair clearance once the organism has entered the blood stream. s. aureus is notorious for the numerous a/b type toxins, cytotoxins, and superantigens it generates during the course of invasion. staphylococci deploy a complex series of quorum sensing signals that coordinate adhesin and invasion genes within biofilms or between planktonic organisms and likely contribute to the success of this pathogen. p. aeruginosa produces an array of extracellular exotoxins and cytotoxins delivered by type iii secretion systems. these include elastase, phospholipases c, a series of apoptotic and anti-phagocytic exotoxins, along with an alginate capsule and an unusual and variable lps structure that participate in microbial invasion. the pathogen expresses at least three interacting, quorum sensing systems to coordinate virulence and biofilm formation. a detailed understanding of these virulence factors is now providing therapeutic options to control these respiratory pathogens. surface expressed and extracellular toxins of pneumococci have been selected as new vaccine targets. inhibitory peptides and small molecule inhibitors of quorum sensing and biofilm formation are under investigation for staphylococcal and p. aeruginosa infections. these innovative and non-antibiotic treatment strategies are gaining greater importance as progressive antibiotic resistance threatens the management of these severe bacterial infections in the future. brucellosis, possibly the commonest zoonotic infection worldwide, has troubled humans since antiquity. recent years have seen the expansion of the animal reservoir of the disease to a wide spectrum of wildlife species, extending to marine mammals, and the recognition of novel brucella species. furthermore, animal and human disease has re-emerged in numerous countries which were brucellosis-free, and currently the most important endemic foci include near east and central asia. complex socioeconomic and political factors may be incriminated for these alterations in endemicity. the complex mechanisms by which brucella evades immune response and survives intracellularly are progressively clarified. novel diagnostic techniques as real time pcr may shed light in the life cycle of brucella inside the human host; preliminary studies have indicated that the pathogen may persist in latent form for years after apparent clinical cure, in asymptomatic individuals. treatment principles have not evolved significantly. the expert guidelines issued recently under the name of "ioannina recommendations" support the need for a six-week combined treatment that includes traditional antibacterials and is modified accordingly in serious complications as spondylitis and central nervous system involvement. the road to the development of a vaccine for humans seems long though. anthrax is ancient diseases and relatively a forgotten disease in western world until 2001 when spores were mailed in usa causing five deaths. currently, human anthrax is seen most commonly in agricultural regions of the world where anthrax in animals is prevalent, in which countries of middle east, in africa, central asia and south america. it is also an endemic disease in turkey. human cases may occur in an agricultural or an industrial environment. the infection is an occupational hazard of workers who process hides, hair, bone and bone products, and wool and of veterinarians and agricultural workers who handle infected animals. the main route of transmission is contact with or ingestion of contaminated meal with or inhalation of bacillus anthracis spores. leptospirosis is a very old disease that has been known for more than a hundred years and possibly even longer since the time of hippocrates. it remains a major cause of illness in many tropical and subtropical countries and thus in travellers. it has also been identified as a zoonosis in europe and north america. it is a disease that can surprise us because the clinical presentations are not always typical. in recent years, pulmonary and other atypical presentations have been more widely recognised. there is no effective vaccine but chemoprophylaxis is effective in selected populations. prompt recognition and early institution of appropriate treatment as with most other infectious diseases appear to be critical in ensuring a good outcome for our patients. there are interesting new developments in diagnostics and molecular epidemiology but clearly there are many challenges remaining in this field. objectives: the spread of carbapenemase genes within gram negative bacteria is of great cause for concern. in 2008, the first report of a blaoxa-58 gene outwith acinetobacter baumannii was reported in acinetobacter genospecies 3. we had also identified a genospecies 3 isolate encoding a blaoxa-58-like gene, and the aim of this study was to examine the genetic environment of the gene to investigate the mobilisation between species. methods: restriction analysis of rrna was used to confirm identity to the species level. susceptibility to imipenem and meropenem was determined through the plate doubling dilution method. screening by pcr for blaoxa-51-like, blaoxa-23-like, blaoxa-40-like and blaoxa-58-like genes was carried out. analysis of the genetic environment surrounding the blaoxa-58-like gene was conducted by sequencing inverse pcr products and gene-walking fragments. the structure of the surrounding sequence was confirmed using internal primers, which were also used to screen other blaoxa-58-like positive isolates in our collection. results: restriction analysis confirmed the isolate belonged to acinetobacter genospecies 3. the isolate showed reduced susceptibility to imipenem and meropenem with mics of 2 mg/l for both antibiotics. the isolate was negative for a blaoxa-51-like, blaoxa-23-like or blaoxa-40-like gene, but positive for a blaoxa-58-like gene. analysis of the genetic environment of the blaoxa-58-like gene revealed the gene was within a novel genetic structure. upstream of the blaoxa-58-like gene was the left-hand end of an isaba3 element, interrupted by an isaba125 element. the elements contained putative promoter sequences. downstream was an arac1 and a lyse gene, followed by a sequence similar to the re27 element described previously. following this was a complex region containing the right-hand end of an isaba3 tnpa gene, interrupted by an incomplete tnpa gene with 99% similarity to isaba3, itself interrupted by an isaba125 sequence. this region was followed by a second blaoxa-58-like gene. all other blaoxa-58-like positive isolates in our collection were negative for isaba125 upstream of blaoxa-58. this study is the first to report multiple copies of a blaoxa-58-like gene in an acinetobacter genospecies 3 isolate, and has identified a novel structure containing two blaoxa-58-like genes and two isaba125 sequences. the isaba125 elements may be responsible for the duplication of the blaoxa-58-like gene. objective: acinetobacter baumannii is an important nosocomial pathogen with wide intrinsic resistance. however, due to the