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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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 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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-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-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-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 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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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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 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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-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 a (h1n1) virus in an immunocompromised patient during treatment with neuraminidase inhibitors influenza c virus surveillance during the first influenza a (h1n1) 2009 pandemic wave in catalonia mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods world health organization (who) tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods the early diversification of influenza a/h1n1pdm guidance for clinical and public health laboratories testing for influenza virus antiviral drug susceptibility in europe differences in patient age distribution between influenza a subtypes agespecific epidemic waves of influenza and respiratory syncytial virus in a subtropical city incidence of 2009 pandemic influenza a h1n1 infection in england: 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-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 comparative epidemiology of pandemic and seasonal influenza a in households simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections excess mortality associated with influenza a and b virus in hong kong virologically confirmed population-based burden of hospitalization caused by respiratory syncytial virus, adenovirus, and parainfluenza viruses in children in hong kong respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa dual respiratory virus infections the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis multiple simultaneous viral infections in infants with acute respiratory tract infections in spain infection with multiple viruses is not associated with increased disease severity in children with 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-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-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-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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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-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 to perform hts should be considered early in the management of encephalitis, meningoencephalitis, and meningitis in as a second-line technique or in parallel to recommended investigations. the authors have no conflicts of interest to declare. no funding was received for this study. beyond viruses: clinical profiles and etiologies associated with encephalitis infectious encephalitis in france in 2007: a national prospective study causes of encephalitis and differences in their clinical presentations in england: a multicentre, population-based prospective study acute bacterial and viral meningitis viral infections of the central nervous system in spain: a prospective study viral meningitis etiology of aseptic meningitis and encephalitis in an adult population guidelines on the management of infectious encephalitis in adults genetic characterization of human herpesvirus type 1: full-length genome sequence of strain obtained from an encephalitis case from india analysis of an echovirus 18 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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-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-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-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 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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-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 assay. clinical microbiology and infection sars-cov-2 onthe-spot virus detection directly from patients. public and global health era of molecular diagnosis for pathogen identification of unexplained pneumonia, lessons to be learned an ultrasensitive, rapid, and portable coronavirus sars-cov-2 sequence detection method based on crispr-cas12 novel coronavirus sars-cov-2 using a crispr-based detectr lateral flow assay. infectious diseases (except hiv/aids) 2020 profiling early humoral response to diagnose novel coronavirus disease (covid-19) evolving status of the 2019 novel coronavirus infection: proposal of conventional serologic assays for disease diagnosis and infection monitoring molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes evaluation of nine commercial sars-cov-2 immunoassays analytical sensibility and specificity of two rt-qpcr protocols for sars-cov-2 detection performed in an automated workflow high-throughput extraction of sars-cov-2 rna from nasopharyngeal swabs using solid-phase reverse immobilization beads. infectious diseases (except hiv/aids) 2020 group testing: an information theory perspective evaluation of group testing for sars-cov-2 rna. infectious diseases (except hiv/aids) 2020 efficient and practical sample pooling high-throughput pcr diagnosis of covid-19. public and global health pooled-sample analysis strategies for covid-19 mass testing: a simulation study efficient high throughput sars-cov-2 testing to detect asymptomatic carriers. infectious diseases (except hiv/aids) 2020 group testing against covid-19 rapid identification of yeast artificial chromosome clones by matrix pooling and crude lysate pcr theoretical analysis of library screening using a n-dimensional pooling strategy the detection of defective members of large populations group testing with prior statistics suppression of covid-19 outbreak in the municipality of vo sars-cov-2 rna in wastewater anticipated covid-19 occurrence in a low prevalence area early sars-cov-2 outbreak detection by sewage-based epidemiology 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 <dl all <dl ebv pcr in serum (dna copies) 1507 (<50-14127) † ; n = 2 <dl all <dl data presented as median (interquartile range). *p < 0.05; † p < 0.01; comparison by mann-whitney u-test. csf: cerebrospinal fluid. ebv: epstein-barr virus y. achermann°, c. spormann, c. kolling, c. remschmidt, j. wüst, b. simmen, m. vogt (baar, zurich, ch) objectives: elbow arthroplasty is increasingly used for treatment of rheumatic or posttraumatic arthritis. data on epidemiology, characteristics and treatment outcome of elbow prosthetic joint infection (pji) is limited. furthermore, no validated therapeutic algorithm exist as for hip or knee pji. we analyzed all pji, which occurred in a cohort of implanted elbow prostheses during a 13-year-period. methods: between 01/1994 and 12/2007, all cases with implanted elbow prosthesis at our institution were retrospectively included. elbow pji was defined as visible purulence, acute inflammation on histopathology, sinus tract or microbial growth in periprosthetic tissue. patients were regularly follow-up at outpatient orthopedic visits and were in addition recently contacted by phone. a kaplan-meier survival analysis was performed. results: during the study period, 358 elbow prostheses were implanted. overall, 25 of 358 cases (7%) developed elbow pji (median age 61 y, range 41−82 y, 40% males). among them, 14 (56%) had a rheumatic disorder and 11 (44%) a posttraumatic arthritis. 12 infections were early ( 3 months), 3 were delayed (3−24 months) and 10 were late ( 24 months). more infections were acquired intraoperatively (n = 15, 60%) than haematogenously (n = 10, 40%). the following pathogens were cultured: staphylococcus aureus (n = 11), coagulase-negative staphylococci (n = 7), streptococcus agalactiae (n = 2), corynebacterium sp. (n = 1), enterococcus sp. (n = 1), enterobacter cloacae (n = 1), mixed infections (n = 1) and culture-negative (n = 1). treatment approaches included débridement with implant retention (n = 19), one-stage exchange (n = 2), two-stage exchange (n = 1), resection arthroplasty (1) and antibiotics only (n = 2). at follow-up, 16 (64%) patients were free of infection (median follow-up time 2.6 y, range 0.7−11.3 y) and 9 (36%) had a relapse (median time to infection 0.4 y, range 0.1−4.0 y). one patient died due to infectious endocarditis with secondary haematogenous elbow pji. the relapse-free survival (95% confidence interval) was 75% (58%-93%) after 1 year, 70% (52%-89%) after 2 years, 62% (39%-85%) after 3 years and 52% (27%-78%) after 4 years. the infection incidence after elbow arthroplasty was higher (7%) than reported after hip (<1%) or knee arthroplasty (<2%). most infections (48%) manifested early ( 3 months). underlying rheumatic disorder were common in elbow pji (58%). the relapse-free survival after elbow pji was 75%, 70%, 62% and 52% after 1, 2, 3 and 4 years. e.a.n. oostdijk°, a.m.g.a. de smet, h.e.m. blok, e.s. thieme groen, j.a.j.w. kluytmans, m.j.m. bonten (utrecht, breda, nl) introduction: selective digestive tract decontamination (sdd) and selective oropharyngeal decontamination (sod) aim to eradicate gram-negative bacteria (gnb) from the intestinal en respiratory tract in intensive-care-unit (icu) patients. we aimed to quantify effects of sdd and sod on bacterial ecology in 13 icus that participated in a clustered group-randomised cross over study in the netherlands, in which sdd, sod or standard care (sc) was used during consecutive periods of 6 months (nejm 2009; 360:20) . the order of periods was randomised per icu. methods: point prevalence surveys of rectal and respiratory samples were performed once monthly in all patients in icu (receiving or not receiving sod/sdd). selective media were used to detect gnb and mic testing was done for ceftazidime (cft), ciprofloxacin (cip) and tobramycin (tob). effects of sdd on rectal carriage were determined by comparing data from 6 sdd months in each icu to data from all months preceding and following (in which sod or sc was given). combined effects of sdd/sod on respiratory tract carriage were determined by comparing data from 12 months sdd/sod (in any order) to data from the 6 sc months before and afterwards. results: 2583 rectal and 2023 respiratory tract samples were analysed. during sdd average proportions of patients colonised with gnb resistant to either cft, tob, cip were 5%, 7% and 7% and these proportions were 15%, 13% and 13% during the post-intervention period (p < 0.05 in each case). this effect is most explicit for cft with resistance levels increasing from 2.6% in the last month of sdd to 11.4% in the 1st month after sdd (p < 0.05). during sdd and sod resistance levels in respiratory tract samples were <6% for all three antibiotics, but prevalence increased gradually (for cft and tob resistance; p < 0.05 for trend). after discontinuation of sdd and sod average proportions increased to levels >10% for all three antibiotics (p < 0.05 in each case). cft resistant isolates in rectal samples mainly included enterobacteriaceae not being escherichia coli and klebsiella spp, whereas tob and cip resistant isolates mainly included e. coli. conclusion: sod and sdd have marked effects on the bacterial ecology in an icu with a rapid and persistent increase in resistance after intervention. antibiotic resistance remains a major concern associated with these infection control measures. o391 throwing caution to the winds? three cases of anaphylaxis to chlorhexidine coated central venous catheters from a regional cardiac centre in northwestern england this blaoxa-58-producing clone showed resistance to several b-lactams (including imipemem), susceptibility to ceftazidime, netilmicin and minocycline, and variable susceptibility to meropenem, cefepime, and aztreonam. mics for colistin and tigecycline ranged from >16 mg/l and from 0.25−4 mg/l, respectively. all oxa-58-producing isolates presented the isaba3 downstream of the blaoxa-58 gene. hybridisation assays revealed a plasmidic location for the blaoxa-58 gene with ca 90kb. plasmid sequencing showed an isaba3-like truncated at the 3 end upstream of the blaoxa-58 gene, a fact that may explain the observed negative carbapenemase-production bioassay. conclusion: blaoxa-58-carrying a. baumannii is, apparently, more ancient than initially imagined. although undetected from 2004 onwards, the fact that it possessed a non-expressible gene, due to alterations in the promoter region, suggests that this information might have been incorporated from a still unidentified source. twenty-seven (45%) were male. isolates were recovered from respiratory secretions (33 isolates, 55.0%), blood (11, 18.3%), urine (7, 11.7%), catheter (5, 8.3%) and other secretions (4, 6.7%). only 24 (40.0%) of 60 patients received appropriate antimicrobial therapy either with polymyxin b (79.2%), ampicillin-sulbactam (12.5%) or tigecycline (8.3%). overall 30-day mortality of patients with crab was 50%. mortality rates were 3.2 per 1000-patient/day. these rates were significantly higher among patients who have not received appropriate therapy (1.2 per 1000-patient/day) compared with those who have received it (0.3 per 1000-patient/day; p = 0.001; figure 1 ). in the cox regression model only receiving appropriate treatment (hazard ratio [hr] 3.29; 95% confidence interval [ci] 1.35−8.02); p = 0.009) was independently associated with 30-mortality. positive blood culture for crab remained in the final model (hr 1.85; 95% ci 0.86−4.00; p = 0.12). all 25 isolates submitted to pcr were positive for blaoxa-23. all these isolates were susceptible to polymyxin b and tigecycline. conclusion: high 30-day mortality occurred in this icu outbreak. many patients did not receive appropriate therapy, which significantly increased mortality. other clinical risk factors for mortality in this outbreak are currently under investigation. acinetobacter baumannii in norwegian strain collections reveal major discrepancies to phenotypic identification and the presence of carbapenemase-producing clonal lineages baumannii isolates the per-1 gene was identified in 18 (23%). the similarity of the bands were calculated according to "dice smilarity coefficients" and all per-1 positive isolates were found as clonally related. conclusion: in our study the prevalence of per-1 was lower than the previous studies. but the presence of high ceftazidime resistance rates among these isolates may indicate the presence of other beta-lactamases. dna analysis by pfge and rapd revealed an outbreak caused by a unique clone. detection of clonal related isolates among different services may be because of the treatment of these patients at the same services before and this may explain the spread of per-1 positive strains.o442 resistance genomic islands related to abar1 are common in acinetobacter baumannii strains belonging to european clone i l. krizova°, m. maixnerova, l. dijkshoorn, a. nemec (prague, cz; leiden, nl) objective: acinetobacter baumannii strains belonging to european (eu) clone i are commonly resistant to multiple antimicrobial agents. a number of resistance genes were recently detected on an 86-kb genomic resistance island (abar1) inserted in the atpase gene of eu clone i strain aye. the aim of this study was to assess the presence of abar1related structures in epidemiologically unrelated strains of eu clone i. methods: the study set included 25 multi-drug resistant (mdr) strains of eu clone i collected in 19 european countries in 1978-2004 and 10 genotypically unique, fully susceptible strains. using pcr, all strains were investigated for the presence of the atpase gene and for nine genes found to be associated with abar1. furthermore, the strains were tested for the disruption of the atpase gene using pcr primers directed against the 3 and 5 ends of this gene. strains with the disrupted gene were investigated for the presence and structure of the atpase gene-abar1 connecting regions using pcr mapping and rflp. pcr primers were derived from the known sequence of strain aye. results: all strains were positive for the atpase gene. the 10 susceptible strains had an intact atpase gene whereas all mdr strains failed to produce the expected amplicon in the atpase disruption test. all eu clone i strains yielded positive results for the atpase gene-abar1 connecting regions, the structure of which corresponded to those of aye. these findings suggest the presence of atpase integrated elements in clone i strains, the integration of which had invariably taken place at the same locus site. none of the abar1-associated resistance genes were found in any of the susceptible strains. in contrast, the mdr strains harboured the following abar1-associated genes (% positive strains): aacc1 (21), aada1 (21), aadb (4), apha1 (21) stra (3), mera (20), teta (18), cat (23), the gene encoding heavy metal detoxification protein (25). individual mdr strains carried from one to nine abar1-associated genes in 11 different combinations. there was a good correlation between the content of resistance genes and resistance phenotypes. conclusion: genetic structures related to abar1 are common in strains belonging to eu clone i. the heterogeneity of resistance patterns in this clone is likely to result from the variations in the content of abar1related structures. supported by grant 310/08/1747 of the grant agency of the czech republic. objectives: to study the differences in mutation frequency and evaluate the possible correlations between drug resistance development and mutation rate in acinetobacter baumannii (ab). the mutation frequency (mf) of rifampicin (rif) resistance was used as a surrogate measure of differences in mutation rate and for detection of the presence of mutator phenotype. 10-and 100-fold higher when larvae were infected with atcc17978 and sdf, respectively. thus, the sdf genome was used as reference genome to identify functions acquired by pathogenic strains with a possible role in antibiotic resistance and pathogenicity. sixty-two clusters, corresponding to almost 870 cdss, were identified in the acicu and aye genomes (and partially in atcc17978) that were absent in sdf. this study found that targeted interventions that reduce the use of quin were associated with a decrease of the quin resistance rate in e. coli. e. velasco°, w. espelage, i. noll, a. barger, t. eckmanns (berlin, de) objectives: growing populations of older and immunocompromised patients, changes in epidemiology and unchecked use of antibiotics can led to a rise in consumption as well as resistance to certain treatments. medical doctors (mds) often have an important role alongside contributing factors. we conducted a national survey of mds in germany on their behaviours and expectations for intervention. we aimed to assess md behaviours with and influences on antibiotic prescribing and the potential for related interventions that address antibiotic resistance.methods: a representative sample comprised 10,610 mds with differing practice specialties, from both stationary and ambulatory settings (respectively: 36% and 0% internists, 0% and 54% general practitioners, 32% and 11% surgery, 3% and 4% ear/nose/throat, 10% and 10% paediatrics, 5% and 3% urology, 9% and 13% gynaecology, 2% and 4% dermatology, 3% and <1% other) in 15 federal states. we developed study questions to capture baseline information on mds and their practice with antibiotics. questions also focused on selected influences that may affect behaviour in practice. other questions solicited opinions about interventions that may improve practice. mailed questionnaires were distributed to participants via state medical associations. results: among survey respondents (n = 3,613; response rate = 34%), 66% reported that they prescribe antibiotics daily, and 90% indicated they do so at least weekly. of all surveyed mds, 60% reported that they think their own prescribing practice has an influence on antibiotic resistance in their region. of all mds, 83% found it "important" to continually improve use of antibiotics through industry independent experts providing consultation, audits and feedback. of all mds, 96% found it "important" to have provision of regional coverage of antibiotic resistance with appropriate feedback for practicing mds, and 82% found it "important" to have provision of antibiotic regulations of prescriptions with appropriate feedback for practicing mds. (results in table 1 .) -a not all results shown and remaining percentages are as follows: a closed three category scale was used for options "yes", "no", "do not know". a closed four category scale was used with options "very important", "important", "less important" and "not important". a closed five category scale was used for options "daily", "weekly", "monthly", "seldom" and "never". a closed five category scale was used for options "strongly agree", "agree", "neutral", "disagree" and "strongly disagree". objectives: to investigate the mlsb and tetracycline resistance and the emm gene distribution among the invasive streptococcus pyogenes (gas) strains. methods: between january 2006 and december 2008, a total of 991 strains responsible for invasive infections for adult patients were sent to the french national reference center for streptococci to be studied. antibiotic susceptibility testing was done by disk diffusion method according to the ca-sfm guidelines. mics were determined by e-test method. streptococcal emm sequence was done according to the cdc protocol. detection of macrolide and tetracycline resistance genes: erm(b), erm(tr), mef(a), tet(m), tet(o), tet(k), and tet(l) was performed by pcr. results: among the 991 streptococcus pyogenes invasive strains; more than ten different emm-types were identified. the most frequent emm sequence types were emm1, emm28 and emm89. a total of 80 strains (8%) were resistant to erythromycin. erythromycin resistance prevalence had decreased during the three years period (12. 2%-2006, 7.6%-2007, 5.5%-2008) . 69 had an mlsb constitutive (65 strains) or inducible (4 strains) phenotype due to erm(b) or erm(tr) resistance gene. 11 with the m phenotype and mef(a) gene were susceptible to clindamycin. among the 132 (13.3%) tetracycline resistant isolates tet(m), tet(o) and tet(l) genes were detected in 86, 27 and 19 strains, respectively. tetracycline resistance prevalence had also decreased during the three years period (16. 8%-2006, 14%-2007, 8.7%-2008) . conclusion: most of the invasive french gas isolates remained erythromycin and tetracycline susceptiple during three years. nontheless, the resistance rates have had the tendency to decrease slightly. taking into account the resistance trends helps to guide the therapy for penicillin-allergic patients. objectives: during a survey on antimicrobial susceptibility in betahaemolytic group c and g streptococci (gcgs) from portugal, a macrolide resistance rate higher than previously reported in other european countries was found (22%) among s. dysgalactiae subsp. equisimilis isolates. to gain further insights into the resistance mechanisms involved and the clonal structure of the resistant population, we undertook the phenotypic and molecular characterisation of macrolide resistant s. dysgalactiae subsp. equisimilis isolates and compared it with the susceptible population. methods: antimicrobial susceptibility testing and macrolide resistance phenotype were determined by disk diffusion. all the macrolideresistant isolates were further characterised by mic testing and genotype determination by pcr. a combination of emm typing and pulsed-field gel electrophoresis (pfge) was used to type the population and the simpson's index of diversity (sid) with 95% confidence intervals was calculated as previously described.results: a total of 69 isolates were resistant to erythromycin (mic range, 4 to >256 ug/ml). the vast majority of isolates presented a mlsb phenotype (n = 64) and carried the erm(a) gene (n = 55), while the mefencoded m-phenotype was expressed by only 5 isolates. among resistant isolates, 13 distinct emm types were found distributed by 10 pfge clusters that overlapped with the main clusters detected in the susceptible population. the emm types stg480, stg6, stg485 and stg2078 accounted for approximately two thirds of the resistant isolates. pfge did not always separate neither macrolide-resistant from susceptible isolates nor erm(b) and mef(a) from the prevailing erm(a) isolates. the sids of emm and pfge calculated for resistant isolates were not statistical different from the overall population. the two most prominent mls resistant lineages were one with stg480/erm(a) isolates (n = 8) and stg485/mef(a) (n = 3), and another including stg2078/erm(a) (n = 8).conclusion: although most of the resistant isolates presented a mlsb phenotype and carried an erm(a) gene, molecular typing revealed extensive diversity in both emm types and pfge clones. macrolide resistance had a polyclonal origin, with resistance emerging among most susceptible clones. monitoring of macrolide resistance patterns in s. dysgalactiae subsp. equisimilis is essential as this pathogen is increasingly recognised as an important human pathogen. a.s. simões°, r. sá-leão, s. nunes, n. frazão, a. tavares, h. de lencastre (oeiras, pt) while performing pneumococcal nasopharyngeal colonisation surveillance studies among children attending day care centres (dcc) in portugal, we observed that the rate of strains with penicillin mic 2 ug/ml more than tripled from 1.8% in 2006 to 6% in 2007 (p = 0.002). the aim of this study was to characterise the 20 isolates recovered in 2007 which had a mic to penicillin 2 ug/ml. methods: pneumococci were isolated and identified on the basis of selective growth on gentamycin blood agar plates, optochin susceptibility, colony morphology, and alfa-haemolysis. susceptibility to antimicrobials agents was performed according to the clsi recommendations and definitions. strains were serotyped by the quellung reaction and/or multiplex pcr using specific primers for each serotype. pulsed-field gel electrophoresis (pfge), after restriction of the total dna with smai, was performed to compare genetic backgrounds. results: sixteen of the 20 isolates belonged to serotype 14, three were serotype 19a and one was of serotype 15a. strains of serotype 14 were also resistant to sulfamethoxazole-trimethoprim and belonged to a single pfge cluster identified as clone spain 9v st156. the penicillin resistant serotype 14 strains were isolated in two dcc, from nine children vaccinated with the 7-valent pneumococcal conjugate vaccine (pcv7), four non-vaccinated children and three children with unknown vaccination status. five of these carriers had received antibiotics recently. in these two dcc the overall proportion of children vaccinated with pcv7 was 64%; 27% of the children had received antibiotics within the previous month and 16% had received three or more courses of antibiotics in the last six months. since the introduction of the pcv7 in portugal, in june 2001, the proportion of penicillin resistant pneumococci recovered from colonisation has been stable (c.a. 2%). the sudden increase in the levels of penicillin resistance observed in the 2007 surveillance study was found to be largely due to the dissemination of clone spain 9v st156 serotype 14 variant in two dcc with high consumption of antibiotics. the observations suggest a combination of high antibiotic selective pressure and transmission rates resulting in an outbreak-like situation with a penicillin resistant vaccine type clone being disseminated among children in day care despite use of pcv7. background: beside target mutation, active efflux is another common resistance mechanism to fluoroquinolones (fq) in s. pneumoniae. two main efflux systems have been described so far, namely pmra (member of the mfs superfamily) and the two abc transporters pata/patb. we have studied the inducibility of pmra, pata and patb genes expression when bacteria are exposed to subinhibitory concentrations of fq. we used a wild-type sensitive strain (atcc49619), two clinical strains resistant to fq (sp13 and sp295), and two efflux mutants (sp334 and sp335; selected in vitro after exposure to ciprofloxacin [jac 2007, 60:965-972] ). mic were determined according to clsi. induction was obtained by growing bacteria in todd-hewitt broth added by half the mic of each fq (cip, nor, lvx, mxf, gmf) for 4 h at 37ºc in 5% co2 atmosphere. expression levels of pmra, pata and patb genes were determined by real-time pcr. reversibility of induction was tested by re-cultivating bacteria for 4 h in drug-free medium. results: antimicrobial susceptibilities for cip and mxf and gene expression at basal level and after exposure to these fq are shown in the in women with single infection, the most common hpv types were hpv-6 and hpv-16, followed by hpv-51, hpv-31, hpv-53 and hpv-66, whereas in women with multiple infections hpv-6 was the most commonly detected type, followed by hpv-66, hpv-31, hpv-16 and hpv-51. a different distribution of hpv types and a higher rate of multiple infections were observed in young vs. older women, suggesting the existence of a natural selection of hpvs which preserve a better fitness. high-risk hpvs were detected in all high-grade cervical intraepithelial lesions, with hpv-16, hpv-18, hpv-31, and hpv-51 as the most frequent types. however, hr-hpv types were detected also in a high rate of women with a negative pap test as well as in women with a negative cervical biopsy, suggesting the need to improve the accuracy of available cervical cancer screening tests. the results of this study, which provide information on the epidemiology of hpv infection and type distribution in women from south italy, should be taken into consideration in the implementation of local vaccination programs. objectives: chromosomal integration of the hhv-6 genome (cihhv-6) into the human genome occurs in 1−2% of healthy individuals and leads to persistently high levels of hhv-6 pcr copy numbers in blood and tissue. consequently, this may be interpreted as persistent active hhv-6 infection. although hhv-6 mrna has been detected in a few individuals with cihhv-6, there is no evidence of replication of viral particles up to now. viral cultures have shown negative results. so, cihhv-6 is thought not to be linked to any disease. methods: we performed hhv-6 antigen detection in pbmcs of 4 individuals with fish proven cihhv-6 by means of antibodies directed against hhv-6 variant a and b (indirect immunoperoxidase staining). results: in 2 unrelated female adolescents (both with cihhv-6 variant a) we detected hhv-6 antigen. one patient is suffering from recurrent parotitis since 5 years and from hypoimmunoglobulinaemia. the other patient (15a) was treated with allogeneic bone marrow transplantation (bmt) for acute myeloid leukaemia (aml) and acquired cihhv-6 from the healthy donor. so, cihhv-6 is only found in blood cells. in the latter patient only symptoms attributable to the post bmt course have been observed (prolonged mixed haematological chimerism, protracted mucositis, transient hypertension and transient neuropathy). at the time of antigen detection 5 years after bmt the patient was clinically well. in 2 individuals (a girl after fatal myocarditis and her healthy father − both with variant b) no hhv-6 antigen has been detected. discussion: up to now cihhv-6 is considered not to cause any disease. for the first time we show the expression of hhv-6 antigen, which indicates the replication of viral particles. this might have a pathophysiological impact. sixty-seven % of cases with ebv meningo-encephalitis have detectable viral dna amounts in csf and serum, whereas neuroborreliosis patients do not. cases with primary ebv meningoencephalitis have increased systemic leukocytosis, with higher lymphocyte, and monocyte levels compared to neuroborreliosis patients.o506 incidence of post-herpetic neuralgia in treated and untreated patients with herpes zoster followed for 1 year in an italian prospective cohort: preliminary results g. parruti°, f. sozio, c. rebuzzi, m. tontodonati, e. polilli, a. agostinone, a. manna, f. di masi, a. consorte, g. congedo, l. cosentino, d. d'antonio, l. pippa, l. manzoli, c. granchelli (pescara, chieti, it) objectives: a large prospective cohort of patients with herpes zoster (hz) was enrolled between may 2006 and june 2008 in pescara, italy, with a planned 1-year follow-up after clinically and/or molecularly assessed diagnosis. aim of the study was to evaluate predictors of prolonged acute course and/or incidence of post-herpetic nevralgia (phn). methods: data from all enrolled patients were collected by a network of 51 general practitioners. suspected cases and patients with intense acute pain were referred to our institution for immediate evaluation. clinical and demographic information was mandatory at baseline, as photographs of enrolled patients. for uncertain cases, varicella-zoster virus (vzv) antibodies and vzv dna pcr on plasma and/or vesicular eluates (whenever available) were performed. follow-up data were collected at outpatient control visits or by phone calls at 1, 3, 6 and 12 months after onset of hz. phn was diagnosed when pain persisted or relapsed at least one month after complete clearing of dermatomeric lesions. adverse events other than pain were classified according to who grading scale and reported if 2. all statistical calculations were performed by stata 8.0 software package. results: 523 patients were enrolled, 306 (58.5%) females, with a mean age of 57.7 years, 1-year follow-up data being now available for 489. hz was localised at thorax in 45.4% and head in 20.7%; pain in the acute phase was reported as intense or very intense by 127 (25.97%) patients; 54 (11.04%) patients were referred for molecular diagnosis as clinically uncertain, 37 (68.5%) being confirmed as vzv-related cases. forty eight (9.82%) patients were not prescribed any antiviral drug at diagnosis by referring physicians, in spite of extensive support in the study plan. during follow-up, 163 (33.3%) patients reported any type of adverse event (at a mean of 91.2±74.8 days), including 93 (19.02%) patients reporting phn. phn was significantly more frequent in untreated vs treated patients (37.5% vs 17.0%, p = 0.001), as were total adverse events (54.2% vs 31.1%, p = 0.001). untreated patients did not significantly differ from those treated by age (56.3% vs 57.7%, p = 0.66) and sex (females vs males 11.9% vs 6.9%, p = 0.056), whereas they complained for more intense pain (15.0% vs 8.0%, p = 0.024) at presentation. conclusion: our study confirms the importance of early diagnosis and prompt antiviral treatment at the onset of hz in order to minimise the risk of phn. methods: faecal specimens (n = 78) from apparently healthy and diarrheic calves (aged <1 year) were collected per-rectally and investigated for detection of group a rotavirus by antigen capture elisa (generic assay, germany). elisa positive specimens (n = 3) were investigated further for molecular characterisation. genotyping of borv-a strains was carried out on dsrna extracted from 10% pbs faecal suspensions by a nested and/or heminested rt-pcr key: cord-019490-m1cuuehi authors: nan title: abstracts cont. date: 2015-12-28 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2005.clm_1134_02.x sha: doc_id: 19490 cord_uid: m1cuuehi nan addition of a biocatalytic oxygen-reducing agent may be required in the absence of fresh media (<12 hours old) when testing tigecycline using broth microdilution t. stevens, b. johnson, s. bouchillon, j. johnson, d. hoban, m. dowzicky, p. bradford (schaumburg, pearl river, usa) objectives: tigecycline (tgc), a new glycylcycline antimicrobial in development has demonstrated excellent in vitro activity against gram-positive and -negative pathogens. recent reports suggest that tgc mic values, against some organisms, may be elevated if broth used in microdilution panels is (>12 hours old (aged). the nccls has tentatively recommended that testing of tgc be performed in broth that is (<12 hours old (fresh). this study looks at the difference between panels using fresh broth, aged broth and broth with a biocatalytic oxygen-reducing agent (bora) added to compensate for any potential broth differences. materials and methods: testing was performed on approximately 120 organisms including: e. coli; k. pneumoniae; m. catarrhalis; s. epidermidis; and s. pneumoniae. the bora used in this study is oxyrase ò for broth at 2% concentration. tig microdilution panels evaluated in this study include: panels and aged broth without oxyrase (aged); panels and aged broth with oxyrase (ao); panels and fresh broth without oxyrase (fresh); and panels and fresh broth with oxyrase (fo). panels and aged broth were prepared by microscan. fresh broth was prepared internally. each organism was tested on all four panel types. quality controls were performed using nccls approved atcc strains. results: combined test results showed an mic correlation (with in 1 log2 dilution) as follows: 97.5% between fresh/ao; 85.6% between fresh/aged; 94.3% between fo/ao; and 87.9% between fo/aged. quality controls ranges for fo, fresh and ao were all in compliance, but aged panels were out of range 29.3% of the time. conclusion: the addition of a bora to aged broth produced results equivalent to fresh broth without a bora. objectives: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many grampositive pathogens including methicillin-resistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillin-tazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from multinational evaluation centres in the test program. methods: a total of 1245 clinical isolates were identified to the species level at each of 15 sites in 8 countries and confirmed by the central laboratory. isolates were collected between january 2004 and november 2004. mics were determined by each participating laboratory using supplied broth microdilution panels from dade microscan. all testing was performed according to nccls guidelines and manufacturer's instructions. results: the mics of tigecycline ranged from 0.06 to 1 mcg/ml for all isolates of s. aureus. tigecycline's mic50/mic90 of 0.12/ 0.25 mcg/ml, respectively, against mssa was similar to imipenem and minocycline and 4/8 fold lower than the remaining comparative agents. tigecycline's mic50/mic90 of 0.25/0.5 mcg/ml, respectively, against mrsa was 8/4 fold lower than vancomycin, 2/4 fold lower than minocycline and 4/8 fold lower than linezolid. all isolates of s. aureus were inhibited by tigecycline at an mic of 1 mcg/ml regardless of methicillin phenotype. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. background: rapid increasing resistance in nosocomial pathogens has always been a challenge for clinicians and hospital infection control. tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of enterobacteriaceae (mainly e. coli, klebsiella spp., enterobacter spp., and serratia spp.) collected from hospitals in north america, europe and asia. methods: a total of 3204 clinical isolates of enterobacteriaceae were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity was equivalent to imipenem presenting a mic50/mic90 of 0.25/1 mcg/ml against all strains of enterobacteriaceae. in comparison to other antimicrobials tested, the mic90 of 1 mcg/ml for tigecycline was also the lowest being 8 fold lower than commonly prescribed broad spectrum antimicrobials such as ceftriaxone, levofloxacin, and minocycline and 16 fold lower than ceftazidime and piperacillin/tazobactam. the frequency of esbl production among k. pneumoniae and e. coli was found to be 10.3% and 2.2%, respectively. tigecycline inhibited >98% of all e. coli and k. pneumoniae esbl producers at an mic of 2 mcg/ml. approximately 20% of enterobacter spp. and serratia spp. presented resistance to third generation cephalosporins (ceftazidime and ceftriaxone) suggestive of ampc-type resistance. tigecycline also inhibited a majority of these isolates with an mic90 of 2 mcg/ml. conclusion: tigecyclines in vitro activity was comparable to the activity of a broad spectrum antimicrobial, carbapenem (imipenem) , and greater than other commonly prescribed broad spectrum agents tested in this study. the presented data suggest that tigecycline may be an effective therapeutic option against both susceptible strains of enterobacteriaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have a potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram-positive, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against isolates of enterobacteriaceae collected from hospitals across the usa. methods: a total of 2446 clinical isolates, collected in 2004, were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: the efficacy of all broad spectrum antimicrobial agents still remain highly active against enterobacteriaceae in the united states. the susceptibility rates for amikacin, cefepime, ceftazidime, ceftriaxone, imipenem, levofloxacin, minocycline and piperacillin/tazabactam are 99.3%, 97.5%, 89%, 92.2%, 98.8%, 85.5%, 85.7% and 91.8%, respectively. tigecycline's activity was similar to imipenem presenting a mic50/mic90 of 0.25/1 mcg/ ml against all strains of enterobacteriaceae. the frequency of esbl production among k. pneumoniae and e. coli was found to be 8.5% and 2.2%, respectively. tigecycline successfully inhibited >98% of all e. coli and k. pneumoniae esbl producers at a mic of 2 mcg/ml. it was also noticed unusual resistance to imipenem in 29 of these isolates. while still under more detailed analysis, preliminary data have shown that tigecycline presented a mic50/ mic90 of 1/2 mcg/ml against these multi-resistant isolates. conclusion: most of broad spectrum antimicrobial agents still remain active against enterobacteriaceae from the us. tigecycline's activity was comparable to the activities of broad spectrum antimicrobials and with greater activity against most esbl and ampc producing isolates. tigecycline also showed in vitro activity against isolates that were intermediate or resistant to imipenem, which in many instances is considered as a last therapeutic option. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible strains of enterobacterieaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered gram-positive and gram-negative species, including anaerobic pathogens responsible for community and hospital infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/ tazobactam against gram negative rods in addition to linezolid, penicillin and vancomycin for the gram positive species. isolates were collected from hospitals in the united states throughout 2004. methods: a total of 4100 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with a mic equal or lesser than 2 mcg/ml. tigecycline also showed in vitro activity with a mic90 2 mcg/ml against 29 imipenem resistant enterobacteriaceae strains. although similar to other classes of broad spectrum antimicrobial agents against non-fermenters, tigecycline was especially active against acinetobacter spp. with the lowest mic90 of 2 mcg/ml. tigecycline inhibited s. aureus with mic90 of 0.25 mcg/ml for both mssa and mrsa isolates. against enterococci, tigecycline's mic90 was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable or greater than most commonly prescribed broad spectrum antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible common gram-positive and gram-negative pathogens, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, and piperacillin/tazobactam against gram negative rods in addition to linezolid, penicillin, and vancomycin for the gram positive species. isolates were collected from hospitals located in germany, italy, spain, and united kingdom throughout 2004. methods: a total of 1064 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with mics equal or lesser than 2 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against glucose non-fermenters, tigecycline was especially active against acinetobacter spp. presenting the lowest mic90 of 1 mcg/ml. tigecycline inhibited s. aureus with a mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. the same results were noticed against enterococci where tigecycline's mic90 of 0.25 mcg/ml was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered spe-cies responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/ tazobactam against gram negative rods in addition to linezolid, penicillin and vancomycin for the gram positive species. isolates were collected from hospitals located in asia throughout 2004. methods: a total of 424 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity was similar to imipenem against enterobacteriaceae with mic50/mic90 of 0.25/1 mcg/ml. resistance to third generation cephalosporin was found in 63.2% of e. coli and 77.8% of k. pneumoniae consistent with esbl phenotype. tigecycline inhibited esbl and ampc producers with mics equal or lesser than 1 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against glucose non-fermenters, tigecycline was especially active against acinetobacter spp. presenting the lowest mic90 of 1 mcg/ml. tigecycline successfully inhibited s. aureus with mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. same phenomenon was noticed against enterococci where tigecycline's mic90 of 0.12 mcg/ml was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram-positive, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against isolates of enterobacteriaceae collected from hospitals in germany, italy, spain and united kingdom. methods: a total of 627 clinical isolates, collected in 2004, were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory abstracts using supplied broth microdilution panels and interpreted according to nccls guidelines. results: it was observed that the in vitro activity of all broad spectrum antimicrobial agents still remains highly active against enterobacteriaceae in europe. the susceptibility rates for amikacin, cefepime, ceftazidime, ceftriaxone, imipenem, levofloxacin, minocycline,andpiperacillin/tazobactamare99.8%,94.6%,84.8%, 87.6%, 100%, 86.1%, 84.5%, and 92.2%, respectively. tigecycline's activity was similar to the most effective antimicrobial agent, imipenem (100% susceptibility) presenting a mic50/mic90 of 0.25/1 mcg/ml against all strains of enterobacteriaceae. the frequency of esbl production among k. pneumoniae and e. coli was found to be 21.7% and 2.4%, respectively. tigecycline inhibited 100% of all esbl producing e. coli at a mic of 0.5 mcg/ml and at a mic of 4 mcg/ml for esbl producing k. pneumoniae. approximately 30% of enterobacter spp. and 6% of serratia marcescens presented resistance to third generation cephalosporins (ceftazidime and ceftriaxone) suggestive of ampc-type resistance. conclusion: most of the broad spectrum antimicrobial agents still remain active against european representatives of enterobacteriaceae. tigecycline's in vitro activity was comparable to the activities of all broad spectrum antimicrobials with greater activity against esbl and ampc producing isolates with a mic 90 of 2 mcg/ml. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible strains of enterobacterieaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, and piperacillin/tazobactam against gram negative rods in addition to linezolid, penicillin, and vancomycin for the gram positive species. isolates were collected from hospitals in north america, europe and asia throughout 2004. methods: a total of 6383 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with a mic equal or lesser than 2 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against non-fermenters, tigecycline was especially active against acinetobacter spp. demonstrating the lowest mic90 of 2 mcg/ml. tigecycline successfully inhibited s. aureus with mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. the same results were noticed against enterococci. tigecycline's mic90 was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: resistance to glycopeptides in enterococci was first recognized in the late 1980s, and since then has been a major challenge to clinicians and infection control. tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to vancomycin, linezolid, ampicillin, imipenem, ceftriaxone, levofloxacin, minocycline, penicillin and piperacillin/tazobactam against members of enterococcus spp. collected from hospitals in north america, europe and asia. methods: a total of 685 clinical isolates of enterococcus spp. were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: of 437 e. faecalis evaluated, resistance to vancomycin in 15 (3.4%) isolates was observed. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.06/0.12 mcg/ml) among all antimicrobial agents evaluated. among 248 e. faecium, 50 (20.2%) were resistant to vancomycin, of which three isolates were also resistant to linezolid. tigecycline also presented the lowest mic50/mic90 of 0.03/0.06 mcg/ml. five isolates of vancomycin susceptible e. faecalis and one vancomycin susceptible e. faecium were nonsusceptible to linezolid. no abnormal resistance phenotype was observed in other enterococcus species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multidrug resistant strains regardless of degree or type of resistance. tigecycline evaluation surveillance trial (test) -global in vitro antibacterial activity against selected species of glucose non-fermenting organisms objective: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many gram-positive pathogens including methicillinresistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillintazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from 23 us centres in the test program. methods: a total of 794 clinical isolates were identified to the species level at each of participating sites and confirmed by the central laboratory. isolates were collected throughout 2004. mics were determined by each participating laboratory using broth microdilution panels from dade microscan. all testing was performed and interpreted according to nccls guidelines and manufacturer's instructions. results: among the 794 isolates, 389 (49.0%) were found to be resistant to methicillin (mrsa). besides the cross resistance of mrsa isolates to imipenem, ceftriaxone, penicillin, ampicillin, and piperacillin/tazobactam, it was observed a high rate of nonsusceptibility to levofloxacin (74.9%). no resistance was observed against vancomycin and linezolid. the mics of tigecycline ranged from 0.03 to 1 mcg/ml for all isolates of s. aureus, and tigecycline presented the lowest mic50/mic90 of 0.12/0.25 mcg/ml against mrsa isolates, being several folds lower than all the comparator agents. the mssa isolates showed the expected profile of high resistance to ampicillin and penicillin. the only unusual pattern was the 20.4% nonsusceptibility to levofloxacin. tigecycline's mic50/mic90 of 0.12/0.12 was also the lowest among all mssa isolates. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. tigecycline evaluation surveillance trial (test) -united states in vitro antibacterial activity against selected species of enterococcus spp. results: of 276 e. faecalis evaluated, it was observed resistance to vancomycin in 8 (4.5%) isolates. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.06/0.12 mcg/ml) among all antimicrobial agents evaluated. as a typical profile of e. faecalis, fluoroquinolone (levofloxacin) and tetracycline (minocycline) had limited activities against this species. among 144 e. faecium, 35 (24.3%) were resistant to vancomycin, of which three isolates were also resistant to linezolid. tigecycline also presented the lowest mic50/mic90 of 0.06/0.12 mcg/ml. five isolates of vancomycin susceptible e. faecalis and one vancomycin susceptible e. faecium were non-susceptible to linezolid. no abnormal resistance phenotype was observed in other enterococcus species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multi-drug resistant strains regardless of degree or type of resistance. background: glucose non-fermenting gram negative rods are known to be highly resistant in hospital settings and have always been a challenge for clinicians and hospital infection control. the degree or type of resistance may be due to several sophisticated mechanisms such as production of broad spectrum beta-lactamases, efflux pumps and altered membrane permeability, inactivating most classes of antimicrobials that are available for treatment (cephalosporins, carbapenems, aminoglycosides, fluoruquinolones). tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae and selected species of non-fermenters, as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of acinetobacter spp. and pseudomonas aeruginosa collected from hospitals in the united states. methods: a total of 1687 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity against p. aeruginosa showed a mic90 of (16 mcg/ml. towards a. baumannii (n = 849), which cephalosporins were ineffective, tigecycline showed the lowest mic50/mic90 of 0.5/2 mcg/ml outperforming amikacin mic50/mic90 4/32, imipenem mic50/mic90 0.5/16 and minocycline mic50/mic90 1/8. similar findings were found in other species of acinetobacter genus. conclusion: the presented data suggest that tigecycline may be an effective and reliable therapeutic option against strains of acinetobacter spp., including multi-drug resistant strains regardless of degree or type of resistance. objective: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many gram-positive pathogens including methicillinresistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillintazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from 5 european centres in the test program. methods: a total of 216 clinical isolates were identified to the species level at each of participating sites and confirmed by the central laboratory. isolates were collected throughout 2004. mics were determined by each participating laboratory using broth microdilution panels from dade microscan. all testing was performed and interpreted according to nccls guidelines and manufacturer's instructions. results: among the 216 isolates, 76 (35.2%) were found to be resistant to methicillin (mrsa). besides the cross resistance of mrsa isolates to imipenem, ceftriaxone, penicillin, ampicillin, and piperacillin/tazobactam, it was observed that all of mrsa isolates were also non-susceptible to levofloxacin. no resistance was observed against vancomycin and linezolid. the mics of tigecycline ranged from 0.06 to 0.5 mcg/ml for all isolates of s. aureus, and tigecycline presented the lowest mic50/mic90 of 0.25/0.25 mcg/ml against mrsa isolates, being several folds lower than all the comparator agents. the mssa isolates showed the expected profile of high resistance to ampicillin and penicillin. opposite to mrsa isolates, mssa presented very little resistance to levofloxacin (2.5%). tigecycline's mic50/mic90 of 0.12/0.25 was also the lowest among all mssa isolates. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. tigecycline evaluation surveillance trial (test) -european in vitro antibacterial activity against selected species of enterococcus spp. been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to vancomycin, linezolid, ampicillin, imipenem, ceftriaxone, levofloxacin, minocycline, penicillin and piperacillin/tazobactam against members of enterococcus spp. collected from hospitals in germany, italy, spain and united kingdom. methods: a total of 146 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: of 82 e. faecalis evaluated, resistance to vancomycin in 3 (3.6%) isolates was observed. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.12/0.25 mcg/ml) among all antimicrobial agents evaluated. among 40 e. faecium, 1 (5.0%) was resistant to vancomycin. tigecycline also presented the lowest mic50/ mic90 of 0.03/0.06 mcg/ml. no abnormal resistance phenotype was observed in other enterococci species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multi-drug resistant strains regardless of degree or type of resistance. tigecycline evaluation surveillance trial (test) -european in vitro antibacterial activity against selected species of glucose non-fermenting organisms background: glucose non-fermenting gram negative rods are known to be highly resistant in hospital settings and have always been a challenge for clinicians and hospital infection control. the degree or type of resistance may be due to several sophisticated mechanisms such as production of broad spectrum beta-lactamases, efflux pumps and altered membrane permeability, inactivating most classes of antimicrobials that are available for treatment (cephalosporins, carbapenems, aminoglycosides, fluoruquinolones). tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae and selected species of non-fermenters, as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of acinetobacter spp. and pseudomonas aeruginosa collected from hospitals in germany, italy, spain, and united kingdom. methods: a total of 826 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity against p. aeruginosa showed a mic90 of (16 mcg/ml. the cephalosporins were ineffective towards a. baumannii (n = 552). tigecycline showed the lowest mics against a. baumannii mic50/mic90 of 0.25/1 mcg/ml, outperforming amikacin mic50/mic90 4/>64, imipenem mic50/mic90 0.5/16 and minocycline mic50/mic90 1/8. similar findings were found in other species of acinetobacter genus. conclusion: the presented data suggest that tigecycline may be an effective and reliable therapeutic option against strains of acinetobacter spp., including multi-drug resistant strains regardless of degree or type of resistance. p818 antimicrobial activity of tigecycline tested against bacterial pathogens from intensive care units h. sader, t. fritsche, r. jones (north liberty, usa) objectives: to evaluate the antimicrobial activity of tigecycline (tig) and selected antimicrobials against bacterial pathogens isolated from patients hospitalized in intensive care units (icus) worldwide. methods: a total of 7129 were consecutively collected in >70 medical centres located in north america (3164), south america (1465), europe (2428) and the asia-australia region (72). the isolates were collected from (no. of isolates/%): bloodstream (5349/75%), respiratory tract (746/10%), skin/soft tissue (323/ 5%), and urinary tract (182/3%) infections in the 2000-2004 period, and susceptibility tested by nccls broth microdilution methods. results: the antimicrobial activity of tig and the frequency of occurrence of bacterial pathogens are summarized in the table: all gram-positive pathogens (4817) were inhibited at <1 mg/l of tig. resistance (r) to oxacillin was detected in 43% of sa and 84% of cons, and r to vancomycin was detected in 19% of enterococci. tig was very active against enterobacteriaceae (ent; 1468) with a mic90 <1 mg/l, except for serratia spp. 10% of e. coli and 30% of klebsiella spp. showed an esbl phenotype while 28% of enterobacter spp. were r to ceftazidime. 14% of ent showed r to ciprofloxacin. tig and trimethoprim/sulfamethoxazole were the most active compounds against s. maltophilia (mic90, 2 and 1 mg/l respectively). tig was also highly active against asp (mic90, 1 mg/l), but psa showed decreased s to tig (mic90, 16 mg/l). non-s to imipenem (mic, >8 mg/l) was observed in 16% of asp and 31% of psa isolates. conclusions: isolates from icu patients showed high rates of antimicrobial r. the most alarming problems detected were vancomycin r among enterococci, esbl mediated beta-lactam r and fluoroquinolone r among ent, and carbapenem r among psa and asp. tig exhibited potent in vitro activity against the vast majority of clinically important pathogenic bacteria (except psa) isolated from icu patients and may represent an excellent option for the treatment of infections in this clinical environment. potency and spectrum of tigecycline tested against an international collection (1999) (2000) (2001) (2002) (2003) of bacterial pathogens producing skin and soft tissue infections t. fritsche, h. sader, m. stilwell, r. jones (north liberty, usa) objective: tigecycline (tig) is the sentinel representative of the glycylcycline class to be developed as a parenteral agent targeting bacterial pathogens responsible for pneumonia, intraabdominal sepsis and ssti. the aim of this study was to evaluate the activity and potency of tig when tested against a large collection of bacterial pathogens causing ssti. methods: consecutive, non-duplicate bacterial isolates (3,421 strains) were collected from 1999 to 2003 from patients with documented community-acquired or nosocomial ssti in >70 medical centres participating in the tig surveillance program in north america (38.6%), europe (54.0%), latin america (3.7%) and the asia-pacific region (3.7%). all isolates were tested using nccls broth microdilution methods against tig and representative comparator agents used for empiric and directed therapy of ssti. results: ssti pathogen rank order (top ten), potency and cumulative inhibition rates for tig are in the table: all sa, streptococci, enterococci, cons, ksp and ec were inhibited by £2 mg/l of tig, along with 98% of asp and 96% of ent. the broad-spectrum of activity exhibited by tig included tetracycline resistant subsets as well as mrsa, vre, and esbl-producing strains. only pm and psa isolates were less susceptible (mic90 values at 8 and 16 mg/l, respectively). conclusions: among the top ten-ranked pathogens producing ssti, 94% of isolates were inhibited by £2 mg/l of tig and 98% were inhibited by £4 mg/l (the current nccls breakpoint for tetracyclines). tig may represent a welcome choice among newer parenteral agents for the common gram-positive and negative pathogens producing serious ssti given in vitro testing results, thus warranting continued investigation for this indication. objective: to assess the activity of tigecycline (formerly gar936), a novel glycylcycline, against recent bloodstream infection (bsi) pathogen isolates from six continents. frequency of clinical occurrence of these pathogens was determined and their antibiograms assessed using nccls reference broth microdilution methods. methods: a total of 22,950 strains were tested by the m7-a6 (2003) method with interpretations from m100-s14 (2004) . a tigecycline susceptible (s) breakpoint was defined as £2 mg/l for comparison purposes only, although £4 mg/l has been used for tetracyclines. the rank order of pathogens was: s. aureus (sa; 34.2%), coagulase-negative staphylococci (cons; 14.1%), e. coli (ec; 13.2%), enterococci (ent; 12.7%), klebsiella spp. (ksp; 5.3%), p. aeruginosa (psa; 4.0%), enterobacter spp. (ebs; 2.9), beta-haemolyticstreptococci (bst; 2.8%); s. pneumoniae (spn; 2.4%), and viridans group streptococci (vgs; 1.6%). more than 20 comparison agents were tested including tetracycline (tc) and ciprofloxacin (cip) . results: bsi pathogens (gram-positive and enterobacteriaceae) tested against tigecycline are shown in the table. tigecycline was consistently active against tc-resistant (r) strains (89-100% s versus 45-90%). mic50:% s results for other bsi species were: p. mirabilis (4 mg/l:10), acinetobacter spp. (0.5 mg/l:77), serratia spp. (1 mg/l:81), s. maltophilia (1 mg/l:77) and indole-positive proteae (1 mg/l:54). tigecycline exhibited a broader spectrum of activity against bsi isolates when compared to cip, tc, older aminoglycosides and imipenem. tigecycline was not active against psa (mic50, 8 mg/l; 4.0% of bsi isolates). conclusions: tigecycline exhibited a wide spectrum of antimicrobial potency versus bsi isolates collected worldwide. serious infections in nosocomial environments should benefit from tigecycline among the investigational phase 3 agents focused on r strains. evaluation of the in vitro activity of tigecycline against gram-negative anaerobic bacteria objectives: the evaluation of the in vitro activity of tigecycline in comparison to tetracycline, penicillin, piperacillin ( tazobactam, cefoxitin, clindamycin, metronidazole and imipenem against recently isolated gram-negative anaerobic bacteria. materials and methods: a total of 185 gram-negative anaerobic clinical strains (116 bacteroides fragilis group, 20 other bacteroides spp. non-fragilis, 34 prevotella spp. and 15 miscellaneous) isolated during the period 11/2002-11/2004 were tested using the e-test and the agar dilution methods on brucella agar plates supplemented with 5% horse blood, vitamin k1 and hemin. incubation in a chellab anaerobic chamber was performed for 48 hours. interpretation of the results was according to nccls guidelines. for quality control the strains b. fragilis atcc25285 and b. thetaiotaomicron atcc29741 were used. results: overall mic90 to tigecycline, tetracycline, penicillin, piperacillin + tazobactam, cefoxitin, clindamycin, metronidazole and imipenem were 8, 128, 256, 2, 64, 256, 1 and 0.5 mg/l, respectively, whereas mic50 were 0.5, 16, 32, 0.125, 8, 1, 0.5 and 0. 064 mg/l, respectively. bacteroides fragilis group mic90 were 8, 256, 256, 4, 128, 256 , 1 and 0.5 mg/l, respectively, whereas mic50 were 0.5, 16, 64, 0.25, 16, 1, 0.5 and 0. 064 mg/l, respectively. at the tigecycline mic90 concentration of 8 mg/ l the compound inhibited a higher percentage of isolates than clindamycin, cefoxitin or tetracycline (which inhibited 76, 56 and 45% of the isolates, respectively). only one strain was resistant to imipenem (mic > 32 mg/l), having a tigecycline mic of 8 mg/l. in addition, six of the eight strains with a metronidazole mic > 16 mg/l had a tigecycline mic < 1 mg/l. conclusions: in general, tigecycline showed better in vitro activity than clindamycin, cefoxitin, tetracycline or penicillin against gram-negative anaerobic bacteria and somewhat lower clinical microbiology and infection, volume 11, supplement 2, 2005 activity than imipenem, metronidazole or piperacillin + tazobactam. nevertheless, among most metronidazole and imipenem resistant isolates tigecycline displayed good activity. the results of this in-vitro evaluation show that tigecycline should be considered as a possible alternative for the treatment of mixed aerobicanaerobic infections. objectives: to ascertain the variation between mics of tigecycline determined in broth and agar, and by nccls and bsac methods. to assess the effect of adding tigecycline to the medium one day before use and of using media that had been aged for one week before addition of tigecycline and inoculation. method: mics for 96 non-fastidious bacteria and 20 streptococci were determined in mueller-hinton broth (mhb), mueller-hinton agar (mha), iso-sensitest broth (isb) or iso-sensitest agar (isa). fresh antibiotic stock solutions (tigecycline, tetracycline and minocycline) were incorporated into bulk media by rolling, to minimise aeration. preparation conditions otherwise were as fol-lows: (a) freshly-prepared media, antibiotic added upon cooling, then inoculated; (b) freshly-prepared media, antibiotic added upon cooling thenstored for 1 day before inoculationand (c) media stored for 7 days before addition of the antibiotic and inoculation. results: mics in fresh mhb (the nccls reference method) were taken as a standard. for all the tetracyclines, these mics were 0-2 doubling dilutions higher than on mha or isa (bsac reference method), or in isb. media with tetracyclines added a day before use gave raised mics, though rarely by more than one dilution. tigecycline mics were increased in 7-day-old mhb or isb. this effect was greatest (2-3 dilutions) for the most susceptible strains (mic £ 0.5 mg/l) and was absent for the resistant organisms (mic ‡ 8 mg/l); it did not occur in agar. minocycline mics were not raised in aged media, and tetracycline mics were only marginally affected. the addition of blood to the mhb largely abrogated the effect on tigecycline mic, as did boiling the broth prior to adding the antibiotic. conclusion: the raised mics of tigecycline in aged broth probably reflect inactivation by dissolved oxygen. this accords with lack of any mic increase in newly boiled (i.e. degassed) mhb or on aged agar (which is boiled to melt before use). the effect of blood may be to add to the reducing capacity, protecting the tigecycline. at a practical level, broth mic methods for tigecycline (e.g. the nccls reference method) require, freshly prepared or steamed medium; this is not a concern if agar dilution methods are used, such as that of the bsac. sexually transmitted diseases p823 prevalence of chlamydia trachomatis and neisseria gonorrhoeae in native and immigrant population (ambits study) in barcelona (spain) testing of all danish gc strains isolated in 2002-2004 (n = 953) . clinical and epidemiological data were obtained when possible. results: the 25 pip-negative gc isolates were all designated as serovar ib-4 and similar mic values were identified in the antibiograms of these isolates fingerprints were identified using spei and three using bglii among the 25 ib-4 isolates. four distinguishable pfge . however, all these isolates differed by less than six bands in both fingerprints. the phylogenetic tree analysis of the porb1b sequences, suggested that these minor differences represented the ongoing evolution of the same strain. during the period jan. 2002 to nov. 2004 the proportions of pip-neg gc per year in denmark were 15/332, 47/261 and 27/360, respectively, and the pip-neg gc infected mostly men (only 5 women). the antibiograms of all danish isolates 2002-2004 indicate a spread of at least one pip-neg gc strain. the results of the present study indicate a circulation of at least one n. gonorrhoeae prolyliminopeptidasenegative strain in the danish homosexual community. an increased awareness for pip-negative n. gonorrhoeae, which create large problems in diagnosis using e.g. api nh, rapid nh, gonocheck ii or neisseria pet, is essential. a new recombinant antigen haemagglutination test for the serological diagnosis of syphilis n. chepurchenko, t. ulanova, v. puzyrev, l. loginova, a. burkov, a. obriadina (nizhniy novgorod, rus) introduction: passive treponema pallidum hemagglutination (ha) -the routine treponemal test has equal sensitivity with the fluorescent treponemal antibody absorption (fta-abs) test and with the enzyme immunoassay (eia) in later stages of syphilis, but some studies have indicated that it is less sensitive in the primary stage of the disease. the use of recombinant t. pallidum antigens instead of a poorly defined mixture of antigens from wild-type t. pallidum has the potential for improving the sensitivity of hemagglutination test. objectives: the purpose of this study was to evaluate the diagnostic relevance of 4 recombinant proteins that efficiently model the antigenic epitope(s) of the treponema pallidum proteins. methods: four full-length recombinant proteins (tmpa, 47, 17, 15 kda) were expressed in e. coli and then used individually to develop the hemagglutination test (ht) for the detection of anti-treponema pallidum (anti-tp) activity in serum specimens. serum samples (n = 267) from patients with clinically proven syphilis in various stages of the disease (primary (n = 36), secondary (n = 70), latent (n = 161)) and from normal blood donors (n = 505) were tested. 24 samples from patients with primary syphilis, 21 samples from patients with secondary and 17 samples from patients with latent stage were tested initially with commercially available hemagglutination test based on mixture of treponema pallidum antigens (http). all specimens were additionally tested for specific antibodies by commercially available enzyme immunoassay (eia). results: the sensitivity of the ht for the detection of anti-tp activity in human serum specimens varied from 38.9% to 97.2% with primary syphilis sera, from 87.1% to 100% with secondary, and from 96.9% to 98.1% with latent stage sera for each protein. the tmpa was found as the most immunoreactive when serum samples from patients with untreated syphilis were tested. the sensitivity of tmpa ht was identical to those of the http in cases of untreated secondary and latent syphilis and significantly higher with specimens from primary stage of disease. the overall specificity and sensitivity of the tmpa ht were comparable to those of eia 99% and 99.6%; 98% and 99.6% respectively. the results of this study indicate that the tmpa ht may be a reasonable alternative to the routine hemagglutination test and the elisa as a confirmatory test for syphilis. use of treponema + vdrl virablot test as a useful ôall in oneõ confirmatory assay for treponema pallidum antibodies a. kostoula, g. vrioni, c. bobogianni, c. stergiopoulou, e. papantoniou, s. levidiotou (ioannina, gr) objectives: as detection of treponema pallidum subsp. pallidum by dark-field microscopy or direct immunofluorescence is possible only in the early stages of the disease, serology has become the method of choice for syphilis diagnosis. except for conventional treponemal tests, the t. pallidum western immunoblot assay has also been used as an alternative confirmatory assay for t. pallidum antibodies. the aim of the present study was the use of a multiparameter immunoassay, which simultaneously detects treponemal specific and lipoid antibodies, for the confirmation of syphilis antibodies. methods: a total of 86 serum specimens were tested including, 56 reactive sera from patients with documented treponemal infection; a specificity panel of 10 sera from normal subjects, 10 sera with rapid plasma reagin (rpr) reactivity (fta-abs and tpha negative), 5 leptospira igm serology-positive sera and 5 borrelia burgdorferi igg or igm serology-positive sera. all serum samples were tested with conventional syphilis tests according to the manufacturersõ instructions: rpr, venereal disease research laboratory (vdrl), t. pallidum haemagglutination (tpha) at a serum dilution of 1:80, fluorescent t. pallidum absorption (fta-abs) and igm capture enzyme immunoassay (eia). subsequently, the results were compared to those of the treponema + vdrl virablot igg, igm test (viramed, labor -diagnostika), a immunoassay for the qualitative detection of specific igg or igm antibodies to t. pallidum using the specific treponemal proteins p47, p44.5, p17 and p15, and the quantitative determination of vdrl reaction on one nitrocellulose strip. results: treponema + vdrl virablot igg and igm tests confirmed the results for 56 serum specimens positive for t. pallidum antibodies: reactive vdrl band and at least two clear bands from p47, p44.5, p17 or p15 for igg treponemal antibodies or at least one clear band p47, p17 or p15 for igm treponemal antibodies. the 10 rpr reactive sera were vdrl virablot reactive, but treponema virablot specific antibodies negative. none of the remaining sera reacted in tests with the treponema + vdrl virablot assay. conclusions: the treponema + vdrl virablot assay combining the necessary serologic markers in one test strip (i.e. specific and non-specific treponemal antigens) is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques. the great imitator: syphilis with predominant acute severe hepatic involvement the ground of a significant increase of serum transaminiases (up to 2200 and 900 u/l of gpt, respectively), frank jaundice, elevated bilirubin (up to 11.7 and 9.6 mg/dl, respectively), and evident rise of serum alkaline phosphatase and gamma-gt. clinical history did not show risk factors for viral hepatitis, and all laboratory examinations for major and minor hepatotropic viruses proved negative, as well as autoimmmune, dysmetabolic, or drug-induced liver damage. repeated ultrasonography detected increased liver and spleen dimensions, in absence of other abnormalities. in the second p, histopathologic study performed after liver biopsy, showed an aspecific cholestatic hepatopathy, a moderate granulocyte and lympho-mononocyte intralobular infiltrate, appreciable necrotic foci, and a mild portal fibrosis (knodell score 5). only syphilis serology, performed despite the absence of significant history or clinical clues, allowed to recognize an igm positive serology associated with tpha titres ranging from 1:1250 and 1:680, respectively. specific chemotherapy (i.v. penicillin g at 24 mu daily for 7 days, followed by 12 mu/day in the second p),led to a complete resolution of hepatic involvement, associated with an improvement of serology after 1 month. discussion: while in the majority of episodes of syphilis liver involvement remains missed or subclinical, an early diagnosis of syphilis with predominant hepatic disease remains an unresolved problem of differential diagnosis, due to the rare occurrence of isolated organ involvement compared with typical syphilis signs and symptoms, and the aspecific clinical, histopathological, and imaging picture (jozsa l,acta hepatogastroenterol 1977; 24:344-7; young mf, j clin gastroenterol 1992; 15:174-6) . during the recent recrudescence of syphilis in italy, a diagnostic suspicion should be maintained by clinicians who face an apparent acute icteric hepatitis, and syphilis serology should be included among initial laboratory workout. the great imitator: syphilis with predominant meningoencephalitic features r. manfredi, s. sabbatani, d. pocaterra, f. chiodo (bologna, i) introduction: a significant recrudescence of syphilis was observed in recent years, not contained by prophylactic measures against hiv and other stds. meningeal and meningoencephalitic involvement may occur also in early stages of syphilis, thus posing problems of differential diagnosis. case reports: a young woman and an hiv-infected male developed a syphilis with predominant meningoencephalitis expression. in the first p, based on an isolated positive borrelia burgdorferi serology (later deemed as a cross-reaction), early ceftriaxone was started, due to a suspected neurological lyme disease. also after the diagnosis of neurosyphilis (on the ground of positive csf serology), antimicrobial therapy was left unchanged for 3 weeks at our day-hospital facilities, until complete clinical-microbiological cure. a second, hiv-infected p was hospitalized owing to mild fever lasting 2 weeks, associated with cephalalgia and anxiety.the favorable virological-immunological status (cd4 + count 439 cells/ll and undetectable hiv viraemia, under an effective haart regimen), did not exclude all searchs for possible hiv-related disorders.although serum examination detected a potential latent syphilis (isolated positive serum treponema pallidum igg, tpha 1:640,igg-positive rpr, and mute history for syphilis), only csf examination disclosed an increased cell content (50/ll), altered brain-blood barrier indexes with increased intrathecal ig synthesis, and frank vdrl,tpha (1:1520), and borderline t. pallidum igm serology.penicillin g at 24 mu/day for 14 days led to a slow resolution of neurological signs and symptoms, and a tendency to improvement of specific csf-serum syphilis serology during subsequent controls. discussion: focusing on differential diagnosis, a luetic etiology should not be underestimated, when facing young p suffering from a meningoencephalitis of unclear origin.our cases were characterized by a young age (34 and 44 years, respectively), when compared with usual mean age of tertiary neurosyphilis. in absence of suggestive history and other syphilis signs, the diagnosis was achieved only after the retrieval of elevated syphilis serology positivity on both csf and serum, together with some clinical signs, such as seizures, altered mentation, cognitive abnormalities, and anisochoria in the first p, and persisting headache and anxiety in the second p. our experience with ceftriaxone (started in the first p when neuroborreliosis was suspected), was favorable like that with high-dose i.v. penicillin g. comparison of the genomes of pathogenic treponemes, treponema pallidum ssp. pallidum, ssp. pertenue and treponema paraluiscuniculi objectives: the cervical human papillomavirus (hpv) infection is common among young sexually active women. because the cytology tests have some limitations, including up to 30% false-negative results, molecular techniques are increasingly used for identification of hpv-induced cell changes in the cervix. the polymerase chain reaction (pcr) for hpv diagnostics was successfully introduced recently in bulgaria. however limited data on specific prevalence and determinants of subclinical hpv infections are available. in this study we designed a multiplex primers pcr system for simultaneous identification of the most common low-and high-risk hpvs in women with cytologically normal cervical smears. materials & methods: dna from 102 cervical cell samples from women aged between 18 and 40 years with normal pap smear tests was extracted by standard procedure and studied for hpv presence. six type-specific primer sets for e6 hpv gene were used in a single-tube reaction for detection and identification of . respective controls and statistic analysis were included. results: the overall hpv dna prevalence was 23.5% (24/102). ten of the hpv positive women (41.6%) were infected with high-risk (hpv-16 or hpv-18) virus types; eight (33.3%) with low-risk types (hpv-6 or hpv-11), and six (25%) were infected with both high-and low-risk types (3 hpv-6/-16; 2 hpv-6/-18 or 1 hpv-6/-33). hpv prevalence among women younger than 23 years was 58.3% (14/24). for low-risk types the peak prevalence was observed in women between 35-40 years. besides age, there was a positive association between the detection rates of hpv infection and number of sexual partners and oral contraceptive use. conclusion: the used technique is simple, economic, rapid and reliable for screening studies of hpv infections. the prevalence of hpv in cytologically normal bulgarian women is similar to the reported in other countries. our findings suggest that molecular techniques for hpv detection might be useful as an adjunct to pap smear screening. were examined. mycoplasmas were determined by use of a commercially available system (liofilchem s.r.l, italy) and c. trachomatis antigen by dif (cellabs pty ltd, australia). results: of 1284 specimens, 1121 were positive. the isolated species in each age group are summarized in table 1 . in 818 cases of lower genital tract (lgt) infection, only one species was isolated. in all three age groups, mycoplasmas were the most commonly identified. in particular, the species of the isolated pathogens were: (1) group a (n = 43): mycoplasmas 52%, streptococcus spp 24%, candida spp 8%, c. trachomatis 8% and anaerobes 8%, (2) in 39 women of this group a mixed infection with three species was found. from 818 women with positive cultures, 562 (68.70%) were symptomatic, while in 256 asymptomatic women (31.20%), sexually transmitted pathogens were identified. noticeably 40.58% and 37.5% of the infected women with candida spp and c. trachomatis respectively, presented no symptoms. conclusions: (1) the rate of mixed lgt infections was 27.02% (2) asymptomatic infections were 31.20% (3) all teenagers in group a were symptomatic. ureaplasma mba size variants in woman with spontaneous preterm delivery j. spergser, a. witt, l. petricevic, p. husslein, a.m. hirschl, r. rosengarten (vienna, a) objectives: despite colonization of the lower urogenital tract, ureaplasmas reach the upper genital tract only in a subpopulation of women, and only in some of these individuals symptoms like preterm delivery ensue. while some studies indicated that certain serovars are more frequently implicated with particular diseases, others have suggested that size variability of the multiple banded antigen (mba) may contribute to ureaplasmal pathogenesis. to address these hypotheses, vaginal and placental samples from women with spontaneous preterm delivery were tested for ureaplasma species, serovars and mba size variants. methods: placenta/amnion specimens and corresponding vaginal samples from women that delivered via caesarean section less than 34 weeks of pregnancy were collected. samples were examined for ureaplasma species and serovars by cultivation and pcr assays. to determine mba size variants, pcr with primers amplifying the non-repetitive and the c-terminal repeat region of the mba gene were performed. results: out of 100 cases examined so far, 32 placenta/amnion specimens and 40 corresponding vaginal samples were positive for ureaplasmas by cultivation and pcr. while ureaplasma positive samples from the lower genital tract were carrying u. parvum (100%) and u. urealyticum (50%), colonization of the placenta/amnion was restricted to u. parvum which was only recovered from women with preterm labor or preterm prom. in contrast, ureaplasmas were not detected in samples from women with hellp syndrome, iugr and preeclampsia. as opposed to some previous studies, particular serovars were not implicated in adverse pregnancy outcome. however, significant differences between number and lengths of mba pcr products among placental and vaginal ureaplasma isolates were obvious. ureaplasma isolates from placenta/amnion membranes gave shorter pcr products than did corresponding vaginal isolates and while a majority of vaginal ureaplasma isolates were composed of multiple size variants, placental isolates were predominantly restricted to a singular mba size variant. conclusion: only u. parvum was found to be frequently present in the placenta/amnion of women with preterm delivery and certain serovars were not more frequently implicated with particular diseases. in addition, mba size variation represents an additional level of phenotypic and genetic variability beyond the serovar category which may provide an important contribution to ureaplasmal pathogenesis. use of kanamicin in the topical therapy of aerobic vaginitis g. tempera, g. bonfiglio, e. cammarata, s. corsello, a. cianci (catania, paternò, i) objectives: demonstrate the efficacy of the topical therapy with kanamicin in a new pathology called aerobic vaginitis (av). methods: eighty-one patients with clinical diagnosis of av in accordance to donders's criteria, have been included in the study. the presence at least of four of the following symptoms were taken as diagnosis of av: objective abnormal yellow vaginal discharge, foul smell (but negative to koh test), elevated vaginal ph (>5), abundant vaginal leukocytes and lactobacillary grade iia, iib and iii in accordance to donder's classification. other characteristics such as vaginal dyspareunia, vulva-vaginal itching, cervical erosion as well as the isolament of vaginal microorganisms were also documented. the patients were randomised treated, 45 with kanamycin (100 mg vaginal ovules per 6 days, consecutively) whereas, 36 with meclocycline (35 mg vaginal ovules per 6 days, consecutively). the patients were visited before starting the study, 1-2 days and 30 days after the end of the study. results: at the first follow-up the patients showed different level of symptoms reduction. particularly, reduction of presence of leukocytes, burning of vaginal mucosa and itching were statistically significant in the group treated with kanamycin with respect to the group treated with meclocycline. moreover, there was also a reduction of isolament of enterobacteriaeae (97%) in the group treated with kanamycin versus 76% treated with meclocycline. at the second follow-up, vaginal homeostasis (normalisation of ph and presence of lactobacilli) was demonstrated only in kanamycin treated group. conclusion: our data suggested that the topic use of kanamycin could be considered a specific antibiotic for the therapy of this new pathology. objectives: establishing the identity of lactobacillus species colonizing the vagina of women is of importance, because clinical studies have demonstrated an association between the presence of h 2 o 2 -producing strains of lactobacillus and a decreased prevalence of bacterial vaginosis (bv). recently, culture based studies using molecular identification methods showed that l. crispatus and l. jensenii are the most common species of vaginal lactobacilli and that colonization by these species was positively associated with a lower frequency of bv. here were report that l. crispatus can be distinguished from other lactobacilli using gram staining of vaginal smears. methods: several approaches were used to characterize 515 vaginal microflora samples obtained from 197 pregnant women at three time points in pregnancy: 1/gram stained smears from vaginal swabs, scored according to modified ison and hay criteria; 2/identification of cultured isolates obtained after anaerobic culture, identified using tdna pcr; and 3/species specific pcr for atopobium vaginae and gardnerella vaginalis. grade i specimens, representing a normal microflora, were further characterized as grade ia when only l. crispatus cell types were present, grade ib when other lactobacillus cell types were present, grade iab when both l. crispatus and other lactobacilli were present and grade ic when either gram-positive rods, small and short, or irregularly shaped gram-positive rods, with clubbing, curved edges and irregular staining (ôdiphteroid cell typesõ) were seen. results: out of the 515 samples, 86.4% showed a normal vaginal microflora (grade i), 8.9% were grade ii, 3.7% grade iii and 1.0% grade iv. based on the presence of different lactobacillus species, grade i specimens were further characterized as grade ia (36.4%), grade ib (40.9%), grade iab (13.5%) and grade ic (8.5%). this classification was supported by the finding that out of respectively 86.0% and 76.6% of grade ia and iab specimens l. crispatus was cultured while this species was present in only 13.1% and 2.6% of respectively ib and ic specimens. in addition, 55.1% of grade ic specimens contained bifidobacterium spp. conclusion: further refinement of gram stain based grading of vaginal smears is possible by distinguishing additional classes of normal microflora. these categories of gram scores may facilitate and improve future studies regarding the interpretation of clinical data and therapeutic outcome. objectives: the importance of the isolation of gardnerella vaginalis in bacterial vaginosis is well known. the involvement in male urogenital infection is uncertain, probably due to low rate of isolation for inadequated specimens process. the objective of this study is to evaluate the possible pathogenic rol of g. vaginalis in male urogenital pathology. semen is a recommended specimen in diagnosing of prostatitis. at the same time it also reflects normal genital tract microflora. although its composition may be affected by the sexual activity, there are no data regarding the changes in genital tract microflora during the sexual debut. objective: to identify possible associations between sexual experience and the reproductive tract inflammatory parameters and microflora in healthy young men. methods: a total of 72 healthy men aged 17-22 years were included and divided into 3 groups based on their sexual experience (virgins n = 13, monogamous n = 11 and polygamous n = 48). a semen sample of each subject was assessed for basic semen parameters (semen volume, sperm concentration, sperm motility), white blood cell (wbc) counts and quantitative cultures for aerobic, microaerobic and anaerobic bacteria, and possible correlations between the type of sexual experience, wbc counts and seminal microflora were calculated. results: basic semen parameters were similar in all groups. 5.5% of men had high (>1 m/ml) and 19.4% of men had medium (0.2-1 m/ml) semen wbc counts. there were no correlations between the type of sexual experience and semen wbc counts. in comparison to sexually active men in virgin subjects the total microbial counts in semen (4.4 vs 5.0 log cfu p < 0.05) and the number of different species (3 vs 5, p ¼ 0.05) were lower, however, no differences between mono-and polygamous men were found. there was a positive correlation between the total microbial concentrations and number of different species (r = 0.54; p < 0.0001). staphylococci, corynebacterium seminale, other coryneforms, gamma-and alpha-haemolytic streptococci and peptostreptococci were the most prevalent organisms. no significant differences in microflora were observed between the groups yet the gram-negative anaerobic rods were more often seen in sexually experienced men than in virgins. the subjects with high wbc counts (>1 m/ml) tended to have higher number of different microorganisms than the rest of the men (median 7 vs 4, p = 0.05). the sexual debut is associated with the enrichment of seminal microflora but not with the influx of wbc or changes in basic seminal parameters. strong positive correlation can be observed between the total microbial concentration and the number of different species in semen. sexually transmitted infections among registered female sex workers in athens under investigation were 122 sexually active non-pregnant women aged from 18 to 40 years with diagnosed cervicitis by cytological examination and presence of muco-purulent discharge. fifty five women were included in studied group -with confirmed c. trachomatis cervicitis and 67 women -in control group (with non-chlamydial/non-gonococcal cervicitis). bv was diagnosed using amsel and nugent (0-3 negative; 4-6 intermediate; 7-10 positive) criteria. by using amsel criteria bv was suggested in 11% and 6% correspondingly among women in studied and control group. by using nugent criteria bv was suggested correspondingly in 5.5% and 3%. these differences were not statistically significant and can be explained by the presence of muco-purulent discharge among studied women. objectives: helicobacter pylori is recognized as an important human pathogen but the differentiation of the species by classical phenotypic methods is really difficult. therefore the objective of this study was the development of an identification method for helicobacter species based on pcr-restriction fragment length polymorphism (pcr-rflp) analysis of the 16s and 23s rrna genes. objectives: since the first identification of helicobacter pylori by warren and marshal in 1983, members of the genus helicobacter increased to more than 26 species, which have been detected in human and animals. the construction of species specific pcr assays is difficult due to the close relatedness between different helicobacter species. the pcr-dgge technique was developed for the identification of helicobacter colonization. the aim of this study was to investigate the effect of multiple regions of the 16 rrna gene on the diagnostic efficiency pcr-dgge. methods: dna was extracted from 40 helicobacter strains, which represent 20 helicobacter species. amplification of 1.2 kb of helicobacter 16s rdna, which contains v3 and v6-7 regions, was done using previously published helicobacter genus specific primers c97 and c05. the pcr product can be used as a template for two helicobacter genus pcr assays, the v6-7 regions 16s rdna was amplified using the primers 1fc254 and 2rc686, the v-3 region was amplified using the primers bsf917 and bsr1114 published at (http://rrna.uia.ac.be/ primers). dgge analysis of the v3 and v6-7 regions was performed on 9% polyacrylamide gels containing urea and formamide gradient from 20-40% or 15-30%, respectively. electrophoresis was performed in a dcode electrophoresis unit (biorad) at constant voltage (200 v) at 60°c for 4 hours. results: dgge analysis of two regions of helicobacter 16srrna gene showed mobility patterns that allowed discrimination of most helicobacter species except those which are closely related such as h. pullorum-h. pametensis, h. ganmani-h. rodentium and h. bizozeroni-h. felis-h. salmonis. conclusions: helicobacter species are widely distributed in the gastrointestinal tract of mammals, birds and other animals. the pcr-dgge technique has proven to be an easy, inexpensive and efficient tool for the identification of helicobacter species and for the detection of colonisation by more than one helicobacter species, without the need for species-specific pcr assays. in addition, the diagnostic efficiency of this technique was increased by analysis of multiple regions of the 16s rrna gene. helicobacter spp. in chronic pancreatitis, pancreatic adenoma and pancreas cancer objectives: helicobacter spp. efficiently colonise various hostile habitats such as the stomach, colon and biliary tract of animals and man. with the exception of h. pylori, many helicobacter spp. are difficult to culture. nucleic acid based detection has thus become a method of choice for analysis of helicobacter spp. in chronic inflammatory gastrointestinal (gi) tract diseases and cancers. pancreatic exocrine cancer, with few established risk factors and very poor prognosis, is a common cause of cancer death. previously, serological studies found an association between h. pylori and pancreas cancer (pc). methods: pancreatic tumour specimens of patients with pc (n = 40), tissues of chronic pancreatitis (n = 6), pancreatic adenoma (n = 8), and benign pancreatic tissue of patients with cancers of the choledochus, colon and duodenum (n = 6), were analysed by semi-nested helicobacter-specific 16s rdna pcr. stomach (n = 23) and gallbladder (n = 12) specimens of the pc-patients were also analysed. were characterized by dna-sequence analysis. results: the helicobacter spp. pcr was positive in 19 of 40 (47.5%) pancreas cancer patients, and in the pancreas in 4 of 6 (66.7%) patients with chronic pancreatitis. adenoma and pancreas tissues of other gi-tract cancers, as well as gallbladder specimens, were all negative. four of 23 (17.4%) stomach samples of the pc-patients were helicobacter positive with sequences homologues to h. bilis by blastn analysis. dna-sequencing of 13 pcr-products amplified in pcs were closely related to h. pylori (n = 9), h. flexispira sp. (n = 3) and h. cinaedi (n = 1). two pancreatitis pcr-products matched h. pylori. conclusions: helicobacter spp. were common in pc compared with benign pancreatic tissue. helicobacter pylori infection and low igg immunoglobulin s. kokkinou, k. pavlou, a. varouta, j. villiotou, k. papaefstathiou, a. moutsopoulou, e. papafrangas (halandri athens, athens, gr) it is established that helicobacter pylori (hp) may cause haematological disturbances such as iron malabsorption and idiopathic thrombocytopenic purpura (itp). the aim of this study is to show that hp is involved in human disease in a more complicated way, producing an abnormal immunologic profile. patients and methods: this work reports the presence of a significant low value of igg immunoglobulin in 52 pts suffering from unidentified iron deficiency (id)or iron deficiency anemia (ida coexisting with megaloblastic anemia(ma), in 12 leukopenic pts and in 3 more pts having thrombocytosis, itp and polycythemia respectively. the ratio male/female was 25/42, and their age ranged from14-80 years. in the blood examination there was a10-fold titre of hp-igg type antibody in all, while in 23 there was also a high titre of hp-iga type using elisa technique. the histopathological examination of an antral biopsy showed diffuse corpus gastritis, atrophic gastritis and achlorydria secondary to hp infection that take place in gastric area. results: 52 pts had unidentified id or ida coexisted with megaloblastic anemia. serum iron, transferrin saturation, serum ferritin and b12levels were found to be significantly low. all of them had also hp infection. their anemia existed for more than 10 years, was unresponsive to treatment and recurrent in nature. twelve pts with undefined eukopenia (l:3.3 · 10 9 /l) lasting for at least 8 years, had also an hp infection. all of our pts had a significantly low value of igg immunoglobulin. mean value ranged from 551 to 868 mg/dl(normal value:850-1517 mg/dl). they subjected to eradication therapy. all but one responded well. conclusions: (1) hp infection was found to coincide with id, ida, ma and leukopenia. (2) the normalization of haematological parameters after eradication started 6 months later. (3) the essential mechanism between the hp infection and low igg is unclear and its value remains low and after the eradication therapy. (4) studies with larger samples should be done for a better evaluation of the hp infection and it's involvement in human disease. (5) hp infection acts in a catabolic way for the humans. it must be considered to search for it in haematological conditions of undefined origin. helicobacter pylori from the dyspeptic patients in thailand: diagnosis, antimicrobial susceptibility and correlation to clinical outcomes c. chomvarin, p. kulsuntiwong, p. mairiang, c. kulabkhow (khon kaen, th) objectives: to evaluate methods for routine clinical diagnostic use of helicobacter pylori infection of gastric biopsies, to study the correlation of h. pylori infection to the clinical outcomes and to study antimicrobial susceptibility by disk agar diffusion in thailand. methods: gastric biopsies, obtained from 210 patients underwent at the endoscopy unit of srinagarind hospital, faculty of medicine, khon kaen university, were diagnosed by culture, rapid urease test (rut, pronto dry) and histological examination. a true positive criteria was indicated when culture or both rut and histology tests were positive. one hundred and fifteen isolates of h. pylori isolates were tested for six antimicrobial agents by disk agar diffusion method. results: the h. pylori infection rate was 44.3% (93/210). the sensitivity vs. specificity of the culture, rut and histology were 88.2% and 100%, 95.7% and 98.3%, and 96.8% and 59.8% respectively. the prevalence of h. pylori in gastritis (gt), duodenal ulcer (du) and gastric ulcer (gu); and gastric cancer (gca) patients were 41.2%, 57.9% and 70.6%, respectively. the chi-squared test showed that gca patients were significantly more often infected with h. pylori than gt patients. the resistance of 115 h. pylori isolates to single antimicrobial agent as metronidazole (mtz), clarithromycin (clr),ciprofloxacin (cip), amoxycillin (aml), and tetracycline (te) were detected in 21.7, 0, 5.2, 1.7 and 0 percent, respectively. the combination resistance to mtz & clr, mtz & cip, mtz & aml, mtz & te; and clr & cip were detected in 1.7, 1.7, 0.8, 1.7 and 0.8 percent, respectively. the 2.5 per cent of h. pylori isolates were resistance to three or more antimicrobial agents. conclusion: rut method was highly sensitive and specific and appropriate for routine laboratory use. the correlation of the gca patients to h. pylori infection was significant difference compared to gt patients. the high resistance rate to metronidazole indicated that the effective for eradication of h. pylori by metronidazole should be considered in the clinical management of h. pylori infection. use of an indirect immunofluorescence test for the detection of caga in h. pylori c. scherer (essen, d) objectives: the cytotoxin associated gene (caga) of h. pylori is a frequently discussed virulence factor which codes for a 128-kda cytotoxin associated antigen (caga). the caga-gene is usually detected by pcr methods. publications about phenotypic methods for the detection of caga are rare. aim of our investigation was to compare a slightly modified, recently published indirect immunofluorescence test (iift) for the detection of caga with commonly used pcr assays. methods: h. pylori atcc 43504 (caga+/caga+) was used as positive control strain, atcc 51932 (caga+/caga+) as negative control strain. nineteen clinical isolates of h. pylori were examined for their caga-and caga-status. caga was detected by iift using monoclonal anti-caga-antibody from mice, which were visualised for immunofluorescence microscopy with fitc-conjugated anti-mouse-antibody from rabbits. dna was extracted by a standard phenol-chlorophorm procedure after proteinase k treatment of the cells from a 4-day old culture. 7 primer pairs and their published protocols were used: f1/b1 and d008/r008 as the most described primer pairs and further five primer pairs. all tests were performed in duplicate. results: 4 from 19 (22%) strains were caga positive in the iift, and also caga positive in each pcr. 13 from 19 (68%) strains were negative in the iift. 10 from these 13 were caga-negative in each pcr and 3 showed varying results in the pcr-assays. 2 from 19 ( 10%) strains could not be interpreted by iift, both showed varying results in the pcr-assays. the iift might be a simple to use tool in the determination of the caga-status, since it showed no false positive result in neither tested pcr. the differing results in iift and pcr suggest that genotypic positive strains may not always express caga in vitro and maybe also not in vivo. therefore the caga-iift may be an interesting parameter for the determination of strain virulence in addition to pcr-assays. evaluation of elisa serology for the primary diagnosis of helicobacter pylori infection in turkish dyspeptic patients: a prospective study from a developing country b. kocazeybek, y. erzin, s. altun, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: hp is recognized as an important human pathogen by virtue of it's association with peptic ulcer disease, gastric cancer and gastric lymphoma and the high prevalence of infection worldwide. both invasive and noninvasive tests are available to diagnose hp infection but there is still no single gold standard. the aim of the present study was to evaluate the diagnostic accuracy of a commercially available anti-hp igg elisa kit for the primary diagnosis of hp infection. materials and methods: a total of 185 patients who were referred to our endoscopy unit were included. according to our gold standard, a patient was classified as being hp(+) if the culture and/or both histology, rapid urease test were positive and as hp()) only if all of these tests remained negative. standard methods were used to calculate sensitivity, specificity, predictive values of positive and negative results and 95% confidence intervals of these values. results: after excluding patients with discordant results131 of 152 patients (86%) were hp (+). the sensitivity, specificity and diagnostic accuracy of the igg-elisa were 97%, 67%, 93% respectively. conclusion: due to it's lack for specificity, we conclude that quantitative anti-hp elisa test may not be a suitable alternative for primary diagnosis of hp in our country, where the prevalence of infection is still very high. comparison of two different stool antigen tests for the primary diagnosis of helicobacter pylori infection in turkish dyspeptic patients b. kocazeybek, y. erzin, s. altun, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: to assess the reliability of two different enzyme immunoassays in detecting the helicobacter pylori(hp) status in stool specimens of turkish dyspeptic patients. materials and methods: 151 patients [74 with non-ulcer dyspepsia(nud), 64 with duodenal ulcer(du),13 with gastric cancer]who were admitted to the endoscopy unit of istanbul university, cerrahpasa medical faculty for upper gastrointestinal endoscopy due to dyspepsia were enrolled in the study. hp infection was confirmed in all patients by histology, rapid urease test(rut) and culture. a patient was classified as being hp-positive if the culture alone or both histology, rut were positive in the absence of a positive culture and as negative only if all of these tests remained negative. stool samples of patients were obtained in order to assess the reliability of a monoclonal (femtolab h. pylori, connex, martinsried, germany) and a polyclonal (premier platinum hpsa, meridian diagnostics inc., cincinnati, usa) stool antigen test and to compare their diagnostic accuracies. the v 2 test was used for statistical comparison of the values. results: using a cutoff 0.19 for femtolab h. pylori and 0.16 for premier platinum hpsa the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy of the former and latter tests were 93%, 90%, 98%, 68%, 93% and 84 %, 67%, 94%, 40%, 81% respectively. the sensitivity, specificity, npv and diagnostic accuracy of the former test were significantly superior to the latter one's (v 2 = 3.98; p < 0.05 for sensitivity and v 2 = 15.67; p = 0.000 for specificity, v 2 = 15.78; p = 0.000 for npv and v 2 = 6.37; p = 0.012 for diagnostic accuracy). the bacterial load did not affect the sensitivity of both tests. conclusions: the monoclonal femtolab h. pylori, using a cutoff 0.19 is a very sensitive, specific and easy to perform diagnostic tool for the primary diagnosis of hp infection in turkish, dyspeptic patients. evaluation of two different enzyme immunoassays for detection of helicobacter pylori in stool specimens of turkish dyspeptic patients after eradication therapy b. kocazeybek, y. erzin, s. altun, s. saribas, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: to assess the reliability of two different enzyme immunoassays(eias) in detecting the hp status in stool specimens of turkish, dyspeptic patients in the post-treatment period. materials and methods: 48 patients with non-ulcer dyspepsia(nud) who were positive for hp underwent a one week regimen of triple therapy. stool samples of patients were obtained 2-6 weeks and 13c-urea breath test(ubt) was performed 6 weeks after eradication therapy in order to assess the reliability of a monoclonal (femtolab h. pylori, connex, martinsried, germany) and a polyclonal (premier platinum hpsa, meridian diagnostics inc., cincinnati, usa) stool antigen test and to compare their diagnostic accuracies. the v 2 and fisher's exact tests were used for statistical comparison of the values. results: using a cutoff 0.19 for femtolab h. pylori and 0.16 for premier platinum hpsa the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy of the former and latter tests were 93%, 67%, 56%, 96%, 75% versus 87%, 33%, 37%, 85%, 50% 2 weeks and 93%, 88%, 78%, 97%, 90% versus 67%, 70%, 50%, 82%, 69% 6 weeks after completion of eradication therapy, respectively. both at the 2nd and 6th weeks in the post-treatment period the diagnostic accuracy of femtolab h. pylori was significantly superior to premier platinum's (75% versus 50%, v 2 = 6.4 ;p = 0.011, and 90% versus 69%, v 2 = 6.316; p = 0.012 respectively). conclusions: the monoclonal femtolab h. pylori, using a cutoff 0.19 is sensitive and specific enough to monitor hp infection in turkish, dyspeptic patients 6 weeks after completion of eradication therapy. carriers develop serious gastroduodenal diseases. so, it is important to define whether strains with specific genotype are associated with the clinical outcome. the aim of this study was to determine the distribution of the caga, cage and vaca subtypes of hp in patients with various gastroduodenal diseases in turkey, and to explore the association between genotype and the clinical outcome of infection. methods: 93 strains of hp were isolated from cultures and pathological archives of 30 patients with non-ulcer dyspepsia (nud), 30 with duodenal ulcer(du) and 33 with gastric carcinoma. the caga, cage and vacas1a/s1b, m1/m2 genotypes were determined by polymerase chain reaction methodology. results: there were no statistically significant difference among three different clinical outcomes according to the positive rates of vaca s1b, s2 and vaca m1/m2. but the positive rates of vaca s1a, caga and cage were 66.7% (20/30), 50% (15/30) and 26.7% (8/30) in nud; 93.3% (28/30), 83.3% (25/30) and 66.7% (20/30) in du; 87.9% (29/33), 84.8% (28/33) and 81.8% (27/33) in gastric cancer group, respectively, showing statistically significant difference (p = 0.015, p = 0.002, p = 0.001, respectively). the prevalence of vacas2 and m2 in nud patients was higher than that in duodenal ulcer group, but this difference was not statistically significant. conclusions: helicobacter pylori caga, cage and vacas1a genotypes have a significant relation with duodenal ulcer and gastric carcinoma in turkish population. there were no statistically significant association of the vaca s1b, s2 and vaca m1/m2 strains among disease groups. clinical relevance of icea1, icea2 and baba2 genotypes of helicobacter pylori in turkish patients with non-ulcer dyspepsia, duodenal ulcer and gastric cancer b. kocazeybek, y. erzin, v. koksal, s. erdamar, s. altun, a. dobrucali, a. oner (istanbul, tr) objectives: the clinical relevance of the helicobacter pylori(hp) adherence factor, baba gene, and the other virulence determinant, icea gene, has not yet been determined in turkish clinical isolates to date. therefore, the aim of this study was to evaluate the prevalence of icea1, icea2 and baba2 hp genotypes in turkish patients with non-ulcer dyspepsia(nud), duodenal ulcer(du) and gastric cancer. methods: 30 strains were isolated from patients with nud, 30 were isolated from individuals with du and 33 were isolated from patients with gastric cancer. bacterial dna was extracted from cultures and hp positive paraffin-embedded biopsy samples and genotyping of icea and baba2 was carried out utilizing polymerase chain reaction molecular technique by using specific primers. results: the icea1, icea2 and baba2 genotypes were detected in 75.3% (70/93), 24.7% (23/93), 53.8% (50/93) respectively, of the hp strains studied. the prevalence of icea1 in gastric cancer patients was higher than that in nud group, 84.8% (28/33) and 60% (18/30), respectively (p = 0.045). the prevalence of icea2 in nud group was higher than that gastric cancer patients, 40% (12/30) and 15.2% (5/33), respectively (p = 0.045). the positive rates of baba2 were 23.3% (7/30) in nud; 46.7% (14/30) in du and 87.9% (29/33) in gastric cancer group, showing statistically significant difference (p = 0.0001). conclusions: baba2 genotype distribution was evaluated in different gastric pathologies in turkish population and was a good marker for the presence of du and gastric adenocarcinoma. thus, our current results confirm previous experimental studies indicating a central role of hp's adhesin and lewis antigens in the pathogenesis of ulcer disease and adenocarcinoma. influences of interleukin 1b and interleukin 1rn polymorphisms on the development of non-ulcer dyspepsia, duodenal ulcer and gastric cancer in turkish population b. kocazeybek, y. erzin, v. koksal, s. erdamar, s. altun, a. dobrucali (istanbul, tr) objectives: il-1b and il-1rn gene polymorphisms (which encode interleukin 1b and interleukin-1 receptor antagonist, respectively) have been associated with development of gastric atrophy and with increased risk of gastric carcinoma. the aim of this study was to determine the influences of host il-1b -31tc, il-1b -511ct and il-1b rn gene polymorphisms on the development of various gastroduodenal pathologies in 93 helicobacter pylori (hp) positive turkish patients [(30 with nonulcer dyspepsia (nud), 30 with duodenal ulcer (du) and 33 with gastric cancer (gc)]. methods: genomic dna was extracted from hp positive paraffin embedded gastric biopsy samples of 30 nud, 30 du and 33 gc patients. il-1b and il-1b rn gene polymorphisms were analysed by polymerase chain reaction-restriction fragment length polymorphism. these polymorphic sites include promoter regions of il-1b at positions-511 (c-t transition) and -31 (t-c transition), and il-1 rn variable tandem repeats (intron2 vntr). results: there were no statistically significant difference among three clinical outcomes in the frequencies of il-1b -511ct + tt and il-1b -31 tc + cc genotypes (as called proinflamatory genotypes) (p = 0.161 and p = 0.077, respectively). the prevalence of the proinflamatory il-1b rn 1/2 + 2/ 2 alleles in gc group was higher than that in nud and du groups. the frequencies of il-1b rn 1/2 + 2/2 alleles were 20.0% (6/30) in nud; 31.0% (9/30) in du; and 57.6% (19/33) in gc group, showing statistically significant differences (p = 0.006). conclusions: there were no statistically significant difference among three clinical outcomes in the frequencies of il-1b -511ct + tt and il-1b -31 tc + cc genotypes, whereas proinflamatory il-1b rn *2 allele (1/2 + 2/2) was associated with gastric cancer in turkey. rapid detection of clarithromycin-resistant helicobacter pylori in patients with dyspeptic by fluorescent in situ hybridisation (fish), in comparison with e-test m. moosavian, s. tajbakhsh, a. samarbafzadeh (ahwaz, ir) objectives: isolation of h. pylori from more than 90% of the patients with peptic ulcer have demonstrated a strong relationship between these bacteria and created ulcers or dyspeptic. recovery of the patients will be accelerated, if clarithromycin be added to therapeutic protocol. the objectives of this study are: 1-rapid detection of the susceptible or resistant strains of helicobacter pylori to clarithromycin, in patients with dyspeptic by fish technique. 2-comparison of the results of fish & epsilometer test ( e-test) techniques. methods: frozen -sections of gastric biopsies of 50 patients with dyspeptic were hybridized in situ by 5 fluorescent oligonucleotide probes (fish). the prepared slides were examined under a fluorescent microscope, after staining with dapi. also, susceptibility and resistance of isolated strains of h. pylori to clarithromycin were determined by e-test. both results of e-test and fish techniques were compared in final. results: 25 out of 50 examined gastric biopsy samples were positive for h. pylori by fish. out of these 25 h. pylori strains, 17 strains (68%) were susceptible, 6 strains (24%) were resistant and 2 strains (8%) were mixture of susceptible and resistance strains to clarithromycin. this study showed no significant different between fish and e-test results, in view of number in susceptible or resistance strains. conclusions: since the patient gastric biopsies are not routinely cultured in the most of clinical laboratories, also, for isolation of h. pylori, it is needed to enriched and selective media and the other way, clarithromycin is an expensive drug, so, it looks like that fish technique is a suitable method for replacing of the culture, antibiotic sensitivity test and or e-test method. diagnosis of atrophic gastritis from a blood sample p. douramani, s. mavrea, a. arkas, a. adamopoulos, e. anastasakou (athens, paros, gr) introduction: the majority of gastritis is related to infectious agents, where h. pylori is the most important and common. h. pylori gastritis could proceed, over time, to atrophic form of gastritis a serious disease that increases the risk of peptic ulcers and gastric cancer. a new test panel was developed for nonendoscopic diagnosis of atrophic antrum and corpus gastritis from a blood sample. aim: to investigate whether atrophic gastritis can be diagnosed and typed non-endoscopically if the serum levels of pepsinogen i (s-pgi) and gastrin-17 are assayed in connection with h. pylori testing. materials and methods: the study population consists of 50 selected dyspeptic outpatients with (cases) or without (controls) advanced (moderate or severe atrophic gastritis) who underwent a diagnostic gastroscopy for dyspeptic symptoms. of the 50 selected patients,28 (cases) had an advanced atrophic gastritis or a resected stomach. of these 28 cases, 4 had an advanced antrum-limited atrophic gastritis, 6 had resected antrum,15 had a corpus limited advanced atrophic gastritis. two patients had an advanced atrophic gastritis in both the antrum and corpus (multofocal atrophic gastritis) and the whole stomach was removed in one patient. the controls comprise 22 patients of whom 10 had a non atrophic h. pylori gastritis; the antrum and corpus were normal and healthy in 12 patients. the sample for ôpostprandialõ gastrin -17 (s-g-17 prand) was taken 20 min after a protein drink. the s-g-17,s-pgi and igg class antibodies to h. pylori were determined using elisa methods. results: a low s-pgi (<25 mg/l) was found in 15 of 18 patients (83%) with and in 2 of 32 patients (6%) without corpus atrophy. a low s-g-17 prand (<5 pmol/l) was found in all 4 patients with h. pylori associated antral-atrophy and in 5 of 7 patients (71%) with resected antrum but in one of 10 patients (10%) with h. pylori-related non-atrophic gastritis. the mean values of both s-g-17 prand and s-pgi decreased with increasing grade of antral and corpus atrophy respectively. among all patients with atrophic gastritis,24 of 28 patients (86%) had a low s-pgi and/or a low s-g-17 prand with positive igg h. pylori antibodies. such low values were found only in one of the 22 (4.6%) control patients. conclusions: low serum levels of g-17 and s-pgi are diagnostic markers of atrophic antral and corpus gastritis, respectively. a low s-g-17prand is a sigh of the multifocal or antrum-limited atrophic gastritis in patients infected with h. pylori. helicobacter pylori in gastric mucosa: an ultrastructural study e. ozbek, a. ozbek, h. dursun, o. vuraler (erzurum, tr) objectives: helicobacter pylori are extraordinary among bacteria in their ability to colonize the human gastric mucosa, an inhospitable acidic environment, and to stay on there for long periods as decades in despite of host immune and inflammatory responses. in this study, we aimed to research where h. pylori are found within the stomach by transmission electron microscopy (tem). methods: gastroscopic biopsy samples from patients suspected with gastritis or peptic ulcer were processed for both of pcr and tem. the presence of h. pylori in biopsy samples was investigated with pcr method by using specific 23 s ribosomal rna primers. the samples for electron microscopic examination were fixed in 3% glutaraldehyde, postfixed in 1% osmic acid, dehydrated in acetone, and embedded in araldite. plastic blocks of positive samples for the presence of h. pylori confirmed by pcr were cut with a nova lkb bromma ultratome. thin sections were stained with uranyl acetate and lead citrate, and examined by using a jeol 100 sx transmission electron microscope. results: we observed that cross-and longitudinal-sectioned h. pylori within the mucus layer overlying gastric epithelium, in the intercellular spaces between gastric epithelial cells (figure 1), and between two microvilli protruding from the apical surface of an epithelial cell into the gastric lumen. apart from the extracellular h. pylori, we found also intravacuolar h. pylori within the epithelial cells of the gastric mucosa. we observed h. pylori engulfed by pseudopod-like structures at the apical part of the epithelial cells. there were also late endosomal vacuoles containing h. pylori within the deeper cytoplasmic parts of the cell. conclusion: although h. pylori is considered generally a noninvasive pathogen, our electron microscopic observations prove that h. pylori is able to invade gastric epithelial cells, and to enter large cytoplasmic vacuoles. that h. pylori is able to be found intracellularly might explain why the eradication of h. pylori infection is difficult with the antibiotics such as gentamicin that cannot easily cross the eukaryotic cell membrane. abbreviations for figure 1: h, helicobacter pylori; n, nucleus of a gastric epithelial cell; e, cross-sectioned epithelial cells; nm, nuclear membrane; is, intercellular space. bar = 0.5 micrometre helicobacter pylori and association with histological findings in endoscopic biopsies in western greece c. petropoulos, e. jelastopulu, m. kardara, s. spiropoulos (erymanthia, patras, gr) objectives: there is evidence that helicobacter pylori (h. pylori) infection is strongly associated with gastric and duodenal lesions in general population. in this study we reviewed gastroscopic and histological records of patients who underwent gastroscopy for multiple reasons in a tertiary hospital in order to evaluate the frequency of h. pylori colonization in biopsy specimens and to examine the association between h. pylori infection and histological findings. methods: the medical records of 251 patients who presented to the general hospital ôagios andreasõ in patras, western greece, during the period 2002-03 for upper gastrointestinal complaints and other specified symptoms were reviewed for the presence of h. pylori. all patients have been submitted to endoscopy with biopsy mainly of the gastric mucosa. the statistical analysis was performed using the spss. results: gastroscopies and biopsies were undertaken in 251 patients (149 men, 102 women; mean age 58.5, range 16-95 years). in 140 (59.1%) of the patients dyspepsia was the main indication for gastroscopy, followed by 44 (18.6%) patients with anaemia, 12 (5.1%) patients with black faeces and 11 (4.6%) patients with reflux. usual macroscopic findings were oedema (70%), hyperaemia (50%), erythema (22.3%), ulcer (16.8%) and erosions (7.5%). the histological examination revealed chronic gastritis in 209 (83.3%) subjects (in 39% of mild form, in 50% of moderate and in 11% of severe form), atrophic chronic gastritis in 8 (3.1%) and adenocarcinoma in 10 (3.9%) patients. the overall colonization of h. pylori was detected in 80 (34.2%) subjects. in case of h. pylori positive patients, chronic gastritis was found in 15.4% of mild form, in 61.5% of moderate and in 23.1% of severe form, whereas in absence of h. pylori infection the histological type of chronic gastritis was 52.5%, 43.2% and 4.3% respectively. in case of chronic gastritis of severe form h. pylori colonization was found in 75% (p < 0.001), whereas regarding the mild form h. pylori presence was observed only in 14% (p < 0.001). conclusions: the study reveals that h. pylori infection is strongly associated with the severe form of chronic gastritis. thus, for the optimal management and treatment of the patients with gastritis a biopsy of the mucosa appears to be necessary. objective: the ultimate aim of the study is to explore potential natural products as alternative for treatment of infections from staphylococcus aureus including methicillin resistant staphylococcus aureus (mrsa). the specific objective of the paper is to assay for molecular changes in the genes of mrsa isolates upon inhibition by natural products. five genes of mrsa isolates study include meca, meci and mecri adab and sav1017 and the non-mrsa isolates were adab and sav1017 genes. the adab gene encodes for methyltransferase activity for dna repair mechanism while the sav1017 is the cell wall protein gene. methods: mrsa and non-mrsa isolates were obtained from patients receiving treatment in malaysian hospitals. these isolates were subjected to growth in methanol extract of marine seaweed. inhibition assay of extract on isolates including several genera of the gram-negative bacteria, were determined by the disc diffusion and minimum inhibitory (mic) methods. treated and untreated isolates inhibited by extract were subjected to pcr amplification, followed by commercial sequencing, rt-pcr assay of the mrna followed by sequencing of the cdna and blastn analysis with the genbank sequences. results: the inhibition assay of methanol extract showed activity only in mrsa and non-mrsa isolates but not in gram negative isolates. the nucleotide sequences changes were seen in four genes of treated mrsa isolates which were the meca, mecri, meci and the adab. the nucleotide changes in non-mrsa and mrsa were only seen in the adab gene but not the sav1017. the blastn analysis showed variation in nucleotide changes in all genes involved in mrsa phenomenon. conclusions: these preliminary results utilizing genomics for study on nucleotide sequences of pathogen can aim at utilizing the biomolecules from natural products to target the affected nucleotides so as to inhibit growth of the organism. the research activity has the potential of speeding up drug discovery programme and the nucleotide changes in several genes treated with the extract indicates the potential target sites of the extracts. the inhibitory effect of extract on the nucleotide of some genes remained unchanged in the pcr and rt_pcr assay, indicating selective effect of extracts on the genes and the extracts has the potential to be applied as antibacterial agents. implication of the findings is directed towards discovery of antibacterial drug target sites, which warrants further study. phenotypic and genotypic characteristics of staphylococcus aureus strains defective in species-specific proteins a. luczak, j. krzyszton-russjan, k. nowak, w. hryniewicz (warsaw, pl) objectives: the objective of this study was to characterise phenotypically clumping factor and/or coagulase defective s. aureus. methods: the study was performed on 170 s. aureus isolates identified between 1996 and 2004 as clumping factor (cf) or free coagulase (fc) defective by clinical microbiology laboratories and submitted to national institute of public health in warsaw for verification. reidentification included detection of clumping factor, coagulase, thermonuclease, and ability to ferment mannitol. antimicrobial susceptibility testing was performed by diskdiffusion method according to the nccls. glycopeptides susceptibility was determined by screening method, mic evaluation and population analysis. pcr reactions were performed to confirm the presence of species specific genes, as cfa, cfb, coa, nuc, spa. to obtain isolates clonality, multiple-locus variable-number tandem repeat analysis (mlva) was applied. results: based on the phenotypic reidentification methods, 114 (67%) isolates were phenotypically defective in respect to clumping-factor or coagulase production. all but one isolate exhibited the presence of the genes encoding cf or fc. seventyfive (67%) isolates were resistant to methicilin (mrsa) and multiresistant. twenty-six (22.8%) isolates showed reduced glycopeptides susceptibility in pap. all hvisa and hgisa isolates were mrsa and all were defective mainly in clumpingfactor production. thirty-one different mlva groups were distinguished. the a and l groups were the most common and variable. the a type was the most prevalent in hvisa/hgisa isolates and found in 18 centres. conclusion: the clonal spread of s. aureus defective in species specific proteins was revealed. the correlation between decreased susceptibility to glycopeptides and defective species specific proteins was observed. detection of panton-valentine leucocidin and other staphylococcal toxins using oligonucleotide arrays s. monecke, r. ehricht (dresden, jena, d) objectives: the recent outbreaks of community acquired mrsa (cmrsa) harbouring panton-valentine leucocidin (pvl) caused serious concern worldwide. in order to screen clinical isolates, we designed and tested an oligonucleotide array which can detect both components of this virulence factor, six staphylococcal superantigens and the two exfoliative toxins as well as several resistance genes and species-specific controls. methods: twenty-two clinical isolates (two suspected cmrsa and twenty randomly selected) from swabs obtained from a dermatological clinic in saxony/germany were cultured. dna was isolated and subjected to a linear multiplex amplification incorporating biotin-labeled dutp into the amplicon. hybridization of the amplicons to the array was detected using an enzymatic precipitation reaction. results: in two cases of chronical furunculosis, pvl-positive cmrsa were identified. one case was a soldier returning from a peace mission to kosovo where he apparently contracted the disease, the other one was his spouse. in three other cases of furunculosis/abscesses, pvl-positive but methicillin-susceptible s. aureus were found. staphylococcal enterotoxins were found in four, toxic shock syndrome toxin in three isolates, and exfoliative toxin a in one isolate. in two cases, multiple toxins were found (pvl + tst + entc and entb + entk + entq + eta). conclusions: while epidemic cmrsa strains often can presumably be identified due to their resistance profile (methicillin, tetracycline, fusidic acid), there is no easy method to detect pvlpositive, methicillin-susceptible s. aureus. therefore, strains carrying pvl are more common than previously suspected. the high prevalence of this virulence factor as well as of other toxins found in the present study warrants further investigations. characterisation of methicillin-resistant staphylococcus aureus harbouring the panton-valentine leucocidine a. anders, m. kaase, s. friedrich, s.g. gatermann (bochum, d) objectives: until now methicillin-resistant staphylococcus aureus (mrsa) have been known only as a major problem in hospitals. lately, a new kind of mrsa has been described. this mrsa is responsible for community-acquired infections (c-mrsa) and harbours the determinant for the panton-valentine leucocidine (pvl).as there are descriptions of c-mrsa possessing the pvl throughout the world with different mlst types we wanted to characterize eleven c-mrsa from our area possessing the gene for panton-valentine leucocidine. methods: the isolates were found from 1999 until the summer of 2004, the gene encoding pvl was verified by pcr.we determined the pfge pattern, the mlst type, the sccmec type and the agr allele type of the eleven isolates. results: so far all isolates belong to the st 80 which has been also described elsewhere in germany and in france but differs from the types found in the us (1 and 8) or in australia (30 and 298). they possess a sccmec element of type ivb and the agr3 allele type. all isolates were resistant to oxacillin, which was determined by meca pcr, and all were resistant to fusidic acid. only two were additionally resistant to erythromycin. no resistance could be found to fluoroquinolones.the average age of patients was 47 years and they all had severe communityacquired abscesses of the skin or soft tissue that caused hospital admission. conclusion: mrsa cannot be longer considered as affecting only hospitalized and elderly patients but we should also be aware of them in community-acquired infections. accessory gene regulator (agr), and its locus of s. aureus is a quorum-sensing gene cluster of five genes (hld, agrb, agrd, agrc, and agra) that upregulates production of secreted virulence factors, including the alpha-, beta-, delta-hemolysins, and downregulates production of cell-associated virulence factors. s. aureus strains can be divided into 4 major agr groups (agr i-iv) on the basis of a polymorphism in agrd and agrc. the purpose of this study is to know the proportion of agr i, ii, and iii polymorphisms and to compare clinical characteristics between group ii and non-group ii polymorphism of methicillin-resistant staphylococcus aureus (mrsa) strains in a tertiary care teaching hospital in korea. methods: isolates were identified as s. aureus by conventional methods. susceptibility to methicillin was determined by the nccls guideline. agr locus was identified using restriction fragment length polymorphisms (rflps) analysis of agr bdc amplicons with drai digestion. the factors assessed included the patients' demographics, comorbidities (diabetes mellitus, congestive heart failure, peripheral vascular disease, dialysisdependent renal failure, cirrhosis, malignancy, and alcoholism), infection site (central catheter-related bacteraemia, bacteraemia of unknown origin, device, endocarditis, intraabdominal, respiratory, skin, urine, and ear), receipt of mechanical ventilation and operation, the presence of nosocomial infection and treatment failure, creatinine level, and mortality. results: a total of 18 strains from 18 mrsa-infected patients (7 males and 11 females) were evaluated. their mean age was 50.5 ± 24.3 years old. there were 4 (22.2%), 12 (66.7%), and 2 (11.1%) strains in agr group i, ii, and iii polymorphism, respectively. only nosocomial infections were statistically significant clinical parameter according to univariate analysis (p = 0.009) and multivariate analysis (or, 22.0 (1.540 ) 314.292); p, 0.023). conclusion: agr group ii was most prevalent in this study and nosocomial infections were correlated with group ii polymorphism. this result suggests virulence and nosocomial spread of mrsa strains are originated from group ii polymorphism. a recruit of more strains will increase and clarify the clinical difference between agr groups. distribution of toxic shock syndrome toxin-1, exfoliative toxins and enterotoxins seg and sei among methicillin-resistant staphylococcus aureus clonal types v. chini, g. dimitracopoulos, i. spiliopoulou (patras, gr) objectives: staphylococcus aureus produces a variety of exotoxins including the toxic shock syndrome toxin-1 (tsst-1), the exfoliative toxins eta and etb and the enterotoxins seg and sei. the seg and sei genes (seg and sei) coexist in the same operon. the aim of this study was to investigate the presence of genes encoding the tsst-1 (tst), eta (eta), etb (etb), seg (seg) and sei (sei) among methicillin-resistant s. aureus (mrsa) clinical isolates, in relation to their clonal types. methods: the mic of oxacillin was determined by the agar dilution method in mueller-hinton agar supplemented with 2% nacl according to the guidelines of nccls. pbp2a production was investigated by a latex agglutination test (biomerieux) in all s. aureus isolates with mic >0.5 mg/l. pcr amplification was performed for the detection of meca gene to the aforementioned isolates, from different patients, during 2001-2003, resulting to 160 mrsa. the presence of tst, eta, etb, seg and sei genes was also investigated by pcr. clonal types were determined by pfge of chromosomal dna smai digests. results: among the 160 mrsa, 35 (22%) carried both seg and sei genes, 24 (15%) isolates carried the tst, 2 (1%) the eta, whereas no strains exhibited the etb gene. precisely, during 2001 the tst gene coexisted in 12 out of 13 seg/sei-positive strains. eleven strains belonged to pfge type a, whereas the other two to type b, mainly associated with wound infections. eight mrsa in 2002 belonging to pfge type a, carried tst, seg and sei. two more strains carrying seg/sei, belonged to pfge type c. two out of 3 seg/sei-positive additional mrsa which were characterized as a new clone f, carried also eta. in 2003, four mrsa carrying the tst, seg and sei belonged to clone a, whereas 5 more carrying the seg/sei were characterized as pfge type c. most isolates were associated with wound infections and abscesses. conclusions: during the study period (2001) (2002) (2003) the incidence of the tst gene carriage among mrsa was reduced from 26% to 6%, while the seg/sei genes were almost constantly present. the distribution of these genes was mainly associated with strains of clonal type a, exhibiting a drift towards clone type c predominant during the last two years. detection of decreased susceptibility to glycopeptides in staphylococcus aureus using tablet (disc) prediffusion s.v. nielsen, j.b. casals (taastrup, dk) objectives: the aim of the study was to develop a screening and confirmatory method to detect staphylococcus aureus with decreased susceptibility to glycopeptides (hvisa/visa and gisa). methods: the glycopeptide resistance of 20 staphylococcus aureus strains from our strain collection including well-known resistant strains (atcc 700788, atcc 700789, atcc 700698, atcc 700699) was examined. we compared: (i) zone sizes of vancomycin 5 lg and teicoplanin 30 lg neo-sensitabs (rosco diagnostica) when prediffused for 2 h (with disc) + 18 h (without disc) on mueller-hinton and bhi, both media supplemented with 5% horse blood, (ii) agar dilution mics (iii) zone sizes around cefoxitin 60 lg neo-sensitabs. the inoculum used was mcfarland 0.5 and the plates were incubated for 24 h at 35°c. results: regression lines were prepared and there was a good correlation between mics for vancomycin and teicoplanin and the corresponding zone sizes. all hvisa/visa/gisa strains tested showed zones less than 15 mm with cefoxitin 60 lg and zones £ 21 mm (teicoplanin) and/or £ 23 mm (vancomycin) on mh blood agar, corresponding to mics > 2 lg/ml for both antimicrobials. on bhi agar the zones were £ 17 mm (teicoplanin) and/or £ 21 mm (vancomycin) for the hvisa/visa/gisa strains corresponding to mics > 4 lg/ml for both antimicrobials. conclusion: zones obtained by agar diffusion methods using prediffusion with vancomycin and teicoplanin neo-sensitabs correlated with mics and predicted hvisa/visa/gisa strains. for screening high resistance to cefoxitin (zones <15 mm) may be used to select strains for further testing in a daily routine laboratory. the system should be evaluated further in the routine diagnostic laboratory. high rate of vancomycin intermediate staphylococcus aureus among methicillinresistant isolates in taiwan from 637 patients in the tri-service general hospital in taiwan were screened for vancomycin resistance and isolates with reduced susceptibility to vancomycin were further studied for their characteristics. methods: brain heart infusion agar plates containing vancomycin (5 lg/ml) (bhia-va5) were using for vancomycin resistance screening. minimum inhibitory concentration (mic) of vancomycin was determined on both bhia and the standard mueller-hinton agar (mha) on screened-positive isolates. for strains with mics equaled to 8 lg/ml, characteristics including population analysis, susceptibility to triton x-100 induced autolysis, van gene and pulsed field gel electrophoresis (pfge) typing were further performed as previously described. background: mrsa isolates with decreased susceptibility to glycopeptides (gisa) have been reported worldwide since 1997. most strains were isolated from catheters and other foreign bodies in patients treated with glycopeptides. we aimed to determine the prevalence of gisa isolates in a belgian hospital where mrsa has been endemic for many years. methods: all mrsa isolates collected between 09/98 and 06/ 04 were screened for reduced susceptibility to glycopeptides on mueller-hinton agar and 5 mg/l teicoplanin (mh-teico) as recommended by the casfm. the macromethod etest (bhi agar, 2 mcfarland (mf) inoculum, 48 h incubation) was performed as secondary screening for all isolates which grew 4 colonies on mh-teico. gisa/h-gisa phenotypes were confirmed by e-test mic determination on mh agar with 0.5 mf and 24 h incubation as well as by vancomycin (v) and teicoplanin (t) population analysis profiles (pap). all confirmed gisa/h-gisa isolates identified were compared by pfge to other strains identified in belgium in order to delineate their clonality. results: among 520 mrsa strains (from 468 patients), 14 isolates showed growth on mh-teico and 8 of these (from 7 patients) yielded a positive macromethod e-test (mic 8 lg/ml for v and t). seven were confirmed by pap as h-gisa and one as a gisa (t mic: 32 lg/ml; v mic: 8 lg/ml). origins of the isolates were: lower respiratory tract (4), urine (2) and wound (2). all h-gisa strains had a similar antibiotic resistance phenotype by disk diffusion (gentamicin, rifampin, erythromycin, clindamycin, ofloxacin) . by molecular analysis, the h-gisa/ gisa isolates clustered in two different pfge groups a (n = 6) and g (n = 1). the a pfge group was previously described in h-gisa isolates in belgium. in this study, it was subdivided in 2 types, both including 3 strains. one pfge type resulted in a small outbreak in 3 patients during a stay in icu. all 5 other isolates were sporadic with no apparent geographic or temporal relationship between cases. conclusion: gisa/h-gisa were found in only 7 out of 468 (1.5%) mrsa-positive patients. none of them presented with severe infection nor had any previous known exposure to glycopeptides. in view of the low prevalence of gisa/h-gisa in our hospital, screening should not be performed systematically but rather on an individual basis taking into account clinical data and risk factors. whenever the occurrence of gisa is considered, teico-mh agar appears as a suitable first screening approach. a case-control study to evaluate the economic outcome of early po linezolid in patients with methicillin-resistant staphylococcus aureus infections oral therapy with linezolid (lzd) has the potential to decrease hospital costs and shorten length of stay of patients with grampositive infections who would otherwise continue to receive iv therapy. objective: to evaluate the impact of early po conversion to lzd in patients receiving traditional iv therapy for methicillinresistant s. aureus (mrsa) infections. methods: patients with documented mrsa infection between 2001 and 2004 were evaluated in a matched case-control study. cases included patients who were converted to po lzd from iv table 1 includes comparisons of cost, duration of therapy and los. predictably, patients receiving lzd in the hospital had higher drug costs; however, a decreased los contributed to a $7000 lower cost of hospitalization. lzd or iv vancomycin (vanc) , and controls were patients who received only iv vanc. patients were paired in a 1:2 ratio of cases to controls and matched based on infection type, age and comorbidities. demographics, antimicrobial agents, concomitant infections, and clinical status were assessed daily from the onset of infection through the end of treatment or hospitalization. antibiotic cost, length of stay (los), and clinical success rates were compared between the groups. results: 78 patients were assessed. demographic characteristics were similar between groups. average age (mean ± sd) was 46 ± 14; apache ii (median, range) was 6 , charlson comorbidity score 2 [0] [1] [2] [3] [4] [5] [6] [7] [8] , and 60% were male. 55% of patients were treated for skin/soft tissue infection, 19% pneumonia, 13% bone/joint infections, 11% bacteraemia, and 2% other infections. clinical and microbiologic outcomes were similar between groups, with 100% clinical success at the end of therapy. objectives: to compare the efficacy and safety of linezolid (lzd) with those of vancomycin (van) for the treatment of infections caused by methicillin-resistant staphylococcus aureus (mrsa) in japan. methods: this was a randomized, open-label study conducted in japan. patients with nosocomial pneumonia, complicated skin and soft tissue infections, or sepsis syndrome, caused by mrsa, were randomized (2:1 lzd: van) to receive lzd, 600 mg q12 h, or van, 1 g q12 h. outcomes were evaluated at end of therapy (eot) and at a follow-up (fu) evaluation 7-14 days after completion of therapy. results: 151 patients received study drug (100 lzd, 51 van); the treatment groups were similar with regards to demographics and risk factors. at eot, clinical success rates in the mrsa-microbiologically evaluable population were 62.9% and 50.0% for the lzd and van groups, respectively; microbiologic eradication rates were 79.0% and 30.0% in the two groups, respectively (p < 0.0001). at fu, the clinical success rates were 36.7% for both groups, and the microbiologic eradication rates were 46.8%, and 36.7%, respectively. clinical success rates in both groups reflect an automatic outcome of failure for patients receiving prohibited antimicrobials prior to fu. reversible anemia (13%) and thrombocyotopenia (19%) were reported as adverse events more frequently in lzd patients. analysis of laboratory data showed that platelet counts decreased more frequently in lzd patients with recovery by fu. the mean platelet count in lzd patients with an adverse event of thrombocytopenia was 101,000/mm 3 . significantly low platelet counts (<50,000/mm 3 ) were more frequently observed in van patients (6 % vs 3%). no difference was observed in mean changes in hemoglobin levels between the treatment groups. conclusions: lzd is as effective as van for the treatment of mrsa infections in seriously ill patients, and may be more effective in achieving microbiologic eradication. although hematologic adverse events were reported more frequently in lzd-treated patients, analysis of laboratory data showed only a mild reversible trend toward lower platelet counts. cost-effectiveness of linezolid versus vancomycin in complicated skin and soft-tissue infection due to suspected methicillin-resistant staphylococcus aureus infection in germany -12, 2003; san diego, calif) . the objective of the present analysis was to evaluate the cost-effectiveness of linezolid compared with vancomycin in the treatment of cssti due to suspected mrsa infection from the german perspective. methods: a decision-analytic model was developed to examine the costs and outcomes of using linezolid versus vancomycin in hospitalized patients with cssti in germany. an expert panel of german physicians experienced in treating cssti provided resource-utilization data through structured interviews. costs from published sources (rote liste, dkg-nt, ebm) were applied to clinical and laboratory tests, adverse events, isolation procedures, intravenous (iv) or oral (linezolid only) drug treatment, hospitalization by ward type (medical, intensive care), and outpatient physician consultation. if appropriate, patients could be discharged from hospital and treated in an ambulant setting. the model assumed 50% of suspected mrsa patients had proven mrsa. outcomes included total costs per patient, cost per death avoided, cost per life-year gained, and cost per cure. results: an additional 4.4% of patients treated with linezolid (98.4%) versus vancomycin (94.1%) were cured. average total cost per episode was 9352 versus 9474 for linezolid-versus vancomycin-treated patients, giving a 121 saving per episode. the model was sensitive to the los in hospital, days in isolation, duration of oral versus iv treatment, percentage of mrsa patients, and price of linezolid. conclusion: in this decision analytic model, more patients with cssti due to suspected mrsa achieved clinical cure in the linezolid group compared with the vancomycin group. linezolid was cost saving versus vancomycin for the treatment of cssti. linezolid resulted in a shorter treatment duration and shorter los that offset the higher acquisition cost of linezolid versus vancomycin in cssti in germany. clinical microbiology and infection, volume 11, supplement 2, 2005 training, scientific output in infectious disease p874 a bibliometric analysis of worldwide trends in research productivity in microbiology p. vergidis, a. karavasiou, k. paraschakis, i. bliziotis, p. papastamataki, m. falagas (athens, gr) background: microbiology contributes significantly to the understanding and control of infectious diseases and has always been a field of extensive research. however, the literature lacks studies estimating the quantity and quality of worldwide research production. we evaluated the contribution of different world regions in research production in the field of microbiology. methods: using the medline database we retrieved articles from 64 journals included in the ômicrobiologyõ category of the ôjournal citation reportsõ database of the institute for scientific information for the period 1995-2002. the world was divided into 9 regions based on geographic, economic and scientific criteria. using an elaborate retrieval system we obtained data on published articles from different world regions. in our evaluation we introduced an estimate of both quantity and quality of research produced from each world region per year using: (1) the total number of publications, (2) the mean impact factor of publications, and (3) the product of the above two parameters. results: data on the country of origin of the research was available for 76,118 out of 77,080 retrieved articles (98.8%). western europe exceeds all other world regions in research production for the period studied, with usa ranking second (table). the difference in production between these two regions increased gradually from 1995 to 2002. however, the mean impact factor for articles published in microbiology journals was highest for the usa (3.34), while it was 2.79 for western europe and 2.31 for the rest of the world (7 regions combined). conclusions: usa and western europe make up a striking 78% of the world's research production in terms of both quantity and quality. all world regions have increased their research production during the period studied. the highest increase was achieved by asia (excluding japan), latin america, and eastern europe. from conference abstract to full paper: differences between data presented in conferences and journals e. rosmarakis, p. vergidis, a. kapaskelis, k. paraschakis, m. falagas (athens, gr) background: on some occasions, we noted differences between data presented in conference abstracts and subsequent published papers. we studied the frequency and the type of these differences in the fields of infectious diseases and microbiology. methods: we reviewed all abstracts from the first session of 7 out of 15 major research categories presented in the 1999 and 2000 icaac. for each selected pair of icaac abstract and related full paper published in journals indexed by index medicus, two independent investigators performed a comparison of all data in the abstract and the corresponding information in the published paper. using cox logistic regression models we analysed variables for association with differences of data. results: from 190 abstracts that were reviewed, 68 (35.8%) were subsequently published as papers by march 2004. from them, 52 referred to the same study period and population for both the abstract and the paper. differences between data presented in conference abstracts and published papers were found in 28 out of 51 pairs which were analysed further (54.9%, 95% c.i.: 41.2%-68.6%). the identified differences were related to the numbers and/or rates of the studied patients (11/28), numbers or rates of isolates (9/28), mics values or ki values (5/ 28), other chemical properties of antibiotics (2/28), odds ratio (1/28), and duration of observation (1/28). the differences were substantial in several pairs. time to publication of full paper was found to be independently associated with presence of differences (p = 0.029, or = 2.043 per year), while the research category, type of presentation (oral or poster), number of publications of the presenting and last author, impact factor of the journal, and country of origin were not. conclusions: while there are several explanations for the noted differences between data presented in conference abstracts and full papers, it is likely that the research community may improve the accuracy of presentation of data. worldwide trends in quantity and quality of published articles in the field of infectious diseases i. bliziotis, k. paraschakis, p. vergidis, a. karavasiou, a. kapaskelis, m. falagas (athens, gr) background: trying to confront with the widespread burden of infectious diseases, the society worldwide invests considerably on research. however, the literature lacks studies estimating the quantity and quality of worldwide research production. we evaluated the contribution of different world regions in research production in infectious diseases. divided into 9 regions based on geographic, economic and scientific criteria. using an elaborate retrieval system we obtained data on published articles from different world regions. in our evaluation we introduced an estimate of both quantity and quality of research produced from each world region per year using: (1) the total number of publications, (2) the mean impact factor of publications, and (3) the product of the above two parameters. results: data on the country of origin of the research was available for 45,232 out of 45,922 retrieved articles (98.5 %). usa and western europe are by far the most productive regions concerning publications of research articles, as displayed in the table. however, the rate of increase in the production of articles was higher in the developing world regions during the study period. the mean impact factor is highest for articles originating in the usa (3.42), while it was 2.82 for western europe and 2.73 for the rest of the world (7 regions combined). conclusions: usa and western europe make up a striking 80% of the world's research production in infectious diseases in terms of both quantity and quality. however, all world regions present a steady increase in the production of infectious disease articles with the developing countries displaying the highest rate of increase. . financial support was obtained from a pharmaceutical company. however, the sponsor had no role in organizing the scientific content of the programme. results: 146 physicians including id specialists were selected from several university hospitals or tertiary care government hospitals where running clinical trials has been a common practice. pre-training queries revealed that among the trainee population, 30.5% never obtained an informed consent from a patient, 47% did not randomize any patients for trials, 44% did not run any phase i-iv trials, 55% did not use placebo for trial purposes and 49% did not report any serious adverse events to local and or international authorities. a 24-question pre-and post-course tests about basic principles of gcp and local and eu regulations for running clinical trials were applied to determine and compare the knowledge of the trainees on these subjects prior and after the course. the percentage of correct answers ranged 35-91% before, and 51-98% after the course was completed (p < 0.01). conclusions: training in gcp and other regulations about running clinical trials may increase the awareness and knowledge of physicians. these educational activities will also help to raise the quality of clinical trials. learning appropriate use of antibiotics (pk/pd and guidelines): a cd-rom course for healthcare professionals and students e. ampe, y. glupczynski, p.m. tulkens, f. van bambeke (brussels, mont-godinne, b) objectives: in a context of growing resistance and limited supply of new molecules, a rational use of antibiotics should be a high priority. our objective is to train healthcare professionals and students in pk/pd and in a correct implementation of guidelines, since this could help to improve antibiotic use in both short and mid-terms. methods: we developed a pk/pd -guidelines course on cd-rom, targeted to both physicians and pharmacists but also usable by students. the course was prepared by a team of 2 pharmacists, 1 clinical microbiologist, and 1 pharmacologist. sources of information were (i) textbooks, review papers and primary papers by internationally recognized experts (ii) materials presented at training workshops of the international society for antiinfective pharmacology (isap; www.isap.org) during the last 3 years, (iii) national and, if not available, international guidelines for the management of respiratory tract or urinary tract infections. results: the course is organized as a series of power point presentations covering in a progressive fashion the followings topics: (1) bases in microbiology (in vitro properties of antibiotics); (2) pharmacokinetics (definition of the main parameters); (3) pharmacodynamics, with (a) the concepts, (b) the methods and pertinent models, and (c) the data, including the parameters to take into account to optimize the dosage of the main antibiotic classes; (4) resistance, including (a) the main mechanisms and (b) the use of pharmacodynamics to avoid the selection of resistance; (5) the appropriate use (including appropriate dosages) of antibiotics in (a) respiratory tract and (b) urinary tract infections. conclusions: this course promotes continuous education in the pharmacology and pharmacotherapy of antibiotics, in a format easily usable for courses and seminars to both students and professionals. background: although macrolides have no intrinsic antipseudomonal activity, these drugs appear effective in controlling infection by p. aeruginosa through mechanisms such as disruption of quorum sensing or anti-inflammatory effects. objectives: the aim of our prospective study was to evaluate the efficacy of macrolides in patients with bronchiectasis infected and colonised by p. aeruginosa. methods: the study included hospitalised patients with ct evidence of bronchiectasis and presenting an acute exacerbation by p. aeruginosa, isolated in sputum. microbiological findings, clinical data and treatment recommendations were recorded at admission and at the end of therapy. patients completed daily diary cards for symptoms and pef values. patients were followed for 1 year. results: twenty-two patients (14 men, mean age 57.3 ± 14.5) with bronchiectasis and p. aeruginosa infection were included. during hospital stay, patients were treated with at least two susceptible antipseudomonal drugs, according to the antibiogram (usually beta-lactam plus aminoglycoside) for a period of 16.9 ± 1.6 days. an oral macrolide (azithromycin 250 mg x 3 days/week in 10 patients, clarithromycin 500 mg daily in 12 patients) was further administrated for 4.9 ± 1.9 months. at the end of long-term therapy, 11 (50%) patients showed no evidence of p. aeruginosa in sputum, 8 (36.3%) patients still presented p. aeruginosa in sputum and 3 (13.6%) discontinued the treatment after less that 1 month because of adverse events. all patients had a significant reduction of sputum volume (83.3 ml/day before therapy vs. 22.5 ml/day at the end of therapy, p = 0.03) and of the mean number of exacerbations during follow-up (2.4 in the previous year vs. 1.2 in the follow-up year, p = 0.01). an increase of the mean pef value was also noted, although statistically not significant (373.2 ± 43.5 l/min before therapy vs. 489.2 ± 23.5 l/ min at the end of therapy, p = 0.3) conclusion: these observational findings suggest that macrolides may have a role in modulation of p. aeruginosa colonisation in patients with bronchiectasis. objectives: there is little information on cap requiring hospitalization in young adults (£ 40 years). we analysed clinical features, causative organisms, and outcomes of cap in this patient population. methods: prospective observational study of non-immunocompromised adult patients with cap (1995 cap ( -2003 . patients with hiv infection, transplantation, and neutropenia were not included. for the purposes of the study, patients were divided into 2 age-groups, £ 40 years and > 40 years. results: we documented 1732 consecutive patients with cap; 150 patients (8.7%) were £ 40 years and 1582 were > 40 years. mean patient ages were 30.3 years in the former group and 69.6 years in the latter group. main reasons for hospitalization in young adults were: hypoxia (po2 < 60) 59 patients, port high severity risk class (iv-v) 13 patients, empyema 9 patients, and/ or shock 7 patients. young patients were more frequently current smokers (63% vs 22%, p < 0.001). comorbid conditions were more common in older patients (20% vs 66%, p < 0.001), mainly diabetes mellitus, copd, and chronic heart disease. multilobar pneumonia was more frequent in young patients (56% vs 32%, p < 0.001). the most common causative organisms were streptococcus pneumoniae (19% vs 24%, ns) and legionella pneumophila (9% vs 7%, ns). atypical agents were more frequent in young adults (11% vs 4%, p = 0.001), while haemophilus influenzae (2% vs 7%, p = 0.026) and gram-negative bacilli (0% vs 1.4%, ns) were rarely identified in this group. the frequency of bacteraemia was similar in both groups (8% vs 12%, ns). icu admission was more frequent in young adults (13% vs 7%, p = 0.006), but no differences were found regarding the need of mechanical ventilation (5% vs 4%). median los was shorter in young patients (7 vs 9 days, p = 0.002). five young patients died; shock (2 patients) and multiorganic failure (3). early mortality (< 48 hours) was similar in both groups (1.4% vs 2.8%). overall mortality (< 30 days) was higher in older patients (3.4% vs 8.7%, p = 0.026). conclusions: cap requiring hospitalization in young adults, mainly smokers, is not uncommon, being respiratory failure the most frequent reason for admission. s. pneumoniae and atypical agents are the most frequent causative organisms in this group. although overall mortality is relatively low, a number of young patients require icu admission and have a complicated course. corticosteroids in patients with severe community-acquired pneumonia: impact on mortality c. garcia vidal, e. calbo, c. ferrer, v. pascual, s. quintana, j. garau (terrassa, e) background: mortality in patients with severe cap has been traditionally related to microorganism virulence and to host characteristics. recently, there has been an increasing interest host-pathogen interaction, and inadequate immunologic response has been shown to be associated with a higher mortality. immune modulation concomitant to antibiotic therapy has been postulated to improve outcome. the aim of our study was to determine the risk factors for increased mortality in patients with severe cap and to evaluate the impact of administration of corticosteroids in outcome. methods: we reviewed the charts of all hospitalized cap patients in our centre between october 2001 and december 2003. severe cap defined by pneumonia severity index (psi) categories 4 and 5 were included. data on demographics, comorbidity measured by charlson score, bacterial aetiology, the presence of immunosuppression, copd, icu admission, antibiotic therapy, use of corticosteroids and mortality were recorded. results: of the 375 severe cap patients included, 258 and 117,were categories 4 and 5, respectively. 256 patients(69%) were treated with standard antimicrobial therapy and116 (31%) received also corticosteroids. mean age (78 vs 77), charlson score (2.5 vs. 2.6), neoplasm (22% vs. 21%), hiv (0.7% vs. 0.8%), chronic liver disease (9% vs.7%), diabetes mellitus ( 19% vs.22%), icu admission (4% vs. 4%), monotherapy (63% vs. 66%), and aetiology (s. pneumoniae 34% vs. 36%; l. pneumophila 3% vs.2%; others 8% vs.11%; unknown 54% vs.51%) were similar. patients receiving corticotherapy had been exposed to another immunosuppressive treatment more frequently (6% vs. 12%; p = 0.04), copd was commoner ( 20% vs. 66%; p < 0.001) and mortality was higher (5% vs. 11%; p = 0.03) than in the group not treated with corticosteroids. in multivariate analysis, severity of disease (or 3.1; ic95% 1.3-7.1; p = 0.07) and the use of corticosteroids (or 3.3; ic 95%, 1.3-8.0;p = 0.009) were found to be related to mortality. conclusions: in our experience corticosteroid treatment in patients with severe cap appears to be associated with higher mortality. use of levofloxacin in community-acquired pneumonia j. olalla, i. escot, j.j. garcía-alegría (marbella, e) introduction and objectives: community acquired pneumonia (cap) is a prevalent and important disease, with an important consume of resources at hospitals. our aim is to study the profile of income patients with cap diagnosis, the year of introduction of levofloxacin in our centre and compare the length of stay in order to different antibiotics treatments, adjusting the results by fine's scale. methods: in our hospital (a second level hospital in marbella, spain) all the diagnosis at discharge are codified and included in the hospital database, so, all the diagnosis of cap between september 2001 and march 2002 were collected, and clinical reports were examined. demographic data were registered, so were all the parameters affecting fine classification, antibiotic treatment, intensive care unit (icu) incomes, deaths and length of stay. results: 129 cases of cap were found (86 males, 43 females), with a mean age of 67 years (ys)-ci 95%: 64-70), no difference between gender. 4 patients (pts) were living in a residence. 11 pts (8.5%) had a previous diagnosis of heart failure -hf-(mean of age 78 vs 66, p < 0.005) and other 11 pts had cerebrovascular disease -cvd-(mean of age 80.7 vs 66, p < 0.01), 12 pts had chronic hepatopathy, 15 -11.5%-any form of cancer, 23-17.8%-pts chronic renal failure (mean of age 74 vs 66, p = 0.049), 41 pts (32%) chronic obstructive pulmonary disease (copd). 3 pts were admitted at icu and 3 deaths were registered. blood cultures were collected in 30% of cases, culture of sputum in 21%, legionella antigen in urine in 13.2%. in according with fine's classification the patients were stratified in group i (10.9%), ii (16.3%), iii (22.5%), iv (27.9%) and v (22.5%). in 40% cases corresponding to fine i and ii levofloxacin was used alone, vs 19% in fine iii, iv and v (p=0.021). when length of stay was analysed in patients fine i and ii, the use of levofloxacin was associated with a non significant reduction (mean + se: 5.86 + 2.7 days vs 7.67 + 4.9 days), in people on groups iii, iv and v use of levofloxacin do not showed any reduction of length of stay (6.67 + 5.06 days in people using levofloxacin vs 6.11 + 4.5 days). no readmissions in relationship with recurrent cap was registered. conclusions: in our experience, use of levofloxacin is associated with less severe cap, in which a non significant reduction in length of stay is observed. telithromycin versus other first-line single-agent antibiotics in the treatment of communityacquired pneumonia: a randomised superiority trial y. mouton, v. thamlikitkul, r.b. nieman, c. janus (tourcoing, f; bangkok, th; bridgewater, usa; paris, f) objectives: macrolides and beta-lactams are commonly recommended for the treatment of outpatients with communityacquired pneumonia (cap). the aim of this study was to demonstrate the superiority of the ketolide telithromycin (tel) to other first-line, oral, single-agent antibiotics (comparators, comp) in achieving clinical cure in outpatients with mild to moderate cap in geographical areas of high pneumococcal resistance (erythromycin resistance rate ‡ 30%). methods: patients with clinical and radiological evidence of cap were randomised centrally to receive either tel 800 mg once daily for 7-10 days or comp (chosen by the investigator in accordance with local treatment guidelines/practices). efficacy was assessed post-therapy (days 17-21). clinical outcomes in this open-label trial were validated by three independent experts who were fully blinded to treatment assigned. results: of the 505 patients enrolled, 482 were included in the modified intent to treat (mitt) and 438 in the per protocol (pp) efficacy analyses. in the comp group, 39% of patients had received macrolides, 44% beta-lactams and 17% fluoroquinolones. resistance to penicillin and erythromycin among streptococcus pneumoniae isolates was 28% and 31%, respectively. clinical cure rates in the tel group were significantly higher than those in the comp group (as shown in the following table). tel and comp were similarly well tolerated. conclusions: in this outpatient cap study, post-therapy clinical cure rates achieved with tel were shown to be statistically superior to those achieved with other usual care first-line antibiotics -in both the mitt and pp populations, and according to evaluations by both the investigators and blinded experts. changing epidemiology, clinical features, and outcome of acute community-acquired pneumonia among adults in india background: acute cap is the third leading cause of mortality in india. two prospective studies from our centre identified common causes of cap in india to be mycoplasma pneumoniae [mp] and legionella pneumophila [lp] by serology in 11% each, and spn in 10% by culture of respiratory secretions/blood/ conclusion: although spn is the most common isolate, the rising numbers of gram negative organisms (38%) and atypical pathogens associated with increasing mortality stress the need for review of initial antibiotic choice for adults with higher port classes. in view of enduring susceptibility of spn to pcn and minimization of resistance with narrow spectrum activity, this should be the drug of choice for spn in india. fqr that has a wide coverage against most respiratory pathogens including atypicals, can be considered as an initial choice in both hospitalized and ambulatory setting. continued surveillance of respiratory pathogens is needed. fluoroquinolones in the treatment of community and hospital-acquired legionella pneumonia objective: community-acquired pneumonia (cap) is a major cause of morbidity and mortality worldwide. inability or failure to comply with standard antibiotic regimens, which may last up to 10 days, may result in patients receiving suboptimal antibiotic treatment. treatment with a single-dose antibiotic regimen maximizes compliance with prescribed therapy. a novel microsphere formulation of azithromycin makes it possible to administer more drug in a single dose while maintaining tolerability. the objective of this study was to test the hypothesis that a single 2.0 g oral dose of azithromycin microspheres was as effective as a 7-day regimen of oral levofloxacin 500 mg/day when used to treat adult patients with mild to moderate cap (fine classes i, ii, iii objectives: many guidelines have been issued for the treatment of cap. we wanted to assess how frequently the national guidelines for treatment of cap were followed in 10 spanish acute care hospitals in patients admitted with cap. methods: retrospective review of the charts of patients admitted to the hospital with the diagnosis of cap over a 1-year period (1nov 01 to 31oct 01) in 10 geographically scattered hospitals in spain. data were available from 3233 patients. results: amoxicillin/clavulanate was the most common antibiotic choice (79.5% was given as monotherapy and 20.4% in association with clarithromycin). levofloxacin (93.1% given only as monotherapy) follows closely. they were followed by third generation cephalosporins (ceftriaxone and cefotaxime) but up to 60% of them were given in association with clarithromycin. clarithromycin was used mainly as a complement to both amoxicillin/clavulanate and cephalosporins since only 8.6% of its 25.8% was given as monotherapy. other antibiotics were very uncommonly prescribed (cefepime 1.9%, and ciprofloxacin 1.4%). conclusions: 1) monotherapy with levofloxacin, followed by amoxicillin/clavulanic acid were the most common first line antibiotic choices in these 10 spanish hospitals. however, amoxicillin/clav alone or in combination with clarithromycin was the most common antibiotic prescribed. 2) monotherapy with clarithromycin was relatively uncommon (2.2%). 3) these choices reflect quite well the recommendations of the different national guidelines issued in our country in an environment of high prevalence of resistance to penicillin and to macrolides in s. pneumoniae in spain and the concomitant coverage of legionella and other atypical pathogens. the microbiology of abecb -potential predictive value of clinical criteria j. kahn (raritan, usa) objectives: to explore the relationship between specific pulmonary disease severity criteria and the microbiology of acute bacterial exacerbations of chronic bronchitis (abecb). methods: this was a double-blind, randomized clinical trial evaluating levofloxacin 750 mg qd for 3-5 days in abecb. all potential subjects had to meet the ats definition of chronic bronchitis but only those patients with anthonisen type 1 or 2 exacerbations (n = 763) were enrolled. stratification by disease severity was determined using parameters suggested by grossman: fev1 % of predicted value, defined co-morbidities, and number of exacerbations during the previous 12 months. because different spectra of etiologic organisms were expected on the basis of the stratification, different comparator agents were used for uncomplicated patients (azithromycin for five days) and those considered complicated (amoxicillin/clavulanate for ten days). results: initial microbiology from all intent-to-treat patients revealed notable differences between the two strata. among isolates from uncomplicated cases, 55% were the ôtraditionalõ triad of s. pneumoniae, h. influenzae, and m. catarrhalis. inclusion of h. parainfluenzae raises this figure to 79%. in the complicated arm the figures were 48% and 68%, respectively. gram-negative bacilli, primarily enterobacteriaceae spp. and several pseudomonads, represented 15% of the uncomplicated and 23% of the complicated isolates. microbiological eradication rates and the percentage of organism persisting after therapy from the uncomplicated stratum were compatible with the unexpectedly large number of macrolideresistant organisms isolated. in the complicated arm, both levofloxacin and amoxicillin/clavulanate had lower, but comparable, eradication and persistence rates. clinical efficacy in microbiologcally-evaluable patients was consistent with these figures. conclusion: while there was clearly overlap among the flora isolated from the two strata defined by application of the grossman criteria, there was separation of the populations. this predictive approach seems to be of value in identifying the optimal antimicrobial regimen, especially for uncomplicated exacerbations. further work to define and validate predictive clinical parameters may help optimize the choice and duration of antimicrobial agents for abecb. objectives: nosocomial pneumonia is the second most common nosocomial infection and, along with primary bacteraemia, the leading cause of death from infection acquired in the hospital. despite the frequency of ventilator-associated pneumonia (vap) and the threat it poses to patient survival, consensus on an appropriate antibiotic treatment of vap has yet to be established. the uncertainty is compounded by a lack of well-controlled comparisons of specific treatment regimens and their impact on relevant outcomes, such as morbidity, mortality, and cost. the comparable analysis of clinical and microbiological efficacy of cefepime and a combination ceftazidime and amikacin in the treatment of vap in severe trauma patients was the aim of present study. methods: this prospective, randomized study was approved by the institutional review board for human research. thirty adult patients who admitted in icu of an 1850-bed emergency municipal hospital with severe trauma complicated with vap were included. the cpis was used for diagnostics of vap. a combination of ceftazidime (2 g tid, iv) and amikacin (1 g once daily, iv) was used as initial empiric therapy of vap in 17 patients and monotherapy with cefepime (2 g bid, iv) -in 13 patients. these regimes of empiric therapy have been chosen according our local microbiological monitoring and p. aeruginosa was the prevalent pathogen of vap. the cost-effectiveness analysis was performed to compare both costs and outcomes of competing regimes. results: there was no difference between groups in patientsõ mean age, average time of mechanical ventilation before onset of vap and cpis. mean apache ii score was 23.1 in the group of combination therapy and 24.0 in the group of monotherapy. the positive clinical outcome was registered in 92.3% patients on cefepime and 88.2% patients on ceftazidime plus amikacin. the rate of eradication was 53.8 and 58.8% respectively. the duration of effective treatment with cefepime was from 7 to 12 days (mean 8.5 days), with combination therapy -from 7 to 18 days (mean 9.1 days). the mean cost of vap treatment with cefepime was 452.8 euro in comparison with 585.4 euro when the empiric therapy was initiated with ceftazidime and amikacin. conclusions: cefepime in a monotherapy regimen has the same clinical and bacteriological efficacy in comparison with a combination of ceftazidime and amikacin in vap patients with severe trauma but the use of cefepime results in lower costs. influence of antibacterial protocol in multiple trauma patients on incidence and mortality of vap d. protsenko, a. yaroshetskiy, s. yakovlev, b. gelfand (moscow, rus) objectives: the aim of present study was the evaluation of antibacterial protocol in multiple trauma patients on incidence and mortality of vap. methods: a comparable analysis of incidence, attributive mortality (chi-square test) and pathogens of vap in multiple trauma patients was performed during two periods: 2001 (before introduction of protocol) and 2003 (after introduction of protocol) years. the developed protocol included: 1. abandoning of antibiotic prophylaxis of vap; 2. intrusion criteria of early diagnostics of vap; 3. exclusion of 1st, 2nd and 3rd generation cephalosporines , aminoglycosides and fluoroquinolones as empiric therapy of vap; 4. cefepime (apache ii <20) or carbapenems (apache ii >20) were used as initial empiric therapy of vap; 5. efficacy of antibiotic treatment was evaluated within 48 hours; 6. carbapenems and/or vancomycin was added if initial therapy was inefficient. in the case of suspected diagnosis of vap microbiological analysis of bronco-alveolar lavage fluid (bal) was performed. a widespread use of broad spectrum antibiotics shifted the structure of nosocomial pathogens. we observed decrease of mrsa rate and significant increase (more than 10 times) in rate of klebsiella pneumoniae (from 0.6 to 18.1%, most of strains were resistant to 3rd generation cephalosporines) and in rate of acinetobacter baumanii (from 1.2 to 12.3%, most of strains were resistant to ceftazidime). conclusions: introduction of strict antibacterial protocol in patients with multiple trauma and vap results in significant decrease of attributive mortality without any change in incidence of vap. antimicrobial prophylaxis in cardiac surgery and postoperative pneumonia rate objective: the purpose of this study was to assess the postoperative pneumonia rate according to antimicrobial prophylaxis regimens in patients after cardiac surgery with cardiopulmonary bypass. patients and methods: cardiac surgery patients were included (n = 86) in the retrospective study. we observed 44 patients with postoperative pneumonia (pneumonia group) and 42 patients without infectious complications in postoperative period. the groups were compared by age, sex, volume of transaction, severity of disease. in a studied period is from 2001 to 2003 two main regimens of antimicrobial prophylaxis were used: ceftriaxone 1-2 g iv once before operation or cefuroxime 1.5 g iv before operation and additional dosage 0.75 mg in 2-3 hours after surgery beginning; then prophylaxis were conducted after operation during 48-72 hours. data were compared by fisher exact; we determined about significantly differences between groups when p < 0.05. results: preoperative prophylaxis by cefrtiaxone was administered more often in pneumonia group than in patients without infection complications (56.3% vs. 33.3%, ns). ceftriaxone was used significantly more often in comparison to cefuroxime in subgroup of patients with cardiopulmonary bypass less than 180 min who developed postoperative pneumonia (63.2% vs 30%, p < 0.05). during cardiopulmonary bypass longer than 180 min we did not reveal difference between antimicrobial prophylaxis regimens. conclusion: cefuroxime is more preferable than ceftriaxone for antimicrobial prophylaxis of postoperative pneumonia in patients after cardiac surgery with cardiopulmonary bypass less than 180 min duration. closed versus open tracheal suction system to prevent ventilator-associated pneumonia objective: the aim of this study was to analyze the incidence of ventilator-associated pneumonia (vap) using a closed tracheal suction system ( objectives: dalbavancin is a novel, second generation lipoglycopeptide that has been shown to have clinical efficacy as a once-weekly treatment for complicated skin and skin structure infections (csssi). the dosage used in clinical studies is 1000 mg day 1/500 mg day 8. in vitro studies have determined dalbavancin mic90s of less than or equal to 0.06 mg/l for target bacteria, principally staphylococci and streptococci, including isolates resistant to other antibiotics. the plasma protein binding of dalbavancin is 93%. as skin infections occur in extravascular space, antibiotic concentrations are assessed in blister fluid as a measure of skin penetration and to obtain pharmacokinetic observations that may correlate with efficacy. methods: a phase i, open label, single-dose study was conducted in 9 healthy subjects. subjects were administered a 1000 mg iv dose of dalbavancin. blisters were induced by applying 0.25% cantharidin ointment to the skin. blister fluid was collected prior to dose, and at 12 hours, day 3, day 5 and day 7. blood samples were collected throughout the study. blister fluid and plasma were assayed for dalbavancin using a validated lc-ms/ms method and pharmacokinetic parameters were determined. area under the concentration-time curve (auc) was determined for blister fluid (aucbf) and plasma (aucp) through the first week. the degree of penetration into skin was determined by aucbf/aucp (%). results: dalbavancin was well tolerated with no serious adverse events; most adverse events were mild and self-limited. maximum observed dalbavancin blister fluid concentrations were achieved by the first collection (12 hours) and concentrations were maintained above 30.3 (±4.4) mg/l through day 7. the mean (sd) aucbf and aucp were 6438 ± 1238 and 10806 ± 1926 mg h/l, respectively. skin penetration was approximately 60%. dalbavancin blister fluid concentrations were maintained well above mics throughout the treatment interval, even when considering the possible effects of protein binding. conclusions: blister fluid concentrations are maintained well above mics of csssi target organisms for a week following a single dose of dalbavancin. these data support a once-weekly regimen and are consistent with the efficacy observed in clinical studies. the safety and pharmacokinetics of dalbavancin in subjects with renal impairment or end-stage renal disease j.a. dowell, e. seltzer, d. krause, t. henkel (king of prussia, usa) objectives: dalbavancin is a novel, second generation lipoglycopeptide antibiotic in late stage clinical development for complicated skin and skin structure infections (csssi). the weekly dosage used in clinical studies is 1000 mg day 1/500 mg day 8. since dalbavancin will likely be used in patients with various degrees of renal impairment, it is important to determine the safety and pharmacokinetics in this population, as well as to evaluate if a dosage adjustment is necessary. methods: single intravenous doses of 1000 mg dalbavancin were examined in subjects with mild (clcr of 50-79 ml/min) and moderate (clcr of 30-49 ml/min) renal impairment. doses of 500 mg dalbavancin were studied in subjects with endstage renal disease (esrd; dialysis-dependent). single doses of 500 mg and 1000 mg dalbavancin were studied in subjects with severe renal impairment (clcr <30 ml/min). subjects with normal renal function were studied and used as controls. pharmacokinetic data were analysed using non-compartmental methods; parameters included maximum concentration (cmax) and area under the plasma concentration time curve (auc). results: a total of 43 subjects received dalbavancin and were included in the pharmacokinetic analysis (6 mild, 6 moderate, 10 severe, 6 esrd, and 15 normal). dalbavancin was well tolerated in each of the renal impairment groups. the majority of adverse events were mild or moderate in severity and unrelated to study drug. there were no related serious adverse events. dalbavancin pharmacokinetics were similar between subjects with mild and moderate renal impairment and subjects with normal renal function. concentrations in subjects with esrd were similar to subjects with normal renal function, indicating compensation in renal insufficiency due to regular dialysis (3 times/week). subjects with severe renal impairment had increased concentrations and exposure. concentrations were increased by no more than 40% through the first week post dose, but differences continued to increase through the rest of the profile. auc was increased almost 2-fold. background: tlv, a novel lipoglycopeptide antibiotic with multiple mechanisms of action, exerts rapid and concentration-dependent bactericidal activity against clinically important gram-positive bacteria, including methicillin-resistant s. aureus. the kidney is the primary route of elimination of tlv in man. phase 3 studies are ongoing in skin and skin structure infections and hospital acquired pneumonia. the lines represents the model-based predictedprobability of the first occurrence of naused. the boxes represent the 25th and 75th percentiles of auc for each dose group. the wiskers extend to the minimum and maximum values. objective: to compare the single dose pk of tlv in subjects maintained on hemodialysis with an aged and sex-matched group of healthy subjects without renal dysfunction. methods: six hemodialysis subjects (5 males, 1 female) received a single 7.5-mg/kg dose of tlv intravenously over 1 hour approximately 2-4 hours prior to a 4-hour hemodialysis session. six subjects (5 males, 1 female) with normal renal function (mean creatinine clearance 94 ml/min) also received the same dose of tlv over 1 hour. blood and dialysate samples were obtained at specified intervals post dose and assayed for tlv with a validated lc/ms/ms assay. pk parameters were determined using non-compartmental methods. results: average values for plasma pk parameters for tlv in both groups of subjects are shown below.the average (range) clearance of tlv via dialysis was 4.6 ml/min (4. 0-5.8 ) and the per cent of dose removed by dialysis was 5.9% (3.0-7.6). subjects tolerated tlv well. conclusions: dose reductions of tlv of 50% are recommended in patients maintained on hemodialysis. supplementation of a tlv dose post dialysis is not needed. pharmacokinetics and tissue penetration of telavancin in healthy subjects background: tlv, a novel lipoglycopeptide antibiotic with multiple mechanisms of action, exerts rapid and concentration-dependent bactericidal activity against clinically important gram-positive bacteria, including methicillin-resistant s. aureus. the mic90 for s. aureus, including mrsa and gisa, from pooled studies is 0.5 lg/ml. phase 3 studies are ongoing in skin and skin structure infections and hospital acquired pneumonia. objectives: to determine the steady-state pk profile of tlv in plasma and skin blister fluid in healthy subjects. methods: 8 subjects (7 males, 1 female) received three daily doses of tlv (7.5 mg/kg) intravenously over 1 hour. cantharidin ointment (0.25%) was applied to the forearms to produce 7 blisters/subject beginning 12-14 hours prior to the third dose of tlv. blood and blister fluid samples were obtained at specified intervals on days 3 and 4 and assayed for tlv with a validated lc/ms/ms assay. pk parameters in both matrices were determined using non-compartmental methods. the average values for pk parameters for tlv in plasma and in blister fluid are shown below. results: after single intravenous infusions of 750 mg equivalent (eq) ceftobiprole for 30 minutes or 500 mg eq ceftobiprole for 60 minutes, mean cmax-values at infusion endpoint are 57.9 lg/ ml and 34.2 lg/ml, respectively. the corresponding mean auc0-inf-values are 154 lg h/ml and 116 lg h/ml. intersubject variability of the parameters auc and cmax of ceftobiprole is below 20%. mean systemic clearance is 4.9 and 4.5 l/h and mean steady-state volume of distribution is 13.7 and 11.0 l for the 750 mg and 500 mg dosing regimen, respectively. after infusion of 500 mg for 60 minutes or 750 mg for 30 minutes, the ôtime above micõ (target mic of 4 lg/ml for methicillin resistant staphylococcus aureus) is 6 and 7 hours for the total plasma concentrations and 3.5 and 5.2 hours for the corrected unbound plasma concentrations, respectively. with twice-daily administration of 500 mg for 60 minutes or 750 mg for 30 minutes, the proportion of the dosing interval above the mic of 4 lg/ml, would be 75% and 87% for the total plasma concentrations and 55% and 65% for the unbound plasma concentrations, respectively. conclusions: ceftobiprole infusions of 500 mg eq for 60 minutes or 750 mg eq for 30 minutes, results in similar time above target concentration of 4 lg/ml well above the minimum efficacy requirement 25-30% required for mrsa. methods: the study was a randomized, three-way crossover study with 30 hours wash-out period in 8 healthy volunteers. each subject received imipenem in three regimens : (i) 0.5 h infusion of 0.5 g every 6 h for 3 doses; (ii) 2 h infusion of 0.5 g every 6 h for 3 doses; (iii) 2 h infusion of 1 g every 6 h for 3 doses. conclusion: the 2 h infusion of 0.5 or 1 g of imipenem both give greater values for t > mic than a 0.5 h infusion and that a 2 h infusion may be a useful mode of administration in tropical countries where drug instability may prevent the use of continuous infusion. pharmacodynamic characterisation of lmb415 against haemophilus influenzae in an in vitro hollow-fibre system a. meagher, p. smith, a. forrest, a. odundele, p. ambrose (buffalo, albany, usa) objectives: lbm415 is a peptide deformylase inhibitor with in vitro activity against those pathogens commonly associated with community-acquired respiratory tract infections. the purpose of these studies was to determine which pharmacokineticpharmacodynamic (pk-pd) measure is most strongly associated with drug response and to examine the relationship between drug exposure and response for lmb415 against haemophilus influenzae (hi). methods: two wild-type hi strains (mic 2 and 8 mg/l) were studied. the hollow-fiber system (hfs) was inoculated with approximately 10e7-10e8 cfu/ml in log-phase growth. simulating human pharmacokinetics (t ½ = 2 hours), bacteria were exposed to escalating free drug lmb415 exposures (auc ranging from 0 to 200 mg · h/l) using a dose fractionation study design and delivering drug q12 hours, q24 hours, and by continuous infusion (ci). serial samples were collected to determine bacterial counts (cfu/ml) and drug concentrations. drug effect was quantified as the log 10 ratio (lr) of the 24 hour area under the bacterial growth/kill curves for drug and growth control (lr = log 10 aucdrug/aucgrowth control). results: overall, the greatest activity (at 6xmic) was seen with the ci regimen (ci > q12 >> q24). due to the short half-life, dosing q24 hours yielded no net kill compared to baseline. neither per cent time above mic, auc:mic ratio, nor peak:mic ratio could co-model the q12 and q24 hour regimens with ci results. separating these into 2 datasets (q12/24 vs ci), only the auc:mic ratio could adequately describe the ci results (emax lr of (2 at an auc:mic ratio ‡50). the q12/24 dataset was reasonably fit by the auc:mic ratio and per cent time above mic (emax lr of (2 to (3 at an auc:mic ratio ‡120 and per cent time above mic of ‡50-60%, respectively). the peak:mic ratio was not informative. conclusion: for intermittent dosing regimens, per cent time above mic and the auc:mic ratio adequately described drug response. percent time above mic and the auc:mic ratio associated with maximal decline were ‡50-60% and ‡120, respectively. ci regimens could not be co-modeled with intermittent regimens, suggesting that neither per cent time above mic nor the auc:mic ratio completely described drug effect. drug effect continued to increase beyond 100% time above mic. q12 hour dosing had more effect than q24 hour dosing, and would probably be an effective lbm415 regimen in humans for hi. pharmacodynamics of antimicrobials for the empiric treatment of nosocomial pneumonia: a report from the optama program objectives: appropriate empiric antibiotic therapy is vital for maximizing patient outcomes in the treatment of nosocomial pneumonia and is dependent on the drug exposure achieved in a patient and the causative pathogen's mic. the purpose of this study was to compare the probability of achieving bactericidal exposures for commonly used intravenous antibiotics against the bacteria most commonly implicated in nosocomial pneumonia. was high, target attainments for fep, caz, and tzp decreased, but carbapenem target attainments remained the same regardless of pathogen prevalence. conclusions: target attainments were greatest for ipm and mem, followed by higher doses of fep, caz and tzp. because these antibiotic regimens provide optimal bactericidal exposure, they would be most suitable for the empiric treatment of nosocomial pneumonia along with an anti-mrsa antibiotic until pathogen identification and susceptibility results are available. minimal inhibitory concentration effect (sme) of 6 antibacterial agents against two strains of b. anthracis. methods: the pa-sub mic and sme were determined by exposing the bacteria to antibiotic concentrations 10 times the mic for 2 h at 37°c. following repeated washings and centrifugations to remove the antibiotic, cultures were divided into four tubes. to three tubes, the tested antibiotic was added to make a final concentration of 0.5 mic, 0.25 mic and 0.125 mic to the fourth tube no antibiotic was added. optical density was determined before exposure, immediately after washing and then hourly up to 9 h. for the determination of sme, the same procedure was performed except that the organisms were not pre-exposed to any antibiotic. the ods were converted to cfus by using a standard curve. the pa-sub mic was defined as pa-sub mic = tpa ) c, where tpa is the time for cultures previously exposed to an antibiotic and then reexposed to different sub-mics to increase 1 log 10 above the counts determined immediately after washing and c is the corresponding time for the antibiotic unexposed control. the sme was defined as sme = ts ) c, where ts is the time for the cultures exposed only to sub-mics to increase 1 log 10 above the counts determined immediately after washing and c is the corresponding time for the unexposed control. methods: serum and csf cefepime pk data was obtained from 7 hospitalized patients with pneumonia and external ventricular drains. the concentration-time profiles in serum and csf were modeled using a three-compartment model with zero-order infusion and first order elimination and transfer. the apparent volume of the central compartment (vc), apparent volume in the csf (vcsf), intercompartmental transfer rate constants (k12, k21, k13, and k31) and plasma clearance (cl) were identified in a population pk analysis (npag) [table 1]. for cefepime 2 g iv q8 h (0.5 h infusion), a monte carlo simulation of 10,000 subjects (adapt ii) was performed to estimate the probability of attaining the targets of free cefepime serum concentration (assumed 20% protein binding) and total cefepime csf concentration 40-100% t > mic for mics 0.25-8 mg/l (table 2) . csf/serum median penetration ratio was calculated. post map-bayesian observed-predicted regression and r 2 for serum and csf were as follows: (serum) observed = 0.984 · predicted + 2.570; r 2 = 0.944. (csf) observed = 0.785 · predicted + 0.868; r 2 = 0.821. the penetration of cefepime as measured by median auccsf/auc serum was 7.8% (25th-75th percentiles 2.9-21.4%). results of serum and csf target attainment analysis for cefepime 2 g iv q8h conclusion: in the setting of non-inflamed meninges, cefepime 2 g iv q8 h does not provide adequate t > mic in the csf for >80% of patients for mics ‡0.5 mg/l. the influence of inflammation on the calculated csf target attainment rates is unknown. the definitive pharmacodynamic target in the csf has not been elucidated and further research is needed. application of microbiological and capillary electrophoresis methods to phenoxymethylpenicillin dissolution assay w. grzybowska, g. pajchel, m. zapasnik, s. tyski (warsaw, pl) objectives: drug absorption from a solid dosage form after oral administration depend on the release of the active substance from the medicinal product. in some cases of drug analysis, especially when the results of tests are out of specification, it is necessary to use another assay, simultaneously with the reference method. in case of phenoxymethylpenicillin tablets, the reference method for dissolution assay is spectrophotometric method. the aim of the study was to apply parallel microbiological and capillary electrophoresis methods and compare the results with those obtained using uv assay. methods: two preparations (tablets), containing 1 00 000 iu/mg of penicillin v: taropen and ospen were examined. the dissolution tests were performed at temperature 37°c ± 0.5°c, in phosphate buffer ph 6.8 (900 ml for ospen and 1000 ml for taropen) using baskets, rotation speed 100 rpm; samples were taken only at: 30 minute or at 10, 20 and 30 minutes in case of profile of dissolution analysis. the amount of penicillin v dissolved was assayed spectrophotometrically at 268 nm. hanson research dissolution system and ce apparatus: quanta 4000 of (waters) were used. pharmacopoeal, microbiological agar diffusion method and staphylococcus aureus atcc 6538 p strain, were applied. results: the dissolution of penicillin v from taropen preparation after 30 minutes was 100%, independently on method applied. in case of ospen tablets these values were different, depending on the analytical assay. the statistical fisher-snedecor test for comparison of dissolution data obtained using three methods was performed. the fisher fcalc. value was lower than theoretical only in taropen case. the dissolution profiles of two examined preparations were also compared and statistically evaluated according to the fda method. the results of these calculations showed that dissolution profiles of phenoxymethylpenicillin from taropen and ospen tablets differ significantly. conclusion: performed analysis proved that capillary electrophoresis and microbiological methods can be used alternatively but only for determination of taropen dissolution. penetration of penicillin g in combination with sulbactam into nasal tissues u. frank, s. wenzler-rö ttele, w. maier, f. knapp, r. trittler, h. egle, k. kuemmerer (freiburg, d) objective: the penetration of antimicrobial agents into target tissues is essential for treatment at the site of infection. we investigated the distribution of penicillin g in combination with sulbactam in human nasal tissues. methods: to determine the pharmacokinetics of penicillin g/ sulbactam, informed written consent was obtained from 22 patients aged between 19 and 73 years scheduled for endonasal operations such as septoplasty and conchotomy. after infusion of 5 m iu of penicillin g and 1 g of sulbactam, the concentrations of these agents were determined in serum and various nasal tissues such as septal mucosa, septal cartilage and bone. serum and nasal tissue concentrations were determined by liquid chromatography-mass spectrometry (lc-ms). results: up to 1, 2, 3, 4 and 5 hours after infusion, the mean serum concentrations of penicillin g were 114.9 lg/ml (sd ± 24.2), 54.0 lg/ml (sd ± 29.8), 33.6 (sd ± 26.9), 12.7 lg/ml (sd ± 9.6), 17.6 lg/ml (sd ± 8.2) and 2.8 lg/ml, while the mean serum concentrations were 17.6 lg/ml (sd ± 8.2) 24.3 lg/ml (sd ± 15.2), 9.3 lg/ml (sd ± 3.5), 3.5 lg/ml (sd ± 0.6), and 1.9 lg/ml (sd ± 0.4). mean penicillin g and sulbactam concentrations in septal mucosa decreased from 141.7 and 128.3 lg/g (1st hour) to 3.0 and 2.7 lg/g (5th hour), respectively. in septal cartilage, the highest tissue concentrations were observed 2 hours after infusion, with mean penicillin g and sulbactam concentrations of 59.4 and 80.1 lg/g, respectively. the mean penicillin g and sulbactam concentrations in bone decreased from 31.2 and 14.8 lg/g (1st hour) to 1.4 and 1.8 lg/g (5th hour), respectively. the regression analysis of the data showed that 3 h after administration of the drug combination, the levels still exceeded the 4-fold mic90 of methicillin-susceptible staphylococcus aureus, streptococcus pyogenes, moraxella catarrhalis and haemophilus influenzae. conclusions: our data support the use of the drug combination in perioperative prophylaxis and the treatment of ent (nasal and paranasal) infections due to common bacterial pathogens. mic vs. kill-curve based pharmacokinetic/ pharmacodynamic modelling of activities of cefpodoxime and cefixime h. derendorf, p. liu, k. rand, b. obermann (gainesville, usa; munich, d) objectives: pharmacokinetic (pk)/pharmacodynamic (pd) modeling of antibiotics usually consists of the comparison between plasma pk and the mic, such as the t > mic, cmax/ mic, and auc/mic. these indices are limited due to the innate inaccuracy of the mic and the fact that it does not reflect the in vivo scenario where concentrations are not static but fluctuate between doses. an alternative is to use time-kill curves that follow bacterial killing and growth as a function of time and concentration. this study provides a systematic comparison of mic and kill-curve approaches to show the potential of both methods for antibiotic evaluation. in an example, we developed a mathematical pharmacokinetic/pharmacodynamic (pk/pd) cefepime population on pk mean (sd) parameter estimates obtained by npag analysis model to integrate the in vitro antimicrobial activity with the pk profile of two oral cephalosporines at the tissue site. methods: kill curves could be described with different combinations of maximum kill rate (kmax), drug concentration at half-maximum effect (ec50) and bacterial growth rate (k0) that resulted in the same mic in each scenario. in the experimental part, bacterial time-kill curves of cefpodoxime and cefixime against four bacterial strains were compared in in vitro kinetic models in which previously measured human pharmacokinetic profiles of unbound antibiotic were integrated. results: different combinations of kmax, ec50 and k0 can yield the same mic and consequently the same mic-based index. however, depending on the combination of pd parameters, kill curves may predict quite opposite outcomes in both scenarios. ec50 values of cefpodoxime and cefixime were consistent with their respective mic values. both antibiotics had similar high potency against h. influenzae (ec50: 0.04 mg/l) and m. catarrhalis (ec50: 0.12 mg/l), while the potency of cefpodoxime against s. pneumoniae strains was about 10-fold higher than that of cefixime (ec50s/ sensitive: 0.02 vs 0.27 mg/l; ec50s/intermediate: 0.09 vs 0.69 mg/ l). simulations showed that cefpodoxime will have higher bacteriological success against s. pneumoniae than cefixime. conclusions: simple comparison of exposure and mic may not be sufficient to evaluate anti-infective efficacy. kill curves provide a more detailed approach in predicting antimicrobial effects. the developed emax model effectively described the pharmacodynamics of cefpodoxime and cefixime. cefpodoxime (200 mg bid) has higher tissue penetration and antimicrobial efficacy than cefixime (400 mg qd) against s. pneumoniae. bacteriologic efficacy of intravenous piperacillin/ tazobactam and ampicillin/sulbactam for infected diabetic foot ulcers objectives: to test the efficacy of single-agent empiric treatment, either intravenously administered piperacillin/tazobactam (tzp) (4 g/0.5 g q8 h) or ampicillin/sulbactam (sam) (2 g/1 g q6 h), of moderate to severe infected foot ulcers in patients with diabetes. in this open-label trial, 314 adults were randomized to receive tzp or sam for up to 21 days. patients with polymicrobial infections involving methicillin-resistant staphylococcus aureus also received vancomycin 1 g q12 h. samples for bacteriologic evaluation were taken at baseline from infected ulcers and blood to document causative pathogen(s) and test for antimicrobial susceptibility; samples were taken as clinically indicated at the end-oftreatment and test-of-cure visits (14-21 days post-treatment). to minimize risk of contamination, samples were to be obtained by aspirate, curettage, or biopsy rather than by swabbing. results: a total of 123 of the 314 patients in the study were bacteriologically evaluable. the most common causative pathogens in monomicrobial infections were s. aureus, streptococcus agalactiae, enterococcus faecalis, and pseudomonas aeruginosa. about 30% of patients in each treatment arm had polymicrobic infections. the combination of s. aureus plus s. agalactiae was most common. the median duration of treatment was 8 days for both groups. the bypathogen bacteriological success rates were similar in both treatment groups for s. aureus, s. agalactiae, and e. faecalis. tzp had an eradication rate of 85.7% for p. aeruginosa; sam is not active against p. aeruginosa, and patients with p. aeruginosa in the sam group (n = 5) were discontinued from the study . conclusions: previous studies have shown that tzp at a dose of 3 g/0.375 g q6 h was effective for treatment of diabetic foot infections. our study confirmed that less frequent administra-tion of tzp at 4 g/0.5 g q8 h was sufficient to attain bacteriologic success rates of 76.9% for s. aureus, 83.3% for s. agalactiae, 71.4% for e. faecalis, and 85.7% for p. aeruginosa in the bacteriologically evaluable population. this study differed from previous studies of infected diabetic foot ulcers, which found gram-negative enterics to be more common than p. aeruginosa as causative pathogens. however, it is consistent with data showing that p. aeruginosa has recently become the gramnegative pathogen most frequently isolated from soft tissue infections in europe, north american, and latin america. penetration of beta-lactamase inhibitors tazobactam and sulbactam in severe acute pancreatitis objectives: despite of high standard intensive care and surgical management, acute necrotising pancreatitis is still related with an extremely high mortality rate. this is determined by local infectious complications, especially in necrotising areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotising pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of beta-lactamases compared to those of beta-lactam antibiotics alone. some bsp has been shown to penetrate rapidly and efficiently into pancreatic tissue. the penetration of bli into inflamed pancreatic tissue has not been investigated yet. methods: addressing the penetration capability of bli, a clinical trial was designed to investigate the penetration of tazobactam (taz, n = 8) and sulbactam (sul, n = 5) in patients with severe necrotising pancreatitis undergoing pancreas surgery. samples were taken from blood, necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of 0.5 g taz or 1.0 g sul. concentrations of bli were determined by hplc/ uv. the aimed concentration for full enzymatic effect of bli should be 4 lg/mg (taz) and 8 lg/mg (sul), respectively. results: mean plasma concentrations at 1.0 h after application were 20.6 ± 9.58 lg/ml (taz) and 48.6 ± 18.8 lg/ml (sul background: uti is one of the most common infectious conditions treated in general practice and represents a major part of abstracts antibiotic use in the community. it is of major importance to know, how we treat these infections correctly, i.e. maximum efficacy with the least amount of drug in order to reduce the risk of development of resistance. materials and methods: uti was induced in anesthetized female nmri mice via intraurethral inoculation of 10-8 cfu of e. coli. one day later, treatment was started with 12-24 hour dosing schedules with 12-16 different doses of each drug securing a wide variation of time > mic and auc/mic. 24 h after the last dose mice were sacrificed and urine collected, and the bladder and both kidneys removed. bladder and kidneys were homogenized before cfu determination. the drugs used were the cephalosporins, cefuroxime (cef, mic = 0 3 mg/l) and ceftriaxone (cro, mic = 0.094 mg/l), and the penicillin, mecillinam (mic = 0.1 mg/l). the pk of the drugs in serum was determined in similar mice as well. the two cephalosporins were studied together (cef has a t ½ of 14 min and cro a t ½ of 56 min, respectively, in mice). pd parameters were calculated from serum total antibiotic concentrations, since protein-binding is a minor issue in the urine, and the relation to cfú s estimated by the hill-equation. results: all drugs reduced cfú s in urine and kidney tissue by 3-4 logs, but little effect was found in the bladder tissue. for the two cephalosporins combined time > mic in % of dosing interval best described the correlation with cfú s in urine (r 2 = 0.69, p < 0.05) and in kidney tissue (r 2 = 0.89, p < 0.05), respectively, while no such correlation was found in the bladder tissue, neither did auc/mic reveal any correlation with cfú s in urine or any organs. the same was found for mecillinam, i.e. no correlation for auc/mic vs. cfú s in urine or organs, while time > mic % significantly (p < 0.05) correlated with cfú s in urine (r 2 = 0.48), and kidney tissue (r 2 = 0.82), respectively. the time > mic% for maximum efficacy was around 30-40% for all three antibiotics. conclusion: the optimal pkpd parameter for efficacy of betalactam antibiotics in uti is the time > mic. maximum effect is seen for time > mic for 30-40% of the dosing interval, when serum total drug concentration is used as surrogate parameter. in humans this would call for tid dosing of mecillinam and cefuroxime, and od dosing for ceftriaxone. comparative performance of different methods to simulate drug exposure variability in a population v.h. tam, g.l. rosner (houston, usa) objectives: stochastic pharmacokinetic (pk) forecasting such as monte carlo simulations (mcs) are increasingly being used to predict the pk variability of antimicrobials in a population, based on data from relatively few subjects. however, various mcs approaches may significantly differ in the accuracy and precision of the predictions. we compared the performance of 4 different mcs approaches using a dataset with known parameter values and dispersion. methods: concentration-time profiles for 10 subjects after an intravenous bolus of 1000 mg were randomly generated using elimination rate constant (k) 0.1 ± 0.025 h)1(mean ± sd) and volume of distribution (v) 20 ± 5 l. normal distribution of parameter values and no correlation between k and v were assumed. system noise was incorporated as a linear function of drug concentration. using these concentration-time profiles, the best-fit parameter estimates were determined by the standard two-stage method. four methods were subsequently used to simulate the auc0-inf of the population by mcs, using the central tendency and dispersion of the following in the subject sample: (1) k and v; (2) clearance and v; (3) dose/clearance; (4) auc0-inf. in each scenario, 10,000 subject simulations were performed with adapt ii, using normally distributed input parameter(s) with means and variances set to the fitted values. results: reasonably good parameter estimates were obtained (k = 0.101 ± 0.034 h)1; v = 18.0 ± 7.2 l). compared to true auc0-inf of the population (581.1 ± 314.9 mg h/l), the simulated auc0-inf by various methods were: (1) 911.1 ± 2416, (2) 1348 ± 23510, (3) 1348 ± 23510, and (4) 713.7 ± 430.1 mg h/l, respectively. conclusions: our results suggest that various mcs approaches may predict pk variability in a population differently. the most realistic approach appeared to be based on the variability of auc0-inf in the subject sample. our observations are consistent with statistical principles concerning estimation. this method did not amplify variability of the model parameters and was the least likely to be associated with model misspecification. in objectives: fluoroquinolone treatment of humans and animals can rapidly select for organisms with increased resistance to these antibiotics. animal models are key to investigating in vivo antibiotic concentrations that prevent selection of resistance (e.g. the mutant prevention concentration (mpc) concept), but use of such models requires validated procedures for extraction and analysis of fluoroquinolones from relevant tissues. here, we report the validation of a method to quantify the concentration of enrofloxacin, and its metabolite ciprofloxacin, extracted from chicken liver, caecal contents and serum following treatment with baytril tm (enrofloxacin). methods: the extraction procedure described by wiuff et al. 2002 was validated for fluoroquinolone analysis, in pig tissues, by fortification of target matrices with enrofloxacin and ciprofloxacin. norfloxacin was added as an internal standard to all samples. samples were homogenised and extracted with acetonitrile (6 ml), centrifuged and the supernatants retained for analysis. the extracts were diluted with distilled water and analysed by hplc equipped with fluorescence detection. the analytes were chromatographed on a c18 reverse phase column and eluted with an isocratic potassium phosphate buffer/ acetonitrile system. results: liver samples were fortified with 0.1, 1.0 and 5.0 lg/g enrofloxacin and provided recoveries of 114.5, 78.6 and 78.2% respectively. quantification of ciprofloxacin at the 0.1 lg/g level was not possible due to interference from sample co-extractives, but the recoveries at 1.0 and 5.0lg/g were 73.7 and 91.1%. for caecal content samples, enrofloxacin recoveries were 70.6 and 69.1% at 1.0 and 5.0lg/g respectively, and ciprofloxacin 65.4 and 63.3%. the recovery from serum was much higher, samples fortified at 0.1, 1.0 and 5.0lg/ml, enrofloxacin and ciprofloxacin yielded recoveries of 94.7, 80.9 and 86.1%, and 91.1, 85.6 and 87.9% respectively. selected samples were also analysed by zonal microbial growth inhibition bioassay and hplc equipped with ms/ms detection; the data were broadly similar between all methods. conclusion: a valid method for the analysis of fluoroquinolones in chicken liver, caecal content and serum has been established, and will be applied to in vivo mpc studies. where sample matrices interfere (e.g. liver, caeca), bioassay or lc-ms/ ms must be used to provide valid results. the ecological effects of norfloxacin and pivmecillinam (piv) on the periurethral and vaginal flora in women with recurrent urinary tract infection objectives: to compare the ecological effects on the periurethral and vaginal microflora and time to normalisation of orally administered norfloxacin (nflx) and pivmecillinam (piv) in women with recurrent lower uti. methods: women with recurrent lower uti participated in a randomized, single blind parallel multi-centre study. twentyfive women, aged 18-55 years, with a positive nitrite test and symptoms (urgency, frequency, dysuria and/or suprapubic pain) of lower uti were included. only patients with an uti caused by e. coli or klebsiella spp were evaluated. key exclusion criteria were; menopause; pregnancy or breast feeding, known hypersensitivity to study drugs, antibiotics within the preceding month, impaired liver or kidney function, known or clinically suspected pyelonephritis, complicated uti and/or gastrointestinal infection. study drugs: seven days treatment of either nflx 400 mg bid or piv 400 mg tid. samples from midstream urine, periurethra and vagina were obtained before start of treatment day 1 and at two follow-ups day 12-4 and 28-35. informed consent was obtained. the ethical review committee and medical product agency approved the study protocol. results: nineteen patients (11 nflx and 8 piv) fulfilled the inclusion and exclusion criteria. no differences of patient characteristics were seen between the two groups. at the initial visit, more nflx patients were colonized with aerobic bacteria, although no differences were seen for anaerobic bacteria. the e. coli strains were suppressed markedly by nflx and piv in both locations. s. epidermidis increased more markedly following piv treatment compared to nflx in the periurethral location. in the piv group e. faecalis was less frequent initially, increased at visit 2, and returned to pre-treatment numbers at visit 3. for the nflx group, more patients were colonized initially with e. faecalis, with a decrease at visit 3. lactobacilli decreased slightly in the periurethra in the nflx group, whilst no changes were seen for piv. bacteroides spp. decreased more markedly for nflx. restoration of the pre-treatment colonization levels occurred gradually, except for e. coli being markedly suppressed by nflx throughout the study period. the bacterio-logical outcome of the urinary tract infection both at the shortterm and long-term follow up was successful. conclusions: limited ecological effects on the microflora were seen following treatment with nflx and piv, a benefit for antibiotics used for treatment of patients with uti. antibiotics influence release of il-6 and il-8 by kb cells and gingival fibroblasts s. eick, m. lausse, s. schwarz, p. wachter, w. pfister, e. straube (jena, d) objectives: in general, antibiotics are used in defense against pathogenic bacteria. nevertheless, side effects such as immunomodulatory activity should be considered. the purpose of this study was to determine the effect of antibiotics normally used in periodontal treatment on the release of il-6 and il-8 by kb cells and gingival fibroblasts. methods: kb cells and primary gingival fibroblasts were infected with actinobacillus actinomycetemcomitans y4 (a.a.) and porphyromonas gingivalis atcc 33277 (p.g.). the antibiotics doxycycline, tetracycline, minocycline, metronidazole, and moxifloxacin were added in concentrations ranging from 0.25 mic to 100 lg/ml. after 1, 6 and 18 h supernatants were obtained and the levels of il-6 and il-8 were measured by elisa technique. additionally, after 18 h the number of surviving bacteria was enumerated. results: minocycline and moxifloxacin in concentrations predicted in serum as well as 100 lg/ml were effective in killing planctonic a.a. and p.g. as well as adherent and intracellular bacteria. low levels of both interleukines released from kb cells and fibroblasts infected by p.g. were found only after addition of tetracycline up to 10 lg/ml. metronidazole, moxifloxacin and tetracycline in subinhibitory concentrations enhanced the release of il-6 from non-infected and a.a.-infected fibroblasts (up to 3200 pg/ml) after 18 h, contradictory 100 lg/ml tetracycline and minocyline reduced the release of il-6. il-6 in the supernatants of kb-cells was detectable only after addition of 100 lg/ml moxifloxacin. each 100 lg/ml of tetracycline and minocycline suppressed totally the release of il-6 and il-8 from non infected and infected fibroblasts, but not from kb cells. metronidazole and moxifloxaxin in high concentrations promoted the release of il-8. conclusions: antibiotics influence the release of il-6 and il-8. besides the good antibacterial effect especially minocycline might suppress inflammation. nevertheless, the killing of bacteria as well as a possible inhibition of virulence factors (bacterial proteases of p.g. by tetracycline) might influence the enhanced or reduced release of these cytokines. exceedingly infrequent infectious complications during orthotopic liver transplantation: tubercular peritonitis introduction: tuberculosis (t) is a very infrequent complication in patients (p) who undergo solid organ transplantation: in liver transplant recipients a 0.9-2.3% rate has been retrieved by a literature update. in these p t occurs within 12 months, and less than 10% of cases are observed after a repeated transplantation. pulmonary localization predominates (50% of p), followed by disseminated t (30%), while extrapulmonary t is an exceedingly rare event, reported only nu anectdotal p descriptions. pathogenetically, a t peritonitis follows disseminated infection or a primary t enteric localization, and is borne by a 80% mortality rate. case report: a 57-year-old female p with a mute t personal and familial history and normal chest x-ray, underwent liver transplantation 9 years ago, because of a hcv-related decompensated cirrhosias. four years later a novel graft was needed, after the development of end-stage hepatic insufficiency due to massive hcv re-infection. one year later (while of cyclosporin treatment), abdominal pain and ascites formation prompted admission, and m. tuberculosis sensible to all tested drugs was repeatedly isolated from an abdominal fluid with an elevated lymphocyte-monocyte count. associated ethambutol, isoniazid, streptomycin and levofloxacin (this one replaced by cycloserine after 2 months), led to a complete clinical and bacteriological cure already achieved after 3 months, although a 3-drug anti-t treatment was continued for 1 year. conclusionas: in our rare case report, the selected anti-t chemotherapy showed a successful efficacy and tolerability profile, with contained untoward events despite a very critical clinical context, due to the severity of t complication, and the broad spectrum of problems related to graft and all associated issues. in order to obtain an early diagnosis, an elevated clinical suspicion should be maintained for this rare complication too, by recommending microscopy and culture search of mycobacteria on ascitic fluid. methods: from january 2000 through december 2001, a prospective cohort study was conducted to determine the rate of bacterial nosocomial infection in renal transplant recipients. the patients were divided in two groups according to the origin of the allograft: cadaver or living donors. enterobacter cloacae strains, the most prevalent multidrug-resistant bacterial organisms isolated from uti were determined using genomic analysis with pulsed-field gel electrophoresis (pfge). results: one hundred sixty-three patients who received renal transplants were reviewed during the hospitalization. one hundred and ten (67.5%) renal transplanted were from cadaver and 53 (32.5%) from living donor. the median of length of stay in hospital was 12 days (range, 9-75 days) from transplant of living donor and 26 days (range, 2-28 days) from transplant of cadaver (p < 0.0001). twenty-one (39.6%) living donors recipients and 68 (61.8%) cadaver donors recipients had bacterial infection episodes (p = 0.019). post-transplant nosocomial infections diagnosed during the hospitalization were uti (44.8%), ssi (11%), pneumonia (6.1%), bloodstream catheter-related infection (4.2%) and others (1.8%). risk factors for uti in the multivariate analysis included cadaver donor recipient; substitution of the initial immunosuppressive regimen; days of urinary bladder catheterization and length of stay in hospital before the infection. substitution of initial protocol of the post-transplant immunosuppressive regimen and the surgeon was also associated with ssi. six different e. cloacae multidrug-resistant to antibiotics dna profiles were detected in uti of our recipients and hospital dissemination was documented. conclusions: uti was the single most important type of hospital infection in renal transplant recipient and a significant difference in the incidence of uti was found comparing living donor and cadaver donor recipient. the high incidence of uti in the early period post-transplant suggested that the operative manipulation of the urinary tract may be an important causative factor for the development of uti. usefulness of cmv antigenaemia assay after liver transplantation recurrence rates were higher (p < 0.001) in symptomatic infections. underlying liver diseases of the recipients, infectious complications other than cmv, rejection rates, and survival rates were not different between symptomatic and asymptomatic cmv infections (p > 0.05).conclusion: about an half of the recipients experienced cmv reactivation, mostly within 180 days post-transplantation. thirty percents of reactivation were symptomatic. peak value and duration of cmv antigenaemia were significantly higher in symptomatic infections than those in asymptomatic infections. toxoplasma gondii infection in bone marrow transplant recipients: the polymerase chain reaction-blood sample combination in diagnosis and early detection a. sensini, r. castronari, c. ferranti, t. aloisi, f. aversa, f. bistoni (perugia, i) objectives: toxoplasmosis is a rare and frequently fatal complication in bone marrow transplant recipients. it presents with local brain lesions or disseminated infection. usually, the diagnosis is based on histologic confirmation and observation of typical lesions on radiologic imaging, which resolve after appropriate therapy. the aim of this study was to stress the pivotal role of the polymerase chain reaction (pcr) in diagnosis and early detection of toxoplasma infection. clinical microbiology and infection, volume 11, supplement 2, 2005 methods: from january 1999 to july 2004 two hundred and thirteen patients underwent allogeneic peripheral blood stem cell transplantation (pbsct). in the presence of symptoms suggestive of cerebral, pulmonary and disseminated toxoplasma infection, 992 biological samples (blood, serum, csf, pleural fluid, bal) were collected and examined by nested pcr. results: fourteen cases of toxoplasmosis were identified, having an incidence of 6.6%. ten patients manifested cerebral toxoplasmosis, two had pulmonary infection and two disseminated. the overall mortality in this group, not necessarily due to toxoplasmosis, was 57% (8/14). the pre-transplant recipient serostatus was known in 9 patients: 5 were seropositive, but it is to be noted that 4 were seronegative, whereas 5 were unknown. mri study was performed in 12 patients. typical lesions were observed in 7 patients (sensitivity: 58%). the pulmonary toxoplasmosis occurred very early (day 9 and 10) and cerebral toxoplasmosis from day 29 to day 240 (mean 80.4) after pbsct. one case of disseminated infection had cns localization (day 21) and then pulmonary (day 42), whereas the second case had first pulmonary involvement (day 8) immediately followed by cns. blood samples demonstrated higher sensitivity (10 pcrpositive/12) than csf (6 pcr-positive/11) in identification of the etiologic agent. all patients with toxoplasmosis were treated with pyrimethamine and sulfadiazine. the study confirms the pivotal role of pcr in early diagnosis of toxoplasmosis after pbsct. in our experience, a positive pcr signal in blood was an early sign of toxoplasma infection, therefore blood appears to be a suitable and reliable biological sample for diagnosis. moreover, the samples were pcr-negative after the start of therapy and therefore pcr can be useful to monitor the effect of treatment. finally, our study indicates that toxoplasmosis is a potential pathology even in pre-transplant seronegative subjects, suggesting primary infection. cryptosporidium associated cholangitis in a livertransplant patient c. denkinger, p. harigopal, p. ruiz, a. tzakis, l. dowdy (wü rzburg, d; miami, usa) cryptosporidium parvum is a parasite of the group coccidia. c. parvum causes self-limiting diarrhoeal illness in immunocompetent hosts and severe long standing diarrhoea in immunocompromised patients. sclerosing cholangitis (sc) caused by c. parvum is frequently found in hiv-infected patients. in the transplant recipients only two case reports exist describing sc due to c. parvum; one in an adult renal transplant patient and the others in three children who underwent liver transplantation. we report herein the first case of c. parvum associated cholangitis in an adult liver transplant patient. this 52-year-old male patient underwent a liver transplantation three years prior to presentation. his initial transplant failed due to acute rejection requiring retransplant within one year. the second transplant was complicated by sepsis, tacrolimus induced hemolytic uremic syndrome, leaving the patient hemodialysis dependent. in 2004, he presented with pruritus, hypotension, severe dehydration and a three-week history of severe diarrhoea. endoscopic biopsy revealed numerous organisms in the stomach and changes in the colon consistent with cryptosporidiosis. stool cultures were negative. in addition, gamma gluytamyl transferase and alkaline phosphatase were elevated while bilirubin and ast were normal. the patient was diagnosed with colitis due to cryptosporidium and treated with azithromycin. immunosuppression with tacrolimus was continued. the patient continued to have diarrhoea, low-grade fevers and fatigue. two weeks later a cholangiogram and liver biopsy were performed. the liver biopsy revealed c. parvum lining the ductal epithelium. sc was diagnosed. the cholangiogram showed no sclerosing changes in the biliary tree, suggesting an early phase of disease. paromomycin was added to the regimen. the patient improved, and was discharged home. we believe this to be the first case of sc associated with c. parvum in an adult liver transplant recipient. objective: human herpesvirus 6 (hhv-6) infection usually occurs during the first 2 years of life, and its prevalence is higher than 95% in adult population. negative serostatus for hhv-6 in solid organ transplant recipients is a risk factor for fungal infection, but the incidence and severity of hhv-6 infections in seronegative patients has not been evaluated. our objective is evaluate prospectively seronegative hhv-6 solid organ transplant patients for hhv-6 infection by means of quantitative pcr for hhv-6. methods: from february to august 2004, we prospectly evaluated all solid organ transplant patients. patients must have a minimum follow-up of 3 months. patients underwent basal determination of igg for hhv-6. seronegative patients were included for follow-up. blood samples were obtained at 7, 14, 21, 28, 45, 60, 75, and 90 days post-transplant. for viral dna analysis, following dna amplification, it was determined by hibridation in microplaque (affigene). results: in the study period, 112 patients were evaluated (58 kidney, 35 liver, 11 heart, 4 kidney and pancreas, liver and kidney 2, pancreas 1, heart and kidney 1). nine patients were seronegative (7 kidney, 1 liver, and 1 heart). we analysed 67 plasma samples. all patients but one had a negative pcr for hhv-6. the patient with primary infection had a maximum of 78,002 hhv-6 dna copies/ml. clinically, the patient suffered from slight abdominal pain and discrete cholestasis in blood analyses; no fever or serious complications were detected. he did not receive pre-emptive treatment with ganciclovir. no fungal infections were detected. conclusion: primary infection by hhv-6 in seronegative solid organ transplant patients is not frequent, and in 1 patient it appeared with slight symptoms. however, a larger study with more seronegative patients is needed. development and validation of a new real-time pcr assay for hhv-6 detection and differentiation of hhv-6a and hhv-6b d. becker, s. ziegelmaier, s. wach, t. laue, t. grewing (hamburg, d) objectives: human herpesvirus 6 (hhv-6) has been identified as the etiologic agent of roseola infantum in infants. in adults hhv-6 causes complications in immunocompromised patients such as aids patients and transplant recipients. two virus variants can be distinguished: hhv-6a and hhv-6b. several studies have shown variant specific clinical outcome. the objective of this work was the development of a reliable, sensitive and specific hhv-6 detection assay, based on real-time pcr technology, which enables the discrimination between hhv-6a and b. this assay ought to run with the same temperature profile as the realart lc pcr assays for hsv1/2, ebv, cmv and vzv (artus gmbh). methods: by sequence alignments, a region of the hhv-6 genome was defined, which allows the specific amplification by real-time pcr, and discrimination between hhv-6a and b by melting curve analysis. assay specificity has been tested by analysing hhv-6 a/b reference material (abi) and cell culture supernatant from clinical isolates. cross reactivity was excluded by testing dna from related viruses and bacteria. analytical sensitivity was determined by probit analysis. a heterologous pcr system was incorporated, which serves as an internal control. a set of quantitation standards was established, for exact quantitation of the viral load. results: with the realart hhv-6 a/b lc pcr assay artus gmbh developed a ready-to-use assay for the detection and distinct differentiation of hhv-6a and b. quantitation of the viral load within a broad linear range is possible for both variants. simultaneous amplification and parallel detection of internal control and specific sample in one reaction tube excludes false negative results. the realart hhv-6 a/b lc pcr assay has the same temperature profile as the realart lc pcr assays for hsv1/2, ebv, cmv and vzv. 0 conclusion: the realart hhv-6 a/b lc pcr assay allows sensitive and specific detection, as well as subtyping of hhv-6. this leads not only to a reliable hhv-6 diagnosis, but also to a better understanding of the role of hhv-6 variants in pathogenesis. due to the same temperature profile, the realart hhv-6 a/b pcr assay completes the realart lc pcr assays for the detection of different herpes viruses. now the parallel detection of hsv1/2, ebv, cmv, vzv, and hhv-6 a/b is possible in a single real-time pcr run. thus, clinicians receive a reliable diagnostic tool for rapid detection of herpes viruses, and especially in transplant recipients. quantitative cmv pcr in allogenic stem-cell transplant patients l. cardeñ oso, c. blazquez, r. rodriguez, j. diaz-regañ on, e. lomas, p. sanchez, r. cámara (madrid, e) objective: to assess the clinical value of a commercial quantitative plasma cmv-pcr assay (cobas amplicor cmv monitor test, roche molecular system) in allogeneic sct patients by comparing the results obtained with the pcr with those obtained with antigenaemia. to evaluate the impact of an automatised dna extraction method and a lower cut-off on pcr sensibility. methods: all patients were monitored until day 100 post-sct with weekly antigenaemia and pcr. a total of 562 blood samples from 30 patients (23 mieloablative and 7 non-mieloablative; 25 hla identical siblings and 5 from unrelated donors) were tested prospectively. twenty-seven cmv seropositive patients (or negative with a seropositive donor) received high dose of acyclovir as prophylaxis. pcr was considered positive when more or equal 600 dna copies/ml were detected. antigenemia was considered positive when one or more positive cells were detected (in 2x105 pmns). positive samples by antigenaemia and/or pcr (n = 54 samples) were tested retrospectively with an automatised extraction method (magnapure). results: all cmv seronegative patients with a seronegative donor (n = 3) were antigenaemia and pcr negative. seropositive patients (n = 27): 14 had a positive antigenaemia and/or pcr, with a total of 23 cmv infection episodes. none developed cmv disease. pcr detected 10 out of 23 cmv episodes. overall there were 44 positive antigenemias (belonging to 13 patients): 23 were pcr+, 20 pcr negative and 1 pcr inhibited. only 1 patient had a positive pcr with a negative antigenaemia. automatised magnapure extraction increased the sensibility of the pcr. with this method pcr detected 19 out of 23 episodes in contrast with only 10/23 with the manual extraction. for samples with less than 600 dna copies/ml a manual calculation of the number of copies was retrospectively done (using a new cut-off of 50 copies/ml). with this lower cut-off, pcr detected 20 out of 23 cmv episodes. none of the negative patients by antigenaemia and/ or pcr gave a positive result with the automatised extraction method or with the lowered cut-off pcr. conclusion: quantitative cmv pcr assay showed a lower sensibility for the detection of cmv infection in allogeneic sct compared with antigenaemia. two technical modifications increased the sensibility without a decrease in the specificity: automatised magnapure extraction, and lowering the cutoff. trichosporon beigelii as a life-threatening pathogen in bone marrow transplantation recipients: the first case report from iran patients undergoing bone marrow transplantation (bmt) are profoundly immunosuppressed as a result of their intensive chemotherapy and are at high risk for opportunistic fungal infections mainly caused by candida spp and aspergillus spp. trichosporon beigelii (t. beigelii) has emerged as a life-threatening opportunistic pathogen in immunocompromised hosts. response to antifungal agents is poor, mortality is high and immunological recovery is the most important factor for a favorable outcome in patients with trichosporonosis. we present the first case of t. beigelii infection in patients undergoing allogeneic bmt in iran. a 12-year-old female presented with aplastic anemia, cough and fever. she had received cytotoxic drug therapy, broad spectrum antibiotics and was neutropenic. t. beigelii was repeatedly demonstrated by appropriate morphological and physiological characteristics in her sputum, nose and mouth ulcers by direct microscopy and culture, and also isolated and histopathologically recognized in post-mortem from lung and liver. trichosporonosis is likely to be recognized more frequently than apparent from the available reports. objectives: trichinellosis is a well-known problem in greenland. in west greenland a number of large outbreaks took place during the 1940's and 1950's, but since then only sporadic cases have been registered. it is unknown whether the decrease in trichinellosis cases reflects the general transition in greenland towards a more western lifestyle with less consumption of meat from wildlife, or whether it reflects insufficient case registration. the objectives of the present study were to determine the prevalence of human trichinellosis from the 1980õies to present times in nuuk, the capital of greenland, and in ammassalik district on the east coast of greenland, where more traditional food is still eaten, and to evaluate if changes in lifestyle had an effect on prevalence. methods: blood samples from 86 persons collected in 1981 and 533 persons collected in 1998 in nuuk and ammassalik district were tested for trichinella-specific igg antibodies using elisa and western blot analyses. background information was obtained from questionnaires from the 1998 study. results: in 1981 the trichinellosis prevalence was 8.7% in nuuk and 23.8% in ammassalik district, while the prevalence in 1998 was 5.2% in nuuk and 19.8% in ammassalik district. persons living in the settlements of ammassalik district had higher trichinella-specific antibody prevalence than persons living in the town of tasiilaq. the following risk factors were significant in a univariate analysis: ethnicity, age, place of living, dietary habits, intake of seal meat, and hunting. a multivariate model was constructed consisting of the variables: ethnicity, age, place of living, diet group, intake of seal meat, and hunting. the variables were removed stepwise in the following order: seal meat (p = 0.73), hunting (p = 0.36), age (p = 0.3), and ethnicity (p = 0.16). the final multivariate model consisted of diet group and place of living, both being significant. thus, living on a greenlandic diet and living in the settlements in ammassalik district implied higher relative risk of being trichinella seropositive than living on a diet dominated by imported food items and living in nuuk. conclusion: as lifestyle in the settlements is more traditional compared both with tasiilaq and nuuk, these results indicate that the decline in human trichinellosis cases in the 19th century most likely reflects the transition from traditional greenlandic lifestyle to more western dietary habits. study on prevalence of toxocara cati in vagrant cats of sari township, iran, 2004 m sharif, p. ziapoor, h. ziaee, s. kholami (sari, ir) objectives: toxocara cati is one of the important and prevalent parasites in cats and common between human and animals. ingestion of larvae of this parasite and lack of it's maturation in human body , the infected persons develop visceral larvae migrans which is known as toxocariasis. considering the significance of transmission of this parasite in human and due to the increase in vagrant cats in sari township, this study was performed in order to determine the severity and prevalence of toxocara cati in these cats in sari township. methods: this cross sectional descriptive study was done on 100 vagrant cats (28 males and 72 females) during april to october 2004. sampling was done randomly at different regions by net. the specification of vagant cats were recorded after hunting and were deeply anaesthetized by chloroform to annihilate them. the intestinal content were examined for the presence of parasites and counting of their number was done finally after fixation and staining by recommended kits of reference book for genus and species identification. results: in all, 42 (42 %) cats were infected with toxocara cati, of which 8(19%)were males and 34 (81%)were females. range of infection was 1-32 and mean severity of infection was 3.14 for each cat. the rate of infection at the west region was more than the other sites, and it was more in the females than males and also it was more in immature than the mature cats. conclusion: considering the merely high prevalence of this zoonotic parasite and it's hygienic significance in causing toxocariasis in human particularly in children, who are in more contact with soil and also lack of control on vagrant cats population, which are the potential risk factors, it is recommended to control personal and dietary hygiene and avoid storing the garbages in unprotected wire boxes on the streets in order to prevent the gathering of vagrant street cats. imaging in 100 patients of pulmonary hydatid disease; review of unusual imaging appearances s. zahirifard, m. bakhshayeshkaram, m. tahbaz, k. kaynama (tehran, ir) objective: to evaluate the chest radiography and ct scan characteristics of pulmonary hydatid disease. material and methods: 100 patients with surgically proven pulmonary hydatid cysts were enrolled for study. we reviewed clinical findings and imaging of patients. the radiological features (localization, diameter, architecture, density and other radiological signs and appearances) were determined. results: on cxr 124 cysts were determined. on 82 available ct scans a total of 112 cysts were detected. no discrete cyst was detected on 11 cxr. on 82 available ct, a total of 112 cysts were detected and in 5 ct scans no cyst was detected. 