dissemination of the acquired resistance mechanisms; such as extended-spectrum beta-lactamase (esbl) and metallo betalactamase (mbl) production, multidrug resistant strains have been isolated more often. per-1 was first detected in turkey and was found to be widespread among acinetobacter spp. and p. aeruginosa. since then, per-1 has been discovered in other countries, and most recently found in northern italy and in korea. in this study, the presence of per-1 type esbl was investigated in caftazidime resistant a. baumannii strains isolated from bloodstream infections by pcr and also the clonal relatedness of the isolates were investigated by random amplified polymorphic dna (rapd) and pulsed field gel electrophoresis (pfge) in all per-1 producing a. baumannii strains. methods: a. baumannii strains isolated from bloodstream infections was included in this study. the isolates were identified as a. baumannii by conventional methods and phoenix 100 bd automated system system (becton dickinson diagnostic systems, sparks). ceftazidime resistance was determined by e-test. per-1 genes were screened by these clusters encode: (i) resistance genes and transporters plausibly involved in drug efflux (30 transporters of the mfs, dmt, abc, rnd, mop and acr3 families were unique of drug resistant strains and absent in the susceptible sdf strain); (ii) pili and fimbriae systems related to biofilm formation and motility; (iii) haemolysin-and haemagglutininrelated proteins differently distributed among the four genomes, (iv) iron uptake and other metabolic genes. conclusion: genome comparison identified unique features of a. baumannii epidemic clones and provided novel insights into the genetic basis of multidrug resistance and pathogenesis in this species. this study may contribute to understand the concept of infection, invasiveness and colonisation in the emergent pathogen a. baumannii. hard to swallow − emerging and re-emerging issues in food-borne infection (symposium arranged with efwisg) s460 mrsa in food products: cause for concern or case for complacency? in 2003 first, a switch from intravenous-to oral medication (01-2006); second, education programs for interns/residents and physicians and the release of a new antimicrobial formulary (05-2006); third, a restriction note was printed on all laboratory rapports (10-2006) and fourth, active monitoring and giving feedback on prescriptions (01-2007). susceptibility patterns for e. coli including ciprofloxacin, cefuroxim, ceftazidim, co-trimoxazole and tobramycin from hospitalised patients were analyzed starting in 2004. statistical analyses were performed using segmented poisson regression models to look at effect of interventions on resistance (both sudden stepwise changes and changes in trends). bayesian model averaging was used to account for model uncertainty. results: before the start of the interventions the resistance rate was increasing by an average of 2.6% per year. the interventions resulted in a significant reduction of quin use from on average 550 prescribed daily doses to 350 pdd per month. in the best fitting poisson model for the resistance data, a significant stepwise decrease was found to be associated with interventions 2 and 4. however, there was substantial uncertainty in the model choice, and after accounting for this there was no conclusive evidence in support of any particular intervention, although there was evidence that at least one of the interventions was associated with the observed reduction in resistance. there were no stepwise decreases or decreasing trends in resistance rates to other antimicrobials during the study period. conclusion: many mds prescribe antibiotics often and believe their practice may have an effect on antibiotic resistance. results indicate that mds value information, interventions and surveillance in order to support responsible use of antibiotics. there is an ongoing effort in germany to address these findings at the national level e.g. by establishing a surveillance system for antibiotic resistance and antibiotic usage. table) . . ir for pn, er and tt were always higher in children (ch) than in adults (ad). significant differences were found for pn (1995), er (1997 er ( , 2004 er ( , 2006 er ( , 2007 er ( , 2008 , tt (2004 tt ( , 2006 . generally, cp-ir was higher in ad than in ch. ir was lower in the north (n) than in the south (s). significant differences: pn (2005 pn ( , 2006 , er (2003 er ( , 2004 er ( , 2005 , tt (2005) . both n and s knew a deceasing ir tendency: pn= n (12.1−8.1), s (18.8−13.2); cp= n (11.6−5.9), s (18.9−5.9); tt= n (27.4−21.5), s (35.9−23.5). er increased in the n (20.9−29.7). total outpatient antibiotic use (did) decreased from 26.2 (1999) to 22.7 (2004) and increased to 24.2 (2006) . did for pn and fq increased, mls stabilised and tt decreased. conclusions: since 2001-2003 an ir decrease was noted for pn, cp and tt. er-ir increased further over the years. the decrease paralleled the start of public campaigns on antibiotic use. ir rates remain higher in ch than in ad. the n/s difference became less marked. objectives: parachlamydia acanthamoebae is a new recognized member of the order chlamydiales. growing evidences suggest that this bacteria may have a pathogenic role in humans causing respiratory diseases. it has also been recently identified as an agent of bovine abortion and may be a cause of miscarriage in women. in contrast, little is known about the pathogenic role of rhabdochlamydia crassificans, another related chlamydiales. molecular diagnostic tools are useful to detect these obligate intracellular bacteria because of their inability to grow on conventional culture media. the aim of this work was (i) to develop a real-time pcr for the diagnosis of rhabdochlamydia infection and (ii) to study respiratory secretions of newborns for the presence of parachlamydia and rhabdochlamydia dna. methods: a new quantitative real-time taqman pcr (q-pcr) to be used on abi prism 7900 was developed. the q-pcr was then blindly applied to 41 consecutive respiratory samples (endotracheal or nasopharyngeal secretions) taken from 29 critically-ill newborns admitted in the neonatology ward of our university hospital. these samples were also tested using a previously developed parachlamydiaspecific pcr. results: most newborns (28/29) were premature (median gestational age: 28.6 weeks; range: 24.6−41.2). initial respiratory distress syndrome was present in 86% of them. positive pcr results were obtained in 12/29 (41%) patients (8 parachlamydia, 3 rhabdochlamydia, 1 both species) at a median of 17.5 days (range: 2-230) after birth. when compared to the control group (17 patients with negative pcr), these 12 newborns had a significantly worse primary adaptation and a higher incidence of resuscitation maneuvers at birth (table) . duration of noninvasive mechanical ventilation and stay in neonatology ward were also significantly longer. a fatal issue was observed in 3 infected cases, as compared to no death in controls (p = 0.06). gestational age at birth