57 cysts were ruptured and 25 patients with ruptured cysts had hemoptysis. single cysts were in 63 patients while multiple cysts were in 37. median ct density was 24hu. respectively, it was seen on ct and cxr waterlily sign in 18 and 22 patients, air-fluid level in 12 and 17 patients and cresent sign on 11 and 5 of patients. inverse cresent sign and calcification were not observed on cxr s but each one was recorded on 4 ct scans. on ct scan 90% of cysts were smooth and 89% were uniloculated. 19% of cysts were infected. conclusion: ct scan should be done to elucidate cystic nature of the lung masses and for accurate localization in the preoperative period. in endemic regions like iran, atypical imaging presentation of complicated pulmonary hydatid disease such as solid masses should be considered in differential diagnosis of pulmonary lesions (abstract truncated at 250 words) the seroprevalence of coxiellosis in farmers and cattle in north-eastern turkey s. seyitoglu, z. ozkurt, u. dinler, b. okumus (erzurum, tr) objectives: coxiellosis is a worldwide zoonosis caused by coxiella burnetii. the aim of this study was to determine the abstracts prevalence of coxiellosis in cattle and farmers in north eastern turkey. methods: a total of 230 cattle and 92 human sera were collected in 2003, and tested for antibodies against c.burnetii by commercial elisa test. results: the antibodies of c. burnetii were detected in 22 (9.56%) cattle and 18 (19.5%) healthy farmers. seropositivity was found in 12 of 53 (22.6%) cattle with abortion history, and 10 of 177 (5.6%) cattle without abortion history (p < 0.05). there was correlation between animals and human seroprevalance in same district. thirteen of 18 farmers who were antibody positive had seropositive animals, and there were seropositive animals in the villages of remaining 5 cases. it was observed that seroprevalance of coxiellosis was higher in north districts (in animals 15.4%, in humans 32.4%), have drier and warmer climate, than in other districts (in animals 6.5%, in humans 12.1%) both for humans and animals (p < 0.05). conclusions: the study show that coxiellosis was an important health problem both in humans and in animals in our region. an unusual case of gramineae in sublingual salivary gland l. doganci, m. calgurel, y. gungen, g. kiran (ankara, tr) foreign bodies of organic nature and parasitic elements are very rare in oral cavity and in salivary glands in humans because of their structural and functional features. here we report a very interesting case, with sublingual salivary gland involvement of gramineae (brachiaria spp.) which was thought to be a parasitic life cycle in the gland. case: 40 y/o female teacher with a recent history of travel to mountains where she drunk and had an exposure to unsafe spring water. she had a strong stinging pain right after she drunk the water in her mouth, radiating to her neck. she developed fever, pain, lymph node swelling, difficulty of eating, weight lost and remarkable eosinophilia. she also noted a self moving small tail-like object on the orifice of the sublingual salivary gland. removal of the object revealed an interesting unusual living object. then, definitive diagnosis is gramineae (brachiaria spp) after parasitological and pathological consultation. comment: to our best knowledge, this unusual case is the first presented patient related to travel in our region. differential diagnosis has several entities including squid sperm bag sting, marine animal larvae and nematod and trematod life stages. evaluation of echinococcus granulosus prevalence using elisa and abdominal ultrasonography in a group of students staying in a state hostel in turkey objective: cystic echinococcosis, caused by larval form of echinococcus granulosus is one of the most important and widely distributed parasitic zoonotic diseases in the world. echinococcosis is a major problem in all regions of turkey but particularly prevalent in rural areas, where domestic livestock raising is common. the reported epidemiologic findings are usually based on retrospective evaluation pf surgical findings or hospital charts. well planned epidemiologic studies are limited. in this study our aim was to analyse the seroprevalance of cystic echinococosis and prevalance of lesions (with abdominal ultrasonography) in a group of young adult university students who were staying in a state hostel in bornova-izmir-turkey. method: the study group consisted 750 students (360 woman, 390 men, aged 20.92 ± 1.82, min 17, max 29). informed written consent was received from each student and they were requested to fill a questionnaire form. blood sampling was performed by iv puncture and sera were obtained after centrifugation. anti echinococcus granulosus antibodies were detected by using elisa tecnique. all participants were gven an appointment for abdominal ultrasonography. results: of 750 students 99 (13.2%) were seropositive for anti echinococcus granulosus ig g (table 1). a total of 250 students (49 seropositive-201 seronegative) were performed abdominal ultrasonography (3.5 mhz transducer, sonoline, elegra-siemens). two of 250 students (1 in liver, 1 in kidney both seropositive) had cystic lesions and were referred to surgery. well designed epidemiologic studies about cystic echinococcosis are lacking in turkey. a previous study showed seropositivity rate of 13.7% for echinococcus granulosus. our findings suggest that cystic echinococcosis is prevalent in turkey and epidemiologic studies combining elisa and abdominal ultrasonograpy are warranted. objective: anthrax is an epidemic zoonosis in eastern part of turkey. this study was aimed to investigate the epizootiology and epidemiology of anthrax in this region. methods: animal and human anthrax cases during 1992-2004 period were included in this study. data were obtained formal records of health directorate, institute of veterinary control and investigation. the diagnosis of anthrax had based on clinical findings and/or microbiological procedures, including gram strains and culture of materials obtained from lesions. results: in a 13-years period, 464 animal cases and 503 human cases with anthrax were detected. of 464 animal cases, 20 (4.3%) were sheep, others (95.7%) were cattle. most cases occurred between july and september in both animals and humans. anthrax was seen most frequently in erzurum, which is the important centre for animal commencement. when the first 6 years period (1992) (1993) (1994) (1995) (1996) (1997) was compared with last 7 years period (1998) (1999) (2000) (2001) (2002) (2003) (2004) ) the number of animal cases were 161 vs. 230 and human cases were 234 vs. 269. an analysis of the yearly case distribution shows that the incidence of anthrax in this region had not been changed. conclusion: anthrax is an important health problem both animals and humans in this region. the preventive measures should be taken for decreasing the incidence of anthrax. objectives: anthrax is endemic in kazakhstan and occurs among humans at an incidence rate of 0.1 to 1 per 100,000 population per year. during the late 1990s, several epidemics occurred associated with home slaughter of infected animals, and a general increase in incidence was seen among animals and humans. historically, kazakhstan accounted for 15% of the anthrax cases reported from the soviet union; and the plains of central asia may have served as the historical source of b. anthracis for the rest of the world. in kazakhstan, the geographic distribution of anthrax among animals and humans is focal and generally associated with areas considered to be ôhigh-riskõ. the determinants of this focal distribution are poorly understood. in addition, strains of b. anthracis isolated from outbreaks within these foci have not been compared on a molecular or pathogenic basis. methods: work has been initiated to study the geographic distribution and potential environmental preferences of b. anthracis using geographic information system (gis) technologies. results: the gis was used to map a set of b. anthracis strains from several outbreaks between 1997 and 2003 to evaluate geographic patterns. the gis is also being used to derive a historical database of spatially explicit human and livestock outbreaks from 1948 to 2003 for spatial analyses on the nationwide distribution of b. anthracis. this database contains information on the location of the anthrax incident, the year and month of the occurrence, whether the incident involved livestock or human, the outcome of the disease, and biological information about the strains. conclusions: although in a preliminary stage of application, the arcgis software can be used to map loci of anthrax occurrence. in the future, these maps will be used to identify factors that influence the occurrence and spread of the pathogen and to prevent infection of livestock or humans. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency (dtra) under the project «kb0-1950-al-03» and administered by u.s. civilian research and development foundation (crdf). objectives: the anthrax situation in the country was unfavorable from the 1950s through the 1970s. until the 1990s, anthrax in kazakhstan was an occupational disease in animal husbandry. after 1991, the practice of raising livestock for private use increased. this report shows that, at the same time, the incidence of anthrax in persons involved in this practice increased significantly. methods: in order to characterize the epidemic process, we used methods of descriptive epidemiology, using epidemiological variables (who became ill, where and when, trends of disease by time, sources, transmission factors of b. anthracis, and the age, gender, and occupation of persons who become ill with anthrax). results: anthrax occurs in rural areas among private owners of livestock and their families and persons hired to assist with the slaughter. in the last decade, 89.4% of cases of anthrax illness in humans were caused by the slaughter of domestic livestock. of these cases, 62.2% were unemployed rural residents, 2.3% were shepherds working for cooperatives, 11.7% were blue-collar workers, 5.4% were white-collar workers, and 18.1% were students. a single source, path and factors of transmission of b. anthracis are typical of this type of the disease. in recent years, the majority of cases involve infection of several persons during the slaughter of a single diseased animal. the source of infection is cattle in 48.2% of cases, sheep and goats in 15.7% of cases, horses in 31.2% of cases, and soil in 4.7% of cases. the factors of transmission of b. anthracis to humans are contact with the meat of livestock during slaughter and butchering in 91.9% of cases, vector-borne transmission in 2.7% of cases, and soil in 5.4% of cases. a large number of human cases occur in july through september. conclusion: in kazakhstan today, the prevalent sub-type of anthrax disease in humans involves non-professional activity at home. this requires changes in the tactics of preventing the disease. the research described in this abstract was objectives: severe acute respiratory syndrome (sars) is an emerging infection caused by a novel coronavirus known as sars-cov. during the early phases of the disease, the presence of the replicative intermediates of sars-cov has been shown in pbmc from patients. no information is available on the ability of sars-cov to stimulate ifn induction, although ifn-gamma may be activated in sars patients. we investigate the capability of sars-cov to give rise to a productive infection in normal pbmc, in parallel with the ability to activate ifn-alpha andgamma gene expression. methods: normal pbmc were infected with a moi 0.1. virus progeny formation was measured at subsequent time points, by back-titration of cell lysates on vero cells. cell mortality and apoptosis were detected by trypan blue and propidium iodide staining. in order to detect virus-specific plus-and minus-rna strand, total rna extracted from sars-cov-infected pbmc was retrotranscribed in the presence of the sense and antisense primers, respectively, targeting the replicase gene. measurement of sars-cov genomic rna was performed by quantitative real time rt-pcr. ifn induction was performed by exposing pbmc to sars-cov at different moi, ranging from 0.1 to 10. induction of ifn-alpha and -gamma was analysed by measuring their mrna levels by limiting dilution rt-pcr. sensitivity of sars-cov replication to exogenous ifn-alpha and -gamma was tested in vero cells pre-exposed to the individual cytokines of to a combination of both. results: in unstimulated pbmc, no infectious viral progeny formation was detected up to day 13 post-infection. nevertheless, minus-strand and genomic sars-cov rna peaked at 24 hours post-infection. dose dependent induction of both ifnalpha and ifn-gamma mrna was observed, that was most evident at moi 10. a combination of these two cytokines was shown to strongly inhibit sars-cov replication in vero cells. our results show that sars-cov is able to infect normal pbmc. however, this appears to be only a transient phenomenon, followed by a progressive disappearance of both virus-specific rna strands, with no infectious virus progeny production. moreover, sars-cov appears to have the intrinsic ability to activate dose-dependently both ifn-alpha andgamma gene expression in pbmc cultures. our results can be pathogenically relevant to the inflammatory events occurring in the diseased tissues that can be mediated by the endogenous activation of inf system. objectives: trichoderma species are potential candidates for biocontrol of plant pathogenic fungi and cellulase producers of biotechnological importance. on the other hand, they are emerging as opportunistic pathogens of immunocompromised and dialized patients. this study was designed to examine the ability of 10 clinical trichoderma isolates to produce extracellular proteases and to screen them for toxicity to mammalian cells. methods: supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. trypsin-and chymotrypsin-like activities were separated by sephadex g-100 column chromatography and their ph-dependence was studied. sperm toxicity bioassays were performed by exposing boar sperm suspension to trichoderma-methanol extracts, the motility of spermatozoa was estimated by phasecontrast microscopy. mitochondrial membrane damage in spermatozoa was studied by epiflurescence microscopy using the jc-1/propidium iodide staining. results: the production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities was common among the examined strains. separation of trypsin-and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. relatively high activity levels were detected between ph 5 and 9 suggesting that the different isoenzymes have different ph characteristics. more than 90% of the spermatozoa were motile in a non-exposed control sample and in a sample exposed to 5 microliter of methanol. more than 90% of the spermatozoa were immotile in samples exposed to t. longibrachiatum uamh 9515, atcc 201044, cm382 and t. harzianum cbs 102174 in a concentration of 1.25 mg biomass ml-1, indicating that these strains produce heat-stable substances toxic to sperm cells. these four strains were found to inhibit the motility of boar spermatozoa even at a low concentration (50% reduction of motility in the case of 5 microgramm ml)1 of extended boar sperm) and to induce dissipation of mitochondrial membrane potential. other studied strains had no toxic effect. conclusions: production of extracellular proteolytic enzymes and toxic metabolites are among the potential virulence factors of trichoderma strains as emerging fungal pathogens. this work was supported by grants f037663 of the hungarian scientific research fund and 53305 of the academy of finland. occurrence of scabies in patients and in the staff of healthcare facilities objectives: scabies is an itching dermatosis which continues to be a frequent health problem. long-term follow-up of the epidemiological situation confirms a thirty-year cycle for which there is no satisfactory explanation. methods: standard methodology of diagnostics is based on a) subjective sensations in the patient b) objective dermatological finding c) positive epidemiological history d) laboratory demonstrations of causative agent e) remission of symptoms upon specific therapy. cases of scabies diagnosed and reported by dermatologists nationwide are included by the public health service in the central system epidat (niph, prague). results: the last two documented epidemic waves of scabies affecting the czech republic peaked in 1970 and in 1993. specific morbidity is the highest among persons 15-24 years of age (it closely connected with sexual activity-the source of infestation in 60% of adults was a sexual partner and that is confirmed by an almost identical trend in reported scabies morbidity and fresh cases of syphilis in 1965-1999 years), but the morbidity trend has been falling sharply after the year 1993. however, morbidity in persons over 65 years of age has been gradually rising over recent years. group epidemics (86) have been repeatedly reported in such facilities as departments of gerio-psychiatry, institutions for long-term chronic patients, old folks homes, social care institutes and charity facilities. that occurs in bed-ridden subjects hospitalized on a long-term basis, the mentally retarded and the superannuated, in whom the scabies is of a nosocomial character. the affected staff of healthcare institution is very often the source of further transmission to others, namely to immobile patients, as well as to especially vulnerable individuals under higher risk of infection. analysis of the reported cases revealed a 13% share of healthcare workers in this infection. under greater risk are senior nurses (62%), junior nurses (21%), auxiliary paramedical staff (13%)and the last of all physicians (4%). scabies as an occupational disease occurs almost exclusively in the healthcare sector. conclusions: scabies poses on increased risk for the younger population (active scabies) and the elderly population (passive scabies). it poses a great occupational risk in the paramedical healthcare staff carrying out nursing or managing services. it is necessary to consistently observe preventive and repressive epidemiological measures in the population. fire ants, the new public health problem in iran k. akbarzadeh, m. nateghpour, s. tirgari (tehran, ir) ants are probably the most successful of all insects. they are present almost in all countries and all places. a few of them can bite, sting and squirt formic acid. formica rufibarbis and a few the others are secondary hosts of dicrocoelium dentriticum in iran. but the newest public health problem depending on ants in the country is biting and stinging of fire ant pachycondyla sennaarensis (formicidae; hymenoptera) in south and southeastern iran. ecology, biology and morphology of p. sennaarensis were considered in iranshahr county (southeast of iran) where has been much infected because of the ant. according to the survey over 92% of questioned individuals had bitten at least once with the ant. usually the effects of the stings are mild but this ant is capable of multiple stinging and this can induce annoyance people especially in children. objective: tick play as important role in diseases transmission to human being. different tick-borne diseasea including borreliasis, cchf have been reported from iran. method: an attempt was made to determine the fauna and geographical distribution of soft and hard ticks in this area from october 2002 until april 2004. about 10% of the villages were selected randomly and tick collection was carried out using standard method. ticks were collected from indoor resting and hiding places in 70 villages as well as inside stables, rodent borrows, on sheep, lamps, goat and cattel by monthly in each season. result: 3382 ticks were collected and identified using national systematic key. the copmosition and frequencies of them were as follows: ornithodoros lahorensis (11%), argas persicus (23.12%), rhipicephalus bursa (4.3%), rhipicephalus sanguines (1.53%), haemaphysialis concinna (9.6%), haemaphysialis sulcata (26.8%), haemaphysialis punctata (1.54%), hyaloma schulzei (8.16%), hyaloma marginatum (7.21%), hyaloma dentriticum (3.08%), hyaloma asiaticum(1.53%). conclusion: distribution and ferquency of tlck was differ in various season and location. evaluation of the use of saliva to soothe bloodsucking arthropod bites (dedicoat m et al, 2004 , j infect dis 190:1068 . the conditions for hhv-8 transmission are met when i) a child's skin responds to the bite of a blood-sucking arthropods, and ii) an hhv-8-seropositive mother (or caregiver) attempting to relieve the child's itching and to reduce scratching applies her infective saliva to the bite's site. the use of saliva is a traditional behavioural practice in sub-saharan africa, for healing with herbal leaves crushed and mixed with saliva and for medical practices and in the premastication of food (wojcicki jm, 2003 , br j cancer 89:2016 . the human behaviour associated with the transfer of saliva from parents to infants and the use of herbal leaves treated with saliva in relation to arthropod bite could be a risk factor for hhv-8 infection and perhaps other infections too (such as the hepatitis b virus). methods: to evaluate to what extent saliva is used to heal insect bites on children's and adolescentsõ skin, we draw up a questionnaire directed at students of elementary and intermediate school. two groups were tested, one from italy (a school near rome, latium, and schools in veneto) and one from a sub-saharan african country known for high transmission levels (uganda, between kampala and entebbe by lake victoria). results: the frequencies of the use of saliva were not significantly different in latium (12.5%) and in veneto (14.6%) so that an average frequency of 13.5% (96 / 712) has been compared with the frequency of 74.8% (119 / 159) from uganda (p << .0001). the frequencies in the two groups are related also to a different cultural and economic development and improvement in hygiene. conclusion: we suggest that blood-sucking promoter arthropods can play a role in the spread of hhv-8 infection particularly in africa. therefore, a behavioural change limiting the use of saliva on the skin and to prepare herbal leaves, could be an attempt to control the spread of hhv-8 infection. aim of study -to find out in csf some baseline markers of inflammation and redox status for the estimation of development tendences and clinical course of pathological process in tbe. patients and methods: 60 patients with meningeal and meningoencephalitic form of tbe treated at the infectology center of latvia were included in this study. diagnosis of tbe was confirmed by elisa, enzygnost, anti-tbe igm in blood and/or csf. traditional clinical criteria were used for the characterization of tbe severity degree. some nontraditional lab methods were used: c-reactive protein (crp) (immunoturbidimetrically) and reduced glutathione (gsh) concentration (colorimetrically) detection in csf. results: concentration of crp in csf was significantly higher in tbe patients in acute phase (5.55 ± 1.0 mg/l; n = 34) than in reconvalescence (2.41 ± 0.5 mg/l; n = 26), p = 99%. crp and gsh in about 50% of tbe cases in csf was under detectable values. the second part of tbe patients demonstrated tendency of the increasing of gsh during acute stage of disease (1.64 ± 0.20 mg%; n = 32) if compared with reconvalescence (1.41 ± 0.20 mg%; n = 24), p = 61%. no statistically significant differences of crp and gsh concentrations in csf were found in cases of moderate meningitis form of tbe if compared to severe meningoencephalitic form of tbe. conclusion: 1) analysis of data obtained from patients with tbe gives evidence of substantial crp and gsh changes in csf in dependence on disease stage, but not on its severity degree. 2) these objective biochemical data from csf investigation give proof that there are no separately moderate and severe tbe cases in clinic: they have to be interpreted always as equally severe. 3) the origin of crp and gsh in csf is unknown (production in cns and/or from blood due to blood-brain barrier permeability increasing?). background: cholera is a severe disease caused by vibrio cholerae. the natural reservoir of this aquatic pathogenic bacterium is referred to as the ôaquatic environmentõ. our group showed that egg masses of the non-biting midge chironomidae spp. (diptera) harbour v. cholerae and provide nutrient source for their multiplication, thereby offering a new natural reservoir for the cholera bacterium. objective: the presence of viable v. cholerae on flying adult chironomids will suggest that v. cholerae can be carried through the air to other water bodies by the adult flying insects and hence a new route of infestation of water sources by the bacteria is suggested. methods: in field studies chironomid egg masses and adults were simultaneously collected from the same environment. in addition fresh, drinking quality water was left exposed or netted in the vicinity of adult chironomid hatching foci. the exposed water was monitored for chironomid egg masses and v. cholerae presence. in the laboratory those experiments were repeated with v. cholerae o9 tagged with green fluorescent protein. results: to date, more then 30 v. cholerae serogroups have been isolated from chironomids egg masses and adults. in the field experiments, a clear-cut connection between netting and v. cholerae absence was noted implying that a relationship exist, between entrance of large invertebrate to water bodies and presence of v. cholerae in that water. in the laboratory experiments, the gfp tagged bacteria were found on the outer surface of the adult chironomid, in locations prone to microbial attachment. conclusions: the evidence shown here suggests that aerial transport of v. cholerae by chironomids flying adults is feasible and does lead to v. cholerae contamination of unprotected water bodies. objectives: cholera is a severe and potentially life-threatening diarrheal disease caused by certain spices of the gram-negative bacterium vibrio cholerae. many millions of cases of cholera occur annually, in epidemic and pandemic forms due to v. cholerae o1 and o139, and also sporadically due to non-o1 and non-o139 v. cholerae strains. in this context, the survival of v. cholerae in nature is of interest from an epidemiological perspective. although the natural reservoirs for survival and multiplication of v. cholerae are far from compeletly disclosed, our previous study has shown that free-living amoebae can be reservoirs for the seventh-pandemic v. cholerae o1 el tor-inaba strain n16961, which can survive and grow intracellularly in acanthamoeba castellanii.the aim of this study was to examine the ability of different strains of v. cholerae o1 el tor to grow and survive in acanthamoeba castellanii, and to examine whether intra-amoebic survival of bacteria alter their pattern of resistance and sensitivity to different antibiotics. methods: v. cholerae o1 el tor strains were co-cultured with a. castellanii for more than two weeks. the interaction between these microorganisms was followed by viable counts of alone-and co-cultivated microorganisms. intra-amoebic growth and localization of each bacterial strain were estimated by gentamicin assay, viable count, microscopy, and pcr to detect cholera toxin gene and amoebic 18s rna gene disclosing symbiont-host association. results: the results show that examined v. cholerae o1 el tor strains multiplied and survived inside trophozoites and cysts of a. castellanii. the bacterial internalization was in cytoplasmic compartment of the amoebae cells. the relation between these microorganisms in co-cultures could be classified as symbiosis, since presence of the amoebae enhanced growth of bacterial strains, and presence of the bacteria did not affect amoebic growth. the intra-amoebic survival of bacteria did not alter their pattern of resistance and sensitivity to different antibiotics such as ampicillin, gentamicin, and tetracycline. conclusions: this study shows a facultative intracellular behaviour of examined v. cholerae o1 el tor strains, which is in contrast to the general held view, which considers the bacterium to be extracellular microorganisms. the clinical importance of free-living amoebae is their possible role as ôtrojan horseõ. the genetic variation of vp7 and vp4 antigenic proteins was studied in 38 g4 strains recovered in palermo from 1985 to 2003. vp7 sequence analysis showed that 14 strains, recovered during 1999-2003, though cross-reacting with g4-st3:1 mab, belonged in fact to genotype g9 and were 99% identical to g9 strains recovered in palermo from 1999. sequence comparison of 23 g4 strains grouped them into three sublineages (ia to ic) containing respectively viral strains collected in 1990-1994, 1995 and 1999-2003 . amino acid sequences were well conserved within each sublineage and a peculiar single amino acid insertion was observed in the variable region 4 of all sublineage ic strains. both g4 and g9 italian strains exhibited p[8] genotype and their vp4-encoding sequences clustered in previously defined lineages following a temporal distribution: strains collected in 1989-1993 fell within lineage p[8]-1, while those recovered in 1995-2003 clustered in lineage p[8]-3. the variety of their deduced amino acid sequences enabled us to describe, respectively, two (1a and 1b) and six (3a-3f) different patterns of substitutions with regard to lineage p[8]-1 and p[8]-3 reference strains. comparison of vp7 and vp4 gene sequences of italian and european g4 strains indicates that different g4p[8] populations have been circulating in europe over the years and that the latest italian strains are more closely related to recent isolates from other countries than to the reference vaccinal st3 strain. these results will help increase knowledge about g4 rotavirus epidemiology and evolution, and might be useful for future rotavirus vaccine formulation. detection of rotavirus, adenoviruses and astrovirus in gastroenteritis cases among young children, first experience with adenovirus identification by pcr and microplate hybridisation assay , adeno (all human serotypes) and astroviruses antigens (seven human serotypes) was performed by enzyme immunoassay method (dako ideia rotavirus, adenovirus and astrovirus tests, respectively). all specimens positive for adenoviruses were confirmed and identified by a pcr-microplate hybridization assay (pcr adenovirus consensus, argene, biosoft), which used adenovirus genus-specific primers and one generic and six species-specific probes defined in the va rna gene. according to our results: rotavirus was detected in 63 cases (9.2%), adenovirus in 15 cases (2.2%) and astrovirus in 15 cases (2.2%). pcr adenovirus consensus assay identified the adenovirus species from all the 15 cases: adenovirus species f (enteric adenovirus serotype 40 / 41) in 11 cases (1.6%), adenovirus species c in 2 cases (0.3%) and adenovirus species a in 2 cases (0.3%). rotavirus gastroenteritis presented a seasonal distribution in spring, while adenovirus and astrovirus gastroenteritis presented no seasonality. conclusions: rotavirus gastroenteritis was significantly more common than adenovirus and astrovirus infections in young children in our territory. pcr adenovirus consensus technique was a useful method for the identification of adenovirus in stool specimens. eventhought enteric adenoviruses 40 / 41 (species f) caused the majority of acute gastroenteritis due to adenovirus, non-enteric serotypes (species a and c) were occasionally involved in the aetiology of acute diarrhoea. the study continues with the examination of great number of specimens and patients for the investigation of more useful and specific results. immigrant inpatients from 1999 until today: 'pressure' exerted over infectious disease wards r. manfredi (bologna, i) objective: foreign individuals officially living in bologna and province are 39186 with predominant origin from northern africa (31.5%), eastern europe (22%), and far east (12%). the temporal trend of admissions of patients coming from outside of the european union was examined according to a broad series of variables. methods: through the electronic databases of our hospital located in a metropolitan area of northern italy, informations related to all foreign patients (p) hospitalized or followed by day-hospital centres have been collected from january 1999 up to march 31, 2004. after recording all discharge diagnosisrelated group (drg) codes we focused attention on those regarding infectious diseases. results: overall discharges were 339051, with 6670 regarding foreign p:a prevalence of the female sex (64.4%) was noticed. in 49.6% of foreign p,the mean age ranged from 20 to 35 years. the rate of foreigners compared with overall admitted p varied from 0.3 of year 1999 to a. maximum of 0.47% of year 2002. a major involvement was borne by obstetrics-gynecology (39.3%), internal medicine (13.1%), specialistic surgery (7.3%), specialistic medicine and pediatrics (7.3% each), and emergency medicine (7.1%). when considering infectious disease-related drgs, tuberculosis (142 cases), hiv-aids (25), chronic viral hepatitis and related complications (22), septicemia, central nervous system infection, and acute viral hepatitis (12 episodes each), were the most frequent discharge codes followed by other infectious disease drgs (72 cases). the duration of admission of foreign p was very short (1-3 days) in 42.1% of cases,while it reached 4-7 days in 22.6% of p,and 8-14 days in 11.2% of cases, while the mean hospitalization time at infectious diseases ward was 8.6 ± 3.2 days (p < .002). even 549 hospitalizations ended with voluntary discharge which occurred during the first day of admission in 61.8% of p. discussion: our pilot study demostrates that the need for health care expressed by foreign p is increasingly frequent and covers a very broad spectrum of disorders with infectious diseases-associated illnesses gaining a increasing role through time. a significant problem is represented by the tendency to very short duration of hospitalization for foreign p, often ending with voluntary discharge while admissions carried out at our specialized infectious disease ward are significantly more prolonged, although they are expected to give an increased benefit in terms of effective management. change of the characteristics of natural focuses of plague and microbiological properties of plague microbes as a result of consequences of the natural calamity on aral sea enzootics of plague as that of any other transmissible feral herd [feral nidal] infection are determined by the interaction of a disease's causative agent and its warm-blooded carriers under specific ecological environment. introduction and objectives: change of the characteristics of natural focuses of plague and microbiological properties of plague microbes as a result of consequences of the natural calamity on aral sea. methods: conventional microbiological research methods were used, when studying microbiological properties of 116 strains of plague microbe from kyzylkum natural focuses of plague isolated from rodents, ticks and fleas. results: strains from the kyzylkum natural focuses of plague have all determinants of virulence, show characteristics that allow attributing them to the continental variety, i.e. glycerin digestion, do not ferment rhamnose and decompose arabinose; they have nutrient need that is typical for the strains from the central asian desert focus. there are differences in the need for amino acid leucine, whereas strains from ustyurt and gissar focuses are dependant on it. out of 116 strains from the kyzylkum natural focus only 8 turned out to be dependant on luecine. there were identified stains from the kyzylkum natural focus that had essentially different properties as compared to the majority of isolated cultures, in particular, those can not synthesize fraction 1 of the plague agent; have growth factor of different nature, i.e. lower dependence on phenylalanine. plague bacteria strains circulating in rodent populations in the kyzylkum natural focus are characterized by low virulence in white mice and guinea-pig, although they have all the determinants of virulence. when studying resistance to antibiotics, there were identified strains non-susceptible to streptomycin (5%). conclusions: thus, studying the properties of the causative agent of plague is of great importance for solving problems of natural focuses and understanding patterns of fluctuations in the epizootic situation in a certain territory. this understanding is an integral part of determining the epidemic potential of natural focuses, and is used in organizing epidemiological surveillance of the infection. objectives: toll-like receptors (tlr) play a key role in the recognition of products from virtually all classes of pathogenic organisms. amyloid peptides also can stimulate the innate immune system. for this reason, we hypothetized that bacterial compounds and amyloid peptides may jointly stimulate the innate immune system. methods: the interaction of endogenous and exogenous stimulators of innate immunity was examined in primary cultures of mouse microglial cells after application of defined toll-like receptor (tlr) agonists [lipopolysaccharide (lps) (tlr4), the synthetic lipopeptide tripalmytoyl-cysteinyl-seryl-(lysyl)3-lysine (pam3cys) (tlr2) and single-stranded unmethylated cytosineguanosine (cpg) oligodesoxynucleotide (tlr9)] alone and in combination with amyloid beta peptide (abeta) 1-40. supernatants from stimulated glial cultures and unstimulated controls were analysed for nitric oxide (no), tumor necrosis factor-alpha (tnf-alpha) and interleukin-10 (il-10) production. results: co-administration of abeta1-40 with lps or pam3cys led to an additive release of no and tnf-alpha. in contrast, coapplication of abeta1-40 with cpg led to a substantial decrease of no and tnf-alpha release compared to stimulation with cpg alone. cpg was the only compound investigated, which induced the release of the anti-inflammatory cytokine il-10. conclusion: the additive effect of lps and pam3cys and abeta may be one reason for the clinical deterioration frequently observed in patients with alzheimer's disease during infections. the substantial decrease of no and tnf-alpha release after cpg and abeta application compared to stimulation with cpg alone suggests that not all microbial products enhance the stimulatory effect of abeta on innate immunity. the cause for the divergent behaviour after activation of tlr9 versus stimulation of tlr2 and tlr4 probably lies in differences of the signaling cascade. hla-b27 is a marker for susceptibility and severity of reactive arthritis after salmonella, shigella, and yersinia infections but not after campylobacter and e. coli enteritis p. schiellerup, h. locht, k.a. krogfelt (copenhagen, dk) objectives: reactive arthritis (rea) is a well-known complication after infections with salmonella, yersinia, shigella and campylobacter. the presence of tissue-antigen hla-b27 is associated with rea, and hla-b27 may be a marker of a prolonged disease course. we designed a case-case comparison study of rea after gastrointestinal infections caused by salmonella, yersinia, campylobacter, shigella and e coli, to evaluate the impact of hla-b27 on attack rate and severity of rea between these 5 bacteria. methods: between january 2001 and november 2002 consecutive patients were included from the danish registry of gastrointestinal infections, when culture positive for salmonella, yersinia, campylobacter, shigella, or e coli. all patients were addressed by a questionnaire. in this study rea includes reactive arthritis and arthralgia. debut of pain in a previously healthy joint was registered and patients were asked to mark swollen or tender joints on a drawing. a vas-scale was used for assessment of pain caused by rea. all patients were encouraged to donate blood samples for serology and hla-b27 typing (pcr). results: a total of 2106 patients returned the questionnaire (response rate 67%). only subjects with mono-infections were included. the male/female ratio was 40/60. blood samples were obtained from 1558 (74%): campylobacter (n = 693), salmonella (n = 503), e. coli (n = 213), shigella (n = 74) and yersinia (n = 75). the overall prevalence of hla-b27 was 9%, corresponding to the rate within the danish population. odds ratios for contracting rea when the individual was hla-b27 positive were; campylobacter 1.70 (ci: 0.89; 3.43), salmonella 3.4 (ci: 1.83; 6.37), e. coli 1.1 (ci: 0.23; 5.04), shigella 33.5 (ci: 2.48; 452.78) and yersinia 5.1 (ci: 1.03; 25.68). thus, hla-b27 was significantly more frequent among rea-patients with salmonella, yersinia, and shigella compared to campylobacter and e coli. the vasscore for rea was significantly higher in the groups with yersinia and salmonella. there was a significant positive correlation between vas-score and the fractions of patients that were hla-b27 + . conclusions: this large comparative study shows that hla-b27 has a significant impact on the susceptibility to contract rea after salmonella, shigella, and yersinia, but not after campylobacter or e. coli. furthermore the severity of rea as measured by vas differed among bacterial species and was strongly correlated to the presence of the hla-b27 antigen. objectives: the normal gut microbiota triggers experimental colitis and large intestinal manifestations of human inflammatory bowel diseases (ibd). however, the availability of animal models for the study of ileitis is limited. thus, only little is known hitherto in small intestinal ibd. recently, parasiteinduced ileitis in the mouse, mimicking characteristics of ileitis in human ibd, was proposed as a model for crohn's disease (cd). oral infection of susceptible c57bl / 6 mice with toxoplasma gondii induces a severe th1-type immunopathology, which is restricted to the terminal ileum. in order to unravel the mechanisms underlying ileitis, we used this model to monitor microflora changes during ileal inflammation and determined contributions of the gut microbiota to ileal disease. methods: c57bl / 6 mice were infected perorally with 100 cysts of t. gondii (me49 strain). ciprofloxacin, metronidazole, ciprofloxacin plus metronidazole (each 50 mg / kg body weight / d, p.o.), piperacillin plus tazobactam (200 mg/kg body weight/d, i.p.) were applied from day 0 until day 8 after t. gondii-infection. the ileal bacterial flora was analysed on day 8 p.i. by microbiological and molecular methods (16s rdna-targeted denaturing gel electrophoresis (dgge), sequencing of clone libraries). results: microbiological and molecular approaches revealed that ileal inflammation leads to an increased total bacterial load and to drastic changes in the flora composition. in the acute stage of disease, the grampositive cocci and rods -predominant in the ilea of healthy mice -are displaced by gramnegatives, identified as escherichia coli and bacteroides sp., respectively. antibacterial treatment (ciprofloxacin, metronidazole, a combination of both, or piperacillin plus tazobactam) ameliorated disease symptoms and reduced ileal inflammation. this was also seen in spf-mice colonized by only one grampositive bacterial species. conclusions: the fact that gramnegative bacteria accumulate during acute ileitis and contribute profoundly to intestinal inflammation is well in line with similar observations in experimental colitis and in human ibd. this provides evidence that gut flora modulation is a valuable therapeutical strategy for ibd treatment. finally, the contribution of gut microbiota to ileal disease supports the use of the parasite-induced small intestinal inflammation as a model for ibd research. objectives: pathophysiologic mechanisms that contribute to brain injury in neuroinflammatory disorders include breakdown of the blood-brain-barrier, extravasation of leukocytes, cerebral hypoperfusion and vasculitis. matrix-metalloproteinases (mmps) including the collagenases mmp-2 and -9 are crucially involved in all these steps. in this study we aimed to assess the extent of in-vivo collagenolytic activity in cerebrospinal fluid (csf) by determination of the amino acid hydroxyproline, a major and exclusive degradation product of collagen. methods: paired serum and csf samples from patients with bacterial meningitis (n = 11), aseptic meningitis/encephalitis (n = 17), multiple sclerosis (n = 13), and normal csf (n = 12) were assessed. degraded collagen was hydrolysed to dissolve its major component hydroxyproline, which subsequently was determined spectrophotometrically. csf levels of mmp-2 and -9 were studied by gel zymography. in a rat model of pneumococcal meningitis localization of collagenolytic activity was performed by in-situ zymography with intramolecularly quenched gelatin. results: hydroxyproline in csf from patients with bacterial meningitis was significantly increased compared to all studied groups (p < 0.001) while serum hydroxyproline did not differ significantly between the groups. the amount of hydroxyproline in csf correlated significantly with the amount of mmp-9 (r = 0.8; p < 0.001). in the rat model in-situ zymography localized gelatinolytic activity to the subarachnoidal and ventricular space inflammation and in association to cortical lesions. the study documents a significant increase of the collagen degradation product hydroxyproline in csf of patients with bacterial meningitis. the close correlation of hydroxyproline and mmp-9 in the csf validates the assessment of hydroxyproline as an index for csf collagenolytic activity in neuroinflammatory diseases and supports a role for collagenases in the pathogenesis of bacterial meningitis. introduction: toxic shock syndrome (tss), is an acute, noncontagious systemic illness. it is caused by the toxin producing strains of s. aureus and the b-haemolytic streptococci and can occur in any non-immune person exposed to a tss toxin. tss is commonly associated with menstruation and tampon use, however can also be related to skin or soft tissue infections, particularly post surgical, skeletal infections or respiratory tract infection. tss is often non-immunising and recurrent menstrualassociated tss is well-described. literature suggests that tss is extremely rare, but diagnostic difficulties can lead to misdiagnosis and tss can be fatal if left undiagnosed. we report a series of three cases of tss, presenting within a short period of time. case reports: case 1. 17 year old female, presented with sudden onset collapse, diarrhoea, vomiting and abdominal pains. she gave no history of menstruation and an initial diagnosis of severe gastroenteritis was made. she failed to respond to conservative management and required itu support. she was discharged with no firm diagnosis and re-presented one month later with similar symptoms, when a diagnosis of staphylococcal tss was confirmed. case 2. 15 year old female, presented during menses with sudden onset rash, rigors, severe diarhhoea, vomiting and abdominal pains. she was diagnosed with staphylococcal tss on admission. case 3. 28 year old female. presented with sudden onset severe diarrhoea, vomiting, pyrexia and rash. she gave no history of menstruation. she responded poorly to treatment, required itu support, high dose steroids and was eventually diagnosed with streptococcal tss. discussion: diagnostic criteria for tss include high fever, hypotension, erythematous rash and a complicated multisystem disfunction. patients often require aggressive management. the public health laboratory service reports an average of 18 cases of diagnosed tss in the uk per year. however, because of the uncommon and difficult nature of the diagnosis, many cases are misdiagnosed and therefore go unreported. it is essential to maintain a high level of suspicion for patients who are epidemiologically at high risk, but importantly, also the less ill patient with suggestive symptoms who fails to meet all diagnostic criteria but are in an at-risk group. high morbidity and mortality has been reported for undiagnosed and untreated cases. objectives: staphylococcus aureus colonization and infection of burn wounds increases morbidity and delays wound healing of patients suffering from burn wounds. a vulnerable moment in the route of transmission of s. aureus is the exposed burn wound during dressing changes. the aim of this study was to evaluate the effect of dressing changes under laminar flow conditions on burn wound patients with regard to s. aureus colonization. methods: from the first of february 2003 to the first of june 2003, 25 patients were included in this study at our 10-bed burn centre. in 12 of these patients the dressings were changed under laminar flow conditions. all patients were frequently screened for s. aureus in nose, throat and burn wounds. all s. aureus isolates were genotyped with pulsed field gel electrophoresis. results: at admission 17 / 25 (68%) patients had no s. aureus colonization in their nose, throat or burn wounds. dressing changes under laminar flow conditions were carried out on 8 / 17 patients of this group. however, 6 / 8 (75%) patients acquired burn wound colonization with s. aureus during their stay at the centre. six out of 9 patients (67%) whose dressing changes were not carried out under laminar flow conditions acquired burn wound colonization with s. aureus. a total of 12 patients acquired burn wound colonization; these patients also acquired carriage of s. aureus in nose or throat. the same type of s. aureus was carried in the nose or throat as well as the burn wounds in 7 (58%) of these patients. the results of this study suggest firstly that dressing changes under laminar flow conditions does not prevent colonization of burn wounds with s. aureus, and secondly, that the exogenous route plays an important role in the transmission dynamics of this pathogen in burn wound centres. future research should be focused on this mode of transmission of s. aureus in this special circumstance. mupirocin prophylaxis to prevent staphylococcus aureus wound colonisation in patients admitted to a burn centre objectives: staphylococcus aureus (s. aureus) colonisation of burn wounds increases morbidity and delays wound healing. many burn wound colonisations with s. aureus are endogenous in nature. the aim of this study was to evaluate the effect of eradication of nasal s. aureus with mupirocin in patients with regard to s. aureus colonisation of their burn wounds. methods: from september 2000 to march 2001, 42 patients admitted to our 10-bed burn centre were screened for s. aureus in nose and burn wounds. isolates were genotyped with pfge. all patients received nasal mupirocin at the time of admission to the burn centre. fifty-five patients from the same unit who were followed from september 1997 till may 1998 and had not received mupirocin prophylaxis served as a historical control group. results: at admission 29 / 42 (69%) patients in the mupirocin trial had no s. aureus colonisation in their burn wounds. of this group 25 patients were non-carriers and 4 patients were nasal carriers of s. aureus. seven (28%) non-carriers and 2 (50%) nasal carriers acquired burn wound colonisation with s. aureus during their stay at the centre. of the historical control group, 39 (71%) patients had no s. aureus colonisation of their burn wounds at the time of admission. of this group 31 patients were noncarriers and 8 patients were nasal carriers; 18 (58%) non-carriers and 7(88%) nasal carriers acquired s. aureus wound colonisation during their stay. the overall probability of wound colonisation in the patients treated with mupirocin was significantly lower than in the historical non-treated group (p = 0.01, chi-square test with yates correction). conclusion: the results of this study suggest that application of nasal mupirocin to all patients upon admission to a burn centre may reduce but not eliminate the risk of subsequent s. aureus colonisation of burn wounds. other measures including improved infection control practices and eradication of exogenous s. aureus reservoirs, including s. aureus carriage among healthcare workers, may be necessary to further reduce the incidence of s. aureus burn wound colonisation. controlling an outbreak of staphylococcus aureus on a surgical ward results: the characterisation of the s. aureus in february showed a cluster of identical strains in three patients and hcw a. the strains of two patients were similar to the strain of hcw b. three patients had a unique strain. in the surveillance period a total of eighteen isolates in sixteen patients were found. in the surveillance period the epidemic-strains were found in other patients but only in patients who have been on the ward during the epidemic period. two patients had an identical new isolate. that indicates that the exchange between patients of s. aureus may occur without notion. another remarkable observation was that some of the patients lost their original strain and acquired a new isolate of s. aureus during hospitalisation. conclusions: a small outbreak of s. aureus could be traced to 2 hcw who proved to be carriers. relieving them from patient contact stopped the outbreak. molecular surveillance is an appropriate way to evaluate the success of measurements taken and to underscore the importance of hygienic measures. risk factors for s. aureus carriage in health care workers a. widmer, m. lang, r. frei (basel, ch) introduction: s. aureus (mssa) carriage is common among health care workers (hcws)and might be associated with type of care provided and other potential risk factors. transmission of mssa has been observed by ''cloud adults''. objectives: to determine risk factors for mssa carriage in hcws, and the percentage of methicillin-resistant s. aureus (mrsa) in a tertiary care centre. methods: retrospective review of all data of staff health department and the microbiology laboratory. all hcws who were working in a high mrsa prevalence country were routinely screened at the time they were hired by the institution. in addition, data were generated from mrsa screenings of hcws if there was evidence for nosocomial transmission of mrsa in patients. mrsa was defined as s. aureus being oxacillin-resistant and expressing the pbp2' and/or being positive for the meca gene by pcr. data of hcws were collected by chart review in a case-report form that included demographic data, work place, skin diseases and other variables. data were compared by univariate and multiple logistic regression analyses. results: a total of 3474 swabs were taken from 1217 hcws between jan 1997 and dec.2001. data were available from 1182 hcws:50% originated from switzerland, 27% from germany, 4% from france, and 19% from other countries. the overall prevalence of mssa and mrsa was 53% and 1%, respectively. more than one sample was available from 332 individuals: of the repeat samples, 38% were negative, 32% were negative, but had one single positive culture, and 30% were consistently positive. sex, profession, work place, number of years at the institution was not associated with s. aureus carriage (p > 0.2). the only significant risk factor for s. aureus carriage was young age, defined <35years (p = 0.02). the prevalence of mssa increased from 51% (1997) to 63% (2001) . eight hcws were carriers of mrsa: they were young and half of the them reported skin diseases. conclusion: s. aureus carriage among hcws is common: only young age remained a risk factor even after adjusting for other variables. mrsa was rare among hcws. prevalence of nasal carriage of methicillinsusceptible and methicillin-resistant staphylococcus aureus among hospital personnel and outpatients st. fokas, sp. fokas, c. tsamolia, m. skoutari (eghio, gr) objectives: to assess the nasal colonization of staphylococcus aureus among hospital staff in the absence of an epidemic and to compare it with the nasal carrier status among outpatients, focusing on the mrsa rate. methods: the study included 150 hospital personnel and 150 adults admitted to outpatient clinics of our hospital. given that a history of hospitalization is the most important facilitative factor for colonization of staphylococcus aureus we exclude individuals with a history of hospitalization in the preceding year of our study. the specimens were obtained with swabs from the anterior nares-ecological niches of s. aureus-and cultured according nccls guidelines. id 32 staph strips were used for identification and confirmed with slidex staph plus kit (both by biomerieux). mrsa strains detection was achieved with slidex mrsa detection kit of the same company. antibiotic susceptibility of mrsa strains was determined with atb staph method (version 2000) according to the recommendations of the producer (biomerieux). results: among hospital staff the carriage rates of mrsa and methicillin susceptible s. aureus were 2.7% and 20% respectively. none of the outpatients was found to be a nasal carrier of mrsa. in addition mssa carriage in this group was 11%. the prevalence of mrsa nasal carriage rate among personnel working in the different medical wards was also examined. the difference in the rates among personnel working in the surgical ward, operating theaters and the rest of the staff was statistically significant (p < 0.001). all mrsa strains were susceptible to oral antibiotics. conclusions: although the prevalence of mrsa is low among hospital personnel, education is needed as they are one of most important reservoirs for nosocomial mrsa infections. the low incidence of mrsa infections around the time of the study suggests that with usual infection control practices mrsa spread was avoided. all mrsa carriers were treated with intranasal muripocin for eliminating the nasal carrier status. continuous monitoring of mrsa carriage is needed to avoid nosocomial spread. aids patients are a special population related with high risk of infection due to staphylococcus aureus. previous colonization is a recognized predisposing factor for development of infection due to this organism. however, nasal carriage of s. aureus in hivpatients is still not completely characterized. objectives: to describe the epidemiological features of community-acquired s. aureus nasal colonization in out care hivpatients based on the molecular genotypes. methods: hiv-outpatients without previous hospitalization within the last two years were screened for s. aureus nasal colonization. three samples from each patient were collected, and the variables associated with colonization were assessed. nasal carriage was classified as: absent, transient colonization or persistent colonization. the persistent colonization was assessed according to the molecular profiles performed by pulsed-field gel electrophoresis, in: simple (same profile), multiple (different profiles) or combined (two with the same e and one with different profiles). results: 111 patients were enrolled in the study. seventy patients (63%) had at least one culture positive for s. aureus and 99 patients concluded three samples collection. only one isolate was a community acquired mrsa. the rates of colonization was higher when two or more samples were taken, ranging from 45.5% in the first sample up to 65.6% when 3 samples were collected. hiv-patients with aids were more likely to be colonized than non-aids patients (p=0.02). among the patients with s. aureus nasal carriage, 39% were transient and 61% were persistent carriers, whose genomic subtype was: 61.5% simple, 20.5% combined and 18% multiple persistent colonization. previous use of antibiotics was associated with absence of s. aureus colonization (p < 0.05). conclusions: hiv out care patients had high rates of oxacillinsusceptible s. aureus nasal colonization. regardless, the recent literature, only one isolate was community-acquired mrsa. the most common characteristic of colonization is simple persistent colonization showing the same genomic profile. objectives: s. aureus bacteraemia (sab) continues to be a major problem related to both community-acquired and nosocomial infection. we undertook an evaluation of a large cohort of patients with sab to assess features of the infection and predictors of mortality. patients and methods: 238 cases of sab admitted to our hospital (2000-3) were prospectively studied. all patients had at least one positive blood culture for s. aureus and clinical symptoms of bacteraemia. sab was considered to be nosocomial if the first positive blood culture specimen was obtained > 72 h after admission and there was no clinical evidence of infection on admission. microbiological studies were carried under standard protocols. epidemiological and clinical characteristics, antimicrobial treatment and outcome (relapse or mortality) were analysed. results: 434 cases of sab were identified but only 238 were prospectively included in the study protocol. incidence was the highest in year 2001 (4.21 per 1000). out of the 238 cases, 167 (70%) were male with a mean age of 51 year (sd 24.89 have not yet implemented similar guidelines. this study evaluated the detection and reporting of mlsb results in staphylococcus using the bd phoenix tm automated microbiology system and bdxpert system with nccls, sfm or din as interpretative standards. methods: comments regarding mlsb resistance as listed in the nccls m100-s14 were converted into expert rules. special bdxpert rules were also constructed for the detection and reporting of mlsb resistance when applying sfm or din guidelines. a total of 177 strains of staphylococcus (146 s. aureus, 12 s. epidermidis, and 19 coagulase-negative staphylococci) were tested in phoenix panels containing erythromycin (e) and clindamycin (cc). nccls, sfm or din breakpoints were used to interpret the phoenix mic results. the double disk diffusion d-test (e and cc) was used as the reference method for the determination of mlsb resistance phenotypes. results: the phoenix e and cc mic values were interpreted based on the standard selected. the bdxpert rules were executed and applicable expert messages were displayed. the phoenix correctly detected 38 out of 43 constitutive mlsb (cmlsb) phenotypes as compared to the d-test results. the remaining 5 cmlsb strains were interpreted by the bdxpert as potential inducible mlsb (imlsb)/efflux phenotypes. a total of 69 imlsb and 22 efflux phenotype isolates were all reported by the bdxpert as imlsb/efflux phenotype and the users were alerted to perform d-test before reporting the cc results. the cc interpretation was suppressed in these isolates. the nccls, sfm, or din showed identical detection and interpretation of mlsb resistant phenotypes by the phoenix and bdxpert systems. conclusions: the phoenix and bdxpert systems can assist laboratories in rapid detection and accurate interpretation of mlsb results for staphylococcus. special messages can be used to communicate timely and accurate information to clinicians for proper therapy of staphylococcal infections with mlsb resistant phenotypes. in vitro activity of new compounds tested against multi-drug resistant s. aureus isolated in latin american medical centres background: oxacillin-resistant s. aureus (orsa) isolated in lamcs shows high rates of co-resistance (r) with most antimicrobial classes used in the clinical setting. we evaluated the mdr patterns of orsa strains collected in lamc by the sentry antimicrobial surveillance program in 2003. we also evaluated the susceptibilities (s) of new, investigational compounds against these important endemic pathogens. methods: among 1.437 s. aureus collected, 507 (35.3%) were r to oxacillin. these strains were isolated from 10 lamcs (5 countries) and tested against > 30 antimicrobials by nccls broth microdilution methods. orsa strains were grouped by the mdr patterns for the 8 primary drugs (47). conclusions: all newer compounds (lzd, q/d, dap and dal), tei and van were very active against endemic and epidemic orsa in lamcs. these s. aureus having mdr patterns (ave. r to 6 agents) will require greater use of newer compounds particularly in brazil. objectives: coagulase negative staphylococci (cons) represent a part of physiological microflora. however, in recent years, they have also emerged as nosocomial pathogens of infections associated with indwelling medical devices (e. g. pleural drains), mainly in immuno-compromised patients. the aim of this study was to determine dynamics of nasopharyngeal colonization by cons in patients with resectable lung cancer during short-term hospitalization, especially those receiving routinely preoperative antimicrobial prophylaxis. methods: in present study cons species were selected from patients divided into two groups: (i) patients who were undergoing pulmonary resection and receiving preoperative prophylaxis (piperacillin, cefuroxime alone or in combination with amikacin) -ôprophylaxisõ group and (ii) control group. throat and nasal specimens were taken from each patient twice, in four-days interval. all isolates were identified by the biochemical microtests api staph (biomerieux) and tested for drugs susceptibility according to nccls reccomendations. results: 187 strains of cons strains were selected from clinical specimens: 137 strains from ôprophylaxisõ group and 50 strains from control group. the most often isolated species was staphylococcus epidermidis with the predominated api numerical profile 6606113. a detailed analyses of the api numerical profiles and resistance to antimicrobial agents indicated that during four-days interval of hospitalization, changes in phenotypes among cons colonizing nasopharynx were observed in both groups of patients. the changes in drug resistance patterns were found more often in ôprophylaxisõ group compared to those in control group -48 / 54 (88.9%) and 11 / 22 (50%), respectively. this difference was statistically significant (p = 0.0007 -chi2 test with yates correction). conclusion: our data suggest that preoperative antimicrobial prophylaxis routinely used in patients with resectable lung cancer may be regarded as an important factor predisposing to changes in drug resistance patterns among cons isolates colonizing nasopharynx. the susceptibility of coagulase-negative staphylococci in a teaching hospital compared with a secondary care hospital results: in total, six different species were identified in 71 isolates, 72% of which were staphylococcus epidermidis. in the lumc the species diversity was greater than in the mca, with five isolates of staphylococcus haemolyticus found in the first. no difference between the two hospitals was found in activity of the antibiotics tested, except for li. the mic90s were as follows: v 2 mg / l, t 4 mg / l, c 16 mg / l, m 2 mg / l, l 8 mg / l. of li the mic90 was 2 mg / l in the lumc and 1 mg / l in the mca. according to dutch guidelines, in total 7 / 71 isolates were found resistant to t and 21 intermediate resistant. all isolates were susceptible for v. conclusion: the first choice glycopeptide for serious cons infections, t or v, has no influence on the susceptibility of cons for these antibiotics. according to dutch guidelines, the activity of t, c, and l against cons is relatively low, whereas the activity of m and le is in the susceptible range. objective: staphylococcus epidermidis is one of the bacterial species mainly implicated in foreign body associated infections. we have characterized several s.epidermidis clinical isolates for their ability to form biofilms and their resistance to antibiotics. methods: 76 s.epidermidis strains isolated from implantable medical devices have been collected from hospitals of central italy. susceptibility to penicillin, methicillin, erythromycin and tetracycline has been determined in vitro by e-test according to nccls guidelines. the specific antibiotic resistance determinants have been checked by pcr (blaz, meca, erma, ermb, ermc, msra, tetk and tetm). the ability to form biofilms has been determined: (i) by pcr, detecting genes specific for attachment and biofilm development (icaadbc operon, aap, and atle); (ii) by congo red agar (cra) plate test to assay the production of polisaccaridic intercellular adhesin (pia); (iii) by crystal violet (cv) stain to determine the biofilm biomass development on polystyrene microtiter plates; (iv) and by cslm microscopy observations to investigate biofilm structure. results: 94% of the strains under study was resistant to penicillin, 87% to methicillin, 72% to erythromycin and 25% to tetracycline. on the side of biofilm-specific genes detection, 66% of strains was positive to ica operon genes, 82% possessed atle gene, and 42% aap determinant. in 89% of the population, the cra test confirmed the correlation between the presence of ica genes and slime expression. the cv assay classified the quasitotality of our strains (97%) as biofilm producers on plastic surface. in addition, the distribution of optical density values (od540) obtained after cv stain, showed a significant statistical difference in biofilm biomass development between the ica-adbc-positive strains and the icaadbc-negative ones. finally, a correlation, although not always present, has been observed between ability of the strains to develop in a high-structureted biofilm and specific biofilm-formation determinants. conclusions: analysing the origin of colonization or infection in 6 patients with cons isolated from the operative site, the bacteria could only be traced in one patient who developed an infection. transmission most probably occurred by direct contact. the low level of bacterial contamination in the uca together with the lack of correlation of genotypes between isolates found in the uca and in the wounds suggest that airborne transmission does not play a major role in the development of swi. more than 90% of staphylococci found in air samples were not traceable to any investigated sources. most probably, they originated from non swabbed parts of the hcw's bodies. since 50% of the traceable isolates from the uca originated from non scrubbed team members, protection of the operative site by lfv appears to be suboptimal. clonality of endemic methicillin-resistant staphylococcus epidermidis strains isolated in a neonatal intensive care unit n. ben saida, a. ferjani, n. sfar, j. boukadida (sousse, tn) objectives: methicillin-resistant staphylococcus epidermis (mrse) is one of the important pathogen responsible for nosocomial infections particularly in newborns. the aim of our study was to establish if these infections are caused by endemic clones or by incidentally occurring bacterial strains of this ubiquitous species and to evaluate the performance of pcr iner-is256 as a novel method for typing mrse strains using pulsed-field gel electrophoresis (pfge) as the reference method. methods: twenty strains of mrse (meca+) has been collected during the period of survey (december 2003 to september 2004) from different pathological products of newborns hospitalized in a neonatal intensive care unit of a public maternity hospital in sousse, tunisia. identification of strains was done by conventional procedures and the api staph (api-biomerieux. sa). susceptibility testing to antibiotics was determined according to ca-sfm recommendations. all strains have been characterized by genotypage in pulsed-field gel electrophoresis (pfge) variety chef after smai macrorestriction. genomic profiles have been interpreted according to criteria of tenover (j. clin. microbiol 1995; 33:2223 -2239 . strains were also characterized by electrophoretic profiles obtained by pcr-based analysis of inter-is256 spacer polymorphisms. results: these mrse represent 41.6% of the total strain of s.epidermidis collected from the neonatology ward during the period of survey. majority of these strains come from blood sample (75%), other strains from vascular catheter (5%), pus (10%), sound (10%). 17 antibiotypes and 6 genotypic profiles according tenover criteria were individualized: type a (with 5 subtypes), type b, type c (with 1 subtypes), type d (with 1 subtypes), type e (with 2 subtypes)and type f. a total of four pcr patterns were obtained based on the interpretation criteria that pcr patterns exhibiting more than one band difference were considered to represent distinct types, type 1, type2, type3, and type4. conclusion: mrse appear to be endemic and multiclonal with a dominant clone in the neonatal intensive care unit. our study demonstrates that a significant proportion of mrse infections may be attributable to transmission among newborns and that certain strain can become endemic over long periods in this setting. clonality of staphylococcus haemolyticus strains isolated in a neonatal intensive care unit n. ben saida, a. ferjani, j. boukadida (sousse, tn) objective: methicilllin-resistant staphylococcus haemolyticus (mrsh) was increasingly important nosocomial pathogens particularly in critically ill neonates. the aim of our study was to establish if these infections are caused by endemic clone or by incidentally occurring bacterial strains of this species. methods: thirteen strains of mrsh according ca-sfm recommendations and meca+ (murakami k. j.clin.microbiol 1991 , 29:2240 has been collected during the period of survey (april 2004 to october 2004). eleven strains come from different pathological products of newborns hospitalized in a neonatal intensive care unit of a public maternity hospital. tow strains come from paediatric and internal medicine wards were used as control to ensure that endemic strains can be differentiated from outbreak strains. all strains were identified with the api-staph method (api biomerieux). strains have been characterized by genotypage in pulsed-field gel electrophoresis (pfge variety chef) after smai macrorestriction. genomic profiles have been interpreted according to tenover criteria (j. clin.microbiol.1995 ; 33 :2223 -2239 . results: s. haemolyticus was quantitatively the second staphylococci (23.6 %) after s. epidermidis isolated in neonatology ward. mrsh represent 85. 7 % of the total strains of s. haemolyticus collected from the neonatology ward during the period of study. the majority of the strains come from blood sample 7 (63.63%), 4 (36.36%) from pus and 1 (9%) from csf. eight resistant antibiotyps profiles and 3 genotypic pattern according tenover criteria were individualized: type a, type b (with 3 subtypes), and type c. the strains coming from paediatric ward present pattern type b3, but strains coming from internal medicine ward present distinct pattern. conclusion: mrsh is a major bacterium of nosocomial infection in neonatal intensive care unit. this bacterium is responsible as shown in our study, of endemic multiclonal nosocomial infections with a predominant clone. results: s. haemolyticus was isolated from 21% of all positive blood cultures and represented the 37.9% of the coagulase negative staphylococci. twenty-nine cases of shb occurred during the study period (5.6 episodes per 1000 patients-days). all cases were hospital-acquired and occurred after a mean hospitalisation of 24.5 days. the mean age was 68.4 years (range 20-90) and 16 patients (55.2%) were males. twenty-eight patients (96.5%) had a central venous catheter (cvc). all patients received previously antimicrobial therapy. thirteen patients (44.8%) had polymicrobial bacteraemia. in 18 cases (62.1%) the source of bacteraemia was the cvc. all isolates were resistant to oxacillin and gentamicin, 94.3% and 34.3% were resistant to ciprofloxacin and teicoplanin, respectively; all strains were susceptible to vancomycin. sixteen patients (55.2%) had sepsis, 3 (10.3%) severe sepsis, and 7 (24.7%), all polymicrobial, septic shock.; in 3 patients (10.3%) shb had no clinical significance. twenty patients died (69%) and deaths were directly related to shb in 1 patient (3.4%), and partially related in 2 (6.8%). nine patients (31%) survived.the patient who died for shb, developed a persistent cvc-related bacteraemia after a 6-week course therapy for streptococcal endocarditis. s. haemolyticus become resistant to teicoplanin during a teicoplanin-based therapy and the patient died 47 days after the onset of bacteraemia. the 2 patients, in which death was partially related to shb, had a polimicrobial bacteraemia due to c. tropicalis and enterococcus spp, respectively, and died after 22 and 6 days respectively, for septic shock, during adequate therapy. among the 17 patients in which death was not related to shb, 15 (88.2%) received adequate therapy that cleared the blood cultures, 2 patients improved after the removal of cvc without antimicrobial therapy. conclusions: s. haemolyticus is an emerging pathogen, responsible of sepsis, frequently cvc-related; most of strains are resistant to many antistaphylococcal agents currently used as empiric therapy, thus constituting a clinical concern. nevertheless, this pathogen shows a low virulence and is associated with a low related mortality, probably representing a marker of severe underlying diseases. staphylococci from the skin of patients: changes associated with treatment by isotretinoin a. thomas, r. williams, n. hepburn, t. watts, r. dixon (lincoln, uk) objectives: isotretinoin, a retinoid has been used to treat patients with moderate to severe acne despite the adverse effects of mucosal surface drying and the contraindication of use during pregnancy. in clinical practice, patients are frequently prescribed systemic isotretinoin because they have not responded to previous treatment with oral or topical antibiotics. broad-spectrum antibacterial therapy has nevertheless promoted changes in the diversity and antibiotic resistance status of the patientsõ skin microbiota and we are studying the effect of isotretinoin on these changes. methods: the present study investigated the recovery and analysis of skin organisms from 53 patients (mean age 23 y range 15-37y) before, during and after treatment with a 16-week course of isotretinoin (1 mg / kg body weight). the number of aerobic bacterial isolates (presumptive staphylococci) recovered from baird-parker agar from each of three specific sites per patient were compared with age-matched controls of healthy volunteers. results: both patients and controls had generally similar numbers of organisms on the nares, cheek and toe webs before treatment, whereas all patients showed a significant reduction in staphylococci recovered from one site during and after 16 weeks of isotretinoin treatment. the majority of the patients had a greater reduction (1-2 log) for the cheek site than either nares or toe webs both of which showed minimal reduction. conclusions: preliminary results from this study show a pronounced reduction in the number of presumptive staphylococci recovered from the treated group immediately following isotretinoin compared to untreated controls. since isotretinoin has little known antibacterial activity against the staphylococci the observed reductions would suggest that the effect is mediated through changes in the skin nutritional micro-environment. objectives: emergence of mutational resistance to linezolid (lin) in staphylococcus aureus has been associated with loss of erythromycin resistance methylase (erm) genes, both in vivo and in vitro. we tested the general validity of this claim, and examined whether the emergence of lin resistance is delayed in the presence of ery. methods: six lin-susceptible s. aureus strains were used: 2 genetically-related, ery-resistant (ermc) and susceptible pairs of emrsa-15, also the isogenic pair rn4220 and its hypermutable rn422muts mutant, which harbours ermb. in 4 replicated experiments, ery-susceptible and ery-resistant isolates were grown on agar containing increasing concentrations of lin. in addition, ery-resistant isolates were grown with 100 mg / l ery and increasing concentrations of lin. the number of days for isolates to develop the ability to grow in the presence of 6 mg / l lin was recorded. the absence of erm genes was checked by pcr; mics of lin and ery were determined by agar dilution, and pfge profiles of mutants were verified. results: thirty-three mutants were obtained from the 4 experiments; 24 from the 4 clinical emrsa-15 isolates, 3 from rn4220 and 6 from rn422muts. lin-resistance emerged more slowly in ery-resistant clinical isolates grown with lin and ery than with the same isolates grown with lin alone: mean 20 days (sd 26.8) to emerge on 6 mg / l lin versus 30 days (sd 18.4) in the presence of lin and ery. growing the hypermutable strain, rn422muts, in the presence of lin and ery did not delay the emergence of lin-resistance (11 days with sd 8.7, in either case). in the absence of ery selection, loss of erm with the emergence of lin-resistance was only noted for one emrsa-15 isolate in 1 of 4 experiments. all isolates grown in the presence of ery retained erm determinants. ery 100 mg / l did not supress growth of ery-resistant strains and linezolid was stable within the ambit of the long-selection experiments. conclusions: growing the clinical isolates in the presence of ery and lin slowed the emergence of lin-resistance. this merits further study with lower, clinically-relevant concentrations of ery. in vitro selection of linezolid resistance in hypermutable and wild-type staphylococcus aureus objectives: resistance to linezolid (lin) is rare, but can arise via mutations in the 23s rrna genes. we hypothesised (a) that resistance would emerge preferentially in staphylococci with a hypermutable phenotype engineered by mutations in the muts gene, and (b) that hypermutability might be co-selected with linezolid resistance. muts is part of the dna mismatch repair (mmr) system, which identifies and corrects genome alterations post-replication, thereby influencing the net mutation rate. method: linezolid-resistant (linr) mutants of staphylococcus aureus rn4220, its hypermutable muts deletion mutant rn4220muts, harbouring an erythromycin resistance marker gene (ermb), and a genetically-related pair of clinical isolates were selected by repeated passage with increasing concentrations of lin. mutant parentage was confirmed by pfge and susceptibility was tested by agar dilution. an amplified 694-bp fragment of 23s rrna genes was studied by sequencing and by rflp analysis. frequencies at which linr mutants yielded variants resistant to fusidic acid and rifampin were calculated in triplicate experiments. results: seventeen linr mutants (mics 8-64 lg/ml) were raised. ten mutants had a g2447t mutation in their 23s rrna genes; 4, all from rn4220muts, had g2576t, typically seen in linr gram-positive cocci from the clinic; 1 had t2504c; 2 had no identifiable mutations. curiously 5 / 7 rn4220muts mutants lost ermb during the course of the experiment, reverting to macrolide susceptible. linr mutants had mutation frequencies for rifampicin and fusidic acid resistance comparable with those of their parents (10-6 to 10-9), implying no co-selection of stable hypermutability. conclusion: more linr mutants were obtained from the hypermutable (rn4220muts) strain than from the wild type strain, and g2576t mutants were only obtained from the hypermutable organism. these data suggest hypermutability facilitates the emergence of linr, nevertheless, linr mutants derived from wild-type parents did not have elevated mutation frequencies. objectives: the mechanism of resistance in glycopeptide intermediate staphylococcus aureus (gisa) and hgisa isolates is unknown. however the inactivation of certain genes has been shown to confer an increase in glycopeptide resistance. two genes, tcaa and mprf, when inactivated in gisa strains cause a reduction in glycopeptide mic. overexpression of tcaa causes an increase in teicoplanin susceptibility and so both genes may play a role in the development of glycopeptide resistance. this study aims to investigate the expression levels of tcaa and mprf in a set of gisa, hgisa and glycopeptide-susceptible s. aureus (gssa). methods: rna was extracted from the following: 1 set of clinically related (cr) gssa and hgisa (lla and lle), 2 sets of cr hgisa and gisa (pc1 and pc3, lim1 and lim3) harvested during exponential phase growth (ex) and during exponential phase growth in the presence of sub-mic levels of vancomycin (exv). rna was extracted from a further hgisa, mu3 and gisa, mu50 as well as 4 clinical gssa strains. rt-pcr was performed with customised primers and the products visualised by electrophoresis. band intensity was taken as indicative of mrna quantity, and hence expression level, at time of cell harvest. results: the expression of tcaa is similar in all strains tested. each strain within the related strain sets all show similar band intensities, despite differing glycopeptide resistance phenotype. both mu3 and mu50 also exhibited similar expression levels to those found for all other gisa, hgisa and gssa strains. levels of expression were not affected by the presence of sub-mic levels of vancomycin in any strain tested. sequence analysis of tcaa in these strains showed no mutations present as previously thought. expression levels of mprf were found to be strain specific. no difference in gene expression was found in the cr related strain sets. conclusions: no reduction in expression levels of tcaa and mprf were found to correlate with clinical strains exhibiting higher glycopeptide resistance. thus it it uncertain what role tcaa ot mprf play with mediating glycopeptide resistance in staphylococcus aureus. objectives: gisa and hgisa strains all have a characteristic thickened cell wall with reportedly fewer cross-links. fewer peptidoglycan cross-links create increased d-ala-d-ala pentapeptide termini and hence more vancomycin targets. pbp4 has been shown to have both transpeptidase and d,d-carboxypeptidase activity, cleaving terminal d-alanine residues from un-cross-linked muropeptides. reduced levels of pbp4 have been reported in laboratory-generated gisa mutants and a few clinical gisa strains. this study aims to investigate the expression levels of pbp4 in a set of gisa, hgisa and glycopeptide-susceptible s. aureus (gssa). methods: rna was extracted from the following: 1 set of clinically related (cr) vssa and hgisa (lla and lle), 2 sets of cr hgisa and gisa (pc1 and pc3, lim1 and lim3) harvested during exponential phase growth (ex), after overnight growth (on) and during exponential phase growth in the presence of sub-mic levels of vancomycin (exv). rna was extracted from a further 8 clinically unrelated (cu) gisa (including mu50), 8 cu hgisa (including mu3) and 8 gssa (ex). rt-pcr was performed with customised primers and the products visualised by electrophoresis. band intensity was taken as indicative of mrna quantity at time of harvest and hence gene expression. results: the expression of pbp4 varies according to strain. the band intensities of the related strain set lla (gssa) and lle (hgisa) reduce slightly with increasing intermediate vancomycin resistance. however in two other cr hgisa and gisa (pc1/ pc3, lim1/lim3) strain sets no variation in expression was observed. both mu3 and mu50 exhibited lower pbp4 expression than any other strain, with the presence of sub-mic levels of vancomycin reducing expression further. pbp4 expression was found to be reduced in 3 other gisa and 1 other hgisa, which showed lower band intensities than gssa. conclusions: the reduced levels of pbp4 suspected to be associated with reduced susceptibility to vancomycin in s. aureus is not exclusive. the expression of pbp4 is reduced in mu50 and 3 clinical gisa, as reported previously. however 5 other gisa strains exhibit pbp4 expression levels similar to gssa isolates. therefore pbp4 expression levels appear to be strain specific and a lowered expression level is not an essential requirement in the development of the gisa phenotype. objectives: the prolonged use of vancomycin is known to be the most important factor in inducing vancomycin resistance to s. aureus. methicillin-resistant s. aureus (mrsa) colonizing in respiratory tracts might be chronically exposed to subtherapeutic concentrations of vancomycin because the level of vancomycin within pulmonary lining fluids was much lower than the concentration of serum. despite the continuous use of vancomycin mrsas were still found to be presented in respiratory specimens of some patients. methods: all mrsas which had been continuously isolated in respiratory specimens during vancomycin therapy were collected from jul 2003-mar 2004 at seoul paik hospital. mrsas were screened on brain heart infusion plates containing vancomycin 4 lg / ml (bhi-4) and 6 lg / ml (bhi-6) for vancomycin resistance. minimal inhibitory concentrations (mics) were determined by e-tests, agar dilution methods, and broth dilution methods. the patterns of pulsed field gel electrophoresis were analysed. results: nine patients and 27 isolates were assessed. five patients had pneumonia and four patients had other mrsa infections. twelve mrsas were isolated from tracheal aspirates and 15 from expectorated sputum specimens. the duration of vancomycin use ranged from 7-22 days. five isolates and one isolate were cultured on bhi-4 and bhi-6, respectively; the vancomycin mics of these isolates revealed 3 lg / ml by e-tests. the mics from agar dilution methods and broth dilution methods showed from 0.5-2 lg / ml. conclusion: vancomycin resistance during the treatment of patients did not develop. objectives: fusidic acid resistance (fusr) in staphylococcus aureus usually results from acquisition of the fusb resistance determinant, or from mutations in the fusa gene encoding the drug target (elongation factor g). following a recent report of fusr in s. intermedius, and the detection in our own studies of fusr s. lugdunensis (unpublished), we have examined whether resistance in these strains results from fusa / fusb-type mechanisms. methods: standard methodology was employed for strain identification, susceptibility testing, southern hybridization (sh), pcr amplification and dna sequencing. primers for pcr amplification of the previously-unsequenced s. intermedius fusa gene were based on portions of the flanking genes (rpsg, tufa) that are conserved between b. subtilis and s. aureus. results: three fusr s. lugdunensis (fus mic = 4 ug / ml) and two fusr s. intermedius (fus mic = 2 ug / ml) were probed for the presence of fusb by sh alongside their wild-type, fuss counterparts (fus mics of 0.064 and 0.125 ug / ml, respectively). the fusr s. lugdunensis all carried fusb, but this gene was not detected in s. intermedius, or in the fuss strains. furthermore, fusb appeared to be chromosomally-located, since no hybridization was observed with purified plasmid dna. pcr analysis mapped fusb to a chromosomal region upstream of the groel gene, the same locus at which this gene is carried in the epidemic fusr european s. aureus clone. to examine whether mutations in fusa are responsible for fusr in s. intermedius, pcr amplification and sequencing of fusa from the resistant and sensitive strains was performed. relative to fuss s. intermedius nctc 11048, the resistant strains carried six coding polymorphisms in fusa, although none of these correspond to reported fusr mutations in the fusa gene of s. aureus. conclusions: fus resistance in recent strains of s. lugdunensis results from carriage of the fusb determinant on the chromosome, probably acquired horizontally from fusr s. aureus strains. the fusb gene was not detected in the s. intermedius strains, although multiple fusa polymorphisms were identi-fied which may be responsible for the observed fusr phenotype. objectives: methicillin-resistant s. aureus (mrsa) and methicillin-resistant coagulase-negative staphylococci (mrcns) are the most important pathogens that cause nosocomial infections worldwide. one of the few antibiotics which is still effective against mrsa is mupirocin. but mupirocin-resistant s. aureus has been increased since first reported in 1987. and more than 5-10% of s. aureus isolated in tertiary hospitals in korea was reported to be high level mupirocin resistant. in this study, we investigated the restriction fragment length polymorphism (rflp) type for mupa gene loci of plasmid dna and sequenced mupr plasmid containing mupa gene of high level mupirocin resistant (muh) staphylococci from tertiary hospitals. methods: susceptibility to antimicrobial agents including mupirocin was tested by the disk diffusion and mic of mupirocin was determined by agar dilution method. the plasmid-encoded mupa gene was detected by pcr. mupa polymorphisms of plasmid dna digested by hind iii, ecor i, and cla i restriction enzymes was investigated with southern hybridization using mupa probe. s. aureus isolate showing the most prevalent rflp type was conjugated by filter mating method and selected the transconjugant showing only mupirocin resistant phenotype. the mupr plasmid of selected transconjugant was purified and analysed the plasmid dna sequence. results: 9 muh-staphylococci were isolated among the 680 staphylococci from tertiary hospital in korea. muh-mrsa (6 isolates), s. haemolyticus (1 isolates), s. hominis (1 isolates) and s. epidermidis (1 isolates) were isolated. 1.65kb mupa pcr product was detected in plasmid of muh isolate. all isolates contained more than two plasmid molecules that were repeatedly purified with different efficiences. three different polymorphs of the mupa loci were investigated. the most prevalent mupa polymorph was 4.2kb ecori-8kb hindiii-2.1kb clai hybridizing fragment. the mupirocin resistant plasmid dna was about 52kb and is257-mupa-is257 sequence was located. conclusion: the high-level mupirocin resistant staphylococci had the multiple plasmids of various size and the diverse rflp type suggested the variation of mupa gene loci. the mupr plasmid contained transferable element such as is257 with mupa gene. genetic basis of macrolide, lincosamide, streptogramin resistance in coagulase-negative staphylococci s. gatermann, t. koschinski, s. friedrich (bochum, d) objective: in s. aureus erythromycin resistance is almost exclusively caused by erma or ermc genes whereas export of macrolide antibiotics by msra or inactivation of lincosamides by lina is rare. resistance may be inducibly (clindamycin appears susceptible) or constitutively (clindamycin appears resistant) expressed. during therapy of inducibly resistant strains with clindamycin mutations may occur that render them objectives: lysostaphin is an antibacterial enzyme which is specifically capable of cleaving the cross-linking pentaglycine bridges in the cell walls of staphylococci. s. aureus cell walls contain high proportions of pentaglycine, making lysostaphin a highly effective agent against both actively growing and quiescent bacteria. relationship between lysostaphin susceptibility, vancomycin susceptibility and cell wall thickness of passage-selected vancomycin -resistant staphylococcus aureus isolates (vrsa) and their parent strains were studied in order to investigate characterization of isolates with decreased susceptibility to vancomycin. methods: the susceptibility of lysostaphin for six vrsa and their parent strains were determined using the spectrophotometric and the macrodilution methods. spectrophotometric determination of turbidity was measured as a decrease in a620 during incubation of the isolates samples at 37°c. mics were determined by a broth macrodilution method in cationadjusted mueller-hinton broth according to standards of the national committee for clinical laboratory standards. cell wall measurement were determined using transmission electron microscopy. results: determination of lysostaphin activity revealed that there are significant decreases in per cent reduction in turbidity (activity of lysostaphin) of the vrsa. vrsa shown to be more resistant to lysostaphin than parent strains. the mics of lysostaphin for the vrsa strains also increase. there were either a 2 or 4 fold increases in the mic to lysostaphin. electron microscopy of the vrsa showed enhanced cell wall thickness, uneven surfaces and irregular shape in contrast of the thin and regular cell wall morphology of the parent strains. those strains acquiring the highest thickness in cell wall demonstrating the highest resistant to lysostaphin. conclusion: the mechanism of lysostaphin resistance and its relationship to vancomycin resistance remains unclear. it is possible that the increase in cell wall thickness we observed prevented lysostaphin access to all pentaglycine targets or increased the number of cross-linkers requiring cleavage before the strain could lyse. typing and epidemiology of mrsa methods: creation and distribution of a consensus document detailing the needed harmonization of sequencing technology in diagnostic microbiology will be followed by a certification program for sequence based typing, using methicillin-resistant s. aureus (mrsa) as a prototype organism. as typing method spatyping will be used. five strains, 5 dnas, and 5 forward and reverse chromatogram files of well characterized mrsa strains will be distributed to all participating laboratories. an annual proficiency testing for sequence based typing of mrsa is planned. staphylococcus aureus is an important human pathogen related to its ability to develop resistance to many antimicrobial agents. methicillin resistance of s. aureus (mrsa) has become a major public health problem especially in nosocomial infectious. more over community acquired mrsa is a growing concern, the rate of mrsa vary importantly within countries, the highest rates were reported especially in southern european countries. a multicentric study was done to establish antimicrobial resistance of s. aureus isolated from clinical specimens of hospitalized patients over five years period (1999) (2000) (2001) (2002) (2003) . s. aureus was identified by conventional methods and antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. a total of 5186 strains of s. aureus were collected, they were recovered mainly from pus (60%), blood cultures (15%), respiratory samples (8%) and urines (7%). 34% of the strains were from in medicine, 18% from surgery, 16% from orthopaedics, 14% from paediatrics and 13% from intensive care units. the rates of resistance to different antibiotics was 15% to oxacilline,9% to gentamicin, 23% to amikacin,8% to ofloxacin, 23% to erythromycin and 8% to clindamycin. all isolates were susceptible to vancomycin. according to this data the rate of mrsa remained relatively low (< 20%) with no significant change during this time period, however, mrsa presented high rates of associated resistance to gentamicin (48%), to amikacin, (91%) to erythromycin (38%), to clindamycin (20%), to ofloxacin (40%) and to trimethoprim-sulfamethoxazole (33%).mrsa were not of great concern in our country and meticillin remains an effective antibiotic for treatment of s. aureus infections. characterisation of gentamicin-susceptible strains of methicillin-resistant staphylococcus aureus in two spanish hospitals c. potel, m. alvarez, c. lopez-melendez, i. otero (vigo, e) objectives: characterize gentamicin-susceptible methicillinresistant staphylococcus aureus(gs-mrsa)by two molecular typing methods. find out whether some gs-mrsa clones are related to gr-mrsa. describe the spread of these clones. methods: one hundred and twelve samr were isolated in two hospitals between 1997-2001. the epidemiological relatedness was studied by repetitive element sequence-based pcr and coa gene restriction fragment length polymorphism. the genes ant(4) and aac(6)-aph(2õ), that codify two aminoglycoside modifying enzymes, were detected by pcr.we analysed the gentamicin consumption and its relation with the emergence of gs-mrsa. results: thirty nine strains were gs-mrsa. the 80% of these gs-mrsa belonged to two epidemic clones, the clone a was tobramycin sensitive and the clone b was tobramycin resistant. the clone a was only isolated at hospital-i and was replaced by an epidemic gr-samr clone (clone c). the clone b was only isolated at hospital-ii and displaced the clone c. objectives: the long-term care facilities (ltcfs) patients are those with serious underlying disease, poor functional status, wounds such as pressure sores, invasive devices of urinary catheters. there is increasing concern about the emergence of resistance to antimicrobial agents including methicillin resistant s. aureus in ltcfs and mupirocin has been used in ltcfs to prevent the occurrence and spread of mrsa. for inducible mls-resistance. nitrocefin discs were used for beta-laktamase detection after induction with cefoxitin and/or oxacillin. the oxacillin (4 mg / l) agar method was used for mrsa screening. mrsa was confirmed by meca and nuc pcr and further analysed by smai-pulsed-field gel electrophoresis (pfge). results: a total of 102 strains (78%) were beta-lactamase positive. other resistance prevalence rates were: tc (29%), em (19%), cm (19%), ci (11%), gm (11%), ts (0%) and fu (0%). thirteen strains (10%) were mrsa-screen positive, both with the oxacillin-agar test and the cefoxitin disc, and confirmed mrsa by meca and nuc pcr. all mrsa strains were isolated from hospitalized patients and expressed multiple resistances. pfgeanalysis revealed that the mrsa-strains belonged three different pfge-types. type 1 consists of 8 strains from the department of general surgery and icu. the type 2 group consisted of 3 strains from general surgery department. finally, one strain from a patient at the department of pulmonary medicine belonged to the type 3 group. we were not able to type one strain. mlsttyping of mrsa-strains will be performed. results: a total of 793 infants were included in this study. mrsa colonization was detected in 331 infants (42%) during nicu stay and 89% were detected by the first 2 samplings. naris (71%) and umbilicus (60%) were the 2 most common sites of colonization. clinical mrsa isolates were identified from 92 infants (12%). among the 92 infants, mrsa colonization, either preceding or later acquiring, was noted in 84 infants (91%). 121 clinical isolates from 64 infants with 84 episodes of possible mrsa infection were available for genotyping analysis. prior colonization was detected in 68 episodes (81%), among which both the colonized and clinical isolates were indistinguishable in 63 episodes (93%), highly related in 2 episodes and distinct in 3 episodes. among the 16 episodes without prior colonization, colonization was acquired following infection in 9 episodes (53%), among which indistinguishable or highly related colonized strain was acquired in 6 episodes. conclusion: surveillance of mrsa strains as a basis for active antibiotic policy has become of increasing concern to both health care providers in hospitals and community general practitioners. there is a need for better awareness of mrsa infections among both health care professionals and the public. the incidence of mrsa infections in last years in the czech republic shows a slightly increasing trend. very interesting is local distribution of mrsa strains. the development of the incidence of mrsa infections in their spread will be the subject of further study. comparison of phenotypic typing methods and pfge of methicillin resistant s. aureus isolated from two university hospitals in thailand c. chomvarin, k. chaicumpar, s. srigulbutr (khon kaen, th) objectives: comparison of phenotypic typing methods and genotypic typing method to differentiate mrsa isolates obtained from the two university hospital in thailand. methods: seventy-four mrsa isolates were randomly collected from the two university hospital (central and northeastern) in thailand. they were confirmed with the presence of meca gene. antibiograms, phage typing and enterotoxin productions were used for the phenotypic typing analysis. pulsed-field gel electrophoresis (pfge) with smai digestion of chromosomal dna was used for the genotypic typing analysis. results: we found 19 distinct profiles by phenotypic typing methods and 18 pfge types designed as 5 major types (a to e) and 13 subtypes. the most frequently types and their related subtypes found in both hospital, were a and c with represented 54.1% and 27%, respectively. all isolates resistant to penicillin, cephalosporin, erythromycin, gentamicin, kanamycin, tetracycline and oxacillin; and variably resistant to cotrimoxazole, lincomycin, chloramphenicol, ciprofloxacin. all isolates were susceptible to vancomycin and fosfomycin. ten (13.5%) mrsa isolates produced enterotoxin a. forty-five (60.8%) isolates were nontypable by phage typing method. no significant correlation between the phenotypic typing methods and the genotypic typing method were found. conclusions: the phenotypic typing methods were not significantly correlated with the genotypic typing (pfge). our results demonstrated that pfge types a and c were the common endemic mrsa clone of both hospitals in thailand. objective: to investigate the molecular characteristics of methicillin-resistant staphylococcus aureus (mrsa) strains isolated from neonates transferred from primary care obstetric clinics. methods: twelve mrsa strains were isolated from 11 neonates in 2004. ten mrsa strains were also isolated from nurses and facilities in corresponding primary care obstetric clinics. molecular features of mrsa strains were analysed by using multilocus sequence typing (mlst, arcc-aroe-glpf-gmk-ptatpi-yqil), spa typing, and sccmec typing. presence of panton-valentine leukocidin (pvl) gene was investigated by pcr method. results: all 22 mrsa strains analysed in this study contained sccmec type iva. of six isolates from neonates of clinic a, five showed st1 (1-1-1-1-1-1-1) and resistance to only gentamicin and tetracycline. the remaining one showed novel st (25-1-1-1-1-1-1), shared with one isolate from nurse of clinic a. four mrsa strains from neonates of clinic b showed st1 (1-1-1-1-1-1-1) and resistance to erythromycin and gentamicin, which is the same characteristics with three isolates from nurses and environment of clinic b. the other two mrsa strains from neonates of clinic b showed st493 (62-1-1-1-1-1-1), which is shared by six strains from nurses and environment of clinic b. spa type of these 22 mrsa strains was identical as ujebkbp and all strains were negative for pvl gene. objectives: over the last years there has been a dramatic increase in the prevalence of methicillin-and multiresistant staphylococcus aureus (mrsa) leading to a major health problem, especially in the nosocomial setting. to support infection control measures typing of mrsa is essential. the present ôgold standardõ for strain typing is pulsed field gel electrophoresis (pfge), but several sequence based methods including spa typing have recently been introduced. therefore this study was initiated to compare typing results obtained from pfge with those from spa typing. methods: 90 well characterized s. aureus strains of different evolutionary origin including methicillin sensitive and resistant isolates were included. strains were selected from a variety of clonal groups prevalent in germany and middle europe during the last ten years. the collection comprised hospital derived strains as well as isolates from the community, including the recently emerging community acquired mrsa. all isolates were subjected to pfge with subsequent cluster analysis; additionally the polymorphic x-region of the protein a gene was sequenced and a spa type was assigned using the ridom staphtype software. the newly developed algorithm burp (based upon repeat patterns) was applied to the spa sequences to cluster the resulting spa types into different groups. clustering results were compared to those obtained from pfge and mlst. results: overall the results obtained from the different typing approaches were in agreement. in some cases where pfge results were inconsistent despite uniform epidemiological background spa typing was able to group the corresponding strains together. only two strongly related epidemic clones could not be separated by spa typing. conclusion: spa typing in combination with burp analysis is an easy, rapid and reliable method for epidemiological analyses in s. aureus. it is superior to pfge in cases where pfge might be ôoverdiscriminatoryõ. in rare cases a second discriminatory locus (for example a single mlst locus) might be helpful for an ultimate classification. the antibiotic susceptibility of methicillinresistant staphylococcus aureus methicillin-resistant staphylococci (mrsa) are often multidrug resistant and represent a major problem for the antimicrobial therapy. glicopeptides are the golden standard for these infections but resistance and toxicity concerns limit their usage. mrsa antibiotic resistance may be divided into two distinctive profiles: multidrug resistance (probably nosocomial infections) and variable resistance, usually to 1 or 2 non-betalactamic antibiotics (often correlated with community-related mrsa infections results: in total, 4.5 % (98) of s. pn was resistant to erythromycin and 2.9% (8) of invasive and 4.6% (10) of non-invasive s. pyo were resistant to erythromycin. none of the isolates were resistant to clindamycin. among the ery-res s. pn, the most frequent gene was mef(a) 83.7%, while erm(b) was found in 13.3%. in two isolates no resistance genes were identified. 81 out of 82 mef(a) carrying isolates had serotype 14.erm(a) and mef(a) was present in 62.5% and 37.5% of the invasive ery-res. s. pyo isolates. none of the isolates were positive for erm(b). in non-invasive ery-res. s. pyo isolates 40%, 10%, 50% were pcrpositive for erm(a), erm(b) and mef(a) respectively. conclusion: in contrast to s. pyo, ery-res. s. pn is dominated by one serotype, carrying mef(a). the overall resistance profile of s. pn and s. pyo is still favourable, but the resistance to macrolides is of growing concern in denmark. resistance in streptococcus pneumoniae: auc/ mic breakpoints differ between gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin k. laplante, m. rybak, b. tsuji, g. kaatz (detroit, usa) objective: the potential for resistance development in streptococcus pneumoniae (sp) secondary to varying exposure to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin was examined at high inoculum (108.5-9 log 10 cfu/ml) over 96 hours in an in vitro pharmacodynamic model. results: the molecular analysis of one hundred and forty serotype 11a dspn isolates revealed six clonal types. however, the vast majority (95%) belonged to a single clone. the prevalence of this dominant clone during the study ranged from 6.3% to 13.9% and it was disseminated in all day-care centres. conclusions: serotype 11a is not a capsular type with an important representation in drug-resistant collections which are the most commonly studied worldwide. however, it seems to be common among dspn strains. this serotype 11a collection showed little genetic variability. in fact, one genotype was found to be dominant and disseminated successfully in all day-care centres studied. these findings suggest that this serotype 11a clone is frequent in colonization and thus its monitoring is of importance particularly after the introduction of the sevenvalent pneumococcal conjugate vaccine. diseases. an abrupt antimicrobial resistance increase and clonal spread of resistant pneumococcal strains has resulted in serious therapeutic problems in recent years. the aim of this study was to analyze resistance patterns and genetic diversity of s. pneumoniae resistant strains isolated in our region during three years (2001) (2002) (2003) . methods: using e-test method and the nccls criteria for benzylpenicillin (p), erythromycin (e), clindamycin (l), tetracycline (t), cotrimoxazole (s), ceftriaxone (c), chloramphenicol (h), vankomycin (w), imipenem (i), fifty nine resistant or intermediate to at least one drug strains were obtained. strains were submitted to molecular characteristic by pfge with smai restriction enzyme and computer analysis using molecular analyst software application. for each strain resistance pattern and pfge profile was determined. results: resistance to 8 out of 9 determined antibiotics (except vancomycin) was described. strains showed 22 different resistance patterns and tsh (13%strains), psi (11.8%) and elts (10.1%) were the most often. resistance degree reached 7 drugs (5% strains) and 69% strains were mdr. we have found 26 pfge profiles: 15 of them (u 1-15) were unique single isolates, 11 clusters (a-k) were represented by 2-10 strains, which were more than 78% of similar. the most numerous clusters (a-k) consists of strains that were isolated over three years of study and showed the same or very similar resistant patterns: a-tsh (8 of 10 strains), th (2); b-psi (7 of 8), s (1); c-pelshi (3 of 6), ptshi (2), ptschi (1); d-elts (3 of 4), els (1); e-sh (2 of 4), ts (1), t (1). all strains of tsh and psi resistance patterns were found in one cluster a(a1-a9) or d (d1-d6) respectively but strains with elts and pelshi patterns belonged to different (2-3) clusters and unique pfge profiles. another clusters (f-k) were represented by two strains with different resistant patterns each. conclusions: population of s. pneumoniae resistant strains in our region presents high genetic diversity and numerous different resistance patterns. the majority of strains with the same resistance pattern showed different pfge profiles but there were observed some strains of the same resistance pattern, which belonged to only one cluster and were isolated over three years of study. ) hlpr strains showed a profile identical or related to two epidemic clones: spain23f-1 and spain9v-3. the remaining hlpr pneumococci belonged to unique or rare clones. llpr isolates were characterised by more variable backgrounds in terms of pfge patterns and serotypes than hlpr strains. in fact, 27.7% showed an identical or related profile (arbitrarily called d, serotype 15a) previously not described in italy and 10 (15.4%) exhibited the pfge patterns typical or related to the spain23f-1 and spain9v-3 clones. the remaining 37 llpr isolates (56.9%) belonged to 22 different profiles and to 11 distinct serotypes. in this study, the 23f ôitalian cloneõ circulating in 1993-1996 was not found. conclusions: the recent increase of total penicillin-resistance in italy can be ascribed to the emergence of a new clone and to the diffusion of two well known international clones, whose ability to spread is higher than that of the autochthonous italian clone described in 1996. changing antibiotic prescribing habits, including the recent strict limitation to the consumption of parenteral drugs, may also explain the variations described. results: during the study period, 143 strains of s. pneumoniae were isolated: 6 (4.2 %) in csf and 137 (95.8%) in blood. total incidence was 6 cases/100,000 inhabitants/year, with maximum incidence in winter and spring. the largest number of cases, 76 (54 %), were in the over 65s. in children, 11 (7.7 %) were detected, all in the under 5s. the total number of strains whose sensitivity to penicillin fell was 43 (30 %), of which 20 (46.5 %) had an mic 2 mg/ml. 46.5% of the penicillin-resistant strains were also resistant to erythromycin. 