as well as the incidence of pulmonary or systemic infections did not differ between cases and controls. conclusion: a high prevalence of parachlamydia and rhabdochlamydia dna was observed in respiratory secretions of premature critically-ill newborns. the presence of dna of these microorganisms was associated with a worse primary adaptation, a more severe respiratory distress syndrome and a trend towards a higher mortality. their pathogenic role should be further investigated. the genus kingella consists of 3 species, k. kingae, k. oralis and k. denitrificans. all are gram negative, sometimes difficult to stain, rod shaped bacteria that are normal respiratory and genitourinary flora. they are slow-growing and fastidious. although improved recovery was shown when using fan or peds-f blood culture bottles, the majority of these infections remain undetected, especially in pre-treated patients. we report the use of real time polymerase chain reaction (rt-pcr) assays for detection of k. kingae and s. aureus in paediatric osteoarticular infections. methods: 116 synovial fluid samples from 97 patients, 1 month and 17 years of age, were collected over 19 months (03/2006 to 10/2007). the samples were from 54 knees, 39 hips, 9 ankles, 6 elbows, 4 shoulders, 2 wrists and 2 femur abscesses. after automated dna/rna extraction, specimens were subjected to 4 hour pathogen-specific rt-pcr. samples were inoculated onto sheep blood and chocolate agar as well as a peds-f bottle. final species identification and antimicrobial susceptibilities were determined by phoenix (tm). results: 45 patients (56 specimens) had positive culture and/or rt-pcr, resulting in an overall positivity rate of 46%. s. aureus was the predominant pathogen accounting for 31 specimens of 23 patients (12 mrsa, 11 mssa) and. 37% of positive specimens (18 patients) were due to k. kingae (n = 21). among children 0−2 years (n = 35), k. kingae was the predominant pathogen accounting for 16 positive patients (46%), followed by mssa in 4 patients (11%). the positivity rate for this age group was 57%. only 2 children >2 years (5 and 9 years) were positive for k. kingae. mrsa was the predominant pathogen in 6−12 year olds, and mssa was evenly distributed among children 3−17 years old. culture detected only 5 of 21 specimens positive for k. kingae and 25 of 31 s. aureus. 4 other pathogens were detected by culture only. the use of these molecular assays enhances detection of organisms, especially for k. kingae (19% vs. 5% for culture). additionally, faster identification (tat 4 hrs) allows for rapid targeted therapy. this improvement in tat could lead to shorter hospital stays in about 54% of cases. results: genotyping revealed a high degree of diversity, indicative of a panmictic bacterial population. further, there was no association between genotype and colonisation frequency, or year of isolation. pcr screening for virulence genes revealed an incidence of 98% for uspa1, 81% for hag, 82% for uspa2 and 18% for uspa2h. no significant difference was observed in the prevalence of virulence-associated genes between isolates originating from children who were colonised only once or children colonised on all 3 occasions (p = 1). pcr-rflp analysis of uspa1, hag and uspa2 showed many gene variants, with no association between pcr-rflp patterns and colonisation frequency, or year of isolation. conclusion: even in relatively localised geographical settings, the genotypic diversity of m. catarrhalis isolates colonising children is large, with no yearly pattern of genotype predominance. children serially colonised with m. catarrhalis isolates appear to clear a particular genotype only to become subsequently colonised with a different genotype. the incidence of virulence genes in this relatively localised study group is remarkably similar to that reported in global m. catarrhalis isolates, possibly indicating that similar selection pressure exists for m. catarrhalis at both the local and global level. virulence gene variation appears to be high, even in this relatively restricted geographical group. these results could have consequences for vaccines designed against virulence genes. a. naessens°, i. foulon, a. casteels, w. foulon (brussels, be) objectives: to evaluate the epidemiology of cytomegalovirus in pregnancy and to evaluate the risk for delivering a child with congenital cmv (ccmv). methods: between 1996-2006, 11825 unselected mother-infant pairs were included. in the mother a serological screening was performed consisting in the detection of cmv igg and igm antibodies at the first prenatal visit and at birth. in the neonate cmv urine culture was performed to diagnose congenital infection. when a pregnant woman was found to have a second trimester spontaneous abortion or a death in utero, an investigation for possible congenital cmv infection was carried out. results: serological screening at the first prenatal visit showed no immunity in 4701 women, evidence of past infection (igg positive igm negative) in 6877 women (58.2%) and in 250 women (2.0%) both igg and igm antibodies were detected. after investigation of stored and follow up samples from these 250 patients, 14 could be classified as having a primary cmv infection during pregnancy, 99 patients had previous immunity before the current pregnancy and from 137 patients the type of the maternal cmv infection could not be determined. follow-up serology of the 4701 women without immunity revealed a seroconversion in 58 of them (1.2%). a total of 61 (0.52%) congenital infections (ccmv) were diagnosed. the incidence of the ccmv among the different groups of women are summarised in the table. conclusion: ccmv infection occurs in 0.52% of our population of pregnant women. ccmv was considered to be due to a primary maternal cmv infection in 54% of the infants; 33% due to a recurrent maternal cmv infection and in 13% the type of maternal infection could not be determined. the risk for a seronegative pregnant woman of acquiring cmv during pregnancy is 1.2%. the transmission risk after a maternal primary infection is 45%. women with prior immunity have a very low risk (0.20%) for ccmv, this risk increases to 3% when igm are find in women with know prior immunity. the risk for women with undetermined infectious status in early pregnancy to give birth to a congenitally infected neonate is 5.8%. this report provides the first data on rotavirus epidemiology and disease burden in norway. further studies are needed to assess the economic impact of rotavirus disease and the cost-effectiveness of vaccination to inform decisions on introduction of rotavirus vaccines into the national program of childhood immunisation. pseudomonas aeruginosa may colonise the lungs of cystic fibrosis patients over years but may also cause acute infections in mechanically ventilated patients and immuno-compromised hosts within a matter of days. despite aggressive antibiotic treatments the organism is rarely eradicated. instead p. aeruginosa adapts to its host environment by developing resistance mechanisms and changing its lifestyle and virulence properties. focusing on mechanically ventilated patients, we will detail the dynamics of resistance emergence and persistence of p. aeruginosa lung populations during antibiotic therapy. we further discuss how p. aeruginosa populations evolve naturally in the absence of any antimicrobial treatment within the lungs of intubated patients by changing their virulence properties. the relevance of these findings both with respect to concepts of social evolution and the development of novel anti-infective strategies will be highlighted. the genome of p. aeruginosa encodes many potential efflux systems. however, only a few of them appear to play a significant role in antibiotic resistance. in this respect, the mex (for multiple efflux) systems are of particular interest because of their ability to extrude a wide range of antimicrobials. these polyspecific machineries result from the assembly of (i) a drug/proton antiporter, (ii) a periplasmic adaptor protein, and (iii) an outer membrane gated channel. it is now well established that the constitutive expression of the tripartite pump mexab-oprm provides p. aeruginosa with a relatively high intrinsic resistance to quinolones, blactams (except imipenem), tetracyclines, macrolides, chloramphenicol, trimethoprim, and novobiocin. this protective mechanism is potentiated by the poor permeability of the outer membrane and activity of another pump, mexxy/oprm, whose expression is induced by substrates targeting the ribosome (e.g., tetracyclines, macrolides, aminoglycosides). accumulating reports indicate that multidrug resistant mutants upregulating one or both of these systems are quite common in the clinical setting. such mutants, which are readily selected by sub-optimal treatments with fluoroquinolones, b-lactams or aminoglycosides, tend to accumulate various resistance mechanisms without loosing the wildtype pathogenicity of p. aeruginosa. whether the low resistance levels (mic x 2-to 8-fold) conferred by efflux may promote second-step mutants with altered drug targets (gyra, gyrb, parc) or derepressed ampc b-lactamase has not been confirmed in vitro. in the specific context of cystic fibrosis (cf), a recent study from our laboratory showed that the mexxy/oprm pump can be responsible for much higher resistance levels to aminoglycosides (64-to 128-fold). this increased efficacy of the system partially results from adaptive mutations in the mexy gene. in contrast, subpopulations deficient in mexab-oprm tend to emerge during long-term colonisation of cf airways. while easily selected in vitro on selective media, mutants overexpressing other mex systems (mexcd-oprj, mexef-oprn, mexghi-opmd, mexjk/oprm, mexvw/oprm) have been rarely described in cf and non-cf patients. some data support the notion that up-regulation of mexcd-oprj or mexef-oprn might be detrimental to the virulence of p. aeruginosa. in conclusion, therapeutic strategies based on efflux inhibitors should target the mexab-oprm and the mexxy/oprm systems in priority. european aspects of malaria s478 rapid diagnostic tests for malaria: twenty years to convince . . . prompt diagnosis and treatment of malaria are critical factors in reducing morbidity and mortality. microscopy has long been the gold standard for malaria diagnosis, but the newer rapid diagnostic tests (rdts) now offer considerable advantages, especially so in endemic countries. after close to twenty years of development and operational research, the diagnostic performance of rdts is now established in all settings. meta-analyses have clearly demonstrated equivalence of rdts over expert microscopy to detect parasites, and clear superiority over routine microscopy. actually, one of the major reasons that have delayed successful implementation of rdt in endemic areas was the use of poor quality microscopy that has impeded reliable measurement of sensitivity and specificity and undermined confidence of health workers in rdts. other factors were poor product performance, inadequate methods to determine the quality of products and a lack of emphasis and capacity to deal with these issues. for the potential of rdts to be realised, it is crucial that high-quality products that perform reliably and accurately under field conditions are made available and that quality insurance is performed on all steps of the procedure. in achieving this goal, the shift from symptom-based diagnosis to parasite-based management of malaria can bring significant improvement for the management of fever in endemic areas. for travelers returning in temperate climates with fever, rdts have also the potential to improve diagnostic procedures, especially so in hospitals where reliable microscopy is not available out of hours. in patients with no danger sign or significant thrombopenia, a negative rdt is sufficient to exclude malaria and allows waiting 12−24 hours for performing or reading the microscopy slide. rdts should be repeated every 12−24 hours for three consecutive days if fever persists and in the absence of alternative diagnosis. rdts represent a revolution in the fight against malaria and will tremendously help to manage appropriately patients with fever, especially so when malaria is declining and hence other causes of fever increasing. the ambitious deployment that is foreseen in the coming years in africa through large grants from the global fund should contribute to achieving the millennium goals. fever is the key symptom of malaria among returning travellers (97%). headache, chills, myalgia, sweating and lack of a focus are frequently recorded, but non-specific. nausea and vomiting are often seen in children. the differential diagnosis of other infections, mainly of viral origin, is further difficult because (dry) cough and (mild) diarrhoea are often present. laboratory findings (thrombocytopenia, low or normal leucocyte count) can be helpful in the assessment of mild to moderate malaria. clinical signs and symptoms, e.g. fever, may be mitigated in semiimmune patients (visiting friends and relatives, foreign visitors) seen in non-endemic countries who represent the majority of cases diagnosed in industrialised countries. caution is warranted in assessing such patients as many of them may no longer be exposed to malaria in their countries of origin, thus no longer partially protected and also at risk of suffering from severe complications. up to 10% of all imported malaria cases may be severe, presenting with jaundice, impaired consciousness to coma, acute renal failure, and, in the course of events, acute respiratory failure. delay in diagnosis and start of treatment is partly responsible for fatality rates of 1% and more in some countries. if you don't look for them, you won't find them: anaerobes revisited s481 anaerobic microbiota of the mouth − friend or foe? anaerobes form a major part of the commensal microbiota in the digestive tract where they constitute an integral component of the function on mucosal surfaces. in the mouth, teeth create a unique, non-shedding