16 (11%)/ 143 had reduced sensitivity to cefotaxime, and most of these (63%) had intermediate sensitivity. resistance to erythromycin was detected in 34 (24%). during the study period, resistance to antibiotics decreased gradually in such a way that the resistance percentages each year were: penicillin 41%, 30%, 27%, 23%; cefotaxime 28%, 14%, 4%, 4%; erythromycin 34%, 19%, 29%, 8%. no differences were observed in the resistance percentage in the different age groups. conclusions: the prevalence of csf and blood strains of s. pneumoniae with reduced sensitivity to penicillin in this study was 30%. the treatment of choice in s. pneumoniae invasive disease was third-generation cephalosporins, due to their low level of resistance. resistance to the main antimicrobials from this type of strain has fallen. during the study period, resistance to penicillin fell by 43.9 %, 58.5 % for cefotaxime 63.4 % for erythromycin. the fall in resistance among invasive s. pneumoniae could be due to vaccination in children and the elderly from 2002 onwards, since the vaccination includes the most common and resistant serotypes. the low number of isolates in children could be due to the low number of samples processed. change in distribution of macrolide-resistant streptococcus pneumoniae genotypes over 4 years -data from protekt 1999-2003 objectives: this analysis of data from 39 countries over the first 4 years (1999) (2000) (2001) (2002) (2003) of the protekt global surveillance study was undertaken to investigate longitudinal and regional trends in the distribution of macrolide resistance mechanisms among streptococcus pneumoniae, and the susceptibility of these isolates to antibacterials, including the ketolide telithromycin. objectives: the 7-valent formulation of the pneumococcal conjugate vaccine (pcv7) was introduced in the usa in february 2000. there is a general recommendation for its use in all children <2 years of age, and the vaccine is licensed for children up to 5 years of age. in europe, the vaccine is licensed for children up to 5 years of age, but most countries do not have a general recommendation for use. protekt -a global, longitudinal study of the antimicrobial susceptibility of bacterial respiratory tract pathogens -has now completed its fourth year. a comparison was made between serotype distribution and pcv7 coverage between y1 and y4. methods: analysis was performed only on streptococcus pneumoniae isolates obtained from paediatric patients at the 31 centres common to both years (y1, n = 553; y4, n = 682). serotyping was performed by neufeld's quellung reaction using statens serum institute antisera (ssi, denmark). results: pcv7 coverage decreased from 67.7% and 62.5% in y1 to 51.0% and 41.0% in y4 in the usa and latin america, respectively (chi-squared; p = <0.001 and 0.007, respectively). serotypes that showed the greatest increase (y1, y4) in the usa were 19a (7.0%, 22.2%), 6a (1.8%, 9.9%), 3 (1.8%, 6.2%), 15 (1.8%, 6.2%), and 11 (0%, 6.2%). in brazil, a similar pattern was seen although there was no change in the proportion of isolates with the 19a serotype. in contrast, no change in serotype distribution was observed in canada, western europe, and the far east, where the pcv7 vaccine is not routinely used for paediatric vaccination. conclusions: results from this analysis of protekt data indicate that the proportion of s. pneumoniae isolates from paediatric patients covered by the pcv7 vaccine has decreased significantly over 4 years in regions where the vaccine is routinely used. other serotypes, such as 19a, which is known to be highly resistant to antimicrobials and is not covered by pcv7, have increased in these regions. this study demonstrates the importance of serotyping antimicrobial surveillance study isolates in order to monitor such changes and any potential future implications for therapy and vaccine formulations. background: sequential alterations in pbp sequences constitute the classical mechanism to acquire penicillin resistance in s. pneumoniae, that is reflected in changes in the cell wall peptidoglycan structure. murm gene controls the addition of the first amino acid of the dipeptide bridge of the pneumococcal muropeptide. mutations in this gene are apparently required for high penicillin and cefotaxime resistance. the aim of this study was to compare the murm gene sequence within the earlier and the latest highly penicillin resistant pneumococcal populations recovered in spain. material and methods: a total of 56 s. pneumoniae isolates (10 of them from 1987-88 and 46 from 2002-04) with resistance to penicillin (mic 2-8 lg/ml) were included. susceptibility to different b-lactams, macrolides, tetracycline, levofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole (sxt) were determined by the agar dilution method. all isolates were grouped according to serotype and pulsotype (pfge-smai pattern) and the murm gene was sequenced. results: a high percentage of resistance (intermediate plus resistant) was observed for cefotaxime (100%) and sxt (96.6%). all isolates remained susceptible to telithromycin but not to levofloxacin (93% of susceptibility). all isolates recovered during 1987-88 were genetically unrelated and mainly belonged to serotypes 23f (40%) and 6b (40%). in contrast, the most frequent serotypes during 2002-04 were 14 (30%), 9v (26%), and 6b (17%). in this period, a predominant spain9v-3clone was detected in 10 out of 14 isolates of serotype 14, and in 11 out of 12 isolates of serotype 9v (capsular switch). all 8 isolates of serotype 6b belonged to a single clone (spain6b-2). the analysis of murm sequences of isolates from both periods revealed the existence of different alleles. homologous sequences to the murma (identical to that described for r6 penicillin susceptible strain), were replaced in part by murmb5, and murmb6 (different variants of these alleles were detected in our collection). conclusion: early high penicillin resistant s. pneumoniae isolates in spain were genetically unrelated and corresponding to 23f and 6b serotypes. on the contrary, recent isolates showed dissemination of selected clones, particularly spain9v-3. many s. pneumoniae isolates with high-level resistance to penicillin retain (or reacquired) the murm gene of susceptible strains, but different allelic variants was also detected. objectives: to determine among 712 s. pneumoniae clinical isolates recovered from different spanish hospitals during three different periods (1999-2000, 2001-2002 and 2002-2003) the prevalence of erythromycin resistance phenotypes and resistance genes associated to this resistance. methods: susceptibility testing was performed by the standard microdilution technique (nccls). a pcr assay was carried out to identified erythromycin resistance genes (erm or mef) and a multiplex-pcr was designed to distinguish between mef(a) and mef(e) genes within mef positive isolates. clonality was studied using the sma-i-pfge technique. during the studied period ranged from 48.3% (first studied period) to 45.2% (third studied period) for penicillin, 39% (first studied period) to 32.9% (third studied period) for erythromycin, and 32% (first studied period) to 30.3% (third studied period) for clindamycin. levofloxacin resistance was only found in 2.4% of isolates and were recovered during the third studied period. m phenotype was observed in 3.2% of all tested isolates. considering non-susceptible erythromycin isolates, ermb and mef genes was found in 87.1% and 9.5% of isolates, respectively, as a sole erythromycin resistance gene. the concomitant presence of both determinants was found in 3.3% of isolates. interestingly, among the isolates with mef pcr positive result, only one (3.2%) carried the mef(a) gene whereas the mef(e) gene was detected in the others (96.8%). an increase in mef prevalence was observed between the first and the second studied periods (2% to 5.8%) and a slightly decrease (4.9%) in the third period. pfge analysis revealed a polyclonal structure of mef positive isolates. conclusions: prevalence of mef positive isolates among erythromycin-resistant strains remains low in our country (4.3%) being the mef(e) gene the most prevalent mef determinant among these isolates (96.8%). results: among young children, single ery resistance was most prominent in all countries, (from 6% in sweden to 38% in belgium), except for spain where the proportion of dual resistance was highest (33%). for the other countries dual resistance among young children ranged from 0% in denmark to 13% in belgium. except for ireland (12%) and spain (21%), single pen resistance remained below 5%. conclusion: between countries, large differences in the patterns of s. pneumoniae resistance were found among young children. ery resistance was most common among young children, which may indicate greater use of macrolides in this age group. in order to assess the effectiveness of interventions like vaccination, resistance and serotype data should be monitored carefully. evolution in the pattern of sensitivity to penicillin and cefotaxime of streptococcus pneumoniae background: the increase in resistance to penicillin, and more recently to cefotaxime, means that it is necessary to study the prevalence of resistance to these antibiotics, giving them huge clinical importance. this study compares the sensitivity of s. pneumoniae to penicillin and cefotaxime during 1991,1995,2000,2001,2002,2003 and 2004. methods: a total of 892 strains of s.pneumoniae from significant respiratory tract isolates were studied. these corresponded to the years 1991 (130) conclusions: in 2004, (1) 14.9 % of the isolates were erythromycin-resistant and clindamycin-and miocamycin susceptible (m phenotype), a smaller percentage than in previous studies; (2) there was a significant increase in 2004 of isolates with the mlsb constitutive phenotype; (3) there was a high prevalence of resistance to telithromycin (88.6 %) in the 35 strains with mlsb constitutive phenotype. patterns of macrolide resistance determinants among s. pyogenes and s. pneumoniae isolates in saudi arabia objective: to characterize the macrolide sensitivity of recent isolates of s. pyogenes and s. pneumoniae collected from different hospitals around saudi arabia and to investigate the resistance determinants carried by macrolide-resistant isolates. methods: susceptibility testing was performed using standard nccls methodology on 335 s. pyogenes and 350 s. pneumoniae isolates. macrolide resistance mechanism phenotypes were identified using double disk diffusion. results: all s. pyogenes were penicillin sensitive, while 6.4% were macrolide resistant, the main mechanism of which was of m-phenotype (96%). approximately 51% of s. pneumoniae was penicillin non-susceptible. macrolide resistance in s. pneumoniae accounted for 18.8%, the majority of which was m phenotype (92%). low-level resistance mediated by mef-bearing strains predominated. conclusions: efforts should focus not only on antibiotic resistance surveillance and the development of guidelines but also on appropriate use of antibiotics. strategies have been proposed which include restricting access, compliance promotion and reduction in our prescriptions and inappropriate use of antibiotics. newer macrolides, including azithromycin, are still considered drugs of choice for empirical treatment of respiratory infection in such circumstances. objectives: mlsb phenotype erythromycin resistance (eryr) is often associated with tetracycline resistance (tetr) and in streptococci is mediated by the methylase genes erm(b) or erm(tr). in streptococcus pneumoniae, erm(b) is carried in transposons such as tn1545 which also carry the tetr gene tet(m). m phenotype eryr is associated with the efflux genes mef(a) or mef(e). these genes are carried in genetic elements that do not commonly include tetr genes, but the mef(e) element in s. pneumoniae can be inserted in a transposon that carries tet(m). our objective was to investigate tetr in eryr beta-haemolytic streptococci of groups a, b, c and g. methods: 129 eryr beta-haemolytic streptococci of groups a (26), b (42), c (9) and g (52) were collected over two years. tetr was determined by disc diffusion and mics by e-test. pcr was performed for tet(m), tet(o) and tet(l); eryr genes had been characterised previously. results: the prevalence of tetr amongst eryr mlsb isolates was high in groups a (73%, 11 of 15), b (92%, 34 of 37) and c (100%, 2 of 2) but lower in group g (38%, 18 of 47). the range of mics in tetr isolates was 4 ‡ 256 (mg/l). the tet(m) gene was responsible for most tetr in groups a (100%, 11 of 11), b (85%, 29 of 34), c (100%, 2 of 2) and g (56%, 10 of 18); tet(l) and tet(m) were both found in the same isolates in groups b (2) and g (1). at least 80% of erm(b) isolates and 57% of erm(tr) isolates of groups a, b and c carried tet(m). in contrast, only 21% of erm(tr) isolates in group g carried tet(m). amongst m phenotype isolates, the prevalence of tetr was high in groups b (100%, 5 of 5) and g (80%, 4 of 5) and lower in group c (14%, 1 of 7). tetr was not detected in m phenotypes of group a. in m phenotypes, tet(m) was responsible for 100% tetr in group b (5 of 5) and 25% in group g (1 of 4); 1 isolate of group c carried tet(o). tet(m) and tet(o) were found in mef(e) isolates but not in mef(a) isolates. there were tetr isolates in groups b (5) and g (11) in which no tetr gene was identified. conclusions: 1) the prevalence of tetr in eryr beta-haemolytic streptococci was high in mlsb isolates of groups a, b and c and in m isolates of groups b and g. 2) tet(m) was the predominant tetr gene. 3) tet(m) was strongly associated with erm(b) and to a lesser extent with erm(tr). 4) tet(m) was found in association with mef(e) but not mef(a). results: reduced susceptibility (mic >1 mg/l) was detected in 44 (2.7%) s. pyogenes isolates. a total of 32 (73%) s. pyogenes isolates demonstrated a mlsb phenotype resistance. the dominating gene was erm(a) (n = 27), encoding resistance in 27 strains, whereas erm(b) was detected in only five of the strains isolates. while one of the cmlsb strains carried both erm(a) and the mef(a) gene, another cmlsb strain was reproducibly negative in erm and mef pcr analyses, and will be further investigated. the remaining 11 (25 %) isolates all carried the mef(a) gene and showed a consistent with their m phenotype resistance. thirty (68%) strains were resistant to tetracycline and carried the erm-positive strains (n = 30) genes, while all the mtype strains were susceptible to tetracycline. typing analyses revealed t-type 4 and st39 dominated in nine 9 out of 10 with mtype phenotype, whereas t-type 3 was associated with st46 in 8 of 9 erm(a)-positive and tetracycline resistant strains. results from emm typing supported these clonal observations. conclusions: (i) the prevalence of macrolide resistance among s. pyogenes in norway is low, and the (ii) mlsb resistance, erm(a) or erm(b) was the most prevalent resistance type and co-resistance to tetracycline was frequently observed. rates of resistance to macrolides vary from region to region and these rates are important in defining the role of macrolides in empiric therapy. macrolide and lincosamide resistance are mediated by 3 distinct mechanisms, (1) mefa encodes resistance to erythromycin but not clindamycin, (2) ermb encodes resistance to erythromycin and clindamycin, (3) erma encodes resistance to erythromycin and inducible resistance to clindamycin. methods: consecutive pharyngeal isolates of streptococcus pyogenes isolates were collected between january 2003 and april 2004. isolates were confirmed as s. pyogenes by lancefield grouping using the pastorex strep rapid agglutination kit. erythromycin susceptibility testing was performed by nccls disc diffusion. erythromycin resistant isolates were tested for susceptibility to erythromycin and azithromycin by etest. the mechanisms of resistance was assessed using the erythromycin/ clindamycin disc approximation test and by pcr, with primers specific for the erma, ermb and mefa genes. positive controls were kindly provided by ralf r. reinert. results: four (4) of 360 consecutive isolates (1%) were resistant to erythromycin. erythromycin resistance was confirmed by etest and these isolates were also resistant to azithromycin. all 4 erythromycin-resistant study isolates and 3 of 4 previously collected erythromycin-resistant isolates were confirmed as erma positive by pcr and exhibited inducible resistance by disc approximation. one previously collected isolate was susceptible to clindamycin and was mefa positive by pcr. conclusion: these data show that macrolide resistance in s. pyogenes is very uncommon in this area and is encoded primarily by erma. objective: aim of this study was to evaluate antimicrobial prophylaxis (ap) practice and its cost in intensive care unit of heart surgery depertment in our hospital. method: study was performed prospectively between january-december 2002. ap lasting >1 day was considered inappropriate, unless the patient was in high risk group (heart transplantation or repeated by-pass). in addition ap with two beta lactam agents; beta lactam+aminoglycoside (only beta lactam+gentamicin was allowed); 3rd generation cephalosporins; quinolones or glycopeptides were considered inappropriate. in high risk patients, ap lasting up to 3 days and ap with glycopeptides were considered appropriate. cost of inappropriate ap was calculated by using august-2004 prices in euro. cost of inappropriate ap in patients who received cefazolin or beta lactam/beta lactamase inhibitors (ampicillin/sulbactam or amoxycillin/clavulanate) was calculated by substraction of cost of 1 day lasting ap from total ap cost. in case of inappropriate glycopeptide or quinolone or aminoglycoside usage, one day lasting cefazolin cost was substracted from total ap cost. in high risk patients cost of inappropriate glycopeptide usage was calculated by substracting 3 days lasting ap cost from total cost. infections were diagnosed according to the criteria of center for diseases control and prevention (cdc). data were analysed with chi square and student's t-tests. objectives: the aim of the present study was to investigate the frequency and the type of wound infections, as well as the bacteria involved, after posterior spinal fusion in children, especially with spinal deformities, over a 6-year period (1999) (2000) (2001) (2002) (2003) (2004) . materials and methods: a total of 133 spinal fusions were performed on 110 children, with or without instrumentation, because of spinal scoliosis (120), spondylolisthesis (4), fractures (4), and for other reasons (5 objectives: therapy of chronic wounds with lucilia sericata presents a promising alternative to the classical approach with antibiotics. larvae are placed in wounds free-roaming or in biobags. the therapy often stimulates the process of wound healing. this effect is based on different mechanisms. maggotsõ saliva contains collagenases, trypsin and chymotrypsin-like enzymes which dilute necrotic tissue. furthermore, an antibacterial effect has been described. in our study, we focused on the question whether this effect could be explained by the ingestion of bacteria by larvae. methods: free roaming maggots were placed on agar plates with escherichia coli. these bacteria had been transformed with green fluorescent protein (gfp)-containing plasmids. gfp-free escherichia coli xl1 blue served as a control. two days old maggots were put on agar plates in groups of ten at room temperature. every thirty seconds, a maggot was displaced from the agar plate and washed with sterile saline (0.9%). dead larvae which could not have taken up bacteria served as a control. maggots were tested for flourescence under a fluorescence microscope [zeiss axioskop; hpo50/ac, axiocam mrmzeiss]. larvae were examined by an independent observer as well. results: maggots that had been placed on gfp-labelled escherichia coli showed an intensive green fluorescence, especially at the back of the head and in the intestines. the controls did not show any fluorescence. after three to four minutes gfp-labelled escherichia coli could be detected inside the maggots' body. at this early stage of digestion gfp-fluorescence could mainly be examined in the area of the head. this fluorescence results of the ingestion of gfp-labelled escherichia coli by larvae. we have proven that the uptake of bacteria represents a further mechanism of antibacterial activity of larvae. not only diluting necrotic tissue, but also minimizing the rate of infections, maggot therapy which is also well-known as biosurgery could be a beneficial alternative to the use of antibiotics. as the mechanical uptake is a non-specific process, there might not be a risk of resistance. antibiotic resistance: nosocomial pathogens p1055 antimicrobial resistance patterns of bacterial pathogens from blood culture of cancer patients in a single cancer institution m. faiz, h. aziz, t. bashir, s. asghar (lahore, pak) objective: the widespread emergence of resistance to antimicrobial agents among bacterial pathogens is well known and has an impact on our ability to treat patients effectively. blood stream infections (bacteraemia) among cancer patients that develop during the course of disease are potentially life threatening because of suppression in their immune systems. the changing spectrum in the incidence and epidemiology of microbial pathogens has resulted in an increase in resistance to many antibiotic compounds emphasizing the need to monitor the prevalence of resistance in these strains. methods: susceptibility and resistance pattern of clinically significant bacterial isolates from positive blood cultures collected during 2000-2004 was studied. the isolated strains were tested against a wide range of antibiotics belonging to cephalosporins, aminoglycosides, carbapenems and quinolones derivative groups by disc diffusion method and the results were interpreted according to british standard for antimicrobial chemotherapy. results: a total of 250 bacterial pathogens were isolated with 60% gram positive and 40% gram negative bacteria. the dominating pathogens were staphylococcus aureus, streptococci, pseudomonas , enterobacter and klebsiella. among gram negative strains, highest level of resistance (98%) to third generation cephalosporins was observed followed by carbapenems and penicillin (79%, 80%) respectively. similarly, high resistance to aminoglycosides were found (69% to ampicillin, 50% to tobramycin and 62% resistant to quinolone derivates group of antibiotics. however only 29% were resistant to ciprofloxacin. in gram positive bacteria, high resistance to ciprofloxacin (40%) was observed as compared to gram negative bacteria. a still higher resistance level (100%) was observed for aminoglycosides and third generation cephalosporins (97%) respectively. conclusion: the spectrum of isolates among our patients were shifting towards gram positive bacteria with high resistance to different groups of antimicrobial agents limiting few choices for alternative therapies for infection control. antimicrobial resistance continues to increase and ongoing surveillance of microbial pathogens is essential. this study also warrants the need of infection control measures, rational antibiotic policies and rapid laboratory detection of resistance to prevent the spread of resistance among these strains. (9), and epidemiological swabs from throat and rectum (21). we found two susceptibility groups: group 1 (7 patients) resistant to amox/ clavul.acid, pip/tazobactam, aztreonam, cefuroxime, and group 2 (11 patients) susceptible for these antibiotics. rapd profiles corresponded with the susceptibility testing results. drug resistance profile in group 1 could be attributed to overproduction of chromosomal encoded k1 beta-lactamase. group 1 was from ru and group 2 from nicu, pu and su. analysis of sociodemographic variables and potential risk factors showed differences between analysed groups in mean birth weight, length of hospital stay, antimicrobial treatment and invasive therapeutic procedures usage. the outbreak analysis revealed concurrent isolation of two k. oxytoca clones, normal and k1 overproducer. rapd results were supported antibiotyping results. results: a total of 3077 isolates was analysed. 51.4% of these isolates were non-susceptible to at least one agent, the highest proportion is due to mono-drug resistance to piperacillin (39.6%). 4.8% of the strains were classified as multi-resistant, the most frequent patterns were pip/caz/ctx/gen (2.1%) and pip/caz/ctx/gen/cip (1.7%). two strains were resistant to all six antibiotics. significant differences in multi-drug resistance rates were associated with ward type with highest rates for icu-patients (8.8%). furthermore, multi-resistance rates varied significantly between the centres involved with a range from 0.9% to 7.2%. the centres differed not only in respect to the multi-drug resistance rates, but also in respect to the dominating phenotype. conclusions: the relevance of multi-resistance in k. pneumoniae as a major clinical problem is proved by an overall rate of almost five per cent for german university hospitals and an even higher proportion for icu-patients. among the agents tested piperacillin plays an eminent role in regard to its mono-resistant rate as well as a component of the most frequent resistance patterns. resistance to the combination of pip with tazobactam however is rare. the development of antimicrobial resistance in german hospitals objectives: quinolones (q) are among the most frequently used agents for treating ltcf-acquired infections. consequently, bacteria exhibit increasing resistance to q. a multicentre survey was conducted in order to determine (i) the prevalence and risk factors for colonization with qrgn in greek ltcf (ii) the corresponding antimicrobial resistant rates (á r). methods: a total of 28 ltcf were randomly selected from the public sanitation list of attica province. urine, nasopharyngeal and wound samples were collected from 668 elderly residents. we chose randomly 35% of the existing population from each ltcf (minimum sum 25 residents, age above 65 years). gram negative bacteria were identified by api strips and underwent antimicrobial disk susceptibility testing, following the nccls guidelines. data were also collected on resident factors and institutional variables. univariate and multivariate analyses were performed. odds ratios (or) and p values were calculated. results: 412 gram negative (gn) strains were isolated from 1448 samples (prevalence rate 28.5%). the majority of them were recovered from urine samples (86.8%). 53% of the residents had been taking systemic antibiotics during the preceding month. q were the leading class (38%). á r of gn to ciprofloxacin (cip) were estimated (table) . multi-drug qrgn accounted for 11.5% of the isolated strains. the most prevalent phenotype was resistance to ampicilline, cip and trimethoprim-sulfamethoxazole. in the multivariate analysis, only prior exposure to antimicrobial agents (p < 0.001; or = 2), specifically to q (p < 0.001; or = 14), and the presence of a urinary catheter (p < 0.001; or = 2) were significantly associated with colonization with qrgn. objectives: colonization and resistance dynamics of gramnegative bacteria in the intestinal and oropharyngeal microflora of patients admitted to intensive care units (icu) and general wards (gw) were investigated during and after hospitalization. methods: specimens were obtained on admission, once weekly during hospitalization, at discharge from the icu, at discharge from the hospital and one and three months after discharge from the hospital. five colonies per specimen were selected for identification and susceptibility testing by the vitek 2 system. results: a total of 3316 specimens from 411 patients were collected. in both patient populations, the gram-negative colonization rates in oropharyngeal specimens increased during hospitalization and did not decrease in the three months after discharge. in rectal specimens, colonization rates decreased during hospitalization and increased after discharge. there was a change in species distribution among the dominant microflora during hospitalization. klebsiella spp., enterobacter spp., serratia marcescens and pseudomonas aeruginosa were more often isolated, whereas the frequency of e. coli declined. the percentage of icu patients colonized with ampicillin and/or cephalothin resistant faecal e. coli was significantly increased at discharge from the hospital and did not change in the three months after discharge. emergence of multi-drug resistance was observed in many gram-negative species during stay on the icu. resistance frequencies in e. coli significantly increased with length of stay on the icu. for the gw population, no significant changes in resistance frequencies were found during hospitalization. conclusion: from a population perspective, the risk of dissemination of resistant gram-negative bacteria into the community through hospitalized patients appears to be low in gw patients, but is noticeably higher among icu patients. association of antibiotic resistance phenotypes and the presence of class i integrons in a sample of bacterial isolates from u.s. hospitals objective: we assessed the association between class 1 integrons and patterns of resistance to aminoglycosides, beta-lactams, quinolones, and chloramphenicol in escherichia coli and klebsiella species isolates from u.s. hospitals participating in project icare. methods: a convenience sample of 320 e. coli and klebsiella species isolates was collected between october 2002 and december 2003 from 19 u.s. hospitals as part of phase iv of project icare and tested for susceptibility to a variety of antimicrobial agents. these organisms were submitted in response to a request for organisms resistant to 3rd generation cephalosporins (ceftazidime, ceftriaxone, cefotaxime) and/or fluoroquinolones. the isolates were screened for the presence of class i integrons using sets of primers specific for 5¢ and 3¢ conserved segment of the integrase gene. we obtained measures of association between the presence and absence of integrons and resistance phenotypes. results: of the 320 e. coli and klebsiella species isolates screened, 181 (57%) contained a class i integron. a significant association was seen between the presence of the class i integron and resistance to amikacin, gentamycin, tobramycin, ampillicin, chloramphenicol, aztreonam, ceftazidime, cefotaxime, and cefpodoxime. resistance to ciprofloxacin and cefepime was not significantly associated with the presence of an integron. the presence of class i integrons in 57% of our convenience sample of e. coli and klebsiella species isolates, along with the differential association of those integrons with resistance to some drugs, but not others, suggests that integrons may play an important role in determining how the epidemiology of bacterial resistance results from antimicrobial agent selective pressure. objectives: the main purpose of this study was to investigate the mechanisms of resistance to fluoroquinolones in two isogenic citrobacter freundii clinical isolates, which led us to characterize the acra and acrb genes of this microorganism. methods: two c. freundii strains (1.44 and 1.38) sequentially isolated from the same patient were characterized. the relationship was determined by rep-pcr and pfge. the susceptibility to ciprofloxacin and chloramphenicol was determined by e-test. mutations in the quinolone resistance determining region of the gyra and parc genes, the outer membrane protein profile and the accumulation of ciprofloxacin was also investigated. the expression of genes in both strains was analysed using dna microarrays for e. coli and the expression of the acra and acrb genes was verified by rt-pcr using the gapa gene as the control. the acra gene was cloned and dna sequenced. results: both c. freundii belong to the same clone by both rep-pcr and pfge. the mic of ciprofloxacin was of 8 (strain 1.44) and 32 mg/l (strain 1.38). the mic of chloramphenicol was of 16 and 96 mg/l, respectively. the two strains showed the same substitutions in the gyra and parc (thr-83-ile and asp-87-tyr in gyra and ser-83-ile in parc). no major differences were found between the outer membrane protein profiles. however, differences were observed in the amount of ciprofloxacin accumulated, with strain 1.38 showing less accumulation. eleven genes were overexpressed in strain 1.38 compared to strain 1.44. among these genes the acra was overexpressed. this result was further corroborated by rt-pcr. the nucleotide similarity between the partially sequenced acra (1027 bp) and acrb (420 bp) genes of c. freundii and e. coli was of 81.5% and 86%, respectively. conclusion: the acra and acrb genes of c. freundii are similar to those described in e. coli and in collaboration with mutations in the gyra and parc genes, their overexpression may play an important role in modulating the final mic of fluoroquinolones. high prevalence of aac (3) objectives: multidrug resistance of gram negative bacilli (gnb) has become a major public health problem in tunisia. surveillance of both antimicrobial resistance and antimicrobial consumption has now been recognised as essential for planning future strategies to control resistance. the aim of our study is to examine for the whole hospital and wards with risks the correlation between resistance of gnb and the consumption of third generation cephalosporins (3rd gc) and imipenem for the period from january 2003 to september 2004. methods: a surveillance of antibiotic resistance and antibiotic consumption was established respectively by the laboratory and the department of pharmacy. data on antimicrobial resistance and antibiotic consumption were collected quarterly and antibiotic consumption was calculated in defined daily doses (ddd) using abc calc. results were expressed per 1000 bed-days (bd). statistical analysis was done using spss software for the calculation of the spearman rank correlation (rs) with an alpha risk of 5%. results: for the whole hospital, resistance to 3rd gc ranged from 0.9 to 1.7 gnb/1000 bd. the highest rates were observed in intensive care unit (icu: 4.7-12.7 gnb/1000 bd) and the lowest rates in orthopaedic ward (0-2 gnb/1000 bd all mrsa isolates were resistant to multiple classes of antimicrobials whereas mssa were sensitive (p < 0.001). all esbl+ bacteria were resistant to multiple classes but sensitive to meropenem, colistin, amikacin (82%), cotrimoxazole (50%) and chloramphenicol (50%). resistance in non-esbl+ bacteria was detected to ampicillin (73%), cefalexin (32%), 2nd/3rd generation cephalosporin (0%), chloramphenicol (32%), cotrimoxazole (11%), ciprofloxacin (5%), trimethoprim (21%), gentamicin (5%), netilmicin (53%) and tobramycin (11%). potentially community acquired isolates showed lower rates of resistance (0% to trimethoprim, ciprofloxacin, gentamicin, amikacin, tobramycin, 2nd/3rd generation cephalosporin).duration of hospital stay was not a risk for acquiring either esbl+ bacteria or mrsa whereas having a surgery was associated. exposure to quinolone was a risk factor for mrsa but not esbl+ bacteria. aminoglycoside and cephalosporin were risks for esbl+ bacteria. acquiring both mrsa and esbl+ enterobacteriacae together did correlated with the duration of hospital stay in addition to exposure to aminoglycoside and cephalosporin. conclusion: majority of patients remained free from resistant bacteria implying that cross-transmission alone was not sufficient in absence of risk factors, particularly exposure to antimicrobials. good hand-hygiene and prudent use of antimicrobials are realistic options for resource poor countries to reduce the burden of resistant bacteria. there is a unique opportunity to sequentially introduce specific infection control measure and evaluate its effectiveness. antimicrobial resistance in an intensive care unit before adopting an antibiotic rotation programme for empiric therapy of ventilator-associated pneumonia results: among gram-negative aerobic bacilli, we found p. aeruginosa strains to be meropenem and piperacillin/tazobactam-resistant in 22% and 14.6% of cases, respectively; p. aeruginosa, e. coli and k. pneumoniae were resistant to ceftazidime in 29.3%, 6.25%, and 0% of cases, respectively, and resistant to ciprofloxacin in 54.9%, 12.5%, and 5.6% of cases, respectively. neither e. coli nor k. pneumoniae were resistant to either meropenem or piperacillin/tazobactam. among gram-positive aerobic cocci, oxacillin-resistant s. aureus and cns were 55.8% and 82.7%, respectively; s. aureus isolates were very susceptible to cotrimoxazole (resistance 7.65%) and rifampin (11%), whereas resistance exceeded 40% for clindamycin, gentamicin, and norfloxacin. ampicillin-and penicillin-resistant e. faecalis were 20.8% and 33.3%, respectively. neither s. aureus nor e. faecalis were resistant to vancomycin, whereas one cns strain was resistant to it (1.3%) and 6 out of 76 to teicoplanin (7.9%). conclusion: in our icu, where a careful policy of antibiotic use with no predetermined restrictions has been applied for years, ar among both gram-negative and gram-positive microorganisms is generally lower than in the icus of italy and other mediterranean countries. we expect that the institution of an arp for empiric therapy of vap can further minimize ar in our icu. background: although newer antibiotics have been introduced to the market during the last years, they have not solved the problems arising in the management of infections due to multiresistant gram-negative bacteria. colistin, an antibiotic almost forgotten for decades has proved itself helpful, when used parenterally in patients where a lot of the classic and newer antibiotics fail. methods: we report our experience with the management of the case of a young patient who, after head trauma, had five episodes of meningitis due to multidrug-resistant gram-negative microorganisms. results: a 28-year-old patient was admitted in our hospital because of coma. three months prior to his admission, he sustained acute brain injury due to a fall and was hospitalized elsewhere for this entire period. a computerized tomography scan of his brain at his admission in the first hospital, showed multiple cerebral contusions, and an intracerebral hematoma causing deviation of the midline. he underwent a decompressive craniotomy with removal of the hematoma. during his long-standing hospitalization, he developed 5 episodes of meningitis, i.e. on day 65, 96, 108, 147, and 165 after head trauma. the pathogens isolated from each episode of meningitis were multiresistant strains of pseudomonas aeruginosa (1st and 2nd episodes), acinetobacter baumannii (3rd episode), enterobacter cloacae (4th episode), and acinetobacter baumannii (5th episode). the two episodes of acinetobacter baumannii meningitis were managed only when colistin and amikacin were given by the intraventricular in addition to the intravenous route for long period of time (six weeks for the last episode) and in high doses (40,000 iu of colistin and 10 mg of amikacin results: eight patients received aerosolized colistin. all were admitted to the icu with a mean acute physiological and chronic health evaluation ii (apache ii) score on the day of icu admission and on the 1st day of aerosolized colistin administration of 14.6 and 17.1, respectively. six of the 8 patients had ventilator-associated pneumonia. the responsible pathogens were acinetobacter baumannii (7/8 cases) and pseudomonas aeruginosa (1/8 cases) strains. half of the isolated pathogens were sensitive only to colistin. daily dosage of aerosolized colistin ranged from 1.5 to 6 million iu (divided into 3 or 4 doses) and the mean duration of administration was 10.5 days. seven out of 8 patients received concomitant intravenous treatment with colistin or other antimicrobial agents. clinical response of pneumonia was observed in 7 out of 8 patients [4 cured, 3 improved (they were transferred to another facility)]. one patient deteriorated and died due to septic shock and multiple organ failure. aerosolized colistin was well tolerated by all patients; no bronchoconstriction or chest tightness was reported. conclusions: aerosolized colistin may be a beneficial adjunctive treatment in the management of nosocomial pneumonia (vap or not) due to multidrug-resistant gram-negative bacteria. to date, susceptibility rates to imipenem and colistin of nosocomial strains are reported as high as 88% and >98%, respectively. in particular, resistance to colistin has been rarely described and the mechanisms of this resistance are poorly understood. we report a case of colistin resistant a. baumannii infection in a patient without previous administration of colistin. case report -methods: a colistin resistant (mic >2 mcg/ml) a. baumannii was isolated from 3 sets of blood cultures in a patient admitted in a burn unit without previous colistin exposure. the patient was treated with ampicillin/sulbactam and colistin, despite the documented resistance to all antibiotics. mdr a. baumannii was repeatedly isolated from different sites and the patient died after 61 days of hospitalization. strain identification and susceptibility tests were performed using phoenix automated microbiology system ò . resistance to ampicillin, ampicillin/sulbactam, ceftazidime, imipenem, meropenem, cotrimoxazole, amikacin, ciprofloxacin and colistin was confirmed by disk diffusion test and broth microdilution, in accordance with the nccls guidelines. the susceptibility results were confirmed by a reference laboratory. conclusion: colistin is one of the few effective drug available against mdr acinetobacter infections and acquired resistance is exceptional. our report points out that colistin resistant a. baumannii may be found in hospitalized patients without previous exposure to colistin. nosocomial infections by colistinresistant a. baumannii could represent a new therapeutic challenge. programmes of active surveillance cultures and contact precautions should be implemented to control the spread of this mdr microorganism. intrathecal colistin treatment of central nervous system infections due to multi-resistant acinetobacter baumannii c. suárez fernández, p. fernández-viladrich, f. tubau, l. corral , a. mateu, j. ariza (barcelona, e) objectives: the optimal therapy of cns infections due to multi-resistant acinetobacter baumannii (mrab) is not established. in 1999 we published the first two cases of ventriculitis due to mrab treated with intrathecal (it) colistin. one patient received 10 mg colistin base/12 hours after delayed csf sterilization was documented with initial dosage of 5 mg/12 hours. here we describe our extended experience with this kind of treatment . methods: we retrospectively reviewed all documented cns infections due to mrab diagnosed in our terciary 1000-bed hospital from 1999 to 2004. mrab was defined as those strains with only susceptibility to colistin (mic < 4 mg/l). cns infections were defined as those cases with suggestive clinical features plus ventricular or lumbar csf with both characteristic cytochemical alterations and positive culture. results: there were 7 cases with documented nosocomial cns infection due to mrab. six of them were acquired after neurosurgical procedure through surgical wound infection or local catheter infection. one case was caused by hematogenous spread after urological manipulation. intrathecal therapy was administered through a ventricular catheter in five cases, lumbar catheter in one case and both ventricular and lumbar catheter in one case. the usual doses administered was colistin base 10 mg (equivalent to colistimethate sodium 24 mg) every 12 hours with csf continuous drainage being interrupted for about 3 hours. length of treatment were 8-21 days with any apparent adverse effect. six of these patients received simultaneous intravenous colistin therapy. culture sterilization was documented in all patients. two patients died (one from unclear cause after initial favorable evolution; one after receiving only two doses of it colistin). the rest of them cured with sequela attributable to their previous neurological diseases. conclusions: in our experience, combined treatment with both intrathecal and intravenous colimicin seems safe and effective. when administered by local route to patients with continuous csf drainage we suggest a dosage of colistin base of 10 mg every 12 hours with temporary interruptions of the drainage. bacillus cereus -the forgotten pathogen. an unusual infection in an orthopaedic patient s. thomas, a. pillai, j. arora, a.c. ogden (dumfries, uk) background: infection with bacillus cereus has been well documented in the literature for over a century. infection is generally associated with the gastrointestinal effects of food poisoning and linked to the consumption of infected rice dishes. however, the bacillus genus is extremely heterogenic. it can occupy a wide variety of ecological niches and bacillus spores are found ubiquitously in the environment. even so, bacillus cereus isolated from the hospital and clinic setting in any material other than vomitus or faeces is commonly dismissed since it is a known contaminant of blood cultures and most bacillus bactereamias are transient and usually insignificant. case report: we report a case of bacillus cereus wound infection in a previously healthy 31-year-old male admitted to the orthopaedic ward with a comminuted fracture of the right distal tibia. he developed compartment syndrome one day later and was taken to theatre for fasciotomy. within 48 hours, he developed chest complications and was admitted to the high dependency unit. after three days he was taken back to theatre for wound inspection and change of dressing. seven days later external fixation was applied. subsequently, the wound site became red, inflamed and tender and was associated with an acute rise in inflammatory markers. microbiological reports showed bacillus cereus growth from the suture sites, sensitive to ciprofloxacin. no source was identified. discussion: this report merits concern, because it highlights the risk of wound cross infection with this unlikely pathogenic species. bacillus cereus has been known to be a contaminant of dressings, intravenous catheters, theatre scrub suits and linens pointing to an array of possible infective sources within the hospital. it emphasizes the need for theatre and hospital sterility and stresses the importance of vigilance against this infrequent cause of a potentially serious non-gastrointestinal bacillus infection. this is of particular importance because alcoholbased cleaning solutions are known to be ineffective against bacillus spores and infections dismissed as contaminants may lead to rapid clinical deterioration. antibacterial effect of phenytoin in wound healing objectives: phenytoin is an antiseizure medication, has since been reported to promote wound healing when applied as a topical agent. this effect is due to rapid infiltration of fibroblasts, collagen deposition, new vessel formation as well as antibacterial activity. this study was undertaken to evaluate it's effect on chronic skin ulcers with different causes and compare it with normal saline. methods: fifty inpatients with chronic skin ulcers were included in this case-control study: diabetic foot (20%), fracture and surgery wounds (40%), decubitus ulcers resulting from warrelated (40%). the patients were matched for age, sex, severity and size of wounds and randomly assigned to two group: 25 phenytoin treated (pht) and 25 control (ctl). each group included 15 men and 10 women, age range was between 20 and 60. surface areas of the ulcers was 12-200 cm 2 . the ulcers were debraded of necrotic tissue (if required). cultures of ulcers were taken at the beginning of treatment and on day 7, 14. in pht, thin dusting of phenytoin powder and dry gauze dressing were applied daily after washing with normal saline. ctl group received only daily washing with normal saline and plain dressing. results: bacteriologic culture in both group confirmed some strains (staphylococcus aureus, kelebsiella, proteus and psudomonas). at the beginning of treatment, there was 17 culture positive in pht. 13 case became negative by day 7 and 4 case became negative by day 14. in ctl, we had 15 culture positive that they didn't become negative by day 7 and 14 (seventy-six per cent of pht had negative cultures by day 7 compared to 0% of ctl). the mean time for appearance of granulation tissue was 13.48 days in pht compared with ctl that was 37.5 (p = 0.006). the average time to complete healing in pht was 24.76 days compared to 43.36 in the ctl (p = 0.0001). in pht only 13 patients required to analgesics (mean 1/day) compared with ctl that all patients required to analgesics about 3-4/day (p = 0.0001). age, sex and kind of wounds didn't effect the healing time in both group. conclusions: this study with statistical analysis demonstrated good improvement and efficacy of phenytoin in treatment of chronic ulcers compared with normal saline. 1. prompt pain relief, 2. reduction in ulcer size, 3. changing bacterial culture to negative, 4. more rapid granulation and healing time, characterized the pht group. we recommend wider use of this safe, inexpensive, readily available and easy to use agent because of it's positive effect on wound healing especially in our country that a lot of patients suffer from decubitus ulcers resulting from war-related wounds and limited access to more expensive wound care therapies. recurrent osteomyelitis secondary to different bacteria i. uckay, m. assal, l. legout, p. rohner, r. stern, d. lew, p. hoffmeyer, l. bernard on behalf of the osteomyelitis group study objective: recurrence of osteomyelitis due to the same bacteria from a prior site of infection without evidence of trauma or bacteraemia is an entity well-known in the literature and clinical practice. the objective of this study is to try and determine if all these reactivations are actually due to the same bacteria, as has been assumed in previous case reports. case reports: we report three cases of osteomyelitis recurring in the same bone in otherwise healthy patients. in all three patients, there was no history of illness or trauma to assume another origin. surprisingly, the strains were different from the two infectious episodes. conclusion: reactivation of osteomyelitis can be caused by different strains of bacteria, many years after the initial episode without trauma or evident bacteraemia. former infected and therefore altered bone surface might be a region of diminished resistance for a new infection during silent transient bacteraemia, reminiscent of the clinical entity of (recurrent) infectious endocarditis on altered valve surfaces. objectives: vascular graft infection proved to be the most dangerous complication in vascular surgery patients. the aim of our study was the identification of microorganisms causing vascular graft infections and the evaluation of their antimicrobial susceptibility. 25 patients with infected vascular grafts, treated in vascular surgery department, took part in our research. in 76% of patients, the late type of infection was recognized, in 24% of patients the infection was qualified as early. methods: purulent discharge obtained from the fistula was inoculated on the bacteriological media. antimicrobial susceptibility was assessed by disc-diffusion method. results: staphylococcus aureus and pseudomonas aeruginosa, present in 16,7% of patients, proved to be the most frequently isolated microorganisms. staphylococcus epidermidis and e. coli were isolated in 13.3% and 10% of patients, respectively. mixed infection, caused by two distinct bacteria, occurred in 20% of patients; in all cases one species belonged to gram-positive, and the second one to gram-negative bacteria. in 50% of patients with early type infection different species of gram-negative rods were present, in 37.5% of patients, staph. aureus and staph. epidermidis were isolated, enterococcus faecalis occurred in 12.5% of patients. in late type infection gram-negative rods were isolated from 54.5% of patients and gram-positive bacteria from 31.5% of patients. the most frequently isolated species appeared to be pseudomonas aeruginosa. in one patient candida crusei was isolated. the isolated species of bacteria varied depending on the degree of infection (according to shilagy and samson). in iiia degree of infection staph. epidermidis and e. coli were present in 67.7% and 33.0% of patients, respectively. pseudomonas aeruginosa and staph. aureus were isolated in 21,1% of patients with iiib degree of infection. various species of gramnegative rods were isolated from 80% of patients with iiic degree of graft infection. conclusions: most isolated bacteria appeared to possess the resistance patterns typical for hospital flora; among them methicillin-resistant staph. aureus (mrsa) and methicillin-resistant coagulase-negative saphylococci (mrcns) strains, gramnegative rods producing extended spectrum of beta-lactamases (esbl) or ampc beta-lactamases, and enterococcus faecalis with high level of aminoglicosides resistance (hlar). treatment failure due to emergence of resistance to imipenem during therapy for shewanella algae bacteraemia objective: we describe here a case of bacteraemia caused by s. algae, which was initially susceptible to imipenem, but the bacteria later became resistant during the treatment. in addition, we investigated the propensity of s. algae to develop resistance to imipenem by using a serial passage technique. method: in vitro test for resistance induction 1. bacterial strains resistant variants selection (a) single step resistant variants were obtained from clinical isolate on mueller-hinton agar containing increasing amounts of imipenem (1xmic, 2xmic, 4xmic, 8xmic), (b) serial passage experiment s. algae strain and p. aeruginosa atcc 27853 were grown overnight in mueller-hinton agar and then grown overnight in mueller-hinton agar and then swabbed onto mueller-hinton agar plates containing one-half the mic of imipenem. the surface growth at 24 h was swabbed to mueller-hinton agar containing twice the prior concentration of imipenem. results: single step resistant variants were selected from isolate 1 at up to 4 times the mic, whereas the resistant variant from p. aeruginosa atcc 27853 could be selected at up to 2 times the mic. all the resistant variants of s. algae selected either by a single step or by a sequential stepwise passage exhibited at up to 8-16 lg/ml of mic, whereas those of p. aeruginosa atcc 27853 showed up to 16 lg/ml of mic conclusion: we documented that s. algae, which was initially susceptible to imipenem, subsequently became resistant to imipenem during the treatment. we also demonstrated in vitro that the initial isolate of s. algae could easily develop resistance to imipenem when the organism was exposed to imipenem, suggesting that s. algae organisms have a propensity toward resistance to imipenem. objective: peritonitis remains a common and serious complication of peritoneal dialysis. in this study, we evaluated the frequency of microorganisms isolated from peritoneal fluids of patients on continuous ambulatory peritoneal dialysis (capd). methods: during a 2.5 year period, 321 peritoneal fluid samples were collected from 107 capd patients with peritonitis symptoms. the specimens were inoculated on appropriate media after 10-min centrifugation. gram stained smears and aerobic, anaerobic and broth cultures were performed. the isolates were identified by commercial id panels. mics were determined with a microdilution method according to nccls guidelines. results: fifty two out of 321 samples were positive (16.2%), six of the positives were polymicrobial. the following microorganisms were obtained: pseudomonas aeruginosa (8), staphylococcus epidermidis (7), escherichia coli (7), staphylococcus aureus (6), streptococcus viridans (6), klebsiella pneumoniae (4), enterococcus faecalis (4), stenotrophomonas maltophilia (4), enterobacter aerogenes (2), acinetobacter baumannii (1), enterococcus faecium (1), proteus mirabilis (1), pseudomonas fluorescens/putida (1), acinetobacter lwoffii (1), salmonella group d (1), bacteroides spp. (2). fungi were isolated in three patients: candida tropicalis (1), candida krusei (1) and candida parapsilosis (1). three out of 6 of s. aureus strains and three out of 7 of s. epidermidis isolates were found resistant to methicillin. all saphylococci and enterococci were susceptible to vancomycin and linezolid. gram-negative pathogens were sensitive to used antibiotics. conclusion: thirty five patients (32.7%) developed peritonitis the most prevalent etiologic agents of peritonitis on capd patients were pseudomonas aeruginosa, staphylococcus epidermidis, escherichia coli, staphylococcus aureus and streptococcus viridans. since capd patients are commonly outpatients, the antimicrobial resistance in the gram-negative strains is low compared to the nosocomial isolates. appropriate antibiotic therapy based on microbiologic results needs for the management of peritonitis on capd patients. objectives: cefepime is a fourth generation cephalosporin with a broader spectrum of activity against gram-negative and gram-positive pathogens in comparison with all other cephalosporins. due to the excellent antibacterial activity including pseudomonas aeruginosa and enterobacter spp. together with low rates of resistance and favourable pharmacokinetic and clinical properties, cefepime is a drug of choice for initial empiric treatment of severe nosocomial infections. nevertheless, in addition to recommendations in guidelines, the surveillance of local resistance data is important for empiric treatment decisions in hospitals. methods: in this study, the in vitro activity of cefepime (cep), imipenem (imi), piperacillin tazobactam (p/t), ceftazidime (caz) and cefotaxime (cft) has been investigated against 1130 bacterial strains, isolated in the year 2004. mics have been determined by microdilution method according to din 58940. reference test strains were e.coli atcc 25922, s. aureus atcc 29213, p.aeruginosa atcc 27853, and s.pneumoniae atcc 49619. results: highest in vitro activity against gram negative enterobacteriaceae were determined for cep and imi with susceptibility rates for enterobacter spp. 98%,98%, m.morganii 96%,92%, proteus spp. 98%,96% and citrobacter spp. 96%,100% whereas the susceptibility rates for p/t, caz and cft were much lower (e.spp. 68%,72%,68%; m.m. 88%,72%,66%; p.spp. 96%,96%,66%; c.spp. 68%,76%,72%). for pseudomonas aeruginosa (n = 100), susceptibility rates were 76% (cep), 73% (caz), 72% (imi) and 55% (p/t) with lowest resistance rates for cep (6%) followed by caz (11%), p/t (14%), imi (23%). the study confirmed excellent cefepime activity against gram positive isolates (pneumococci, streptococcus viridans and b-hemolytic streptococci) and high cefepime susceptibility rates for staphylococci (mssa 99% and msse 98%) on contrary to ceftazidime (mssa 44% and msse 42%). conclusion: regarding our in vitro data, cep is a reliable treatment option for empiric therapy in patients with severe nosocomial infections, demonstrating high activity against gram positive isolates, comparable activity in gram negative enterobacteriaceae to carbapenems (imi) and lowest resistance rates in pseudomonas aeruginosa. antimicrobial activity and spectrum of cefepime tested against 65,746 clinical strains from north american medical centres: report from the sentry antimicrobial surveillance program, 1998 program, -2004 h. sader, d. biedenbach, t. fritsche, r. jones (north liberty, usa) objectives: to evaluate antimicrobial spectrum and potency of cefepime (cpm) and selected comparators against clinical bacterial strains collected in north america (na) over a 7-year period (1998) (1999) (2000) (2001) (2002) (2003) (2004) . methods: isolates were consecutively collected from bloodstream (44%), respiratory tract (41%), urinary tract (6%) and skin/soft tissue (5%) infections in 48 medical centres. 75% of isolates were from hospitalized patients. isolates were susceptibility (s) tested by reference nccls broth microdilution methods in a central laboratory. oxacillin-resistant (r) staphylococci (ors) and enterococci were excluded. results: the activity of cpm against the key organisms tested is summarized in the table. overall, 99.8% of gram-positive cocci (gp) tested were s to cpm. imipenem (imp; mic90, 1 mg/l; 99.9% s) was the most active compound tested against ent, followed by cpm (mic90, 0.25 mg/l; 99.5% s) > amikacin (amk; 99.4% s) > ceftriaxone (95.6% s) > aztreonam (95.1% s). the lowest s rate for ent was observed with ciprofloxacin (cipro; 92.8%). imp was also the most active compound against esbl-producing ksp and e. coli (99.3 and 100% s, respectively), followed by amk (81.4 and 97.2% s) and cpm (92.5 and 93.8% s). cpm activity against psa (85.2% s) was similar to that of imp (86.9% s). against ossa, cpm was 4-fold more potent than ceftazidime (caz; mic90, 16 mg/l, 86.4% s) and showed higher activity than cipro (93.2% s). cpm was the most active compound against spn after gatifloxacin and levofloxacin (99.2% s). against vgs, cpm was 8-fold more potent than caz and 4-fold more potent than piperacillin/tazobactam. regionally, mdr ranged from 9.1% in los angeles to 32.2% in south florida. r to pen, ery, and sxt was the most common mdr phenotype in all regions except baltimore/dc (r to ery and sxt was most common). by specimen source, 15.5% of blood, 21.0% of lr, and 29.0% of ur isolates were mdr with r to pen, ery, and sxt being the most common phenotype regardless of specimen source. for the 30 strains tested, the fluoroquinolone mic ranges were: 0.008-0.03 mg/l (gem), 0.12-0.25 mg/l (gat), 0.06-0.25 mg/l (mxf), 0.5-1 mg/l (lfx). conclusions: mdr among sp is a phenotype that is widely dispersed geographically and is likely to be encountered regardless of the site of infection. fluoroquinolones show activity against most mdr isolates. as the use of fluoroquinolone compounds increases, surveillance to monitor the prevalence of mdr and track the in vitro activity of agents such as gem used for these resistant strains must be continued. background: multi-drug resistant (mdr) s. pneumoniae strains are increasing at an alarming rate worldwide. the therapy of respiratory tract infections due to these strains is challenging with an urgent need for antimicrobials with reliable activity against the mdr strains. comparative activity of oral cephalosporins and parenteral cephalosporins against various pneumococcal mdr phenotypes was analysed in a large, multi-year international collection of clinical strains of s. pneumoniae. methods: s. pneumoniae strains (21,605) collected during 1997-2003 from worldwide participants of the sentry program were tested and interpreted using nccls (m7-a6, m100-s14) guidelines. the antimicrobial agents analysed included penicillin (pen), erythromycin (er), clindamycin (cm), tetracycline (tet) and trimethoprim/sulfamethoxazole (ts); cephalosporins monitored included oral (cefpodoxime, cefuroxime) and parenteral (ceftriaxone, cefepime) agents. results: the rank order of occurrence rates of the various resistance phenotypes were: pen only (32.0%) > pen and er (17.6%) > pen, er and cm (8.6%) > pen, er, cm and tet (7.6%) > pen, er, cm and ts (6.5%) > all five drugs (5.7%). the susceptibility rate of all strains to the orally administered cephalosporins, cefpodoxime (77.6%) and cefuroxime (77.3%), dropped to only 16.1% and 14.5% respectively, for the five-drug mdr phenotype. the parenteral cephalosporins retained excellent activity for all mdr phenotypes, with resistance rates being lower for cefepime than ceftriaxone (cefepime, 1.3-1.9%; ceftriaxone, 3.0-4.4%) or the oral cephalosporins (cefpodoxime, 64.4-74.1%; cefuroxime, 69.3-79.1%) using respiratory infection breakpoints (nccls). conclusions: our in vitro findings confirm that the parenteral cephalosporins, cefepime and ceftriaxone, retain excellent activity against the mdr phenotypes analysed, and remain useful drugs in the armamentarium to treat mdr pneumococcal respiratory tract infections. antibiotic resistance surveillance over a 5-year period in spain: results of the mystic programme results: three icus, two neutropenia units and two general wards provided a total of 4.022 gram-negative and grampositive isolates during the period 1999-2003. the most common species tested were escherichia coli (14.3%), methicillin-susceptible staphylococcus aureus (13.8%) and coagulase-negative staphylococci (13.5%), pseudomonas aeruginosa (12.2%), enterobacter cloacae (7.2%), klebsiella pneumoniae (5.7%) and a.baumannii (5.6%). in general, the carbapenems (mem and ipm) were the most active antimicrobial agents tested against the common organisms (range of %s:100% to 71% and 100% to 67%,respectively). among enterobacteriaceae, 100% of enterobacter spp, citrobacter spp and serratia spp were susceptible to carbapenems. e. coli and k. pneumoniae susceptibility to carbapenems were 100% and >98% respectively. the %s of mem was the same as or higher than ipm against every organism tested. cip resistance in e. coli was around 20%. caz and taz were the least active antimicrobial agents against enterobacter sp (74% and 77% s, respectively) and citrobacter spp (67% and 68% s, respectively). mem and taz were the most active agents against p. aeruginosa (89% and 88%s, respectively). a. baumannii were 61-90% susceptible to carbapenems, but only 6-1% susceptible to cip. in this period, around 100% of methicillinsusceptible staphylococci were susceptible to mem. conclusion: there was no significant decrease in susceptibility to the carbapenems throughout the five-year period. mem and imp appear to remain reliable options for the treatment of serious nosocomial infections. the clinical use of mem did not increase bacterial resistance to this agent in the spanish centres evaluated. surveillance of antimicrobial susceptibility of anaerobes in a belgian university hospital y. glupczynski, h. nizet, c. berhin (yvoir, b) background: antimicrobial resistance is becoming a growing concern among anaerobes involved in human infections. since there are only scarce data on the in vitro susceptibility of anaerobes in our country, we aimed to determine the susceptibility and resistance profiles of anaerobic bacteria isolated in our hospital. table. conclusion: b-lactams-b-lactamases inhibitors, carbapenems, and met remain highly active against anaerobes including the more resistant bfg of organism. resistance in cli actually almost reaches 30% and is observed among almost all different species of anaerobes. mox, as a representative of the newer fluoroquinolones exhibits a broad spectrum and potent activity in vitro against all anaerobes tested and looks promising for the treatment of mixed infections involving anaerobes. pharmacokinetics/pharmacodynamics of quinolones objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens, including anaerobes. some of the fluoroquinolones have the potential to prolong qtc. to determine the effect of grn on qtc, a retrospective analysis was performed on manually read ecgs from five phase i studies. methods: serial ecgs were collected in 5 randomized, doubleblind, placebo-controlled studies in healthy adult subjects administered oral (po) or intravenous (iv) grn 50-to 1200mg doses (therapeutic dose £600 mg) with dosing duration 1 to 28 days (therapeutic duration £14 days). the qt interval was corrected for heart rate using bazett's (qtcb) and fridericia's (qtcf) formulas, and the effect of grn was assessed by counts of outliers and linear regressions. single-and multiple-dose pharmacokinetics of po and iv grn were derived from plasma concentration versus time data. results: a total of 149 subjects received grn and 55 received placebo. grn plasma exposure (auc and cmax) increased with increasing dose. no subjects experienced a prolonged qtcb interval (>450 msec for males, >470 msec for females). only one subject experienced a prolonged (>60 msec) qtcb change from baseline which occurred on day 7, but not on subsequent days despite continued dosing. the incidence of borderline qtcb (change between 30-60 msec) was comparable between placebo and grn. means for qtcb max, qtcb avg and qtcb at tmax and their changes from baseline for grn were similar to those for placebo, with the exception of qtcb max, qtcb at tmax, and their changes for 600 mg iv grn on day 14. although 1200 mg po and 800 mg iv grn produced higher plasma exposures than 600 mg iv on day 14, the means for the derived qtcb values were comparable to placebo. all 95% confidence intervals for the linear regression slopes of derived (delta)qtcb on cavg(0-12 h) were equal to or less than 0, except for (delta)qtcb at tmax on day 7, indicating that grn had no effect on qtcb. results obtained for qtcf were similar to those for qtcb. conclusions: grn does not appear to have dose-, route of administration-or concentration-dependent effects on qtc interval in healthy subjects. objective: garenoxacin (grn) is a novel des-f(6)-quinolone being developed for a variety of indications because of its efficacy against a broad spectrum of pathogens, including anaerobes. the objective of this study was to assess the tissue or fluid to plasma ratios of grn following a single oral dose. method: an open-label study was conducted in subjects ‡18 years of age with a body mass index £33 kg/m2 undergoing abdominal surgical procedures that would permit the removal of tissue or fluid without increased risk to the subject. a single 600 mg oral dose of grn was administered based on the scheduled operative time. the tissue or fluid (with corresponding plasma sample) was collected 3 to 5 hours post-dose. concentrations of grn in biological fluids and tissues were determined using validated lc/ms/ms assays. safety was assessed by measurement of vital signs, physical examination, and electrocardiographic and clinical laboratory evaluations. adverse event (ae) monitoring was performed from time of consent until discharge from the study. results: thirty-one subjects were enrolled in and completed the study. mean fluid or tissue grn concentrations greater than that found in plasma (mean fluid or tissue to plasma ratio >1) occurred in large and small bowel tissue, gallbladder, and liver. mean fluid or tissue grn concentrations less than that found in plasma occurred in adipose tissue, bone, sinus mucosa, striated muscle, incisional skin, and subcutaneous tissue. mean grn concentrations in bile and lymph node tissue were roughly similar to that found in plasma. tissue and fluid concentrations of grn exceeded the mic90 of most target organisms involved in skin and soft tissue, bone, and intra-abdominal infections and sinusitis ‡2-fold. no treatment-emergent aes or serious aes were considered to be related to grn. conclusion: a 600 mg oral dose of grn penetrates well into most of the tissues and fluids studied, with concentrations exceeding the mic90 of most pathogens causing sinusitis, skin and soft tissue infections, bone infections, and intra-abdominal infections. these results suggest that adequate concentrations of grn can be achieved to treat infections at these sites. penetration of garenoxacin into lung tissues in patients undergoing lung biopsy or resection objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens, including those commonly found in the respiratory tract (rt). this study was conducted to determine the penetration of grn into lung parenchyma (lp) and bronchial mucosa (bm) following a single 600 mg oral dose. penetration of grn into bone was also assessed. this was an open-label, nonrandomized study in subjects undergoing an invasive procedure to the lung (other than percutaneous lung biopsy) which facilitated the removal of macroscopically normal healthy lung tissue without exposing the subject to undue risk. penetrance of grn into bone was determined when removal of rib bone was part of the normal surgical procedure. a single 600 mg oral grn dose was administered based on the scheduled operative time. lp and, if possible, bm and/or bone samples and corresponding plasma samples were removed at 2 to 4, 4 to 6, 10 to 12, or 20 to 24 h post-dose. concentrations of grn in these samples were determined using validated lc/ms/ms assays. safety was assessed based on the occurrence of adverse events (aes) and changes in physical examinations, vital signs, clinical laboratory results, and electrocardiographic results. results: twenty-seven subjects were enrolled; samples were taken from a minimum of 6 patients at each time interval. mean grn concentration in lp increased between the 2-4 and 4-6 h post-dose, suggesting rapid penetration into lp, then declined similar to the decrease seen in plasma concentration. concentrations of grn in lp exceeded the mic90 of organisms associated with rt infections by 62-to 466-fold over a 24 h period. grn concentrations in bm over a 24 h period exceeded the mic90 of respiratory pathogens 51-to 380-fold. concentrations of grn in bone exceeded the mic90 of the organisms associated with bone infections (except pseudomonas aeruginosa, mrsa, fusobacterium species, and enterobacter species) by 6-to 101-fold over a 12 h period. across the 4 time intervals, mean ratios of tissue to plasma grn concentration in lp, bm, and bone reached 2.80, 0.99, and 0.56, respectively, suggesting adequate penetration. no grn-related aes were reported, indicating that a single 600 mg oral grn dose was well tolerated in subjects undergoing invasive procedures. conclusions: grn penetrates rapidly into lp, bm, and bone tissue, producing sustained concentrations that predict adequate coverage of grn-susceptible pathogens at these sites. bioavailability and safety in healthy volunteers unaltered when crushed garenoxacin tablets are administered via a nasogastric tube with or without enteral feeding r. noveck, r. vargas, g. krishna, d. grasela (new orleans, kenilworth, princeton, usa) objective: the purpose of this trial was to assess, in healthy volunteers, the bioavailability and safety of single doses of the novel des-f(6)-quinolone garenoxacin (grn) administered as crushed tablets with and without concomitant enteral feeding compared with intact tablets. methods: in this randomized, crossover, single-dose study, healthy adult volunteers (aged 18 to 45 y) received grn 600 mg (one 200 mg and one 400 mg tablet) orally in 3 regimens: a) intact tablets; b) crushed tablets suspended in water and delivered via nasogastric (ng) tube; c) regimen b plus concomitant enteral feeding (osmolite, 600 ml at 100 ml/hr). subjects were randomized to receive all 3 regimens in 1 of 6 crossover sequences (abc, acb, bac, bca, cab, cba) separated by 7-day washout intervals. for each treatment, subjects entered the facility 1 day before and were confined for 72 hours after drug administration. subjects were discharged from the study after final assessments on day 4 of the third treatment. post-screening assessments included vital signs; plasma drug levels for pk analysis; and physical, laboratory, and ecg examinations for drug safety. results: 18 male subjects were enrolled (4 white, 14 black; mean age, 30 y). pk analysis for grn administered as crushed tablets with and without concomitant enteral feeding vs intact tablets showed no differences in the adjusted geometric means for cmax (8.5 and 8.3 lg/ml vs 8.3 lg/ml) or auc(inf) (97.2 and 93.4 lg*hr/ml vs 103.3 lg*hr/ml). the 90% ci for logtransformed cmax and auc(inf) for b and c compared to a were contained within the protocol-specified ôno effectsõ limit of 70% to 143%. mean grn half-life was similar among the 3 groups (mean range of 12-13 h). tmax was 1 hour following administration of intact tablets compared with 0.5 hour in the other 2 groups. a single oral 600 mg dose of grn was well tolerated whether administered as crushed tablets suspended in water and delivered via ng tube with or without enteral feeding, or as intact tablets. three adverse events (aes) were reported; 1 (nausea) was deemed probably related to study drug. there were no serious aes, meaningful changes on safety examinations, or discontinuations due to aes. conclusions: the bioavailability of grn was similar for crushed versus intact tablets, regardless of whether an enteral feeding was given with crushed tablets. these results show that grn may be administered as crushed tablets with or without concomitant feeding. garenoxacin pharmacokinetics in subjects with severe renal impairment objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens. the absolute bioavailability of grn after oral intake is 92% and approximately 40% of grn is excreted unchanged in the urine. the objective of this study was to evaluate the pharmacokinetics of grn in subjects with severe renal impairment. methods: this non-randomized, open-label study enrolled patients into 1 of 4 groups: healthy control subjects (hc) with normal renal function (clcr > 80 ml/min); subjects with severe renal impairment (sri) (clcr < 30 ml/min) not requiring dialysis; subjects with sri receiving hemodialysis (hd); and subjects with sri receiving continuous ambulatory peritoneal dialysis (capd). a single 600 mg oral dose of grn was administered on day 1 for all groups except hd patients. hd patients received a single 600 mg oral grn dose both before (hd1) and after (hd2) hd, with dosings separated by a 14-day washout. blood, urine, and dialysate samples were collected up to 144 h post-dose and concentrations of grn were determined using validated lc/ms/ms assays. safety was assessed by physical examination, vital signs, and electrocardiographic and laboratory evaluations. results: twenty-five subjects received grn (hc, n = 6; sri, n = 6; hd1, n = 7; capd, n = 6). six subjects in hd2 received grn. mean grn exposure [auc(i)] was similar in hc and hd1 but was 51%, 15%, and 21% higher in sri, hd2 and capd, respectively, than in hc. however, many of the individual values were within the range observed for hc, and these average increases in auc(i) did not exceed values previously shown to be well tolerated. decreases in cmax were observed in sri, hd1, capd, and hd2, but were not considered clinically relevant. approximately 11%, 1.5%, and 3% of grn dose was removed in the dialysate for hd1, hd2, and capd, respectively. a total of 34 treatment-emergent adverse events (aes) were reported, all mild or moderate in severity; 5 were considered to be probably or possibly related to grn. one treatment-emergent but not treatment-related serious ae was reported. conclusions: a single dose of 600 mg grn was well tolerated in patients with sri, including those requiring hd and capd. there was no clinically significant increase of grn exposure in patients with sri based on the broad therapeutic index of grn. therefore, no grn dosage adjustment is recommended in subjects with sri. the effect of omeprazole on the bioavailability and safety of garenoxacin in healthy volunteers j. kisicki, g. krishna, s. olsen, d. grasela (lincoln, kenilworth, princeton, usa) objective: the novel des-f(6)-quinolone garenoxacin (grn) is more soluble in acidic conditions than at neutral ph. therefore, this study was designed to determine if omeprazole affects the bioavailability of grn. methods: this non-randomized, open-label pharmacokinetic study was conducted in healthy adult subjects. on day 1, the single-dose pharmacokinetics of 600 mg of oral grn (one 200 and one 400 mg tablet) were determined in fasting subjects, with serial blood samples obtained through 72 hours post-dose. omeprazole 40 mg was administered once daily on days 4 to 7 to achieve steady-state inhibition of gastric acid secretion. on day 8, single doses of grn and omeprazole were administered concomitantly. omeprazole treatment was continued on days 9 and 10 throughout the period of pharmacokinetic blood sampling. study assessments included vital signs and physical, laboratory, and electrocardiographic examinations for safety. the gm cmax value for grn and grn/omeprazole co-administration was 9.6 and 9.3 mcg/ml, respectively, with 90% ci of 90% to 104%. half-life (mean range 12.9 to 14 h) and tmax (median range 1.5 to 1.8 h) were similar after administration of grn either alone or concomitantly with omeprazole. concomitant administration of garenoxacin and omeprazole was well tolerated. nine of 14 subjects (64%) experienced a total of 33 adverse events (aes). the majority of aes were mild, and only 12 were deemed possibly or probably related to the study drug. the most frequently cited aes, headache, nausea and abdominal pain, were mild to moderate in severity. two subjects withdrew from the study; neither discontinuation was due to aes. conclusions: the concomitant administration of omeprazole had no effect on garenoxacin bioavailability. these findings indicate that garenoxacin can be administered with omeprazole or other agents that affect gastric ph to a similar or lesser extent. the effect of maalox on bioavailability and safety of garenoxacin in healthy volunteers j. kisicki, g. krishna, s. olsen, d. grasela (lincoln, kenilworth, princeton, usa) objective: garenoxacin (grn) is a novel broad-spectrum des-f(6)-quinolone antibiotic. quinolones are known to chelate with cations such as aluminum, impairing antibiotic absorption. this study was therefore designed to assess the effect of a 20-ml dose of maaloxò (containing 900 mg aluminum hydroxide and 800 mg magnesium hydroxide) on the pharmacokinetics of grn when administered concomitantly with, 2 and 4 hours before, and 2 and 4 hours after, maalox. methods: this was a randomized, open-label, 6-treatment, control-balanced, residual effects design pharmacokinetic study. the 6 oral treatments consisted of 600 mg grn (one 200 and one 400 mg grn tablet) given alone, with concomitant maalox, 2 h before maalox, 4 h before maalox, 2 h after maalox, and 4 h after maalox. each healthy adult subject received 3 of the above treatments, with a 7-day washout period between treatments. in each treatment, serial blood samples for pharmacokinetic analysis were collected before and up to 72 h after grn dosing. study assessments included vital signs and physical, laboratory, and electrocardiographic examinations for drug safety. results: twenty subjects (12 male, 8 females; mean age, 27 years) were enrolled. exposure to grn [auc(inf)] was reduced by 58% when coadministered with maalox. exposure to grn was also reduced when administered 2 or 4 hours after maalox, by 22% and 16%, respectively. in contrast, when administered 4 hours before maalox, grn exposure was not affected. when grn was administered 2 hours before maalox, a small reduction (12%) in grn exposure was observed that is unlikely to be clinically relevant. half-life (mean range 11.5 to 14.2 hours) and tmax (median range 1.5 to 2 hours) were similar across treatment groups. a single oral 600 mg dose of grn was well tolerated. mild adverse events were reported by 2 subjects; 1 subject discontinued due to mild abdominal pain and blood in the stool. conclusions: maalox does not affect grn bioavailability when grn is administered at least 2 h prior to maalox. however, a reduction in grn exposure is observed when grn is administered concomitantly or up to 4 h after maalox. therefore, grn can be administered 2 h before or 4 h after administration of maalox or other products containing a high content of cations, particularly aluminum. objectives: it has been demonstrated that fluoroquinolone treatment of humans and animals can rapidly select for bacteria with reduced susceptibility to fluoroquinolone antibiotics. in recent years, there has been considerably interest in the concept of the mutant prevention concentration (mpc). the mpc has been defined as that concentration of antibiotic at which no mutants are selected from 10,000,000,000 organisms. in previous studies mpcs of ciprofloxacin and enrofloxacin were determined in vitro against salmonella enterica serovar typhimurium dt 104. here the use of a chick model to investigate the mpc of enrofloxacin in vivo is reported. methods: chicks were experimentally infected with s. typhimurium and treated with baytril (enrofloxacin) at 50 ppm/10 mg/kg for 5 days (recommended therapeutic dose) or 2 days or 125 ppm/25 mg/kg for 2 days or 250 ppm/50 mg/kg for 1 day. chicks received the dose in the drinking water (ppm) or by oral gavage (mg/kg). during and 24 hours after dosing, birds were killed and tissue samples (caecal contents, liver and sera) were taken and the levels of enrofloxacin and ciprofloxacin were determined in these tissues by hplc or bio-assay. caecal contents were also monitored for the presence of salmonella during dosing and for up to 4 weeks after dosing. selection of resistance was monitored by replica plating of colonies to media clinical microbiology and infection, volume 11, supplement 2, 2005 containing 4x the ciprofloxacin and nalidixic acid mic of the strain before treatment.results: the only conditions where the antibiotic treatments did not select for reduced susceptibility (e.g. 4x mic of parent strains) were in birds that received the antibiotic at 2.5x the dose for 2 days by oral gavage or 4x the dose for 1 day in the water. this oral gavage treatment also significantly reduced the counts of salmonella compared to all other oral gavage treatments. for the oral gavage study, concentrations of fluoroquinolones in caecal contents were above the mpc for at least 6 hours after dosing for all treatment regimes. conclusion: it was of interest to note that whilst the fluoroquinlone antibiotics are regarded as concentration rather time dependent antibiotics, the 2 day at 2.5x the dose was more effective at reducing the counts of salmonella in both experiments than the 1 day at 5x the dose. these results would suggest that length of treatment as well as dose and route of administration, is critical in minimising the selection of fluoroquinolone resistance in vivo. effect of levofloxacin coadministration on the international normalised ratios during acenocumarol therapy j. de otero, m. payeras, c. carratala, a. artigues, m. sanz, p. nadal (manacor, e) levofloxacin (l) showed no effect on warfarin pharmacokinetics in healthy volunteers but several recent descriptions reveal elevated international normalised ratios (inr) in patients receiving concurrent l and warfarin. to our knowledge no cases of l-acenocumarol (a) interaction have been reported. objectives: to report 6 cases of hypoprothrombotic response resulting from addition of l therapy to chronic a therapy. methods: a 9-month retrospective analysis of adult patients receiving a who were admitted to our ward under l prescription on the basis of clinical diagnosis and judgement. descriptive statistical measurements were performed. results: six patients, 74-89 years old (median 81.5), were included. four were men (66.6%). they suffered from a median of 4 (range 2-6) concurrent chronic medical problems (including coronary heart disease, atrial fibrillation, hypertension, obstructive pulmonary disease, renal insufficiency, cardiomyopathy and heart failure) and took a median of 7 (range 4-10) different kind of drugs (including digoxin, furosemide, antihypertensives and inhaled bronchodilators). types of infection for which the patients were treated with l included acute bronchitis (4 cases) and pneumonia (2 cases objectives: cip is substrate for an mrp efflux pump in j774 mouse macrophages (michot et al, aac 48:2673-82), which reduces its cellular accumulation. we have recently shown that cip-ôresistantõ macrophages can be selected upon chronical exposure of these cells to cip, in which cip accumulation becomes negligible because of an increased activity and/or expression of the efflux transporter (icaac 2004 (icaac , abstract a-1304 . we have now examined whether this reduced accumulation affects cip intracellular activity using a model of intracellular infection by l. monocytogenes (l.m.) and comparing resistant cells to the wild type parent cell line. methods: infection was carried out using a bacteria: macrophage ratio of 7:1, according to the procedure described in seral et al (jac 51:1167-73) . cfu/mg cell protein was determined after 5 h exposure to increasing concentrations of cip ± 10 mm probenecid (inhibitor of mrp transporters). cip cellular concentration was measured by fluorimetry (aac 48:2673-82) . results: in wild cells, an extracellular concentration corresponding to 3 x the mic was sufficient to obtain a static effect, and this value rose to 30 x in cip-resistant cells. this value was decreased to 1 x and 1.5 x the mic, for wild and cip-resistant cells respectively, in the presence of probenecid. in cells incubated with 20 mg/l cip (10 x mic), cip accumulation was 4.8 ± 0.3 and 0.3 ± 0.1 in wild and cip resistant cells, but increased to 21.3 ± 0.8 and 10.6 ± 0.5 in the presence of probenecid. conclusions: increase in expression and/or activity of the cip efflux transporter causes a reduction of the antibiotic efficacy against intracellular infection, which can be reversed upon inhibition of the efflux transporter. objectives: quinolones can cause tendinitis and tendon ruptures. retrospective studies and case reports reveal that an additional therapy with glucocorticoids increases the risk of quinolone-induced tendon disorders. methods: we investigated possible combination effects of quinolones and glucocorticoids in an in vitro model with human tendon cells. tenocytes were cultured for 1, 2, 3 and 4 days as monolayers and incubated with (a) ciprofloxacin or levofloxacin (3 mg/l), (b) 0.1 nm dexamethasone, and (c) ciprofloxacin or levofloxacin (3 mg/l) plus 0.1 nm dexamethasone. immunoblotting was used to quantify changes in the amount of beta1-integrin, activated src homology collagen (shc) and of activated caspase-3 (casp-3). furthermore, ultrastructural alterations were analysed by electron microscopy. results: at 3 mg/l, ciprofloxacin caused a significant decrease of the amount of beta1-integrin from day 3 onwards, while no effect was seen with 3 mg levofloxacin/l medium or 0.1 nm dexamethasone compared to the untreated control. the combination of both quinolones (3 mg/l) with 0.1 nm dexamethasone resulted in an earlier onset of the beta1-integrin reduction: for ciprofloxacin + dexamethasone from the first day of incubation, for levofloxacin + dexamethasone from day 3 onwards. quinolone-induced changes in signalling proteins, such as activated shc, are not fortified by dexamethasone at the concentration tested. interestingly, the time and concentration dependent increase of the apoptosis marker activated casp-3 was intensified with dexamethasone. the results of immunoblotting with respect to the induction of apoptosis were confirmed by electron microscopy. conclusions: our results show that quinolone-induced changes in the amount of the beta1-integrin as well as of the apoptosis marker activated casp-3 are enhanced by dexamethasone in vitro. the addition of the glucocorticoid caused an earlier onset of the quinolone-induced changes. our results support the clinical observations that glucocorticoids are an additional risk factor for quinolone-induced tendopathies. pharmacokinetics and pharmacodynamics of a novel extended release formulation of ciprofloxacin as compared to levofloxacin against extended spectrum beta-lactamase producing enterobacteriaceae s. schubert, h. stass, u. ullmann, a. dalhoff (kiel, wuppertal, d) background: an extended release formulation of ciprofloxacin (cip xr, 1000 mg qd, p.o.) has been developed, delivering peak concentrations which are 40% to 50% higher than following the administration of the conventional formulation (cip ir, 500 mg bid, p.o.) while the areas under the curve (auc) were comparable. objectives: first, cip is used for the treatment of infections due to enterobacteriaceae, frequently being esbl producers. thus, the pk/pd characteristics of cip xr vs. ir against esbl strains was examined. second, cip is used in urinary tract infections (utis). thus, the pk/pd profiles of cip xr vs. ir in serum and urine were studied. third, the pk/pd characteristics in either bhi or artificial urine were compared. methods: genotypically characterised esbl producing e. coli (ec) and k. pneumoniae (kpn) and their wild type counterparts were exposed to the geometric mean plasma and urine concentration profiles following cip 1000 mg xr qd, 500 mg ir bid and lev (500 mg qd). the cip and lev urine concentrations were fitted from phase i study data by compartmental modeling using topfit 2.0. bacteria were cultivated in a modified grasso model at 37°c in brain-heart infusion broth as well as artificial urine acc. to griffith et al.; viable counts were quantitated at 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 24 h. the areas under the bacterial kill curves normalised to the inoculum were calculated. results: first, irrespective of the pk profiles simulated, the wt were eliminated rapidly. the esbl producing ec and kpn were moderately to negligibly affected upon simulation of serum pk profiles of both cip and lev. second, simulation of urine concentrations by using artificial urine ir and xr resulted in an elimination of the esbl producers from the test system. lev eliminated the esbl producing k. pneumoniae but not the e.coli strain, which regrew. cip xr killed the esbl kpn strain more rapidly than cip ir or lev. third, pk/pd surrogates derived from studies in conventional media or artificial urine are significantly different. conclusions: this model and the use of artificial urine predicts a more rapid and pronounced effect of cip xr as compared to cip ir or lev. objectives: the antibacterial spectrum of moxifloxacin includes all the major respiratory pathogens and its pharmacokinetics demonstrates high peak concentrations in plasma as well as at respiratory sites. nevertheless, the tonsillar tissue concentrations have never been investigated. in the present study we determined the moxifloxacin concentrations in plasma and tonsils after the administration of three doses of 400 mg to adult patients with chronic or recurrent tonsillitis undergoing tonsillectomy. methods: this was an uncontrolled, open-label, randomised, parallel group study including 35 patients randomly assigned to 5 groups of 7 patients each, depending on the time between the last dose of moxifloxacin and that of plasma and tissue sampling (2, 3, 6, 12 and 24 h). moxifloxacin was given orally od for 3 days. moxifloxacin concentrations were measured by a validated hplc assay and fluorescence detection. each sample was analysed twice and the mean value obtained used for statistical analysis. pharmacokinetic data were analysed by presenting descriptive statistics of moxifloxacin levels in plasma and tonsillar tissue. results: cmax occurred at 3h in plasma (mean value 3.20 mg/ l) and in tonsillar tissue (mean 8.96 mg/l). tonsillar tissue/ plasma ratios (mean values) were constantly >2 mg/l at any collecting time, ranging between 2.37 mg/l (after 2 h) and 2.93 mg/l (after 24 h), which indicates a prolonged maintenance of moxifloxacin level in tonsillar tissue compared to plasma. variability among pts was present at 6 h, the tonsillar tissue / plasma ratio ranging between 0.8 and 3.4 mg/l. conclusions: moxifloxacin achieves a good penetration in tonsillar tissue, which favourably compares with that reported for other fluoroquinolones. the moxifloxacin concentrations that we observed exceed the mics of the usual respiratory tract pathogens. objectives: acute necrotizing pancreatitis is still related to a high mortality rate, based on local infectious complications, particularly due to bacterial infections of the necrotic pancreatic and parapancreatic areas. limited penetration of antimicrobial drugs in these areas/compartments is considered to be a major cause of failure of therapy of severe local pancreatic infections. however, fluoroquinolones (e.g. ciprofloxacin, levofloxacin) have been shown to penetrate sufficiently into pancreatic tissue. on that score, the value of new quinolones such as moxifloxacin (mxf) has not been investigated yet. using a rat model of acute necrotizing pancreatitis mxf has been demonstrated to penetrate rapidly and efficiently into pancreatic tissue, also into the inflamed and necrotic pancreas. methods: addressing the penetration capability of mxf following intravenous (iv) or oral (po) administration with respect to the human pancreas, a prospective clinical trial was designed using a single iv (20 patients) or po dose (40 patients) of 400 mg mxf for antimicrobial prophylaxis in patients undergoing pancreas resection. samples were taken from blood and from resection area of pancreatic tissue at two time points after application (1st sample at the beginning, 2nd sample at the end of resection). concentrations of mxf were determined by hplc/uv using ofloxacin as internal standard. results: mean plasma concentrations of mxf iv and po at first sampling time (3 -3.7 h) were 1.8 ± 0.5 and 1.2 ± 0.6 lg/ml, respectively. at the end of resection (4.3-5.3 h) 1.5 ± 0.4 and 1.0 ± 0.5 lg/ml were measured. corresponding mean concentrations of mxf in pancreatic tissue were 2 to 3times higher (3.1 ± 0.9 and 2.7 ± 1.4 lg/g 1st sample; 3.6 ± 1.5 and 3.1 ± 1.8 lg/g 2nd sample). the mxf concentrations in pancreatic tissue exceeded the mic90 s of the relevant pathogens encompassed by mxf (e.g. e. coli 0.008-0.06 lg/ml, klebsiella spp. 0.13 lg/ml, and s. aureus 0.06-0.12 lg/ml) for at least five hours at the dosing interval. conclusion: mxf has been demonstrated to penetrate efficiently into human pancreatic tissue following iv as well as po administration. in this study mxf concentrations after po administration were found to be slightly lower than those observed in healthy subjects probably due to diminished or delayed intestinal absorption prior and during surgery. objective: in clinical practice on icu wards moxifloxacin (mfx) may be occasionally administered through a nasogastric (ng) feeding tube. in absence of an oral liquid formulation and since the divalent cations contained in enteral feeds may potentially impair absorption of mfx given via this way, we wanted to study the effect of concomitant enteral feeding on the pharmacokinetics and tolerability of mfx passed as a crushed tablet through a ng tube, firstly in healthy volunteers. methods: in a randomized 3-way crossover study the oral bioavailability of mfx was investigated in 12 healthy volunteers (9 f, 3 m). each subject received separately an intact 400 mg mfx tablet (a), a crushed tablet as a suspension through an ng tube with water (b), and while receiving enteral feeding (c). the concentrations of mfx in serum were measured by a validated hplc. auc and cmax were analysed statistically assuming log normally distributed data using anova. results: all mfx treatments were well tolerated. the auc was slightly, but not significantly decreased in treatments b and c [geo means ( . thus, rate of absorption was not affected by ng tube administration. this route of ingestion seems to be associated only with a slight loss of bioavailabilityindependent of the carrier medium used (water vs. enteral feed). no clinically relevant interaction with the multivalent cations contained in the enteral feed was observed indicating that mfx and enteral nutrition can be administered concomitantly. conclusion: there was no clinically relevant effect of enteral feeding on the pharmacokinetics of oral mfx in healthy volunteers. this results has to be evaluated in patients, especially those from icu, who are characterised by severe infectious and/or concomitant diseases, which might influence absorption of mfx. in the healthy volunteer studies, compliance with dosing regimens was strictly enforced and participants had no significant underlying diseases or concomitant medications that could confound interpretation of safety data. therefore, for the purpose of this analysis, the healthy volunteers served as a control group for the patient population. in both groups, most subjects received pos 800 mg/d in divided doses. results: treatment-related aes (traes) occurred in 44% (196/ 449) of healthy volunteers; the most common were headache (17%), dry mouth (9%), and dizziness (6%). in patients, many aes were consistent with underlying diseases. traes occurred in 38% of patients (164/428); the most common were nausea (8%), vomiting (6%), headache (5%), abdominal pain (4%), and diarrhoea (4%). notably, gastrointestinal (gi) traes occurred in 18% of healthy volunteers and 19% of patients. treatmentrelated abnormal liver function test results were observed in 2% of healthy volunteers and up to 3% of patients. there were no clinically significant differences in mean qtc interval change from baseline in either population. serious aes (sae) considered possibly or probably related to pos occurred in 35 (8%) patients and 1 healthy volunteer. the most common saes in patients were altered drug level, increased hepatic enzymes, nausea, rash, and vomiting (1% each). no significant trends related to age, sex, or race were observed in either group. additionally, no unique traes were identified in patients during long-term exposure (>6 months) compared with those identified during shorter-duration therapy. conclusion: the safety profile of pos in patients was similar to that observed in a controlled population of healthy volunteers and is likely indicative of what will be observed in the clinical setting. headache and gi events (nausea, vomiting, abdominal pain, diarrhoea) were the most common traes observed in patients. given its favorable safety profile, pos should provide an additional treatment option for severely ill patients with ifis. objective: we evaluated the drug interaction (di) potential of posaconazole (pos), an extended-spectrum triazole antifungal agent, in 7 phase 1 studies. pos is an inhibitor of cytochrome p450 (cyp) 3a4 activity, but it does not affect the activity of other cyp enzymes. these studies were conducted to evaluate potential changes in the pharmacokinetics of pos and the coadministered medication, when given in combination. methods: seven open-label di studies were conducted in which subjects received pos (200-800 mg) for 8 to 14 consecutive days, alone or in combination with single or multiple doses of cimetidine, cyclosporine, glipizide, phenytoin, rifabutin, tacrolimus, or antiretroviral medications (zidovudine, lamivudine, ritonavir, and indinavir). in the cyclosporine and antiretroviral studies, patients received established doses (ed) of these drugs, and plasma concentrations were presumed to be at steady state (ss) before pos administration. bioavailability, based on logtransformed auc values, was expressed as the ratio of the combination treatment to pos or concomitant drug given alone. the effects of coadministration on the auc values of pos and each concomitant medication are summarized in the table. conclusion: no dose adjustments are required for glipizide, zidovudine, lamivudine, indinavir, or ritonavir when coadministered with pos, as the small differences in exposure are not considered clinically significant. however, when pos was given with glipizide, glucose concentrations decreased in some healthy volunteers more so than after glipizide administered alone. monitoring of cyclosporine and tacrolimus blood levels is warranted with pos coadministration, and dose adjustments of cyclosporine and tacrolimus should be made accordingly. concomitant use of pos with rifabutin, phenytoin, or cimetidine should be avoided unless the benefits outweigh the risks, due to the decrease in pos concentrations. objectives: the pharmacokinetics (pk) of many medications may be altered in the elderly population. because alterations in pk can potentially affect clinical outcomes, the pk parameters of posaconazole (pos), an extended-spectrum investigational triazole antifungal agent, were examined in healthy elderly persons ( ‡65 years), and the clinical implications of age-related pk differences were evaluated. methods: we conducted a meta-analysis of pos steady-state pk data from 5 clinical studies in healthy volunteers. pk data from elderly healthy volunteers were compared with those of elderly patients with invasive fungal infections ( objective: to develop a bioassay for measurement of csp blood levels using the hypersusceptible c. albicans cell wall sensor mutant delta-mid2. methods: the c. albicans mutant delta-mid2 (mic for csp: 0.008 mg/l) was constructed by targeted deletion of the gene encoding for a cell wall sensor putatively involved in the maintenance of cell wall integrity. the bioassay was validated according to international guidelines (shah et al, 2000; fda, 2001) . standard curve included 8 csp concentrations over the range 0.4 to 25 mg/l. four quality controls were used (0.5, 2, 8, 16 mg/l) to study: i) stability of csp concentrations over time at different pre-analytical storage conditions, ii) accuracy (measured/nominal value x 100, validation range 85-115%), iii) precision (coefficient of variation: sd/mean of measured values x 100, validation range 15%). the validation procedure included 6 intra-run and 6 inter-run measurements. results: the limit of detection and quantification was 0.2 and 0.4 mg/l (corresponding to 5 and 10 ng of csp in a sample volume of 25 ml), respectively. reproducible standard curves were obtained over the clinically relevant concentration range (0.4 to 25 mg/l) (r ‡ 0.99). analytical time was 16 h. csp concentrations with a deviation from nominal values < 15% were measured: i) over 4 days in plasma, and 3 days in whole blood, at 4 c, ii) over 3 and 2 days, respectively, at 21 c, iii) over 6 months in plasma at -80 c, iv) after 5 freeze/thaw cycles. intra-and inter-run validation with the four quality controls (mean % value, ± sd) are shown in objective: to identify the pharmacokinetic and pharmacodynamic (pk-pd) parameters of antifungal therapy predictive for intra-and inter-run bioassay validation i intra (n = 6) inter (n = 6) accuracy 93.1 ± 1.9 96.0 ± 2.5 precision 1.3 ± 1.7 5.8 ± 3.0 invasive fungal infections in patients admitted to an intensive care unit (icu). the medical records of all ptt admitted to a tertiary hospital icu in a 6 month period in 2002 were retrospectively reviewed, and the antifungal therapies were recorded and correlated to the findings of fungi from the ptt. the pk-pdparameters for each pt was estimated from the treatment given and the mic of the isolated candida, i.e. auc/mic-ratio (in h) for the azoles, and peak/mic ratio for the free fraction of amphotericinb. candida isolated from various infectious foci, or from the surveillance samples taken routinely three times a week, were susceptibility tested using e-tests for fluconazole, itraconazole and amphotericinb. results: in total 120 ptt were included.among the 14 ptt with invasive fungal infections, 3 ptt had bloodstream infections at admission. one of the remaining 11 (9%) received systemic prophylactic therapy before getting colonized, and 3 ptt (27%) were prophylactic treated when colonized, 3 ptt with fluconazole (auc/mic: 66-1600), and one pt with liposomal ampho-tericinb (peak/mic = 3 objective: to predict the pharmacokinetic properties of bal4815 a new azole antifungal, in humans.methods: bal8557 (the water-soluble pro-drug of bal4815) was incubated at 10 lg/ml in pooled heparinized rat, cynomolgus monkey and human plasma for 5 minutes at 37°c. bal4815 was incubated at 1,10 and 100 lg/ml equivalent bal4815 with rat, cynomolgus monkey and human liver microsomes for 120 min at 37°c in the presence of 1 mg/ml proteins. the extent of plasma protein binding of bal4815 in rat, cynomolgus and human plasma was measured using the red blood cell partitioning method and 14c-bal4815. rats received a single oral dose of 10 mg/kg equivalent bal4815 and a single intravenous dose of 5 mg/kg equivalent bal4815 as bal8557. cynomolgus monkeys received single oral and intravenous doses of 3 mg/kg as bal8557. serial blood samples were obtained. plasma concentrations of bal4815 and bal8557 were quantified using an hplc/fluorescence method or a lc-ms/ms assay. results: in plasma, bal8557 is converted within minutes to bal4815 in all species investigated. in the presence of rat, cynomolgus monkey and human liver microsomes, less than 10% of bal4815 is metabolized during a 120 min period. bal4815 is bound to plasma proteins with a free fraction of 3.5%, 3.6% and 2.2% in rat, cynomolgus monkey and human, respectively. after intravenous administration, the pro-drug bal8557 is converted to bal4815 within minutes. bal4815 has a large volume of distribution with values greater than the total body water: 15 l/kg and 5 l/kg in rat and cynomolgus monkey, respectively. bal4815 is slowly eliminated with halflives of 5 h in rat and 10 h in cynomolgus monkey. after oral administration of the pro-drug, bal4815 reaches cmax-values between 2.0 to 3.5 hours. we observed high bioavailability of bal4815: 62% in rat and 87% in cynomolgus monkey. conclusions: based on these in vitro results and animal data, we predict that the pro-drug bal4815 is very rapidly converted to bal4815 in man. due to the protein binding and in vitro metabolic stability, bal4815 is expected to behave as a low intrinsic clearance drug in human (clearance < 10 % of liver blood flow). this combined with a large volume of distribution explains the long half-life > 30 hours observed in man. animal data suggest a good oral bioavailability as is confirmed in humans. pharmacokinetics and fungicidal activity of aminocandin (hmr3270), a novel echinocandin in healthy volunteers b. sandage, g. cooper, n. najarian, j. lowther (lexington, usa; romainville, f) objective: aminocandin (ac), an echinocandin, is being developed for treatment of systemic fungal infections. a study was carried out to determine the pharmacokinetic (pk) and fungicidal activity of ac in plasma samples against 2 strains of c. albicans and 1 strain of a. fumigatus following single iv doses. methods: single iv doses of 75-300 mg of ac were administered to 12 healthy, male volunteers in a single-blind, randomized, placebo-controlled trial. individual plasma samples were collected from active and placebo (pbo) treated volunteers, serially diluted in 25% control plasma/75% growth medium, and the fungicidal titre determined on 2 c. albicans strains and inhibitory titre against 1 a. fumigatus strain. titres and pk results are the mean values (n = 3). due to the dilution factor in test medium, the limit of detection was a titre of 4 the germicidal effect of ceiling-and wall mounted ultraviolet c (uvc) light at 254 nm in isolation units with negative pressure ()45 pascal) was examined and compared with disinfection with chloramine during end-disinfection after patient stay. microbial samples were taken from surfaces before and after disinfection with uvc (33-47 min) and chloramine (5%, 1 h exposure) using standard contact plates (20cm 2 ). the uvc-distribution in the isolation units was monitored at 165 positions. the output on the floor varied between 0.08 and 3.2 w/m 2 , with an average ( ± sd) of 2.2 ± 0.5 w/m 2 in the patient room, 2.0 ± 0.7 w/m 2 in the sluice and 1.4 ± 0.5 w/m 2 in the bath/ decontamination room. on other places, the values varied from 0.08 to 6.82 w/ m 2 . the units were uvc-disinfected for 33-47 min, corresponding to doses ranging from 160 j/m 2 in shadowed area to 19 230 j/m 2 at the highest exposed site. according to published uvc-dosimetry, the survival of bacteria and bacterial spores are reduced by 90% with doses ranging from 4-120 j/m 2 and 100-365 j/m 2 , respectively. thus, uvc doses used in this study should be high enough to inactivate most bacterial organisms, including spores, even on surfaces not directly exposed to uvc. uvc-disinfection significantly reduced the bacteria on surfaces directly or indirectly exposed to uvc to a very low number (from ca 30 to 1-2 cfu/plate), as did 5% chloramine disinfection (fom ca 30 to 1-2 cfu per plate) alone; p < 0.001,and p < 0.001, respectively. since cleaning before disinfection may be a risk for the staff in isolation units, disinfection with uvc-or chemicals should always be performed first. the presence of completely shadowed areas in the isolation unit (the bed rail, lockers, matresses etc.) still needs disinfection by chemicals before cleaning. therefore, uvc may not be used alone, but is a good additive to chemical disinfection, to lower the biological burden of infectious agents in isolation units for high risk infectious patients. disinfection of medical equipment, using dry mist of hydrogen peroxide objective: to compare the antimicrobial efficacy before and after disinfection of pine core wood surfaces with surfaces made of polyethylene and synthetic laminate against organisms typically causing hospital-acquired infections. methods: s. aureus (mrsa), e. faecium, e. coli, p. aeruginosa. c.albicans, m. terrae and p camembertii were uses as test organisms. after inoculation of the test organisms on surfaces made of pine core wood, polypropylene and synthetic laminate, samples were collected on rodac plates before and after disinfection with commonly used hospital germicides (alcohol, aldehyde, glucoprotamine and a quaternary ammonium compound). bacterial colonies were counted after 0 min, 30min, 1 h, results: substantially lower colony counts were measured on pine core wood surfaces before application of the disinfectant compared with counts on polypropylene and synthetic laminate surfaces. after bacterial contamination of the surfaces, significant colony counts were not observed on the surfaces after disinfection with alcohol, aldehyde and glucoprotamine. however, after disinfection with a quaternary ammonium compound, significant elevations in colony counts were found on the pine core wood surfaces in comparison to the polypropylene and synthetic laminate surfaces. discussion: the poor efficacy of quaternary ammonium compound on wooden material might be explained by the interaction between wood and the anionic polyphenols and cationic surfactants contained in the disinfectant. the study findings corroborate the antimicrobial effect of pine core wood against organisms typically causing nosocomial infections. with the exception of a quaternary compound, all the germicides displayed good disinfecting properties. from a hygienic point of view, pine core wood is suitable for use in hospitals. survey of the microbiological quality of drinking water supply in hospitals. drinking water cooler vs tap water fed dispenser vs bottled water objectives: hygienic, economic and ecologic comparison of drinking water coolers versus tap (mains) water-fed dispensers versus bottled drinking water. methods: samples were taken from each system without preflow or cleaning of the nozzle. these comprised 2 samples from 20 drinking water coolers each over 2 months, 1 sample per week from the tap water dispenser over 6 months, and samples from 6 different bottles of drinking water. total colony counts were analysed at 22°c and 36°c, respectively. total p. aeruginosa, e. coli, and coliforms, as well as sulphite-reducing clostridia were examined according to european norms (en iso) for compliance with the german drinking water ordinance 2001 (limit value for total colony counts at 22 ± 2°c: 100 cfu/ml, at 36 ± 1°c: 100 cfu/ml and 20 cfu/ml for bottled water). costs per litre were calculated and compared based on the economic data supplied by the manufacturers. the ecological impact of the different systems was evaluated based on expert opinion. results: all the samples from the water cooler system exceeded german drinking water ordinance threshold values. all the samples taken from the tap water dispenser, and, with one exception, all the samples from the bottled water complied with the german drinking water ordinance. if more than 400 l of water are consumed per month, the tap water dispensing system and bottled water are more economical than the water cooler system ( 0.57 per l for water cooler, 0.45 for month tap water dispenser, 0.49 per l for bottled water). in economic terms, the water cooler system can only be recommended for consumption levels below 400 l per month. from an ecological point of view, the tap water dispenser is most favourable (expressed in co2emissions per year for 403.720 l at freiburg university hospital), followed by the water cooler system and finally the bottled water. conclusion: for reasons of hygiene, the use of water cooler systems cannot be recommended in hospitals. furthermore, drinking water cooling systems are only economic for low consumption rates. tap (mains) water-fed dispensers feature the best hygienic, economic and ecologic properties. objectives: the application of antimicrobial finishes to textiles can prevent bacterial growth and might reduce the risk of infection resulting from fabrics that are contaminated with pathogenic microorganisms in hospitals. the main aim of this study was the determination of the antibacterial activities of chemical treatments applied to textiles. comparison of testing methods assessing antibacterial efficiency was conducted. these studies were performed in order to select the right methods of evaluating the antibacterial and bacteriostatic activity of finishes. methods: fibers and fabrics made from cotton (100%) applied with quaternary ammonium salts were tested. finishes treated with bactericidal agent were compared with samples (not treated with the disinfectant). the european standards iso / dis 20645/2002 and aatcc 147/1998 suitable for assessment of antibacterial activity were applied. the bacterial strains recommended by the above standards such as: klebsiella pneumoniae (atcc 4352), staphylococcus aureus (atcc 6538), escherichia coli (atcc 11229) were tested. due to its high resistance to quaternary ammonium salts, additionally p. aeruginosa (atcc 9027) was examined. bacterial suspension 1 -2,9 x 10 8 cfu/ml was prepared according to the standardsõ requirement. the samples impregnated with biocidal products were examined with agar diffusion plate test. the specimens were located transversely across the bacterial inoculum on the nutrient agar. the level of antibacterial activity was assessed by examining the extent of bacterial growth in the contact zone between agar and the specimens. additionally, the extent zone of inhibition (minimal 1 mm) around the specimens was considerate. results: tested fibers did not show antibacterial activity. the fabrics showed antibacterial activity against k. pneumoniae and s. aureus (inhibition zone 4 mm). the examined specimens showed no bacteriocidal activity against escherichia coli and pseudomonas aeruginosa. the results obtained in two applied methods were comparable in assessing antibacterial activity of finishes. the antymicrobial finishes did not fulfill requirements of the standards. conclusion: based on the obtained result the tested antimicrobial finishes may not be considered effective in preventing bacterial transfer at contaminated areas in hospitals. a new interactive approch to improve hand hygiene compliance j. holt, e. tvenstrup jensen, d. buhl (copenhagen, dk) introduction: the compliance of guidelines for hand hygiene is well below 50 % despite several at-tempts to improve. when planning an educational outline to promote a behavioural change in the hand hygiene practice in the aim of lowering the incidence of nosoco-mial infections the mapping of critical factors and implications that affect this practice is of utmost importance. objectives: this study was an empirical investigation of the aspects of the hand hygienic practice as it takes place in the danish health care system today. the study attempted to answer the question:''how can the health staff's lack of compliance with hand hygiene be explained and understood as basis for planning an educational material to support a behavioural change.'' methods: literature studies of hand hygiene, a questionnaire based done survey (1700 per-sons/294 respondents) and qualitative interviews (n = 15) was performed in order to uncover the explanations for this low rate of compliance.the data was analysed and discussed on the basis of theories on action, experience, reflective thinking, control and rituals in order to aim for a broader view and under-standing of this field. results: both the questionnaire and interviews showed that hygiene still is a field that has great implications on the interactions between individuals. furthermore it seemed difficult for the staff to draw a professional and not a private line between ''the clean and the unclean'' and thereby perform hand hygiene without compromising each other. further the interviews showed that it is difficult for the staff to react on something they cannot see and that not gives an immediate result when staff does not act as pre-scribed. introduction: leishmaniasis has increased in importance in recent years as hiv-infected patients have emerged as a risk group for the disease. however, real prevalence in the general population is unknown. objetive: the objective is to know the antibody seroprevalence for infection by leishmania infantum in the general population of castilla-leon (spain). methods: a random sample from the general population (4825 sera) and from hiv-infected patients in the autonomous community of castilla-leon was collected in 1996. seroprevalence of antibodies against l. infantum was determined by an indirect enzymoimmunoanalysis (eia) test designed in the laboratory. results: anti-leishmania infantum antibody seroprevalence in the general population was 4.9%. there is a significant increase in seroprevalence with age (p = 0.001), from 3.96% in the 14-20 years group up to 7.2% for those over 70 years old. there were no significant differences between women and men (5.0% versus 4.9%; p = 0.9534). seroprevalence was significantly higher in people from rural areas than in those from cities (6.0% versus 3.4%; p = 0.001). hiv-infected patients had a seroprevalence against l. infantum of 64.0%. no differences were observed between women and men, nor was there a prevalence increase with age. efficacy of intralesional pentavalent antimony in combination with oral azole for treatment of oldworld cutaneous leishmaniasis results: a 30-year-old man presented with a 6-month history of slowly growing round lesions on his right arm and leg. he had a history of travel in the mediterranean area (including north african and middle east countries) 6 months prior to presentation. examination showed one nodular lesion (1 · 1 cm) with margin induration and depressed central ulceration on the right arm, a second nodular lesion (0.5 · 0.5 cm) on the right leg, and a red papule (0.5 · 0.5 cm) on the left arm. a 52-year-old man with an history of a 4-month stay in iraq until 1 months prior to presentation, showed 6 lesions, which had grown during the last three months: two nodular lesions (3.5 · 1 cm, 4.5 · 2 cm) with erythematous margins and a thick crust on the left arm, 4 round lesion (2.5 · 2.5 cm) with depressed central ulceration, at the left arm, right arm, right leg and second finger of the right hand. a 49-year-old man with a 5-month stay in iraq until 5 months prior to presentation, showed two painless confluent nodular lesions (5.5 · 3.5 cm) with depressed central ulceration at the right elbow, which had grown over 4 months, two round lesions (1 · 0.5 cm and 0.8 · 0.7cm) with a thick crust on the left poplitea fossa and one nodular lesion (1.5 · 1 cm) at right leg. all patients received 12 alternate-days intralesional injections of meglumine antimoniate and 6 weeks of oral fluconazole. all lesions healed completely at the end of therapy and at the 3-month follow-up. none of the patients complained of any adverse events during treatment or follow up. conclusion: treatment with intralesional pentavalent antimony in combination with azole is effective and absolutely safe, in that it reduces the total amount of antimonial exposure. entomological study of sandflies (dipthera: psychodidea) in three foci of endemic cutaneous leishmaniasis in iran human leishmaniasis is a globally widespread parasitic disease caused by members of the genus leishmania. currently, leishmaniasis is considered to be endemic in 82 countries including iran. phlebotomine sandflies the vectors of leishmaniasis have received considerable attention in recent years due to the resurgence of leishmaniasis in some endemic areas of iran ,so extensive studies have been conducted on the ecology of sandflies in different parts of the country in recent years. a total 8026 sandflies were collected by using funnel traps of rodent burrows in rural district of the 3 province of iran (shiraz, yazd, khozestan). these sandflies identified in 3 maine croups: phlebotomus. papatasi(61%) , p. sergenti (10%) , p. sergentomya (29%). these findings indicate that p. papatasi could be a vector of humans and the gerbils (merion libycus). the close contact between vectors and resevoirs have created a very efficient cycle for the transmission of the disease in these areas and the villages around these 3 provinces. erysipelas like cutaneous leishmaniasis: a case report a. erdogan, d. balaban, e. dervis, g. sengoz, g. barut, a. karaoglu (istanbul, tr) cutaneous leishmaniasis is an infectious disorder of the skin caused by leishmania major, tropica, aethiopica and infantum which are transmitted by phlebotomine sandflies. we present here a patient who demonstrated a morphologically rare form of erysipelas like cutaneous leishmaniasis. a 74-year-old woman with a four-month history of erythema and edema on her nasal and left malar area was referred to our clinic for further evaluation. her dermatological examination revealed an erythematous, edematous and fine desquamative plaque on her nose and left malar region resembling erysipelas. other physical and laboratory findings were normal. punch biopsy taken from the lesion and dyed with h.e revealed dense lymphocytic infiltration between the layers of just beneath the epidermis and deep dermis. in addition, a huge number of amastigots were found in macrophages and histiocytes that formed granulomas. in light of these findings the patient was diagnosed with cutaneous leishmaniasis and commenced a regimen of meglumin antimonat 425 mg b.i.d. after two weeks of therapy the lesion was gradually disappeared without any scar formation. in conclusion, cases with cutaneous leishmaniasis are still observed in our country not always with usual clinical appearance, but also like the present case, it may clinically resemble erysipelas and provides difficulties in the differential diagnosis. study of human infection of cutaneous leishmaniasis in a focus of the disease, southern iran ) with scar and 14 cases (1%) with ulcers. the mean of acute lesions and scars per infected cases was 1.64 and 1.87, respectively. totally 129 cases were observed with sores and scars, and 122 out of them were infected in the area. the highest rate of the acute lesion was observed in 0-4 years old age group (4.5%); meanwhile the highest rate of scars was in 10-14 years old age group (12.9%). sores were located on hands (8.7%), foot (65.2%) face (4.4%) and other parts of body (21.7%). in the study of schools, 1201 students (53.5% boys, 46.5% girls) were visited. the infection rate to acute lesion was 0.6% and 22.6% of students had scar. there is no significant difference between males and females based on the acute infection (p > 0.05), but this difference was significant based on scar (x 2 = 9.84, p < 0.05). the highest infection rate was observed in tashkooieh village (2.7%). two injected mice were developed the acute lesion and the agent of the disease was identified as leishmania major by pcr test. conclusion: the infection rate of the sores and scars shows that the disease is located in the studied area in recent years. the disease had one peak during 1995-2000 and has been increased from 2002 up to now. this is the first time that the parasite isolated from human in the area. therefore, this focus must be added to the zoonotic cutaneous leishmaniasis foci of iran. the disease is endemic in the area because more than 95% of