environment for bacteria to attach and to form biofilms. there is an age-related succession order of species in bacterial colonisation of the mouth, and once established, individual anaerobic species tend to remain as members of the oral microbiota. the agerelated pattern of the colonisation of anaerobic bacteria is partly connected with the development (or loss) of the dentition. interactions between different bacteria residing in the same microenvironment influence the composition of the microbiota − or the development of pathologic conditions. although commensal bacteria are regarded beneficial to the host, some anaerobic members of the oral microbiota contain characteristics potentially detrimental for the health status of an individual. molecular means of characterisation have resulted in increased knowledge about the "normal" microbiota of the mouth and in detection of new species and genera as well as phylotypes, which can be associated with infectious situations in the mouth. oral infections are multifactorial and polymicrobial in nature, and their aetiologic organisms originate mainly from the oral resident microbiota. the involvement of anaerobes is most obvious in infections of root canals, periodontal tissues, and tissues surrounding erupting wisdom teeth where typical anaerobic findings are gram-negative rods. in addition, gram-positive anaerobic cocci and non-spore-forming gram-positive anaerobic rods are common in odontogenic infections. on some occasions, anaerobes of localised dentoalveolar infections can spread to adjacent tissues and even to the bloodstream, resulting in severe complications in extraoral sites. interestingly, a relatively limited number of anaerobic species are involved in clinically severe infections, however, microbial findings seem to vary depending on geography. concomitant with the increase in the number of immunosuppressed patients, the number of opportunistic infections caused by commensal anaerobes may increase. identification to the species level will help to establish associations between individual anaerobic species and specific disease states. studies on the bacteriology of diabetic foot infections (dfi) have yielded varied and often contradictory results. the role of anaerobes is particularly unclear, often because the type and severity of the infection is poorly defined, recent antibiotic therapy is unknown, and specimen collection and culture techniques are inadequate. when optimal collection, transport, and culture techniques are used, multiple organisms including aerobes and anaerobes are usually recovered from severe dfi. interactions within these polymicrobial soups lead to production of virulence factors, such as haemolysins, proteases, collagenases, and short chain fatty acids, which promote inflammation, impede healing and contribute to the chronicity of the infection. to better define the bacteriology of diabetic foot infections, we analyzed our data from a large prospective u.s. multicentre trial of patients with moderate to severe infection that required initial parenteral antibiotic therapy and used optimal post-debridement sample collection, transport and culture procedures. of the 427 culture-positive specimens (of 454 total), only 16.2% were pure cultures while 30.4% yielded 5 or more organisms. a total of 462 anaerobes (range 0−9, average 2.3, per specimen) were recovered from 49% of patients, with gram-positive cocci (gpc) accounting for 45.5% of all anaerobic strains. s485 is culture still the gold standard, really? tremendous technological advances are made in culture-independent methods of detection and identification of human bacterial pathogens, such as pcr or hybridisation of their genomic dna. yet, time honoured pastorian bacterial culture in liquid and solid nutritive media still remains the gold standard for the laboratory diagnosis of a majority of bacterial infections. this unusual robustness of a 19th century technology stems from its unmatched operational characteristics: 1. broad range of detected agents, depending on adequate combination of media/incubation conditions; 2. unlimited source of clonal population for individual isolate, allowing versatile characterisation of antibiotic susceptibility and/or pathogenic factor production and/or epidemiological subtyping; 3. possibility of storage/bio-banking of cells for complementary clinical testing, research and diseases surveillance collections; 4. proof of pathogenic role of agent at the time of viable cell isolation from the site of infection, in contrast to false-positive results with molecular tests (tissue translocation or persistence of bacterial dna, soluble antigen,. . . ). major drawbacks of bacteriological culture include long turn-around time, cost and labour/skill intensity. these are partly alleviated by new technologies, including automated processing, physical/chemical growth detection and rapid molecular fingerprinting (maldi-tof, raman spectrometry, 16s rdna snp detection). it is likely that the next decade will see a complete redefinition of the place of direct detection methods and culture-based confirmation methods in clinical bacteriology, enabling a rejuvenation rather than elimination of culture as a daily diagnostic tool. the advent of real-time pcr revealed instrumental to the successful implementation of molecular methods in routine clinical microbiology laboratories. automated nucleic extraction platforms can now be coupled to robotic handling for large-scale detection and quantification purposes, mostly in virology. i will review here the attempts of implementing home-brew and commercial nucleic-acid based detection methods directly from blood samples and highlight hopes and pitfalls. i will then expand on two promising nucleic acid amplification methods: lamp (loop mediated isothermal amplification) and a protein-free method called dnazyme. these isothermal amplification methods share several strengths: robustness across highly diversified physico-chemical conditions, versatility in assay development and minimal requirements (if any) for sample preparation. they will definitely compete against current real-time pcr assays and might become a novel standard, due to lower costs and improved performances. the ribosomal rna (rrna) approach to microbial evolution and ecology has become an integral part of microbiology. rapidly growing databases exist that encompass besides the 16s rrna sequences of almost all validly described bacteria and archaea also numerous 16s rrna sequences of so far uncultivated microbes, directly retrieved from the environment by pcr or metagenomics. based on the patchy evolutionary conservation of rrna genes oligonucleotide probes can be designed in a directed way with specificities ranging from species up to large evolutionary entities like phyla or even domains. when such probes are labeled with fluorescent dyes or the enzyme horseradish peroxidase they can be used to identify single microbial cells by fluorescence in situ hybridisation (fish) directly in complex environmental samples. an update on recent applications and methodological improvements will be given which includes the identification of small bacterial cells by catalyzed reporter deposition (card)-fish. with optimised methods and proper controls fish yields exact cell numbers and spatial distributions for defined bacterial populations also in highly complex mixed microbial communities. r. amann & b.m. fuchs (2008) nature reviews microbiology 6:339-348. quick and reliable species identification of microorganisms is of great importance in medical microbiology. several bacterial and fungal species can be identified only using laborious and time-consuming methods. furthermore, in many cases misidentification occurs due to e.g. limited biochemical reactivity, different morphotypes or limited information in reference panels. in this talk, matrix-assisted laser desorption/ionisation time-of-flight (maldi-tof) mass spectrometry will be presented as a method for species identification. this technology applies protein pattern matching based on mass spectrometry. during the identification process, a mass pattern is generated for each organism. the subsequent comparison of this pattern with a database comprising reference patterns derived from well-characterised reference strains leads to species identification. as examples, the identification of various nonfermenting bacterial strains isolated from clinical specimens in comparison to partial 16s rdna sequencing will be shown. moreover, speed, accuracy in comparison to other methods, and inter-and intra-laboratory reproducibility of maldi-tof ms-based species identification will be discussed. o489 trends in invasive streptococcus pneumoniae serogroup 1 sequence types in belgium t. goegebuer, k. van pelt, j. verhaegen, j. van eldere°(leuven, be) objectives: s. pneumoniae serogroup 1 (sg1) isolates frequently cause invasive pneumococcal disease, particularly in children. from 2003 onwards a marked increase in sg1 isolates was observed; overall prevalence increased from 8. 2% (1998-2002) to 13.6% (2003) (2004) (2005) (2006) . we determined the sequence types (st) in sg1 isolates in order to better understand trends in sg1 resistance and spread. methods: as national reference centre, we receive all invasive isolates from more than 100 of 182 laboratories in belgium. 124 randomly chosen sg1 isolates from all ages from 1998 to 2006 were analysed via multi-locus sequence typing (mlst) as described by enright & spratt (microbiol. 1998; 144: 3049−60) . we also included data on strain characteristics and patient characteristics. results: 10 different sequence types (st) were identified: st350 (n = 66), st306 (n = 24), st304 (n = 13), st227 (n = 10), st228 (n = 5), st2915 (n = 2), st305 (n = 2), st612 (n = 1), and st217 (n = 1 mutations usually increase the mic slightly, but enhance the probability of further mutations. efflux pumps like pmra reduce antibiotic concentrations in the bacterial cell, enabling longer survival. we hypothesised that efflux positive bacteria are more likely to develop resistance than efflux negative bacteria. the following questions were addressed: 1. do the efflux pump inhibitors reserpine and verapamil reduce the mutation frequency? 2. do fluoroquinolone-susceptible efflux positive pneumococci exhibit higher parc or gyra qrdr mutation frequencies than efflux negative isolates? 3. does efflux phenotype impose a fitness cost? methods: matched efflux positive and negative pneumococcal isolates with identical or similar genotype according to multi-locus sequence typing collected by the german community acquired pneumonia network capnetz were analysed (n = 17). strains tigr4 and r6 were included as efflux negative controls. ciprofloxacin (cip) mics and efflux phenotype were measured by agar dilution method, for efflux detection reserpine (10 mg/l) was added and a fourfold decrease in mic was considered as efflux positive. mutation frequencies were determined by plating bacterial suspensions onto agar with and without cip. after incubation colonies were counted and the ratio of cfu/ml yielded the mutation frequency. equally, the mutation frequency was determined adding different concentrations of verapamil (10, 25, 50, 100, 500 mg/l) or reserpine (0.01, 0.1, 1, 5, 10 mg/l). biological fitness was calculated as the maximum slope of growth curves recorded in a microtitre plate reader. results: 1) even at low concentrations, reserpine clearly reduced the mutation frequency of efflux positive and, to a lesser extent, efflux negative pneumococci when exposed to cip (figure 1); verapamil exhibited this effect merely at high concentrations. 2) efflux positive isolates produced more frequently mutants (8/9) than efflux negative isolates (2/10) (p = 0.005, fisher's exact test). 3) efflux phenotype had no measurable impact on the biological fitness. conclusion: a positive efflux phenotype increases the qrdr mutation frequencies in the presence of fluoroquinolones and this effect can be inhibited by very low concentrations of reserpine. as a matter of concern, efflux is not associated with decreased biological fitness. background: use of fluoroquinolone (fq) has been associated with increasing fq resistance in s. pneumoniae. because respiratory fqs (levofloxacin (levo) and moxifloxacin (moxi)) are first line therapy for serious respiratory infections, increasing fq resistance (fqr) in sp is a concern. levo targets parc, and moxi targets gyra, which may permit differentiation of degree of selective pressure. we examined fq use, and changes in the prevalence of fqr and qrdr mutations in canadian isolates of sp. methods: cbsn is a canadian collaborative network of microbiology laboratories that has performed surveillance for antibiotic resistance in sp since 1988. antimicrobial resistance is performed in a central lab to clsi standards. we sequenced qrdr regions of all fqr isolates and a stratified sample of fq susceptible isolates. population fq use was obtained from ims canada. results: from 1995 to 2007, fq use increased from 53 to 97 rx/ 1000pop/yr; levo use from 0 to 10 rx/1000pop/yr, and moxi use from 0 to 17 rx/1000pop/yr. 31081 isolates were available for testing. levo r rates increased from 0 in 1993 to 1.8% in 2002 then remained stable until 2008 (1.6% in 2008). moxi r rates increased to 0.6% in 2004, then stabilised (0.7% in 2008). the prevalence of parc only mutations has not increased significantly in the last decade (see table) . the prevalence of isolates with both parc and gyra mutations increased until 2002, but has decreased in 2008. the first gyra only mutant was detected in 2000; the prevalence of gyra only mutants since then has increased, but remains very low (7/2044, 0.34% in 2007) . conclusion: despite increasing use of respiratory fqs, fqr in pneumococci is very low and not increasing in canada. the prevalence of isolates with parc mutations is decreasing. isolates with mutations in gyra alone remain extremely rare, suggesting that moxi exerts minimal selective pressure for resistance. in