cases were infected locally. giardia and cryptosporidium in the netherlands objectives: • the studies were designed to get an insight into the incidence of protozoal-, bacterial-, and viral infections in patients with diarrheal complaints in different groups: patients consulting their general practioner and the dutch population. here we focus on giardia and cryptosporidium • to decrease diagnostic deficit • study the risk factors methods: three studies were designed and conducted : • ôhaarlemõstudy: 1994-1996, general practitioners, haarlem region • nivel: case-control study in sentinel general practitioners practices (1996 general practitioners practices ( -1999 • sensor: prospective population based cohort study with a nested case-control study in the dutch population. (1999)the studies differ in inclusion criteria and the diagnostic laboratory techniques used, esp. virological stool examinations results: incidence of gastroenteritis in the nivel (gp) study (after correction) was 79.5 per 10,000 personyears. this means that 80.000-130.000 persons will consult a gp annually for gastroenteritis. incidence of gastroenteritis in the populationbased study was 283 per 1000 personyears. giardia was detected in 14.8% of the cases in haarlem, in 5.4% of the cases in the nivel study and 3.3% of the controls. for cryptosporidium this was resp 3.3%, 2.1% and 0.2%. the diagnostic deficit decrease substantialy by testing for viral pathogens like nlv. detection of pathogens was influenced by age, season and duration of symptoms. we were able to construct an algoritm for diagnostic workup in gi patients. statistical surveys have been made for the effect of the climate on the epidemic diseases in tropical area, but not so much has been clarified on the relation between the variations of meteorological elements and of the number of patients. in this paper, we apply eof analysis method to time series of diarrhoea patients and meteorological elements in bangladesh, to understand effects of the meteorological variation to the prevalence of the diarrhoea disease. the eof analysis of the time series of patients and meteorological elements averaged every two weeks for 21 years from 1981 to 2001 shows that in the dominating component, the anomaly of the number of the diarrhoea patients has different signs for the periods before and after june, corresponding to the two seasonal peaks of the number of the patients. higher maximum temperature and more humidity in the pre-monsoon period are found to have a tendency to enhance the first peak of the diarrheal occurrence. we will also report the result for the different types of diarrhoea as v. cholera, rota and etec. serologic evidence for babesiosis in the northern and eastern tyrol (austria) and the southern tyrol (italy) methods: leptospirosis was diagnosed by leptospiral igm enzyme linked immunosorbent assay (elisa) and the serovar was confirmed by the microscopic agglutination test (mat) using a battery of 12 live leptospiral strains as antigens. results: most of them were outdoors manual workers (52%), housewives (29%), indoors non-manual workers (16%) and unknown (3%). mean duration of symptoms was 12.4±21.2 days. majority of the patients presented with fever (89%), jaundice (55%), chills and rigors, vomiting (47% each), cough (28%), abdominal pain (25%), diarrhoea, and altered sensorium (19% each), purpura/bleeding (11%). salient laboratory abnormalities included anaemia (50%), leucocytosis (59%), thrombocytopaenia (64%), elevated erythrocyte sedimentation rate (esr) (55%), hyponatremia (50%), hypokalemia (23%). acute renal failure occurred in 47%. hepatic function derangement occurred in 64%. three patients had pulmonary infiltrates and sputum revealed haemosiderin laden macropahges in them. two others manifested exudative pleural effusion which was bilateral and sequential in one of them. the common serovars encountered included l. autumnalis (30%), l. hebdomadis (22%), l. grippotyphosa (18%), l. icterohaemorrhagiae and l. javanica (11% each), l. australis (8%). all patients were treated with parenteral crystalline penicillin, oral doxycycline and were managed conservatively. haemodialysis was required in four patients. two patients died, both of whom developed multiorgan system failure. conclusions: leptospirosis is an important cause of acute febrile illness with renal, hepatic dysfunction and bleeding abnormalities. a high index of clinical suspicion is required confirm the diagnosis early as the condition responds well to conservative management. determination of fr900098, a promising antimalarial agent, in human serum by capillary electrophoresis for pharmacokinetic studies the acetyl derivative of fosmidomycin, fr900098, was demonstrated to be twice as active against plasmodium falciparum in vitro and in the plasmodium vinckei mouse model. fr900098, as fosmidomycin, is an inhibitor of 1-deoxy-d-xylulose-5-phosphate (doxp) reductoisomerase, an essential enzyme of the non-mevalonate pathway of isoprenoids biosynthesis. the biosynthesis of isoprenoids in plasmodium is depending on this doxp pathway, as found in eubacteria, algae and plants, but not in human. in plasmodium, the doxp pathway is localised inside a plastid like organelle, the so-called apicoplast. here, we report on the development of a high-performance capillary electrophoresis (hpce) technique for the determination of fr900098 in serum. various instrumental setting for migration (voltage, capillary temperature) were tested and the buffer properties (ph values, component molarities) were optimized. the assay was submitted to standard quality control procedures: within-, between-days reproducibility, accuracy, limits of detection (lod) and quantification (loq), linearity, short and long term stability. finally, the working buffer used was 14 mm kh2po4 / 56 mm k2hpo4, 15% methanol, and 0.2 mm hexadecyltrimethylammonium bromide (htab). the ph was adjusted to 10.9. the electroosmotic flow was modified by the cationic ion pairing reagent, htab. the assays were linear over the large concentration range tested, from 0.25 to 100 lg/ml. the recovery of the sample pre-treatments was higher than 100%. good precision was obtained, with betweendays reproducibilities resulting in coefficients of variation (cv%) of 2.1, 2.0 and 3.2%, and within-days reproducibilities of 0.6, 1.8, and 4.9% (cv%), for 100, 10, and 1 lg/ml, respectively. the lod was 0.25 lg/ml, and the loq was 0.5 lg/ml. the studies of short and long-term stability of fr900098 in serum showed a good stability of the molecule, at room temperature, +4°c or )80°c. moreover, fr900098 was resistant to numerous cycles of freezing and thawing. indeed, after four freeze-thaw cycles (4 days), 94.9%, 101.0%, and 97.5% of fr900098 were recovered from the 100, 10, and 1 lg/ml samples, respectively. in conclusion, we have developed a convenient ce technique for the determination of fr900098 in serum which offers advantages of speed, sensitivity, and accuracy. at present, the procedure is applied for pharmacokinetic studies in an animal model of gö ttingen mini-pig. present and future of malaria in kahnouj endemic area, southern iran objective: kahnouj district is associated with one of the malaria regions in southeast of iran. the anopheleine fauna does not appear to have changed much over several decades. methods: entomological studies and mosquito collection were performed every 15 days from indoor and out door shelters. malaria surveillance was carried out by health centers, ministry of health. microscopy was performed on out patients with fever or suspected malaria. malaria cases detection were mostly performed passive and rarely actively. the positive cases were treated with chorolquien/primaquen. results: in the present study five vectors species of malaria were found in this study which had been previously recorded 3 decades ago. this district like other malaria endemic areas in iran has been under pressure of anti malaria programmes including case finding and residual spraying insecticide as well as larviciding against the vectors since 1958. annual incidence of malaria declined from 10.27 to 3.86 during ten years. the most transmission occurred in october to december when the temperature was suitable for vector activity in kahnouj area. electricity was recently supplied into the rural area where most malaria cases were found. therefore, most houses equipped with air conditioner and the resident keep windows close, so they are secured from mosquitoes bites during hot season but not in beginning of spring and autumn when temperature is mild enough, in order to save the electricity cost, the residents do not use air conditioners whereas they leave windows open and no other protection against mosquito bites is provided. residual spraying insecticide of indoor shelters have been stopped for five years and the most activity of antimalaria programme set up base on case finding. conclusion; in order to control malaria, indoor residual spraying of insecticide would not assist effectively, so given knowledge to people to use bed net particularly in season people are more expose to mosquitoes bite may be considered as an effective measure in controlling malaria in khanouj district. also development in this area is effectively pushing back malaria in near future. entomological studies and mosquitoes collection were performed every 15 days from indoor and outdoor shelters as well as breeding places with the aid of suction tube and dipper. results: entomological researches were found that five vectors species of malaria in this study had been previously recorded 2 decades ago. anopheles stephensi was recognized as the main vector of malaria in this area with two peaks, one in may and the other in december. the most malaria transmission occurred in june and december. the larval habitats includes draying river bed with pools, rocky river pools, stagnant streams, slow foothill streams, temporary pools, slow moving water with or without vegetation. conclusion: operational of insecticides for adult and larval control, as well as surveillance of malaria cases, would not assist effectively to control of malaria, so given another malaria control methods as impregnation of bed nets as well as repellent particularly in seasons that people are more expose to mosquitoes bite, may be considered as an effective measure in controlling malaria in this area . case report: a 16 year old boy presented with fever and a generalized exanthema at the local dermatology clinic. the mixed appearance of pustules and umbilicated non purulent vesicles led to the suspicion of an orthopoxvirus infection. the patient reported that little red dots had occurred 2 weeks earlier at the extremities after contact with a sick pet rat. 1-2 weeks later a generalized pustular exanthema appeared, which was associated with high fever (>39°c) and a pronounced feeling of sickness. histologically no viral inclusion bodies were found in pustule material but poxvirus particles were detected by elmi in swabs from pustule ground. following the anamnestic suspicion of transmission by an infected rat, which unfortunately had perished soon afterwards, and the experience made at the recent monkeypox outbreak in the usa initiated by imported rats, one of the major aims was the exclusion of monkeypoxvirus. this was accomplished by pcr typing, which like elmi had been established on occasion of the implementation of the austrian plan for poxvirus preparedness (ôpocken-alarmplanõ) and despite the diagnostic means for detection of variola vera also included the ability to discriminate between animal orthopoxviruses. additionally serological diagnosis was performed and presumably due to the protracted course of infection cowpox specific igg antibodies were already present at admission with a titre of 1:800. interestingly the humoral immune response initially seemed to be rather cowpox specific as there were no antibodies crossreacting with vaccinia virus detectable in indirect immunofluorescence. as an exclusive immune reaction with one species alone would be a very rare situation observed with orthopoxviruses we did more extensive testing by western blot analysis. although not exclusively directed against cowpoxvirus antigens a very restricted reactivity of the patient serum was observed in the early course of infection using different recombinant and virus-derived orthopoxvirus antigens. this would suggest that also serological methods might be able to give a hint towards rapid determination of orthopoxvirus species, which certainly is also the case with immunological antigen detection methods. crimean-congo haemorrhagic fever in results: forty veterinerians from endemic region (tokat), and 43 from non-endemic region (aydin) were included. demographic characteristics in two groups were similar, whereas professional activities of veterinarians in non-endemic region were more intense (p = 0.001). the cchf igg positivity (2.5% vs 0%), brucella agglutination titre of >1/160 (27.5 vs 4.6%) were more common in endemic region than non-endemic region. three veterinarians in tokat had malaise, myalgia, and fever. in multivariate analysis to detect the risk factors for serum tube agglutination of > 160, the veterinarians living in endemic area were found to have 7.8 times higher risk of brucella infection than the ones living in non-endemic area (or; 7.8, confidence interval; 1.4-40.9, p = 0.015). the prevalence of coxiella burnetii serology was equal in both regions as 7%, and none of the seropositives had complaints. the history of tick bite was significantly more common in the endemic region than the nonendemic region (35% versus 12%, p = 0.011). conclusions: cchf and brucellosis compose infection risks for veterinarians in endemic region, despite the veterinarians in endemic region perform less riskfull professional activities. veterinarians in cchf endemic regions should be warned to protect themselves against tick bites according to universal precautions. the use of masks should be employed to prevent inhalational transmission of brucellosis in endemic regions. changes in temperature and the crimean congo haemorrhagic fever outbreak in turkey o. ergonul, s. akgunduz, i. kocaman, z. vatansever, v. korten (ankara, istanbul, tr) objective: to investigate the role of the climatic factors that could effect the activation of hyalomma marginatum marginatum population, and consequently the emergence of crimean-congo hemorrhagic fever (cchf) epidemic. methods: the meteorological data were obtained from three meteorology stations (tokat, sivas, and yozgat), where the majority of the cchf cases were reported in the last 2 years. these 3 provinces are located at the northern parts of eastern anatolia and southern parts of black sea region. temperature data have been observed and recorded by the turkish state meteorological service (tsms), and were available for 70 years in sivas, for 40 years in tokat, and for 60 years in yozgat. meteorology stations are located at the city centres, not at the airports. temperature variations and trends for turkey were analysed by using a data set including monthly averages of daily mean, and minimum temperatures. annual rainfall, and the number of days in april with the temperature of > 5°c were also included in the analysis. in order to detect homogeneity in mean annual series, first a homogeneity analysis was performed by using the non-parametric kruskal-wallis (k-w) test. the non-parametric mann-kendall (m-k) rank correlation test (13) the interest in the coordination properties of acrylate acid and its homologues was generated by the facile synthesis of the ômetal-containing monomersõ (mcm) materials. these compounds can be polymerised with a lot of organic monomers leading to various metal-containing polymers. polymeric transformations of mcm led to a new research field of current interest due to the practical importance of the obtained products which exhibit a number of unique features: high catalytic activity, unusual magnetic, electro-physical and biological properties.these polymers are especially appropriate for biological applications (tissue engineering, implantation of medical devices, dentistry, bone repair etc.) because of their molecular weight, compositions and architectures which can be regulated through controlled reactions. we report here the antibacterial and antifungal activity of new complexes of type m(phen)(c3h3o2)2(h2o)y ((1) m: mn, y = 0; (2) m: ni, y = 2;(3) m: cu, y = 1;(4) m: zn, y = 2; phen = phenantroline and c3h3o2 is acrylate anion), representing the first step products in the synthesis of polymeric materials. the in vitro antimicrobial testings were performed by broth microdilution method, in order to establish the minimal inhibitory concentration (mic), against gram-positive (bacillus subtilis, listeria monocytogenes, staphylococcus aureus), gram-negative (psedomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, both concerning the microbial spectrum and the mic value. the mics values widely ranged between 4 mg/ml and 16 mcg/ml. all the tested compunds were highly active against salmonella and listeria (mic = 16 mcg/ml objectives: c. glabrata is innately less susceptible to azole than most other species of candida and it acquires azole resistance after short-term exposure to fluconazole, as recently noted in oropharyngeal isolates. since c. glabrata has emerged as a significant cause of candidemia, we examined the change in azole mic and karyotype in sequential isolates of c. glabrata during the course of fungaemia, and its relationship to antifungal therapy. methods: serial bloodstream isolates of c. glabrata were obtained from 15 patients with fungaemia over periods of up to 38 days. forty-seven c. glabrata isolates from 15 patients (6 patients who received antifungal therapy and 9 patients who did not receive antifungal therapy) were analysed using electrophoretic karyotyping (ek) and tested for antifungal susceptibility to fluconazole, voriconazole, and itraconazole. results: the overall rates of resistance to fluconazole (mic ‡ 64 lg/ml) and itraconazole (mic ‡ 1 lg/ml) for all 47 isolates were 8 and 50%, respectively. for most patients, the sequential strains from each patient exhibited the same or similar azole susceptibilities. however, sequential isolates from two patients showed three-or four-fold increases in the mics of all three azole antifungals, while they retained the same karyotypes. azole-resistant strains were isolated from both patients after fluconazole therapy was discontinued and the intervals after the first blood isolation were 24 and 38 days, respectively. the isolates from one of these patients exhibited increased expression of the cgcdr1 efflux pump. the sequential strains from each patient had identical karyotypes in 10 (67%) patients, but two or four different karyotypes in 5 (33%) patients. the sequential isolates from these five patients exhibited the same or similar antifungal susceptibilities, showed only one or two chromosome band differences, and had no association with previous antifungal therapy. conclusion: this study showed that sequential bloodstream isolates of c. glabrata were able to acquire azole resistance in association with fluconazole therapy, and that they developed two or four different karyotypes in some patients, during the course of fungaemia. results: the next table shows the mics obtained for the tested drugs. from the three isolates with voriconazole mic > 1 mg/l, two of them had a fluconazole mic ‡ 16 mg/l. six isolates with voriconazole mic = 1 mg/l were found: the fluconazole mic for one of them was 4 mg/l, for another four was 8 mg/l and for the last one was 32 mg/l. conclusions: a. there wasn't found any fluconazole or voriconazole ôresistantõ isolate in the haart era. b. the percentage (11.84%) of isolates with voriconazole mic ‡ 1 mg/l is higher than it has been described in other works. c. the 75% of the voriconazole ôresistantõ isolates were fluconazole ôresistantõ too, so a cross resistance mechanism could be implicated. in vitro susceptibiility testing of micafungina (fk-463) and anidulafungin (ly303366) against candida spp. objectives: despite antifungal therapy, mortality in disseminated zygomycosis is still too high. we analysed the in vitro activity of posaconazole (pos), voriconazole (vrc) and caspofungin (cas) against 59 strains of the genera rhizopus, mucor, absidia, and cunninghamella in different media compositions. methods: the following five media compositions were compared: rpmi 1640 ± 2% glucose, am3 ± 2 % glucose and hr-medium. mics were determined by microdilution method following nccls guidelines with minor modifications. each well was read visually, the growth in each well was compared with the growth control. two endpoints were evaluated for each drug: an inhibition of growth >90% war recorded as mic1 and an inhibition of growth >50% was recorded as mic 2. the final concentrations of the antifungal agents were 0.015-8 mg/l for pos and vrc, and 0.03-16 mg/l for cas. results: pos was significantly more active than cas and vor, both in r + g and in hr media (p < 0.05 when comparing the mic1 of pos in hr medium to that of vrc; p < 0.001 for all other comparisons). growth in rpmi and am3 media supplemented with glucose was more robust than in the corresponding media lacking glucose. glucose had little influence on mic values. the agreement when comparing the mic1 evaluated in am3 ± glucose was > 85%, while the use of mic2 endpoint yielded 100% agreement for all genera. when comparing the data obtained in rpmi ± glucose to that in hr, the agreement was good. the percentage agreements using the mic2 and mic1 endpoints were 100% and > 70%, in contrast, the agreement between am3 ± glucose and the other media was generally poor. moreover, the average mics obtained in am3 medium were lower than those obtained in either hr or rpmi which was due to a difference among genera. conclusions: our results suggest the following: a) pos is active in vitro against zygomycetes at clinically relevant concentrations. b) within zygomycetes, there are differences between genera in terms of their antifungal susceptibilies. c) growth medium is an important variable for mic determination in zygomycetes, and the more relevant medium appears to be rpmi supplemented with 2% glucose. objectives: here we describe first clinical case of a caspofungin resistant c. glabrata infection. the patient was a 43 year old male with aml. he received a matched unrelated hsct, 9 months prior to his death. he had a complicated hospital course which included c. glabrata sepsis. c. glabrata was cultured from his stool, from the time of transplant to death. initial blood culture isolates were azole resistant and amphotericin b was started. this was stopped due to renal insufficiency and caspofungin was started (mic 0.12 lg/ml). he had a prolonged duration of therapy which comprised of alternating courses of iv voriconazole and caspofungin. four months after initial fungaemia c. glabrata was cultured which was both caspofungin resistant (mic > 8lg/ml) and azole resistant. despite the addition of amphotericin b the patient died 8 weeks later. c. glabrata was isolated from the bone marrow at autopsy. methods: the series of patient isolates, from time of transplant to death, had susceptibilities performed as per nccls document m27-a. the isolates were typed using cg6 probe and mlst. results: he susceptibilities demonstrated caspofungin resistance in the blood isolate ( > 8 lg/ml) and azole resistance in all but a few of the initial stool isolates. all the isolates were shown to be identical by cg6 and were determined to be mlst group 1, which has previously been shown to be the most prevalent clade of c. glabrata worldwide. discussion: this describes the first caspofungin resistant clinical isolate of c. glabrata. it has been recently reported in c. albicans. this data details an isolate that has developed resistance in the presence of therapy. resistance to caspofungin is not common and is thought to be due to mutations in the beta-1-3-glucan synthase (fks) gene. there are 3 fks genes in c. glabrata. preliminary sequencing data of one of these genes has so far revealed no non-conservative mutations. the 2 remaining genes are yet to be sequenced and the presence of other mechanisms cannot be ruled out. objectives: the aim of this study was to investigate the production of slime factor among candida species which were isolated from hospitalized patients. another aim of this study was to see in vitro activities of antifungal agents and to compare these results with slime production. methods: total 116 candida spp (79 c. albicans and 37 nonalbicans candida spp) isolated from various specimens were included to the study. fluconazole, itraconazole, amphotericin b and caspofungin susceptibilities of these strains were determined by broth microdilution method according to nccls m27-a2 standards. biofilm production of candida spp. was determined by microplate method on polystyrene microtiter plates using brain heart infusion broth supplemented with 0.25% glucose as a growth medium. results: caspofungin and amphotericin b was the most active agents which mic90 values 1mg/ml and 0.5 mg/ml respectively. fluconazole resistance (mics > 64) was obtained from 24 of the isolates (21%). biofilm formation was detected in 33 of the total strains (28%). statistically important difference (p < 0.05) was determined between the biofilm production of c. albicans (21.5%) and nonalbicans species (%48.4). significant correlation between biofilm production and amphotericin b mic values was established (p < 0.05) conclusion: candida species are one of the most important etiologic agents of catheter related infections especially biolfilm producing strains. this study showed us biofilm production rates are too high particularly in non-albicans strains. this will explain the rising incidences of non albicans strains especially c. parapsilosis in serious infections. our study also implicated that the mic values amphotericin b which was one of the most active agent against candida infections had a significant correlation with slime production. this will be a problem in treatment of slime producing candida infections with this drug. conclusions: (i) scedosporium spp. carry on with high mics of antifungal agents, even for new antifungal agent as pos. (ii) s. prolificans is a multi-resistant organism. (iii) s. apiospermum is resistant to itc and tbf, but 15% of strains are susceptible in vitro to amb (mic < 2 mg/l), 75% to vrc (mic < 2 mg/l), and 60% to pos (mic < 2 mg/l). (iv) these findings reinforced the need of continued surveillance programmes that analyze antifungal susceptibility profiles of medically important fungal isolates. assessing susceptibility of fungi isolated from hospital environment to disinfectants objectives: the past decade has witnessed a worldwide increase in serve invasive gas infections. these rapidly progressive infections are associated with high mortality rates despite prompt antimicrobial therapy. the aim of this study was to characterize gas isolates causing severe invasive disease in different regions of poland by emm-typing, multilocus sequence typing (mlst), pfge, virulence genes distribution and their susceptibility to antimicrobial agents. methods: a total of 42 gas isolates from blood (54.7%), pus (33.3%), sputum (7.1%), peritoneum fluid (4.7%) and other sources were examined. susceptibility to penicillin, erythromycin, clindamycin, telithromycin, tetracycline, levofloxacin, chloramphenicol, quinupristin/dalfopristin and linezolid was determined by the microdilution method according to the nccls guidelines. clonality of all isolates was studied by emm typing, pfge of sma i-restricted bacterial dna as well as mlst. the strains were also tested for the presence of spea, speb, spec, spef and ssa genes by pcr. results: resistance to erythromycin was found in four isolates (9.5%), two of them exhibited the imlsb and two the cmlsb phenotype. twenty-one (50%) and five (11.9%) were resistant to tetracycline and chloramphenicol, respectively. all tested isolates were fully susceptible to penicillin, levofloxacin, quinupristin/ dalfopristin and linezolid. twenty different emm types were detected, of which emm1 (23.8%) and emm12 (21.4%) were most common, followed by emm85, emm60 and emm81. all emm1 types and emm12 types corresponded to the st28 and st36, respectively. altogether, 24 different pfge patterns, designated a-y were discerned among the isolates, with two predominant profiles a (n = 10) and b (n = 9). our study showed that all isolates possessed speb and spef genes, while the frequencies of spec and spea were 59.5% and 26%, respectively. conclusion: two clones predominated among gas strains causing severe invasive disease in poland; these clones were of emm type 1 and 12. we present 45 actinomyces spp isolations, their identification to a species level and their clinical sources. in addition, we perform susceptibility testing of 23 of those strains to 13 drugs. the identification of actinomyces spp was done taking into account their cultural features, growthõs atmosphere and biochemical and enzimatical tests, according to schemes proposed by sarkonen n., funke g., moncla b.,hillier s., and bernard k. we studied too, the clinical sources of actinomyces spp, if they were isolated lonely or in association with mucosa's normal flora. the susceptibility testing was performed by the agar dilution method, with mueller hinton agar supplemented with 5% sheep blood. the reading of minimum inhibitory concentration (mics) were done after 48 hours incubation at 37°c in at atmosphere enriched with co 2 . all strains were grown at 37°c on sheep blood agar plates with co 2 added. a. radingae, a. europaeus, and a. odontolyticus were the most frequent isolated species ( 7 each one) followed by a. israelii, a. graevenitzii, a. turicensis and a. viscosus. in 27 isolations, actinomyces spp were recovered as sole microorganism, and in the 18 remainder, in association with mucosa's normal flora. there were not relations between actinomyces species and the clinical sources of the samples. mics for penicilin, ampicilin and cefotaxime were from <0.016 to 0.25 lg/ml. there was a bimodal behaviour with macrolides : erythromycin, azithromycin and clarithromycin (mics from 0.032 to 128 lg/ml) and the same was observed with quinolones: levofloxacin, ciprofloxacin and moxifloxacin (mics from 0.032 to 8 lg/ml). all isolations presented mics for vancomycin <0.125 lg/ml. the identification of the actinomyces genus presents diagnostic difficulties due to its growth requirements. some species are not so infrequent, and the sources from which they are recovered suggest that they may be of clinical relevance, for they are often isolated as sole pathogen. isotretinoin versus tetracycline: a comparative study with regard to efficiency in the treatment of acne vulgaris c. oprica, l. emtestam, c.e. nord (stockholm, s) tetracyclines are most commonly used for treatment of moderate and severe inflammatory acne, and systemically administered isotretinoin has proved to be the most efficient treatment, used in patients with moderate or severe acne that fails to respond to other therapies. objectives: a randomized clinical trial was conducted to compare the clinical efficacy and the antimicrobial susceptibility of propionibacterium acnes strains isolated before, during and after treatment with either tetracycline or isotretinoin in patients with acne vulgaris. methods: 52 male and female patients, 15-35 years of age, with moderate or severe acne, were randomized into two groups of 26 patients each. they received oral tetracycline hydrochloride 1 gram/day together with topical retinoid (differine gel 0.1%) or isotretinoin (roaccutane) 1 mg/kg/day. the therapy was given for a 6-month period. clinical evaluation (leeds acne grading system and lesions counting) and bacterial samples were taken before the treatment started, during the treatment and 2 months after the treatment had stopped. dermatology life quality index questionnaire was completed by patients before and after treatment, in order to determine impairment of life quality. results: acne severity was significantly reduced by both regimens during therapy, and patients in the isotretinoin group continuously improved the acne scores after the treatment had stopped. after 6 months of treatment, isotretinoin produced greater lesion reductions than tetracycline. the mean per cent reduction in the different lesion counts was as follows: 83% versus 45% for non-inflammatory lesions (p < 0.01) and 80% versus 56% for inflammatory lesions (p < 0.01) in isotretinoin or tetracycline group, respectively. in the drug-free period, the group of patients treated with isotretinoin presented significantly less inflammatory lesions compared to the group treated with tetracycline (p < 0.001). both treatments had improved the life quality (p < 0.01), independent of acne severity. resistant p. acnes strains were isolated after treatment in both the tetracycline group (53%) and isotretinoin group (25%). conclusions: both treatments were effective during treatment period. more resistant strains were recovered from the tetracycline group. psoas abscesses. differences between pyogenic and tuberculous abscess objectives: to compare the demographic characteristics, clinical features, laboratory, microbiologic and imaging data, therapeutic options and outcome of pyogenic and tuberculous psoas abscesses. patients and methods: retrospective descriptive study of the medical records of the patients diagnosed of psoas abscess in our institution, in the period between january/1994 and october/2004. results: in this period 14 patients were diagnosed of psoas abscess, all of them secondary to a adjacent source of infection. nine patients had pyogenic abscess, and m. tuberculosis was the causal microorganism in 5 patients. in 3 patients there were bilateral involvement, and all three were tuberculous abscess. an underlying disease was present in 67% patients of pyogenic abscess, and only in 20% of tuberculous abscess. these one were malignancy, intravenous drug use, cirrhosis, use of steroids, and 3 with inflammatory bowel disease. patients with tuberculous abscess were younger (37 vs 49 years), and they presented with a longer duration of symptoms from presentation to diagnosis (145 vs 15 days) than patients with pyogenic abscess. abdominal pain was the most frequent symptom at diagnosis in pyogenic abscess (78%), whereas lumbar pain (80%) was in tuberculous abscess. other clinical manifestations were similar in both groups. the source of infection in pyogenic abscess was gastrointestinal in 7 patients (4 polymicrobial), and in 1 patient infection of an aortobifemoral bypass and sacroilitis (both caused by s. aureus). all tuberculous abscess were secondary to spondilitis. there were no differences between both groups in analysis or imaging alterations. culture of the abscess was positive in all cases practised. drainage was performed in 12 patients (7 percutaneous), without differences in both groups. clinical improvement was more delayed in patients with tuberculous abscess than in patients with pyogenic abscess (18 vs 10 days). there were no deaths in any group. conclusions: tuberculous psoas abscesses presents in younger patients with lesser underlying diseases and a more prolonged clinical presentation than pyogenic abscesses. these tuberculous abscesses are secondary to spondilitis, usually with bilateral involvement. prevalence of micro-organisms isolated from wounds of inpatients versus outpatients in a spanish teaching hospital objectives: hidradenitis suppurativa (hd) is a chronic disorder characterized from dilatation of sweat glands and recurrent bacterial infections. vitamin e was administered in several patients as an antioxidant in an attempt to relieve tissue function probably altered by the locally increased oxidant status. methods: twenty nine patients with hd, 15 male and 19 female, were enrolled over a period of twelve months. all have presented with more than three episodes of bacterial exacerbations for at least two years. a detailed medical history was taken upon first evaluation and patients were examined for areas affected by the disease. they were asked to self-evaluate the severity of their condition on a scale of 1 to 10 (1 representing intact skin and 10 maximum severity). patients were divided into three groups of treatment: a (n = 6), controls; b (n = 8), vitamin e orally 200 mg bid; and c (n = 15), vitamin e 200 mg tid orally. patients were withhold from any antimicrobial regime. patients were followed-up at three-month intervals; they were asked to re-evaluate their condition, and to provide details regarding frequency of relapses before and after the initiation of treatment. results: mean ± se duration of the disease was 11.7 ± 1.9) years and of involved areas 3.84 ± 035, with axillas and groin being involved in the majority of cases. mean interval between exacerbations before initiation of therapy with vitamin e was 33.98 ± 9.43 days and after initiation of therapy 91.35 ± 21.64 days (p: 0.042). for group b, mean ± se time interval between exacerbations before initiation of treatment was 45.00 ± 27.39 days and after initiation of treatment 69.00 ± 38.42 days. for group c, respective values were 36.30 ± 9.42 days and 132 ± 12 days. mean ± se of self-evaluation scores before therapy with vitamin e was 9.00 ± 0.71 and 4.33 ± 1.76 for patients of groups b and c respectively. they were 4.75 ± 1.31 and 2.75 ± 1.55 respectively after twelve months of follow-up. the latter changes constituted a significant improvement (p: 0.027). conclusion: vitamin e seems to improve the overall clinical condition of patients with hd substantially. further placebocontrolled studies are necessary to confirm these results. clonal diversity and toxin genes of staphylococcus aureus causing infections in a clinical ward over a 12-year period (1992-2003) s. pollini, g. zanelli, a. sansoni, s. cresti, c. cellesi, g.m. rossolini (siena, i) objectives: s. aureus remains one of the leading bacterial pathogens worldwide, representing a major challenge for antimicrobial chemotherapy. the aim of this study was to evaluate the molecular epidemiology of s. aureus infections observed in an infectious disease ward during the past 12 years. clinical microbiology and infection, volume 11, supplement 2, 2005 methods: 38 nonreplicate s. aureus isolates were collected from patients with staphylococcal infections (including food-borne infections, osteomyelitis, skin and soft tissue infections [ssti], pneumonia, meningitis and bacteraemia/endocarditis; mostly community-acquired) at the infectious diseases clinic, university of siena, during the period 1st feb 1992-31st march 2003. all isolates were analysed for antimicrobial resistance, clonal diversity and toxin genes (sea-e, eta-d, tst, luks-pv and lukf-pv). susceptibility testing was performed according to the nccls guidelines. clonal diversity was determined by pfge and by analysis of the coagulase (coa) and protein a (spa) genes polymorphism. toxin genes were analysed by pcr and restriction mapping. results: most isolates (35, 92%) were methicillin-susceptible (mssa), while 2 were methicillin-resistant (mrsa, mecapositive) and 1 borderline. genotyping revealed a single mrsa clone and remarkable clonal diversity among the mssa isolates (at least 20 distinct clonal lineages). several clones (including 6 mssa and the mrsa clone) were detected over prolonged times (2-8 years). the most prevalent clone was detected over a 8-year period and was exclusively associated with ssti (mostly bullous impetigo). toxin genes were overall detected in 87% of isolates, the most frequent being sea (45%), tst (32%) and eta and/or etb genes (29%). six isolates (all mssa) harboured the luks-pv and lukf-pv genes. conclusions: mrsa were rare and appeared only since 1997. significant clonal diversity was observed within mssa. a significant relationship was observed between toxin production and some infections (e.g. ssti). severe group a streptococcal infections in romania. a surveillance within the strep-euro project b. luca, m. straut, v. ungureanu, c. schalen, a. jasir on behalf of the strep-euro study group objectives: in the last 20 years, severe invasive streptococcal infections, often afflicting otherwise healthy subjects and yielding high mortality rates, have been increasingly recorded in europe and other areas. our main objective was to investigate the situation in romania lacking earlier data in the field. methods: the strains used in this study were clinical isolates from nine regions in romania. the strains were mainly blood isolates, but some were from other sterile sites. the in vitro susceptibility to antibiotics was tested by disk diffusion on pdm agar following the standard instruction. t-typing was performed by slide agglutination using sera from sevapharma, prague. spe gene detection and emm sequence typing was performed according to provious publications. results: during eighteen months 33 cases were reported. most prevalent t-type was 3,13 followed by types 12 and 1. more than fifteen different emm types were recognised, most of them unusual types; only four were type 1. half of the reported cases were from children below 12 years. out of all strains, 50% harboured spec gene, and 25 harboured spea. erythromycin resistance was uncommon (one isolate), whereas an overall high rate of tetracycline resistance was found (54%). conclusion: this is the first report on severe invasive streptococcal infections since no surveillance has been previously done in romania. the population covered was from eight out of 42 provinces. the emm type distribution might be of concern since many unusual types, not previously associated with severe disease, were found. this may be partly related to the dissemination of new types in populations immune to classical types. since tetracycline is not used in the treatment of streptococcal infections the level of tetracycline resistance among clinical isolates, appeared comparatively high. in contrast to several european countries, macrolide resistance seems not to be common among invasive group a streptococci in romania. acknowledgment: the strep-euro project is funded by the european commission. severe soft tissue infection and secondary bacteraemia due to community-acquired mrsa in a traveller returning from the congo r. padmanabhan, r. shrestha, g. hall, s. gordon, l. saravolatz (cleveland, detroit, usa) objectives: community acquired mrsa is increasingly becoming a global problem. these isolates possess the staphylococcal cassette chromosome (scc) mec type iv in their genome and a different pattern of antibiotic susceptibilities than hospital acquired strains. they are frequently virulent and cause predominantly skin and soft tissue infections by virtue of a panton valentine leukocidin (pvl) toxin gene. a returning traveler, who had spent the preceding four weeks in congo, presented with a staphylococcal bacteraemia and a large cutaneous ulcer of the right lower extremity that progressed to extensive soft tissue necrosis. he first experienced symptoms in kinshasa international airport, while awaiting his return flight to the united states, approximately 48 hours prior to presentation at our facility. methods: mrsa that was isolated from blood was susceptible to vancomycin, clindamycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and gentamicin. pulse field gel electrophoresis (pfge) analysis was performed using sma1 on the patient's isolate along with another strain from our hospital and compared to other strains isolated from the midwestern united states. pcr amplification of the genes encoding panton-valentin leukocidin (pvl) was performed using primers described elsewhere. results: pfge demonstrated that the congo strain was different from any of the other midwestern united states strains. scc mec typing of this isolate revealed that this strain was type iv. pcr analysis did not detect sequences specific for the pvl gene. conclusions: the differential diagnosis of an ulcer in a returning traveler is large and includes bacterial, fungal, helminthic, protozoal, viral and arthropod borne causes. mrsa should be added to this list. the widespread occurrence of ca-mrsa in many parts of the world would support the finding of this organism on the african continent. to our knowledge, this represents the first ca-mrsa with the scc type iv mec gene reported from congo. ten-year of community-acquired methicillinresistant staphylococcus aureus st80-iv clone in denmark objectives: to determine the epidemiology in denmark of the pandemic european ca-mrsa clone st80-iv with respect to subtypes, dissemination, acquisition and types of infection. methods: all methicillin resistant staphylococcus aureus (mrsa) isolated in denmark between 1999-2003 were referred to and stored at the staphylococcus laboratory, statens serum institut (n = 617). the isolates were characterized by macro-restriction (smai) analysis using pfge, sccmec typing, dru sequence typing, antibiotic susceptibility testing and pcr amplification of the panton valentine leukocidin gene (pvl). clinical and epidemiological information from all patients were obtained from discharge summaries and registered. results: between 1999 and 2003, the mrsa st80-iv clone accounted for 28% (172/617) of the total number of mrsa in denmark and 30% (146/490) of infections due to st80-iv had primarily community onset (122/146) and thereby st80-iv accounted for 67% of all community onset mrsa in the period. more than 80% of the st80-iv infections were skin and soft tissue infections. by comparing the antibiogram of st80-iv (streptomycin-, kanamycin-, tetra-cycline-and fusidic acidresistant and gentamicin sensitive) with the isolates stored in our s. aureus bacteraemia collection, we traced the st80-iv clone back to 1993. before 1999 one to five st80-iv isolates were encountered pr. year, which increased to 25, 26, 50, 26 and 45 isolates in the years after. by pfge, the st80-iv isolates exhibited nine different patterns (a1-9), of which 60% were type a1. nine type a1 isolates, isolated 1993-2003 were subjected to dru typing, which has earlier enabled separation of pfge clones into subtypes. this was however not possible for the st80-iv isolates. conclusions: st80-iv isolates cause a large proportion of the mrsa infections in denmark and in particular among the infections with a community onset. the success of st80-iv as an infective clone outside the hospital environment may in part be due to its small sccmec type iv and its ability to cause skin and soft tissue infections probably associated with the expression of pvl. the dru sequence typing could not subdivide the major type of st80-iv found in denmark pointing out the strict clonality of this clone, which may indicate that it is a very well adapted pathogen. epidemiology and management of pressure ulcers in an acute care hospital objectives: the aims of the study were to determine pressure ulcer prevalence in an acute care hospital, to identify risk factors, to evaluate the medical and non medical management of pressure ulcers and to study the microbiological contamination of pressure ulcers. methods: for each patient, a standardized questionnaire was completed. the questionnaire included demographic data (age, sex, previous hospitalizations…) and the risk factors of the braden scale. detection of pressure ulcer was performed by skin examination of patients by two experts in skin care. management of ulcer pressure was evaluated by reviewing the clinical charts of each patient with pressure ulcer. each pressure ulcer was swabbed and inoculated on selective media. all the data were entered and analysed with epiinfo 6.04d (cdc, atlanta, ga). results: a total of 549 patients (59 ± 19 years old) were included : 75 ulcer pressures were observed in 37 patients (prevalence = 6.9%). heel anckle was the most frequent localization (40%), followed by sacrum (20%), elbow (11%), spinous processes (7%) and ischial tuberosities (7%). pressure ulcers were stage i (24%), stage ii (29%), stage iii (31%) and stage iv (16%). eighty percents of pressure ulcers were acquired within the hospital. using univariate analysis, risk factors significantly associated with pressure ulcer were : braden cutoff < 15 (or = 6.56, p < 0.0001), neurological disorders (or = 2.30, p = 0.009), previous ulcer pressures (or = 6.36, p < 0.0001), and hospitalization in an intensive care unit (p = 0.0003). among the criteria used for braden scale, humidity, activity, motility, nutrition friction and shear were significantly associated with ulcer pressures (p < 0.05). among the 75 pressure ulcers, 9.3% were diagnosed only by the experts in skin care and 83.8% were treated. treatment was considered inappropriate in 31.5% (mostly in stage i and iii) according to the french guidelines. microbiological results showed that 24.5 % of ulcer pressures, mostly from stage iii and iv were colonized with multiple resistant bacteria (i.e. methicillin resistant staphylococcus aureus, extended spectrum beta-lactamase enterobacteriaceae). conclusion: this prospective prevalence study led to a better awareness of patients at risk for pressure ulcer. this surveillance also contributed to a better knowledge of the mobile unit of geriatrics recently created within the hospital. background: following surgery for a musculoskeletal infection (mi), a positive suction drainage culture (sdc) is consistent with persistent sepsis and an unfavorable evolution in the majority of cases. the objective of this study was to determine the effect of a negative sdc obtained in a subsequent operation or repeated operations on the outcome of mi. methods: one hundred patients treated surgically for mi utilizing suction drainage for 24-48 hours postoperative and appropriate antimicrobial therapy were enrolled in this prospective study. surgical treatment consisted of the routine practice developed for the treatment of mi, consisting of drainage of purulent material, débridement, and prosthetic exchange or implant removal. the accumulated drainage fluid in the reservoir was cultured. sdc was considered negative if all bottles resulted in negative cultures. patients were placed into one of three possible mi treatment groups according to the sdc results identified after the first surgical procedure, as follows: (i) patients with a negative sdc and no new operation was performed; (ii) patients with a positive sdc and a new operation(s) was performed until the sdc was negative; and (iii) patients with a positive sdc and there was no new operation. the duration of antibiotic therapy for those with osteomyelitis ranged from 6 to 12 weeks, while others with mi (soft tissue infection) were treated for 10-21 days. results: the 3 groups were similar with regards to gender, age, underlying conditions, mi, bacterial organism, surgery, and antibiotic therapy. the majority of patients (84%) had osteomyelitis in the presence of an implant. at final review, a cure was obtained in 90% of patients (45 of 50) in group i, 87% of patients (20 of 23) in group 2, and 44% of patients (4 of 9) in group 3. conclusion: a negative sdc following mi surgery is a strong indication as to the eventual outcome of the infectious process. microbial aetiology of osteomyelitis of the foot in diabetic patients related hospitalization and lower-extremity amputation.acute infections in pts who have not recently received antibiotic therapy are predominantly caused by aerobic gram-positive cocci, often as a monomicrobial infection. although chronic wounds tend to develop polymicrobial flora. objective: to know the microbial etiology of osteomyelitis of the foot in diabetic pts, to determine the best choice empirical antibiotics combination. methods: diabetic pts with osteomyelitis of the foot admitted in our unit between 2002 and 2004 were included. the infection was documented with intraoperative samples and osteomyelitis was histolopathologically prouved. clinical and biological data were collected. results: 87 diabetics pts (64 males, 23 females) with a median age 65 years(23-94) were included.the co-morbidities were chronic renal failure (n = 35), arteriopathy (n = 34), obesity (n = 13), alcoholism (n = 10), immunodepression (n = 10). the clinical data were fever (40%), shock (2%), local signs as erythema (90%), fistula (86%), necrosis (43%) and foul odor (54%). the median rates of neutrophils polynuclear, c-reactive protein and erythrocyte sedimentation were respectively 9.7 g/l, 131 mg/l, 82 mm. 59 pts (68%) had a polymicrobial infection.the intra-operative samples (n = 87) yieled a majority of gram positive cocci [mssa:n = 30;mrsa:n = 15; cns:n = 15; streptococcus sp:n = 34; enterococcus sp: n = 15], following with gram negative bacteria [ pseudomonas sp: n = 12; proteus sp: n = 11; morganella sp: n = 11; e. coli: n = 8; klebsiella sp: n = 6; e. cloacae: n = 6; citrobacter sp: n = 4; serratia sp: n = 3, others: n = 3] and anaerobes (n = 17). 10 pts had positive blood samples. s. aureus is the most frequent pathogen isolated especially in monomicrobial infections and infections with positive blood samples. pseudomonas spp. seems to be correlated with chronic renal failure and immunodepression. no clinical data was significantly associated with a special bacteria , except necrosis and foul odor with gram negative bacteria or anaerobes. conclusion: our results are according with clinical studies in the literature. the major difficulty to treat osteomyelitis of diabetics foot is to well documented the infection. selecting an appropriate antibiotic to treat is particularly important because of the prolonged duration of therapy required and potential resistance of pathogens. the duration of hospitalisation (length of stay) in patients hospitalised with complicated skin and skin structure infections: identifying clinical and microbiologic risk factors in a comparison of tigecycline with vancomycin/aztreonam 42% of patients had polymicrobial infections. about 18% had a gram-negative causative pathogen, mostly escherichia coli. gram-negative causative pathogen prevalence was greatest in asia (39%). conclusions: this analysis showed significant global variations in csssi subdiagnoses, etiologies, comorbidities and causative pathogens. consistent with findings of the sentry antimicrobial surveillance program, s. aureus was identified as the predominant pathogen in csssi, especially in the us. gramnegative pathogens were also identified as causative in a substantial proportion of complicated skin infections. these findings may offer guidance in selecting effective empiric therapy, especially regarding newer agents with expanded broad-spectrum activity. a novel approach for evaluating the microbiological efficacy of tigecycline in patients with complicated skin and skin-structure infections a. meagher, p. ambrose, j. passarell, b. cirincione, t. babinchak, e.j. ellis-grosse (buffalo, albany, collegeville, usa) objectives: tigecycline (t) is a glycylcycline in development for the treatment of patients (pts) with serious infections, including complicated skin and skin-structure infections (csssi). while csssi can be caused by a mixture of grampositive and -negative bacteria, staphylococcus aureus and streptococci are the predominant pathogens. previous analyses combining all pathogens have failed to identify an exposureresponse (er) relationship. a method was developed to create more homogenous pt populations for the microbiological (m) er analysis of t in the treatment of csssi. methods: pts from 3 csssi clinical trials (one phase 2 & two phase 3) with t pharmacokinetic data and classified as both clinically and m evaluable, were pooled for analysis. pts received 100 mg loading dose (ld)/50 mg q12h (100/50) or 50 mg ld/25 mg q12h (50/25). at the test of cure visit, m (eradication or persistence) response was evaluated. indeterminate responses were excluded. non pathogenic baseline isolates were excluded. five homogeneous pt cohorts (c) were created based on baseline pathogens: s. aureus only (c1); s. aureus or streptococci (c2); 2 gram-positive pathogens (c3); polymicrobial (c4); other monomicrobial infections (c5). prospective step-wise procedures for combining c to increase sample size were used. logistic regression was used to evaluate steady-state 24 hr area under the concentration-time curve (auc) to mic ratio (auc/mic) to predict response. results: the dataset included 58 pts with 88 observations. c1 (n = 20) and c2 (n = 9) could not be evaluated due to small sample size. analysis began with pooled c2 + c3. continuous auc/mic ratio was marginally significant (p = 0.1130); a pt was 5.1% more likely to have successful response for every oneunit increase in auc/mic. adding c4, including pathogens with mic values up to 16 mcg/ml, decreased auc/mic, added cures to the lower end of the distribution, and added significant noise to the analysis. adding c5 increased sample size and further decreased the ability to detect a relationship. conclusion: analysis of all pathogens combined could not identify an er relationship. polymicrobial infections with gramnegative and anaerobic pathogens, associated with high mic values, added noise to the analysis and decreased the predictive capability of the model. the approach of creating homogenous populations based on two key pathogens in csssi, s. aureus and streptococci, was critical for identifying significant er relationships. exposure-response analysis of the efficacy of tigecycline in patients with complicated skin and skin-structure infections a. meagher, j. passarell, b. cirincione, s. van wart, k. liolios, t. babinchak, e.j. ellis-grosse, p. ambrose (buffalo, collegeville, albany, usa) objectives: tigecycline (t), the first glycylcycline to reach clinical trials, is in development for the treatment of patients (pts) with serious infections, including complicated skin and skin-structure infections (csssi). pharmacokinetic-pharmacodynamic (pk-pd) relationships, including pt covariates, for microbiological (m) & clinical (c) efficacy of t were evaluated in pts with csssi. methods: pts from 3 csssi clinical trials (one phase 2 & two phase 3), with pk data and classified as both c & m evaluable, were pooled for analysis. only those pts with infections due to staphylococcus aureus and/or streptococci, the predominant pathogens in csssi, were prospectively evaluated. pts received 100 mg loading dose (ld)/50 mg q12h (100/50) or 50 mg ld/ 25 mg q12h (50/25). at the test of cure visit, m (eradication or persistence) & c (cure or failure) outcomes were assessed. indeterminate responses were excluded. steady-state 24 hr area under the concentration-time curve (auc) and auc/mic ratio were evaluated as predictors of response. pt covariates included: age, weight, country, baseline pseudomonas aeruginosa or anaerobes, & comorbidities (diabetes, peripheral vascular disease). classification and regression tree (cart) analyses determined auc/mic breakpoints (bp). logistic regression (one observation/pt) was performed to determine predictors of efficacy. results: the dataset included 35 pts with 40 s. aureus and/or streptococcal baseline pathogens. mic values ranged from 0.06 to 0.5 mcg/ml. clinical cure was achieved in 30 (85.7%) pts and 35 (87.5%) pathogens were successfully eradicated. the median auc/mic ratio was 13.5 and 29 for the 25 and 50 mg dose groups, respectively. covariates were not significant predictors of efficacy. cart identified a significant auc/mic bp of 12.5 (p = 0.0177 for m and 0.0341 for c). the continuous auc/mic ratio was marginally significant based on sample size (p = 0.0563 for m & 0.1960 for c) and was deemed the most informative model. for each unit increase in auc/mic, within the observed range, pts were 3.7% more likely to have a successful c response and 17.1% more likely to have a successful m response. conclusion: pts with auc/mic ratios ‡12.5 were 13.5 times more likely to have successful m response. at the median auc/ mic ratio of 13.5 & 29 for the 50/25 & 100/50 dose groups, the model-predicted probability of c success was 0.66 & 0.96, respectively. t is likely to be an important treatment option for csssi. introduction: vertebral osteomyelitis and/or sacroiliitis due to streptococcus pneumoniae are extremely rare. although it is one of the most common pathogen isolated in blood cultures, there are only about 30 cases reported in the literature. in the main series of vertebral osteomyelitis (vo) spsi represents less than 1% of cases (1/587). material and methods: we report 6 cases of spinal infection due to s. pneumoniae (5 vo and 1 si with psoas abscess) seen between 1999 and 2004 at 4 different acute care hospitals. results: the 6 cases reported were part of a total of 1227 episodes of pneumococcal bacteraemia (0.48%).mean age of the patients was 57.1 years (range 37-82); there were 4 men. alcohol abuse 2/6, immunosupression 2/6 and smoking 4/6 were the most common comorbilities. blood cultures were positive in all cases, epidural pus and vertebral biopsy were also positive in 3/ 6. all strains were susceptible to penicillin. diagnoses were made by mri in 4 and ct scanning 2. all patients recived betalactam agents during a mean of seven weeks (3.8-10.7) in monotherapy (cefotaxime 4/6, ampicillin 1/6) or in combination (with vancomicin+gentamicin in 1 case). one case needed surgery, because of progression of a psoas abscess. other foci were found in three cases (psoas and paravertebral abscesses and acromio-clavicular arthritis). conclusions: spsi is a rare presentation of invasive pneumococcal disease. underlying disease is comnmon and other territories are frequently involved. infective endocarditis must be ruled out. its course appears to be benign. beta-lactam antibiotics are a good option, and surgery is occasionally indicated for progressive disease despite appropriate antimicrobial therapy. lumbar spine spondylitis due to staphylococcus schleiferi following coronary angioplasty: isolation of the pathogen in peripheral blood cultures a. karakolios, c. karagiannidou, t. kallinikidis, f. markou, v. kontos, g. tsinopoulos (serres, gr) a 70-year old man presented with acute, severe lower back pain 3 weeks after having been subjected to coronary angioplasty for ischaemic heart disease. he was treated with nsaids but not antibiotics without any improvement. ct scan of the lumbar spine showed changes in l5-s1 vertebrae that were consistent with spondylitis. mri imaging, as well as confirming spondylitis at l5-s1, it also revealed the presence of a left paravertebral abscess. on admission to hospital, low grade fever was documented. clinical examination of the lower limbs was normal. staphylococcus schleiferi, sensitive to several antibiotics, was isolated from 3 sets of blood cultures taken from both arms. initially, ciprofloxacin 200 mg bd iv was administered for 4 weeks. despite continuous clinical improvement, mri imaging 8 weeks after the commencement of antibiotic treatment showed no improvement of the radiological findings. thus, levofloxacin 500 mg bd p.o. rifampicin 300 mg bd p.o. were given for further 16 weeks. at the end of the treatment, the patient was symptom-free, resumed full activity and mri imaging showed considerable improvement. multiple blood cultures taken from both arms at different time points can help isolate pathogens causing spondylitis averting thus the need for difficult, interventional sampling of the focus of infection. diabetes and use of topical ocular antibiotics: a population-based case-control study objectives: we conducted this population-based case-control study to examine whether patients with diabetes mellitus have an increased relative risk of being treated with topical ocular antibiotics, as compared with population controls. methods: incident cases were defined as 24,559 individuals who redeemed a prescription for a topical ocular antibiotic in 1999 in north jutland county, denmark. ten gender-and agematched population controls per case were selected, using a unique personal identifier. diabetes prior to the topical ocular antibiotic prescription was determined by record-linkage with the county prescription database and hospital discharge registry. we did conditional logistic regression to estimate odds ratios (ors) for ocular antibiotic use among diabetic individuals and population controls, with adjustment for a range of comorbid diseases. results: among individuals treated with topical ocular antibiotics, 2.5% had diabetes as compared with 2.0% among control subjects. the overall adjusted or for use of ocular antibiotics in diabetic individuals was 1.24 (95% confidence interval [ci]: 1.13-1.35). stratified analyses showed that children between 3 and 15 years with diabetes had a 60% increased relative risk for ocular antibiotic use, whereas the effect of diabetes as a risk factor was low in individuals over 65 years and in those with presence of other diseases. conclusions: these results suggest that diabetes is a risk factor for the use of topical ocular antibiotics, especially in younger individuals. objectives: determination of bacterial species and their susceptibility to antibiotics in infectious conjunctivitis in general practice. methods: 179 patients suspected of having infectious conjunctivitis were included from 25 general care centres in the netherlands. from both eyes a swab was taken and pathogens found in the bacterial cultures were tested for their susceptibility using the etest for chloramphenicol, fusidic acid, trimethoprim, ciprofloxacin, and gentamicin (mics according to dutch national criteria). results: 80/220 affected eyes were culture positive. streptococcus pneumoniae (n = 55) was the most predominant pathogen, followed by haemophilus influenzae (n = 19), and staphylococcus aureus (n = 18). only for chloramphenicol and ciprofloxacin the mic90s were in the susceptible or intermediate susceptible range for all three pathogens, whereas this was not the case for the other antibiotics tested, of which at least one mic90 was in the resistant range. conclusion: in only one thirdth of the patients suspected of infectious conjunctivis in general practice a bacterial pathogen was found. although the prescription of antibiotics can be debated on the basis of our results, chloramphenicol and ciprofloxacin seems to be superior above fusidic acid, trimethoprim, and gentamicin. investigation of the aetiology of a trachoma-like condition in a rural population in guangxi province, prc p. cho, m.v. boost, w. ng (hong kong, hk) objectives: to determine the presence of trachoma in primary school children of the zhuang and yiao ethnic minority groups in du¡an county in guangxi, china. method: primary school children were examined using a slit lamp and several were noted to display follicular/papillary conjunctivitis suggestive of trachoma. to confirm the etiology of the condition, children were examined on a follow-up visit. for each subject, after examination with the slit lamp, the upper lid was everted after the application of a topical anesthetic (novesin), and a dacron swab was passed along the upper tarsal lengthwise four times. strict aseptic precautions were employed to prevent cross-contamination during sample collection. swabs were placed in dna-free tubes, transported to hong kong, and tested using the roche amplicor kits for detection of chlamydia trachomatis following the manufacturer¡s instructions. twenty children displayed significant conjunctival signs and photographs of the everted upper lids were taken to determine if these visual signs correlated with presence of trachoma as confirmed by pcr. results: sixty-five of 120 primary school students were willing to be tested, and 29% of these were positive for trachoma by pcr. comparison of photographs and pcr result indicated that not all children who showed signs of conjunctivitis were positive for trachoma. in addition, some children whose eyelids displayed milder form for conjunctivitis were positive by pcr. no obvious corneal involvement was observed for these children. conclusions: it was confirmed that hyper-epidemic levels of trachoma can be found among ethnic minority children in du¡an county. this may be related to the poor hygiene standards and the scarcity of water in this limestone region. pcr was not positive for all children showing signs of follicular/papillary conjunctivitis, which may indicate that some cases were in a dormant stage. it is also possible that failure to detect the organism was due to sampling error or delay in pcr analysis. nevertheless, the high level of infection is a cause for concern as trachoma remains one of the leading causes of blindness, and further investigation and treatment are warranted. clinical and epidemiological considerations about anthrax in constanta county, romania s. rugina, i.m. dumitru, c.n. rugina, e. dumea, e. basca (constanta, ro) introduction: anthrax is by primarily a disease of herbivores but humans can become infected as they come into contact with infected animals or their products. objective: to study the cases of human anthrax diagnosed in the last twelve years and their characteristics: epidemiology, clinical forms and evolution. material and method: it is a retrospective study on a period of twelve years performed in infectious diseases clinical hospital. the diagnosis was based by smear, and culture of vesicular fluid or csf. results: during this period fifteen cases of anthrax in humans were identified. the repartition of cases according to sex groups revealed a high predominance of male cases (13 cases). the disease was most prevalent in 35-44 age group and after 55years old (6 cases), although anthrax can be observed between 15 years and 65 years. in all cases anthrax was occupational disease and the provenience of patients was rural area. the annual incidence of anthrax was characterized by a low value and irregular frequency of the disease. the most cases were in 1994 (6 cases) and 2004 (5 cases). according to the seasonal repartition, anthrax was most prevalent in the summer months (11 cases). the most frequent clinical form of anthrax was the cutaneous anthrax (14 cases), and only one patient achieved anthrax meningitis. under specific treatment with penicillin g, all the patients with cutaneous anthrax recovery complete, only the patient with anthrax meningitis died. conclusions: in constantza county, anthrax remained sporadic in the last years. after a free period (2001) (2002) (2003) 5 cases of coutaneous anthrax were registered in 2004. a good cooperation between veterinary and physicians and individual awareness of the materials is the key to prevention of anthrax. hemolytic activity. all of the 30 strains tested have hemopeptic activity, which is indirect evidence of their virulence. the strains have no phosphatase enzymes or lecithinase. the strains we studied ferment glucose, sucrose, glycerin, and rhamnose, and they form acid without gas. the strains all grew in synthetic "a" medium, although growth was weak. when l-tyrosine, ltryptophan, l-threonine, and l-methionine were added to the synthetic "a" medium, the pathogen grows in the typical manner. two anthrax strains are moderately resistant to rifampicin (mic 1.5-16), and three are not sensitive. the anthrax strains are highly sensitive to tetracycline, benzylpenicillin, gentamycin, and oxacillin. conclusions: regardless of their source of isolation and time of storage, the anthrax strains isolated during outbreaks are virulent, spore-forming, and capsule-forming cultures. this study confirms that virulent forms of b. anthracis are still circulating in kazakhstan. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency under the project «kb0-1950-al-03» and administered by u.s. crdf. the importance of laboratory methods in diagnosing anthrax in humans l. lukhnova, a. aikimbayev, t. meka-mechenko, g. temiraliyeva, s. zakaryan, m. sagatova (almaty, kaz) objectives: isolation of anthrax bacillus using bacteriological methods is slow and frequently misleading. the literature suggests anthrax bacillus is isolated in 13% to 48% of patients infected with bacillus anthracis. the pathogen quickly perishes or transforms into atypical forms. the number of bacilli in a clinical specimen is often low or below the sensitivity of culture. this suggests other methods would be useful in establishing the diagnosis of anthrax, specifically; serological methods would be beneficial in diagnosing infection with bacillus anthracis. methods: we conducted studies to assess various methods for diagnosis of bacillus anthracis infection in patients. we reviewed archival data and our own results on pathological material from patients infected with bacillus anthracis in the southern and eastern kazakhstan oblasts. results: the most informative test for detection of anthrax in patients was the delayed type hypersensitivity (dth) or dermatoallergic test with anthraxin. eighty one per cent of patients with signs and symptoms of anthrax gave a positive reaction with anthraxin. the ascoli reaction complete with scab was positive in 72.1% of patients, and the indirect hemeagglutination (iha) test was positive in 65.6% of patients. the clinicoepidemiological diagnosis of anthrax was confirmed with culture in 44% of cases. serum samples from patients diagnosed with anthrax in the eastern kazakhstan oblast were evaluated using iha in 2001-2004. high titres of specific antibodies (1:640-1:5120) were detected in patients with dermatologic anthrax lesions from which anthrax was isolated. conclusion: our data suggest the anthraxin dth test and iha are good methods for establishing a diagnosis of anthrax. cultures for anthrax bacillus were the least efficient method for establishing the diagnosis. when results of laboratory tests are negative or inconclusive, one still has to rely on the clinicoepidemiological diagnosis. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency (dtra) under the project «kb0-1950-al-03» and administered by u.s. civilian research and development foundation (crdf). objectives: the fluoroquinolones are bactericidal agents frequently used in the treatment of respiratory tract infections. the viridans streptococci (vgs), although associated with disease, generally exist as part of the normal human oral flora. as such, they may exposed to antibiotics with greater frequency than oppurtunistic pathogens. preliminary data suggest that the vgs may act as a reservoir for fluoroquinolone resistance in s.pneumoniae through horizontal transfer. here, we compared the killing kinetics of moxifloxacin (mxf) and levofloxacin (lev), against vgs at simulated saliva concentrations. methods: eight strains of vgs, 4 s. mitis (2 with parc mutation) and 4 s.oralis, (2 with parc mutation) were challenged with mxf and lev in a pharmacodynamic model utilizing mueller-hinton broth supplemented with 5% lysed horse blood. cultures were inoculated at a density of 1 · 108 cfu/ml, incubated at 35ordm;c, and examined for viable growth at 0, 1, 2, 4, 6, 12, and 24 hrs after exposure to the antibiotics at concentrations simulating salivary levels. salivary concentrations of mxf (400 mg), and lev (500 mg) were 3.19 and 3.71 lg/ml respectively. protein binding of mfx and lev were taken to be 50% and 30%. adjusted salivary levels were 1.6 (mxf) and 2.6 (lev) ug/ml. pcr was used to amplify parc and gyra, and mutations detected by dna sequencing. results: both mxf and lev were highly bactericidal against susceptible strains of vgs at salivary concentrations. mfx typically eradicated the vgs by 6 hours and lev by 12-24 hours. for parc mutants, both mxf and lev were consistently bactericidal resulting in eradication after12 hours for mxf and 24 hours for lev. regrowth was observed in both strains of s.mitis (parc) exposed to lev (2 of 10 experiments). the mic of these strains to lev increased 4 fold and dna sequencing revealed the selection of a gyra mutation in each strain. no regrowth was detected in the s. oralis strains. conclusions: both mxf and lev are extremely active against susceptible vgs at concentrations of drug found in saliva, however, superior killing kinetics were consistently observed with mxf. mxf consistently eradicated strains with a parc mutation, however, regrowth was observed on 2 occasions with lev suggesting that mxf may be less likely to drive resistance among vgs. again, this may have implications due to the ability of vgs to exchange its genetic determinants with s. pneumoniae. moxifloxacin and ciprofloxacin uptake by human neutrophils and their influence on viability, phagocytosis, oxidative burst, and chemotaxis a.m. berg, j. altrichter, b. drewelow, r.g. mundkowski (rostock, d) objectives: the influence of antibiotics on the immune defence is of increasing interest. the purpose of this study was to set up a cellular model for correlating the intracellular concentrations of the fluorochinolones moxifloxacin and ciprofloxacin with selected cell functions of early immune response. a human promyelotic cell line which is used for sepsis treatment by the extracorporeal immune support system (eissò, teraklin gmbh) was chosen to establish the model. the second stage involved native human polymorphonuclear neutrophiles (pmns) as target cells. methods: the promyelotic model cell line was differentiated towards neutrophils with all-trans retinoic acid, human pmns were isolated using dextran sulfate sedimentation and density gradient centrifugation. the cells were incubated with antibiotic concentrations within the therapeutic range and above (0.5-2 cmax) for time periods up to 8 hours. the extra-and intracellular concentrations were determined by hplc-uv. the viability of the cells was controlled using the trypanblue-exclusion assay. phagocytosis and oxidative burst were assessed by a combined flow cytometric assay after stimulation of the cells with serum-opsonised fluorescein-isothiocyanate-conjugated e. coli in the presence of dihydroethidium as an indicator of superoxid production. a two-compartement chamber system was used to study the influence of either fluorochinolone on the chemotaxis. results: with either cell type, moxifloxacin accumulated rapidly with peak concentrations within 15 min whereas intracellular maximum concentrations of ciprofloxacin were achieved after 2 h. the accumulation rate of both antibiotics inside the native pmns was more than twice as high as in differentiated eiss ò cells. neither moxifloxacin nor ciprofloxacin showed a significant influence on viability and the immune response parameters phagocytosis and oxidative burst of native pmns; chemotaxis ability was reduced to a minor degree.viability and phagocytosis of the diffentiated eiss ò -cells remain unaffected whereas the oxidative burst appeared decreased. chemotactic acticity of eiss ò -cells could not be stimulated. conclusions: a significant accumulation of moxifloxacin and ciprofloxacin was determined in both cell types. however, no relevant influence on viability, phagocytosis, oxidative burst, and chemotaxis was observed. hence no hints were found suggesting a restricted use of moxifloxacin and ciprofloxacin for immunocompromised patients. prospective, multicentre in vitro study to determine resistance rates and comparative activity of moxifloxacin vs. clinical bacterial isolates from patients with respiratory tract infections (moxiaktiv study) a. dalhoff, g. korfmann, e. jacobs (kiel, leverkusen, dresden, d) objective: to determine the prevalence of resistance in current clinical isolates of s. pneumoniae, h. influenzae, m. catarrhalis, s. aureus and k. pneumoniae as well as the in vitro susceptibility to moxifloxacin and other iv antibiotics in germany. methods: up to 20 isolates of each of the above-mentioned species were cultured on standard media and tested for in vitro susceptibility using etestò in each of the 29 study centres in germany. for validation, an atcc control strain was included for each species. drugs tested were moxifloxacin, levofloxacin, amoxicillin/clavulanate, cefuroxime, clarithromycin and penicillin g. the latter two were not tested for klebsiellae. s. aureus was additionally tested for oxacillin resistance and k. pneumoniae was tested for production of extended spectrum beta-lactamases (esbl). din breakpoints were used where applicable. results: overall, 1859 pathogens of 1,811 hospitalised patients with respiratory tract infections were analysed. results for moxifloxacin are summarized in table 1. of s. pneumoniae isolates 93.3% were susceptible to penicillin g and 85.7% to clarithromycin. with an mic90 of 1 mg/l, the in vitro activity of levofloxacin was markedly lower than that of moxifloxacin. h. influenzae showed almost 100% susceptibility to fluoroquinolones, but only 4.5% to clarithromycin (mic90 32 mg/l). as beta-lactamase production is common among h. influenzae isolates in germany, amoxicillin/clavulanate showed far higher susceptibility rates than penicillin g (87.9% vs. 1.0%). due to a high rate of beta-lactamase production only 9.8% of moraxella isolates were susceptible to penicillin g, compared with 99.1% to amoxicillin/clavulanate. moraxella isolates were fully susceptible to the fluoroquinolones. overall, 13.0% of klebsiella isolates produced an esbl. interestingly, esbl prevalence was 38.8% in eastern germany, but below 10% in western germany. esbl producers of klebsiella were less susceptible to antibiotics other than cefotaxime or ceftazidime than non-esbl producers. conclusion: for all species tested, the fluoroquinolones achieved the highest overall susceptibility rate (92.8%) compared to the other antibiotics (penicillin g: 36.9%, clarithromycin: 60.5%, ampicillin/clavulanate: 85.7%, cefuroxime: 89.6%). moxifloxacin showed a high activity against current respiratory pathogens in germany and was the most active fluoroquinolone, in particular against gram-positive pathogens. objectives: drug combinations have become the best choice in treatment of serious infections with resistant microorganisms including p. aeruginosa (pa). we tested ciprofloxacin (cip) in double and triple combinations with ceftazidime (caz), imipenem (imp), piperacillin (pip), and amikacin (amk) against 20 mdr clinical isolates of pa. methods: the mic for each drug alone was determined by broth microdilution technique described by nccls. double and triple combinations of cip with other drugs were tested by using 96-well plates and by time-kill assay. rows a to g were activity of moxifloxacin on s. pneumoniae (sp), h. influenzae (hi) and m. catarrhalis (mc) clinical isolates collected from the respiratory tract by determination of the mic's using the etest and to compare these results against 9 other antimicrobial agents.these results were also compared for sp versus those of the previous study (winter periods 2000-2002) for interpretive criteria s -i -r (according the nccls -breakpoints). methods: 10 belgian university and non-university medical microbiology laboratories participated during the past winter period in this multicentre in-vitro evaluation of moxifloxacin. each laboratory included 40 strains of sp and 20 strains of hi and mc. the total number of evaluable strains (with the exclusion of duplicate isolates) was 758. testing conditions: method and antibiotics: susceptibility testing was performed in the participating centres by using the e-test. the antibiotics tested were penicillin, ampicillin, amoxi/clav., doxycycline, clarithromycin, cefuroxime, ceftriaxone, ciprofloxacin, levofloxacin and moxifloxacin.medium : sp and mc : mueller-hinton agar with 5% blood; hi : htm agar. inoculum : direct suspension and 0.5 mc farland standard.incubation : 35°c for 24 hours in 5% co 2 . quality control was performed using atcc reference strains (s. pneumoniae atcc 49619 ; h. influenzae atcc 49766). results: see graphs 1 and 2. conclusions: this follow-up belgian multicentre study confirms the excellent in-vitro potency of moxifloxacin against clinical isolates of sp, hi and mc and it demonstrates that it is the most potent fluoroquinolone available in belgium for the treatment of lower respiratory tract infections. according the nccls -breakpoints, there are no major s -i -r differences for sp between these results (2003) (2004) and the results of the previous study (2000) (2001) (2002) . this means that the use of new fluoroquinolones like moxifloxacin and levofloxacin has not led, to date, to an increase of resistance in sp. however, repeated follow-up surveys will continue to be needed in order to assess the activity of fluoroquinolones and to monitor the ecological impact of the recently increased fluoroquinolone usage for respiratory tract infections in belgium in the forthcoming years. objectives: the objective of several experiments was to evaluate the in vitro activity of moxifloxacin against porphyromonas gingivalis (p.g.). methods: mics of moxifloxacin against 16 strains of p.g. were determined by e-test (ab biodisk, solna, sweden). furthermore the spontaneous mutation rate and the induction of resistant strains by the 0.25-fold mic of the antibiotic were determined. to find the target of resistance fragments of gyra and gyrb were sequenced. finally the efficacy of moxifloxacin against p. gingivalis atcc 33277 considering special conditions such as effects on the strain in a biofilm or within epithelial cells (kb cells) was evaluated. results: moxifloxacin had very low mic values (ranging from 0.006-0.032 lg/ml), but subinhibitory concentrations of the fluoroquinolone induced very fast mutations. the spontaneous mutation rate was up to 5 · 10 )8 after the two-fold mic and 1.2 · 10 )8 after the eight-fold mic. often the mutants exhibited a high resistance mics >32 mg/l. all these mutants bore ser-83->phe substitution in gyra. the 5-fold mic eliminated p.g. atcc 33277 within biofilms after 24 h, and the 100-fold mic was able to kill all intracellular p. gingivalis within kb cells. conclusions: moxifloxacin showed a very good activity against planctonic p. gingivalis and p.g. within a biofilm. this antibiotic in a higher concentration was also efficient to intracellular bacteria. but a rapid development of resistance was observed in laboratory. moxifloxacin might be an alternative in the antibiotic treatment of p. gingivalis associated periodontitis, nevertheless clinical studies should focus not only on improvement of clinical parameters but also on occurrence of resistant strains. levofloxacin activity on respiratory isolates of streptococcus pneumoniae with decreased susceptibility to ciprofloxacin in spain j. garcía-de-lomas, j.l. juan-bañ ó n, c. garcía-rey, r. dal-ré on behalf of the spanish surveillance group for respiratory pathogens objectives: to test the activity of levofloxacin against recent respiratory isolates of s. pneumoniae with reduced susceptibility to ciprofloxacin (mic ‡ 2 mg/l; n = 607) belonging to the national surveillance sauce-3 (n = 2721; nov01-oct02) and to identify chromosomal mutations in isolates with a levofloxacin mic ‡ 4 mg/l. methods: the activity of levofloxacin was tested by broth microdilution following nccls recommendations. genotypic sequenciation of the qrdr (gyra, gyrb, parc and pare) was done on all the strains with a levofloxacin mic ‡ 4 mg/l. results: levofloxacin mic distribution, mic50 and mic90 (in bold) are given in the table for the whole sample and broken into ciprofloxacin mic categories. mutations in gyra (e85k) were found in 8 (18.6%) strains. no gyrb mutations were found. mutation in parc occurred in 100% these strains (26 s79f; 12 s79y; 2 s79i; 1 s79r; 1 d831; 1 d83n). finally, mutation of pare was found in 17 (39.5%) strains and all of them had the change i460v. one single parc mutation occurred in 24 (55.8%) strains, (1999) (2000) (2001) (2002) ; >99.9% were at £0.12 mg/l conclusions: gemi continues to be a highly potent fq when tested against the two most commonly isolated gram-negative carti pathogens; h. influenzae and m. catarrhalis. this potency and spectrum was consistent across six years and three continents. norfloxacin as a marker of decreased susceptibility to fluoroquinolones in enterobacteria using the vitek 2 system l. ló pez, j. alcala, f. torres, e.j. perea, a. pascual (seville, e) objective: the detection of nalidixic acid resistance acquires importance in enterobacteria since already resistant strains develop more frequently resistance to fluorquinolones than susceptible strains. many clinical laboratories have incorporated the vitek 2 system because their benefit in routine, but nalidixic testing is not included for enterobacteria cards. the aim of this study was the use of norfloxacin vitek mics value as alternative marker to detect nalidixic acid resistant enterobacteria strains. material and methods: a total of 1013 isolates of enterobacteriaceae were analyzed and routinely inoculated into the vitek 2 id-gnb and ast-n020 cards (biomérieux) and then loaded into the vitek 2 system, as recommended by the manufacturer. ninety-six strains with a higher norfloxacin vitek mic value (1-4 mg/l), but still within the susceptibility range established by the nccls breakpoints, and 100 strains with a norfloxacin vitek mic £0.5 mg/l were selected. they were further tested with a 30 microg-nalidixic acid-disk (nal) (oxoid) according to nccls reference. results: the norfloxacin 1-4 mg/l and susceptible to ciprofloxacin phenotype represented the 9% of total isolates loaded in the vitek system during the study, similar to the prevalence observed in previous analysis in spain. both selected groups, one with the highest and the other with the lowest norfloxacin vitek mic value, included 60% and 58% e. coli isolates, 8% and 20% s. enterica isolates, respectively. among the norfloxacin vitek mic 1-4 mg/l strains 93 isolates (98%) were nal resistant, and 76 (79%) were ciprofloxacin susceptible. the agreement between norfloxacin vitek mic £0.5 mg/l and nal susceptibility was foun in 97 (97%) strains. two isolates (one e. cloacae and one k. pneumoniae) with norfloxacin vitek mic 1-4 mg/l were nal-s and 3 isolates (two e. coli and one p. mirabilis) with norfloxacin vitek mic £0.5 mg/l were nal-r. conclusion: when using vitek 2 system for enterobacteria, the consideration of mic of norfloxacin >0.5 mg/l could be an alternative marker of nalidixic acid resistance (and decreased susceptibility to fluoroquinolones). antibacterial susceptibility studies -i objectives: p. aeruginosa is an important nosocomial pathogen, which causes serious and often lethal infections in immunocompromised hosts. this pathogen is intrinsically resistant to many antibiotics and can easily develop resistance towards many currently available agents. natural resistance can be attributed to the low permeability of the p.aeruginosa outer membrane to a variety of antibiotics including glycopeptides (glys). since glys are powerful antibiotics against grampositive bacteria and resistance is very rarely develop, it seemed interesting to evaluate the effect of combining these antimicrobial agents with antibiotics that might disorganize the structure of the outer membrane allowing the entrance of glicopeptides into the gram-negative cells. in order to verify this hypothesis, ceftazidime (caz) has been tested in association with vancomycin (van) or teicoplanin (tei). the same experiments have been carried out also in the presence of azithromycin (azi), which is normally a non anti-pseudomonal agents but has been shown to interfere with some virulence factors. methods: a bacterial suspension of about 10 9 cfu/ml was seeded on plates containing a fixed concentration of glys (500 mg/l) and increasing doses (2x,4x,8x,16x) of caz. survivors were counted after 48 hr at 37°c. results were interpreted as synergism (99%), additivity (90%), and indifference (10%) of the cfu/ml reduction found in the drugs combination in comparison to the drug alone.the same experiments have been repeated adding azi (16 mg/l) and using glys at concentrations ranging from 500 to 300 mg/l. results: caz in combination with glys reacted synergically in 20 out of 59 cases, additivity was found in 31/59 interactions and indifference was noted in 8/59 tests. preliminary results (12 tests performed) indicated that the addition of azi increased the incidence of synergisms and additivities even when using glys concentration of 300 mg/l. conclusions: caz combined with glys gave additive or synergistic results in the great majority of experiments, while the simultaneous combination of azi, caz and a gly always produce an additive or synergistic effect against p. aeruginosa. these data, given the high concentration of glys employed, could be of particular interest in clinical situations where the drugs could be topically administered. a multicentre correlation study of vitek 2 with reference methods for telithromycin, mupirocin, and daptomycin against staphylococcus spp. k.e. aldridge, a. dizney, k. thomson, g. procop (new orleans, omaha, cleveland, usa) objective: clinical studies have shown that appropriate antimicrobial therapy based on in vitro susceptibility data correlates with better patient outcome. vitek 2 is a fully automated susceptibility testing methodology providing rapid(6-8 h) results for a variety of aerobic and facultatively anaerobic gram-positive and -negative pathogens which have been shown to correlate to established reference methods. the objective of this study was to correlate the susceptibility results of vitek 2 testing of telithromycin, mupirocin, and daptomycin against staphylococcus spp. with those from reference methodology. methods: three geographically distinct clinical microbiology laboratories participated in the testing with each site testing 100 consecutive clinical islates and 84 blinded challenge isolates of staphylococci sent to each site.vitek 2 testing was performed according to the manufacturer's recommendations. the challenge isolates were also tested using a manual preparation of the inoculum. nccls recommended reference agar and broth microdilution methods were used to test telithromycin, mupirocin, and daptomycin. mic results were collated to determine the per cent (%) of isolates susceptible ( in vitro efficacy of tigecycline tested against community-acquired respiratory tract infection and nosocomial pneumonia pathogens conclusions: among r subsets of commonly occurring pathogens, 97% were inhibited by £2 mg/l of tig and >99% were inhibited by £4 mg/l (the current nccls breakpoint for tet). tig is highly stable to most tet-r determinants, including protected ribosomes and efflux mechanisms, and may represent a superior choice among parenteral agents for broad-spectrum coverage, including the most commonly occurring-and problematic-r phenotypes. determination of the minimum inhibitory and mutant prevention concentration of telithromycin against clinical isolates of streptococcus pneumoniae s. borsos, c. hesje, l. blondeau, j. blondeau saskatoon, can objective: telithromycin (tl) is a novel ketolide antimicrobial agent that has recently been approved for use in north america for respiratory tract infections of which sp is a principal pathogen. the global escalation of penicillin (p) and macrolide (m) resistant sp has compromised the use of beta-lactam and macrolide compounds. tl was reported to be active against p and m resistant sp. objectives: garenoxacin (grn) is an investigational des-f(6)quinolone with enhanced in vitro activity against gram-positive cocci. the global escalation in antimicrobial resistance amongst respiratory tract pathogens -particularly sp-has highlighted the importance of quinolone compounds for treating sp infections. the mutant prevention concentration (mpc) is a novel in vitro measurement that defines the drug concentration threshold that would require an organism to simultaneously acquire two or more resistance mutations for growth in the presence of the drug. we measured the mics and mpcs for grn against clinical isolates of sp, including multidrug resistant strains and those with elevated mpcs to levofloxacingrn. methods: microbroth dilution in accordance with nccls guidelines was followed for mic testing using todd-hewitt broth and two-fold concentration drug increments. for mpc testing, ‡1 billion organisms were applied to agar plates containing grn, incubated at 35-37°c and 5% co 2 and screened for colony growth at 24 and 48 hours. the mpc was the lowest drug concentration that completely prevented growth. select organisms with grn mpc values >2 lg/ml were screened for amino acid substitution in the quinolone resistance determining region. great concern nowadays because the therapeutical alternatives are limited. fusidic acid inhibits protein synthesis. it has only a few side effects and is usually well-tolerated by patients as an oral drug. fusidic acid can be considered as an alternative drug for the treatment of infections due to both methicillin susceptible and resistant s. aureus strains. evaluation of synergy between glycopeptides, levofloxacin and beta-lactams against methicillinresistant staphylococcus aureus (imi) + va/tp, lvx + piperacillin/tazobactam (ptz) + va/tp] were considered. synergy was evaluated by means of checkerboard assay against 50 (double combinations) and 25 (triple combination) mrsa strains isolated from respiratory tract infections and by means of time kill curves (5 strains). in checkerboard assay synergy was defined as fractional inhibitory concentration index (fici) of <0.5, additivity as fici >0.5 and >1, indifference as fici >1 and antagonism as fici >2, while in time kill studies synergy was defined as a >2 log decrease in bacterial count of combinations in respect to the most active single drug. moreover, mutational rates of single and combined drugs at antimicrobial concentrations equal to the resistance breakpoints were calculated in 10 strains results: synergy and additivity were the prevalent effects, while no antagonism was observed in checkerboard assay. in particular, lvx + va/tp and ctx + tp/va gave synergy in 16/50, 9/50, 43/50 and 23/50 strains, respectively. combinations of lvx + va + caz/cpm/ptz and of lvx + tp + caz/cpm/ptz yielded synergy or additivity in all the tested strains, with combinations of lvx + tp + caz / ptz giving synergy in 20/25 strains. time kill curves evidenced synergy for lvx/ctx + va/tp after 12 and 24 h of incubation. synergistic triple combinations occurred more frequently with lvx + tp than with lvx + va after 12 and 24 h. for single antibiotics, mutational frequencies ranged between 10-5 and <10-9 for lvx, ctx, amk and imi, and <10-9 for va and tp. when tested in double and triple combinations, mutational frequencies fell below 10-9 for all the combinations. conclusion: the study provide in vitro evidence of synergy between glycopeptides with fluoroquinolones, cephalosporins and beta-lactams and of limitations of occurrence of mutations in mrsa strains responsible of respiratory infections, thus underlining a potential role in therapy of such infections. comparative activity (mic and mbc) of daptomycin, quinupristin/dalfopristin, linezolid, vancomycin and teicoplanin against a large worldwide collection of visa/hvisa a. bolmströ m, a. engelhardt, å . karlsson, e. edling, p. ho (solna, s) objective: the worldwide emergence and possible dissemination of vancomycin (va) resistance in staphylococci involving vana (vrs), visa and hetero-visa phenotypes comprise formidable clinical threats. comparisons of the in vitro activity of newer gram positive agents using larger worldwide collections of visa strains become increasingly important both for individual therapy guidance and for epidemiologic purposes. method: a 10 country collection of 243 staphylococci (217 mrsa, 26 mssa), of which 150 were visa (e-test macromethod and pap-auc, walsh et al, jcm 2001) was assessed. mic of dp, qd, lz, va and tp were determined using the nccls broth microdilution (bmd) and e-test. mbc for all agents for a subset of 46 visa and non-visa strains were determined using an etest mbc procedure and bmd. results: inter-method agreement (n = 2430) for all antibiotics were ±95% ±1 dilution for mic and ±80% for mbc results. the continuous gradient format in e-test allowed detection of subtle upward shifts in mic and mbc distributions of visa, as well as the detection of persister colonies in mbc assays seen with the less bactericidal compounds. conclusion: mic distributions showed that daptomycin and linezolid maintained potency against the large collection of visa while quinupristin/dalfopristin and glycopeptides showed a shift towards reduced activity. mbc results showed daptomycin to be superior to linezolid (bacteriostatic) and the other agents. the potential usefulness of newer agents for visa should be continually assessed with respect to both their static and cidal activities in order to detect subtle shifts in susceptibility as early as possible. performance of a unique e-test daptomycin gradient + calcium gradient on isosensitest compared to nccls broth microdilution in mueller hinton objective: daptomycin (dp) requires physiologic levels of free calcium ions (ca 2+ ) for expression of adequate activity. susceptibility testing methods can be significantly influenced by ca 2+ variations in agar and broth. nccls broth microdilution (bmd) uses a final physiologic level of 50 lg/ml calcium for dp testing. e-test dp is a unique gradient which incorporates a constant level of calcium. e-test dp has been shown to be equivalent to bmd when tested with commercially available mueller hinton (mh) agar. since isosensitest (iso) is used in some parts of europe, and has an inherently low level of calcium (approximately 5-10 lg/ml), it is important to evaluate the performance of e-test dp on iso in comparison to bmd. method: a 3 lab study compared e-test dp on iso to nccls bmd. a total of 760 clinical and stock strains comprising clindamycin (cc), ciprofloxacin (c) and tetracycline (t) and also to determine whether there are any differences in the antimicrobial susceptibility patters among the difference species of the vgs and s. bovis. methods: the 145 vgs and 10 s. bovis were isolated from blood and identified at species level by id 32-strep system (68 s. mitis, 44 s. anginosus, 25 s. sanguis, 7 s. salivarius and 1 s. mutans). mics were determined by the agar dilution method and the percentages of resistance determined according to nccls criteria for streptococci other than s. pneumoniae. results: overall, 64.5% of the streptococci were resistant at least to one of the antibiotic tested and of these, 32% was resistant to 3 or more. the most susceptible species were s. anginosus and s. salivarius (57% susceptibility to all antibiotics tested). the only s. mutans strain was susceptible to all the antibiotics tested. the resistance phenotypes for the different species are shown in the table. conclusions: as expected cc resistance was always associated with e resistance. overall, the phenotype most frequently founded was ecct in 24 strains (15.5%) and it was present in the 70% of s. bovis and 27% of s. anginosus. the pattern eccpt was the second more common phenotype (12.3%) and the most frequent in s. sanguis and s. mitis with 20% and 17.6% respectively. s. mitis was the specie that showed more number of phenotypes (17). heterogeneity in the susceptibility patterns in the species of vgs indicates the need for accurate identification. in vitro antibacterial activity of temocillin against extended spectrum beta-lactamases enterobacteriaceae producing clinical isolates objectives: temocillin is a methoxy-derivative of ticarcillin, active on enterobacteriaceae and stable against b-lactamases, including ampc and extended spectrum b-lactamases (esbl). however data concerning its activity on esbl producing strains are limited. the aim of this study was describe the range of minimal inhibitory concentration (mic) of temocillin in different species of esbl producing enterobacteriaceae from a single tertiary care centre in belgium . methods: esbl producing enterobacteriaceae (172 e. aerogenes, 164 e. coli, 104 e. cloacae, 58 k. pneumoniae and 35 other ) strains from patients hospitalised in our institution during the period of 1 january 2000 to 31 december 2003. esbl production was screened by double-disk test and confirmed by oxoid combined disk method. characterisation of enzymes was performed by multiplex pcr for bla tem, bla shv and bla ctx-m gene families detection and by dna sequencing and/or iso-electric focusing. mics (mg/l) were determined by agar dilution method according to nccls guidelines. susceptibility was determined according to breakpoints provided by fuchs et al: susceptible mic £ 16 mg/l. (eur j clin microbiol 1985) results: isolates originated from screening rectal swabs (28%), urinary tract (25%), respiratory tract (19%), wound (10%), blood (6%), gastrointestinal tract (4%), or other sites (8%) from 135 male and 233 female patients with a mean age of 61 (range, 0-94) years. the antimicrobial activity of temocillin against esbl producing enterobacteriaceae is summarised in the table. conclusions: this study confirmed the good in vitro activity of temocillin against multiresistant esbl-producing clinical isolates of enterobacteriaceae. its activity included enterobacter ampc depressed mutant strains that co-produced multiple esbl. these data suggest that temocillin could be a valuable drug to be considered in the treatment of infections by some esbl-producing enterobacteriaceae. in vitro activity of ertapenem against cefpodoxime-resistant gram-negative bacilli from urine introduction: resistance to commonly used antibiotics for urinary tract infection (uti) is of growing concern. this is due to the emergence of extended-spectrum beta-lactamase (esbl) producing bacteria in the community with reports from different parts of the world. the appearance and dissemination of these resistant bacteria cause serious concerns for the treatment of urinary tract infections (utis). objectives: ertapenem is the latest member of the carbapenem class of antimicrobials. the aim of this study was to assess the activity of ertapenem against a collection of cefpodoximeresistant gram-negative bacilli isolated from urine samples of community and hospital-based patients. materials and methods: between june and october 2004, our laboratory received 10,234 urine samples. of these 2246 gave a positive culture, of which 446 from hospitalised patients and 1800 from the community. of these 258 were gram negative bacilli resistant to cefpodoxime 1 mg/l. the identity of the isolates was confirmed using api 20e or api 20ne (biomerieux, france). esbl production was sought using the mastdd and the identity of the resistant determinant(s) was carried out using a combination of pcr and nucleotide sequence analysis techdifferent antibiotics in order to evaluate alternatives for intrapartum chemoprophylaxis to women allergic to penicillin and colonized by a gbs strain resistant to macrolides and/or lincosamides; (2) characterize the mechanisms of resistance to macrolides and lincosamide; (3) evaluate the clonal relationship between these strains. methods: a total of 610 strains collected in a multicentre study: 131 isolated between 1997 and 2002 from newborns diagnosed of early-onset gbs disease and 479 strains collected in 2002 from vagina or rectum of pregnant women. microdilution method was used to study the antimicrobial susceptibility and disk diffusion to define the macrolide-lincosamide resistance phenotype. pcr was performed to determine the presence of ermb, ermtr and mefa resistance genes. to evaluate the clonal relationship, pfge of total dna was done using smai. results: all the strains were susceptible to penicillin, ampicillin, vancomycin, quinupristin/dalfopristin, levofloxacin and teicoplanin. the 12.45% were resistant to erythromycin and azithromycin, 11.80% to josamycin, 11.31% to clindamycin, 1.80% to telithromycin and 0.32% to fosfomycin. among the macrolide resistant strains, the 90.8% presented a constitutive mlsb phenotype (cmlsb), 5.26% an inducible mlsb phenotype (imlsb) and the remaining 3.94% a m phenotype. three strains were susceptible to macrolides and resistant to clindamycin. the ermb, ermtr and mefa genes were presented in 63.2%, 30.3% and 3.9% of the macrolide resistant strains. two strains did not amplified none of the studied genes. the 100% of the strains harbouring the ermb gene showed a constitutive phenotype. all the strains showing an inducible phenotype harboured the ermtr gene. the 11 telithromycin resistant strains presented a cmlsb phenotype; of them 10 posses the ermb gene and one the ermtr. conclusion: in spain, there is a high rate of resistance to macrolides and lincosamides that makes mandatory to perform susceptibility testing to gbs strains isolated from pregnant women allergic to penicillin. in our region ermb methilase is the main cause of macrolide resistance, followed by the ermtr and the macrolide eflux pump in a low proportion. there is a wide clonal diversity among resistant strains to macrolides, lincosamides and telithromycin. detection of the ermx determinant of macrolide resistance in clinical strains of corynebacterium urealyticum a. ortiz, j.i. garcía-cía, r. fernández-roblas, j. esteban (madrid, e) objectives: to detect the presence of the ermx macrolide resistance gene in clinical isolates of c. urealyticum. methods: 57 c. urealyticum strains were isolated from clinical samples in the fundació n jiménez díaz hospital. the strains were maintained frozen until the studies to evaluate macrolide resistance were performed. antibiotic susceptibility testing was performed by agar dilution assay in 14 cases and by disc diffusion assay in 43 cases. detection of ermx gene was performed by using primers cerm-1 and cerm-2 according to the protocol described by rosato et al. dna was extracted by boiling a suspension of bacteria in distilled water. amplification products were analysed by agarose gel electrophoresis and molecular weight of the amplicons were calculated by using the photocapt software (biogene, usa). results: 46 strains were resistant to erythromycin and clindamycin, and 11 were susceptible to both antimicrobials. we detected the gen ermx in 34 cases (all of them resistant). 12 macrolide-resistant strains gave negative results for ermx detection. the susceptible strains did not show any amplification product. the ermx gene is detected in 73.9 % of macrolideresistant corynebacterium urealyticum strains. however, there were 26.1 % macrolide-resistant strains in which ermx gene was not detected. other resistance determinants must be involved in macrolide resistance among corynebacterium urealyticum. characterisation of phenotype and genotype of macrolide-resistant streptococcus pyogenes objectives: the resistance of streptococcus pyogenes to macrolide is a world wide problem. the aim of present study was to determine the prevalence of antibiotic resistance rate of erythromycin (em) resistant isolates from korea, and evaluate their genetic mechanism, phenotype distribution and clonal relationship. methods: total 51 em resistant s. pyogenes from clinical specimens from 1996 to 2003 were studied; agar dilution method to determine minimal inhibitory concentrations (mics) to 9 antimicrobial agents was performed. resistance phenotypes were determined by triple-disc test and resistance induction experiment with sub-inhibitory concentration of erythromycin (0.05 lg/ml). their genetic mechanisms of resistance were determined by pcr. the genetic relatedness of them was also investigated by means of emm genotyping and random amplified polymorphic dna (rapd) analysis. results: an average em resistance rate was 23% and their cross-resistance rate to clarithromycin, azithromycin, and spiramycin were 100%, 98%, and 67%, respectively. the most common resistance phenotype was inducible mls (imls; 51%), followed by constitutive mls (cmls;31%), and m type (18%). 18 of 26 imls isolates (69% ) showed novel imlsd phenotype, which had small (9-12 mm) inhibition zones around all three discs on triple-disc test and high level resistance to clinamycin and spiramycin after erythromycin induction. all isolates of the cmlsa and imlsd harboured ermb gene, while imlsc and m isolates harboured erma and mefa gene, respectively. all imlsd isolates were emm12 except one while all multidrug resistance cmlsa isolates were emm28 genotype. imlsd isolates made a tight cluster on phylogenetic tree and 78% of them showed a common pattern by rapd analysis. conclusion: imlsd was most common macrolide resistance phenotype in korea and emm gene and rapd analysis was suggestive of its clonal relationship. detection of inducible clindamycin resistance in cutaneous staphylococci clinical isolates by phenotypic and genotypic method , and 17 (14.5%) isolates with constitutive clindamycin resistance (7 s. aureus and 10 coagulase-negative staphylococci). the mrsa gene was present in all the clindamycin susceptible isolates except for 1 cns. among the 40 isolates with clindamycin inducible resistance, 37 isolates possess either erma or ermc while 1 cns isolate possess both ermc and mrsa. one cns and 1 s. aureus isolates were negative for all the genes tested. all 17 isolates with constitutive clindamycin resistance amplified to any of the genes tested. among the s. aureus isolates, 2 have the erma gene, 4 the ermc and 1 both ermc and mrsa genes. all the cns isolates possess the ermc but 3 of them possess also the mrsa gene (table 1) . the disk-diffusion test detected all the clindamycin resistant strains, and none of the clindamycin inducible resistant strains was detected by the microscan. the ermc gen was the most prevalent among the clindamycin resistant, in both cns and s. aureus isolates. ribosomal protein l22 mutations in bacillus anthracis associated with cross-resistance between macrolide, lincosamide, streptogramin and ketolide antibiotics rob chemother 2004; 54: 424) . the aim of this study was to determine ribosomal mutations associated with this resistance. methods: primers were designed and used to amplify and sequence the l4, l22 riboprotein genes and the 11 copies of the 23s rrna gene using b. anthracis str. ames complete genome (genbank accession no. nc_003997). results: (mics in mg/l) two significant mutations associated with resistance were found. a 4 amino acid (aa) insertion (a tandem duplication of 90mgra93) into the l22 gene at position 94 was found in the st-1 strain with a quinupristin/dalfopristin (q/d) mic of 128 selected after 18 passages in q/d. this strain was cross-resistant to telithromycin, erythromycin, clarithromycin and clindamycin with mics of 16, 64, 16 and 8 respectively. an 8 amino acid insertion (mgramgra) into the l22 gene (also at position 94) was found in the sterne strain with a clarithromycin mic of 64 after 18 passages in clarithromycin. this strain was cross-resistant to telithromycin, q/d, erythromycin and clindamycin with mics of 16, 8, 32 and 1 respectively. conclusions: tandem duplications of l22 90mgra93 were found to be important in mlsk resistance in 2 separate strains of b. anthracis demonstrating that this locus is important for resistance development under selective pressure of q/d and clarithromycin. importantly, these mutations were also associated with cross-resistance to other mlsk antibiotics. horizontal transfer of the mlsb resistance gene erm(b) between the human pathogen clostridium difficile and the ruminal anaerobe butyrivibrio fibrisolvens p. mastrantonio, f. barbanti, p. spigaglia (rome, i) objectives: the aim of this study was to investigate the possibility of in vitro transfer of the mlsb resistance gene, erm(b) between the human pathogen clostridium difficile and the ruminal commensal butyrivibrio fibrisolvens. methods: the erm(b)-positive c. difficile strain cd51 was used in filter matings as donor, whereasthe b. fibrisolvens strain 1.230, tetracycline resistant, and the b. fibrisolvens strain 2221r, rifampicin resistant, were used as recipients. the strains were cultured in m2gsc broth, with 30% of sterile rumen, in anaerobic conditions and the filter matings were performed on sheep blood agar plates, supplemented with hemin (0.1%) and vitamin k (0.1%). the same medium, supplemented with erythromycin (20 mcg/ml) and tetracycline (10 mcg/ml), were used to select the transconjugants. mic values were assessed by e-tests. the erm(b) transfer was confirmed by pcr and by hybridisation assays, using an erm(b) probe on transconjugant genomes digested with pvuii and with smai (pfge). results: the transfer average frequency was 5 · 10 )7 . mic values for erythromycin were >256 mg/l both in donor and transconjugants. an internal fragment of the erm(b) gene (730 bp) was amplified in all the transconjugants and a specific band was visualised in hybridisation assays on transconjugant genomes digested with both pvuii and smai. the re-transfer of the erm(b) gene from b. fibrisolvens transconjugants to c. difficile 1977, an erythromycin susceptible strain, was also obtained. conclusion: the in vitro transfer of the mlsb resistance gene erm(b) from c. difficile to b. fibrisolvens and vice-versa, is an important result supporting the possibility that horizontal transfer of resistant genes between human pathogenic bacteria and animal commensal microorganisms could occur, under natural conditions, more easily than expected. candida spp. attach on polymers, create a biofilm protecting the yeast and pose a reservoir for entering the bloodstream. candida spp. in biofilm are less susceptible to the antifungal drugs currently in use. we investigated the in vitro antifungal activity of micafungin (mcf) against biofilms of c. albicans, c. parapsilosis, c. dubliniensis, c. tropicalis, c. glabrata and c. kefyr. methods: biofilm formation was performed in vitro on polystyrene 96-well plates and on hickman catheter discs with minor modifications to kuhn (1). mcf was tested at six concentrations (0.25-32 lg/ml). measurements were done by xtt reduction assay at 490nm, % inhibition was calculated in comparison to the growth control. the biphasic structure of biofilms were imaged by reverse light-through microscopy. results: on polystyrene plates and on hickman catheters all candida spp. produced intermediate biofilm after 24-48h incubation. at all concentrations on polystyrene plates micafungin reached at maximum 50% inhibition in biofilm against all candida spp.: c. albicans (47% at 16 lg/ml), c. parapsilosis (49% at 16 lg/ml), c. dubliniensis (42% at 16 lg/ml), c. tropicalis (28% at 16 lg/ml), except for c. glabrata (73% at 8 lg/ml) and c. kefyr (53% at 1 lg/ml) methods: this multicentre, randomized, double-blind study compared mem with ipm (both 500 mg iv every 8 hours) in patients hospitalized with csssi. the primary efficacy endpoints were clinical and bacteriological responses at follow-up in the fully evaluable (fe) patient population (all patients meeting eligibility criteria and who had an identified pathogen prior to treatment) polymicrobial infections were seen in 38% of all documented infection s. agalactiae (n = 59; 100% s to mem and ipm), and e. faecalis (n = 52; 82% s to mem and 100% s to ipm) pseudomonas aeruginosa (n = 53; 94% s to mem and 92% s to ipm), bacteroides spp. (n = 74; 97% s to mem and 100% s to ipm), and peptostreptococcus spp 3% (mem) and 81.2% (ipm) in patients with polymicrobial infections; bacteriological (documented or presumed eradication, colonization) response rates were 79 we used a cox proportional hazard (cph) model adjusting for potential clinical and microbiologic risk factors, to evaluate los. results: among the 1116 modified intent-to-treat patients, the most common infection diagnoses at baseline were cellulitis (58.9%) and major abscess (27.9%). the most common underlying etiologies were spontaneous infection (52.3%) and trauma (27.6%). about 20.2% of patients had diabetes; 6.9% had peripheral vascular disease. staphylococcus aureus was the most common causative pathogen (about 50% of patients) among 540 patients with confirmed microbiology and complete hospitalization data. however, about 18% of patients had a gramnegative causative pathogen among 540 patients with confirmed microbiology, staphylococcus aureus was the most common causative pathogen but varied significantly in prevalence across regions (about 50% of patients overall, 82% in the us, 67 to 68% in europe and asia, and 39% in south africa). about assigned for the drugs as follows: row a for cip alone, b for second drug, c for third drug, d for cip + second drug, e for cip + third drug, f for second drug + third drug and g for the three drugs together. row h was left for drug-free control. briefly, all wells were filled with 50 ll of cation adjusted muller hinton broth. the drugs at 2x of the highest tested concentrations in 50 ll of the medium (alone, in double or in triple combinations) were delivered to wells a1-g1. two-fold serial drugs. results: the best synergistic effects were observed in combination of amk with caz (80%) or pip (80%) and in combination of caz with pip (60%) or cip (60%) as quinolones are widely used for treating sp infections and quinolone resistance is a concern, we tested gm-a newly approved compound-by mic and mpc against ps and prsp. methods: mic testing was by microbroth dilution in accordance with nccls guidelines. for mpc testing, 10 billion organisms were applied to agar plates containing drug and read at 24 and 48 hours following incubation. the lowest concentration preventing any growth was the mpc. organisms with high mpc values ( ‡2 lg/ml) were screened for amino acid substitution in the quinolone resistance determining region. results: a total of 402 clinical sp isolates were tested: 320 ps, 69 penicillin intermediate (pi) and 13 pr. the following represents the mic50/90 (lg/ml) and range (lg/ml) respectively against pr tr) objective: to determine the in-vitro activities of moxifloxacin and ciprofloxacin against methicillin-susceptible s. aureus (mssa) and methicillin-resistant s. aureus (mrsa) strains. methods: the study was conducted in a university hospital in turkey. a total of 440 non-repeat isolates (196 mssa and 244 mrsa) from various clinical specimens were included in the study. all isolates were identified as s. aureus by phenotypic characteristics, gram staining, catalase and tube coagulase tests. mics of the drugs were determined by an agar dilution method, according to the nccls criteria. the drugs were obtained from their manufacturer (bayer inc). s. aureus atcc 29213 was used as quality control strain. results: twenty-seven (14%) of 196 mssa strains and 239 (98%) of 244 mrsa strains were resistant to ciprofloxacin. mic50 and mic90 values of 27 ciprofloxacin-resistant mssa strains for moxifloxacin were 0.25 mg/l and 4 mg/l, respectively. mic50 and mic90 values of ciprofloxacin-susceptible mssa strains for moxifloxacin were £0.125 mg/l and 0.250 mg/l, respectively. moxifloxacin was 4-fold more active than ciprofloxacin against mssa strains. there was not any difference between these two quinolones against mrsa strains (mic90s: ‡16 mg/l). conclusion: mrsa strains are major causes of nosocomial infections. despite advances in antibacterial therapy, treatment of infections caused by mrsa is still troublesome. new quinolone-derived compounds such as moxifloxacin are reported to have improved activities against s. aureus including mrsa. in this first study on in-vitro activity of moxifloxacin against s. aureus from turkey, the moxifloxacin mics were higher than those reported from different countries objectives: a multicentric study during the winter period of 2003-2004 was performed in belgium to assess the in-vitro two mutations in 15 (34.8%) (3 parc+gyra and 13 parc+pare), and 3 mutations in parc+pare+gyra in 5 (11.6%) strains. conclusions: 1) levofloxacin mic50 and mic90 for pneumococcal strains with decreased susceptibility to ciprofloxacin were 0.5 and 1 mg/l, respectively. resistance to levofloxacin (mic ‡ 8) occurred in 32 isolates (1.2% of the sauce-3 collection). 2) activity of levofloxacin was negatively affected only among pneumococcal isolates with a very high mic of ciprofloxacin ( ‡8 mg/l). in this case, levofloxacin mic50 and mic90 were 4 and 8, respectively, and 44.4% of these strains were resistant to levofloxacin. 3) all sequenced strains with a levofloxacin mic ‡ 4 mg/l had a mutation in the parc gene (single in 56% strains), but none of them in the gyrb. the most common mutations identified were parc/s79f, pare/i460v, parc/s79y and gyra/e85k. 4) one single parc mutation was organism/year (no. tested) gemi cipro levo gati moxi 008/2 (na) methods: the organisms numbered 9,347, a total of 2,004 from np and 7,343 from carti. the most frequent carti pathogens were: s. pneumoniae (spn; 2,865), h. influenzae (hi; 4,011) and m. catarrhalis (mcat; 607) usa) objective: to evaluate the activity and potency of tigecycline (tig) when tested against a large international collection of common bacterial pathogens displaying increasing and worrisome resistance (r) profiles. tig is a novel glycylcycline derivative of minocycline that has demonstrated activity against a variety of gram-positive and -negative pathogens mic 50/90 (mg/l) 275 strains) were collected from 2000 to 2004 in >70 medical centres participating in the global tig surveillance program results: tig results for the r organism subsets are in the table: tig was highly active (mic50 and 90, £0.25 and £0.5 mg/l, respectively) against all resistant sa, cons, esp, spn and vgs with >99% of strains inhibited by £2 mg/l. while potency of tig against r subsets of ent was less (mic50 and 90, £0.5 and £4 mg/l, respectively), the vast majority of tet-r isolates remained s to tig (>98% of isolates were inhibited by £4 mg/l). no geographic differences in tig potency among esbl-producing, cip-r or tet-r ent strains were noted. grn drug concentrations in serum would be expected to remain above the mpc90 value for >24 hours. conclusion: grn exhibits potent activity against sp, as measured by both mic and mpc, and is active against strains with high mic/mpc values for other quinolones. grn is less likely to select for quinolone resistant sp isolates s. agalactiae (119) and s. dysagalactiae (30) were used. interlaboratory reproducibility was evaluated using 25 bias/precision strains tested at all sites and 3 nccls qc strains were used to compare e-test dp on iso values to nccls mh values. the various methods were used according to standard recommendations. nccls susceptible breakpoints ‡1 lg/ml for staphylococci and streptococci and ‡4 lg/ml for enterococci were used as is and as adjusted upwards by 1 dilution. results: percentage essential agreement (ea), category agreement methods: e test strips were used to establish the fici of the antibiotic combination pairs. combination testing was carried out on 125 strains of pseudomonas aeruginosa (pa), 69 strains of burkholderia cepacia (bc) and 29 strains of stenotrophomonas maltophilia (sm) from 96 cf patients. 19, 13 and 6 combination pairs were tested between 10 and 69 times against pa, bc and sm respectively. the fici results were categorised as synergistic (fici £ 0.5), additive (fici = 0.5 to £1.0), indifferent (fici = 1.0 to £2.0) and antagonistic (fici > 2.0). results: 1107 combinations were tested. synergy was found in 3.1% of pa, 9.7% of bc and 8.6% of sm. 31.6%, 32.6% and 25.8% of pa, bc and sm respectively demonstrated an additive effect. 64.2% of pa, 54.5% of bc and 62.4% of sm demonstrated an indifferent effect. antagonism was found in 1.1%, 3.2% and 3.2% of pa, bc and sm respectively results: of the 258 isolates resistant to cefpodoxime, 83 were positive for esbl production and the enzymes produced were: tem-1, shv-5, shv-12, ctx-m-15, ctx-m3 and ctx-m9 singly or in combination. ertapenem proved active against all enterobacteriaceae regardless of production of esbls (mic range 0.003-0.125 mg/l). for acinetobacter sp giamarellou on behalf of the hellenic study group for the susceptibility of streptococcus pneumoniae results: of the 275 strains studied, 76 were ery s and 159 were ery r. all strains but two (mic 4 and 8 mg/l) were susceptible to tel. in particular, results for telithromycin for the ery s strains, showed an mic range for tel between 0.008-0.5, with mic50 of 0.015 and mic90 of 0.06. for ery r in total, mic50 and mic90 were 0.125 and 0.5 (range 0.008-8). with respect to macrolide resistance phenotype, the distribution per type was: m 54.5% (ery mic range 1-32), and cmlsb and imlsb (ery mic range 4 to (256) 39% and 6.5% respectively. the two strains resistant to tel displayed the cmlsb phenotype (ery mic ( 256). for each different macrolide resistance phenotype no isolates demonstrated an inducible mlsb phenotype by this method. forty-nine of these carried the ermam gene (34.3%) and three carried both ermam and mefe genes (2.18%). eighty-seven isolates (63.5%) had the m phenotype (macrolide resistance) and all of these contained the mefe gene. conclusions: our findings are in contrast to european studies and to previous greek studies, where the erm gene prevailed or at least equaled the prevalence of mef gene. this is the first report of isolates carrying both ermam and mef genes in greece. knowledge of the resistant mechanisms to macrolides is crucial for the empiric antibiotic treatment of community acquired infections. p1225 antibiotic susceptibility, mechanisms of macrolide resistance and clonal relationship in group b streptococci microbiological characteristics of anthrax strains l. lukhnova, g. temiraliyeva, t. meka-mechenko, y. pazylov, s. zakaryan, j. denissov (almaty, kaz)objectives: the kazakh science center for quarantine and zoonotic diseases (kscqzd) has a collection of pathogens of especially dangerous infections that have been isolated in kazakhstan. by kazakh government resolution, the kscqzd's collection is the official collection of pathogenic bacteria for kazakhstan. kscqzd has 71 strains of b. anthracis that were isolated in various years . methods: we studied the biological properties of 30 anthrax strains from our collection: 16 from humans, 4 from soil, 5 from sheepskins, 2 from sheep organs, and 3 from the external environment. we tested the anthrax strains using standard identification tests. results: the growth of the bacillus anthracis strains on the hottinger's agar was typical (r -form), and no distinctions based on the origin of the strains were noted. all of the strains form spores and are immobile. when stained with specific antibody directed toward anthrax antigens, all strains stained positive. three of the b. anthracis strains did not have capsules when originally studied, but after 7 cultures on the hottinger's agar, the strains' ability to form capsules in a co 2 atmosphere was restored. the anthrax strains are sensitive to anthrax bacteriophage and penicillin. the strains have lipolytic and background: we studied the application of pk/pd modeling techniques as an alternative method to define pharmacodynamic breakpoints complementary to conventional microbiological breakpoints for fluoroquinolones (fqs). methods: 8 staphylococcus aureus strains with mics ranging from 0.03-8 mg/l were exposed to fluctuating concentrations of levo-(lfx) gati-(gfx) and moxifloxacin (mfx) mimicking their total serum concentration profiles following oral doses of 400 mg mfx, 400 mg gfx, 500 and 750 mg lfx over 24 h using the sedimentation model. strains were cultivated in brain heart infusion broth at 37°c. viable counts were quantitated at 14 sampling points. as pd-measure the aubkcnorm (i.e. area under the bacterial kill curve normalized to the initial inoculum [h] ; aubkcnorm <24 indicates a bactericidal activity, >24 bacterial growth) was determined for the time kill experiment and plotted as a function of mic for each treatment. results: the aubkcnorm vs mic function was similar for all treatments following a bimodal profile. while the slope of all fqs studied was shallow for mics below 1 mg/l with au-bkcnorm <5 h, aubkcnorm started increasing steeply beyond this point. it intercepted the 24 cut off line at concentrations of 1.5 mg/l for lfx 500 mg, 2 mg/l for gfx, 2.4 mg/l for mfx and 3.1 mg/l for lfx 750 mg. this indicates that s. aureus with susceptibilites beyond these mics would no longer be eradicated by the corresponding treatments. thus, according to our investigations a breakpoint for fqs integrating both microbiological and clinical pharmacokinetic relation-ships results in a value of 1 mg/l for lfx 500 mg, and 2 mg/l for mfx, gfx, and lfx 750 mg. conclusion: pharmacodynamic breakpoints determined by pk/pd m+s which are taking into respect the fluctuating concentration time profiles encountered in patients are suitable surrogates complementing the data obtained in microbiological experiments. a multicentre agar and broth dilution susceptibility testing study of fluoroquinolones against the bacteroides fragilis group k.e. aldridge, d. snydman, d. hecht, c. edlund, j. herrington (new orleans, boston, chicago, usa; stockholm, s; west haven, usa)objectives: the development of fluoroquinolones (fq) antimicrobials has resulted in effective therapy for a variety of infections due to aerobic and facultatively anaerobic grampositive and -negative pathogens. fq activity against anaerobes has not been as promising with only trovafloxacin receiving fda approval for anaerobic infections. moreover, more recent reports of increased resistance to fq among the b. fragilis group has prompted interested to determine if these increases are due to susceptibility testing methodology problems. this study was designed to compare the susceptibility results from five laboratories that tested the same 70 isolates against fq using agar dilution(ad) and broth microdilution (bmd) testing. methods: seventy clinical isolates of the b. fragilis group identified by only a sequential number were distributed to each of the five participating laboratories. four of the laboratories performed ad and one laboratory performed bmd susceptibility testing using nccls recommendations including breakpoints where appropriate. serial two-fold dilutions of moxifloxacin (mox), gatifloxacin (gate), trovafloxacin (trov), and metronidzaole (metro) were prepared in the test medium within a range of 0.03 to 64 mg/l. plates were inoculated with 1 · 10 5 cfu per spot or well, incubated anaerobically for 48 hours and the mic read. qc was performed using nccls-recommended atcc strains. results: mic values for each antimicrobial were collated as mic90 and per cent (%) of isolates inhibited at £2 and £4 mg/l: these data indicate that inter-laboratory testing of the same test isolates gave very similar results for each antimicrobial. bmd gave slightly lower mic results than ad but was not significantly different. we conclude that susceptibility methodology is not responsible for unusually high resistance rates. global evaluation of gemifloxacin activity tested against community-acquired respiratory tract pathogens, haemophilus influenzae and moraxella catarrhalis (1999) (2000) (2001) (2002) (2003) (2004) fluoroquinolones are a therapeutic option in treatment of infections caused by non-fermentative gram-negative bacteria.however, resistance to these antibiotics rapidly increase in clinical isolates. the efflux mechanism is the most common cause of multidrug resistance of strains p. aeruginosa, a. baumanii and s. maltophilia. these mdr pumps belonging to the rnd family are inhibited by phe-arg-beta-naphthylamide [mc207, 110] . however, different results of reserpine effect on the activity of rnd pumps was established. in our study, we searched for effect of the efflux pump inhibitors (mc207,110, reserpine and rescinnamine) on the mic of quinolones for mdr clinical isolates of p. aeruginosa, a. baumanii and s. maltophilia. we looked for a interdependence between structure of quinolones and ability of tested inhibitors to inhibit efflux of these antibiotics. results: the mdr strains were resistant to all tested quinolones (nalidixic acid-nal, pipemidic acid-kp, norfloxacin-nor, ciprofloxacin-cip and ofloxacin-of). among studied inhibitors only mc207,110 affected the susceptibility of all tested strains to quinolones, first of all to nal. effect of mc207,110 on the mic depended on its concentration, but did not depend on the temperature. for majority of p. aeruginosa and a. baumanii strains the mic of all quinolones decreased in the presence of mc207,110. the highest decrease of the mic of quinolones was obtained for p. aeruginosa (4-512-fold). for a. baumanii strains the mic of kp, nor, cip and of decreased only from 2-to 4-fold but the mic of nal decreased to 16-fold. moreover, the presence of this inhibitor increased the sensitivity of 80% of s. maltophilia only to nal (4-to 16-fold). reserpine and rescinnamine did not effect on the mic of quinolones for all studied mdr strains. however, reserpine alkaloides showed inhibitors activity against control strains of s. aureus (decrease mic of nor). conclusions: our data confirm that mc207,110 inhibited efflux pumps not only in p. aeruginosa but also in a. baumanii and s. maltophilia strains. additionally, obtained results suggested possibility that for quinolones, depending on their structure, are different binding sites in efflux pumps. furthermore, probably also mc207,110 binds to specific site of pumps depending on the efflux systems. so, the effect of this inhibitor on mic of quinolones depends on genus of bacteria, kind of pumps and structure of agents. in vitro activity of fucidic acid against staphylococcus aureus strains objectives: viridans group streptococci (vgs) resistant to penicillin, macrolides and other antimicrobial are increasingly found. linezolid and quinupristin-dalfopristin exert their mechanism of action by protein synthesis inhibition and are agents with activity against multidrug resistant gram-positive cocci. this study was aimed to determine, by time-kill curves, the activity of linezolid and quinupristin-dalfopristin against five vgs expressing various degrees of resistance to erythromycin and harbouring different erythromycin resistance determinants methods: the species, erythromycin mics (mg/l) and the erythromycin resistance genes present for each of the five isolates tested were: s. anginosus/0.06/none, s. sanguis/4/mef(a), s. sanguis/64/erm(b), s. mitis/256/erm(b) +mef(a), s. anginosus/256/erm(b) +mef(a). quinupristin-dalfopristin and linezolid mics for all strains were 1 and 0.5 mg/l respectively. strains were incubated in cation-adjusted mueller-hinton broth supplemented with 5% lysed horse blood. each of the five isolates were exposed to linezolid and quinupristin-dalfopristin for 24 hours in a shacking water bath in presence of 2 · mic. colony counts were performed at 0, 4, 8 and 24 hours. results: time-kill studies showed that for linezolid, the antibiotic concentration of 2 · mic was bacteriostatic for each strain irrespectively of the resistance to erythromycin and the genes harboured, showing a 90% of killing at 24 h. the in vitro activity of quinupristin-dalfopristin was predominantly bactericidal with a 99.9% of killing at 3 h. the exception were the 2 strains with erm(b) + mef(a) determinants, in one of them, a s. milleri, the bactericidal activity was delayed to 24 h, and in the other strain, a s. mitis, the activity was bactericidal at 3 h but with regrowth at 24 h. conclusions: when vgs are implicated in infections in neutropenic patients and in cases of endocarditis where bactericidal activity is usually considered mandatory, we have to considered that: linezolid was bacteriostatic, at the concentration tested, against all strains and the rapid killing of quinupristin-dalfopristin against vgs was lost when the strain presented the erm(b) and mef(a) genes together. comparative antimicrobial susceptibility patterns in different groups of species of viridans group streptococci and streptococcus bovis isolated fom blood cultures i. rodriguez-avial, c. rodriguez-avial, j.j picazo (madrid, e)objectives: viridans group streptococci (vgs) and s. bovis are part of the normal human microbiota and are implicated as a cause of endocarditis and sepsis in neutropenic patients. because of their resident nature are frequently exposed to the antimicrobial agents. this study was performed to study the resistance phenotypes to penicillin (p), erythromycin (e), key: cord-022501-9wnmdvg5 authors: nan title: p1460 – p1884 date: 2015-12-28 journal: clin microbiol infect doi: 10.1111/j.1470-9465.2006.12_4_1431.x sha: doc_id: 22501 cord_uid: 9wnmdvg5 nan resistance rates (rr) over a period of 5 years were generally better than those reported for a total of 40 icus. the mean mrsa rr was 22.1%, for all the sari icus, whereas it was only 2.7% in the study icu. by the end of 2003 duration of treatment for pneumonia had been reduced to 5-7 days and written guidelines on empiric antibiotic treatment and prophylaxis were revised with respect to the resistance situation of the study icu. the significant decrease between 2000 and 2004 in total antimicrobial ad from 1,099 to 607 in the study icu resulted mainly from the reduced consumption of 2nd generation cephalosporins, carbapenems and imidazoles. ni did not change significantly over time. compared to the year 2000, the costs for antibiotics were halved from €51,102 to 22,324, which corresponds to €18.7/pd and €6.6/pd, respectively. the percentage of antibiotics in the total icu budget for pharmaceuticals decreased from 14.6% to 10.4%. conclusion: surveillance and feedback of antibiotic use and resistance can serve as a valuable quality control instrument and can have an impact on antibiotic treatment. from 2000 to 2004, antibiotic use was reduced by 45% and costs for antibiotics/pd were cut by two third in the icu study without any increase in device associated nosocomial infection rates. the resistance situation was generally better than in all sari icus, but showed heavy fluctuations. similar illness burden but different antibiotic prescription to children: a population-based study k. hedin, m. andre, a. håkansson, n. rodhe, s. mö lstad, c. petersson (växjö, falun, malmö, linköping, se) objectives: respiratory tract infections are the most common reason for antibiotic prescription in sweden as in other countries. the prescription rates vary markedly in different countries, counties and municipalities. the reasons for these variations in prescription rates are not obvious. the aim of the study was to find possible explanations for different antibiotic prescription rates in children. therefore a prospective population based log book study was conducted in four municipalities which, according to official statistics, had high and three municipalities which had low antibiotic prescription rates. methods: during one month, parents recorded all infectious symptoms, physician consultations and antibiotic treatments, from 848 18-month-old children in a log book. the children's parents also answered a questionnaire about socioeconomic factors and concern about infectious illness. results: antibiotics were prescribed to 11.6% of the children in the high prescription area and 4.7% in the low prescription area (crude or 2.67 (95% ). after multiple logistic regression analyses taking account of socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations, differences in antibiotic prescription rates remained (adjusted or 2.61 (95%ci 1.14-5.98)). the variable that impacted most on antibiotic prescription rates although it was not relevant to the geographical differences was a high level of concern about infectious illness in the family. conclusion: the differences in antibiotic prescription rates could not be explained by socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations. the differences may be attributable to different prescription customs, in which case physicianś prescription patterns are not always rational. decreasing outpatient antibiotic prescribing in germany, 1995 germany, -2004 , does not include newer macrolides, fluoroquinolones and extendedspectrum beta-lactams w.v. kern, k. de with, k. nink, h. schrö der (freiburg, bonn, de) objective: the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project has shown that outpatient antibiotic prescribing in germany has been comparatively low among european countries. we assessed trends over time and regional variation of outpatient antibiotic use in germany, and wondered if the observable decreasing trend included all drug classes to a similar extent. methods: prescription data (compulsory health insurance covering >90% of the population, sample of 0.4% until the year 2000, all prescriptions thereafter) were analysed using the atc/who methodology and current ddd definitions. we specifically defined the following drug groups: ''basic'' penicillins (bpens, oral penicillin or aminopenicillins), extended-spectrum betalactams (esbls, oral cephalosporins, staphylococcal penicillins, aminopenicillin/betalactamse inhibitor combinations, parenteral cephalosporins and broadspectrum betalactams), newer macrolides (nmls, roxithromycin, clarithromycin, azithromycin) versus older macrolides (omls). quinolones (fqs), folate synthesis inhibitors (t/ss) and tetracyclines (tets) were also assessed. data were expressed in yearly ddd/1000 persons covered by the insurance (ddd/1000). findings: outpatient prescribing in 1995 was 6140 ddd/1000 (corresponding to 16.8 did = ddd/1000 and day) and decreased to 5430 ddd/1000 in the year 2000 and to 4672 ddd/1000 in 2004. the decreasing trend over the last 4 years was observed in all regions. the decrease was most significant for omls ()55%), t/ss ()48%), tets ()36%), and bpens ()13%) while there was no decreasing use of esbls (±0%) and increases in the rate of prescribing nmls (+13%) and fqs (+43%). tets and bpens, however remained the most prescribed antibiotics in 2004. regional variations in 2004 remained large for bpens (>3-fold) with very low prescribing rates in the eastern region, but were small for t/ss, nmls and fqs (<2-fold). conclusions: over a decade we observed a 24% decreasing outpatient antibiotic prescribing that included relevant antibiotic drug classes except esbls, nmls and fqs. the relative increase was most significant for fqs. severe community-acquired pneumonia admitted to the intensive care unit: impact of antibiotic therapy delay on hospital mortality antibiotic therapy were enrolled in the study. pts were divided in 4 groups according to time to treatment (<2 h gi, [2] [3] [4] [4] [5] [6] [7] [8] . baseline severity scores (apache ii, sofa, psi, curb-65), microbiological documentation, and hospital outcome were compared for all groups. results: 152 pts were included in the study. microbiological documentation was achieved in 80% of all pts, positive blood cultures in 54/152 (35.5%), s. pneumoniae in 64/152 (42%). mean age was 66 ± 13, apache ii 25.8 ± 7.6, sofa 8.8 ± 3.7, psi 158 ± 40, curb-65 3.2 ± 1, mechanical ventilation in 67.7% and vasopressors use in 63.1%. overall icu and hospital all-cause mortality were 27.5% and 35.5%, respectively. baseline severity scores were comparable in all 4 groups and their respective hospital mortality is provided in table 1 . conclusions: in severe cap, treated with a combination therapy, time to treatment seems to have an impact on hospital all-cause mortality. based on our results, antibiotic treatment should be initiated within the first 2 hours after hospital admission. (period ii) -cefoperazone/ sulbactam (8 g a day) as monotherapy were used as the empirical antibacterial therapy of vap. the rotation from cefepime to cefoperazone/sulbactam was performed due to our previous study demonstrated high frequency of esbl producers among enterobacteriaceae. the samples from the lower respiratory tract were obtained by mini-bal. the sensitivity of microorganisms to the antibiotics studied (ceftazidime, cefepime, cefoperazone/sulbactam and carbapenems) was determined by the disk diffusion method. results: the main pathogens of vap were s. aureus (20%), p. aeruginosa (19%), enterobacteriaceae (23%) and this structure did not changed during both periods. the antibiotic sensitivity of p. aeruginosa and enterobacteriaceae (k. pneumoniae and e. coli), was studied separately. a high level of resistance of enterobacteriaceae to cefepime can be explained by the strains prevailing in the given icu, which produced extended spectrum beta-lactamases (ctx-m). the resistance of enterobacteriaceae to cefepime was 57.5% in i period and 80.5% in ii period, to ceftazidime -90.0 and 100%, meropenem -0 and 0%, imipenem -5.1 and 2.1%, cefoperazone/sulbactam -10.9 and 20.8%, respectively. a change of cefepime for cefoperazone/sulbactam was not followed by any decrease of enterobacteriaceae resistance level to cefepime during ii period. the resistance level of p. aeruginosa to cefepime was 20.5% in i period and 24.1% in ii period, to ceftazidime -22. 5 and 33.0%, meropenem -44.7 and 39.5%, imipenem -50.1 and 39.5%, cefoperazone/ sulbactam -16.9 and 12.5%, respectively. conclusion: the exclusion of cefepime for 9 months didn't improved the sensitivity of enterobacteriaceae to this medication. the level of resistance of p. aeruginosa and enterobacteriaceae to cefoperazone/sulbactam did not increased despite a wide use of this antibiotic during 9 months. antibiotic consumption in german acute care hospitals m. steib-bauert, k. de with, e. meyer, p. straach, w.v. kern (freiburg, frankfurt, de) objective: outpatient antibiotic use in germany differs substantially between eastern and southern parts of the country (relatively low use) and western part (relatively high use). there is no nationwide estimate of hospital antibiotic use and its geographic variation if any. the aim of the present study was to provide an estimate of recent hospital antibiotic use density in germany and to identify basic unit/hospital characteristics associated with excess use. methods: data on hospital consumption of systemic antibiotics in anatomical therapeutic chemical (atc) class j01 were obtained from a convenience sample of 145 acute care hospitals in germany that participated in an ims survey in the year 2003 and had complete data (dispensed drugs and patient-days per year) for at least one non-paediatric, non-psychiatric department or ward. a total of 275 non-icu surgical departments/wards, 229 non-icu non-surgical (general medicine, oncologyhaematology, neurology/stroke) departments/wards, and 184 icus covering >16 million patient-days were analysed. data were expressed in ddd (who/atc definition version 2001) or ''prescribed/recommended daily doses'' (pdd, better reflecting [high] dosages given to hospitalized patients) per 100 patient days (ddd/100 and pdd/100). findings: the weighted mean over all departments/wards incl. icus was 49.8 ddd/100 (31.4 pdd/100). as expected, icu antibiotic use density was much higher than use in non-icu areas, and use in haematology-oncology was higher than in other non-surgical departments/wards. in univariate analyses, bed-size category and university affiliation (icus, surgical wards), region (icu, surgical and non-surgical wards) and haematology-oncology as specialty (non-surgical wards) were associated with use density, but these associations were only partly confirmed in multivariate logistic regression analyses of factors associated with excess ( ‡75%) use density which showed university affiliation and haematology-oncology to be independently associated with high use. conclusions: based on this hospital sample, antibiotic use in german hospitals shows little, non-significant regional variation and appears to be similar to what has been described from other european countries. adjustment of the data at least for university affiliation and haematology-oncology is important in comparative analyses of hospital antibiotic consumption. impact of formulary change in medical intensive care unit on outcome of infection and antimicrobial resistance sought to evaluate a formulary change and impact it has on infection and resistant. methods: prospectively, all patients in a 20-bed icu were followed for a period of 4 months in phase i (390 patients per 2379 patient days) and to collect baseline data after a decrease in the use of piperacillin-tazobactam (pt) when substituted by cefepime for a period of 4 months in phase ii (383 patients per 2260 patient days). results: total infections in phase i vs. phase ii were lower respiratory tract (lrti) 214 patients (55%) vs. 203 patients (53%); urinary tract infection (uti) 94 patients (24%) vs. 96 patients (25%); and sepsis of undetermined aetiology 70 patients (18%) vs. 65 patients (17%), respectively. there were no significant differences in death (22% vs. 19%) , cure or improvement of infection (53% vs. 56%), readmission to the unit (3.5% vs. 3.2%), hospital risk of death (29.8% vs. 30.2%), mean length of icu stay (6.1 days vs. 5.9 days), or rates of nosocomial infection (6.3% vs. 5.1% for lrti; 4.0% vs. 4.2% for uti; 0.8% vs. 0.0% for soft tissue infection; 0.8% vs. 1.0% for bacteremia; 2.1 vs. 1.0 per 1000 patient days for intravenous catheter infection) in phase i and ii respectively. the cost of antimicrobial acquisition in phase i and ii were $548 and $433 per patient respectively (p < 0.001). the mean antimicrobial treatment costs per patient for pt were $145 vs. $100 and cefepime were $80 vs. $105 in phase i and ii respectively (p < 0.01). the in vitro susceptibility and rate of infection and colonization with escherichia coli were unchanged in both study periods. there were 68 vs. 39 staphylococcus aureus (p < 0.001); of these 94percnt vs. 87percnt were methicillinresistant s. aureus and 11 vs. 9 enterococcus faecium (82% vs. 77% vancomycin-resistant enterococci) in phase i and ii respectively. there were 73% vs. 31% pseudomonas aeruginosa and 70% vs. 4% klebsiella pneumoniae extended spectrum beta lactamases in phase i and ii respectively. conclusion: the implementation of formulary substitution of pt to cefepime in the medical icu had resulted in a decrease in the use of pt. in addition, there were decreased costs and less s. aureus infections without adversely affecting the outcome of infection or antimicrobial resistance. intravenous antibiotic use in scottish hospitals; evaluation of the glasgow antimicrobial audit tool r.a. seaton, d. nathwani, p. burton, e. douglas (glasgow, dundee, uk) introduction: there are few data on antibiotic prescribing within scottish hospitals and a coordinated multisite point prevalence survey had not been performed before. there is concern that antimicrobials are overused in hospitals. methods: antibiotic use in acute medical and surgical units in 10 scottish hospitals across 5 trusts, was investigated using a point prevalence survey. data were collected by pharmacists. appropriateness of the iv route of administration was determined by review of data by an infectious diseases physician (idp) and compared with a specifically designed computerised algorithm. the idp also judged the appropriateness of the chosen iv agent against local guidelines. 3826 patients from 10 hospitals in 5 regions were surveyed on a single day. 1079 (28.3%) were receiving an antibiotic, 381 (35.3%) intravenously. 197 receiving oral antibiotics had received an iv previously. median duration of iv therapy was 4 days (iqr 2-7 days) and time from iv to oral switch was 3.5 (2) (3) (4) (5) (6) . the idp judged appropriate iv route in 84% patients compared with 84.8% by the algorithm. the sensitivity of the algorithm was 93.4% and specificity 60.7%. the positive predictive value was 92.6% and the negative predictive value was 63.8%. the idp judged iv agents to have been chosen and administered appropriately in 80%. most frequently prescribed iv agents were 3rd generation cephalosporins (3gc) (28.3%), co-amoxiclav (20.2%), metronidazole (19.2%), glycopeptides (18.6%). significant regional differences were seen for most antibiotic groups including 3gcs (49.2% (site 3) vs 24.4% (sites 1, 2, 4, 5) , p < 0.001) and glycopeptides [31.8% (site 1) vs 9.3% (site 2, 3, 4, 5) , p < 0.001]. it is possible to coordinate, collect and compare data from scottish hospitals. the gaat gives a good estimate of the appropriateness of iv therapy. significant differences in prescribing patterns between similar patient groups across different hospital sites were demonstrated. such data may usefully inform local and national audit and support prescribing initiatives. associations between continuous variables were tested in univariate analysis with the spearman correlation test (r). multiple linear regression analysis was performed in a backward stepwise approach. results: the median rate of total hospital glycopeptides use was 4.11 (range 0.21 to 27.22) ddds per 1,000 pd with higher consumption in large public hospitals. consumption was higher in intensive care areas (median 46.51; range 7.19 to 134) than in surgery areas (median 4.5; range 0.17 to 24.76 ) and in medicine (median 4.26 ; range 0 to 41). glycopeptides use correlated with number of central line per 1,000 pd (r: 0.44; p: 0.03) and with size of the various areas in the hospital (for intensive care, r: 0.50; for medicine areas, r: 0.33 and for surgery areas, r: 0.42; p < 0.05). median incidence of mrsa was 0.87 per 1,000 pd. incidence of mrsa explained a small proportion of the variation in hospital glycopeptides consumption (r2: 0.13). in a multivariate linear regression model, incidence of mrsa and number of beds in surgery areas were independent predictors of total glycopeptides use in the hospital (r2 adjusted: 0.39). after controlling for these factors, number of central-line per 1,000 pd was no more associated with glycopeptides use. conclusion: in our hospitals, total glycopeptides use was not heavily determined by incidence of mrsa. although glycopeptides use in surgery areas was not the highest, the total number of surgery beds in the hospital explained a large variation of the total hospital glycopeptides use. therefore we had to take it into account to interpret these consumption and to decide further evaluation. antibiotic management of acute lower respiratory tract infections among dutch elderly patients in primary care j. bont, c. birkhoff, t. verheij, e. hak on behalf of esprit objectives: acute lower respiratory tract infection (lrti) can cause various complications leading to morbidity as well as mortality notably among elderly patients. antibiotic treatment of lrti is common, despite dutch clinical guidelines recommending antibiotics only in case of pneumonia or high risk of serious complications. we assessed the course of illness and outcome of pneumonia, acute bronchitis and exacerbations of copd or asthma among dutch elderly patients in primary care and assessed whether gps were inclined to prescribe antibiotics more readily to patients with potential risk factors for complications in acute bronchitis or exacerbations of copd/ asthma. methods: we retrospectively analysed medical data from 3,166 episodes of lrti among patients ‡65 years of age presenting in primary care to describe the course of illness and outcome. the relation between prescriptions of antibiotics and patients with risk factors for a complicated course was assessed by means of multivariate logistic regression. risk factors for a complicated course included heart failure, history of myocardial infarction, angina pectoris, diabetes, history of stroke, dementia, malignancy, and history of pneumonia or hospitalisation in preceding year. results: one or more complications arose in 17% of episodes of lrti. among these, 8% suffered from pulmonary complications, 5% had cardiovascular complications (heart failure, myocardial infarction etc.), 5% had a protracted course and 0.6% had a diabetes event. in 6.9% of the patients complications led to hospital admission and in 2.4% lrti were fatal. antibiotics were more readily prescribed to patients aged ‡90 years, when heart failure was present and in patients with diabetes. no significant association was observed in patients with other co-morbid conditions. patients diagnosed with an exacerbation of copd or acute bronchitis with a history of pneumonia or hospitalisation in the preceding year were not more likely to receive antibiotics. conclusions: a considerable part of elderly patients with a lrti suffers from a severely complicated course in primary care. although gps are inclined to prescribe more readily antibiotics in the very old and those with heart failure or diabetes, other potential risk factors are not taken into account. objectives: in this study it was aimed to analyse the infectious diseases (id) trainees' night/weekend shift consultation process in terms of patient and consultant characteristics, types of recommendations, and compliance with recommendations. methods: all consultations performed by id trainees in night shift and at the weekends between june 10th-august 10th 2004 were analysed in terms of consultation type [treatment continuation (tc), consultation for surgical antibiotic prophylaxis (pa), and consultation with or without a request of a specific antibiotic (others)]. appropriateness of recommendations was assessed the day after the consultation by infectious diseases specialists (ids). adherence to recommendations was assessed 3 days after the consultation by idss. recommendations including antibiotics were considered appropriate, if they were appropriate according to national and international guidelines. recommendations were considered complied, if they were done in up to 72 hours after the consultation (except the consultations in the emergency medicine and the consultations in which antibiotics were started by the counselling idss). results: of 436 consultations 79 was for tc, 74 was for pa and 290 was for others. the clinic where id consultations were requested mostly was general surgery clinic (152/436, 34.9%) . in 2% of all consultations trainees consulted the specialists. overall 146 consultations (74 for sp, 69 for a clinical infectious disease diagnosed clinically, 9 for an infectious disease diagnosed microbiologically) were for requesting spesific antibiotic(s). pa were approved in 68 of 74 consultations. antibiotic was not recommended in 14 of 290 other consultations. in six of 74 consultations for pa antibiotic was changed for a clinically diagnosed infectious disease. in one of 79 consultations for tc antibiotic was changed due to lack of response to the given antibiotic, in others tc was approved. inappropriate antibiotic recommendation rate was 2.4% (8/32, 4 inappropriate choice, 3 inappropriate dosage, one antibiotic unnecessary). overall compliance to id recommendations was 76.1% (476/608). rate of compliance to antibiotic recommendations was evaluated in 240 consultations and was found 98.2% (277/282) and was higher than compliance to other (microbiology etc.) recommendations (60.8%, 199/327, chi square p < 0.05). conclusion: methodologies to improve the compliance to nontreatment based recommendations and optimizing antibiotic selection is necessary. study of the influence of online practice guidelines on the appropriateness of antibiotic prescribing in a university-affiliated psychiatric hospital j.f. westphal, c. nonnenmacher, d. gregoire, m. hittinger, c. oulerich, f. jehl (brumath, strasbourg, fr) background: problems with the dissemination of guidelines are frequently cited as a major reason for failure to impact practice. reviews of the effectiveness of various methods of guideline dissemination show that the most predictable impact is achieved when the guideline is made accessible through computer-based reminders that are integrated into the clinician's workflow. we report a time-series prospective investigation aimed at comparing the appropriateness of antibiotic (ab) orders for pneumonia at the treatment initiation level after vs. before having embedded our current ab guidelines for pneumonia in the computerized physician drug-order entry system of our teaching psychiatric hospital comprising 410 adult beds. methods: in total, 160 consecutive ab orders for pneumonia were evaluated by the pharmacy department, including 80 orders just before and 80 orders just after implementation of online ab guidelines. appropriateness of ab orders relative to the guidelines was assessed according to 3 criteria: (1) the choice of ab with respect to the mode of acquisition (community-or hospital-acquired) of pneumonia or the presence of clinical risk factors for involvement of gramnegative bacilli, (2) the daily dosage, (3) the planned duration of treatment. data were extracted from the computerized infection declaration system that recorded all ab-requiring infections in our hospital. results: the number of ab orders with at least 1 criterion of inappropriateness tended to decrease, yet not significantly (p = 0.11), after vs. before implementation of online guidelines: 30/80 (36.5%) and 40/80 (50.0%), respectively. the number of criteria of inappropriateness relative to all ab orders for pneumonia was significantly lower in the post-implementation period: 43.8% vs. 65.0% before implementation (difference 21.2%, 95% ci 6. 1-36.3, p < 0.01) , with a trend to a decreased number of orders containing more than 1 criterion of inappropriateness. analyzed separately, the numbers of inappropriate orders for the choice of the ab, or the daily dosage, or the planned duration of treatment decreased, yet not significantly (p > 0.1 for each criterion), in the post-vs. preimplementation period : 11 vs. 18, 12 vs.15, 12 vs.19, respectively. conclusion: in this study, the moderate impact on ab prescribing practices of online guidelines available at the time of drug order shows that additional types of intervention are needed to improve further the quality of ab prescribing. material: the pilot hospitals had a median capacity of 654 (range, 154 to 1597) beds; their regional distribution was representative of population size; 18 were general hospitals, 8 teaching hospitals and 10 general hospitals with teaching beds. results: ams were internists (28), microbiologists (13) and pharmacists (13). amts included a mean of 10 members who met every 6 weeks on average. all hospitals irrespective of size or affiliation had undertaken a wide range of antibiotic management interventions in 2003, which increased in 2004; these included (in 2003 and 2004, respectively) : major review of formulary (in 10 and 23 hospitals), development of clinical guidelines (69 and 215 topics), restricted access to selected antibiotics (carbapenems, glycopeptides, quinolones, new drugs; in 25 and 33 hospitals). in 2003, antibiotic consumption databases were established in 35 hospitals and antibacterial susceptibility databases in 31 hospitals. in 2004, cross-analysis of these databases was performed in 28 hospitals. in 2004, prescribing assistance, antibiotic stop orders, treatment streamlining and iv/po therapy switch were implemented in 32, 21, 24 and 24 hospitals, respectively. in 2004, 26 hospitals reported a better use of target antibiotics, 15 hospitals a decrease in consumption of restricted antibiotics, 5 hospitals a decrease of total antibiotic consumption, 2 hospitals a decrease in high consumer departments. conclusion: all hospitals participating in the amt pilot scheme have developed multiple antibiotic policy interventions and established monitoring and guidance of antibiotic prescription. preliminary data from some hospitals indicated success in meeting self-defined targets of appropriate use and reducing the consumption of selected antimicrobial agents. more systematic evaluation using standard quantitative and qualitative indicators is planned. antibiotic prescribing practices at two linked london teaching hospitals p1476 comparison of different antibiotic consumption measurement methods in large multidisciplinary hospital e. pujate, i. apine, u. dumpis (riga, lv) objectives: antibiotic selection pressure is determined by the total amount of antibiotics, number and density of patients treated with antibiotics in the particular geographical area. several antibiotic consumption detection methods should be combined in the hospital setting. our objective was to evaluate efficacy of different approaches in large multidisciplinary hospital. methods: point prevalence studies were repeated annually at [2002] [2003] [2004] in stradins university hospital (1000 beds) in latvia. all patients receiving antibiotics on the day of the survey were identified and their medical records were reviewed. data on antibiotics, dose and route of administration were collected. in addition, annual data on antibiotics dispensed to the departments were collected from pharmacy. total used grams for each antibiotic were expressed into defined daily doses (ddd-who). bed days (bd) and admission days (ad) were used as denominators. results: table 1 total use of antibiotics in stradins university hospital 2002 hospital -2004 the most commonly used antibiotic groups in the pharmacy study were 1st generation cephalosporins (13.35 ddd/100 bd in 2002 , 11.6 in 2003 , 10.8 in 2004 ) and penicillin's with extended spectrum (11.20, 11.98, 13.15 ) followed by fluoroquinolones (6.26, 8.13, 8.69 ) and metronidazole (4.42, 4.59, 5.61 ). there was no significant difference between distribution of different antibiotics from prevalence and pharmacy studies if calculated in ddds. in contrast, distribution of antibiotics calculated per patient in the prevalence study was quite different; 1st generation cephalosporins (8. 71%, 8.63%, 5.84% in 2002, 2003, 2004 respectively) and fluoroquinolones (3.05%, 6.69%, 5.63%) with smaller proportion of extended spectrum penicillins (4.36%, 3.88%, 3.93%) and metronidazole (4.03%, 3.56%, 4.68%). conclusions: there were no differences in the distribution of antibiotics calculated in ddds per bed days and admissions. distribution of antibiotics in annual pharmacy studies and point prevalence studies if calculated in ddds were also similar. in contrast, the prevalence data expressed as a proportion of patients with selected antibiotics showed quite different distribution. studies using only ddds may overestimate use of certain antibiotic groups in our setting where who ddds are significantly different from actual pdds used. a study of prescribing patterns and errors of antibiotics in a saudi hospital m. al-jamal, m. al-barrak (riyadh, sa) background: the term ''prescribing patterns'' has been used extensively in studies to describe different aspects of the prescribing process. antibiotics as well as other drugs are prescribed for the purpose of achieving definite therapeutic outcomes that improve a patient's quality of life while minimizing risk. in the clinical literature, the incidence of antibiotics prescribing errors ranges between 0.5% and 18.8%. objective: in this study we will address antibiotics prescribing patterns and the incidence of prescribing errors in a tertiary hospital and the potential relationship between them. methods: a prospective study of all prescriptions in a 3-month period (june to august 2003) in a tertiary hospital has been analysed. the hospital provides both primary and secondary levels of care. criteria used include frequency of selected prescribed drugs, average number of items per prescription, compliance to the hospital formulary, frequency of prescriptions for antibiotics, generic prescribing and diagnosis. the prescribing patterns and the incidence of prescribing errors were performed. results: total number of prescriptions for the 3-month study was 24,404. emergency room (er) and primary care have the highest number of prescriptions (37.1%). the average number of items per prescription is 2.1. the most prescribed drugs by primary care (25.3% errors), emergency are antibiotics (28.2%), medicine (3.7), ophthalmology (22.3), gynaecology (7.8), and paediatrics (17.8). the prescription errors were 13.6% in primary care and 22.3% in emergency department. discussions and conclusions: over 24000 prescriptions were included in this study. the incidence of prescribing errors was 18.8% the average number of items per prescription was 2.1. total prescription errors are also related to frequency of prescribing antibiotics. there was a relation between prescribing of antibiotics and prescribing of trade names (p < 0.01), and compliance to the hospital formulary (p < 0.001). several factors influence prescribing patterns and variations in prescribing rates has been identified. these include general physician behavior, differences in morbidity and mortality patterns, social perception toward illness, and physician clinical skills, experience and qualification, as well as physician continuing education and training. special antibiotic prescribing guidelines and restrictions should target primary care and emergency department physicians. effect of a policy for restriction of selected classes of antibiotics on antimicrobial drug cost and resistance of the non-restricted antibiotics. the logistic regression model we performed showed that the new policy had an independent positive effect on the in vitro antimicrobial susceptibility of pseudomonas aeruginosa (p = 0.051) but not of acinetobacter baumannii and escherichia coli isolates. conclusion: our data suggest that there are considerable limitations of the programs aiming to reduce the consumption of restricted antibiotics through the approval of their use by specialists, at least in a proportion of settings. education programs that aim to involve the medical staff directly responsible for the care of patients in voluntary decisions regarding the appropriate use of antimicrobial agents may have more profound and sustainable success, and thus, deserve to be studied. estimating hospital versus ambulatory care consumption of antibiotics in southwestern germany k. de with, m. steib-bauert, h. schrö der, k. nink, w.v. kern (freiburg, bonn, de) objective: preliminary data from the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project indicated that the proportion of hospital care (hc) antibiotic use on total antibiotic use in several european countries ranges between 5 and 20%. only few countries, however, have so far been able to report representative countrywide information on both hc and ambulatory care (hc) antibiotic consumption. we estimated ac versus hc consumption of antibiotics for one of the 16 german federal states located in the southwestern part of the country with a 10.5 million population. methods: data on hc consumption (atc class j01) were obtained from a convenience sample of acute care general hospitals (n = 42), extrapolated to state-wide consumption (using official statistics for the total state-wide 152 general plus 77 special non-psychiatric/non-paediatric/non-radiotherapy hospitals), expressed in defined daily doses per 1000 inhabitants and day (did), and finally compared to ambulatory care antibiotic use density in the same region and period of time (years 2001 and 2002) . findings: the estimated state-wide hc consumption of antibiotics was 2.1 did (95% confidence interval, 1.9 to 2.2 did) in both years. state-wide antibiotic consumption in the ac setting during the same time was 12 did (~85% of total consumption). ac consumption of fluoroquinolones (1.1-1.2 did, 79%) and macrolides/clindamycin (1.8 did, 95%) made up a major proportion of total use of that drug classes. conclusions: hospital antibiotic use in the southwestern part of germany can be estimated to contribute~15% to overall antibiotic consumption in the general population. antibiotic use profile and temporal trends during a 5-year period at a greek university hospital: implications for antibiotic policy changes e.i. kritsotakis, p. assithianakis, p. kanellos, n. tzagarakis, m.c. ioannides, a. gikas (heraklion, gr) objectives: to investigate the profile and temporal trends of inpatient antimicrobial use over a 5-year period at the university hospital of heraklion crete, greece. further, to examine the way in which frequency of data collection and stratification by different patient-care areas provides guidance to antibiotic policy changes. methods: retrospective monitoring of antimicrobial consumption was carried out according to the who anatomic therapeutic chemical classification (atc) and defined daily dose (ddd) measurement methodology. pharmacy records were used to obtain aggregate data of drug deliveries to individual wards. results were expressed as usage density rates in ddds per 100 bed-days (ddd/100bd). linear regression was used in order to assess the statistical significance of a temporal trend in usage densities. results: during 1998 -2002 , hospital-wide antimicrobial use (atc group j01) significantly increased by 22%, from 86.97 to 106.24 ddd/100bd. the annual average increase rate was 4.8 ddd/100bd. stratification by clinical service demonstrated differences in the intensity and profile of class use, as well as varying temporal trends (figures 1, 2) . pooled usage rates in ddd/100bd, overall percentage increases and annual average increase rates were respectively 109. 97, 35.6%, 8.1 for medical wards; 98.21, 48.7%, 9.1 for icu's; and 74.46, 34.3%, 5.7 for haemato-oncology wards. surgical wards had a fairly constant usage rate (98.36). a shift towards the newer broad-spectrum antibiotics to the detriment of the older penicillins and cephalosporins was noted in all hospital areas. conclusion: surveillance of aggregate data on the consumption of antimicrobials using the atc/ddd system provided a clear picture of the profile of hospital usage. monthly data over a sufficient surveillance period allowed the assessment of temporal trends. stratification of usage rates by clinical service allowed areas of concern to be specified. thus, surveillance of monthly antimicrobial consumption rates stratified by patientcare area can provide a simple, rapid and efficient tool for triggering antibiotic policy changes in the hospital and targeting more detailed quality-of-use audits. appropriate use of aminoglycosides: the impact of an antibiotic control team c. rioux, p. lesprit, j.r. zahar, a. hulin, a. bernier-combes, c. brun-buisson, e. girou (créteil, paris, fr) objectives: many factors are involved in the appropriate use of aminoglycosides (ag), such as modalities of administration, serum monitoring and duration of treatment. we assessed prospectively the risk factors and the impact of an antibiotic control team on the appropriateness of ag prescriptions. methods: in a setting of a restricted delivery system of ag in our hospital, we first performed an observational audit (oa) to assess the appropriateness of prescriptions including justification of prescribing, adequacy of drug choice, adequacy of administration modalities, modalities of serum monitoring and duration of treatment. after implementation of specific guidelines hospital wide, we then performed an interventional audit (ia) where an antibiotic control team could interfere when ag prescriptions were considered inappropriate. appropriateness of ag prescriptions between the 2 audits was then compared. results: 200 prescriptions were analysed. during the ia, 32% of prescriptions were modified by the control team. as compared to the oa, prescriptions in the ia were significantly more appropriate with regard to treatment duration (73 vs 55%, p = 0.009) and serum monitoring (61 vs 40%, p = 0.05). median treatment duration was shorter in the ia (4 d) than in the oa (6 d) (p < 0.0001). a logistic regression model showed that risk factors for appropriate treatment duration were (adjusted or, 95% ci, p value): hospitalization in intensive care unit (4.39, 1.57-12.2, 0.005) , polymicrobial infection (4.08, 1.38-12.08, 0.01) and antibiotic control team intervention (2.41, 1.23-4.72, 0.01) . table: conclusions: despite a restricted delivery system, ag use was frequently associated with excessive treatment duration and errors in monitoring modalities. reinforcing practice guidelines through direct counselling improved appropriateness of prescriptions. hospital antibiotic consumption in southern and eastern mediterranean countries: preliminary results from the armed project p. zarb, m.a. borg, h. goossens, m. ferech for the armed participants introduction: armed is an international research project investigating antimicrobial resistance and consumption in 7 southern and eastern mediterranean countries through the collection of comparable and validated antimicrobial resistance data as well as information about antibiotic consumption patterns and infection control initiatives. objectives: the consumption part of the study aims to collect data on antimicrobial use within participating hospitals in the region, which information is currently unavailable. methods: data collection is planned over a 24-month period using anatomical therapeutic chemical (atc) classification, a validated methodology adopted by the european surveillance of antimicrobial consumption (esac -www.ua.ac.be/esac). 23 hospitals are participating: cyprus (5 hospitals); egypt (7); jordan (1); malta (1); tunisia (3); turkey (6). results are expressed in ddd/1000 bed-days. results: data from 2004, the first year of data collection, indicates that turkish hospitals seem to show the lowest overall consumption [230-480 ddd/1000bed days], whilst the cypriot hospitals show highest values [2900-7500 ddd/ 1000bed days]. the most common antibiotics used are the beta-lactams, especially the penicillins although in jordan and turkey cephalosporin consumption is very close to the penicillins. broad-spectrum penicillins [j01ca] are the mostly utilised penicillins in cyprus, jordan and tunisia whereas in malta and turkey the combination penicillins [j01cr] are the most widely used. there is more variability where cephalosporin consumption is concerned. cyprus utilises mainly first generation, jordan and malta the second generation. in egypt, tunisia and turkey there is significant variability between hospitals; nevertheless use of third generation cephalosporins appears to be significant. conclusion: a significant variability was evident between countries. this is likely to be multifactorial depending on the antibiotics licensed in a country, the national and/or hospital formulary, the type of hospital as well as any antibiotic donations that are relevant in some of the study hospitals. nevertheless, the preliminary results suggest that trends within hospitals of the same country tend to be similar. furthermore, the region as a whole seems to utilise a considerable quantity of broad-spectrum antimicrobials. this can be a factor in the high prevalence of resistance already documented in the study. russian pharmacoepidemiology study of the antibiotic prescription during pregnancy results: mean age of the patients was 25.6 ± 6.0 (min -14, max -52) years, mean gestational ages at admission to hospital was 27. 0 ± 10.8 (3 to 42) weeks. most often (77.5%) infection was community acquired and 2.1% -nosocomial, in 20% patients there was not to estimate origin of the infection. the most prevalent infections during pregnancy in russia was urinary tract infections -39.9%, std -19.3%, candidiasis -17.4%, rti -6.6% therefore the most interest was analysing the antibiotic prescription for uti in pregnancy (table) . in 28% cases were used topical (intravaginal) antimicrobial administration. most often of topically administrated antimicrobials (19.02% of all prescriptions) were prescribed combined drugs included antibacterials and amtimycotics. in 80.98% cases antimicrobials were prescribed systemically. mostly prescribed antimicrobials were beta-lactams (16.2% for outpatients and 57.7% for inpatients), ampicillin was prescribed more often (4.6% for outpatients and 31.5% for inpatients). amoxicillin + clavulanic acid was prescribed in 6.4% of outpatients and 5.6% inpatients pregnant women with uti. cephalosporins were prescribed in 5.2% and 20.6% for outpatient and inpatient uti (mainly iii-and ist generations). mitroimidazoles -1.8-7.4% (in general metronidasole), nitro-furanes -8. [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] .0%, aminolglycosides -4.6-3.8% were prescribed quite often but unjustified. other antimicrobials (fluoroquinolones, doxicicline, antiviral drugs, antifungals) were prescribed relatively rarely. despite the fact that most prescribed drugs were class b by fda, 5.8% all antimicrobials prescribed to pregnancy were class c, 0.8% class d and 9.1% were unclassified. conclusions: most often prescribed antimicrobials for uti (the most prevalent infections during pregnancy in russia) are betalactams and combined topical antibacterials. in 15.7% cases were prescribed antimicrobials of class c, d or unclassified by fda. in 25% outpatient and 53.6% inpatient were used antibiotics with low in vitro activity for uropathogens. objectives: to study the dynamics of the antibiotic usage in children from orphanages located in different russian cities as the result of interventions with the increased use of the most active antimicrobials and restrictions on use of the least active. methods: the study was performed in 12 orphanages (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) from 5 cities of european russia (moscow, saint-petersburg, smolensk, karachev, bryansk) . use of antimicrobials during the previous 12 months was analysed upon reviews of medical records of 743 children <7 years in 2003. appropriate recommendations on predominant use of selected beta-lactams (e.g. amoxicillin/clavulanate -amc) with restriction of antimicrobials of other classes (e.g. co-trimoxazole -sxt) were made where applicable on the basis of the expert analysis of antibiotic usage and pneumococcal nasopharyngeal resistance rates. repeated antibiotic usage analysis was performed 7 months later in 2004 upon reviews of medical records of 752 children <7 years. results: total usage of antimicrobials increased 1. increase of resistance to pen and aminopenicillins. enhanced use of cephalosporins led to increase of resistance to these drugs. in spite of recommendations to restrict usage of am/ox, aminoglycosides and sxt, the analysis showed that these antimicrobials still accounted for 13.2%, 7.8% and 7.5% of all prescriptions, respectively, thus dictating the need for further enforcement measures. antibiotic consumption in ambulatory care in latvia, 2004 s. berzina, m. ferech, g. ozolins, h. goosens (riga, lv; antwerp, be) objectives: to collect data on antibiotic consumption in ambulatory care (ac) in latvia according to the esac data collection protocol. esac (european surveillance of antimicrobial consumption, granted by dg sanco of the ec) is an international network of national surveillance systems, aiming to collect reliable and comparable data on antibiotic consumption in europe. methods: the data on ac antibiotic consumption for 2004 have been collected using atc/ddd classification (who, version 2005) and expressed in defined daily doses per 1000 inhabitants per day (did). data were obtained from the state medicinal agencybased on the reports of the wholesalers for ac. results: the overall use of antibiotics in ac was 11.7 did in 2004, which positions latvia to countries with comparatively low antibiotic consumption in europe. the mostly used class of antibiotics in ac were penicillins with extended spectrum (mainly amoxicillin) -3.97 did (33.9%). other frequently used antibiotics were tetracyclines (mainly doxycycline), representing 2.41 did (20.6%), combinations of penicillins/with betalactamase inhibitors (essentially co-amoxiclav) -1.18 did (10.1%), macrolides (mainly clarithromycin) 0.85 did (7.26%), fluoroquinolones (essentially ciprofloxacin) -0.84 did (7.17%) and combinations of sulphonamides and trimethroprim, incl.derivatives -1.02 did (8.71%). the most frequently used antibiotics in ac in latvia, in 2004, were amoxicillin (3.97 did) , doxycycline (2.41 did) , and co-amoxiclav (1.18 did) . conclusions: valid data on outpatient antibiotic use in latvia has been for the first time collected and delivered to european surveillance of antimicrobial consumption. this allows international comparison of the pattern of antibiotic consumption in latvia with other european countries. trends in glycopeptide antibiotics consumption over a 6-year period in a general hospital, athens, greece introduction: glycopeptide use is under restriction in hellenic hospitals since late '80s. the aim of our study was to record trends in their consumption over the last 6 years in our hospital (''a. fleming'' general hospital -300 beds) and to correlate these data with the numbers of important gram (+) strains isolated in our hospital during the same time period. methods: we measured glycopeptide use for the period 1999-2004 by using data from the pharmacy computer. consumption was expressed as ddds/1000 patient days (abc calc 3.0). furthermore we correlated these data with data from the microbiology department concerning numbers of mrsa, mrse and enterococci isolated during the same period. results: glycopeptide consumption was 1.3, 6.1, 6.3, 7.9, 4.5 and 13 ddds/1000 patient days for the years 1999, 2000, 2001, 2002, 2003, 2004 (900% increase) . at the same time the cumulative number of mrsa, mrse and enterococci isolated were 175, 224, 207, 415, 338, 479 respectively (170% increase) . when both types of data were put on the same graph, glycopeptide consumption correlated well with the number of important gram(+) strains isolated (figure). furthermore vre percentage among enterococci was 0, 0, 3.0, 0.6, 3.5, 1 for the study years respectively. it is worth noting that 87% of our mrsa strains were sensitive to rifampin, 83% to clindamycin, 74% to cotrimoxazole, 80% to clindamycin + rifampin and 74% to cotrimoxazole + rifampin. linezolid has not been introduced in our hospital yet. conclusions: (a) despite the restriction policy, a tremendous increase in glycopeptide use was recorded in our hospital during the study period and this correlated to the number of the important gram(+) strains isolated; (b) nevertheless, vre is not a significant problem for our hospital yet; and c. the huge increase in glycopeptide use could be avoided at least in part, since other, older and simpler antibiotics could substitute for glycopeptides in many cases. an audit of linezolid use in a university teaching hospital, galway, ireland objective: to audit linezolid use over a six-month period among the in-patient population of a 504-bed teaching hospital that includes most medical and surgical specialties with the exception of nephrology, rheumatology and orthopaedics. methods: a prospective audit was carried out of the prescribing of linezolid to in-patients from october 2004 to april 2005. the ward pharmacist recorded the details of all patients who were prescribed linezolid. a chart review was performed to assess the profile of patients prescribed linezolid, clinical and microbiological indications for treatment, adherence to treatment guidelines and documented adverse events. results: over the 6-month period 53 courses of linezolid were prescribed. fifty two percent of the patients for whom linezolid was prescribed were from surgical specialties; half of these patients were under the care of one surgeon. pneumonia was the clinical indication for use in 32% of cases and soft tissue infection in 36% of cases. the microbiological indication was clear in 64% of cases where mrsa or vre had been isolated. in 36% of cases therapy was either (1) empiric with no significant organisms isolated prior to prescription of linezolid or (2) therapy was directed against an organism that could have been treated with an alternative agent. duration of treatment exceeded 10 to 14 days in 40% of courses. an adverse event was recorded in the case of only one course of linezolid. conclusion: in more than a third of cases linezolid use was prescribed without clear justification. avoidable use of linezolid is associated with increased costs and risks of acquired resistance. participants prior to on an oral interview during which the interviewer filled in the answers. results: a total of 62 cm specialists completed the inquiry. this represents approximately 25% of the national quorum. mean age was 48 years (32-63). 22 of the 62 interviewed cm specialists worked full-time with more full-time employment in hospital labs (hl). next to routine microbiology, other activities performed by cm specialists are mainly the other domains of clinical biology, hospital hygiene and to a lesser extent quality control and lab management. almost two thirds of the interviewed cm specialists believes that their training hasn't prepared them properly for the tasks they are performing now. most desired changes include more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. cm does not exist as a separate speciality in belgium but is included in the 'clinical biology' speciality training. the majority of the respondents thinks that cm should become a sub-speciality (still part of clinical biology) but with a specific minimal training that needs to be defined. the majority of the cm specialists also believes that cm can share lab infrastructure with other disciplines and that the essential aspect of cm lies predominantly in the medical expertise. conclusion: cm training should put more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. the majority of the respondents feels that cm should become a sub-specialty (still part of clinical biology), with a well defined training curriculum. objectives: since more than 10 years, all infectious disease consultations have been recorded in a computerized database (epi info 6.04d, cdc). here we report on 1163 consultations of a fellow, conducted during 1 year, compared with 5708 consultations conducted by two veteran board-certified infectious disease consultants during the same period. methods: we analysed computerized consultation records, including demographic details of patients; referring department; initiative for, route and purpose of the consultation; and recommendations; and compared between the different consultants. results: a larger percentage of veterans' compared to the fellow's consultations, were requested by attending physicians (72% vs. 50%, p < 0.001), while follow-up (15% vs. 23%, p < 0.001), laboratory results (11% vs. 23%, p < 0.001) or prescription for a restricted antimicrobial agent (1.3% vs. 3 .3%, p < 0.001) were more prevalent in fellow consultations. the fellow had a higher rate of additional consultations (in which the patient was seen more than once) (40% vs. 30%, p < 0.001), and performed more bedside consultations (68% vs. 47%, p < 0.001) or consultation by curb side discussion (25% vs. 13.2%, p < 0.001), and less consultations by telephone (6.6% vs. 40.1%, p < 0.001). diagnosis and prophylaxis were more often the purposes of the veterans' consultations (44.8% vs. 36.1%, p < 0.001, 3.9% vs. 2.1%, p < 0.01, respectively), and they also offered new diagnoses more frequently (p < 0.005). the veteran consultants more often conducted consultation for communityacquired infections (53% vs. 44%, p < 0.001), and more often started antibiotic treatment (19% vs. 8 .9%, p < 0.001). conclusions: significant differences were detected between consultations conducted during the first year of a fellow compared to those of veteran infectious disease consultants. these differences reflect the changing demands and activities in the consultant's work as experience and knowledge accumulate. periodic analysis of computerized data of consultations facilitates supervision as well as direction of consultants' work, addressing issues such as antibiotic use and patterns of microbial resistance. bridging the gap between health care and public health; capacity building in infectious disease control objectives: in recent years, the european union (eu) has developed and supported many activities in the field of communicable diseases. these activities not only concern surveillance networks of specific infectious diseases (e.g. enter-net for salmonella and escherichia coli infections, ewgli for legionella infections, eiss for influenza infections), but also eu training programmes like epiet (european programme for intervention epidemiology training) and a eu communicable disease bulletin. even more recent are the eu's initiative bichat to improve preparedness and response to bioterrorism, and the development of a eu cdc. moreover, a major part of the new programme of community action in the field of public health (ph) (2003) (2004) (2005) (2006) (2007) (2008) concerns id, with not only a commitment to improve information and information exchange, but particularly to strengthen the international rapid response capacity.all this, to illustrate the importance of idc on the eu agenda. methods: the european public health association (eupha) is an umbrella organisation for ph associations in eu. in 2003 eupha has created an eupha section idc bringing together eupha members with expertise in this field and representatives of the various above mentioned eu initiatives in order to: promote and strengthen research in the field of idc; provide a platform for the exchange of information, experience and research in idc; bring together researchers, ph practitioners and policymakers active in idc; encourage joint activities in idc; and improve idc training. results: by now the section has 307 members from 50 different countries. as of 2005 the section is represented in the ecdc advisory forum. different section activities: organising 8 workshops, a breakfast meeting and a pre-conference meeting on timely idc topics during eupha conference. objectives: to establish a cross-border dutch-german network (www.mrsa-net.org) providing a user-friendly knowledge centre for hospitals, public health authorities, gps, nursing homes and laboratories. primary purpose is to aid in the reduction of mrsa-rates and limit the cross-border transmission of mrsa. guidelines and their implementation play a significant role in reaching these aims. cross-border (ca-) mrsa guidelines will be redesigned according to international standards and socio-cultural differences between the nations. methods: based on quality standards for safety and healthcare documentation used in high risk chemical organizations, a framework for a systematic content analysis of national mrsaguidelines was developed. national guidelines were analysed on the basis of this framework. results: a content analysis of the current national mrsaguidelines showed five dominating mrsa-perspectives: rule-, expert-, risk-, demand-and community-driven. german guidelines are mainly dominated by the rule-and expertdriven perspectives (guidelines are literally derived from law and follow the infection transmission route), in contrast to the dutch which focus on the demand of the user and the community (addressed to public health and acceptability of guidelines by users). conclusion: the analysis showed that the fact that there are different guideline-perspectives results in an enormous, confusing set of guidelines. the management and use of guidelines becomes uncontrollable and leads to an illusory organisation where healthcare workers don't act in accordance with the guidelines and start applying their own insights. this might lead to cost-increasing and contrasting situations. to implement guidelines successfully in a cross-border situation, a cultural and technical synchronisation alongside an integrated approach of the different perspectives of guidelines is necessary, inline with the current disease management models. further research about the redesign and the evaluation of those guidelines in practice will help achieving this. r. tsiklauri (tbilisi, ge) background: animal bites are a common but under recognized public health problem. it has been estimated that there are 8-10 000 bites each year in georgia, and based on an average visit and post-exposure treatments cost at list $120 000 per year. despite the frequency and expense of these injuries, there is little information about the incidence of animal bites because of a lack systematic reporting and a lack of measurement of the quality and completeness of reported data. objectives: to investigate animal bites and rabies reported cases, revealed unreported cases, analyse and based on study results find more effective epidemiological measures of animal bites and deaths (due to rabies) prevention in georgia. methods: the capture-recapture method was used, along with log-linear modelling. for sources were used to identify victims: policlinic/ambulatory reports, hospital reports, animal control reports and victim reports. results: in 1980-2001 years 150 700 dog and other animal bites were reported. the capture-recapture method estimated that there were 146 200 unreported bites. during these period 118 deaths due to rabies was registered in georgia and 67 (57%) cases among them have been registered during the last 6 years. the reasons of fatal cases were untreated (47%), uncompleted treated (34%) and late began post-exposure treated (19%) cases of bites (mostly dog bites). about 56% of bitted persons did not know about rabies and it's prevention measures. about 32% had incorrect information about prevention and only 12% of them knew epidemiological and clinical aspects of disease. about 16% of physicians who were responsible on quality post-exposure treatment had not an adequate knowledge. conclusion: dog and other animal bites are common but preventable injuries. to improve surveillance and prevention of rabies in georgia, the focus should be on educating the general public about the serious consequences of animal bite injuries and developing the animal's vaccination strategy. pharmacoeconomics and electronic resources p1493 the expected economic burden of methicillinresistant staphylococcus aureus in complicated skin and skin structure infections: a modelling approach a. kuznik, r. mallick, d. weber (collegeville, chapel hill, us) objective: to model the expected rate of clinical failure of initial empiric therapy and economic burden likely to be associated with the increasing prevalence of methicillinresistant staphylococcus aureus (mrsa) in patients hospitalised with complicated skin and skin structure infections (csssi) in the united states. methods: using published data on (1) the prevalence of mrsa and other bacterial pathogens causing csssi in the us, (2) the in-vitro susceptibility rates of commonly used regimens in csssi in the us in relation to the most pervasive pathogens identified above, and (3) estimated costs of failure of initial, empiric treatment from a recent study of a large us multi-hospital database, we developed a model to predict the expected clinical and economic impact of increasing prevalence of mrsa. specifically, clinical failure of 5 of the more commonly used initial regimens in csssi was modeled in terms of their in-vitro susceptibility rates with respect to mrsa, weighted by mrsa prevalence. varying the rate of mrsa further yielded projected clinical failure rates and costs attributable to increasing levels of methicillin resistance over time. results: given current 55% prevalence of s. aureus pathogens in csssi, half of them methicillin-resistant (base case mrsa = 27%), the model projected an overall clinical failure rate of 35.9% for 5 of the more commonly used initial regimens, with an expected overall treatment cost (in us dollars) of $5,492 per patient (range, $4,566-5,633) . if none of the s. aureus pathogens were resistant (mrsa = 0%), clinical failure rate was projected to be 18.4% and treatment cost to be $4,869 per patient. the differences in the two scenarios translated to an expected clinical failure rate of 17.5%, an incremental cost of $623 per patient, and for the 800,000 patients hospitalised for csssi annually in the us, an expected health care system burden of $498 million attributable to mrsa. under a "worst-case" scenario in which mrsa was the only causative pathogen (mrsa = 100%) in csssi, clinical failure rate was projected to be 79.3%, and treatment cost per patient was expected to be $7,035. conclusions: going beyond existing estimates, our model generated a substantial expected clinical failure rate and economic impact attributable to current mrsa levels, as well as simulations of the expected impact of increasing mrsa prevalence over time, varying levels of mrsa across regions and choice of initial empiric regimens. treatment of complicated skin and skin structure infections in the us: expected cost differences between tigecycline and vancomycin/aztreonam r. mallick, a. kuznik, d. weber (collegeville, chapel hill, us) objective: to compare tigecycline and vancomycin/aztreonam in terms of treatment-related costs for patients hospitalised in the united states with complicated skin and skin structure infections (csssi). methods: we conducted a retrospective analysis of pooled data from us centres in two randomized, double-blind clinical studies comparing tigecycline and vancomycin/aztreonam in the treatment of csssi. using regression analysis, we estimated the effect of tigecycline treatment on hospital length of stay (los), controlling for other significant predictors. using published estimates of daily hospitalisation cost of csssi in the us from a multi-hospital audit, we then translated the estimated impact on los into economic terms. this analysis was repeated for the subgroup of patients in which the primary pathogen was methicillin-resistant staphylococcus aureus (mrsa). clinical efficacy (tigecycline 81%, vancomycin/aztreonam 83.1%; p = 0.376) was similar across treatments and was not included as a model parameter. results: our retrospective analysis of the pooled clinical data from us centres found that tigecycline was associated with a shorter los [)1.85 days (p = 0.015)] compared with the combination of vancomycin/aztreonam in the treatment of patients with csssi. at a mean daily hospitalisation cost (in us $) of $794, excluding antibiotic costs, this translated into expected medical cost savings of $1,469 per patient for tigecycline compared with vancomycin/aztreonam. in the mrsa subgroup, comprising 26% of the clinical study sample, tigecycline was associated with a greater reduction in los [)2.82 days (p = 0.011)] compared with vancomycin/ aztreonam, translating to expected medical cost savings of $2,239 per patient treated with tigecycline. these expected medical cost savings more than offset the higher average daily drug acquisition costs of tigecycline ($118/day) relative to the vancomycin/aztreonam combination ($110/day). conclusion: in a retrospective analysis of pooled clinical data of patients with csssi treated at us centres, tigecycline was associated with a significantly reduced length of hospital stay relative to vancomycin/aztreonam; this translated into substantial cost savings, especially in the subset of csssi patients with mrsa. the economic impact of linezolid in the treatment of skin and soft tissue mrsa infections in italy m. eandi, p. dale, s. sorensen, t. baker, m. procaccini, s. duttagupta (turin, it; london, uk; bethesda, us; rome, it; new york, us) objective: linezolid has been shown to be highly effective against infections caused by methicillin-resistant staphylococcus aureus (mrsa) in patients with complicated skin and soft tissue infections (cssti). the objective of this study was to evaluate the clinical and economic consequences of using linezolid for the empiric treatment of cssti from the italian hospital perspective. methods: a decision-analytic model was developed to calculate the clinical and cost outcomes of empiric treatment of hospitalized patients with cssti in italy prescribed linezolid, vancomycin or teicoplanin. efficacy data were derived from clinical trials. costs from published sources were applied to tests, adverse events, and days of intravenous and oral (linezolid only) treatment and hospitalization by ward type (general, intensive-care). resource use and utilization patterns were obtained from a combination of clinical trial data and expert opinion. outcomes included total costs per patient, cost per cure and cost per death avoided. uncertainty surrounding the ce ratio was tested using one-way sensitivity analysis. results: starting empiric treatment with linezolid resulted in 98.4% of patients cured from mrsa compared to 98.0% with vancomycin. the average cost per patient treated with linezolid was €6,305 versus €6,228 for patients treated with vancomycin. this resulted in a cost per cure of €22,404. in a separate analysis more patients were cured using linezolid (97.6%) compared to teicoplanin (94.7%). the average total cost per episode was €6,401 for linezolid treated patients versus €5,897 for teicoplanin treated patients, resulting in a cost per cure of €17,100. the most sensitive parameters included hospital los and mrsa resistance rate. conclusions: in the treatment of cssti due to suspected mrsa in italy, the empiric use of linezolid is cost-effective when compared to vancomycin and teicoplanin p1496 outpatient and home parenteral antimicrobial therapy for the treatment of cellulitis: evaluation of efficacy and cost h. ziglam, r. tilley, c. wootton, j. morrison, d. nathwani (dundee, uk) objective: outpatient and home parenteral antibiotic therapy (ohpat) programmes are effective, well tolerated and economically advantageous in carefully selected patient populations. skin and soft tissue infections represent a high burden disease which in amenable to treatment by ophat programmes. we retrospectively analysed our outcomes registry to evaluate the clinical and health economic impact of treating cellulitis in this setting. methods: we have reviewed 465 patients with cellulitis and erysipelas who were treated with ohpat. each patient treatment has a full integrated care pathway (icp). the icp documents the microbiological outcome, drug and vascular access complication rates, impact on drug costs and in-patient bed days on the 465 number of patients treated from april 1998 to march 2005 are presented here. we also reviewed using the smr1o inpatient discharge diagnosis data from the information statistics division scotland (isd) and the dundee infectious diseases units (didu) outcomes registry database. the key diagnosis (icd 10 codes) groups considered were cellulitis (lo30, 31, 32, 33, 38, 39) and erysipelas (a46x) over eight consecutive years (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) . results: the patients received intravenous antibiotic therapy for a mean duration of 5.64 days. the two primary agents administered were once-daily ceftriaxone in 84%of patients and teicoplanin in 9.8% of patients. of the 465 patients, 431 (92.6%) were cured or improved; 5 worsened and required surgery. tinea pedis was found in 31% of patients treated for cellulitis. economic benefits were realized despite use of more expensive agents. data from the dundee outcomes registry revealed a mean reduction in length of hospitalization from 5.8 days (1996/ 1997) to3.2 in 2003-2004-a reduction of 48% compared to scottish data from isd which did not show any changes in length of hospitalization between year 1996/1997 (5.33 days) and year 2003-2004 (5.92 days) . conclusions: we have found that ohpat is clinically effective and can be administered safely and successfully in an outpatient setting. the majority of complications were minor, and 93% of patients were cured. tinea pedis and were found to be significant risk factors for acute cellulitis and indicate that improved awareness and management of toe web intertrigo might reduce the incidence of cellulitis. this analysis also supports the premise that an adult ohpat programme can substantially reduce healthcare resource use in the european healthcare setting. cost-effectiveness analysis of intravenous moxifloxacin compared to levofloxacin in hospitalised elderly patients with communityacquired pneumonia objective: to evaluate the cost-effectiveness of moxifloxacin compared to levofloxacin in hospitalised patients aged ‡65 with community acquired pneumonia (cap). methods: a randomised double-blind parallel group study was conducted in 47 us hospitals. patients had radiological evidence of bacterial pneumonia confirmed by at least 2 other signs, were aged ‡65 years and were managed as inpatients on initiation of treatment. patients initially received moxifloxacin 400 mg iv o.d. or levofloxacin 500 mg iv o.d., and once stabilised were switched to oral therapy with the same agent. the effectiveness endpoint for the economic analysis was the percentage of patients successfully treated, defined as patients with marked improvement, resolution or clinical cure at test of cure visit after 7-14 days of therapy who did not experience a serious cardiac adverse event. total costs were estimated from the perspective of the treating hospital and included antibiotic drugs, hospital stay, hospital re-admission within 28 days and cost of managing treatment failures. results: 394 patients were included in this analysis, 195 randomised to moxifloxacin and 199 to levofloxacin. 82% (95% ci: 77-88%) of moxifloxacin and 75% (69-81%) of levofloxacin treated patients were successfully treated (resolution, clinical cure at toc and no serious drug related aes). in the moxifloxacin group patients reported a mean of 3.5 days of iv antibiotic treatment and 5.7 days inpatient stay with 3.4 days iv antibiotic treatment and 6.1 days inpatient stay in the levofloxacin group. mean per patient drug cost was $257 in the moxifloxacin group, $222 in the levofloxacin group. mean total cost was $8,339 in the moxifloxacin group and $8,354 in the levofloxacin group. findings were consistent across a range of patient subgroups. costs were sensitive to length of hospital stay. conclusions: patients in the moxifloxacin group had higher rates of successful treatment at slightly lower average costs than the levofloxacin group. this confirms the results of the target study where moxifloxacin showed superior clinical efficacy in comparison to co-amoxiclav with or without clarithromycin in hospitalised cap patients. antibiotic costs were slightly higher in the moxifloxacin group than the levofloxacin group but total costs were slightly lower, due to reduced hospital stay. economic impact of invasive fungal infections in icu patients in a tertiary care hospital in switzerland a. imhof, w. zingg, r. laffer, c. ruef (zurich, ch) objectives: invasive fungal infections (ifi) cause significant morbidity and mortality. the management of invasive fungal infections is currently undergoing important changes due to the availability of new therapeutic agents with improved safety profiles but the acquisition costs of these new agents are high. we evaluated the average overall cost of management (microbiological diagnosis and treatment) of invasive fungal infection in critically ill patients at a large university hospital. methods: a retrospective (2003) (2004) , pairwise-matched cohort study was performed on 4 surgical icus and one medical icu at our university hospital. icu patients with documented ifi (n = 23) were matched with control subjects (n = 46) on the basis of disease severity, sex and age (±5 years). clinical outcome was principally evaluated by in-hospital mortality. the economic impact of microbiological studies and antibiotic treatment was assessed. calculations were based on the period between admission and diagnosis of ifi in cases and the duration of hospital stay in controls, respectively. results: the median length of hospital and icu stay differed significantly between cases and controls (43 vs 14 days, 19 vs 3 days, p < 0.001, respectively). ifi occurred after a median hospital stay of 22 (range 2-95) days. the mortality rate for patients with ifi and matched control subjects were 30.4% and 10.9%, respectively (p = 0.04). there was no significant difference between cases and controls for charlson index, mccabe and saps ii score. median number of antibiotic treatment courses was 3 for cases and 1 for controls (p = 0.003), with a median duration of therapy of 6 days vs 1 day (p < 0.0001), respectively. microbiological studies (mis) were conducted 78 times/100 patient-days (pd) in cases and 17 times/100 pd in the control group (p = 0.03). the most frequent samples were bloodcultures in both groups. swabs were ordered significantly more frequently in cases (median 25 (range: 1-150) vs. 2 (0-23); p = 0.04). the cost for mis was 13'728 euro/100 pd in cases vs. 3742 euro/100 pd in controls, and the costs for antifungal therapy 4'373 euro/100 pd vs. 99 euro/100 pd, respectively. conclusions: ifi is associated with excess length of icu and hospital stay, increased use of antibiotics and microbiological diagnostics. the microbiological studies have a significant economic impact on the treatment of ifi. cost-effectiveness of voriconazole to amphotericin b deoxycholate in early and late treatment of invasive aspergillosis r. greene, j. mauskopf, c. roberts, t. zyczynski, h. schlamm (boston, research triangle park, new york, us) objective: we estimate the cost-effectiveness of alternative initial drug treatments of invasive pulmonary aspergillosis (ipa) in suspected earlier and later lung involvement, based on the presence or absence of the halo sign on thoracic computed tomography (ct). methods: we constructed a decision analysis model comparing 12-week treatment outcomes for a subset of patients enrolled in a clinical trial of initial treatment of ipa with amphotericin b dexoycholate (ambd) vs voriconazole (vor). patients included those with suspected lung involvement who underwent a baseline thoracic ct. the subset was subdivided into two groups based on the presence or absence of a characteristic ct halo sign, a perimeter of ground glass ct opacity surrounding a solid lung nodule ‡1 cm diameter, known as an early indicator of ipa. healthcare resource use and survival data were obtained directly from the clinical trial. us unit costs for drugs and health care services were applied from standard data sources. cost and survival at 12-weeks were estimated for those with and without a halo sign at baseline. incremental cost-effectiveness ratios comparing vor to ambd were calculated for both patient subgroups. sensitivity of results to uncertainty in health care use and cost estimates was tested. results: patients in the halo subgroup had better survival than those in the no-halo subgroup (70.6% vs 54.4%), with lower total treatment cost ($44,352 vs $47,077) . survival was higher for vor than for ambd in both patient subgroups (halo: 75.3% vs 65.2%; no-halo: 68.3% vs 39.5%). in the halo subgroup, total costs were lower for those treated with vor than for those treated with ambd ($40,380 vs $48,985) . in the no-halo subgroup, total cost per patient was slightly higher for those treated with vor ($48,133 vs $45,938) . accounting for the difference in survival, the incremental cost-effectiveness ratio for vor compared to ambd was $7,625 per additional 12-week survivor in this subgroup. conclusions: earlier identification and treatment of ipa appears to result in better survival and potentially lower costs than later treatment. initial treatment of ipa with vor improves survival in patients with early or late disease compared with ambd, is cost saving in the halo sub-group, and is cost-effective in the no-halo subgroup, within the constraints of our analysis. objective: to describe and compare the nursing labor time required for preparation and administration of liposomal amphotericin b (l-amb), amphotericin b deoxycholate (ambd), and voriconazole (vor). methods: activities associated with nurse preparation and administration of the three study drugs were timed by trained observers at five hospitals (one in italy, three in france, and one in the united kingdom). target tasks were classified as those likely to be affected by the difference between the drugs and excluded those tasks likely to differ because of site-specific factors (e.g., travel time to a patient room in different hospitals). target tasks included: obtain supplies and medications; prepare medications; educate patient; administer medications; monitor for adverse events; and prepare follow-up medications. the mean times for administration of a single day of study drug were summarised and compared, accounting for a single daily dose of l-amb and ambd and 2 daily doses of vor iv or oral. results: sixty-nine patients were observed receiving 256 doses of study medications at the five hospitals. time of administration in minutes per day was 20, 16, 14, and 3 for l-amb, ambd, vor iv, and vor oral, respectively. administration time was significantly lower for vor iv compared with l-amb (p < 0.05) and for vor oral compared to all iv regimens (p < 0.05). the task of preparation of medications required the most time for iv formulations, and was longer in the l-amb group than the others (l-amb: 12 mins vs ambd: 7 mins; vor iv: 9 mins). ambd required more time for patient monitoring and administration of followup drugs than other formulations (ambd: 7 mins vs l-amb: 4 mins; vor iv: 2 mins). conclusion: vor iv required significantly less time to prepare and administer on a daily basis compared to l-amb. measurements of iv antifungal versus oral vor administration suggest the opportunity to save 10-17 minutes per day by switching to oral therapy when possible. need of cost-effectiveness investigation focused on diagnosis, management and prevention of osteopenia and osteoporosis in the setting of hiv disease treated with haart: when to act, how to act, which patients are the first target of intervention r. manfredi, l. calza, f. chiodo (bologna, it) background: osteopenia/osteoporosis are emerging untoward effects of hiv infection/haart. the pathogenesis is multifactorial, involving all classes of anti-hiv drugs, although protease inhibitor use, overall haart duration, and the male sex, seem related to a greater risk.epidemiologicalclinical data. in an ongoing study at our centre where >1000 hiv-infected patients (p) are followed, bone mineral density was assessed in lumbar spine/femural head by a dual energy xray absorptiometry (dexa) exam to estimate the prevalence of osteopenia/osteoporosis. in a screening of~100 p, the frequency of osteopenia and osteoporosis (based on lumbar t-score) was 38% and~10%, respectively. an increased risk was found in p treated with protease inhibitors versus those receiving nonnucleoside reverse transcriptase inhibitors or triple nucleoside/ nucleotide combinations. discussion and future insights: prospective studies of extensive p samples are needed, to elucidate the epidemiology, pathogenesis, clinical issues and evolution of hiv-associated bone metabolism anomalies. when planning strategies for their early diagnosis, prevention and management also cost-effectiveness issues should be considered, since no pharmacoeconomic data still exist in this setting. although severe consequences (e.g. pathological fractures, prosthetic implants) are expected to be infrequent their consequences in terms of length and intensity of hospitalization, related costs, and especially severe consequences on the p's quality of life, play a notable role. anyway, the most reliable diagnostic procedure (dexa) has affordable costs (around eur 43.40 for a total-body scan which also offers a body composition assessment), as well as the first-line drugs for osteopenia, e.g. supplementation with calcium (eur 5-6.5/month), and vitamin d (eur 7/month). these costs cannot be compared with the costs of a standard care of an asymptomatic haart-treated p (eur 471 to 774/month) and the immunological, virologic, laboratory and clinical controls made at least quarterly. like postmenopausal osteopenia/osteoporosis (burdened by a greater risk of bone mass anomalies) also hiv disease should be investigated from multiple cost-effectiveness points of view to establish which p are the preferred candidates for a dexa screening when this examination is more useful during hiv disease course and therapy, when the exam should be repeated and when and how to intervene pharmacologically to prevent serious and potentially invalidating complications. a comparative study on the cost of new drugs in different therapeutic categories m. falagas, k. fragoulis, g. zouglakis, i. karydis (athens, gr) objectives: drug treatment is becoming more expensive due to the increased cost for the introduction of new drugs and there seems to be an uneven distribution of medication cost for different therapeutic categories. we hypothesized that the cost of new antimicrobial agents may differ from that of other therapeutic categories and this may play a role in the stagnation of development of new antibiotics. methods: we performed a pharmaco-economical comparative analysis of the drug cost of treatment for new agents introduced in the united states drug market in various therapeutic categories. we calculated the drug cost [in us dollars (usd)] of a 10-day treatment of all new drugs approved by the fda during the period between january 1997 and july 2003, according to the 2004 red book pharmacy's fundamental reference. results: new anti-neoplastic agents were found to be the most expensive drugs in comparison to all other therapeutic categories with a median 10-day drug-treatment cost of 848 usd compared to the median 10-day drug-treatment costs of all other categories ranging from 29 to 301 usd (table) . on the other hand, new antimicrobial drugs were found to be much less expensive with a median 10-day drug-treatment cost of 137 and 85 usd for all anti-microbial agents and for anti-microbial agents excluding anti-hiv medications, respectively. conclusion: the drug-treatment cost of new medications varies considerably by different therapeutic categories. this fact may influence industry decisions regarding the development of new drugs and may play a role in the shortage of new anti-microbial agents in the fight against the serious problem of anti-microbial resistance. usage and expenditure of 4f-quinolones in a tertiary hospital in 2004 t.a. peppas, k. malengou, n. zachos, d. voutsinas, o. kosmopoulou, n. galanakis (piraeus, athens, gr) objective: our aim was to assess 4-f-quinolone (4fq) usage, distribution and expenditure over 2 years. objective: to analyse antiobiotic utilization in croatia using anatomical-therapeutic-chemical (atc) drug classification system and number of defined daily doses (ddd). methods: data on the number of packages and purchase price were collected for each individual drug. these data were used to calculate the number of defined daily doses (ddd) and ddd per 1000 inhabitants per day (ddd/tid). data obtained from 80% of pharmacies and 65% of hospitals were extrapolated to the total number of pharmacies and hospitals in croatia. drug utilization 90% (du90%) segment was used as a prescribing quality indicator. results: in 2004, the overall utilization of antibiotics in croatia amounted to 23.35ddd/tid. according to drug groups, penicillins (j01c) showed highest utilization (12.24 ddd/tid), predominated by the subgroup of penicillin combinations (including beta-lactamase inhibitors, j01cr) with6.86 ddd/ tid, within which the combination of amoxicillin + clavulanic acid accounted for99.9% with 6.85ddd/tid. broad-spectrum penicillins (j01ca) accounted for 43.2%(5.28 ddd/tid) of total penicillin utilization, with a 97.7% predominance of amoxicillin (5.16 ddd/tid). cephalosporins (j01d) ranked second with 4.00ddd/tid, followed by macrolides and lincosamides (j01f) with 2.41 ddd/tid, with an 86.84%predominance of macrolides (j01fa) with 2.09ddd/tid. among the latter, azithromycin showed highest utilization with 1.44 ddd/tid, accounting for 68.66% of total macrolide utilization. tetracyclines (j01a) ranked fourth with 2.02ddd/tid, accounting for 8.65% of overall antibiotic utilization, followed by quinolones with 1.65 ddd/tid, other antimicrobials with 0.91 ddd/tid, and aminoglycosides with 0.16ddd/tid. sulfonamides (j01e) accounted for a negligible proportion of overall utilization. du 90% segment included 9 of 43 antibiotics registered in croatia, with amoxicillin + clavulanic acid as the leading one, followed by cephalexin with 1.83, cefuroxime with 1.53, azithromycin and norfloxacin with 1.3each, nitrofurantoin with 0.56 and clarithromycin with 0.53ddd/tid. hospital utilization accounted for 7.1% of overall antibiotic utilization expressed in ddd/tid and 22.7% of the respective financial cost, predominated by aminoglycosides (j01g) with 72% and 94.2%, and lowest proportion of tetracyclines (j01a) with 4.3%, and 2.4%, respectively. conclusion: the utilization of antibiotics in croatia is among the highest in europe, mostly due to overuse of amoxicillin + clavulanic acid, which has no rational ground in professional guidelines. objective: the objective of the research was to analyse antibiotics prescripsion behavior by family doctors and specialists treatments prior to and after the introduction of the health care reform in poland. materials and methods: prescriptions from the first six months of 1999 and 2005 were compared. the data was collected from two randomly chosen pharmacies in the city of zabrze that supply the citizens of the silesian agglomeration from various social backgrounds. taking into account the value of a single antibiotics package and the price a patient has to pay for it an average price of medications prescribed by family and specialist doctors was calculated. results: a total of 30793 prescriptions were analysed out of which 24834 dated from 1999 and 5959 from 2005.in the first half-year of 1999 the percentage of prescriptions for antibiotics reached 7.76% on average, and in the year 2005 -the average was 11.6%. in the first half-year of 1999 family doctors mostly prescribed: penicyllins (43%), makrolids (26%), cephalosporins (16%), tetracyclins (11%), chinolons (3%). in the same period spcialist doctors prescribed: penicyllins (41%), cephalosporins (17%), makrolids (15%), tetracyclins (13%), lincozamids (9%), chinolons (4%). in the first half-year of 2005 family doctors most often prescribed penicyllins -(44.8%), makrolids -(27.1%), cephalosporins -(12.5%), tetracyclins -(9.4%) and lincozamidsbased (3.1%) treatments specialist doctors, on the other hand, prescribed penicllins (41.7%), makrolids (17.9%), cephalosporins (17.7%), tetracyclins (12.1%), lincozamids (5.2%) and chinolons (3%). the average prices of the prescribed medications in the years 1999 and 2005 were, respectively: for family doctors-eu 3.08 and 4.01, for specialist doctors eu 4.44 and 3.87. conclusions: there has been a considerable increase in the percentage of prescriptions for antibiotics from 7.45% (in 1999) to 11.3% (in 2005). the tendency towards prescribing antibiotics in the specific groups of doctors has not changed significantly. in both years prescriptions for antibiotics were in line with the recommendations. also, prices of medications prescribed by family doctors have risen. internet guide on antimicrobial resistance a. sosa, f. traub, s. valovic, p. chea (boston, us) objectives: (1) to organize the plethora of information available, providing clinicians the tools to easily access available online resources that include academic institutions, professional societies as well as sites maintained by private individuals; (2) to inform clinicians of new advances in the epidemiology, diagnosis, treatment, and prevention of most common infections; (3) to inform on subjects such as clinical trials in antimicrobial resistance, information about specific pathogens and their infections, genomic resources, culture collections, electronic images of pathogens and antimicrobial agents, antimicrobial resistance lecture and teaching materials, environmental health and safety information, and a listing of websites of infectious disease and clinical microbiology professional societies. methods: we defined four inclusion criteria after extensive consulting with apua staff and scientific advisory members: (1) recognized/reputable source; (2) high quality of information presented; (3) potential usefulness to medical professionals and the general public; and (4) ease of navigation. ideal parameters were determined for the guide's scope and the appropriate sources identified online were subsequently reviewed. a set of broad categories was established to organize the topics and the online resources. sites reviewed included those maintained by the federal government, academic institutions, nonprofit organizations, and commercial entities. some personal websites were included because of their quality and their association with academic institutions. this review is intended as an introduction to amr websites. results: with use of popular search engines, such as google, yahoo!, and altavista, we initially identified a great number of websites. using broad search terms, such as "antibiotic resistance," we identified 1,310,000 web addresses. the term "antimicrobial resistance" generated 578,000 hits, and the term "drug resistance" generated 6,190,000 hits. conclusions: websites found were classified following a systematic topic structure. each website listed describes: (1) full citation of the resource: author/editor and title of website; (2) date of publication or last revision; (3) methods and results: the portal is free to use but requires registration and has more than 1850 registered users as of november 2005. of these, 20% are in-training positions, 28% hold a faculty position in infectious diseases (id), microbiology or hemato-oncology, 24% are specialists in a non-university, but a teaching setting. the remaining are a mixed group of primary physicians, other speciality doctors and pharmaceutical company workers. running costs of the portal are partially covered by educational grants from pharmaceutical sponsors who have no role in organization of the site, but their names are acknowledged. articles chosen by the two faculty members, one id physician and one haematologist, are sent to registered users by daily e-mail postings. these are selected from toc alerts of various core clinical journals and well known educational web sites (e.g. cdc, who, medscape). a short turkish summary is provided and the reader is referred to the abstract and if available, to the free full text of original article with a link. other materials included guidelines, free slide sets, study protocols and updates from the group, cme activities and meeting announcements. registrants may also use the site for expert opinion. during the trial period, the site has been visited 30740 times with 282239 hits. approximately 5.5 gb material was downloaded. the frequency of readings are related with the time (highest between 9.00-11.00 am during weekdays and lowest during weekends), the type of documents (i.e. educational materials and guidelines), popularity of the news (e.g. peaked during an epidemic of avian influenza when related news and articles announced). the pages are most frequently visited by id specialists followed by clinical microbiology, haematology and pharmaceutical company workers (33%, 19%, 9% and 8% respectively). conclusion: timely published medical data have high attraction rates among physicians. our results also indicated that, a web page gets ''old'' after about a month of publishing, emphasizing the importance of well-timed announcements of the portal material. neli and nric survey: information needs of infectious disease professionals p. kostkova, s. d'souza, g. madle, j. mani-saada, s. wiseman, a. roy, j. weinberg (london, uk) healthcare professionals are increasingly facing the problem of information overflow. it is getting impossible to keep up-to-date with the latest research findings, care guidelines and pathways, government strategies and national and local policies. internet enables an instant information dissemination enabling access to the latest results at any time as well as informal knowledge exchange by using chat rooms and discussion forums. however, it is getting increasingly difficult for busy professionals to find reliable quality-assured information on the internet when they need it. national internet libraries in the uk are addressing this problem: the umbrella portal national electronic library of infection neli (http://www.neli.org.uk) providing a single access portal to quality-assured information on treatment, diagnosis, prevention and management of infection diseases, and the national resource for infection control nric (http:// www.nric.org.uk) -a single-stop shop for policies, guidelines and research around infection control, hosted by neli. to better meet the information needs of these internet portals, accessed by 2000 unique users per month, we are conducting an information needs study to explore clinical questions, user needs and disease priorities of users seeking answers on neli and nric. a pilot qualitative online questionnaire-based study revealed that our users come from the variety of professionals: clinical scientist, consultant, registrar, psychotherapist, lecturer, gp, medical librarian, information scientist, health protection. these have questions mainly around hiv, tinea, molluscum contagio, meningitis, cold, mrsa, lyme, toxoplasma, chicken pox, influenza, diarrhoea and vomiting, rash, staph. aureus, traveller infections antibiotics resistance, malaria, mmr, meningitis, viral myocarditis, anthrax, smallpox, and tb. this is in line with our quantitative weblog-based evaluation of the most commonly access topics on neli by nhs-based users: antimicrobial resistance and hae (10.27%), tb (9.54%), meningitis (9.47%), hiv (8.95%), chlamydia (6.31%), e. coli (5.54%), staph. aureus (5.26%), adenovirus (4.84%), blood borne infections (4.44%). the results of the ongoing analysis of google search keywords that brought users to neli and nric will be discussed. further results identifying the needs specific to the infection disease professions will be discussed in relation to differences in the national variations in information needs and priorities. training in infection s. d'souza, p. kostkova, f. cooke, a. holmes (london, uk) specialists today require prompt access to quality information in order to work effectively. the diversity of specialist interests in the field of infection has led to the formation of a large number of professional and scientific societies. these play an increasingly important role in ensuring that the trainee is effectively supported, not only during the period of training, but also in longer-term personal development. details of relevant societies, conferences, grants etc are, on most society websites, confined to those for either that society or others in that specialty only, and knowledge of the numerous places in which to look for this information is necessary to find out the latest information. training in infection (tii -www.trainingininfection.org.uk) is an online resource, primarily aimed at infection specialist trainees but useful throughout the career path, which brings together this information into one central access point, so that users from all infection specialties can find the appropriate information for their specialty quickly and easily. it identifies and links to the key relevant resources covering a broad range of infection related disciplines in a dynamic database structure. information on societies, conferences, grants, journals, textbooks and more are available on the site, and have been put together to create a one-stop infection training portal. online discussion forums to be implemented will allow trainees' to share ideas and make the most of their combined expertise, and users will be able to receive alerts on new information in their specialty as well as be reminded of conference deadlines, journal submission deadlines etc. the ability to discuss regional issues online within specialties also aims to promote greater local and international collaboration. training in infection is endorsed by the national electronic library of infection (neli -www.neli.org.uk), an established digital library bringing together the best available online evidence-based resources on the investigation, treatment, prevention and control of infectious diseases. research designs and statistical methods in medical abstracts m. kompoti, m. matsagoura, a. koutsovasilis, a. koutsovasili, s. drimis (athens, gr) statistical methods used in biomedical research articles are being increasingly scrutinized in medical journals. however, no such strict policy is generally applied in abstracts presented in medical congresses. objective: this study aimed at assessing the frequency of research designs and statistical methods reported in abstracts presented in two successive years of the european congress of clinical microbiology and infectious diseases (eccmid). material and methods: we reviewed all abstracts included in the abstract book of the 14th eccmid (prague 2004) (pg) and the 15th eccmid (copenhagen 2005) (cp). all abstracts of original research studies but no abstracts of lectures were included in our study. two independent investigators read all abstracts and extracted information concerning origin, type (clinical, laboratory, animal model), research design, sample size and statistical methods used in the study. data analysis was performed with logistic regression and pearson's chi-square test for categorical variables and student's t-test for continuous variables. statistical significance level was set at p < 0.05. results: a total of 4178 abstracts were included in the analysis according to eligibility criteria (2110 from pg and 2068 from cp). laboratory studies prevailed (61%) followed by clinical studies (35%) and experimental studies with animal models (4%). the majority (79.3%) of the studies were observational (retrospective, prospective, cross-sectional) of which 6.3% concerned diagnostic accuracy testing of laboratory methods and 2.3% were pharmacological studies, 3.8% were randomized controlled trials. statistical evaluation was clearly described in 26.5% of abstracts (26.1% in pg and 19.8% in cp, p < 0.001), while the rest of abstracts included only descriptive statistics or no statistics at all. the proportion of statistical methods reporting varied according to the type of the study (animal model studies 49.4%, clinical studies 38.9% and laboratory studies 12.0%, p < 0.001). multicentre research studies reported statistics more frequently than single-center studies (26.5% vs. 21.5%, respectively, p = 0.005). conclusions: statistical analysis is an inseparable part of original research. research design as well as the implemented statistical methods should always be reported in an adequate manner, thus improving the scientific quality of abstracts. antimicrobial pk/pd p1512 nxl103-oral streptogramin: a phase i, doubleblind, single escalating oral dose study to evaluate safety, tolerability and pharmacokinetics in healthy adult male volunteers m. rangaraju, j. rey, j. hodgson (romainville, vitry sur seine, fr) background: nxl 103 (formerly xrp 2868) is a novel semisynthetic oral streptogramin that consists of a 30/70 (w/w ratio) association of a pristinamycin ia (pi) derivative and a pristinamycin iib (pii) derivative. nxl 103 is being developed for the treatment of respiratory tract and skin and skin structure infections. methods: 60 healthy male subjects were enrolled in this study. 