streptococci, two well characterised macrolide resistance have been described: target modification and active drug efflux. target site modification is mediated by the erm genes -erm(b), erm(a), erm(c)which confers the mlsb phenotype. target modification by mutations in 23s rrna as well as mutation in l4 and l22 ribosomal proteins have also been reported. expression of mef(a) genes activate an efflux mechanism responsible for m-type resistance we characterised a clinical isolate of s. agalactiae mb56gbs022 exhibiting the mlsb phenotype and tetracycline resistance. in this study, we determined the resistance genes, their association, and their localisation and mobility by conjugation. methods: the macrolide and tetracycline resistance genes were confirmed by pcr. the association between macrolide and tetracycline genes was investigated by long-pcr and sequencing. conjugation experiments were performed by filter matings. the genetic localisation of resistance genes was determined by endonuclease i ceui -followed by pfge and southern blot. the hybridisation study was performed using three specific probes for the 16s and 23s rrna genes, for erm(b) and tet(o) genes. results: s. agalactiae mb56gbs022 carried erm(b) and tet(o) genes on the same amplicon of 7 kb in size. the nucleotide sequence analysis of the entire product was identical to the peoc01 of 11 kb from pediococcus acidilactici that contains four orfs, of which orf2 and orf3 encode a putative resolvase and topoisomerase type i, respectively. the endonuclease i ceui method, that easily distinguishes between plasmid and chromosomal localisations as i-ceui only cuts chromosomal dna, revealed the localisation of resistance genes on the plasmid. all attempts to transfer erm(b)-tet(o) structure by conjugation from s. agalactiae mb56gbs022 to og1ss e. faecalis as recipient failed. conclusion: our results show the first case of the association between erm(b) and tet(o) genes on the unique mosaic structure in s. agalactiae, probably on the plasmid, as demonstrated by the i ceui-assay. further studies are on going to characterise the entire genetic element carrying resistance genes. o499 improving influenza pre-analytic collection systems: alternative collection systems to inactivate, preserve, or extract influenza for rapid testing s. castriciano°, k. luinstra, m. ackerman, a. petrich, m. smieja (brescia, it; hamilton, ca) objectives: in this study, 3 alternative influenza sample collection systems were evaluated for potential use in a pandemic situation. the objectives were to develop: 1) a non-temperature dependent swab collection and transport system, that inactivates influenza virus infectivity but preserves cell morphology and nucleic acid (na) for the detection of suspected influenza infections and/or 2) a system compatible with direct na testing without the need for purification prior to detection by a rapid real-time rt-pcr. methods: flocked nasopharyngeal swabs (nps) collected in utm (u) were compared to nps collected in a cymol (c), m-swab (m) or dry (d) flocked swab collection system (copan, italia). cymol is an alcoholbased medium that preserves cells for dfa testing. the m-swab contains 600 ul of medium and 150 ul of glass beads, and requires no na purification step. shell vial culture was used to assess influenza virus inactivation after 30 minutes exposure to the collection media. a mockinfected influenza a virus sample was absorbed to duplicate swabs then placed into the 4 collection systems. the infected collection media were held at rt for 30 minutes and then inoculated in duplicate into shell vial culture and stained after 48 hours. influenza a stability and na recovery after mock infection of each collection system was assessed after 1, 7, 14 and 21 days (d) at 4ºc, −20ºc, room temperature (rt) and 37ºc. aliquots of infected collection media were extracted by easymag and 5 ul of purified na tested by a quantitative influenza a rt-pcr on the roche lightcycler. m-swab collected samples were also tested directly or after boiling, without na purification. results: shell vial culture found that influenza a virus was inactivated after 30 minutes exposure to the c medium but not when exposed to the u and m media. influenza a was detected by dfa from the u and c cell smears. quantitation of influenza a rna was constant after 1, 7 and 14 d in u, c, m and d collection systems at −20, 4ºc and rt. the quantity of rna recovered declined significantly after 14 d at 37ºc in all 4 collection systems. m with boiling yielded data comparable to the easymag extraction. the copan cymol medium inactivates influenza infectivity, preserves cells and stabilises rna up to 14 days at −20, 4ºc and rt. cymol medium is a potential alternative for safe sample collection during a pandemic influenza situation. the m-swab presents a rapid testing alternative. luminex respiratory viral panel in respiratory specimens from children r. selvarangan°, s. selvaraju, d. baker, k. estes, l. hays, d. abel, s. hiraki (kansas city, us) objective: luminex respiratory viral panel (rvp) is a multiplex pcr capable of detecting and differentiating twelve different respiratory viruses and their subtypes; influenza a (flu a) (subtypes h1 and h3), influenza b (flu b), respiratory syncytial virus (rsv) (subtypes a and b), adenovirus (adv), parainfluenza 1 (piv 1), parainfluenza 2 (piv 2), parainfluenza 3 (piv 3), human metapneumovirus (hmpv) and rhinovirus (rhv). the aim of this study was to evaluate the analytical performance characteristics of rvp assay and to evaluate its ability to detect respiratory viruses from nasopharyngeal aspirates obtained from children. method: analytical sensitivity, specificity, accuracy and precision of the luminex rvp assay were determined using control viral stocks and respiratory specimens previously tested by rmix shell vial culture. result: luminex rvp assay reliably detected atcc viral stocks of flu a, flu b, rsvb, rhv and piv3 in the range of 10e-2 to 10e-4 tcid50/ml. no cross reactivity was noted with cmv, hsv, hhv6, ebv, vzv, piv4, cornoavirus 229e and oc43. among 146 respiratory specimens previously characterised by culture 138 specimens were accurately detected with overall accuracy of 95%. the median coefficient of variation in mean fluorescent index values of signals from replicate analyses of influenza a, b and rsv was 9% (7% to 25%). the 146 clinical specimens tested by rvp assay included 109 culture positive and 37 culture negative specimens. respiratory viruses isolated from the culture positive specimens include the following; 19 adv, 11 flu a, 10 flu b, 19 rsv, 9 piv1, 6 piv2, 5 piv3, 19 hmpv and 14 rhv. rvp assay detected all of the respiratory viruses except one each of rsv, piv1 and piv2 virus with overall sensitivity ranging from 88% to 100% for the different respiratory viral groups. among the 37 culture negative specimens 20 respiratory viruses were detected by rvp of which 15 were subsequently confirmed by repeat analyses. conclusion: luminex rvp assay is a highly sensitive and specific test useful in the detection of commonly encountered respiratory viruses in respiratory specimens. the addition of rvp assay to the viral testing algorithm of respiratory infections in children provides rapid results, improves diagnostic yield and may result in decreased antibiotic usage, reduced diagnostic testing and reduced hospital stay. m. savvala, i. daniil, i. berberidou, a. koutsibiri, a. stambolidi, m. papachristodoulou, n. spanakis, d. petropoulou°, a. tsakris (athens, gr) objective: in developed countries, viruses, particularly noroviruses, are recognized as the leading cause of acute gastroenteritis. we determined the aetiology, prevalence and seasonal distribution of viral gastrointestinal infections in hospitalised patients with acute diarrhoea. methods: during one-year period (november 2007-november 2008), a total of 201 faecal specimens were collected from 165 children, 21 premature neonates and 15 adults who were hospitalised with symptoms and signs of acute gastroenteritis. stool samples were tested for the presence of rotavirus, adenovirus, astrovirus and norovirus. rotavirus, adenovirus and astrovirus antigen detection was performed by chromatographic immunoassays (rotavirus and adenovirus, vikia ® -biomerieux, france; h&r astrovirus-vegal farmaceutica, spain). noroviruses were detected by an enzyme immunoassay (ridascreen ® rbiopharm, germany) and confirmed by reverse transcription-pcr. data were analyzed for seasonality of infection and possible transmission mode. the overall incidence of viral identification in acute diarrhoeal stool was 24% (48 of 201 patients). fifty one viral antigens were detected one patient with positive antigen detection is suffering from a disease of unclear aetiology. so, an association of replication of cihhv-6 with the disease might be considered. in contrast, the other patient did not show any symptoms at the time of antigen detection. this patient shows a special mode of acquisition of cihhv-6 (by bmt) possibly resulting in differences in the immunological priming and response. in addition, in the latter patient cihhv-6 is restricted to blood cells. two other patients did not show antigen expression. so, it is unclear how the transcription and translation of viral genes is influenced? furthermore, is there a pathophysiological impact of viral replication in individuals with cihhv-6? objectives: several case studies have reported on meningo-encephalitis caused by a primary epstein-barr virus (ebv) infection. we aimed to investigate the viral loads, and the inflammatory characteristics of this thus far poorly defined disease entity. we evaluated all cases from 2003-2008, in which an ebv polymerase chain reaction test (pcr) was requested on a cerebro spinal fluid (csf) sample. primary infection was defined as a clinical presentation with sore throat/pharyngitis/malaise in combination with lymphocytosis, and detectable heterophile antibodies or positive ebv igm antibodies. patients with proven neuroborreliosis served as control group. leukocyte response and ebv viral loads in csf, and serum were compared between primary ebv and neuroborreliosis cases. results: we identified six cases with a primary ebv infection (median age: 22, male: 4) with neurological symptoms ranging from meningeal signs to encephalitis. these were compared to 14 patients with neuroborreliosis (median age: 27, male: 6). in four out of six patients with a primary ebv infection with neurological symptoms ebv dna was detected in csf and in serum, whereas all neuroborreliosis cases were ebv pcr negative in both compartments. viral loads were lower in csf as compared to serum. in blood, leukocytes, lymphocyte, and monocyte counts were significantly increased as compared to the neuroborreliosis cases (see table 1 ). specific for vp7 and vp4 genes, using pools of g and p type specific primers. all strains (niv/brv/68, niv/brv/79, and niv/brv/86) were not typeable for the vp4 and vp7 genes. after purification by "qiaquick gel extraction kit" (qiagen, germany), the vp4, vp6, vp7, and nsp4 first amplicons of the borv-a strains were subjected to sequence analysis with automated sequencer abi 3130 xl dna analyzer (applied biosystems, usa). phylogenetic analysis was performed using mega version 4.0. objectives: dengue is a flavivirus and is among the most widely-spread viral diseases. our previous report demonstrates existence of live dengue virus in blood and urine even in the convalescent postfebrile period. in some cases, excretion in the patient's urine can be detected as late as 28 days after the onset of illness. this goes along with the model of west nile virus, another type of flavivirus, which can be excreted in the urine for months after acute infection in both animal studies and human case report. here we report a pilot study to address a magnitude of such findings. methods: between april 2007 and october 2008, paediatric and adult febrile patients suspected of dengue infection were enrolled. diagnosis of dengue was based on standard specific serology on paired sera. patients with negative serology served as controls. blood and urine specimens were collected at several time points. whole blood was separated into plasma and peripheral blood mononuclear cells (pbmc). these have been aliquoted and used for earlier studies and some stored in freezers. available plasma, pbmc, and urine were processed and inoculated into aedes aegypti. surviving mosquitoes at 14 days after inoculation were employed for viral detection by dengue-specific rt-pcr. indirect fluorescence antibody (ifa) staining of mosquito heads was performed on all positive rt-pcr specimens, except for the one from pbmc (awaiting ifa result). results: 5 and 45 cases of primary and secondary infections, respectively, and 4 negative controls were included. these translated into 55 and 59 early and late dengue specimens, and 6 and 4 early and late negative-control counterparts, respectively. dengue virus were isolated in some blood and urine specimens as late as 46 days after the onset of illness. no virus was isolated from control specimens. all but 5 positive rt-pcr specimens also demonstrated positive ifa. 4 out of 5 negatives were from early-phase specimens. conclusion: our study demonstrates prolonged survival of dengue virus after clinical recovery. this finding has pathologic and epidemiologic significance, adding a potential role of urine in the transmission of the disease. spread of the virus to humans might occur through infectious urine with help from arthropod vectors. this research could provide new insights into our understanding of the pathogenesis of denv infection. isolation of dengue virus from blood and urine specimens during early (days 1−7 after onset of illness) and late (days 8−46) phases of infection (specimens with dengue isolated/total specimens for mosquito inoculation) early phase late phase plasma 16/25 (64%) 0/13 (0%) pbmc not performed 1/2 (50%) urine 8/29 (28%) 12/44 (27%) all specimens 24/54 (44%) 13/59 (22%) dna copies) 226 (<50-1461) † ; n = 2