10 subjects in each of 6 cohorts (125 mg, 250 mg, 500 mg, 1000 mg, 1500 mg and 2000 mg) received either nxl103 (8) or placebo (2) . an additional cohort of 10 subjects received a single dose of 500 mg nxl103 in fasting and fed conditions. blood and urine samples for pk analysis were collected at multiple time points. safety was assessed via adverse events, physical examination, clinical laboratory data, ecg and cardiac monitoring. results: nxl103 administered as 125 mg capsules at single doses from 125 mg to 2000 mg was well tolerated and safe. there was no serious or severe adverse event, no dosedependency in the number of aes or their severity, no significant variation in blood pressure or heart rate, no abnormality on ecg recording, and no clinically significant changes compared to baseline for laboratory parameters. both components were rapidly absorbed; pi being slightly more rapidly absorbed than pii. the cmax and auc (0-t) increased approximately in proportion with dose. the proportion of pi and pii components estimated on mean exposure values was approximately comparable to that administered (30/70), indicating that the relative bioavailabilities of pi and pii are simila. elimination half-life ranged from between 2 to 3 hours for pi to 4 to 6 hours for pii. food increased the bioavailability of pi and pii by approximately 20%. conclusions: nxl103 is safe, well tolerated and exhibits predictable pk properties in healthy volunteers in doses up to 2000 mg administered as a single dose. correlation of vancomycin and daptomycin susceptibility in staphylococcus aureus in reference to accessory gene regulator polymorphism and function w. rose, m. rybak, b. tsuji, g. kaatz, g. sakoulas (detroit, new york, us) objective: polymorphism at the accessory gene regulator (agr) locus in s. aureus (sa) defines 4 groups (i-iv). agr group ii sa have been associated with glycopeptide treatment failure in patients. sa with loss of agr function appear to have a higher tendency to become vancomycin (v) resistant. it is unknown whether this association only pertains to glycopeptides. we examined the effect of varying v and daptomycin (d) against agr+ and agr null pairs in an in vitro pharmacodynamic model (ivpm). methods: agr group i and ii wild-type prototype and knockout (tetm::agr) pairs were evaluated. mic values were determined according to clinical laboratory standards institute. ivpm glass and hollow fibre models were used to simulate dosages and auc/mic exposures for v ranging from 62.5 mg-1 g q 12 h fauc/mic range 31-665 mg/l*h, and d 0.75 mg/kg-6 mg/ kg/day fauc/mic range 37.5-346. the dosage regimen and auc/mic breakpoints that produced resistance was then evaluated in the hollow fibre ivpm. all ivpm simulations were performed in duplicate over 72 h. resistance was evaluated using 3 and 6 x mic screening plates at 0, 8, 24, 48, 56 and 72 h. results: pre-exposure mic values for agr i± and agr ii± were 0.75/1 and 1 for v and 0.25 lg/ml for d respectively. vintermediate resistance (mic = 8 mg/l) was detected in both agr i and ii null strains at a simulated v dosage of 62.5 mg q 12h (auc/mic 31), representing an mic increase of 8-10 fold. this breakpoint for resistance was verified in the hollow fibre model. although significant regrowth was noted with suboptimal dosing of d, no resistance was detected on d screening plates for any daptomycin regimen evaluated. conclusions: exposure of sa to v approximating 1/6 of optimal serum concentrations resulted in the development of heteroresistance in the agr null group i and ii. loss of agr function did not correlate with the development of d resistance despite suboptimal simulations of d exposures. these results implicate loss of agr function important to the development of glycopeptide resistance but not to loss of susceptibility to d. teicoplanin efficiently penetrates into the rabbit infected vitreous but may enhance expression of virulence factors at sub-inhibitory concentrations e. forestier, f. jehl, c. gallion, r. andres, s. bronner, l. leininger, g. prévost (strasbourg, fr) objectives: fluoroquinolones are the antibiotics that most efficiently penetrate inside vitreous. however, alternative treatments for endophtalmitis may be required in some cases for example resistant bacteria. we used a rabbit experimental model of endophtalmitis to evaluate the penetration of teicoplanin in different conditions. the influence of subinhibitory concentrations of teicoplanin was also evaluated on the expression of s. aureus virulence factors. methods: new zealand rabbits (>3 kgs) received one or repeated doses of intra-venous (iv) teicoplanin (60 mg/kg) every 12 hours for 3 days plus one dose a day for 2 more days. another group of rabbits was infected by 1000 cfu of a methicillin resistant s. aureus ibs 3284 (cmi = 1.5 mg/l) producing enterotoxin a, panton-valentine leucocidin and luke-lukd. they were administrated 24-36 hours later with 60 mg/kg teicoplanin, as a single dose or as to reach the steady state. vitreous (200 ll) was sampled before new injections of teicoplanin or at indicated time as well as blood before or 15 min after teicoplanin injection. teicoplanin concentrations were measured by hplc. bacterial counts were recorded and expression of virulence factors was semi-quantified by dedicated competitive rt-pcr tests. results: in safe eyes, teicoplanin penetration remains moderate reaching about 4 mg/l within about 6-8 h after one iv injection. the half-life of teicoplanin in the rabbit vitreous is about 25 hours. after 5 days of repeated injections, intra-ocular concentration stabilises around 4 mg/l while residual blood concentrations were comprised between 30-40 mg/l. in infected eyes, teicoplanin, when repeatedly administrated after the beginning of clinical signs, i.e. 24 h postinfection = 106 cfu/ml, reaches intra-vitreal concentrations of 9.3 ± 2.3 mg/l 40 h post-infection, and increases to 14.3 ± 5.4 mg/l 84 h post-infection and 12 h after a fourth injection. however, at sub-inhibitory concentrations (~2 mg/l), it may be responsible for a significant increase of agr, gammahemolysin hlga, luked and panton-valentine leucocidin luk-pv expressions with ratio ranging from 15 to 100 folds. conclusion: these preliminary results strongly suggest that teicoplanin iv administration constitutes an interesting alternative therapy for endophtalmitis provided high intraocular concentrations are rapidly obtained. investigations now concern optimisation of teicoplanin dosage regimen. pharmacokinetics of temocillin in intensive care patients and monte carlo simulations to evaluate susceptible breakpoints j.w. mouton, r. dejongh, v. basma, p. tulkens, s. carryn (nijmegen, nl; genk, brussels, be) background: temocillin (tmo) is a narrow spectrum penicillin with good activity against gram negative micro-organisms including esbl and ampc producers. little pharmacokinetic data are available however. we performed a pharmacokinetic study in 6 icu patients receiving tmo 2g q12h. parameter estimates were used to predict concentrations during continuous infusion (coinf) and compared with data obtained from 6 other icu patients receiving coinf to validate the model. the model was then used to perform monte carlo simulations (mcs) to determine probabilities of target attainment (ptas) for pharmacodynamic indices (pdi) in order to evaluate and suggest clinical breakpoints. methods: blood samples were taken from icu patients prior to (t = 0) and after (t = 1, 2, 3, 6, 12 h) a 30 m infusion of 2 g tmo (n = 6) or after 48 h during coinf with 4 g/24 h (n = 6), and then cooled, centrifuged and stored at )70°c until analysis by hplc. protein binding was determined using an ultrafiltration method. results were used to estimate population pharmacokinetic parameters by winnonmix including the covariance matrix. miclab233 was used to perform simulations for coinf as well as to perform mcs (10000 cycles) and obtain ptas for the unbound fraction including 95% confidence intervals (ci) for the target concentrations. ft>mic was chosen as the pdi because of the pharmacodynamic properties of tmo. results: protein binding was 75%. a one-compartment model best fitted to the data, with estimates (se) of vc = 14.3 (0.87) l and k10 = 0.172 (0.059) 1/h corresponding to a mean half-life of 4.03 h. using these estimates, the predicted unbound concentration during coinf was 16.9 mg/l, while the mean concentration in the 6 other patients was 17.03 mg/l, a bias of less then 1%. the breakpoint mic for a mean ft>mic of 50% was 16 mg/l. however, mcs -taking the variation in the population into account -indicated that 100% ptas of a 2g q12h dose were obtained at 4, 4, and 2 mg/l for 40, 50 and 60% ft>mic, respectively. the 95% ci at 50% ft>mic indicated a clinical breakpoint of 8 mg/l. the 95% ci was relatively large, as expected from data obtained in patients rather than volunteers. conclusion: the population pharmacokinetic estimates from 6 icu patients were very well in agreement with the validation study, with a bias of <1%. the mcs indicate a susceptible breakpoint for temocillin of £8 mg/l provided an administration of 2 g q12h is used. tissue penetration and pharmacokinetics of moxifloxacin in diabetic foot infections:an interim analysis j. majcher-peszynska, k. karrasch, m. saß, r. mundkowski, a. gussmann, p. kujath, b. ruf, w. schareck, h. koch, b. drewelow (rostock, bad saarow, lubeck, leipzig, beeskow, de) objectives: with its broad spectrum of activity against grampositive, gramnegative and anaerobic organisms moxifloxacin covers the pathogens of the mainly polymicrobial infections associated with the diabetic foot. inflammatory and fibrotic processes in diabetic foot infections (dfi) contribute to impaired tissue penetration of antibiotics. in addition, diabetic patients represent a pharmacological risk population, physiological changes in diabetic patients may alter the pharmacokinetics of antibiotics. the study was designed to investigate the penetration of moxifloxacin into perinecrotic tissue in 60 patients with dfi and the pharmacokinetic properties of moxifloxacin in diabetic patients. methods: the interim analysis of this open, multicentre study included 30 adult, hospitalized male and female patients (mean age: 69.8 years) with type 2 diabetes mellitus and dfi. the pharmacokinetic parameters of moxifloxacin and penetration into dfi tissue at steady state (day 4 to 8) following once daily administration of 400 mg iv or po were evaluated. correlations between penetration of moxifloxacin and clinical and laboratory parameters were examinated. results: in all 30 patients the moxifloxacin concentrations measured in infected diabetic foot wounds 3 hours after administration exceeded the in vitro mic90 values of susceptible staphylococci (0.125 mg/l). the moxifloxacin concentrations achieved in dfi tissue correlated more strongly with the auc0-24 (r = 0.659; p < 0.01) than with the corresponding plasma concentrations (r = 0.492, p < 0.01), but not with the extent of the systemic inflammation and the blood glucose level. taking into account the predictive pk/pd parameters for moxifloxacin (based on an in vitro mic90 value of 0.125 mg/l for staphylococcus aureus) a therapeutic success can be expected (auc24/mic: 204.3; cmax/mic: 22.9). significant differences between the routes of administration (iv vs po) were only observed for tmax (p < 0.01) and t1/2 (p < 0.05), but not for other clinically relevant parameters (auc0-24, cmax, moxifloxacin tissue concentration). this allows sequential therapy i.v./p.o. in this indication. conclusion: based on adequate plasma concentrations in diabetic patients, the sufficient penetration into dfi tissue and the possibility of a sequential therapy, moxifloxacin representsfrom a pharmacological point of view -a valuable therapeutic option in the treatment of diabetic foot infections caused by susceptible organisms. fluoquinolones effects on patient lymphocytes during prolonged treatments e. bertazzoni minelli, a. benini, d. doria, p. franceschetti, m.e. fracass (verona, it) fluoroquinolones (fq), widely used in clinical practice, are well tolerated. the most common adverse reactions are those affecting gastrointestinal tract, phototoxicity and allergy. the aim of the study is to evaluate the possible cellular damage in lymphocytes of patients treated with different fqs according to pharmacokinetic data. blood samples obtained from thirty-six patients treated with ciprofloxacin (cpx, 14 pts), levofloxacin (lvx, 15 pts.) and moxifloxacin (mfx, 7 pts) at different doses were analysed. patients treated with cpx and lvx were in therapy with other drugs (diuretics, cardiovascular drugs, omeprazole, antiinflammatory drugs, etc.). mxf treated patients were not in therapy with other drugs. samples were collected at time 0 (before fqs administration) and after 3 and 9 days of treatment. serum levels of fqs were determined with microbiological method and hplc. comet test was performed on lymphocytes, to evaluate dna damage. gsh levels were determined as efficiency marker in metabolic process of detoxification. cpx showed good serum concentrations; its levels increases proportionally with administered doses (from 250 to 1000 mg). lvx concentrations resulted in good inhibitory levels after 3 treatments (500 mg) both per os and i.v. patients orally treated with 250 mg showed similar serum levels (from 0.8 to 2.1 mg/l). mfx levels were between 3.6 and 1.6 mg/l after 3 and 9 days. repeated cpx administration induced a dose-dependent increase in all dna damage parameters, with statistical differences after 9 treatments. mfx (400 mg) and lvx administration didn't induce dna damage after 3 and 9 days. intracellular levels of gsh were similar in all treated groups, even if cpx treated patients showed the lowest concentrations. no statistical correlations were found between all parameters studied. these data indicate that cpx induce dna damage in lymphocytes in combination with a reduced efficiency in detoxification system. this effect does not seems to depend on high intersubject variability for fqs administered doses, co-administration of other drugs, different ages of patients and low samples numbers. effects of single fqs molecules seems to be structurespecific and selective. objectives: ertapenem is a carbapenem commonly used to treat intra-abdominal infections. the antibacterial spectrum includes the major causative pathogens. clinical trials proved excellent clinical and microbiological efficacy in peritonitis. on the other hand in inflammatory pancreatic diseases sufficient antibiotic concentration in the inflammatory tissue is vital for the outcome of the disease. we therefore investigated ertapenem concentrations in pancreatic tissue and juice in comparison to the plasma levels measured at the same time. methods: in a prospective clinical trial ertapenem was given in a dosage of 1 g i.v. 30 minutes prior to operation in patients (18-70 years) suffering from chronic pancreatitis or pancreas carcinoma undergoing pancreas resection. blood samples were collected every 60 minutes during the operation. moreover we collected pancreatic tissue and pancreatic juice shortly before resection and shortly before finalisation of the anastomosis. the samples of ertapenem (blood, juice, tissue) were determined by hplc. results: in 13 patients (5 female, 8 male, mean age 54.8 ± 11.2 years) ertapenem blood concentrations were determined and demonstrated intraoperatively high concentration (20 ± 8 mg/l) above mic90 values for major expected pathogens. concomitantly in 10 of these patients ertapenem concentration was determined also in pancreas tissue and pancreas secretion (in further 3 patients in pancreas secretion only). in 7/10 patients sufficiently high ertapenem levels were detected in pancreatic tissue. in 3 patients with chronic pancreatitis no accumulation was seen. mean pancreas tissue concentration was 0.8 ± 0.4 lg/g tissue. 5 of 6 patients with pancreas carcinoma had increased ertapenem levels in pancreas secretion but only 2 of 6 patients with chronic pancreatitis. conclusion: in patients with pancreas carcinoma, ertapenem levels were measured in pancreatic tissue as well as in pancreatic secretion and penetration seems to be similar to imipenem. due to chronic inflammation and possibly altered microcirculation only in one half to one third of chronic pancreatitis patients ertapenem levels were detected. bacterial strain-independent pharmacodynamics of linezolid/doxycycline combinations with staphylococcus aureus: 5-day simulations using an in vitro dynamic model m. smirnova, i. alferova, i. lubenko, y. portnoy, s. zinner, a. firsov (moscow, ru; cambridge, us) objective: to delineate the possible advantages of linezolid (l)/doxycycline (d) combinations over monotherapy, the pharmacodynamics of l, d and l+d were studied with s. aureus. methods: s. aureus atcc 43300 and a clinical isolate s. aureus 479 were exposed to twice-daily l (half-life 6 h) and once-daily d (half-life 15 h), alone and in combination (1:3 ratio based on 24-h auc/mics), for five consecutive days. to provide simultaneous mono-exponential elimination of l and d with different half-lives, a previously described dynamic model was modified according to blaser and zinner. the mics of l were 1.56 and 1.56 mg/l and mics of d were 0.1 and 0.05 mg/l for s. aureus atcc 43300 and s. aureus 479, respectively. nine dosing regimens were simulated with each organism exposed to different auc/mics (in hours): l30, l60 and l200; d90, d180 and d520; l30 + d90, l60 + d180 and l200 + d520. the cumulative antimicrobial effect was expressed by its intensity (ie) measured from the start of treatment to the time after the last antibiotic dose when numbers of antibiotic-exposed bacteria reached at least 108 cfu/ml. emergence of resistance was monitored daily by quantitating surviving organisms on agar plates containing 2x and 4xmic of l or d. results: with both s. aureus atcc 43300 and s. aureus 479 exposed to l or d, ie increased with increasing simulated auc/ mic ratios, although significantly higher ies were produced with l30, l60 and l200 treatments relative to d90, d180 and d520 treatments. each of the combined treatments, i.e., l30 + d90, l60 + d180 and l200 + d520, produced much greater ies than the sum of l and d ies observed in the respective mono-treatments with both s. aureus strains. based on population data, a pronounced selection of s. aureus resistant to d occurred in all mono-treatments with d. it was also observed with l30 + d90 and, to a lesser extent, with l60 + d180 but not with l200 + d520. no resistance to l was observed with l mono-or combination treatments. conclusions: these data predict a synergistic interaction of l with d against s. aureus. anti-staphylococcal effects of telavancin in an in vitro dynamic model: impact of different half-lives and initial concentrations that simulates normal (nek) and impaired elimination kinetics (iek). materials and methods: a glycopeptide intermediately susceptible strain of s. aureus (gisa) mu-50 with a telavancin mic of 0.5 mg/l was selected for the study. with both nek and iek simulations at a starting inoculum of 6 log cfu/ml, gisa mu-50 was exposed to different ratios of the peak concentration (cmax) to the mic of telavancin (as a single dose), i.e., 2.6, 7.8 and 14. based on time-kill data, the intensity of the antimicrobial effect (ie -the area between control growth and time-kill curves) was determined from time zero to the time when the effect no longer could be detected, i.e. the time after the last dosing at which the number of antibiotic-exposed bacteria reached 7 log cfu/ml. results: in each treatment, bacterial regrowth followed gradual reduction in the starting inoculum during the first 12 h (similar in nek and iek simulations) that led to significantly lower minimal numbers of surviving organisms in iek simulations compared to nek simulations. despite similar rates of initial killing, times to regrowth were much longer in iek than nek simulations. at a given cmax/mic ratio, the ies observed in iek were greater than in nek simulations (figure). conclusions: these findings demonstrate pharmacokineticdependent pharmacodynamics of telavancin with staphylococci. pharmacokinetics of amoxicillin in pregnant women with pre-term premature rupture of the membranes objectives: amoxicillin is widely used during pregnancy, in particular to treat group b streptococcus. insufficient knowledge on the pharmacokinetics just before and during delivery, could pose patients with preterm premature rupture of the membranes (pprom) at serious risk for under dosing. we investigated the pharmacokinetics in patients with pprom in this critical situation. methods: seven healthy women at 34-37 weeks of gestation were included. they received 1 g (first dose 2 g) amoxicillin for pprom according to local guidelines. from each patient 14-28 blood samples were taken. antibiotic serum concentrations were determined by a validated hplc method. pharmacokinetic parameters were estimated by population pk modeling using nonmem. to discriminate between various models the minimum value of the objective function (mvof) was used. a reduction of >10 in mvof was considered significant. results: a three-compartment pharmacokinetic model best described the time course of amoxicillin. the clearance and volume of distribution of the central compartment (vc) were estimated at respectively 23 ± 1.8 l/h and 6 ± 0.8 l (mean ± se). estimates of the parameters and model discrimination improved when we assumed the size of the third compartment to be equal to the first compartment. the residual error was found to be proportional to the serum concentrations. most of the inter-individual variability could be explained by variation of clearance. the mean volume of distribution at steady state (vss) and terminal half-life were 23.9 l and 1.34 h respectively. estimated values of elimination and distribution rate constants were: k10 = 3.9 h-1, k12 = 1.7 h-1, k21 = 0.8 h-1, k13 = 8.8 h-1 and k31 = 8.8 h-1. as was to be expected due to the small population size, no significant relationship was observed between the individual posthoc estimates for clearance and patient characteristics. conclusion: here we describe the pharmacokinetics of amoxicillin in pregnant women with pprom. it was found that the pharmacokinetics clearly differs from that in nonpregnant individuals. clearance and vss were significantly higher and the terminal half-life was shorter. furthermore, a 3-compartment model was found to describe the data better than a 2-compartment model. it is an intriguing question whether this 3rd compartment is a unique feature associated with pregnancy. these data offer a theoretical basis to make proper dose-adjustments in a particular patient group in a critical condition. penetration of piperacillin and tazobactam in severe acute pancreatitis objectives: acute necrotizing pancreatitis is still related to an extremely high mortality rate, based on local infectious complications, particularly in necrotizing areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotizing pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of betalactamases. on that score, the penetration of co-administrated pip and bli into inflamed or necrotic pancreatic tissue has not been investigated yet. methods: adressing the penetration capability of bsp and bli a clinical trial was designed to investigate the penetration of piperacillin (pip) and tazobactam (taz) in patients with severe necrotizing pancreatitis undergoing pancreas surgery. samples (n = 21) were taken from plasma (pl), necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of 4.0 g pip and 0.5 g taz. concentrations of pip/ taz were determined by hplc/ uv. results: mean plasma concentrations at 1.5 h after application were 56.4 ± 29.5 mg/l (pip) and 16.9 ± 15.9 mg/l (taz). exceeded in pl and bs, nearly reached in pn but not in pft. the concentration of pip in combination with taz exceeded or reached the mic90 in pl, pn and bs against e. coli, klebs. spp., enterobacter, proteus spp. and clostr. spp., in pl and bs even against pseudomonas and bacteroides. conclusion: given in combination both -pip and taz -have been demonstrated to reach rapidly effective inhibitory concentrations in inflamed and necrotic compartments of pancreatic and peripancreatic tissue. co-administration of piperacillin and tazobactam may have a potential clinical benefit in prevention and treatment of local infectious complications of severe necrotizing pancreatitis. pk/pd challenges of in vitro time-kill curves -a new modelling approach s. schmidt, o. burkhardt, w. treyaprasert, h. derendorf (gainesville, us; bangkok, th) objective: in vitro pk/pd models, based on time-kill curve data, have become a powerful tool to predict the in vivo situation. up to date, several modelling approaches have been undertaken to develop suitable pk/pd models that fit in vitro data sufficiently well. widely used simple sigmoid emax models meet these criteria only partly. a further approach was undertaken to address the weak points of currently used models and applied to model the effects of ceftriaxone against escherichia coli. methods: constant concentration time-kill curves were performed in mueller-hinton broth (mhb, difco) with and without bovine serum albumin (bsa) 40 g/l. using concentrations of ceftriaxone, ranging from 0.25 mic to 16 mic, the change in number of bacteria (cfu/ml) versus time was linked to its effect. escherichia coli atcc 25922 was employed as the test organism. samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 hours. the data were modelled simultaneously, using a modified sigmoid emax model and the software scientist ò for windows tm . results: a differential equation, characterized by growth rate constant (k0) times the starting number (n) of bacteria represent the simplest case. barging from log-growth phase to stationary phase can be described by an additional nmax term. however, bacteria do not necessarily start growing in the loggrowth phase. this delay in onset of growth can be modeled by an exponential term, characterized by a factor beta (b) and time (t). to describe the overall change in number of bacteria not only growth but also concentration (c) dependent kill has to be taken into account. from certain drug specific concentrations on, a maximum effect is reached, described by the maximum kill rate constant kmax. however, it may be necessary to model a delay in the onset of kill with an additional exponential term, characterized by a factor alpha (a) and time (t). finally, a hill factor/shape factor (h) is necessary to smooth the predicting curves out. as shown in figure 1 this new model meets the in vitro time-kill curve data sufficiently well. the final equation including all parameters described above is: conclusion: the proposed model was able to describe the observed data much better than a simple emax-model. incorporating two additional terms into the model, the in vitro situation could be described much better, taking the delay in onset of growth and kill into account. objectives of this study were (1) to describe the pharmacodynamic (pd) profiles of bpr 500 mg iv q8h as a 2hr infusion & 500 mg iv q12h as a 1-hr infusion; (2) to determine the overall probability of target attainment (pta) by weighting for the expected distributions (dis) of renal function (rfx) in the populations (pop) of interests; (3) to determine the organism-specific pta against the pathogens encountered in phase ii trials. methods: 150 subjects (total 2443 samples) were studied (phase i/ii subjects). samples were analysed using bignpod. to assess the impact of differing degrees of rfx impairment on pta, crcl (crcl-cockcroft & gault method) was employed as a covariate in the pop pk analysis. monte carlo simulation (mcs) (9999 subjects) was performed with adapt ii. overall pta was calculated for 30-60% ft>mic. weighting for the expected dis of rfx in the pop of interests was accomplished by using the dis of crcl observed in 2 previous registration studies of the same indications (cssti and np). dis of mics for pathogens was supplied by sponsor. results: in the pop pk analysis, the pop mean (sd) values for volume, clslope, clintercept, kcp and kpc were:7.65 (3.89) l, 0.51 (0.32) l/hr, 2.35 (1.08), 3.05 (5.14) hr-1 and 1.10 (0.95) hr-1, respectively. the obs-pred plot was obs = 1.003 x pred + 0.627; r2 = 0.947 after the bayesian step. in the mcs analysis of bpr 500 mg iv q12 h, the pta of achieving 30% ft>mic & 50% ft>mic exceeded 90% for mics values £2 mg/l & £0.5 mg/l, respectively. for bpr 500 mg iv q8h, the pta of achieving 60% ft>mic exceeded 90% for mics values £2 mg/l. in the organism-specific analysis, the pta of a static effect (30% ft>mic) exceeded 90% for both mssa & mrsa for bpr 500 mg iv q12h. bpr 500 mg iv q8h provided a >90% pta of a cidal effect (50% ft>mic) for both mssa & mrsa. for gnb, the pta of bpr 500 mg iv q8h in achieving a cidal effect (60% ft>mic) exceeded 90% for non-ampc-bearing gnb. for ampc-bearing gnb, the pta of achieving a cidal effect was 84.8%. conclusions: an extensive evaluation of the pd of bpr was performed to estimate the overall activity of bpr against target pathogens. these findings need to be validated in the clinical trial arena. investigation of different levofloxacin regimens in patients with acute complicated urinary tract infections p. tenke, r. benko (budapest, szeged, hu) objective: in the present study we aimed to find out if a continuous or an intermittent levofloxacin (1 · 500 mg) treatment is more advantageous for patients with acute complicated urinary tract infection (uti) caused by urinary obstruction. we investigated if levofloxacin adsorbs to the surface of the foreign body, which was inserted with the aim of temporary resolution of ureteral obstruction. preventive effect of levofloxacin on bacterial biofilm formation and incrustation was also evaluated. methods: we enrolled and randomised 24 patients who had acute uti caused by urinary obstruction. obstruction was resolved with double j stent (djs) insertion or percutaneous nephrostomy (pcn) and meanwhile, antibiotic treatment was started in all patients. 12 patients (group 1) were on antibiotics till the day of definitive curative operation when all foreign bodies were removed. in the other 50% of the patients (group 2) the antibiotic therapy was stopped 7 days after the djs or pcn insertion. short term antibiotic course -which is advisable for prevention of uti before invasive endoscopic treatmentwas administered in both groups from the day of the operation (after djs or pcn removal) and it was continued until the removal of all possible urinary foreign bodies used during the operation. in both groups of patients we recorded and evaluated early and late clinical and microbiological recovery. retrieved stents were sectioned for further laboratory examinations. adsorbed levofloxacin in the conditioning film layer and on the stent surface was detected by hplc. rasterelectron microscopy (rem) was used to examine biofilm formation and encrustation. results: we did not find any significant differences between the two groups of patients, neither in clinical (presence of fever, back pain, flank pain, leukocyte count) nor in microbiological recovery. statistical analysis showed that significantly greater amount of levofloxacin adsorbed to the conditioning film than to the stent surface in both groups of patients (0.72 ± 0.83 vs. 0.20 ± 0.34 in group 1 and 0.24 ± 0.28 vs. 0.1 ± 0.15 in group 2). no viable, adherent bacteria were recovered by sonication and culture in any of the patients, and no biofilms or encrustation were seen under rem either. conclusion: our data prove the hypotheses that continuous antibiotic treatment does not have any clinical or microbiological advantages in patients with indwelling ureteral stents compared to intermittent therapy. objective: the prophylaxis of bacterial infections during cardiac surgery is widely used in clinical practice. staphylococcus aureus, staphylococcus epidermidis and enterococcus spp are the pathogens most frequently involved in infective complications of cardio-pulmonary bypass (cpb) surgery. it is generally agreed that the success of prophylaxis is dependent on the ability to reach and maintain free antibiotic concentration in tissues higher than the mics for the most common pathogens. so we estimated the tissue concentrations of linezolid into sternal bone of patients undergoing cpb surgery. methods: six patients undergoing routine cpb surgery were given 600 mg linezolid as a 30 min iv infusion along with conventional prophylaxis of 1.5 g of cefuroxime immediately before surgery. two hours after the end of infusion blood samples and sternal bone tissues were collected. the local medical research ethics committee approved this study and all patients gave written informed consent. samples were assayed for the presence of linezolid by a high-performance liquid chromatography (hplc) method. results: following a 600 mg infusion of linezolid, mean serum concentration for the six patients were 8.48 mg/l (range 7.3-12.36 mg/l) 2 hours after the end of infusion. the concentration of linezolid into sternal bone was 4.35 mg/l (range 3.9-5.2 mg/ l) 2 hours after the end of infusion. the penetration of linezolid into sternal bone was 52.2%. conclusion: the penetration of linezolid into bone was 52.2% of the simultaneous blood levels. in all bone samples the concentration of linezolid exceeded the mic for susceptible pathogens (<4 mg/l). although these data have been obtained from healthy, well-perfused bone the values suggest that linezolid may be a useful agent in the management of multidrug-resistant gram-positive bone infections. the antibacterial effect of daptomycin, teicoplanin and vancomycin against s. aureus studied in an in vitro pharmacokinetic model of infection a. noel, k. bowker, a. macgowan (bristol, uk) objectives: daptomycin (dap) is the first cyclic lipopeptide antibiotic approved for parenteral use in gram-positive infection. as yet, no comparative pharmacodynamic studies have been performed using dap and the two most common iv therapies teicoplanin (tei) and vancomycin (van). we used a pharmacokinetic (pk) model to study the antibacterial effect (abe) of these agents against two typical mrsa strains (ukemrsa15 & 16) and a hetero vancomycin intermediate mrsa (hvisa). methods: an in vitro dilutional model was used to simulate the free drug concentration over 48 h associated with doses of -dap 6 mg/kg 24 hrly (cmax 6.4 mg/l, t 1/2 8 h); tei 400 mg 24 hrly (cmax 4.5 mg/l, t1/2 24 h); van 1 g 12 hrly (cmax 13.5 mg/l, t1/2 6 h). an inoculum of 10 6 cfu/ml was used and the experiments performed in triplicate. abe was assessed by area-under-the-bacterial-kill-curve 0-24 h (aubkc24) and logcfu/ml.h objectives: linezolid, the first oxazolidinone, is active against methicillin resistant staphylococcus aureus and has been effective in a variety of acute infections. however long-term administration, although desirable in bone infections caused by resistant gram-positive organisms, is hampered by the occurrence of anaemia and thrombocytopenia. administration of vitamin b6 has been reported to prevent myelosuppression. methods: patients attending the infectious disease clinic with bone infections caused by resistant gram-positive bacteria and treated with linezolid (600 mg b.d. orally), received vitamin b6 (125 mg o.d. orally) for the period of administration of linezolid. full blood counts were followed-up weekly. linezolid treatment was discontinued if haemoglobin declined below 9 mg/dl or platelets below 120000/ll. data from sixteen patients with osteomyelitis and 8 with prosthetic joint infections were evaluated. comparisons were performed with 20 matched historical controls receiving linezolid without b6 by kaplan-meyer curves with the log-rank test. results: the median follow-up of patients receiving b6 was 3.5 weeks and of controls 4 weeks. in the b6 group 63% of the patients discontinued while in the control group 44% of the patients discontinued treatment because of side effects (p ns). 47% of patients receiving b6 discontinued due to thrombocytopenia and 20% due to anaemia. respective percentages in the control group were 21% and 38% (p ns for all comparisons). mean time to the occurrence of thrombocytopenia was 4 weeks in the patients who received b6 and 3.25 weeks in the control patients. respective times to occurrence of anaemia were 3.4 and 3.85 weeks. all cases of myelosuppression were reversible. conclusions: administration of b6 failed to prevent or delay both thrombocytopenia and anaemia in patients receiving linezolid. other methods should be investigated to facilitate longer administration of linezolid in this group of patients. therapeutic drug monitoring of colistin -a 7-year review from a uk clinical antibiotic assay service k. bowker, a. noel, s. tomaselli, a. macgowan (bristol, uk) objectives: over the last 5 years there has been increased use of colistin (col). however, little clinical data is available on the therapeutic levels of colistin. monitoring of col is useful in terms of therapeutic levels and avoidance of toxicity for patients with cystic fibrosis and complicated gram-negative infections. previous in vitro data has shown that col is bactericidal at col concentration is ‡3 mg/l. here we assessed col data collected from our antibiotic assay service from the last seven years in order to establish if such levels were obtained. methods: col levels were determined by bioassay. data was retrospectively collected from the hospital information management system. data was assessed collectively and stratified by known cystic fibrosis patients, sex and age. objectives: ßlactams like ceftriaxone (cfx) and quinolones such as moxifloxacin (mox) are widely used to treat pneumococcal infection. we studied the antibacterial effect of (abe) after the first dose exposure to free drug serum concentrations of iv cfx 2 g and po mox 400 mg against s. pneumoniae strains with typical mics at low and high inocula. methods: a hollow fibre in vitro model was used to simulate free drug concentrations over 24 h of cfx (2 g 24 h iv, cmax 26 mg/l, auc 56 mg/laeh, t1/2 6 h) and mox (400 mg, 24po, cmax2.0 mg/l, auc20 mg/laeh, t1/2 10.5 h). the cfx mic was 0.06 mg/l (t>mic cfx 100%) and mox mic 0.06 mg/l (auc/ mic 334). initial abe was measured by the slope of the log viable count 0-5 h and total abe over the dose interval (24 h) by log reduction in viable count at 24 h (d24) and the area-underthe-bacterial-kill-curve (aubkc24). inocula of 106 cfu/ml and 108 cfu/ml were used. results: the initial and total abe at low and high inocula were: given the pk/pd indices modelled both drugs showed a maximal effect. clearance from the model occurred at 12 h (106 inoculum) and 24 h (108 inoculum). there were no significant differences in speed or extent of abes comparing cfx and mox. conclusion: the abes of iv cfx and po mox against s. pneumoniae is similar in the first 24 hrs of drug exposure. emergence of resistance in e. coli and ent. cloacae after exposure to ceftriaxone or ertapenem in an in vitro model of infection k. bowker, a. noel, a. macgowan (bristol, uk) objectives: emergence of resistance (eor) is an emergent factor in therapeutic choice. we studied eor to ceftriaxone (cfx) and ertapenem (ert) in e. coli (ec) and ent. cloacae (entclo), a more challenging species within inducible blactamases. methods: an in vitro dilutional model was used to simulate free drug concentrations associated with 1 g 24 hrly cfx (cmax 22 mg/l, t½ 8 h) and ert (cmax 13 mg/l, t½ 5 h) over 72 h. two inocula 106 and 108 cfu/ml were used and eor assessed by population-analysis-profiles (pap). the area under the pap (auc-pap) was used to measure eor. ert mics were 0.018 mg/l ec and 0.25 mg/l entclo, cfx mic 0.14 mg/l ec and 0.5 mg/l entclo. experiments were performed in triplicate and mean values presented. results: observations at 72 h were similar to those at 48 h, hence data to 48 h is given. at 106 and 108 cfu/ml, ec viable counts were reduced by 2 fi 4 logs, there was no eor. against entclo inoculum 106 and 108, cfx resulted in an initial 0-2 log drop, then regrowth, ert produced a >4 log reduction. eor as measured by the mean auc-pap (n = 3) with entclo is shown below: dosing with cfx resulted in eor to cfx and ert at high and low inoculum. dosing with ert resulted in no eor at 106 inoculum, at 108 resistance emerged to both cfx and ert. conclusions: eor depends on species (entclo > ec); duration of exposure (long > short) and agent (cfx > ert). ert appears to induce less eor both to itself and cfx than cfx does to itself and ert. however, initial use of cfx may reduce the effectiveness of ert. comparative serum activity of telithromycin, azithromycin, and amoxicillin/clavulanate against aerobic and anaerobic respiratory pathogens objectives: the purpose of this investigation was to study the clinical potential of telithromycin, a new ketolide antibiotic, for the treatment of mixed aerobic/anaerobic respiratory infections. in this study, we compared the pharmacodynamics (duration of inhibition/killing) of telithromycin (tel) to azithromycin (azi) and amoxicillin/clavulanate (a/c) against aerobic and anaerobic pathogens associated with mixed respiratory infections. methods: following written informed consent, ten healthy adult subjects (ages, 20-47 yrs) received single doses of tel (800 mg), azi (500 mg), and a/c (875 mg) one week apart following a 12-h fast. venous blood samples were obtained at 2, 6, 12, and 24 h after the dose and stored at )70°c. inhibitory and bactericidal titre s were determined by microdilution (s. pneumoniae & h. influenzae) and agar dilution (peptostrep. magnus, peptostrep. micros, prev. bivia, & prev. melaninogenica) procedures following clinical & laboratory standards institute methodology. bactericidal titres in serum endpoint was determined as the highest dilution of serum yielding 99.9% killing. the median titres at each time point were calculated and the duration of activity was used for comparison of these agents. conclusions: in this ex-vivo study, we found that tel can provide prolonged (50% of the dosing interval) inhibitory activity in serum against macrolide-resistant strains of s. pneumoniae, bl pos. and neg. strains of h. influenzae, and common respiratory anaerobic pathogens. these findings suggest that tel could have clinical utility in the treatment of community-acquired mixed aerobic-anaerobic respiratory tract infections, including sinusitis, bronchitis, and pneumonia. objectives: increasing resistance in isolates of e. coli (12%) and p. aeruginosa (40%) to fluoroquinolones (fq) is a concern since these antibiotics are commonly used in the treatment of complicated urinary tract infections (utis). currently, no interpretive standards exist for ''susceptible'' isolates in urine for the newer fq. the purpose of this investigation was to evaluate the activity of high-dose levofloxacin against fqresistant urinary pathogens. methods: in this study, we determined the serum and urine levels of high dose (750 mg) levofloxacin (l) as well as its bactericidal activity in urine (uba) against l-resistant isolates of e. coli (mics = 8 to 64 lg/ml) and p. aeruginosa (mics = 8 to 64 lg/ml). following written informed consent, blood and urine samples were collected from 10 healthy adult (ages, 20-47 y/o) fasting subjects (5 m and 5 f) prior to and at 1.5, 4, 8, 12 , and 24 hours after a single 750 mg dose of l. serum and urine concentrations were measured by a validated hplc assay (0.9-2.1% cv). the testing methodology for uba was similar to the microdilution assay used for serum bactericidal testing (clsi) with the exception that antibiotic-free urine was used to dilute these samples. the median titre (1:2-1:32) at each time point for the 10 subjects was used to determine uba. results: the mean serum pharmacokinetic parameters were similar to previously published values: cmax = 9.2 lg/ml, auc = 95 mgaeh/l, and t1/2 = 7.6 h. mean urine concentrations ranged from 475 lg/ml (4 h) to 186 lg/ml (24 h). uba (titres >1:2) was maintained for at least 12 hours in all subjects for e. coli isolates with mics = 8, 16, and 32 lg/ ml. for the e. coli strain with a mic = 64 lg/ml, 8 subjects exhibited uba at 8 h but only 2 subjects exhibited uba at 12 h. similar results were observed against the p. aeruginosa isolates. conclusions: the results from this ex-vivo pharmacodynamic study in healthy volunteers found that 750 mg of l provides prolonged (at least half the dosing interval) uba against l-resistant strains of e. coli and p. aeruginosa up to 32 lg/ml. this suggests that a separate urinary susceptibility breakpoint is indicated for urine isolates treated with 750 mg doses of l. objectives: exposure of methicillin-resistant s. aureus (mrsa) to acid ph restores its susceptibility to beta-lactams (sabath and al., aac, 1972) . in macrophages, s. aureus is mainly confined within phagolysosomes where the ph is acidic. we showed that meropenem (mem) displays similar intracellular activity against mrsa atcc 33591 and mssa atcc 25923 in macrophages. in the present study, we have investigated the intraphagocytic activity of mem and cloxacillin (clx) against 3 mrsa clinical isolates (including one visa strain), in comparison with the reference mrsa atcc 33591 and mssa atcc 25923 strains. methods: mic's were determined in mhb (plus nacl 2%) by micro-dilution method. meca expression was examined at neutral and acidic ph by a semi-quantitative rt-pcr (16 s rrna as housekeeping gene). intracellular activity was assessed in human thp-1 macrophages exposed to extracellular concentrations equivalent to human cmax (total drug; mem: 50 mg/l; clx: 8 mg/l) by examining the decrease in cellassociated cfu after 24 h from the original, post-phagocytosis inoculum (controls [no antibiotic]; approx. 1 log cfu increase). results: the table shows the mics (in broth) at neutral and acid ph and the intracellular activity for the 5 strains studied. in atcc 33591, meca expression was similar for bacteria maintained in broth at ph 7. conclusions: the intracellular environment markedly enhances the activity of beta-lactams against mrsa, probably through exposure to acid ph, although the latter does not affect meca expression. comparative activity of dalbavancin against european gram-positive isolates i. morrissey, c. dencer, j.w.t. dallow, j. childers, a. brook, j. cowan (london, uk) objectives: dalbavancin (dal) is a new semisynthetic lipoglycopeptide with a half-life of 8.5 days, enabling onceweekly dosing. this study compared the activity of dal with other agents against gram-positive isolates from europe. methods: isolates from belgium, the czech republic, denmark, finland, france, germany, hungary, italy, the netherlands, poland, spain, sweden and the uk were included. the clsi broth microdilution method was used to determine mic using dried microtitre plates. the following antimicrobial agents were evaluated: dal, vancomycin (van), teicoplanin (tei), daptomycin (dap), linezolid (lzd), dalfopristin/quinupristin (syn), erythromycin (ery), levofloxacin (lev) and tetracycline (tet). results: selected data are shown in the objectives: dalbavancin (dal) is a next generation lipoglycopeptide antibiotic in development for the treatment of complicated skin and skin structure infections (csssi). a population pharmacokinetic (pk) analysis was performed to estimate patient parameters and to determine significant covariates. incorporating the pk model, pharmacodynamic (pd) parameters were simulated to support the effectiveness of a weekly dosage. methods: the pk analysis included 1668 dal concentrations from 532 patients across 3 clinical trials. most patients received 1000 mg on day 1 and 500 mg on day 8. possible covariates examined included demography and concomitant medications, including medications that are considered inhibitors, inducers, and substrates of cytochrome p450 enzymes. the pk-pd analysis employed monte carlo simulations of time-dependent and concentration-dependent parameters. distributions of mics were obtained directly from clinical studies, and were also simulated to explore the effect of higher mics. results: dal pk fit a 2-compartment model with interpatient variability (ipv) on all parameters. the typical value and ipv (cv%) of clearance (cl) was 0.0571 l/h (18.0%), influenced by body surface area (bsa) and creatinine clearance (clcr). volume of distribution (v1) was 4.15 l (24.5%) and influenced by bsa. the inter-compartmental clearance and peripheral volume were 0.476 l/h and 11.4 l, respectively. free drug concentrations were simulated using a dal protein binding of 93%. for a weekly 2-dose regimen, free dal remained above 1 mg/l for the majority (>90%) of patients for more than 14 days. using previously described area under the curve (auc)/mic targets for staphylococcus aureus, a proposed mic of at least 0.5 mg/l was associated with a greater than 90% probability of target attainment. conclusions: dal pk were predictable, demonstrating low ipv. bsa and clcr were the only sources of variability, but described less than 25% of the ipv. pd simulations support the use of dalbavancin in a weekly regimen. objectives: methicillin-resistant staphylococcus joint infection due to peri-operative contamination is a complication after arthroplasty. the objective of this study was to assess the distribution of radioactivity in bone and related structures using quantitative autoradiography after administration of [14c]dalbavancin in rabbits. methods: new zealand white male rabbits were given a single intravenous (iv) bolus dose of 20 mg/kg [14c]-dalbavancin (n = 18) or control vehicle (n = 3). plasma, cerebrospinal fluid (csf), bone, bone marrow, and nucleus pulposus were collected at 12, 24, 72, 120, 168 and 336 h post-dose by necropsy, homogenized, combusted, and analysed for total drug-derived radioactivity using liquid scintillation counting (lsc). in addition, the left hindlimb from 1 rabbit/time point was flash-frozen and cryosectioned for quantitative autoradioluminography. results: [14c]-dalbavancin-derived radioactivity was rapidly and widely distributed into bone, bone marrow and, to a lesser extent, in csf and nucleus pulposus. autoradioluminography data indicated that concentration of radioactivity was highest in bone marrow, whole blood, articulate cartilage, ligament, epiphyseal plate, periostium, and meniscus. at 336 h postdose, [14c]-dalbavancin-derived radioactivity was measurable in all tissues, and remained at relatively high concentrations in bone marrow (30.27 lg equiv/g), epiphyseal plate (8.42 lg equiv/g), periostium (5.18 lg equiv/g), and articular cartilage (4.84 lg equiv/g). in homogenized bone using lsc, mean concentration after 336 hours was 1.96 lg equiv/g. conclusion: [14c]-dalbavancin-derived radioactivity rapidly penetrated knee joint tissues and persisted at relatively high concentrations for at least 336 h after a single iv dose in rabbits. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase 3 trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound (93%) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution (0.15 l/kg) means that it may be removed by cvvh. cvvh is widely used in the management of critically ill patients. the objective of this study was to determine cvvh telavancin transmembrane clearance (cl) with 2 commonly used hemofilters (an69, polysulfone) at conventional ultrafiltrate flow rates. methods: tlv cl was assessed in our in vitro cvvh model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run using an69 (m100, gambro) and polysulfone (f160nr, fresenius) hemofilters. ultrafiltrate (uf) flows were 1, 2, 3 and 6 l/hr with sufficient blood flows [(qb) 200-350 ml/min] to maintain uf rates. blood samples were collected from the pre-filter line and uf samples from the post-filter uf port. concentrations of tlv in plasma and uf samples were assayed using validated lc-ms/ms methods. tlv cl was determined using the following formula:cl = (uf flow rate) [tlv]uf/[tlv]arterial. cl differences between the filter types were compared using a two-tailed, unpaired t-test. conclusion: tlv is substantially cleared by cvvh and cl increases significantly with increasing uf rate. cl did not differ by hemofilter type. cvvh cl at higher uf flows exceeds the total cl reported in patients with normal renal function. tlv likely will require dose adjustments in patients receiving cvvh. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase 3 trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound (93%) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution (0.15 l/kg) means that it may be removed by cvvhd. cvvhd is used in the management of critically ill patients. the objective of this study was to determine cvvhd tlv transmembrane clearance (cl) with 2 commonly used hemodialyzers at conventional cvvhd dialysate flow rates. methods: tlv cl was assessed in our in vitro cvvhd model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run 5 times using an69 (m100, gambro) and polysulfone (f160nr, fresenius) hemodialyzers. dialysate flows were 1, 2, 3 and 6 l/hr with sufficient blood flows [(qb) 200-350 ml/min] to maintain appropriate transmembrane pressures. blood samples were collected from the pre-filter port (a) and post-filter port (v), and spent dialysate samples (d) from the post-filter d port. plasma tlv concentrations (arterial and venous) and dialysate samples were assayed using validated lc-ms/ms methods and tlv cl was determined using the following formula: cl = (d flow rate) [tlv]d/(([tlv]arterial+[tlv]venous)/2). dialytic cl between filter types was compared using a two-tailed, unpaired t-test. conclusion: tlv is effectively cleared by cvvhd. the higher permeability polysulfone dialyzer was associated with significantly increased cl vs. the an69 dialyzer as dialysate flow increased. the degree of tlv cl seen with cvvhd suggests that dose adjustments will be necessary in patients receiving cvvhd. objective: it is still a subject of controversy that only free, unbound drug is responsible for antibacterial activity of antibiotics. to provide further proof, that only free drug contributes to antimicrobial efficacy a comparative, doseranging time-kill curve study was performed. to exclude influence factors resulting from different mechanisms of action this was done within the antibiotic class of carbapenems, using compounds with different serum protein binding. methods: constant concentration time-kill curves were performed in 50% serum for the slightly serum protein bound mer-openem (~2%) and imipenem (10%) as well as for the highly serum protein bound ertapenem (95%) and faro-penem (95-96%), ranging from 1 · mic to 16 · mic. the change in number of bacteria (cfu/ml) versus time was linked to their effect. escherichia coli atcc 25922, klebsiella pneumoniae bay 63, staphylococcus aureus bay 133 and streptococcus pneumoniae atcc 6303 were used as the test organisms. samples were taken at 0, 0.5, 1 2, 4, 6 and 8 hours. the data were modelled simultaneously using the software scientist ò for windows tm and a modified sigmoid emax model characterized by growth rate constant (k0), maximum kill rate (kmax), and concentration at half maximum effect (ec50). results: for all four bacterial strains investigated, there were dramatic increases (400-700%) in ec50 for the highly se-rum protein bound carbapenems (ertapenem, faropenem) in the presence of serum proteins (fig. 1) . for both substances no significant differences in k0 and kmax were determined. in contrast, imipenem and meropenem showed only minor differences in ec50 in the presence and the absence of 50% serum. conclusion: only free, unbound drug is responsible for the antimicrobial activity. analysis of these time-kill curves clearly showed that the antibacterial efficacy was significantly decreased in the presence of 50% serum for the highly bound ertapenem and faropenem while being unaltered for the slightly bound meropenem and imipenem. objective: numerous in vitro experiments have shown that protein binding (pb) is an important factor for antimicrobial activity, especially for highly bound antibiotics. however, the experimental conditions that simulate the in vivo situation best are still subject of controversy. therefore, an in vitro microdialysis experiment was performed that evaluates various influence factors on the pb of the highly bound betalactams ceftriaxone (pb 95-98%). methods: a comparative, dose-ranging in vitro microdialysis study was conducted to determine free, unbound ceftriaxone concentrations in lactated ringer's solution and todd hewitt broth (thb) both with and without bovine serum albumin (bsa; sigma, st. louis) 40 g/l and human plasma at 37°c. furthermore, in vitro constant concentration time-kill curves were performed, using escherichia coli atcc 25922, streptococcus pneumoniae atcc 6303 and streptococcus pneumoniae atcc 49619 as the test organisms. the data was analysed using an appropriate pk/pd model, characterized by growth rate constant (k0), maximum kill rate (kmax), and concentration at half maximum effect (ec50) and correlated to free ceftriaxone thb concentrations determined by hplc-uv. results: there were only minor differences in both unbound drug concentrations and anti-infective activity when bsa 40 g/ l was added to either lactated ringer's (pb 0% and 12.5±2.5%, with and without bsa respectively) or thb (pb 27% and 25.5±2.5%, with and without bsa respectively). no significant changes in k0, kmax and ec50 were observed. however, using human plasma, unbound concentrations (pb 60%)88%) were altered dramati-cally. conclusion: only free, unbound drug is responsible for the antimicrobial activity. however, one cannot rely on that binding to commercially purchased bsa is consistent with reported protein binding values. unbound concentrations should be measured under the respective experimental conditions to be able to correctly interpret the experimental results. in vitro postantibiotic effect of faropenem on penicillin-resistant streptococcus pneumoniae and beta-lactamase-producing haemophilus influenzae c.l. young, i.a. critchley, u.a. ochsner, n. janjic (louisville, us) objectives: faropenem (far) is an oral penem with potent activity against respiratory pathogens such as penicillin (pn)resistant streptococcus pneumoniae (sp) and beta-lactamase (bla)-producing haemophilus infleunzae (hi). the postantibiotic effect (pae) is a pharmacodynamic (pd) parameter that monitors suppression of bacterial growth following short exposure and removal of the drug. paes are clinically important for agents such as far with short half-lives (1 h) . the aim of the study was to determine the pae of far on resistant phenotypes of sp and hi. methods: nine clinical isolates of sp, 3 pn-s, 3 pn-i, and 3 pn-r and six clinical isolates of hi, 3 bla-negative and 3 blapositive were tested in pae studies. paes were determined in cation-adjusted mueller-hinton broth with 3-5% lysed horse blood for sp and haemophilus test medium for hi. exponential cultures (106 cfu/ml) were exposed to far at 1, 4 and 10 x mic. far was removed by serial washing (10,000-fold dilution) prior to transfer to fresh media. control cultures were treated in the same way. bacteria were incubated with shaking and viable cfus determined at 1, 4, 6, 8, 16 and 20 h. counts of log10 cfu were plotted against time and pae defined as the difference is the time required for count in test culture and control (untreated culture) to increase 1 log10 above the count observed immediately after removal. results: significant paes of >0.5 h were observed for all strains of sp at 4 and 10 x mic. however, the pae was more prolonged on the pn-r strains with mean paes of 3.9 h at 4 and 10 x mic. among hi, little or no pae was observed on bla-negative strains but a significant pae was observed on the bla-positive isolates (mean paes of 5.9 h and 8.9 h at 4 and 10 x mic respectively). conclusions: far demonstrates a prolonged pae on key resistant phenotypes of sp (pn-r) and hi (bla-positive) compared with susceptible strains. the observation of pae in bla-positive hi is unique in the class of beta-lactams. far exhibits in vitro pd properties that may contribute to its clinical efficacy against pn-r sp and bla-positive hi. telavancin is more efficacious than vancomycin in a murine model of bacteraemic peritonitis induced by methicillin-resistant staphylococcus aureus s. hegde, n. reyes, b. benton, r. skinner (south san francisco, us) objective: telavancin (tlv) is a novel lipoglycopeptide that operates through multiple mechanisms to produce potent and rapid bactericidal activity against clinically relevant grampositive bacteria including methicillin-resistant staphylococcus aureus (mrsa). the present studies evaluated the in vivo efficacy of tlv vs vancomycin (van) in a model of mrsa induced peritonitis in neutropenic mice. methods: female nsa immunocompromised mice were inoculated intraperitoneally with atcc mrsa 33591 and treated, beginning at 4 h post-infection, with 2 subcutaneous doses (q 12h) of vehicle (veh) or test compound. mouse pharmacokinetic data were generated and used to choose doses of tlv (40 mg/kg) and van (110 mg/kg) in order to equate clinical exposures (auc's (free drug) of 44 and 132 lg.hr/ml, respectively). in survival studies, deaths were recorded for 14days post-infection and survival curves were compared using log-rank test. in bacterial titer determination studies, designated groups of control and drug-treated surviving animals were humanely euthanized at various times post-treatment and their blood and spleen were harvested to determine bacterial titers. results: mics of tlv and van were 0.5 and 1.0 lg/ml, respectively. mortality was 100% in animals treated with veh or van. mortality was 7% in tlv-treated animals (p < 0.05 vs veh and van). the pre-treatment bacterial titres were 4.3 log cfu/ml and 8.7 log cfu/g in the blood and spleen, respectively. analysis of the time kill curves for both blood and spleen revealed that tlv exhibited significantly greater killing activity than van (p < 0.05, two-way anova). at 6 hrs after the first dose, the titers in the blood were reduced to a greater extent by tlv ()2.5 log cfu/ml) compared to van ()1.2 log cfu/ml). at 6 hrs after the second dose, the splenic titers were reduced to a greater extent by tlv ()3.8 log cfu/g) when compared to van ()1.4 log cfu/g). conclusions: the data described here demonstrate that tlv's in vivo bactericidal activity is superior to that of van against mrsa and results in successful infection resolution and, consequently, improved survival in the murine peritonitis model. proper use of carbapenems for blood-derived clinical isolates of pseudomonas aeruginosa y. kobayashi (tokyo, jp) methods: regimens of carbapenems were given to 1500 healthy adult subjects. changes in their blood concentrations of carbapenems were compared by using pharmacokinetic parameters (two-compartment model analysis) of meropenem (mepm), imipenem (ipm), and panipenem (papm) and by applying the lognormal distribution to the probability distribution of distribution volume and plasma half-life with monte carlo simulation (mcs). based on the data on distributions of the minimal inhibitory concentrations (mic) of various carbapenems for 77 blood-derived clinical isolates of pseudomonas aeruginosa isolated/identified at keio university hospital between october 1997 and october 2004 (mic90: mepm 8 mcg/ml, ipm 16 mcg/ml, papm 32 mcg/ml), the mics in the 1500 subjects were obtained with mcs. from the changes in blood concentrations and mics in the subjects, the probability of achieving t>mic was calculated for each carbapenem regimen, using the formula reported by kuti et al.: %t>mic = ln (dose/vd*mic)*(t1/2/0.693)*(100/di) <<ln: natural logarithm, dose: 1 dose (mg), vd: distribution volume (l), t1/2: plasma half-life (h), di: dosing interval (h)>>. based on craig's data, the maximum bactericidal effect on gram negative bacilli is attained when %t>mic is approximately 50%. we focused on this information and analysed our data. results: the probability of achieving t>mic50% was 43.2% for mepm 500 mg bid, followed by 4.2% for ipm 500 mg bid and 3.6% for papm 500 mg bid. when the dose was increased from 500 mg to 1000 mg, it was 60.7% for mepm 1000 mg bid, followed by 10.3% for ipm 1000 mg bid and 9.9% for papm 1000 mg bid. when the dose remained at 500 mg and the dosing frequency was increased to three times daily, it was 76.5% for mepm 500 mg tid, followed by 43.0% for ipm 500 mg tid and 18.8% for papm 500 mg tid. regarding mepm, it was 85.9% for 500 mg gid? and 86.7% for 1000 mg tid showing higher probabilities. discussion: in severe sepsis caused by pseudomonas aeruginosa, remarkably higher t>mic50% was achieved with carbapenems at 500 mg tid, although the daily dose (1500 mg) was lower, compared to 1000 mg bid. carbapenems with a low mic distribution, i.e. a superior antibacterial activity, showed higher probability of achieving t>mic. therefore, the optimal treatment for such sepsis is mepm 500 mg tid. mepm 500 mg qid appeared to provide comparable therapeutic effects with those at 100 mg tid, the usual dose in foreign countries. penetration of moxifloxacin into normal and infected subcutaneous tissue in patients with spinal cord injury measured by microdialysis background: skin breakdowns, also termed decubitus ulcers or pressure sores, are a major complication associated with spinal cord injury, resulting in infection and tissue death. moxifloxacin (mfx) is approved for the treatment of sssi. our objective was to construct a population pk model for mfx disposition in plasma, normal and infected subcutaneous tissue in spinal cord injured patients with infected decubitus ulcer. methods: 4 patients receiving 400 mg mfx orally daily were enrolled in this study. blood, saliva and interstitial tissue fluid samples (microdialysis in normal and infected tissue) were collected over a time period of 8 hrs. mfx concentrations were measured by a validated hplc. concentration-time data obtained in the present study were pooled with previously published mfx data (n = 12). population pk modelling was performed with nonmem. results: the concentrations of mfx achieved in plasma, saliva, normal subcutaneous tissues and infected decubitus ulcers showed parallel profiles versus time. the pk was best described by a 2-compartment model with a link to interstitial tissue fluid. the population pk parameters were as follows (given as estimate with percent interindividual variability in parentheses): cl 10.2 l/h (3%); central vd 83.2 l (15%); intercompartmental cl 31.9 l/h (16%); peripheral vd 92.2 l (17%); and elimination rate constant for interstitial tissue fluid 1.57 h)1 (17%). with a conservative mic90 of 0.25 mg/l, the peak/mic ratios were higher than 10 and the auc24/mic ratios were higher than 100 for plasma, saliva and interstitial tissue fluids. conclusions: this study showed the good diffusion of mfx into subcutaneous tissue in spinal cord injured patients with decubitus ulcers. the interstitial tissue fluids reached bactericidal levels for common bacteria found in infected skin lesions. objective: investigations of pharmacodynamic parameters such as postantibiotic effect and postantibiotic subminimum inhibitory concentration effect have been employed for design of dosing schedules of antimicrobial agents. in this study we compared postantibiotic effect and postantibiotic subminimum inhibitory concentration effect of ciprofloxacin, levofloxacin, and moxifloxacin for clinical isolates of methicillin susceptible staphylococcus aureus, methicillin resistant staphylococcus aureus and pseudomonas aureginosa. methods: the following strains were tested in this study: methicilline-susceptible staphylococcus aureus (n:4), methicilline resistant -staphylococcus aureus (n:2) and pseudomonas aeruginosa (n:2). the pae was determined by viable plate count method using mueller hinton broth. tubes containing 5 ml of broth and the antibiotic to be tested at 1, 2, 4 and 8x the mic were inoculated with approximately 5 · 106 cfu/ml. growth controls with an inoculum but not antibiotic were included with each experiment. result: postantibiotic effects of ciprofloxacin, levofloxacin and moxifloxacin increased with increasing concentration of the drug. the longest postantibiotic effect was observed for moxifloxacin. moxifloxacin showed no postantibiotic effect one p. aureginosa at all concentration and had no post antibiotic effect to another p. aureginosa at x2mic and mic. in our study the longest postantibiotic subminimum inhibitory concentration effect against mssa was determined with moxifloxacin. similarly the moxifloxacin induced the longest effect against mrsa. however, this time frame was shorter than that of mssa. conclusions: all three antibiotics, showed for longer postantibiotic subminimum inhibitory concentration effect in all submic concentrations, immeasurable within the study period i.e. 24 hours. lack of horizontal transmission of fluoroquinolone resistance between s. mitis and s. pneumoniae objectives: fluoroquinolone (fq) resistance can arise in s. pneumoniae through acquisition of dna from s. mitis and subsequent homologous recombination. the frequency at which this occurs is unknown, and while likely a rare event, increases in fq resistance among s. mitis may increase the rate at which horizontal transmission occurs. we sought to determine the frequency at which fq resistance could be transferred from s. mitis to s. pneumoniae or from s. pneumoniae to s. mitis. methods: s. mitis (either fq^r,tetracycline[tet^s], fq^r,penicillin[pen^s], or fq^s,pen^r) and s. pneumoniae (either fq^s,tet^r, fq^s,pen^r or fq^r,pen^s) were grown in co-culture using a pharmacodynamic model in the presence of either moxifloxacin (mxf) or levofloxacin (lfx) at salivary drug concentrations. after incubation, aliquots were plated onto either tet or pen containing sba plates to select for the recipient strains. fq susceptibility was performed using microbroth dilution. the entire parc and gyra genes were amplified and sequenced to determine if horizontal transmission occurred. results: in initial experiments tet was used as the selective agent. however tet resistance was transferred and therefore pen^r was used as a selective marker. an increase in the lfx mic in was observed in 3 s. pneumoniae and 1 s. mitis strain. sequencing of the parc gene revealed the selection of 2 ser79phe and 1 ser79tyr mutations in s. pneumoniae and ser79phe in s. mitis consistent with fq resistance. sequencing of the entire gene failed to uncover evidence of horizontal transmission. no mutations were detected in gyra. selection of 1st step parc clinical microbiology and infection, volume 12, supplement 4, 2006 mutations occurred only after exposure to lfx. mxf eradicated both s. mitis and s. pneumoniae and failed to either select for resistance or support horizontal transmission. conclusions: although 1st step parc mutations were selected in 4 strains (3 s. pneumoniae, 1 s. mitis), we failed to find evidence of horizontal transmission between s. pneumoniae and s. mitis under our laboratory conditions. the phenomenon of horizontal transfer resulting in fq resistance has been described, however, based on our results, we must speculate that it is an extremely rare event and not likely to be a major driver of fq resistance. of interest, the parc mutations were selected only under the selective pressure of lfx. mxf completely eradicated both s. pneumoniae and s. mitis and did not select for the development of fq^r mutations. objectives: the aim of the present study was to assess the killing activity of ertapenem (ert) and metronidazole (mtr) against four selected bacteroides fragilis strains with different mic values in an in vitro pharmacokinetic/pharmacodynamic (pk/pd) model. since anaerobes are often present in mixed infections, kill kinetics were also established for mixed inocula employing the b. fragilis strains together with four selected escherichia coli strains. the killing activity was analysed for kinetic concentrations of the antimicrobial agents simulating human serum kinetics. methods: a pk/pd in vitro model was established by adding appropriate amounts of broth every half hour. at the same time intervals samples were obtained and plated. after incubation colony forming units were counted. human serum concentrations were simulated with cmax = 100 mg/l and t1/2 of 5 hours for ert and cmax = 14.0 mg/l and t1/2 of 7 hours for mtr. mann trend test was used for statistical analysis. results: as to be expected the e. coli strains were not killed by mtr both in pure as well as in mixed cultures whereas the susceptible e. coli strains were effectively killed by ert. in pure cultures the b. fragilis strains were effectively killed by mtr and the growth of the susceptible b. fragilis strains was reduced by ert by about two to four logs. however, in some mixed cultures the killing activity of mtr against the b. fragilis strains was significantly reduced. conclusion: the in part moderate in vitro activity of ert against the b. fragilis strains and the reduced activity of metronidazole in mixed cultures against the b. fragilis strains may explain some of the difficulties in treating mixed aerobic/ anaerobic infections. penetration of ciprofloxacin into human cerebrospinal fluid and brain tissue a. tsona, s. metallidis, e. koumentaki, j. nikolaidis, p. kollaras, g. lazaraki, p. nikolaidis (thessaloniki, gr) objectives: the aim of the present study was to determine the penetration of ciprofloxacin into cerebrospinal fluid (csf) and brain tissue of humans. methods: a total of 10 patients undergoing brain tumor excision were evaluated. the patients received a single intravenous dose of 400 mg ciprofloxacin. samples of blood, cerebrospinal fluid and brain (brain-adjacent tumour tissue) were collected during surgery 2 h after drug administration. ciprofloxacin concentrations in serum, cerebrospinal fluid and brain homogenate were analysed by means of a validated hplc method. results: ciprofloxacin concentrations in plasma (mcg/ml), cerebrospinal fluid (mcg/ml) and tissue homogenate (mcg/g), respectively, after 2 h ranged 0.87-2.67 mcg/ml, 0.07-0.37 mcg/ ml and 0.65-4.02 mcg/g. csf-to-serum ratio ranged between 0.06 and 0.14. tissue-to-serum ratio ranged between 0.41 and 4.25. mean (±s.d.) csf/serum concentration ratios and brain tissue/serum concentration ratios were respectively 0.10 ± 0.03 and 1.70 ± 1.17. conclusion: these findings suggest that valuable informations on brain tissue penetration can be obtained only from brain material. data from csf penetration cannot be extrapolated to the brain since the blood: sf barrier differs from the blood:brain barrier. concentrations of ciprofloxacin in cerebrospinal fluid were lower than those in serum, in contrast to the brain tissue concentrations that exceeded serum concentrations. the achieved concentrations in brain tissue were generally above the mic90 of common pathogens in central nervous system infections (h. influenze, n. meningitidis, s. pneumoniae, l. monocytogenes, escherichia coli, aerobic gram-negative bacilli, group b streptococci, mssa). cerebrospinal fluid concentrations exceed the mics of neisseria meningitidis and most gram-negative aerobic bacilli. our findings suggests that ciprofloxacin may be an acceptable alternative for the treatment of meningitis due to susceptible gram-negative aerobic organisms and for the treatment of brain abscesses. objective: to model the performance of imipenem (imi), meropenem (mem), and ertapenem (etm) against esbl producing e. coli and klebsiella spp in order to identify possible pd differences among compounds. methods: minimal inhibitory concentrations (mics) were generated for 133 randomly selected esbl producing isolates of ec (n = 29) and kl (n = 104) collected during 2004 from brazilian hospitals as part of the mystic program. mic testing for imi, mem, etm, ceftazidime (ctz), and cefotaxime (ctx) were done by e-test methodology. esbls were confirmed via ctz/clavulanate and ctx/clavulanate e-test. pd exposure, measured as percent time above the mic for free drug (ft>mic), was modelled via a 5000 subject monte carlo simulation for the following 30-minute infusions:imi 1 gram every 8 hours, mem 1 gram every 8 hours, and etm 1 gram every 24 hours, using pharmacokinetics from healthy volunteers. the bactericidal cumulative fraction of response (cfr) was calculated for each regimen against the populations of ec, kl, and against all esbl isolates together. bactericidal cfr was defined as 40% ft>mic for all agents. results are reported as cfr (95% confidence interval). results: isolates were 100% susceptible (s) to imi and mem (mic range 0.125-1.5 and 0.023-4 mg/l, respectively), and 97% s to etm (mic range 0.008-32 mg/l conclusions: these findings support other data that although etm is likely to be an effective empiric agent against most esbl producing ec and kl, its ability to achieve high bactericidal pd exposure will be dependent on the presence of less susceptible organisms in the population. imi and mem should remain first line for esbl infections. objectives: this study analyses eradication and resistance selection in streptococcus pneumoniae with moxifloxacin, levofloxacin and azithromycin, using a parental serotype 3 infecting strain (a) and subsequent resistant step-mutants (isolates b, c and d) selected in vivo in a patient with pneumonia. methods: moxifloxacin, levofloxacin and azithromycin mics were 1, 2 and 0.5 lg/ml for the parental strain, 4, 16, and 4 lg/ ml for isolate b, and 4, 16 and >128 lg/ml for isolates c and d, respectively. a pharmacokinetic computerized device was used to simulate serum and epithelial lining fluid (elf) concentrations. initial inocula was approx. 108 cfu/ml. population analysis profiles were performed using plates with increasing antimicrobial concentrations on a mic basis. results: in serum, moxifloxacin eradicated the parental isolate (isolate a), with an auc0-24 h/mic value of 39.6. serum auc0-24 h/mic values of 26.5 and 5.5 for levofloxacin and azithromycin, respectively, were not able to eradicate isolate a. in elf, moxifloxacin showed a bactericidal pattern against all isolates with a minority (approx. 100 cfu/ml) of the survival population (isolates b, c and d) growing in plates with moxifloxacin concentrations higher than those obtained in elf. levofloxacin and azithromycin showed a bactericidal pattern only against isolate a, with the whole population of isolates b, c and d growing in plates with levofloxacin concentrations higher (16-64 lg/ml) than those obtained in elf, and in plates with azithromycin concentrations as high as 2048 lg/ml (for isolates c and d). in elf, moxifloxacin auc0-24 h/mic values were 201.0 for isolate a, and 50.3 for isolates b, c and d. levofloxacin auc0-24 h/mic values were 94.0 for isolate a, and 11.8 for isolates b, c and d. azithromycin auc0-24 h/mic values were 8.0 for isolate a; 10.0 for isolate b; and 0 for isolates c and d. conclusion: if prevention of resistance depends more on the eradication of possible emerging mutants in pulmonary tissues than of the parental susceptible strain, moxifloxacin concentrations in elf may provide advantages over previous quinolones and macrolides in preventing clinical failures. objectives: to explore how antimicrobial pressure influences the evolution of streptococcus pneumoniae populations sharing the same ecological niche. methods: an in vitro computerized pharmacodynamic model simulating physiological concentrations obtained over 24 h after 500 mg o.d levofloxacin, 750 mg b.i.d ciprofloxacin, and 500 mg o.d azithromycin was used to investigate its effect on a mixed culture of five s. pneumoniae serotypes (s) as an approach to ecology of population dynamics. resistance patterns were: s12 was susceptible to study drugs, s31 was low-level macrolideresistant (efflux phenotype), s11 was high-level macrolideresistant (erm genotype), s9v was low-level quinoloneresistant, and s3 was high-level quinolone-resistant. initial mixed inocula (time 0) included similar percentages of each serotype. results: mean colony counts in antibiotic-free plates (whole pneumococcal population) increased (from 0 to 24 h) from log10 6.9 to 8.8 in drug-free simulations (control), from log10 6.8 to 7.9 in levofloxacin simulations, from log10 6.8 to 8.6 in ciprofloxacin simulations, and from log10 7.1 to 8.8 in azithromycin simulations. at 24 h of control drug-free experiments, dominant strains were s9v (57.4%) and s12 (41.8%) with marginal populations of s31, s3 and s11. azithromycin selected in a much higher extent the strain with low-level resistance to macrolides (s31) than the strain with high-level resistance (s11) (accounting for 99.9% vs. 0.1% of total population at 24 h). ciprofloxacin selected in a higher extent low-level (s9v) than high-level (s3) quinolone resistance (72.4% vs. 27.6%). levofloxacin decreased the proportion of the predominant s9v in controls to 22.2% (an intermediateresistant strain with mic = 4 lg/ml), and unmasked the highlevel resistant strain (mic = 32 lg/ml) up to 77.8%. conclusion: strain distribution in antibiotic-free environment depends on bacterial fitness in mono-and multi-strain niches. the selective pressure of antimicrobial regimens eradicate some populations and unmask minor populations, thus redistributing the whole population. selective potential only for resistance phenotypes with very low prevalence (as high-level quinolone resistance) in the community should be preferred to that selecting more prevalent resistance phenotypes. re-evaluation of the role of broad-spectrum cephalosporins against staphylococci applying contemporary in vitro results and pharmacokinetic-pharmacodynamic principals h. sader, s.m. bhavnani, p.g. ambrose, r. jones (north liberty, us) objectives: to re-evaluate the current in vitro activity and to assess the pk-pd target attainment of cefepime (cpm), ceftriaxone (cro) and ceftazidime (caz) against staphylococcus spp. methods: the potency of cpm, cro and caz against staphylococci was accessed through the sentry antimicrobial surveillance program database, worldwide. during the 1998-2004 period 41,883 s. aureus (sa; 63% oxacillin [oxa]-susceptible [s]) and 14,349 coagulase-negative staphylococci (cons; 22% oxa-s) were s tested against cpm, cro, caz and numerous comparators by clsi broth microdilution methods. using volunteer pk data and a linear intermittent intravenous infusion model, and an animal-derived pk-pd target of 25% time above mic, expected probabilities of target attainment (pta) for cephems were evaluated using monte carlo simulation. pta were determined for the following dosing regimens: cpm 1gm q12 and q8 hours, caz 1 gm q8 hours and cro 1 gm q24 hours, each representing the most common dosing patterns applied clinically. cephem susceptibility (%s) was calculated based on the current clsi (2006) breakpoints (bkps) and also on bkps derived from a pta >90%. results: against oxa-s sa, mic50/90 values were (in mg/l): 2/4 for cpm, 4/4 for cro and 8/16 for caz, respectively; and against oxa-s cons mic50/90 values were (in mg/l) 0.5/2 for cpm, 2/4 for cro, and 4/8 for caz, respectively. the calculated %s of these cephems are summarized in the table: twenty year-old clsi bkps would rank the tested agents cpm ‡ cro > caz and by pk-pd pta cpm ‡ caz > cro. cpm has a potency advantage over caz (4-to 8-fold) and superiority at the usual dosing over cro (22.7-66.1%) for oxa-s staphylococci. caz pk overcomes by-weight activity disadvantages, while a low proportion (<5%) of active freedrug penalizes cro in the pta calculations. pta remained at >90% to a bkp of 16 mg/l for cpm (1 gm q8) and caz and to a bkp of 2 mg/l for cro. conclusions: regardless of applied bkp (clsi or pk-pd), cpm has the widest and more potent anti-staphylococcal activity among commonly used ''third-or fourth-generation'' cephems. when used at doses ‡ 3 gm/day, cpm assures maximal coverage of oxa-s staphylococci whether using existing (clsi) or modified (pk/pd) bkps. cro should be used with caution. methods: the mic for all strains were determined by serial two-fold macrodilutions. an in vitro kinetic model was used to investigate the antibacterial efficacy of constant drug concentrations during 6 hours. the selection of the doses of azithromycin tested in each bacterial strains was based on their mic values. bacterial counts were determined on appropriate agar plates using an adapted drop-plate method. twelve different pk/pd models were fitted and compared to the time-kill data by using non-linear regression. results: a simple pk-pd model was not sufficient to describe the pharmacodynamic effects for the four bacterial strains. appropriate models that gave good curve fits included a saturation term for the number of bacteria (nmax), delay terms (1-e-zt) for the initial bacterial growth phase and/or the onset of anti-infective activity as well as a hill factor (h) to capture the steepness of the concentration-response relationship. azithromycin had high potency against s. pneumoniae strains and m. catarrhalis while the potency of azithromycin against h. influenzae was poor. conclusions: the developed pk/pd models are suitable for describing the pharmacodynamics of azithromycin. applications of these pk-pd models will eventually provide a tool for rational antibiotic dosing decisions. objectives: optimal antimicrobial dosage regimens aim to achieve successful clinical outcomes without drug toxicity or emergence of bacterial resistance. for concentration dependent antibiotics, such as the fluoroquinolones, in humans a cmax:mic ratio of >10 is considered more important for efficacy and reduced selection of resistance than prolonged antibiotic concentrations just above the mic. fluoroquinolone resistance in zoonotic bacteria is a matter of public health concern, and fluoroquinolone treatment of poultry can rapidly select for bacteria with reduced fluoroquinolone susceptibility. in this study we compared basic pharmacokinetic parameters for the recommended dose of baytril (enrofloxacin) 10% oral solution in poultry to 2.5x this dose for birds dosed by continuous water (standard) compared to pulsed water treatments and dosing by gavage.methods. for the pulsed versus continuous water treatments, groups of chickens received baytril 10% oral solution at 50 (recommended) or 125 ppm continuously in the water or at 10 (recommended) or 25 mg/kg pulsed in the water. for each group, three birds were killed at 0, 2, 4, 6, 8, 10 and 24 hours after start of antibiotic treatment and caecal contents, liver, lung and sera were taken and the concentration of fluoroquinolone determined by fluorescence hplc. for gavage treatment, dosing was at 10 and 25 mg/kg by crop intubation and four birds were killed in each group at 2, 6 and 24 hours after gavage; caecal contents, liver and sera were taken and analysed as above. basic pharmacokinetic parameters were determined using pk solutions software. results: the mean fluoroquinolone cmax in caecal contents (and sera) for gavage, pulsed water and continuous water treatments respectively was 78.01 (1.81), 53.19 (1.17) and 24.73 (0.73) mg/ml after the recommended dose and 115.86 (3.86), 115.63 (2.74) and 68.02 (1.17) mg/ml after 2.5x the recommended dose. cmax of antibiotic in liver and lung was increased by the modified regimens in similar proportions to above. both pulsed water and gavage treatment not only resulted in higher cmax values, but also a faster rate of fluoroquinolone clearance than continuous water treatment ( figure 1 ). dosing by gavage is not practical for thousands of chickens. however, pulsed dosing at 2.5x the recommended dose can increase cmax values about fourfold and so could improve efficacy and reduce selection of resistance, compared to the current recommended treatment regime. objectives: nephrotoxicity is the major concern arising with the use of intravenous colistimethate sodium. methods: a prospective cohort study was performed at ''henry dunant'' hospital, a 450-bed tertiary care center in athens, greece. patients who received intravenous colistin for at least 7 days for the treatment of multidrug resistant gram-negative bacterial infections were included in the study. the development of nephrotoxicity through evaluations of serum creatinine, blood urea, serum electrolytes, urinalysis, and creatinine and sodium in 24-hour urine collection during intravenous colistin therapy was the primary end point of the study. results: twenty-six patients were included in the study, 21 of whom received colistimethate sodium (cms) for at least 7 days and were evaluated further. the mean (± sd)/median daily dose, cumulative dose, and duration of treatment of intravenous cms was 5.5 (± 1.9)/6 million iu, 90.2 (±52.0)/72 million iu, and 17.7 (±11.7)/15 days (range 7-54 days), respectively. three of the 21 evaluable patients (14.3%) developed nephrotoxicity during the intravenous treatment with cms. the cumulative dose of the administered cms was statistically correlated with the difference between the end and start of cms treatment values of serum creatinine (r = 0.6, p = 0.004 by spearman's test). a statistically but not clinically significant decrease of the mean baseline serum sodium concentration was observed between start and end of treatment [mean 144.2 (±6.9) to 142.1 (±6.1) mmol/l, p = 0.04]. no other toxic events were noted during the intravenous administration of colistimethate sodium. conclusion: although this is an evaluation of a small number of patients, our prospective study shows that nephrotoxicity was not commonly observed in this group of patients who received intravenous colistimethate sodium. however, caution should be taken to avoid the prolonged administration of the antibiotic. objectives: the objective of the present work is quantitative structure-activity relationship (qsar) analysis of antimicrobial activity of the 4-thiazolidone derivatives and consequent computational design of new antimicrobials. methods: for the achievement of the formulated objectives the qsar investigation has been carried out using computational chemistry approach based on simplex representation of molecular structure (sirms). on the framework of sirms it is possible to develop the molecular design of the new effective antimicrobials. results: systematic researches of relationship between antimicrobial activity (staphylococcus aureus -methicillin-sensitive (mssa) strain, pseudomonas aeruginosa -r and s strain, klebsiella pneumoniae, candida albicans s and ñ itrobacter freundii) and a structure of about one hundred fifty compounds (4-thiazolidone derivatives and analogs). the elucidation of structure-activity relations allows predicting biological properties of such compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of their biological effect. completely adequate statistical partial least squares models (r2 = 0.841-0.990, q2 = 0.600-0.814) have been obtained for all of the studied cultures. on the base of the first ones the molecular fragments both promoting and interfering the given antimicrobial activity have been determined. they give a possibility to realize the computer high throughput screening and molecular design of active compounds. the results of prognosis are verifying by the experimental investigations. also the influence of heterocycle system evolution on antimicrobial activity has been revealed. conclusion: qsar analysis of antimicrobial activity of 4-thiazolidone derivatives allows us to discover that the presence of naphthalene-substituted fragment (independently on its location in molecule) has distinctly negative influence on antimicrobial action. the requirements to molecular design have been formulated. for example, high active compounds must include 3-indolyl fragment. computational design of the new antimicrobials based on the substituted crown ethers activity of the row of substituted crown ethers and consequent molecular design of new antimicrobials. methods: the well-known hierarchic system of qsar models based on simplex representation of molecular structure has been used for the solution of the formulated problem, within the framework of which one it is possible to develop the molecular design of the new effective antimicrobial agents. results: we tried to conduct systematic researches of relationship between antimicrobial activity (planococcus citreus, streptococcus lactis, micrococcus lysodeiktious, staphylococcus aureus, streptococcus faecalis, bacillus subtillum) about two hundred fifty crowns ethers including aromatic, cyclic and heterocyclic etc. fragments and a structure of these molecules, in particular -macro cycle size, it dentacy, lipophily, nature of the substituents, and other factors. the elucidation of similar relations allows predicting biological properties of crown compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of biological effect of such kind of compounds. completely adequate qsar models (r2 = 0.841-0.995, q2 = 0.660-0.954) have been obtained using partial least squares method for all of the studied cultures. on the base of the first ones the molecular fragments with positive or negative influence on the explored properties have been determined. they give a possibility to realize the virtual screening and molecular design of compounds with the high level of target activity. the results of prognosis are verifying by the experimental investigations. conclusion: qsar analysis of antimicrobial activity of crown ethers allows us to suppose the presence of two different mechanisms of their antimicrobial action. it is discovered that the presence of diphenyloxide and tert-butyl fragments promotes; diphenyl-sulphide and diamino-biphenyl -prevents the antimicrobial action. it is shown that the hexadenthal crown ethers containing aromatic fragments with a tert-butyl group are the most perspective antimicrobials. objectives: methionyl trna synthetase (mrs) catalyses the covalent attachment of methionine to its cognate trna. rep8839 is a synthetic inhibitor of mrs with potent antibacterial activity against staphylococcus aureus including clinically-relevant resistant strains (mic90 equals 0.06 to 0.5 lg/ml). we determined the biochemical potency and mechanism of action of rep8839 and related compounds with respect to s. aureus mrs enzymatic activity. we also evaluated the enzyme kinetic properties of mutated forms of s. aureus mrs. methods: the mets gene from s. aureus was expressed in e. coli and mrs was purified to near homogeneity by ammonium sulfate fractionation and anion exchange chromatography. aminoacylation of trnamet was measured using scintillation proximity assays (spa). the kinetics of the atp:ppi exchange were determined using thin layer chromatography (tlc). mutants of s. aureus mrs were selected by serial passage and spontaneous resistance in the presence of rep8839. results: rep8839 exhibited strong inhibition of s. aureus mrs in the aminoacylation reaction, having an ic50 limited by the enzyme concentration. in order to estimate the true inhibition constant (ki), we utilized an atp:ppi exchange assay. rep8839 showed potent inhibition of s. aureus mrs, with a ki of 10 pm. related inhibitors were analysed, and a correlation was observed between the ki for mrs and the mic for s. aureus. rep8839 was found to be competitive with methionine binding, but uncompetitive with atp binding (i.e., increasing the atp concentration resulted in tighter binding of rep8839). the majority of analogs exhibited comparable mechanism of action; altered mechanism of action was observed with a subset of analogs. mutated s. aureus mrs variants (derived from strains with elevated mics) showed substantially weaker binding by rep8839. all of the mutated enzymes exhibited impaired trna aminoacylation activity, with defects ranging from reduced turnover rates to weaker affinities for one or more substrates. conclusions: rep8839 is a potent inhibitor of s. aureus mrs. enzymatic potency of this class of inhibitors correlates with microbiological potency. mutations that confer resistance to rep8839 result in functionally impaired mrs, encompassing a wide variety of enzymatic phenotypes. we report here the antibacterial and antifungal activity of 8 newly synthesized and physico-chemically characterised thioureides of 2-(4-chlorophenoxy)-benzoic acid. the new compounds were prepared in three stage. firstly, the 2-(4-chlorophenoxymethyl)-benzoic acid was prepared by treating the phtalide with p-chlorophenol potassium salt in xylene. the second stage was the synthesis of 2-(4-chlorophenoxymethyl)benzoyl chloride by treating the corresponding acid with thionyl chloride using anhydrous 1,2-dichloroethane as solvent, followed in the third stage, by the treatment of the above-mentioned chloride with ammonium thiocyanate. the 2-(4-chlorophenoxymethyl)-benzoyl isothiocyanate resulted after refluxing the reaction mixture in dry acetone. the new compounds were prepared by refluxing the isothiocyanate with primary aromatic amines in dry acetone. the obtained compounds have been characterized by their physical properties and their chemical structures were confirmed using the spectral analysis. the aim of this study was also to evaluate the in vitro antimicrobial activity of the new compounds. the in vitro antimicrobial testing was performed by binary microdilution method, in 96 multi-well plates, in order to establish the minimal inhibitory concentration (mic), against gram-positive (listeria (l.) monocytogenes, staphylococcus (s.) aureus, bacillus (b.) subtilis), gram-negative (psedomonas (p.) aeruginosa, escherichia (e.) coli, salmonella (s.) enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, depending on the nature of the substituents and their position on the benzene ring, both concerning the microbial spectrum and the mic value. the mics values widely ranged between 1024 mcg/ml and 32 mcg/ml. the most active proved to be n-[2-(4-chlorophenoxymethyl)-benzoyl]-n'-(2,6-dichloro-phenyl)-thiourea and n-[2-(4-chlorophenoxymethyl)-benzoyl]-n'-(4-bromo-phenyl)-thiourea, showing a large spectrum of antimicrobial activity against enterobacterial strains (e. coli and s. enteritidis), l. monocytogenes, s. aureus and candida sp. all the tested compounds were highly active against s. aureus (mic = 32 mcg/ml). four of the tested compounds exhibited antifungal activity (mic = 256-32 mcg/ml), and p. aeruginosa as well as b. subtilis were resistant to all tested compounds. in vitro antimicrobial activities of novel dianthraquinones produced by a marine streptomyces sp. against clinical staphylococcus aureus and enterococcus faecium isolates k.l. laplante, k. lor, a. socha, d.c. rowley (providence, north kingston, us) objectives: the escalation of antibiotic resistance among grampositive pathogens presents increasing treatment challenges and requires the development of new therapeutic agents. recently we discovered a new class of dianthraquinone antibiotics produced by a marine streptomycete. the inhibitory and bactericidal activity of four dianthraquinone secondary metabolites and four semi-synthetic derivatives were measured against clinical strains of vancomycin resistant e. faecium (vre), methicillin susceptible and methicillin resistant s. aureus (mssa and mrsa, respectively). two compounds, daq550a and daq552, were tested against an expanded panel of pathogens. methods: thirty-two clinical strains of vre (n = 10), mssa (n = 12) and mrsa (n = 12) were obtained from patients at the veterans affairs medical center in providence, ri. mic's were performed using methodologies described by clsi. control isolates were atcc52923 and atcc29213. the bactericidal activity of each antimicrobial agent was evaluated with time-kill experiments using 6 randomly selected mssa (n = 2), mrsa (n = 2), and vre (n = 2) isolates tested at 4 times the respective mic. conclusions: the potent activities and unusual structures of the dianthraquinones tested here suggest that these may provide a new molecular scaffold for the development of novel antimicrobial agents. more biological testing is warranted to more fully explore the clinical potential of these antibiotics. efficacy of the novel antimicrobial peptide plectasin to staphylococci objective: the purpose of the investigation was to investigate the in vitro efficacy and kill kinetics of plectasin against staphylococcus aureus. plectasin is a newly discovered defensintype antimicrobial peptide found in the fungus pseudoplectania nigrella which showed activity against several gram-positive bacteria including drug resistant strains (mygind ph. et al. plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. nature 2005; 437:975-980). methods: all experiments were determined according to clsi/ nccls guidelines. bactericidal activity was characterized by time kill experiments at 2 and 10 times the mic. staphylococcus aureus (s. aureus) atcc29213 were used as the test organism and vancomycin was used for comparison. the kill kinetics and post antibiotic effect (pae) were evaluated by cfu determination. inoculum sizes ranging from 10e4 to 10e7 cells were used to test the inoculum effect. 10e10 cells were employed for determination of mutant prevention concentration (mpc) and the frequency of spontaneous resistance. results: plectasin is bactericidal as evidenced by kill kinetics showing a 2.1 log reduction in cfu/ml after 1 hour of incubation and a reduction of 3.1 log cfu/ml after 2 hours. this is superior compared to the activity of vancomycin. no inoculum effect was observed in the employed range of cells. the observed pae had a duration of 2 hours and 42 minutes. no spontaneously resistance mutation was observed among 10e10 cells of staphylococci and the mpc were determined to be 8 times mic. conclusions: plectasin is a novel antimicrobial peptide that shows potent antimicrobial activity against gram-positive bacteria including drug-resistant organisms. the potent, excellent bactericidal activity in vitro, lack of cross-resistance to clinical used antibiotics, low spontaneously resistance mutation frequency and good pae properties, suggest that plectasin may have potential as a therapeutic agent against staphylococci. in vitro antimicrobial activity of the novel polymeric guanidine akacid plus ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: cationic antimicrobials are widely used for disinfection within clinical settings. in the present study the bactericidal and fungicidal activity of akacid plus ò , a novel polymeric compound of the cationic family of disinfectants, was evaluated against quality control strains of staphylococcus aureus, enterococcus hirae, escherichia coli, pseudomonas aeruginosa, candida albicans and aspergillus niger in comparison to chlorhexidine digluconate. methods: the in vitro activity of akacid plus ò and chlorhexidine was determined by quantitative suspensions tests according to the european committee for standardization at concentrations of 0.01-0.5% against bacterial strains and c. albicans and at concentrations of 0.5-4% against a. niger after exposure for 5, 15 and 60 min in the presence and absence of 0.3% bovine albumin and dilution in distilled and hard water. results: in the basic quantitative suspension test akacid plus ò destroyed all bacterial pathogens at a concentration of ‡0.1% in £5 min contact time. chlorhexidine was also highly active against s. aureus, e. coli and p. aeruginosa, but failed to eliminate e. hirae within 5 min. under high organic burden, the bactericidal activity of both disinfectants was slightly reduced. akacid plus ò showed fungicidal activity against c. albicans within 15-60 min and eliminated a. niger at a concentration of ‡1% in 5 min contact time. chlorhexidine was fungicidal against c. albicans, but did not achieve biocidal activity against a. niger. conclusion: the novel polymeric guanidine akacid plus ò when compared to chlorhexidine digluconate showed similar bactericidal activity against s. aureus, e. coli and p. aeruginosa and superior biocidal activity against e. hirae and a. niger. investigation of emergence of bacterial resistance to the novel antibacterial photodynamic agent xf-42 are novel, light activated antibacterial agents (1) active against gram-positive bacteria, which have greater potency than antibiotics. the emergence of resistance to xf-42 has been investigated. methods: 0.382 mg/l of xf-42 was added to 108 cells/ml of mrsa. after 5 minutes incubation in the dark the unbound xf-42 was removed and the culture illuminated with 13.7 j/cm 2 of light at 422 nm and cfu analysis undertaken to determine the number of viable cells remaining. 5 surviving clones of the treatment were cultured and subjected to further treatment. 10 cycles were undertaken to determine whether the number of surviving cells increased, suggesting resistance build up to xf-42. results: the survival of methicillin-resistant staphylococcus aureus (mrsa) (atcc baa-44) is expressed as log n0/n, where n0 and n are the cfu of untreated and treated suspensions, respectively. the results demonstrate that no detectable resistance build up to the activity of xf-42 was seen after 10 successive treatments. a low propensity for emergence of resistance is a valuable attribute for new anti-bacterial agents. xf-42 might be effectively employed in the clinical setting for prophylactic use to decolonise skin and nares and therapeutic use to treat infected wounds/ulcers. objectives: the xf drugs are novel, light activated antibacterial agents (1) active against gram-positive bacteria which have superior potency to antibiotics but possess a low propensity to induce resistant bacterial strain emergence. a novel ex-vivo porcine skin model has been developed to test the antibacterial activity of xf-73 on the surface of skin. methods: 10x7 cells of methicillin-resistant staphylococcus aureus (mrsa) were inoculated onto a 1.32 cm 2 area of ex-vivo porcine skin samples, immobilised in agar. after drying, solutions of xf-73 were applied and after 60 minutes, the samples were illuminated for 15 minutes with blue light (422 nm) with various total light doses using a lumacare tm lc-122m lamp. cfu analysis were undertaken to determine the number of viable cells remaining after treatment. controls of drug alone and light alone were included. results: using 7.64 mg/l of xf-73, cfu analysis demonstrated that at a total light dose of 5 j/cm 2 , there was~90% kill of bacteria. at 10 j/cm 2 , there was 99.9% kill of bacteria, and 99.99% at 20 j/cm 2 and 40 j/cm 2 . at a total light dose of 20 j/ cm 2 , it was found that there was a <99% kill by xf-73 at concentrations of 0.76, 1.53 and 3.8 mg/l. at a concentration of 7.64 mg/l, there was a >99.9% kill. this kill did not significantly increase at 22.93 and 76.4 mg/l. conclusions: the results demonstrate that xf-73 has exceptional activity at low concentrations against mrsa on the surface of porcine skin. xf-73 and light are non-toxic to skin at therapeutic concentrations. work is in progress to clinically evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objectives: the rise of epidemic methicillin-resistant staphylococcus aureus (emrsa) and the emergence of mupirocin resistance means that it is essential to develop new therapies that cannot be readily overcome by microorganisms. the xf series of novel light activated antibacterial agents (1) active against gram-positive bacteria addresses this issue and have superior levels of activity to antibiotics but with less likelihood of resistance emergence. the antibacterial activity of the xf drugs against emrsa has been investigated. methods: mic and mbc assays were used to investigate the antibacterial activity of xf-73, a novel antimicrobial photodynamic agent against a range of staphylococcus aureus strains. a concentration range of 2-0.001 mg/l was investigated. 15 minutes of 420 nm light activation (13 j/cm 2 ) was applied. light alone had no effect. results: conclusions: the results demonstrate that xf-73 has exceptionally low mic and mbc values against all of the s. aureus strains tested. the results also demonstrate that xf-73 is equally effective against mrsa and methicillin-sensitive staphylococcus aureus (mssa) indicating its mode of action is independent of antibiotic resistance. xf-73 may therefore be useful in prevention and treatment of emrsa. xf-73 is non-toxic to skin at prophylactic/therapeutic concentrations and has potential for the treatment of skin sepsis and the eradication of nasal and skin mrsa carriage. work is in progress to evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objective: nxl101 is a novel antibacterial currently in preclinical development. the mechanism of action is directed against topoisomerase, and the spectrum of activity is exclusively against gram positive organisms. the goal of the study was to characterise the activity and time/kill kinetics against common aerobic cocci in comparison to currently marketed molecules: linezolid (lin), vancomycin (van), quinupristin/dalfopristin (q/d) and moxifloxacin (mox). methods: (i) in vitro susceptibility tests: the strains used were from the culture collection of novexel and were of clinical origin. mics were determined by an agar dilution technique. mueller hinton agar medium was used, supplemented with 5% horse blood for group a streptococci (gas), group b streptococci and s. pneumoniae. overnight cultures were diluted to obtain the final inoculum of 10 4 cfu/spot. the mic was the lowest concentration which inhibited all visual growth (3 or less colonies were ignored). (ii) time/kill kinetics: experiments were performed against strains of s. aureus (n = 8) and s. pneumoniae (n = 4) in 20 ml volumes of appropriate growth medium with initial inoculum of around 10 6 cfu/ml of logarithmically growing culture. timed samples over a 24 hour period were enumerated using a spiral plating method. nxl101 was compared to linezolid and vancomycin and the concentrations tested were 4, 8 and 16-fold the mic90 for both species. results: (i) the mic90s of nxl101 versus comparators are shown in the table. (ii) time kill experiments showed that nxl101 was bactericidal against s. aureus, including methicillin resistant strains (>3log10 reduction within 6-8 hours) compared to a slowly bactericidal effect for vancomycin (24 hours). nxl101 and vancomycin were both bactericidal against s. pneumoniae within 6-8 hours. linezolid was bacteriostatic against all strains tested. conclusion: nxl101 exhibits bactericidal activity against common gram positive cocci, including strains which exhibit resistance to methicillin, vancomycin and fluoroquinolones. nxl101 warrants further investigation. objectives: the aim of this study was to identify bacterial proteins as targets of the endogenous antiseptic n-chlorotaurine (nct), which is a promising microbicidal agent for topical treatment of infections. in addition, a combination of nct with ammonium chloride which enhances the microbicidal activity significantly was investigated. methods: escherichia coli and staphylococcus aureus were treated with nct and nct plus ammonium chloride for different incubation times between 1 and 30 min -a period where killing takes place. to find out protein changes, 2d-page of bacterial proteins followed by mass spectrometry was performed. results: incubation in 1% nct revealed a change of the charge and a separation of numerous proteins into a series of spots with a different isoelectric point. moreover, in e. coli heat shock protein 60 appeared, while ribosome releasing factor, d-ribose periplasmic binding protein, and malonyl-coa transacylase spots decreased. in s. aureus, enolase and a translation elongation factor decreased. these changes appeared more rapidly in the presence of ammonium chloride, which can be explained by formation of the more lipophilic and microbicidal monochloramine. molecular mechanisms of attack comprised mainly oxidation of thio and amino groups as confirmed with model peptides. conclusion: these results fit very well to previous preclinical and clinical findings. they indicate both surface attack and penetration of oxidation capacity into the bacteria and destruction of essential proteins by nct and nct plus ammonium chloride, respectively. objectives: ceftobiprole is a new extended-spectrum cephalosporin with activity against methicillin-susceptible and methicillin-resistant staphylococci, as well as against most enterobacteriaceae. in this study the anti-staphylococcal activity of ceftobiprole is reported from a set of isolates from a recent clinical trial. methods: consecutive clinical isolates of staphylococci from 340 patients enrolled in a multicentre clinical trial involving complicated skin infections were examined for their susceptibility to ceftobiprole and selected anti-gram-positive agents. mics were determined using clsi methodology. results: among these isolates, 525 staphylococcus aureus and 84 coagulase-negative staphylococci (cons) were identified. the percentages of methicillin-resistant strains were 43% for s. aureus and 50% for cons. all strains (except one cons with a linezolid mic of 8 mg/l) were susceptible to vancomycin and linezolid, with mics <2 mg/l.against methicillin-susceptible s. aureus, ceftobiprole mic50 and mic90 values were 0.25 and 0.5 mg/l, respectively, and against methicillin-resistant s. aureus, ceftobiprole mic50 and mic90 values were 0.5 and 2 mg/l, respectively. ceftobiprole mics ranged from £0.06 to 1 mg/l against methicillin-susceptible-cons (ms-cons) and methods: consecutive, non-duplicate bacterial isolates (10,068 strains) acquired from patients with bloodstream, respiratory, and skin and skin structure infections both nosocomial and community acquired were submitted from >70 medical centres in europe, the americas and the asia-pacific region. all isolates were tested using clsi/nccls broth microdilution methods against grn, the currently marketed fluoroquinolones (fq) including cipro, levofloxacin (levo), gatifloxacin (gati) and representative comparator agents. oxa-and cipro-s and -r subsets were included. a grn-s breakpoint of £0.12 mg/l was applied for comparative purposes only and was based upon the mic population distributions of strains that included quinoloneresistance determining region (qrdr) mutations. results: potency for grn and comparator fqs tested against sa: (see table) . key resistance patterns (%) among this sa collection included oxa (40.3), cipro (36.4), erythromycin (47.5), clindamycin (13.4), tetracycline (9.7), and trimethoprim/ sulfamethoxazole (4.8%); gram-positive-targeted comparator including vancomycin, linezolid, daptomycin and quinupristin/dalfopristin all remained >99% s. compared with currently marketed fqs when tested against all sa, grn was 2-to 16-fold more active (mic50, £0.03 vs. 0.06 or 0.5 mg/ l). against both oxa-s and -r sa, grn displayed markedly enhanced potency compared with cipro and levo ( ‡4-fold), and gati (2-to 4-fold). among cipro-r isolates, grn also maintained ‡4-fold greater potency (mic50, 1 vs. ‡4 mg/l) although overall s for all fqs was 0-1%. compared to the fq agents tested against sa, grn was the most potent agent and maintained the broadest coverage against oxa-and cipro-r strains even when applying a very conservative epidemiologic breakpoint. when a fq is indicated for staphylococcal coverage, this des-f(6) quinolone may represent a superior alternative among fq class agents, while minimizing selection of resistance. objective: to assess the garenoxacin (grn) potency against a vast number of international respiratory tract infection (rti) pathogens, especially versus phenotypic (high mic) or genotypic (sequence change) qrdr mutants. a total of 40,423 isolates from 6 continents were analysed (1999) (2000) (2001) (2002) (2003) (2004) (2005) table) conclusions: grn maintains clinically usable activity (mic, £1 mg/l) against important community-acquired rti pathogens having r to presently marketed fluoroquinolones and against those isolates with documented qrdr mutations. continued development of this novel des-f(6) quinolone agent appears desirable. in vitro activity of garenoxacin tested against ciprofloxacin-susceptible and -resistant enterobacteriaceae and acinetobacter spp. strains collected worldwide by the sentry antimicrobial surveillance program (2004) (2005) h. sader, t. fritsche, p. strabala, r. jones (north liberty, us) objective: to evaluate the contemporary activity of garenoxacin (grn) against ciprofloxacin (cipro)-susceptible (s) and cipro-resistant (r) enterobacteriaceae (ent) and acinetobacter spp. (asp). unlike recently marketed fluoroquinolones (fq), grn, a des-f(6) quinolone lacks the c-6 fluorine. methods: a total of 9,017 isolates (8,247 ent and 770 asp) were consecutively collected from > 70 medical centres from bloodstream, respiratory, urinary and skin and soft tissue infections and tested by reference broth microdilution methods according to clsi/nccls methods and interpretative criteria. a grn s breakpoint of £1 mg/l was applied for comparison purposes only. results: the results of the major organism groups tested: (see table) . grn showed excellent activity against this large collection of ent (mic50, 0.12 mg/l) and 82.7% of isolates were inhibited at £1 mg/l. objectives: garenoxacin (grn) is a novel, broad-spectrum des-f(6)-quinolone with activity against gram-negative and grampositive aerobes and anaerobes including quinolone-resistant staphylococcus aureus. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in complicated skin and skin structure infections (csssi). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn (600 mg iv to po qd) or piperacillin/tazobactam (3.375 g iv q6h) with transition to po amoxicillin/clavulanate (500 mg po q8h). in the second study, subjects received grn (600 mg po qd) or ciprofloxacin/metronidazole (500 mg q12h/500 mg q8h). all antimicrobials were administered for 7 to 14 days. subjects were adults ( ‡18 y) newly hospitalized or ambulatory outpatients with evidence of csssi who did not have underlying osteomyelitis. microbiologic efficacy was determined 5 to18 days post-therapy. results: a total of 567 subjects were microbiologically evaluable (grn, n = 283; comparators, n = 284). the disease diagnosis was similar between grn and comparators and included infected pressure sore (5% vs 3%), infected diabetic foot ulcer (18% vs 19%), major abscess (66% vs 67%), or postsurgical wound infection (11% vs 12%). the majority of common skin pathogens were eradicated by grn background: acute bacterial sinusitis (abs) is a common infection world-wide, with many patients having an associated an allergic component/history. however the role of antibacterials in these patients (pts) has not been examined. as some fluoroquinolones (fq) have an in-vitro immunomodulatory effect (ie) the clinical efficacy of gem was compared other agents in abs pts with or without allergic rhinitis (ar). methods: 5 phase 3 clinical trials were pooled and pts where allergic rhinitis was identified (584 pts) were compared with pts not reporting ar (1834 pts). clinical response (success or failure) at end of therapy (eot) & at follow-up (fu, approx. 1-3 weeks after treatment) was studied. comparators (cmp) were cefuroxime (cef) and trovafloxacin (tro). results: % success based on clinical outcome at eot and fu for ar and non ar pts are shown in the table. for all treatments eot success was high for the non ar pts, but at fu this was reduced, especially with both fqs. in contrast, gem retained a high clinical success rate in pts with ar unlike cef or tro. conclusion: gem has been shown to be very efficacious in a sub group of problematic abs pts. this advantage may be due to the high antibacterial activity of gem vs key abs pathogens and/or a stimulatory ie. both being important with pts having decreased local immune defences. these data also show that not all fluoroquinolones have immuno-stimulatory properties. garenoxacin efficacy against multidrug-resistant streptococcus pneumoniae: retrospective analysis of community-acquired pneumoniae isolates obtained from nine phase ii and iii clinical studies (1999) (2000) (2001) (2002) (2003) t. black, h. waskin, r. hare (kenilworth, us) objective: garenoxacin (grn) is a novel, des-f(6)-quinolone with excellent activity against s. pneumoniae, one of the most common pathogens causing community-acquired pneumoniae (cap). the incidence of infections caused by antibiotic-resistant isolates of streptococcus pneumoniae is on the increase, therefore information regarding the activity of new anti-infective drugs against populations of s. pneumoniae that are multi-drug resistant (mdr) is critical. mdr s. pneumoniae (mdrsp) includes isolates previously known as prsp (penicillinresistant s. pneumoniae), as well as strains resistant to two or more of the following antibiotics: second-generation cephalosporins, macrolides, tetracyclines, and trimethoprim/ sulfamethoxazole. methods: pretreatment sputum and blood isolates collected worldwide during grn phase 2/3 clinical cap trials (1999) (2000) (2001) (2002) (2003) were retrospectively analysed for the mdrsp phenotype. of the 352 s. pneumoniae isolates originally identified, 208 from 180 subjects were subjected to secondary mdr susceptibility testing by central laboratories. confirmed mdrsp isolates were matched to individual subjects to assess clinical and microbiological outcomes for mdrsp-infections treated with grn. results: expanded susceptibility testing identified 53/208 mdrsp isolates from 44 unique subjects. the lowest mic50 and mic90 values for mdrsp isolates tested against a panel of representative drugs were observed for grn (table 1 ; 0.03 lg/ ml and 0.06 lg/ml, respectively). the incidence of resistance to the five classes of drugs was 11%, 12%, 21%, 19% and 18% for penicillin, 2nd generation cep., macrolides, tetracycline and tri/sulf, respectively. no isolates were resistant to grn using a proposed susceptibility breakpoint value of £1 lg/ml. thirtyfive percent, 28%, 15%, 9% and 13% of isolates were resistant to 1, 2, 3, 4 and 5 drug classes, respectively. the worldwide incidence of mdrsp was 18% with an equivalent geographic distribution of 19%, 21% and 16% among north america, europe and the rest of world. overall, grn provided clinical and bacteriological success for 32/35 (91%) cap evaluable subjects with mdr infection, which was similar to clinical success for evaluable subjects with non-mdrsp cap infections 151/165 (92%). conclusions: these data demonstrate the ability of grn to successfully eradicate mdrsp associated with cap. . per cent success is shown in the table (ab, antibiotics, copd, chronic bronchitis and obstructive lung disease, hd, heart disease). results: although gemifloxacin showed lower % success than comparator against cap patients with no defined risk factor, gemifloxacin was considerably more successful than comparator against patients associated with risk factors, especially diabetic patients where comparator success was low. this advantage was often more prominent at fu than at eot. patients with other comorbidities such as renal failure or malignancy were not recruited in sufficient number for analysis. conclusions: these data support the use of gemifloxacin in the treatment of cap, especially where the patient has recognised idsa risk factors. microbiologic efficacy of garenoxacin vs. comparators against common pathogens associated with community-acquired pneumonia objectives: garenoxacin (grn) a novel, broad-spectrum des-f(6)-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. grn has dual sites of inhibition (dna gyrase and topoisomerase iv) and may be less likely to promote resistance. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in community-acquired pneumonia (cap). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn (400 mg po qd for 5 d) or amoxicillin/clavulanate (a/c; 500 mg po q8h for 7-10 d). in the second study, subjects received grn (400 mg po qd for 7-10 d) or levofloxacin (lev; 500 mg po qd for 7-10 d). adults (18 years of age or older) were enrolled with clinical and radiologic evidence of cap [new infiltrate(s) on chest radiograph and fever, leukocytosis, cough, chest pain, auscultatory findings, or sputum production]. the majority of subjects were fine class i/ii in both studies. bacteriologic eradication was assessed 5 to18 days post therapy. results: a total of 377 treated subjects had pretreatment pathogens (grn, n = 179; comparators, n = 198) . the overall eradication rate in all treated subjects was 91% (129/142) for grn and 85% (123/145) for the comparators. eradication rates for s pneumoniae were 92% (24/26) for garenoxacin and 96% (49/ 51) for the comparators. eradication of s pneumoniae was 100% and 89% for a/c and lev, respectively. in strains with reduced susceptibility to penicillin eradication rates were 86% (6/7) vs 67% (2/3) in favour of grn. eradication rates for h. influenzae were 89% (40/45) and 75% (30/40) for grn and comparators, respectively. lev eradicated 71% of h. influenzae isolates and a/c eradicated 77% of the strains isolated. there were very few isolates (4) of moraxella catarrhalis in the 2 studies. in 1 study grn was 100% effective against 3 strains of m. catarrhalis and in the other a/c was 100% effective against the 1 strain isolated. grn eradicated 95% (19/20) of the staphylococcus aureus isolates vs 100% (11/11) for the comparators. conclusions: grn was highly active against pathogens commonly associated with cap including drug-resistant strains of s pneumoniae and represents an effective therapeutic option for this patient population. objectives: garenoxacin (grn) a novel, broad-spectrum des-f(6)-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. there is a growing problem of resistance in strains of s pneumoniae, with multi-drug-resistant s pneumoniae (mdrsp) becoming increasingly more common. the objective of this study was to evaluate the clinical and microbiologic efficacy of grn in the treatment of communityacquired pneumonia (cap) caused by mdrsp. methods: this was a multinational, open-label, noncomparative study. subjects were adults ( ‡18 and <75 y) with clinical (clinical signs, sputum production), radiologic (new infiltrates on chest radiograph), or microbiologic (predominance of gram-positive cocci in pairs on sputum gram-stain or a positive blood culture for s. pneumoniae) evidence of cap caused by s. pneumoniae. subjects received grn 400 mg po qd or grn 400 mg iv with transition to 400 mg po qd for 7 to 14 days. clinical and microbiologic responses were determined at a test-of-cure visit 7 to 14 days posttherapy. results: a total of 121 subjects were enrolled. of these, 47 (17 po only, 30 iv to po) were clinically and microbiologically evaluable. clinical and microbiologic success rates were 91% (43/47) and 89% (42/47), respectively. clinical success rates were 94% (16/17) and 90% (27/30) for po and iv to po, respectively. documented s. pneumoniae bacteremia was present in 28% (n = 13) of subjects with a clinical success rate of 92%. among evaluable subjects, resistance rates for s. pneumoniae were penicillin 13%, second-generation cephalosporin 17%, macrolides 21%, tetracyclines 21%, and trimethoprim/ sulfamethoxazole 17%. twelve evaluable subjects had pneumonia caused by mdrsp. clinical success rate was 92% (11/12) in subjects with mdrsp and 91% (32/35) in non-mdrsp subjects. clinical success of grn for strains resistant to 2, 3, 4, or 5 antimicrobial drug classes, were 100% (5/5), 100% (1/1), 100% (2/2), and 75% (3/4), respectively. microbiologic success was 83% (10/12) and 91% (32/35) for mdrsp and non-mdrsp (susceptible or resistant to 1 class) strains, respectively. grn was generally well tolerated with drug-related adverse events (ae) reported in 14% (8/58; po) and 21% (13/63; iv to po) of subjects. conclusions: grn (po or iv to po) is an effective treatment for cap caused by mdrsp and non-mdrsp. grn is well tolerated. in vitro bactericidal activity of daptomycin against staphylococcus aureus and enterococcus spp.: comparison with vancomycin, teicoplanin and linezolid h. drugeon, m. juvin (nantes, fr) objectives: the aim of this study was to evaluate the bactericidal activity (by killing kinetics) of daptomycin (dap) against staphylococcus aureus (sa) clinical isolates with different teicoplanin mics and against enterococcus faecalis (efl) and e. faecium (efm) with different mechanisms of glycopeptide resistance. dap has been compared with teicoplanin (tei), vancomycin (van) and linezolid (lin). methods: 6 sa strains (2 mssa and 4 mrsa) with tei mic distributed from 0.5 to 8 mg/l, 6 enterococcus (3 efl and 3 efm) with glycopeptide phenotypes [s, r-vana, r-vanb] were studied using a killing curve method. 7 antibiotic concentrations were used from 1 mg/l to 64 mg/l in two fold dilutions. surviving bacteria were counted at t0, t30', t1, t3, t6, t12 and t24 hours using agar plates with inhibitors to prevent antibiotic carry-over. antibiotics tested were daptomycin (dap), teicoplanin (tei), vancomycin (van) and linezolid (lin). results: all the sa isolates were susceptible to dap (mic = 0.25-1 mg/l), to lin (mic = 1-2 mg/l), to van (mic = 1-2 mg/l) regardless of susceptibility to methicillin. dap showed the same strong concentration dependent bactericidal activity with mssa and mrsa: at t30' bactericidal activity (ba) (decrease of 3 log10 cfu/ml) was observed with 8-16 mg/l of dap; at t3 hours, 1-4 mg/l of dap was sufficient and at t6 hours, ba was obtained with 1 mg/l of dap. the other antibiotics showed a time dependent bactericidal activity but ba was observed only with long exposure ( ‡12 hours) and with high concentrations. all the enterococcus isolates were susceptible to dap (mic = 1-2 mg/ l) and to lin (mic = 1-2 mg/l) regardless of the resistance to glycopeptides. ba of dap was also concentration dependent. ba was obtained with 4-8 mg/l after 6 hours of contact and with 1 mg/l after 12 hours of contact for efl. ba was observed with 8-16 mg/l after 6 hours of contact and with 1-2 mg/l after 24 hours of contact for efm. the other antibiotics had a time dependant activity but didn't show bactericidal activity with concentrations 32 mg/l. conclusion: the bactericidal activity of daptomycin was very strong, concentration dependent, and not influenced by the level or mechanism of glycopeptide resistance the bactericidal activity of linezolid was time dependent and observed only with the highest concentration and the bactericidal activity of vancomycin and teicoplanin was time dependent but was influenced by the mechanism of glycopeptide resistance. objectives: telavancin (tlv) is a bactericidal lipoglycopeptide with multiple mechanisms of action that is in phase 3 clinical trials for the treatment of complicated skin and skin structure infections and hospital-acquired pneumonia with a focus on infections due to methicillin-resistant staphylococcus aureus (mrsa). this study evaluated and compared the antibacterial activity of tlv with that of other antibacterial agents against recent gram-positive clinical isolates from germany. methods: a total of 363 aerobic gram-positive bacterial strains recently collected were included. antibiotics tested were tlv, vancomycin (van), teicoplanin, penicillin, oxacillin, ampicillin, cefuroxime, ceftriaxone, daptomycin (dap), linezolid (lzd), quinupristin-dalfopristin, clindamycin, ciprofloxacin, levofloxacin, gentamicin, streptomycin, erythromycin, telithromycin, co-trimoxazole and tetracycline. mics were determined by the broth microdilution procedure according to the guidelines of the clsi. results: tlv exhibited potent activity against all grampositive bacteria including resistant isolates such as mrsa, van-resistant enterococci, pneumococci (including multiple resistant strains with various antibiotic resistance phenotypes) and other streptococcal species. tlv showed excellent in vitro activity against the species irrespective of the antibiotic phenotype tested. for methicillin-susceptible s. aureus (mssa, n = 33) and mrsa (n = 42) mic90 of tlv for both phenotypes were 0.5 mg/l. for coagulase-negative staphylococci (n = 72, incl. msse, mrse, mssh, mrsh and others) mic90s were 0.5 or 1 mg/l. mic90s of tlv for enterococcus faecalis (n = 30) and e. faecium (n = 32) were 1 and 2 mg/l, respectively. for van-resistant strains of e. faecalis (n = 2) or e. faecium (n = 7) mics for tlv ranged from 0.12 to 4 mg/l. against streptococcus pneumoniae (n = 60) tlv mics ranged from £0.001 to 0.03 mg/l. all streptococcus pyogenes, streptococcus agalactiae and all viridans group streptococci (n = 94) had mics of £0.125 mg/l. conclusion: based on mic90, tlv was more potent than van, dap or lzd against staphylococci, streptococci and e. faecalis. it was superior to dap and lzd against e. faecium and at least as active as dap or lzd against most van-resistant enterococci. tlv appears to be a promising new antimicrobial agent for the treatment of infections caused by gram-positive organisms including multiply resistant isolates. the extent of protein binding (pb) of dap is still under investigation and data available so far indicate pb of either 92% or 63%. therefore we tested two fscs: 22.0 (corresponding to 63% pb) and 4.8 (corresponding to 92% pb). the activity of dap was determined in mueller-hinton broth supplemented with 50 mg/l calcium. viability counts were performed at 0.25, 0.5, 1, 3, 6, 12 and 24 h. one methicillin-susceptible staphylococcus aureus (mssa), two methicillin-resistant s. aureus (mrsa), one vancomycin-susceptible (van-s) and one van-resistant (van-r) enterococcus faecalis, one van-s and one van-r enterococcus faecium were tested. bactericidal activity was defined as >99.9% killing during incubation. results: dap was bactericidal at concentrations of 60.0 mg/l and 22.0 mg/l in all seven strains. the concentration of 4.8 mg/l was bactericidal against the two mrsa and against the van-s e. faecium. in the other four strains the maximum reduction of initial inoculum ranged from 1.52 to 2.53 log10 cfu/ml. in six strains a bactericidal effect at 60.0 mg/l and 22.0 mg/l of dap, respectively, occurred between 15 minutes and 3 h and after 6 h in the van-s e. faecalis. van at 40.0 mg/l or 18.0 mg/l was bactericidal in only two strains after 24 h (1 mssa, 1 mrsa). against the other five strains, van was bacteriostatic with maximum reduction of initial inoculum between 1.91 and 2.78 log10 cfu/ml at 40 mg/l after 24 h, respectively. both tpl and lzd were consistently bacteriostatic against the test strains. conclusion: dap at psc of 60.0 mg/l as well as at fsc of 22.0 mg/l showed a pronounced bactericidal effect within 3 h in 6/7 strains. van was bactericidal in only 2/7 strains after 24 h. compared to van bacterial killing by dap was very rapid. tpl and lzd were bacteriostatic only. the effect of human serum on the bactericidal activity of daptomycin and comparators against staphylococcus aureus and enterococcus spp. background: daptomycin is a new cyclic lipopeptide antibiotic that shows rapid bactericidal activity and has high protein binding when assessed by standard methodology. this study investigated the bactericidal activity of daptomycin and the effect of protein binding by the addition of 50% human serum (hs). methods: exponentially-growing methicillin-susceptible andresistant s. aureus (mssa, mrsa) and vancomycin-susceptible enterococcus faecium (vse) and -resistant enterococcus faecium (vre) (ca. 106 cfu/ml) were exposed to daptomycin (dap), vancomycin (van), teicoplanin (tei), piperacillin-tazobactam (ptz) or linezolid (lzd) at peak (p) and trough (t) serum concentrations in mueller hinton broth supplemented with ca2+ to 50 mg/l with or without hs. viable count was determined at 0.25, 0.5, 3, 6 & 24 h. plots were made of log reduction in viable count over time and the area-under-thecurve measured to calculate bactericidal indices (bis) from these plots (j antimicrob chemother 1997, 39: 713-717). results: daptomycin reduced viable count of mssa & mrsa by approx. 5 logs or more within 0.25 h and vse or vre within 3 h at p. other agents either did not achieve this or required 24 h to do so (not shown). bi data are shown below (>represents kill beyond the limit of detection). hs had little effect on dap kill, except against the vre at t. nevertheless, dap at t against vre was more bactericidal than any other antibacterial except dap at p. conclusions: dap was the most bactericidal agent tested as measured either by bi or rate of kill. dap at p reduced mssa and mrsa to below detection within 15 min. the effect of hs was minimal which suggests that protein binding is either weak or highly reversible. these data support the use of dap in the treatment of infections caused by these organisms. daptomycin activity against multi-resistant staphylococcus haemolyticus bloodstream isolates from severe infections objectives: daptomycin, a new cyclic lipopeptide with activity against multidrug-resistant gram-positive pathogens including mrsa, is approved for use in cssst infections (us-fda) and is being reviewed by emea for approval in eu member countries. the rapid bactericidal activity of daptomycin, due to its unique mechanism of action, makes it an attractive antibiotic for serious gram-positive infections. the study was performed: (i) to evaluate the activity of daptomycin and other drugs against 50 multi-resistant clinically relevant staphylococcus haemolyticus (mrsh), isolated from bloodstream infections in various hospitals in italy (ii) to determine epidemiologic and genetic correlation among strains, and (iii) to characterize the sccmec dna of these strains. methods: the mrsh strains were tested against a panel of antimicrobial agents, by broth microdilution method performed according to clsi (clinical laboratory standards institute) guidelines, including supplementation of 50 mg/l calcium for daptomycin. moreover, phenotypic tests and antibiotic susceptibility profiling were carried out and the results compared with molecular typing analysis by using smai-pfge fingerprints and pcr to characterize the mec-complex. results: all isolates were resistant to erythromycin, gentamicin, ciprofloxacin, 16 strains showed reduced susceptibility to vancomycin (mics 2 mg/l), 24 strains were resistant to cotrimoxazole, 16 strains to clindamycin, 6 strains to chloramphenicol and 3 strains to tetracycline. almost all isolates were inhibited by £1 mg/l of daptomycin, and only four strains exhibited a mic value of 2 mg/l. pfge analyses showed the existence of at least two multi-resistant s. haemolyticus clones widespread in different hospitals. methicillin-resistance was correlated to the presence of the meca and preliminary results regarding the genetic element carrying the gene, showed an organization of the mec-complex of class a and class c. conclusions: our results suggest that daptomycin has excellent activity against multiresistant mr s. haemolyticus isolates, which represent a serious threat in catheter-related bloodstream infections. furthermore, the emergence of s. haemolyticus exhibiting reduced susceptibility to vancomycin is of particular concern, probably due to the common use of vancomycin as initial therapy for such infections. moreover, the use of additional molecular techniques to fingerprint isolates makes this study of clinically important cons more accurate. objectives: ceftobiprole is a new cephalosporin with a broad spectrum of action including methicillin-resistant staphylococci (mrs) as well as many other gram-positive and gram-negative pathogenic bacteria. this study investigates the structural basis for the good activity against mrs. methods: the primary beta-lactam resistance determinant of mrs, penicillin-binding protein pbp 2' (or 2a) has been cloned and expressed as a soluble form in which the amino-terminal residues forming a membrane-anchor have been deleted. the soluble form has been crystallized and the structure of the complex formed after soaking crystals in a solution containing ceftobiprole has been determined at 2.8 angstrom resolution. additional data on the structure of the ceftobiprole-pbp2' complex formed in solution has been obtained using spectroscopic methods such as uv-circular dichroism. results: ceftobiprole reacts rapidly with pbp2' to form a stable acyl-enzyme complex. the ceftobiprole moiety is positioned deep within the active site of the acyl-enzyme complex formed with pbp2', where it forms several hydrogen bonds and hydrophobic interactions. in particular, the 7-aminothiadiazolylhyroxyiminoacetyl side chain of ceftobiprole sits more deeply within the side-chain binding pocket of pbp 2' than does the 7-acylamino side chain of nitrocefin in the previously determined complex structure. the additional interactions probably add to the enhanced stability of the acyl-enzyme complex formed with ceftobiprole, compared to complexes formed with other betalactams that are inactive against mrs. significant structural rearrangements between apo-enzyme and acyl-enzyme are evident in the crystal structure and in solution. conclusion: ceftobiprole readily forms a stable inhibitory acylenzyme complex with the pbp 2', the beta-lactam resistance determinant of mrs. this, together with potent inhibition of the normal complement of beta-lactam sensitive penicillin-binding proteins, accounts for its excellent activity against staphylococci and probably accounts for the low rates of resistance development observed in experimental conditions. incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in a french hospital (november 2004 -april 2005 c. morate, a. charron, c. bebear, j. maugein (bordeaux, fr) staphylococcus aureus are a major cause of nosocomial infections around the world. glycopeptides remain the drug of choice for severe infections caused by mrsa. however, after the emergence of vancomycin resistance in enterococcus and in the coagulase negative staphylococcus, strains of staphylococcus aureus with reduced susceptibility to glycopeptides (gisa) have been reported in different countries like japan, france, spain, the uk and the united states. the aim of our study was to determine the proportion of vancomycin resistance in clinical s. aureus isolates in a french university hospital, between november 2004 and april 2005, then we wanted to define if there was an epidemic clone and study the clinical impact of these gisa strains. the protocol of detection was, first, a screening test on bhi agar containing 4 mg/l of teicoplanin, then, the vancomycin and teicoplanin mics were determined by the method of etest with an inoculum of 2.0 mcf on the selected strains. finally, the isolates with mic of the teicoplanin ‡8 mg/ l and mic of the vancomycin ‡4 mg/l or mic of the teicoplanin ‡12 mg/l and mic of the vancomycin £4 mg/l were studied on population analysis. after that, pulsed-field gel electrophoresis (pfge) was performed on the different isolates and the pulsotypes were compared. from november 2004 to april 2005, 468 s. aureus isolates were collected from 331 patients and screened for glycopeptide resistance on an initial agar screening test containing 4 mg/l of teicoplanin. the teicoplanin mic was >4 mg/l for 65 isolates (13.9%) from 59 patients and these strains were selected for the determination of the mics by ''macromethod'' etest. by this technique, 39 strains were selected and studied by population analysis. all the profiles were compared to the reference strain mu3 profile. this procedure detected 5 isolates (from 5 patients) with heterogeneous reduced susceptibility to glycopeptides (hgisa). so the incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in our hospital was found to be 1.1%. four strains were resistant to methicillin and 3 were also resistant to gentamicin. the diversity of the strains was confirmed by pfge: there was not an epidemic clone in the hospital. the clinical history showed that 4 patients had received a prior treatment with vancomycin, and that 3 patients had a failure in treatment: 2 of them had cystic fibrosis. objectives: enterococcus faecalis was the most prevalent organism (72.5%) involved in enterococcal infections at tehran hospitals followed by e. faecium (22.5%). due to widespread expansion of aminoglycoside modifiying enzymes (agmes) genes, the rate of resistance to high level concentration of aminoglycosides has increased in these years. the rate of high level gentamicin resistant isolates of enterococci (hlgr) is high in iran (46%). the aim of this study was to determine the genes encoding resistance to aminoglycosides among enterococci in iran. methods: disks containing 120 lg gentamicin were used to detect hlgr isolates. primers specific for aac (6') aph (2") and aph (3') iiia genes were used in pcr to possibly detect acetyltransferases and phosphotransferas, the common agmes among 113 isolates of enetrococci. theses isolates were resistance to different concentration of gentamicin. results: a 222 bp region of the aac (6')-aph (2") gene was amplified by pcr in 95% hlgr isolates as well as in 40% of low level getamicin resistant isolates (llgr). moreover the gene aph (3') iiia was detected in 92.5% and 72% of isolates of hlgr and llgr respectively. differences between isolates of e. faecalis and e. faecium were found in term of prevalence of aph (3') iiia gene. conclusion: the bifunctional enzyme aac (6')-aph (2") is the main cause of resistance to high concentration of aminoglycosides in our collection of enterococci. this enzyme confers resistance to all clinically useful aminoglycosides with the exception of streptomycin. in the absence of aac (6')-aph (2"), gentamicin could be used in combination therapy. prevalence and genetic analysis of methicillinresistant staphylococcus aureus expressing highlevel and low-level mupirocin resistance m. kural, t. us, y. akgun (eskisehir, tr) objectives: to investigate the genetic location of mupa gene which encoded mupirocin resistance and characterize mupirocin-resistant methicillin resistant staphylococcus aureus (mrsa) isolated from patients in a turkish university hospital by polymerase chain reaction (pcr) and plasmid analysis. methods: methicillin and mupirocin resistance were detected by disk diffusion (oxoid, uk). the etest (ab biodisk, sweden) was performed to determine mupirocin minimum inhibitory concentrations (mics). the presence of mupa and meca were detected by pcr using specific primers. plasmid analysis were used to study the genetic location of mupa gene. results: a total of 595 (44.9%) mrsa strains were identified by disk diffusion in 1324 s. aureus. of the 595 clinical isolates 394 (66.2%) were from wound, 91 (15.3%) from blood, 41 (6.9%) from catheter, 36 (6.1%) from lower respirator tract (bronchoalveolar lavage, pleural fluid and transtracheal aspirates), 13 (2.2%) from sputum, 12 (2.0%) from urine and 8 (1.3%) from other (serebrospinal fluid, parasynthesis fluid, peritoneal fluid, and bone marrow) clinical samples. among the mrsa isolates, mupirocin resistance was detected in 35 (5.9%) strains with disk diffusion and etest. of the 35 mupirosin-resistant isolates 23 (3.9%) expressed high-level (muh) and 12 (2%) expressed lowlevel (mul) mupirocin resistance. all isolates were vancomycin, teicoplannin susceptible and chloramphenicol resistant with disk diffusion. isolates with high-level and low level mupirocin resistance due to the mupa gene were also detected with pcr. plasmids were detected in all of the 35 isolates. however only the muh isolates contained a 38 kb plasmid that encoded highlevel resistance. all of the isolates contained a 4.4 kb plasmid and resistant to chloramphenicol. conclusion: our results indicated that the mrsa clones detected in the hospital had acquired a high-level mupirocin resistant plasmid. the past observations and recent studies suggested that the numbers of such strians have increased following extensive topical use of mupirocin. the usage of mupirocin in our hospital has not yet been systematically implemented. it is frequently prescribed for the treatment of staphylococcal skin infections and less to eliminate nasal carriage of mrsa. in our hospital we should be aware of the possible emergence and increase of mupirocin highly resistant mrsa strains in the future so that we should be considered when using mupirocin to control the spread of mrsa in hospital. emergence and spread of acquired fusidic acid resistance in staphylococcus aureus objectives: a major route to fusidic acid resistance (fusr) in s. aureus involves acquisition of fusb, a resistance determinant first clinical microbiology and infection, volume 12, supplement 4, 2006 identified on plasmid pub101. here we show that (i) the two currently-circulating major clones of fusr s. aureus identified to date have acquired fusb from pub101 (or from the same ancestral source as pub101), and (ii) that the pub101-encoded fusb is only one of at least three lineages of this protein that appear to have evolved since recruitment of the original, ancestral fusb to the staphylococci. methods: plasmid purification, dna sequencing, pcr amplification, and cloning in s. aureus rn4220 using shuttlevector pcu1, were all performed using established methods. antibiotic susceptibility testing was performed by agar dilution. results: the epidemic european fusidic acid-resistant impetigo clone (eefic) and community-acquired mrsa strain st80 have been shown to carry chromosomal and plasmid-encoded fusb, respectively. dna sequencing of fusb and its surrounding regions in these backgrounds revealed that they are identical to sequences on pub101. however, acquired fusr does not always result from acquisition of the prototypical fusb gene. a gene encoding a fusb homologue was recently identified during sequencing of s. aureus strain mssa476, and we identified an additional homologue encoded in the genome of s. saprophyticus strain atcc15305. the products of these genes exhibit~40% homology to fusb and to each other. cloning of pcr amplicons corresponding to these genes and their upstream expression signals into s. aureus established that they both confer resistance to fus. since these functional homologues are more closely related to each other than to those from other gram-positive organisms, it is highly likely that they evolved from an ancestral fusb after its recruitment to the staphylococci. conclusions: the three members of the staphylococcal family of fusb proteins appear to have evolved from the same ancestral protein, which, based on the low level of sequence homology between fusb genes at the nucleotide level, clearly occurred well before the introduction of fus into the clinic. of the three, the fusb protein encoded by pub101 is by far the most successful, and this gene/plasmid represents the source of (or shares a source with) the major fusr strain lineages. telithromycin activity is reduced by efflux in streptococcus pneumoniae c. benvenuti, r. koncan, g. bahar, a. mazzariol, g. cornaglia (verona, it; ankara, tr) objectives: telithromycin shows an excellent activity against m-type erythromycin-resistant streptococcus pneumoniae, thus is commonly regarded as being capable of overcoming the efflux resistance mechanism. nevertheless, telithromycin mic values in those strains appear to be distinctly higher than in the erythromycin-susceptible ones. the possibility of telithromycin acting as an actual efflux substrate, as it was already demonstrated in streptococcus pyogenes, seemed worth investigating. methods: telithromycin mic distribution was analysed in a collection of 423 italian s. pneumoniae strains originating from multi-centre studies (2000) (2001) (2002) (2003) . the effect of an efflux mechanism was investigated using [3h]-telithromycin. results: telithromycin mic ranges were £0.002-0.12 mg/l (mic50 0.008 mg/l and mic90 0.03 mg/l) in erythromycinsusceptible strains (lacking both mef and erm genes) and 0.016-1 mg/l (mic50 0.25 mg/l and mic90 0.5 mg/l) in strains endowed with the m phenotype. a distinct telithromycin efflux was detected in the strains expressing the mef gene, but not in those expressing the erm(b) gene, nor in the susceptible strains lacking mef or erm genes. efflux reversibility by addition of an inhibiting compound (sodium arsenate) was demonstrated. an msr-like sequence was also found in all strains effluxing telithromycin, but not in the others. conclusions: this is the first time that telithromycin has been shown to be effluxed by s. pyogenes isolates. that the efflux is related to the presence of both the mef and the msr-like genes is clearly demonstrated, but -owing to the increasingly evident complexity of s. pneumoniae efflux systems -other genes might also contribute to the efflux. an unusual phenotype of enterococcus faecalis in greece expressing low-level resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin m. maniati, f. kontos, p. liakos, e. petinaki, i. spiliopoulou, a. maniatis (larissa, patras, gr) objectives: to investigate the resistance mechanism of a new described phenotype among enterococcus faecalis expressing lowlevel resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin (q-d). methods: in greece, during 2005, three enterococcus faecalis isolates, expressing this unusual phenotype, were recovered from urine samples. the isolates were studied by pcr for the lsa-gene and by pfge. nucleotide sequencing analysis of lsa and 309 bp of the upstream region was performed. the isolates were also tested by rt-pcr for the expression of the lsa-gene. results: the isolates belonged to three distinct clones and carried the lsa-gene. no stop codons were found in any strain, while some point mutations in the lsa-gene were detected. comparing the lsa mrna production of these unusual strains with that obtained from fully q-d resistant ones no quantitative differences were found. conclusions: the findings of the present study clearly show that the resistance mechanism of quinupristin-dalfopristin is not only correlated with the presence and the expression of the lsagene. some mutations detected in the lsa gene probably are responsible for the production of an lsa protein with decreased activity, resulting to the q-d susceptibility. the presence of erm tr gene is responsible for the macrolide-resistance of streptococcus agalactiae objectives: to investigate the mechanism of resistance to macrolides in strains of streptococcus agalactiae in the area of thessalia, greece during the period 2000-2005. methods: the subject of this study were 147 strains of s. agalactiae which were collected from clinical specimens (90% vaginal swabs) from pregnant and non pregnant women. the strains were identified by gram stain, the lancefield b antigen, and by api strep system (biomerieux, france). susceptibility to macrolides, lincosamides and streptogrammines b was studied by the disk diffusion method. the mics were also measured by the use of e-test. the differentiation between m and mlsb inducible type was tested by the double disk synergy test (ddst). the detection of the genes mef a, erm tr, and erm b was performed by polymerase chain reaction (pcr). the clonality of the resistant strains was studied by pulse-field gel electrophoresis. results: of the 147 strains, 15 were resistant to erythromycin, lincosamid and streptogrammines b. none was found to be resistant to erythromycin only (m-phenopype). 60% of the strains were mlsb constitutive phenotype, while 40% were mlsb inducible. all strains were found to carry the erm tr gene. only one strain was found to carry both erm tr and erm b genes. pfge analysis revealed the emergence of multiple resistant clones. conclusions: the resistance of s. agalactiae to mlsb antibiotics is related with the presence of erm tr gene in central greece. emergence of novel clindamycin resistance phenotype among invasive streptococcus pyogenes isolates in sweden a. jasir, b. luca, c. schalen (lund, se) objectives: in some recent throat group a streptococci (gas) isolates from our diagnostic laboratory total resistance to clindamycin but susceptibility to erythromycin and other 14-as well as 15-membered macrolides was found. the isolates were susceptible to 16-membered macrolides and streptogramin b. these atypical strains thus did not agree with previously known mls resistance phenotypes. the main objective was to characterize theses resistance phenotype and genotypes. method and results: the isolates were examined for resistance genes by pcr. out of 14 strains one harboured an erma gene. the gene was sequenced and showed a mutation in regulatory part and was localized on a transposons. all other strains were negative for any erm genes and were also tested for 23s rrna mutations with negative outcome. strains were t-and emm typed and showed to belong to different types. conclusions: gas account for common human infections such as acute pharyngotonsillitis and impetigo, which untreated may be followed by the nonsuppurative complications rheumatic fever and acute poststreptococcal glomerulonephritis gas may also give rise to invasive, often life-threatening acute disease, such as scarlatina, erysipelas, endometritis, necrotising fasciitis and sepsis, often accompanied by toxic shock. without known exceptions, gas are fully susceptible to betalactams, which are first-choice drugs for treatment. in cases of allergy or intolerance to penicillins, macrolides are most used, and possibly as a consequence, a significant resistance development to these agents has evolved in many parts of the world. though the role of clindamycin for treatment of streptococcal disease is more limited this drug was shown to be particularly effective in eradicating streptococci after penicillin treatment failure of pharyngotonsillitis. clindamycin, often as a supplement to betalactams, also may have a life-saving effect in the treatment of fulminant streptococcal infections. due to its important role in the treatment of invasive streptococcal disease, resistance development to clindamycin in gas is considered highly undesirable. the alarming finding of a possibly new phenotype of selective clindamycin resistance in gas will motivate a thorough analysis of the phenotype as well as identification of its resistance determinants. a. al-lahham, m. van der linden, r.r. reinert (aachen, de) objectives: telithromycin is a novel ketolide antibiotic with significant in-vitro activity against streptococcus pneumoniae. the aim of this study is to characterize the resistance mechanisms of clinical isolates of s. pneumoniae with reduced susceptibility to telithromycin (>1 mg/l) and to perform the time-kill kinetics with telithromycin. methods: determination of mics was performed by the microbroth dilution method according to the clsi and the serotyping by the neufeld quellung reaction. multilocus sequence typing, sequencing of the 23s rrna, sequencing of genes encoding ribosomal proteins (l4 and l22), and ermb were performed according to standard methods. four isolates were selected for time-kill, two of which with a telithromycin mic 2 mg/l and two strains with a telithromycin mic of 8 mg/l. results: in two nation-wide studies and one european surveillance study (n = 6604) performed at the national reference center for streptococci (nrcs) in germany, reduced susceptibility to telithromycin (>1 mg/l) was detected in 17 isolates (0.3%). mic50/mic90 (mg/l) of the strains to other antibiotics were as follows: telithromycin 2/8, penicillin g 2/2, cefuroxime 8/8, erythromycin a >32/>32, clindamycin >32/>32, tetracycline 32/32, and gatifloxacin 0.25/0.25. two major serotypes were observed, serotype 14 (58.8%) and serotype 19a (29.4%). all isolates possess the cmlsb phenotype (ermb positive). the isolates showed a wide range of combinations of resistance determinants including multiple alterations in the 23s rrna (a138g, c150t, a260g, a1745t, and c2216t), a s20n alteration in the ribosomal protein l4 (n = 9), and a n100s alteration in the erm(b) gene (n = 14). the predominant clone was serotype 14 sequence type 143 (8 of 17 isolates), which was seen in france (n = 7) and germany (n = 1). telithromycin-resistance has also spread to the spain23f-1 clone (st 81; n = 1) and its serotype 19a variant. in vitro time-kill showed a minimal kill from 0-8 hours and then regrowth. bactericidal activity was achieved only with 8 times the mic in all strains. conclusions: although the incidence of telithromycin resistance remains rare world-wide, the spread of telithromycin resistance to multi-drug resistance clones with world-wide distribution is worrisome. gbs obtained from non-pregnant women. the erythromycin resistant-gbs were identified, phenotypically analysed, screened by pcr for mre(a) gene and for erythromycin resistance genes: erm(b), erm(tr), mef(a) and mef(e), and serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of gbs, 161 (28.65%) were erythromycin-resistant: 86 (24.29%) erythromycin-resistant gbs were isolated from vaginal swabs of pregnant women and 75 (36.06%) from non-pregnant women. the frequency of serotypes in 151 erythromycin-resistant gbs tested, the distribu-tion of their resistance genes and the distribution of serotypes among the different genotypes are illustrated in the table. nt, nontypeable, the mre(a) gene was found in all the gbs strains tested. mics of erythromycin in erythromycin-resistant gbs were: mic50 and mic90, >128 mg/l; range, 4 to >128 mg/l for gbs harbouring erm(b) and erm(b)+erm(tr) and mic50 and mic90, 8 mg/l and >128 mg/l, respectively; range, 0.5 to >128 mg/l for gbs harbouring erm(tr). conclusion: erm(b) was the erythromycin-resistant gene most prevalent among the gbs isolates and these isolates showed the highest mics of erythromycin. the commonest serotypes among erythromycin-resistant gbs isolated were iii, ii and i, and showed genotypic variability harbouring either of the two most prevalent genes, erm(b) or/and erm(tr). methods: we studied the rates of resistance to tetracycline and minocycline among 161 erythromycin-resistant gbs strains isolated at the university hospital lozano blesa of zaragoza, spain. isolates were subsequently phenotypically analysed by means of the disk diffusion method and screened by pcr for erythromycin and tetracycline resistance genes [erm(b), erm(tr), mef(a/e), tet(m) and tet(o)]. the susceptibility to erythromycin, josamycin, tetracycline and minocycline was tested by the agar dilution method according to the nccls. the strains were serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of 161 isolates of macrolide-resistant gbs collected from may 2002 to april 2004 in our hospital (28.6% of the total sgb isolated), 124 (78.85%) were tetracyclineresistant. the distribution of tet(m) and tet(o) among the erythromycin-resistant gbs harbouring erm(b) was (66.1% and 54.2%, repectively) and harbouring erm(tr) was (63.8% and 34.5%). the distribution of tetracycline resistance genes and serotypes among the different genotypes in 121 gbs are illustrated in the table.*nt: non typable isolates carrying tet(m) or tet(m)+tet(o) presented the following mics: tetracycline (mic90 64, mic50 32, range 16-128 mg/l); minocycline (mic90 32, mic50 16, range 4-32 mg/l). isolates carrying tet(o) presented the following mics: tetracycline (mic90 64, mic50 32, range 32-64 mg/l); minocycline (mic90 16, mic50 16, range 4-32 mg/l). conclusion: the majority (61.1%) of tetracycline-resistant gbs harboured tet(m) alone or in combination with tet(o). the most prevalent serotypes among the total of tetracycline-resistant gbs was the serotype iii (38%) and the serotype ii (28%). serotype iii was more prevalent among the gbs harbouring the tet(m) gene and serotype ii was more prevalent among the gbs harbouring the tet(o) gene. objectives: to know the prevalence of resistance to macrolides in viridans streptococci, its mechanism and the genetic elements which are involved. methods: we studied 89 viridans streptococcus pharyngeal isolates from different patients. mics for macrolides were determined by the agar dilution method. the presence of mef and erma, ermb and ermtr genes, and the presence of mega (macrolide efflux genetic assembly) or tn1207 in resistant, mef + isolates was determined by pcr with specific primers. the similarity to mef genes first described in pneumococci (mefe) and streptococcus pyogenes (mefa) was determined by sequencing. results: 48 viridans streptococci isolates were resistant to macrolides (53.9%). 42 out of the 48 resistant isolates harboured mef genes (87.5%), one harboured ermb (2.1%), and 5 isolates harboured both mef and ermb genes (10.4%). no isolates harboured erma or ermtr genes. we studied genetics elements which harbour mef genes in other streptococci, in 15 mef (+) isolates. we found mega insertion element in 14 of 15 isolates (93.3%), all of them harbouring mefe. the only isolate in which we found mefa, did not harbour mega, but tn1207. conclusions: m phenotype is frequent in viridans streptococci, and all of them harbour mef genes. most mlsb phenotype viridans streptococci do not harbour erm genes alone; most of them combine erm and mef genes. most isolates contained the mef sequence corresponding to mefe, and the genetic element (mega) usually described in pneumococci as harbouring this gene. one isolate contained the sequence corresponding to mefa, and the genetic element usually described in s. pyogenes (tn1207). the increasing presence of macrolide-resistant pneumococci harbouring mega element might be related with its wide presence in viridans streptococci. the acquisition of mega by pneumococci from viridans streptococci through transformation is being studied. objectives: the principal mechanism of macrolide resistance in streptococcus pneumoniae in italy is target site modification mediated by erm(b). the erm(a) gene is common in streptococcus pyogenes but rare in s. pneumoniae, even if recent studies have demonstrated an increased detection of this resistance determinant. recently, a clinical s. pneumoniae isolate carrying erm(a) has been obtained from a patient with meningitis in italy. the aim of this study is the molecular characterization of this isolate. methods: antimicrobial susceptibility tests were determined by etest. the presence of erythromycin resistance determinants was detected by pcr assays. genotyping was performed by pfge and mlst. the flanking regions of erm(a) were analysed by sequencing a fragment amplified by inverse pcr. transformation and conjugation experiments were carried out. transformants were analysed by pfge and hybridization with an erm(a) probe. results: the isolate belonging to serotype (st) 11a, was resistant to erythromycin, inducibly resistant to clindamycin and susceptible to penicillin and tetracycline. by pcr, the only macrolide resistance determinant detected was erm(a). pfge analysis revealed the genetic correlation of this strain with other st 11a s. pneumoniae italian isolates. mlst confirmed this data since the isolate belonged to st2003, which is a single-allele variant of st62, the most common st among italian st 11a isolates. a 5200 bp dna fragment, obtained by inverse pcr and containing the erm(a) gene, was sequenced. this fragment contains an orf upstream erm(a), the gene erm(a) identical to that described in s. pyogenes and 3 orfs, downstream erm(a), one homologous to a hypothetical kinase and the other two to transposases of other gram-positive species. in transformation experiments the gene erm(a) was transferred to an erythromycin susceptible recipient. hybridization analysis of one transformant revealed that the size of the transferred dna fragment was approximately 50 kb. no transconjugant was obtained in mating experiments. conclusions: this is the first italian report of an s. pneumoniae isolate carrying erm(a). erm(a) appears to be contained in a genetic element that includes two transposases, although the gene is not transferable by conjugation. objective: treatment with the first oxazolidinone antibiotic, linezolid, of infections caused by staphylococci has proved effective in most cases. in the present study we present the first three cases of linezolid resistant staphylococci in our hospital. methods: we examined three coagulase negative staphylococcus strains isolated from blood cultures (bactec, becton dickinson). identification and susceptibility testing were performed by the vitek ii automated system (biomerieux) and the results were confirmed by the api system (biomerieux) and e-test (ab biodisk, solna, sweden), according to nccls guidelines. results: three linezolid resistant staphylococci were isolated from blood cultures. identification showed that all three isolates were staphylococcus cohnii subsp. urealitycum and the mic values were 12 lg/ml (n = 1) and 24 lg/ml (n = 2) which are much higher than the value of 4 lg/ml that characterizes sensitive strains. the isolates derived from three patients in different wards of our hospital. the first two isolates were recovered from two icu patients in april and august 2005 and the last staphylococcus cohnii was isolated from a patient in the neurosurgery ward, who is still hospitalized. all patients received prolonged treatment with linezolid. conclusions: although six linezolid resistant clinical isolates of s. aureus were previously reported in the literature, these three isolates are the first coagulase negative staphylococcus isolates resistant to linezolid. it is imperative to screen for resistance to linezolid all staphylococci and take the necessary precausions in order to prevent the spread of a linezolid resistant strain in other wards of our hospital. correlation between mic and number of mutated 23s rrna genes in oxazolidinone-resistant staphylococcus aureus objectives: to determine the number of mutated 23s rdna alleles present in clinical and laboratory-generated linezolidresistant staphylococcus aureus isolates. methods: 36 linezolid-resistant isolates were tested, 9 of them clinical isolates (mics 16-32 mg/l) and 27 mutants selected in-vitro (mics 8-64 mg/l). the mutants were raised by repeated passage on increasing linezolid concentrations and their parentage was verified by pfge. mics were determined by agar dilution. genomic dna was digested with ecori and hybridized with a 420 bp probe corresponding to domain v of the genes encoding 23s rrna, to determine gene copy number. pyrosequencing was used to quantify the proportions of wildtype and mutated alleles present; assays were designed to detect the presence of 7 mutations conferring oxazolidinone resistance. pyrosequencing and hybridization data were combined to determine the number of mutated alleles present. results: resistance selected in-vitro proved less stable than that in the clinical isolates. pyrosequencing showed that all 9 clinical isolates had the g2576t mutation, 16 of the in-vitro selected mutants had g2576t, 7 had t2504c, 2 had t2500a, 1 had a2503g and 1 had g2505a. the 23s rdna copy number in the oxazolidinone-resistant clinical isolates varied from 4-6, and from 5-9 in the laboratory-generated mutants; 14/27 laboratoryselected mutants had changes in copy number, compared with their parent strains, and 3 had changes in fragment size, but not number. the number of mutated copies in lin-resistant isolates ranged from 1-5 in laboratory-selected mutants and from 2-4 in clinical isolates. an increasing number of mutated genes correlated with increasing linezolid mic. conclusions: in combination, pyrosequencing and hybridization successfully determined the number of mutated 23s rdna alleles. exposure to linezolid selected changes in 23s rrna gene copy number as well as sequence in 50% of in-vitro selected mutants. there was a positive correlation between both the number and proportion of mutated 23s rdna copies and mic, previously unproven for staphylococci. objectives: linezolid (lzd) is an important antibiotic for the treatment of enterococcal infections, especially when the corresponding strain possesses multiresistance including resistance to vancomycin (van). we report the emergence lzd resistance in clonally related van-susceptible and vanresistant enterococcus faecium isolates originated from an icu patient only 12 days after initiation of linezolid therapy. patient and methods: van-resistant e. faecium was repeatedly isolated from intraabdominal cultures of a 76-year-old female icu-patient with infected necrotizing pancreatitis after pancreaticoduodenectomy (whipplés procedure). antibiotic susceptibility testing of the bacteria was performed by e-tests; vana gene were detected by pcr. the possible lzd resistance mechanism (mutation in the 23s rdna of one or more of the six 23s rrna alleles of e. faecium) was examined by a pcr-based method. molecular typing of the strains was performed by smai macrorestiction analysis. results: van-resistant but lzd-sensitive e. faecium (vrlse) were initially detected in intraabdominal cultures, however, already twelve days after initation of lzd therapy, van-and lzd-resistant e. faecium (vrlre) strains were detected. resistance to lzd was confirmed: mics ranged from 16 to 32 mg/l. all e. faecium isolates showed identical or closely related pfge patterns. throughout the icu period, van-and lzd-susceptible e. faecium (vslse) strains were repeatedly detected in the same specimens from which the vrlse and vrlre were isolated. additionally, van-susceptible e. faecium isolates with resistance to lzd (vslre) were detected. mutations in the 23s rdna of three out of six alleles led to lzd resistance in the e. faecium isolates examined. two weeks after termination of the lzd therapy, no lzd-resistant strain could be detected in follow-up swabs. conclusions: resistance to lzd in e. faecium can occur already shortly after the initiation of lzd therapy. assessment of antibiotic susceptibilities of all isolates at the start of therapy and regularly during the therapy is advisable, especially during therapy of severe infections. the epidemiological and clinical repercussions of resistance to lzd among enterococci cannot be predicted at this time. attention to proper dosing and prompt removal of infected devices, when feasible, could limit occurrence and spread of lzd-resistant e. faecium. objectives: to investigate the mechanisms of resistance to tetracycline in shigella spp. methods: one hundred and eleven tetracycline-resistant shigella spp strains (67 s. sonnei, 44 s. flexneri), 6 were isolated as a cause of enteritis in our geographical area and the remaining recovered from patients with traveller's diarrhoea. antimicrobial susceptibility to tetracycline was determined by the kirby-bauer method. presence of teta, tetb and tetg genes was established by pcr. sequencing of amplified products were used to corroborate the reliability of the pcr results. maentel haenszel test was used to establish the statistical significance. results: the statistical analysis showed that the teta gene was more frequent in s. sonnei (p < 0.05), while tetb was more usual in s. flexneri (p < 0.0005). although without statistical significance (p:0.07), presence of non-determined mechanisms of tetracycline-resistance seems to be more frequent among s. sonnei. conclusions: species-specific differences in the distribuition of the teta and tetb genes has been shown. moreover 22.5% of the analysed strains did not show any of the analysed determinants of tetracycline-resistance. the concomitant presence of more than one of the analysed genes is a rare event. distribution and genetic determinants of tetracycline resistance in laribacter hongkongensis isolates from humans and fish objectives: to study the distribution of tetracycline resistance and to clone and characterize a tetracycline resistance determinant in laribacter hongkongensis, a recently discovered bacterial genus and species associated with communityacquired gastroenteritis. methods: twenty-four l. hongkongensis strains isolated from patients with community-acquired gastroenteritis and 24 l. hongkongensis strains isolated from freshwater fish in hong kong were used in this study. genetic determinants for tetracycline resistance were looked for by screening a genomic dna library of l. hongkongensis. the prevalence of teta gene in other strains of l. hongkongensis was studied by pcr using laboratory-designed primers. the presence of the tetracycline resistance determinants in plasmid was examined by southern blot analysis. results: among 24 human and 24 fish isolates tested, 3 human and 1 fish isolates were tetracycline-resistant. a 3566-bp gene cluster, which consists of 2 putative transposases, a tetr and a teta gene, was cloned by inserting restriction fragments of genomic dna from a resistant strain, hlhk5, into pbk-cmv. the 1266-bp teta and 636-bp tetr genes shared significant nucleotide sequence homology with known teta and tetr genes. while the flanking regions and 3' end of the teta were identical to the corresponding regions of a tetc island in chlamydia suis, the teta was almost identical to that of transposon tn1721 and plasmids found in many gram-negative bacteria, suggesting that illegitimate recombination may have occurred to produce the present tetracycline resistant determinant. southern hybridization suggested that the teta gene of hlhk5 was plasmid-encoded. the tetracycline resistance in l. hongkongensis was associated with teta. pcr amplification of the teta gene in the 48 isolates of l. hongkongensis, including hlhk5, showed the presence of teta in all the four tetracycline resistant isolates but none of the tetracycline susceptible ones. in contrast to strain hlhk5, the teta of two strains were identical to that of tn1721, while that of the other strain was more closely related to other gram-negative bacteria plasmids. conclusion: our results indicate that horizontal transfer of genes, especially through tn1721 and related plasmids, between l. hongkongensis and other gram-negative bacteria is probably a frequent event and is an important mechanism for acquisition and dissemination of tetracycline resistance in l. hongkongensis. succesful treatment of infective endocarditis with linezolid t. hryniewiecki, u. lopaciuk, j. stepinska (warsaw, pl) objectives: there is an increasing proportion of resistant strains causing infective endocarditis in recent years. it has changed the approach to choice of antibiotic therapy. linezolid (zyvoxid ò ) is a new bacteriostatic antibiotic with a wide spectrum of activity against gram-positive organisms and with good efficacy in experimental animal models of endocarditis. unfortunately clinical experience with linezolid in the treatment of endocarditis is limited. the aim of the study was to observe efficacy of linezolid in the treatment of infective endocarditis. methods: the study group consisted of 8 patients hospitalised in institute of cardiology in warsaw (2003 warsaw ( -2005 due to clinically resistant infective endocarditis. the diagnosis of endocarditis was established according to the duke criteria by clinical examination, echocardiography, laboratory investigations and positive blood cultures with vancomycin mic estimation (in 5 pts). all patients were treated surgically (valve replacement, artificial material removal) in conjunction with different conventional antibiotics and afterwards with 600 mg of linezolid every 12 hours intravenously. results: infective endocarditis was diagnosed as caused by mrcns in 2 pts, mssa in 1 pt, enterococcus faecalis in 1 pt and staphylococcus epidermidis mr in 1 pt. vancomycin mic vary from 2 to 4 mg/l. in 3 pts culture-negative endocarditis was diagnosed. all patients were treated with linezolid intravenously 2 to 4 weeks (average 3,1). clinical response and eradication of bacteremia were achieved in all patients. leukopenia nad thrombocytopenia as an adverse reaction occurred in 1 patient. conclusions 1. linezolid is effective in patients with grampositive endocarditis. 2. linezolid could be also effective in some patients with culture-negative endocarditis. 3. linezolid may provide an alternative in the treatment of infective endocarditis due to multi-resistant bacteria, in patients with resistant course or with adverse reaction to conventional antibiotics. objectives: to evaluate the safety and efficacy of lzd in a chinese population. methods: this randomized, double-blind, multi-centre study was conducted in china. after obtaining written informed consent, patients from 18 to 75 years of age with pneumonia (pneu) or skin and soft tissue infection (ssti) known or suspected to be caused by a gram-positive pathogen were randomized 1:1 to receive either lzd, 600 mg, or vancomycin (van), 1 g, each given iv q12h. patients were to be treated for 7 to 21 days, and outcomes were assessed at end-of-treatment (eot) and follow-up (f-u) visits. results: one hundred forty-two patients were enrolled and received study medication, 80 with pneu and 62 with ssti. clinical assessments (effective = ''cured'' plus ''marked improvement'') for patients in the fully evaluable population are summarized in the table. the most frequently isolated pathogen was staphylococcus aureus: all isolates were susceptible to both study drugs. the eradication rates for all pathogens in evaluable patients at the f-u evaluation were 54/58 (93.1%) in lzd-treated patients and 42/56 (75.0%) in van-treated patients (p = 0.0072). all patients receiving study drug were evaluated for safety. drug-related adverse events (aes) were reported in 18 (25.4%) lzd-treated and 12 (16.9%) van-treated patients. the most commonly reported drug-related aes in lzd-treated patients were mild abnormalities in liver function tests and leucopenia (4.2% each); rash (7.0%) was the most commonly reported ae in van-treated patients. seven (9.9%) lzd-treated and 10 (14.1%) van-treated patients discontinued study drug because of an ae. conclusions: linezolid is an effective drug for the treatment of infections caused by gram-positive pathogens and is welltolerated. eradication in one patient, by rifamycinlinezolid, of a methicillin-resistant staphylococcus aureus producing panton-valentine leukocidin, responsible for 7 relapses over 18 months, and decolonisation of her family by mupirocin objective: we report the case of the mother who experienced 7 relapses over the period. methods: pvl-mrsa were isolated on routine and mrsa agars (biorad). antibiotypes were studied by disk diffusion method. genetics and pulsotypes were studied by the french centre national de référence des staphylocoques in lyon (cnr). results: mrs kym had her 4th child on october 8, 2003 in the hospital of orléans. she was healthy and presented no risk factor for delivery. three weeks later she was addressed for surgical treatment of an abscess on buttocks. cultures yielded the special antibiotype: methicillin-r, kanamycin-r and tobramycin-s, of the pvl-mrsa currently spreading across europe and maghreb. the cnr found the luks-pv and lukf-pv-genes.through march 2005, she relapsed 7 times and was treated by pyostacin for a total of 32 weeks. two of her children were addressed for abscesses (1 buttocks, 1 thumb) yielding the same bacteria. in march 2005, mrs kym was addressed to the infectious diseases ward because of nasal furonculosis. samples yielded a pvl-mrsa with mls-b phenotype. treatment by rifamycin-linezolid 14 d was initiated. the whole family was screened. the father and the boy, 9, who had the infected thumb 17 months earlier, were carriers. the girl, 12, who had an abscess on buttocks 8 months earlier was not. in april the whole family accepted an attempt for decolonisation by 2% nasal mupirocin 2 or 3/d for 5 d. cure and decolonisation were confirmed by nares and cutaneous folds samples in may and june. they missed an additional appointment in the beginning of term, but a phone call to the social worker confirmed none of them relapsed. the cnr studied 7 strains (3 from mrs kym 2003 , 2005 , 2 from children abscesses in 2003 and 2004, 2 from boy's and father's nares 2005), and confirmed that they were all identical along the period and across the family. conclusion: short treatment with linezolid-rifamycin in the relapsing case associated with familial decolonisation by nasal mupirocin was an effective strategy to stop a time-prolonged familial outbreak of pvl-mrsa infection. multiple brain abscesses and purulent meningitis by listeria monocytogenes in an otherwise healthy man. favourable linezolid response, hampered by a suspected early drug myelotoxicity introduction: l. monocytogenes cns infection in immunocompetent adults remains rare. meningitis is the most common cns manifestation, with brain abscesses being <1% of overall episodes. anecdotal episodes of cns l. monocytogenes infection were reported from immunocompetent patients where both diagnosis and treatment may be hampered by low clinical suspicion and a frequent non-specific presentation. in a 15-yearlong survey conducted in dallas (us), only 4 cases of nonneonatal l. monocytogenes meningitis were found (estimated incidence rate: 0.3%). case report: a 50-year-old male with a negligible history and no obvious exposure to l. monocytogenes was hospitalized owing to dizziness. a brain ct scan showed a small, late ischemic lesion. a few days later hyperpyrexia, headache, vomiting and altered mentation occurred. the csf study detected an elevated albumin content (217 mg/dl), low glucose (4 mg/dl) and a wbc count of 256 cells/ll (60% neutrophils) so that ceftriaxone-chloramphenicol were immediately started. clinical-neurological conditions deteriorated while l. monocytogenes was cultured from the csf so that treatment was changed towards high-dose ampicillin-gentamicin. the persistence of severe clinical-neurological conditions and altered csf assay prompted the introduction of rifampicin-cotrimoxazole after 10 days, but 8 days later other focal neurological deficits appeared and a mri showed small, hyperintense focal lesions involving the medulla oblongata, interpreted as multiple abscesses. the introduction of linezolidmeropenem, despite anemia (requiring 2 rbc transfusions after 7-14 days) led to a progressive clinical-csf improvement. our patient recovered completely and a control mri carried out 1 month after discharge confimed the complete disappearance of the multiple brain listeria abscesses. discussion: the l. monocytogenes meningitis and multiple subtentorial abscesses (including rare localizations at cerebellum, bulb, and pons varolii), had an evolving cumbersome presentation. despite the in vitro activity of a broad spectrum of agents, multiple therapeutic changes became necessary, until the last linezolid-meropenem combination, which was proved very effective, although it was affected by relapsing anemia probably attributable to linezolid. linezolid, due to its elevated csf-brain penetration, and its activity against a broad spectrum of cns pathogens (including the intracellular l. monocytogenes), is expected to become a key antimicrobial compound, waiting for rct. discrepancy between favourable in vitro microbiological data and a severe clinical course of a staphylococcal knee and soft tissue infection responsive to oxazolidinone linezolid only after failure of all other therapeutic attempts introduction: to offer therapeutic alternatives for the emerging, multiresistant, serious gram-positive infections, novel molecules (quinupristin/dalfopristin, linezolid, daptomycin) were introduced and are made available when multiresistant gram-positive cocci are documented as no more susceptible to all available drugs including glycopeptides. however, inezolid encompasses unique tissue penetration and diffusion features (regarding soft tissues, lungs, joints and central nervous system) which make this last drug extremely promising in all circumstances where the penetration rate into infectious foci becomes critical. clinical experience: a very intriguing case report of a severe, staphylococcal knee arthtiris associated to an extensive local cellulitis/fasciitis and haematogenous dissemination occurring after a surgical curettage was characterized by a complete lack of response to a prolonged vancomycin/teicoplanin plus rifampicin therapy based on the apparently favourable in vitro sensitivity assays of methicllin-resistant staphylococci, but rapidly responded to i.v. (followed by oral) linezolid administration. the complete lack of clinical activity of a 2-week glycopeptiderifampicin administration cannot be explained by the in vitro measured mic90 values of isolated pathogens which showed complete sensitivity of staphylococcus aureus against vancomycina/teicoplanin and rifampicin and susceptibility of a concurrent hematogenous s. epidermidis strain to glycopeptidesrifampicin. since an abscess formation and an underlying osteomyelitis were carefully excluded by adequate instrumental examinations, from a theoretical point of view the active glycopeptide-rifampicin molecules should have been provided appropriate cure. on the other hand, from a strictly clinical issue, only a 2-week administration of i.v. linezolid followed by one more week of oral linezolid allowed to obtain a complete clinical-bacteriological cure and a complete function recovery without any sequelae after a 1.5-year follow-up. conclusions: when the management of severe, multiresistant gram-positive infections is of concern, the in vitro activity of single drugs and therapeutic classes should be carefully evaluated in relation with the expected penetration and diffusion rates of these drugs into the relevant organs and tissues involved by the ongoing infectious localizations. otherwise, apparently unexplained failures may occur also when in vitro studies point out a complete activity of the tested compounds. epidemiology of resistance to antibiotics -ii p1610 contemporary prevalence of bro betalactamases in m. catarrhalis: report from the sentry antimicrobial surveillance program (usa; 1997 l. deshpande, h. sader, r. jones (north liberty, us) objectives: to evaluate the prevalence of bro-1 and bro-2 among b-lactamase (bl)-producing m. catarrhalis (mcat) in the usa. although the bl-mediated penicillin (pen) resistance (r) in mcat has been stable at 95%, the bro-1 and -2 occurrence has not been determined in usa isolates since 1998. bro-2 rates have been reported at <15 (1980s), 4.8 (1984-94), 2.1 (1994-95) and 3.1% (1997-98) . methods: community-acquired mcat isolates (sentry program 1997 were tested by clsi broth microdilution methods including: 7,860 worldwide and 3,671 in north america (na). bro-1 and -2 was detected by pcr methods (levy and walker; 2004), compared to epidemiologic tests, and mic values. 100 b-lactamase-positive (bl+) mcat samples per year from usa (39 sites) and canada (ca; 7 sites) were tested for the odd-numbered years. results: the bro-2 rate was 4, 4, 3, and 3% for 1997, 1999, 2001 and 2003, respectively; rates in ca (8 isolates) > usa (6). several agents remained active: amoxicillin/clavulanate (mic90, £0.25 mg/l), ceftriaxone (ctri; 0.5), cefuroxime (2), erythromycin (£0.25-0.5), levofloxacin (£0.03-0.06), tetracyclines (2) and trimethoprim/sulfamethoxazole (tmp/ smx; £0.5/9.5). pen mic distribution was tri-modal (£0.03, 1-2, >4 mg/l) and ctri bi-modal (0.016, 0.5), yet bro-1 and -2 mic/zone distributions overlap (best discriminated by methicillin (mean zone, 10.6 vs. 19.4 mm) and pen (13.9 vs. 24.1) disks). possible bro-2 epidemic clusters could not be excluded due to a very common ribotype in 3 centres (ca, 2 sites; usa, 1). conclusions: this bro-1 and -2 enzymes na prevalence update in mcat isolates (1997) (1998) (1999) (2000) (2001) (2002) (2003) shows stability at 96-97% and 3-4%, respectively. phenotypic tests (zones or mics) cannot easily distinguish between these b-lactamase types, necessitating the use of molecular applications. objective: although resistance to penicillin in beta haemolytic streptococci has not been reported yet, increasing resistance rates for alternative drugs, such as erythromycin, clindamycin or tetracycline is an emerging concern which brings the necessity to carefully monitor penicillin susceptibility. materials and methods: in order to detect any changes in penicillin mics, we performed antimicrobial susceptibility testing for all isolated beta haemolytic streptococci in our hospital between january 2000 and november 2005. identification to serogroup level was done using a commercial latex agglutination kit (avipath strep, omega diagnostics ltd., scotland, united kingdom). results: a total of 1890 isolates were identified, distribution of groups for serogroup a, b, c and g were 75.6%, 18.9%, 2.8% and 2.7%, correspondingly. penicillin susceptibility was determined using etest (ab biodisk solna, sweden) strips according to manufacturers' instructions. when results are evaluated in 2year periods, mic90 increased from 0.012 to 0.016 mg/ml for group a, from 0.064 to 0.094 mg/ml for group b, from 0.012 to 0.064 mg/ml for group c and from 0.016 to 0.023 mg/ml for group g (table) . conclusions: even though highest mic90 values were to be found in group b (0.094 mg/ml), our results indicate the steady increase in penicillin mic for all serogroups. three group a and six group b isolates with penicillin mic of 0.125 mg/ml, reaching susceptibility breakpoint concentration according to clsi, and also highly elevated mic90 concentrations for group b streptococci may be messengers of possible forthcoming resistant strains. objective: to study trends in macrolide resistance rates among s. pneumoniae isolated from children aged 3 to 40 months attending day-care centres in france following implementation of prudent antibiotic use campaigns (alpes maritimes 2001 , france 2003 and pneumococcal conjugate vaccine (pcv) (2003) . method: nasopharyngeal aspirates were obtained from a random 2-stage cluster sample of children attending day-care centres in the nord (n) and alpes maritimes (am) areas during 3 consecutive surveys between january and march 1999 march , 2002 march and 2004 . susceptibility to erythromycin and clindamycin and resistance phenotype were analysed by disk diffusion method. serotypes were determined using the quelling reaction. pneumococcal immunization status and antibiotic prescriptions over the 3 previous months were recorded. results: sp was isolated from 278/548, 289/534 and 269/567 children in 1999, 2002 and 2004 , respectively (p < 10 )3 ). resistance to macrolides declined overall from 77.0% to 64.3% of strains between 1999 and 2004 (p < 10 )2 ). among erythromycin-resistant (e-r) isolates, percentage of erm-b phenotype increased from 81.0% to 90.2% (p = 0.001). while the proportion of penicillin non-susceptible strains declined from 33.6% to 24.7% of sp isolates (p < 10 )3 ), erythromycin resistance remained stable among these strains at 92.0%. overall proportion of treated children fell from 64.3% to 48.7% (p < 10 )3 ) between 1999 and 2004; in am this reduction was observed in 2002 (16.0%; p < 10 )3 ), while in n it occurred in 2004 (24%; p < 10 )3 ) and the percentage of macrolides among prescriptions fell from 13.0% to 9.0% (v 2 for trend: p = 0.06). serotype distribution showed most e-r isolates were 6b, 14, 19f and 23f. a 50% reduction in serotype 23f was observed in am in 2002 and in n in 2004. immunisation with pcv concerned at least 33.5% of children in 2004. conclusion: macrolide resistance has followed a parallel decline with penicillin resistance as a result of antibioticprescription reducing campaigns and pneumococcal immunization against the most prevalent macrolide-resistant serotypes. objective: to evaluate the prevalence of resistance of invasive strains of s. pneumoniae to erythromycin after decline in macrolide consumption. methods: the number of packages of antibiotics was obtained from the institute of public health of slovenia. for the period 1999-2004 the data on outpatient antibiotic consumption were collected using the atc/ddd classification (who version 2005) and the results were expressed in ddd/1000 inhabitants per day (did). all invasive strains of s. pneumoniae isolated from sterile body fluids in all slovenian hospitals were included in the study. susceptibility testing was performed using nccls approved disk diffusion test. results: during 1999-2004 the total use of antibacterials in slovenia decreased for 17.4% from 20.23 did to 16.71 did. the consumption of macrolides which constituted 17.1-19.4% of total use of antibacterials decreased for 21.3% (3.81 to 3.0 did). short-acting (erythromycin, miocamycin), intermediate-acting (midecamycin, roxithromycin, clarithromycin), and long-acting (azithromycin) decreased for 33.4%, 25.8% and 13% respectively. in all years the use of intermediate-acting macrolides was the most prescribed subclass of macrolides corresponding for 1.50-2.21 did, followed by long-acting (1.25-1.47 did) and short-acting (0.07-0.12 did). the resistance of s. pneumoniae strains to erythromycin increased from 5.4% (6/110) to 11.2% (19/170); in children from 2.9% (1/ 34) to 18.1% (6/33) and in adults from 6.5% (5/76) to 9.5% (13/137) respectively. rates of the isolates resistant to erythromycin and at least 2 of the following agents: penicillin, tetracycline, tmp/smx, chloramphenicol increased from 0.9% (1/110) to 7.7% (13/169); in children from 0% (0/ 34) to 15.1% (5/33) and in adults from 1.3% (1/76) to 5.8% (8/ 137) respectively. conclusion: despite a reduction of macrolide consumption in outpatients the resistance of invasive strains of s. pneumoniae was increasing during the observation period especially in children. multiple drug resistance explains best the changes in s. pneumoniae resistance in a ten-year surveillance study in belgium objective: belgium is located between countries with very high and very low antibiotic resistance rates. modeling how resistance changes over time and place in belgium provides insights into correlates of s. pneumoniae resistance at the population level. methods: surveillance data consists of 14,488 s. pneumoniae invasive isolates from 1994-2004, identified by postal code as well as clinical and demographic information. antimicrobial consumption (ims health services) is expressed in defined daily doses (ddd) per 1000 inhabitants per day. changes in resistance by month and postal code were evaluated using mixed effects models for repeated measures, using mathematical models of transmission for the curve shape, and taking into account seasonality. resistance to penicillin, erythromycin, tetracycline, and ofloxacin was considered in the analysis. results: resistance to penicillins, macrolides and tetracyclines peaked in the year 2000, and their levels in 2004 were 11.6%, 35.6% and 28.7% respectively. the shape of the curves is similar for most of the antibiotics studied, with a steep rise from 1994 to 2000 and a plateau thereafter. resistance to two or more antibiotic classes corresponded to 72% of all resistant isolates and in a multivariate model explains most of the variability through time and place of the antibiotics studied. resistance to only one antibiotic (any) decreased from 16.3% in 1994 to 11.7% in 2004, while resistance to two or more increased 2.5 times (95% ci 1.95-3.12, p < 0.0001) from 14.1% in 1994 to 28.8% of all isolates 10 years later. more than nine out of ten isolates that were macrolide or tetracycline resistant were also multiply resistant (mr). mr increases 0.57% for each ddd of overall cumulative antimicrobial consumption, and out of all antibiotic classes, macrolides and broad-spectrum penicillins are most associated with resistance. conclusion: resistance to two or more antibiotics is the most important factor in understanding the changes over time for all studied antibiotic classes in belgium. the cumulative impact of antimicrobial exposure of separate antibiotic classes at the population level facilitates the survival and transmission of any isolate that is resistant to two or more antibiotic classes. methods: the isolates were identified by biochemical tests and specific serotyping. antimicrobial susceptibility to ampicilllin (amp), amoxicillin plus clavulanic acid (auc), cloramphenicol (cm), gentamicin (gm), cotrimoxazole (sxt), nalidíxic acid (nal) and tetracycline (tc) were established by the method of kirby bauer. the presence of beta-lactamases encoding genes (tem, carb, oxa2-like) as well as the teta, tetb, tetc, tetg, cmla and flor genes, and integrons type 1 was established by pcr, while the presence of plasmid-mediated dhfr was determined by pcr-rflp and the cat activity by a colorimetric assay. results: seven different resistance patterns were identified: i. susceptible (38 strains); ii. amp, sxt, gm, a/c (37); iii. amp, tc, cm (2); iv. amp, tc, sxt, cm (2); v. amp, a/c (4); vi. amp, sxt, a/c (1); vii (1). -sxt. no isolate resistant to nalidixic acid was detected. resistance to beta-lactam agents was due to the presence of beta-lactamases type tem-like (pattern v), carb-2 (iii) and tem-like plus oxa-30 (ii, v, vi). meanwhile resistance to cloranphenicol and tetracycline was associated to cat activity (iii, iv) and flor (iii), and tetb (iv) and tetg (iii) respectively. no mechanism of cotrimoxazole resistance was detected in the isolates of the patterns ii, vi and vii, while dfra1 was detected in the isolates of the group iv. resistance to gm was associated to the presence of the gene aadb, detected in the analysis of integrons type 1. type 1 integrons were detected in isolates belonging toi the pattern ii (750 bp -aadb; 2000 bp -oxa30, aada1), iii (900 bp -carb2, 1100 -aada1), iv (1500 bp -dfra1, aada1), v and vi (2000 bp -oxa30, aada1). conclusions: a great diversity of resistance mechanisms has been detected. those mechanisms might spread among microorganisms resulting in a serious health problem due to the limited number of antibiotic treatments available in the area. small outbreaks of veb-1 esbl producing acinetobacter baumannii in belgian nursing homes and hospitals through cross-border transfer of patients from northern france methods: from 01/04 to 03/05, all belgian acute hospitals were invited to report cases of nosocomial infections/colonisations due to mdr ab isolates presenting a resistance profile similar to the french epidemic strain (resistance to all agents except carbapenems and colistin) and to send such isolates to the reference laboratory for phenotypic confirmation and for genotypic characterization (pcr of veb-1 and class 1 integron, pfge typing). guidelines for detection of the epidemic strain, screening for carriage in patients transferred from hospitals or nursing homes (nh) close to the french border as well as infection control measures were sent to all hospitals. results: overall 73 ab strains from 30 hospitals were sent to the reference laboratory. only, 26 of these fulfilled the phenotypic resistance patterns and 6 were definitely confirmed as veb-1 ab and had a pfge pattern identical to the french epidemic clone. two mini-outbreak clusters (each involving 3 cases) were documented in hospitals from two cities (tournai and chimay) closed to the french border. two patients died from their infection. in the first outbreak, all 3 patients were residents who lived in the same nh. two of them were french citizens who had been hospitalised in different acute care hospitals in the north of france within the last year. in the second outbreak, the index case had also been previously hospitalised in a french hospital. secondary transmission to two other hospitalised patients occurred in this outbreak. conclusion: despite the large extension of the veb-1 ab outbreak in france no similar problem occurred in belgium. however, this national alert allowed to detect two small outbreaks in belgian institutions located close to the french border. in both outbreaks the epidemic strain was imported from france through patient circuits. this study illustrates that transfers between acute care hospitals and nh may explain cross-border spread of multi-resistant epidemic strains. types of extended-spectrum beta-lactamases in salmonella spp. and decreased susceptibility to fluoroquinolones objectives: the aim of this study was to determine the rate of esbl production in clinical isolates of salmonella spp. and to detect decreased susceptibility to fluoroquinolones in esbl positive isolates in turkey. methods: a total of 620 salmonella spp. isolated from clinical samples from thirteen centres between 2000 and 2002 were included in the study. in vitro susceptibility to ampicillin, amoxicillin/clavulanic acid, cefotaxime, gentamicin, chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and ciprofloxacin were determined using the agar dilution method on mueller-hinton agar following the clinical and laboratory standards institute (clsi) guidelines. decreased susceptibility to ciprofloxacin was defined as an mic of 0.125-1 mg/l. salmonella isolates were screened for esbl production by double disk synergy method using amoxicillin/clavulanic acid, cefotaxime and ceftazidime disks. types of esbl enzymes were analysed by pcr for tem, ctx-m, shv and per-1 genes. results: in salmonella spp. the highest level of resistance was observed against ampicillin (41.3%) followed by chloramphenicol (36.6%), tetracycline (34.2%) amoxicillin/ clavulanic acid (32.4%), trimethoprim/sulfamethoxazole (2.3%), gentamicin (1.6%), and cefotaxime (0.8%). ciprofloxacin resistance was observed in one isolate (0.2%). among 620 salmonella isolates, 6 (0.97%) were shown to produce esbl by double disk synergy testing. these isolates were salmonella typhimurium (n = 2), serogroup c1 (n = 2) and salmonella enteritidis (n = 2). three isolates were from fecal samples two were from urine and one was from blood. one of the esbl producing isolates were susceptible to cefotaxime in vitro. two isolates showed decreased susceptibility to ciprofloxacin. all the esbl producers were resistant to ampicillin, amoxicillin/ clavulanic acid, chloramphenicol and harbored ctx-m type enzymes. in three isolates a tem-type enzyme was also present. conclusion: albeit being rare, esbl production is an important resistance factor among salmonella spp. in order to prevent treatment failures, decreased susceptibility to fluoroquinolones should be investigated routinely in invasive isolates as well as esbl production. incidence of faecal carriage of esbl-producing enterobacteriaceae in hospital and community patients during two non-outbreak periods of time the identities of the esbl-producing isolates recovered during 2002 were: e. coli (n = 98), k. pneumoniae (n = 2), p. vulgaris (n = 2) and e. cloacae (n = 1), and isolates recovered during 2004 were: e. coli (n = 340), k. pneumoniae (n = 3), k. oxytoca (n = 1), p. vulgaris (n = 1), p. mirabilis (n = 2), e. cloacae (n = 1) and e. aerogenes (n = 1). conclusions: a dramatic, significant increase in the frequency of faecal carriage of esbl-producing isolates was demonstrated in 2004 among hospitalized (8.1%) and ambulatory patients (7.2%).the results revealed that the prevalence of faecal carriage among ambulatory patients and hospitalized patients was not significantly different in both periods of time. outpatients came from the community carrying enterobacteria harbouring esbl in the intestinal tract, suggesting that the community could be a reservoir for these microorganisms and enzymes. methods: a total of 71 k. pneumoniae, 3 k. oxytoca, 11 e. coli, 4 c. freundii, 3 s. marcescens, and 3 e. cloacae from 6 university hospitals, isolated from blood, wound, urine, sputum and other clinically significant specimens were proven to produce esbls. antimicrobial susceptibility was determined according to clsi, 2002 ; conjugation on a solid medium was performed; isoelectric focusing was followed by bioassay; pcr with beta-lactamase group-specific oligonucleotides was applied, followed by nucleotide sequencing; rapd with eric-1a and eric 2 primers was performed. results: mic of ceftazidime varied from 4 to >512 mg/l, mic of cefotaxime -2-512 mg/l; the addition of sulbactam 1: 1 reduced mic > 3-fold. transconjugants exhibited resistance both to extended-spectrum cephalosporins and aminoglycosides in 37 of 39 strains. according to their pi, two clusters of betalactamase producers could be described: first one -esbls focussed at pi 8.2, and the second -pi at 6.3. results from pcr confirmed the presence of two groups esbls: tem and shv. sequencing of representative strains showed the presence of shv-12 in two participating hospitals and of shv-5 in only one strain e. cloacae, while tem-3 like enzyme was found in 2 centres and had a clonal dissemination. objectives: during treatment with selective decontamination of the digestive tract (sdd), four strains of multidrug-resistant (mdr) gram-negative bacteria (three escherichia coli strains and one klebsiella pneumoniae) were isolated at the intensive care unit (icu) in the academic medical center (amc) in amsterdam. these isolates were extended spectrum betalactamase (esbl) positive. we investigated whether this was due to interspecies transfer of resistance genes. methods: the strains were typed by amplified fragment length polymorphism analysis. the plasmids from these strains were characterized by restriction fragment length polymorphism. resistance genes of the mdr-strains were characterized by pcr and sequence analysis. results: aflp analysis confirmed that the three mdr e. coli isolates represented three different strains. the mdr-strains were shown to harbour the same plasmid with identical extended-spectrum â-lactamase (esbl) genes; ctx-m-15 and shv-5. conclusions: identification of the emergence of such mdr gram-negative bacteria and recognition of resistance plasmid transfer during sdd treatment is crucial for optimal application of this regimen in icus. the use of the third generation cephalosporins in sdd may associate with emergence and increase in the prevalence of esbls. therefore, for optimal screening of resistance to cephalosporins in icus, the screening for esbls should be included. objective: carbapenems are the drugs of choice for the treatment of serious infections caused by esbl-producing enterobacteriaceae and the emergence of carbapenem resistance is rarely documented. we investigated pairs of carbapenem-susceptible and resistant k. pneumoniae isolates from three patients, collected before and after therapy with carbapenems. methods: pre-and post-therapy pairs of esbl-producing k. pneumoniae isolates were from three patients with urinary catheter-associated infections who were treated with ertapenem (erp, 2 cases) or meropenem (mem, one case) in a district general hospital with a low incidence of esbl-producing organisms (0.43/1000 bed days), and meropenem use of 1160 ddd/year. isolates were compared by pfge of xbai-digested genomic dna. mics were determined and interpreted by british society for antimicrobial chemotherapy methodology. blactx-m alleles were sought by multiplex pcr. outer membrane proteins (omps) were extracted, and analysed by sds-page. results: the three patients relapsed following erp or mem therapy, and the post-therapy isolates from repeat urine samples were resistant (table), with mics erp>mem>ipm. all six isolates from the three patients belonged to the same pfge strain, but transmission of the resistant variants is unlikely as the patients were geographically and temporally unrelated and separate selection of resistance in individual patients seems more likely. all isolates had a group 1 ctx-m esbl; the resistant isolate in each pair had lost a major omp, consistent with a porin, compared with its susceptible 'parent'. all three patients were successfully treated with amikacin. the emergence of carbapenem resistance in ctx-m-producing k. pneumoniae following therapy severely limits treatment options. whilst unusual in general, such selection has occurred repeatedly with this strain. wide geographic spread of diverse acquired ampc beta-lactamases in escherichia coli and klebsiella spp. in the uk and ireland objective: to determine the distribution of genes encoding acquired ampc beta-lactamases in cephalosporin-resistant isolates of e. coli and klebsiella spp. submitted to the uk national reference laboratory. methods: mics were determined by agar dilution and interpreted according to breakpoints of the british society for antimicrobial chemotherapy. isolates of e. coli or klebsiella spp. resistant to cefotaxime and ceftazidime, irrespective of addition of clavulanic acid, were inferred to have possible ampcmediated resistance. genes encoding six phylogenetic groups of acquired ampc enzymes were sought with a multiplex pcr assay (perez-perez & hanson. j clin microbiol 2002; 40:2153-62) . selected isolates were compared by pfge, and selected blaampc amplicons were sequenced. results: 47 e. coli isolates and 8 klebsiella spp. from separate patients yielded pcr amplicons indicating the presence of genes encoding acquired ampc enzymes. forty of these e. coli isolates (from 20 hospitals) produced cit-type enzymes, 6 (from 3 irish hospitals) produced acc types, and 1 a dha type. the klebsiella spp. produced acc (5 isolates from 2 irish hospitals), fox (2 isolates from 2 welsh hospitals) or dha (1 irish isolate) enzymes. genes encoding ebc-/ent-and moxtype enzymes were not detected. twelve e. coli isolates from one hospital all produced a cit-type enzyme; these 12 isolates belonged to an epidemic uk strain, designated strain a; 8 isolates also contained blactx-m-15 linked to an upstream copy of is26, as is characteristic of strain a; 4 isolates lacked blactx-m-15. sequencing of a representative blaampc amplicon indicated production of a novel cmy-2 variant in these isolates. conclusions: diverse acquired ampc enzymes are present in e. coli and klebsiella spp. in the uk and ireland, with cit-types the most common, and acc types linked to ireland. the broad resistance profiles of ampc enzymes compromises patient management. hence, the acquisition of a cmy-2-like enzyme by epidemic e. coli strain a suggests that acquired ampc enzymes are poised to become an important public health issue in the uk. objective: to characterize the spectrum of activity and potency of dor (formerly s-4661) and comparator agents against contemporary wild-type bacterial isolates from medical centres in europe and the middle east in 2004. dor is a novel parenteral 1-b-methyl carbapenem in late stage clinical development whose molecular structure confers stability to b-lactamases and resistance (r) to renal dehydropeptidases. methods: the collection included 6480 non-duplicate, consecutive clinical isolates from patients in 24 medical centres in europe (21), turkey (2) and israel (1) that were submitted to the dor surveillance program (2004) for identification confirmation and susceptibility (s) testing. mic values for >30 antimicrobials were determined using nccls broth microdilution methods (2003) . a tentative dor susceptible (s) breakpoint of £4 mg/l (£0.25 mg/l for s. pneumoniae) was used for comparative purposes; clsi (2005) criteria were used for other tested agents. results: antimicrobial activities of dor and other carbapenems vs. selected isolates. dor consistently displayed activity against staphylococci and streptococci (mic90, 0.06 and 0.5 mg/l) most similar to that of imipenem, and against e. coli and klebsiella spp. (mic90, 0.06 and 0.5 mg/l, respectively, including 8.7 and 26.9% of strains that met esbl screening criteria), most similar to that of meropenem. enterobacter spp. isolates, including 35.8% that were ceftazidime-r (indicative of ampc production), were also highly s to dor and other carbapenems (0.5 to 1.4% r). dor also provided slightly enhanced coverage against p. aeruginosa (82.7% s) and acinetobacter spp. (54.4% s) compared to other carbapenems. carbapenem r among these latter strains is, however, a particularly worrisome development. conclusions: dor is a new carbapenem with a competitive profile that incorporates both potent gram-negative and grampositive activity, with enhanced activity against the commonly occurring non-fermentative gram-negative bacilli. carbapenems are assuming a greater therapeutic role in many nations as multi-drug resistance (including emergence of ambler class a, c and d b-lactamases) spreads, necessitating their accelerated development. phenotypic and genetic characterisations of enterococcal isolates in tehran sewage, with emphasis on detection of vana and vanb genes objectives: enterococci are members of the normal gut flora of animals and humans and are thus released into the environment directly or via sewage outlets, where they can survive for long time periods. during the last decade the concern has been focused on enterococci that are resistant to the glycopeptide antibiotic vancomycin [vancomycin-resistant enterococci (vre)]. the aim of the study was to detect and to analyse the biochemical diversity of the entrococci strains in tehran sewage and to determine the genetic characterization of vre. methods: a total of 410 isolates of enterococci were selected on me agar medium. all of the isolates were identified at the species level by the common biochemical tests. drug susceptibility test of isolates was done by disk diffusion method with 6 antibiotics vancomycin, erythromycin, gentamicin, tetracycline, chloramphenicol and ciprofloxacin. the mic was also done by macrobroth dilution assay. analysis of the plasmid profiles and the pcr tests for vana and vanb genes were done. methods: we studied 153 vre isolates collected in the north and center of portugal (1996 portugal ( -2004 from: (i) 81 clinical isolates from 3 hospitals in different cities, (ii) 4 faecal samples from healthy volunteers, (iii) 2 river water samples, (iv) 6 samples collected downstream of hospital sewage water, (v) 2 samples from urban sewage water, (vi) 4 swine faeces (vii) 27 poultry food samples for human consumption. identification and characterization of vancomycin resistant genes vana, vanb, vanc1 and vanc2 were determined by a multiplex pcr. the backbone structure of tn1546 was characterized by a pcr overlapping assay (10 overlapping fragments), and further sequencing. conclusion: beta-lactamase production among hi strains has declined significantly since 1999 among children attending daycare centres as antibiotic prescriptions fell among this population. results: the mic distribution of am showed 4% of strains (n = 332) with a mic > 4 mg/l and 7% with a mic of >2 mg/l, indicating that resistance to am is still relatively rare and does increase as compared to nethmap2004. the lognormal distribution of both am and amc (1 strain r) extended to 1 mg/l but showed tailing to 4 mg/l. this may indicate hidden less susceptible strains but could equally well be explained by testing circumstances, since the left part of the mic distribution showed comparable tailing. all strains were susceptible to moxifloxacin, levofloxacin and cefotaxim. the lognormal distribution of sxt extended to 0.25 mg/l with 10% of strains showing higher values. doxycyclin resistance was less than 5%. most of the strains were resistant to clarithromycin and azithromycin with a mic10 >0.5 mg/l for both. conclusions: resistance of hi to common antimicrobials in the netherlands is still low and does not increase. objectives: s. pneumoniae (sp) and h. influenzae (hi) are the two most common pathogens associated with community-acquired pneumonia. changes in the prevalence of resistance or multidrug resistance (mdr) among these pathogens have important therapeutic ramifications. the global surveillance initiative is a longitudinal study that benchmarks antibacterial resistance among respiratory pathogens. methods: during 2005, 964 sp and 924 hi were isolated from patient specimens collected at hospital laboratories in france (fr), germany (ger), italy (it), spain (spa), and the united kingdom (uk). isolates were centrally tested by broth microdilution against lev, penicillin (pen; sp only), azithromycin (azi), erythromycin (ery), clindamycin (cli), ceftriaxone (ctx), cefuroxime (cfx), and trimethoprimsulfamethoxazole (tmp-smx) (nccls, 2005) . data were analysed according to pen resistance, mdr, and b-lactamase status. mdr was defined concurrent resistance to ‡2 of the following agents: ctx, cfx, ery, lev, pen, and tmp-smx. results: for sp, pen r was 1.8% in ger, 2.6% in the uk, 8.5% in it, 15.7% in spa, and 27.1% in fr. azi r was 13.0% in the uk, 22.1% in ger, 27.8% in spa, 44.3% in it, and 46 .3% in fr. overall, lev r was rare (£1%) and mic90s = 1 mg/l in all countries. 62.9% of isolates were susceptible to all of the drugs tested, the most common phenotype encountered. the prevalence (%) of mdr among sp ranged from 4.7 in uk to 32.3 in fr. resistance to pen, ery, cfx, and tmp-smx was the most prevalent mdr phenotype found in europe. overall 97.4% of mdr sp were susceptible to lev. for hi, b-lactamase rates varied by country from 4.5% in it to 21.9% in fr. based on mic90 lev and ctx were the most active agents tested against hi, regardless of b-lactamase status. conclusions: lev showed potent activity against sp and hi. for sp, lev activity was independent of resistance to pen or mdr phenotype. lev maintained consistent activity against sp based on mic90, regardless of country studied. antimicrobial surveillance data from studies such as the global offer guidance to physicians for empiric prescribing. sxt was obtained (2001 sxt was obtained ( -2004 . conclusions: our results suggest that beta-lactamase production does not constitute a threat in hi therapy since values were almost constant. although with an unregulated fluctuation on arnblp percentages, it seems that this mechanism is gaining importance in relation to beta-lactamase production. thus, we conclude the need to be aware of arnblp, as these strains are difficult to detect using the nccls (2004) breakpoints. further molecular studies of the resistance genes responsible of this resistance mechanism are needed. resistance of beta-lactamase producer strains, to other antibiotics decreased during the period of study, due to the diminished use of these antibiotics. this study shows the importance of monitoring antibiotic resistance in hi in order to detect emerging mechanisms. antimicrobial susceptibility of respiratory haemophilus influenzae strains in northern greece k. koraki, p. karapavlidou, d. sofianou (thessaloniki, gr) objectives: to investigate the antimicrobial susceptibility of haemophilus influenzae, one of the most frequent bacterial pathogens of respiratory tract infections. treatment of these infections is most often empirical and considerable geographical resistance variation has been reported. methods: eighty h. influenzae strains were collected from respiratory tract specimens (sputum, bronchoalveolar lavages, endotracheal secretions) in a 4-year period (2000) (2001) (2002) (2003) (2004) . identification was made by colonial morphology, gram staining characteristics, x-and v-factor requirements and api nh (biomerieux, france). antibiotics were selected to reflect representative current treatment options and susceptibility was determined by kirby-bauer disc diffusion method on haemophilus test medium according to nccls guidelines. results: out of the 80 h. influenzae strains 58 were isolated from children and 22 from adults. 25% of isolates came from children admitted to the intensive care units and 18.7% from cystic fibrosis patients. a seasonal trend was reported for infections since 41.2% of isolates were collected during springtime and 25% during autumn months. overall ampicillin resistance was 13.3% and resistant strains were isolated exclusively from children. ampicillin resistance was doubled among cystic fibrosis patients (27.3%). all isolates were susceptible to amoxicillin/clavulanate, chloramphenicol, ciprofloxacin and imipenem. the rank order of cephalosporin activity was cefotaxime and ceftriaxone (100%) followed by cefuroxime and cefaclor (95.8% and 88.9% respectively). trimethoprim/ sulfomethoxazole was active against 95.2% of isolates while erythromycin was the least potent antimicrobial agent with 75% of isolates being susceptible to it. no multiresistant phenotypes were detected. conclusion: our results demonstrated that ampicillin resistance among h. influenzae in our area is still relatively low and overall antibacterial susceptibility rates are high. knowledge of antimicrobial resistance among these pathogens is imperative for physicians to choose the most appropriate therapeutic agent. results: 396 nosocomial gram-negative uropathogens were studied. most common uropathogens were p. aeruginosa (30.8%), e. coli (25.8%), k. pneumoniae (11.1%), followed by a. baumannii (7.3%), enterobacter spp. (5.8%), s. marcescens (4.5%), proteus spp. (3.8%) and other gram-negative rods (10.9%). resistance rates (i+r, %) among p. aeruginosa were: gentamicin -87%, levofloxacin -84%, ciprofloxacin -81%, cefoperazone -79%, cefoperazone/sulbactam -71%, cefepime -62%, piperacillin -55%, amikacin -48%, ceftazidime -46%, imipenem -45%, meropenem -43%, piperacillin/tazobactam -43%, polymyxin b -7%. resistance rates (i+r, %) among e. coli were: piperacillin -75%, ticarcillin/clavulanic acid -69%, amoxicillin/clavulanic acid -62%, ciprofloxacin -54%, gentamicin -54%, moxifloxacin -54%, levofloxacin -53%, cefoperazone -48%, ceftriaxone -45%, cefepime -40%, ceftazidime -32%, cefoperazone/sulbactam -19%, piperacillin/tazobactam -18%, amikacin -17%, all strains were susceptible to ertapenem, imipenem, meropenem. resistance rates (i+r, %) among k. pneumoniae were following: piperacillin -82%, cefoperazone -75%, ceftriaxone -71%, gentamicin -71%, amoxicillin/clavulanic acid -66%, cefepime -64%, ceftazidime -57%, ciprofloxacin -53%, piperacillin/ tazobactam -46%, moxifloxacin -43%, cefoperazone/sulbactam -43%, levofloxacin -41%, amikacin -27%, ertapenem -7%, imipenem and meropenem were active against all isolates. conclusion: p. aeruginosa, e. coli and k. pneumoniae are the main gram-negative uropathogens in russian icus patients. imipenem, meropenem, ertapenem showed prominent activity against e. coli and k. pneumoniae. cefoperazone/sulbactam, piperacillin/tazobactam, amikacin exhibited considerable activity versus e. coli, while k. pneumoniae were more resistant to them. p. aeruginosa were highly resistant to all tested antimicrobials except polymyxin b, thus leaving virtually no choices for therapy in terms of acceptable patient safety. results: overall 69 gram-negative anaerobic bacteria from 41 patients were studied. isolation sites were represented by intraabdominal -25 (60.9%), soft tissue -7 (17.1%), prostate fluid -5 (12.2%), bone -3 (7.4%), and dental -1 (2.4%) infections. susceptibility of 31 (44.9%) prevotella spp., 23 (33.3%) bacteroides spp. (predominantly b. fragilis group -20 strains), 7 (10.2%) fusobacterium spp., 4 (5.8%) porphyromonas spp., and 4 (5.8%) veillonella spp. to ampicillin, clindamycin, metronidazole, imipenem, ertapenem, amoxicillin/clavulanic acid and cefoperazone/sulbactam was determined. all species were susceptible to carbapenems. in prevotella spp. there were 64% and 3% strains resistant to ampicillin and clindamycin and 4% of strains with intermediate resistance to metronidazole. among bacteroides spp. 92% of strains were resistant to ampicillin and 22% to clindamycin. no resistance to metronidazole was detected in bacteroides spp. objectives: the objectives of this study were to: analyse our current blood culture practice; describe the frequency of occurrence and antimicrobial susceptibility of bloodstream infections (bsi) isolates; determine the contamination rate. methods: we performed a prospective survey of all positive blood cultures received in the department of microbiology of tartu university hospital (938 beds) in 2004. blood culture system used was bactec 9120. duplicates within one week were excluded. isolates were identified using conventional microbiology methods and susceptibility tests were those recommended by nccls. to determine extended spectrum beta-lactamase (esbl) producers an e-test with cefepime and cefepime combined with clavulanic acid was used. nosocomial infections were defined according to cdc criteria. results: during study period 7230 blood culture bottles were received, comprising 2840 blood culture sets (10.4 sets per 1000 patient-days). these resulted in 427 (17.2%) positive blood cultures, 127 (29.7%) were considered contaminants and contamination rate was 5.1%. a total of 273 bsi episodes involving 246 patients were identified and 161 (59%) of these were nosocomial. the incidence of nosocomial bsi (n-bsi) and community-acquired bsi (ca-bsi) was 0.6 and 0.4 per 1000 patient-days, respectively. polymicrobial bsi was detected in 25 patients. among n-bsi dominated coagulase-negative staphylococci (49/30.4%), staphylococcus aureus (19/11.8%), klebsiella spp. (21/13%), and escherichia coli (16/9.9%). the most frequent pathogens of ca-bsi were e. coli (31/27.7%), s. aureus (14/12.5%), haemophilus influenzae (11/9.8%), and streptococcus pneumoniae (10/8.9%). susceptibility to oxacillin of s. aureus and cons was 100% and 21.4%, respectively. all s. pneumoniae isolates were susceptible to penicillin. 97.8% of e. coli strains were susceptible to ciprofloxacin, 67.4% to ampicillin, and 100% to gentamicin. susceptibility of klebsiella spp. to both ciprofloxacin and gentamicin was 96.7%, and to ampicillin 6.7%. 14.3% of klebsiella spp. and none of e. coli isolates were esbl-producers. the susceptibility patterns of n-bsi and ca-bsi pathogens were similar to each other. conclusion: compared to west and north european countries our number of blood culture sets per 1000 patient-days is low. this may explain the relatively low incidence of bsi. the interventions to reduce contamination rate need to be implemented. the susceptibility among bsi isolates was high. recent outbreaks of c. difficile associated diarrhoea (cdad) reported in north america, united kingdom and the netherlands have emphasized the importance for an ongoing surveillance of cdad. the aims of the present study was to determine the epidemiology of cdad over the past 5 years and the rate of nosocomial transmission in our acute care hospital (750-beds). materials and methods: all the cases of cdad diagnosed between january 1st 2000 and december 31st 2004 were retrospectively reviewed. a cdad case was defined as diarrhoea in hospitalised patients with a positive result for c. difficile cytotoxin or with a positive toxigenic culture. cdad was considered as severe if patient fulfilled at least 2 of the following 3 criteria: fever > 38.5c abdominal pain or leukocyte count >10,000/mm 3 or if the patient had an endoscopically proven pseudomembranous colitis or complications (toxic megacolon, perforation…). cdad was considered as community acquired if the diarrhoea occurred in patients within 72 h after admission and if the patient had no history of hospitalisation in the previous 2 months, otherwise cdad was considered as nosocomial. all the strains were serogrouped and characterized by toxinotyping and pcr-ribotyping. detection of toxin a, toxin b and binary toxin was performed by pcr. results: 151 cases of cdad were diagnosed: 147 clinical charts could be reviewed and 131 strains were studied. global incidence of cdad was 1.1 per thousand discharges with higher rates in 2003 and 2004. diarrhoea was community acquired in 19% of patients. for patients with nosocomial cdad, transmission of the strain from patient to patient (i.e. strain with the same serogroup and pcr-ribotype than the strain from another patient hospitalised in the same ward in the previous 2 months) was demonstrated in 10.1% of cases. binary toxin was positive in 11% of strains. binary toxin was associated to a more severe diarrhoea (p < 0.01) and to a higher case fatality (p < 0.01). a specific clone accounted for 25% of all the strains (serogroup h, pcr-ribotype ''026'') but this clone was found both in nosocomial or community cases. three strains belonged to toxinotype iii but further investigations are needed to know whether these strains correspond to the hypervirulent strains involved in recent outbreaks. conclusion: incidence of cdad is low in our hospital and cross infection is limited. these results also suggest that strains with binary toxin might be more virulent. the development and application of a new exact typing method for clostridium difficile: multilocus variable number of tandem repeat analysis objectives: to study the epidemiology of clostridium difficile, a typing method with a higher discriminatory power, typeability and reproducibility than currently available methods is required. multi-locus variable number of tandem repeat analysis (mlva) is a new candidate technique, that has already been tested successfully on a number of bacterial and fungal species. using the whole genomic sequence, we developed mlva for c. difficile and compared the method to standardized pcr-ribotyping. additionally, mlva was tested on a collection of the new emerging hypervirulent pcr-ribotype 027 strains. methods: short tandem repeat loci (3 to 9 bp) were identified using tandem repeat finder v3.21 on the genome of c. difficile strain 630. amplification of the repeats was performed using a single pcr-protocol. pcr-fragments were analysed using multicoloured capillary electrophoresis on an abi3100, with a rox500-marker as internal marker for each sample. the number of repeats per fragment was subsequently determined.the discriminatory power of the mlva was tested on 23 reference strains representing 11 serogroups and 12 toxinotypes. the ability to subtype specific pcr-ribotypes was investigated with 7 subtypes of pcr-ribotype 001 (rep-pcr types 1-7), 6 tcda-/tcdb+ strains of pcr-ribotype 017, and 11 strains belonging to pcr-ribotype 027. of these 11 type 027 strains, 9 were isolated from 3 outbreaks and 2 from endemic cases. results: a total of 7 regions with short tandem repeats were identified. mlva discriminated all 23 reference strains and the 7 known reference strains of pcr-ribotype 001 (rep-pcr 1-7). two mlva-types were recognized among 6 tcda-/tcdb+ strains; the differences were present in only one of the 7 repeat-regions. of 11 pcr-ribotype 027 strains, 9 outbreak-related strains were identical to each other. interestingly, two endemic type 027 strains differed from the other strains in 2 of the 7 regions. conclusion: mlva is a highly discriminatory genotyping method for c. difficile and is capable to subtype various crribotypes. mlva is also an important new tool to study the epidemiology of the emerging pcr-ribotype 027 strains. comparative study of clostridium difficile diarrhoea in elderly patients treated with moxifloxacin versus amoxycillin for lower respiratory tract infections l. mooney, m. wilcox (leeds, uk) fourth generation fluoroquinolones such as moxifloxacin have improved anti-anaerobic activity. consequently, these new agents could induce c. difficile infection (cdi) by inhibition of 'protective' anaerobic flora. recent reports have suggested such an association. however, further studies are warranted to determine the risk of cdi in elderly in-patients treated with these agents, and notably where exposure to cd is measured/ controlled. methods: we prospectively investigated the propensity of moxifloxacin (mox) or amoxycillin/macrolide (aml/mac) to induce cdi when used to treat lower respiratory tract infections (lrtis) in elderly in-patients, using a 4-ward, crossover design (18 months total). patients prescribed mox or aml/mac were monitored for gastrointestinal symptoms. diarrhoea was assessed as due to cd, viral or other cause. relevant clinical data were collected. concurrent epidemiological surveillance was also performed to determine environmental exposure to cd. results: 128 patients were studied, 24 receiving mox and 104 had aml/mac. univariate analysis indicated that there was no significant difference between mox and aml/mac patients in gender, age (83.6 vs 86.6 mean years, respectively), or duration of hospitalisation (total, prior to and post diarrhoea). duration of antibiotic therapy did not differ significantly between mox and comparator patients (either total days or days before diarrhoea onset). there was a significant association between mox and overall risk of diarrhoea. however, there was no significance between mox treatment and cd, viral or other cause of diarrhoea. risk factor analysis to inform on possible confounders was performed. initial epidemiological survey results indicate that there was no change in environmental exposure levels to cd on each hospital ward. molecular typing of all clinical and environmental isolates of cd is ongoing. conclusions: although recent reports have highlighted a risk of cdi associated with fluoroquinolones (and increased age), none have specifically studied hospitalised elderly populations prospectively and controlled for exposure to cd. diarrhoea occurs relatively frequently after antibiotic therapy in the elderly. mox was associated with an increased rate of diarrhoeal symptoms, but causes other than cdi explained this association. mox treatment was not significantly associated with cdi when compared with amox/mac treatment for lrti in elderly in-patients. prevalence and association of macrolidelincosamide-streptogramin b resistance with resistance to moxifloxacin in clostridium difficile strains isolated from symptomatic adults and children hospitalised in two university hospitals in warsaw h. pituch, d. wultanska, g. nurzynska, p. obuch-woszczatynski, f. meisel-mikolajczyk, m. luczak (warsaw, pl) objectives: clostridium difficile is the main aetiological agent of nosocomial diarhoea. clindamycin, penicillins, and cephalosporins have been associated with cdad. however, several case reports of fluoroquinolone-associated c. difficile diarrhea have been published. c. difficile strains usually exhibits susceptibility to metronidazole, and vancomycin. we describe prevalence and association of macrolide-lincosamide-streptogramin b (mlsb) type resistance with resistance to moxifloxacin of c. difficile strains isolated from adults and children. methods: eighty-three c. difficile strains recovered from adults and children hospitalised in two university hospitals were investigated (hospital 1: adults n = 38, and children n = 30; hospital 2: adults n = 15). toxin types were determined by commercial test for toxin a and cytotoxicity test for toxin b. tcda, tcdb were detected by pcr. mics of erythromycin, clindamycin, moxifloxacin, vancomycin and metronidazole were determined by e-test (ab biodisk, sweden). the ermb gene was detected by pcr. results: sixty-seven (81%) c. difficile strains were toxigenic. among these, 44 were a+b+, and 23 were a-b+. all 83 strains were susceptible to vancomycin and metronidazole. high level resistance to erythromycin, clindamycin and moxifloxacin was found in 46%, 42%, 27% of the tested strains, respectively. twenty-one c. difficile strains harboured high level resistance to erythromycin, clindamycin and moxifloxacin, simultaneously. among these, all were a-b+ and were isolated from adults, only. twenty-one of the macrolide-lincosamide-streptogramin b (mlsb)-resistant a-b+ strains carried the erythromycin resistance methylase gene (ermb). conclusion: resistance against clindamycin, erythromycin and moxifloxacin among polish a-b+ c. difficile strains was very frequent. fluoroquinolone resistance is associated with resistance to mlsb antimicrobials. we suggest that increasing use of fluoroquinolones is selective pressure for clonal dissemination of a-b+ c. difficile strains. fluoroquinolones use is a strong risk factor for cdad in our hospitals. acknowledgement: this work was supported by the ministry of scientific research and information technology, grant no. 2 p05d 074 27. national surveillance to the incidence of clostridium difficile-associated diarrhoea in the netherlands s. paltansing, r. guseinova, r. van den berg, c. visser, e. van der vorm, e.j. kuijper (leiden, amsterdam, nl) objectives: the recent outbreaks of clostridium difficileassociated diarrhoea (cdad) due to the new emerging pcrribotype 027, toxinotype iii strains has renewed the interest of cdad as an important nosocomial infection. to determine the incidence of cdad in the netherlands, we conducted a prospective surveillance study in 13 hospitals in the netherlands. clinical microbiology and infection, volume 12, supplement 4, 2006 methods: from may 1st to july 1st of 2005, 13 participating hospitals registered all patients diagnosed with cdad. a standardized questionnaire was devised to obtain patient information. faeces samples or isolated strains were sent to the reference laboratory at the lumc for culture and the presence of genes for toxins a and b (tcda and tcdb). pcrribotyping was performed according to the method of bidet and toxinotyping as described by rupnik et al. results: routine methods to diagnose cdad in 13 laboratories included combinations of cytotoxicity tests (59%), enzymeimmunoassays (56%) and culture of toxinogenic strains (31%). in total, 91 patients with cdad were reported. the overall incidence (median) of cdad was 17 for 10,000 patient admissions and varied from 1 to 75. of 91 patients with cdad, 41% was community acquired. the median age of 54 patients with nosocomial acquired cdad was 59 years. of 54 patients with cdad, 7 (13.9%) died during the study period. at least 41 different pcr-ribotypes could be recognized among 91 strains. type 027 was identified in 9 patients from 1 hospital. toxinotyping revealed the presence of at least 7 different types. of 91 strains, 87% were tcda+/tcdb+, 10% tcda-/tcdb-and 3% tcda-/tcdb+. conclusions: the incidence of cdad in the netherlands is lower than reported in usa and canada, but varied considerably per hospital. the new emerging type 027 was found in 9 patients from 1 hospital with a high incidence of cdad (39 per 10,000 admissions). outbreak of clostridium difficile pcr-ribotype 027 toxinotype iii in harderwijk, the netherlands objectives: since 2002, several epidemics of clostridium difficileassociated diarrhoea (cdad) caused by c. difficile pcr-ribotype 027 toxinotype iii have occurred in usa, canada, and the uk. in april 2005, the first outbreak encompassing 45 patients was observed in a medium large hospital of 350 beds in the netherlands. the isolated 027 strain was completely resistant to erythromycin and ciprofloxacin. the patient characteristics, predisposing factors and outcome of cdad were studied. methods: a case-control study was performed in 45 patients and 90 at random selected controls without diarrhoea who stayed at the same department as the patients when the diagnosis of cdad was made. standardized questionnaires were designed to collect data from the patient records and all surviving patients were interviewed 3 months after the diagnosis. faeces samples were cultured for the presence of c. difficile and isolates were typed. results: the incidence of cdad increased from 4 per 10,000 patient admissions in 2004 to 81.5 per 10,000 admissions in 2005. between april and september 2005, 45 patients with cdad due to type 027 were identified. of 45 patients, 9 (20%) died of which 3 (7%) as a direct result of cdad. eleven (24%) patients experienced one or more relapses. the average age of the cases was 72 yrs, 42.2% of the patients was male. in a multivariate analysis, antibiotic use (or 15.3, p < 0.001), duration of hospital stay (cases 31 days, controls 13 days; p < 0.001) and tube feeding (or 3.5, p = 0.03) were found to be significantly associated with cdad. in particular, the use of ciprofloxacin (or 11.8, p < 0.001) and cephalosporins (or 5.8, p < 0.001) were associated. no association was found between the use of protonpump inhibitors and the risk of cdad. the use of erytromycin was significantly higher in cases (11.1%) than in controls (2.2%) in a univariate analysis (p < 0.041), but this relation was not significant in a multivariate analysis. conclusion: antibiotic use (especially ciprofloxacin and cephalosporins), duration of hospital stay and tube feeding were significantly associated with cdad caused by c. difficile type 027, toxinotype iii in the netherlands. we could not confirm the previously described relation between use of protonpump inhibitors and risk of cdad. clostridium difficile pcr ribotype 027, toxinotype iii in the netherlands objectives & methods: shortly after the reports in june 2005 of clostridium difficile pcr ribotype 027, toxinotype iii in england, this more virulent type was also detected in the netherlands. in response, the dutch centre for infectious disease control has undertaken measures to monitor and control the outbreak. c. difficile 027 guidelines for infection control and treatment were formulated, separately for hospitals and nursing homes. the leiden university medical centre serves as a reference centre for diagnostics and typing of c. difficile. laboratories are encouraged to send in samples for typing in case of a clear rise in the incidence in c. difficile, rapid spread, or several clinically suspect cases.organisation-based surveillance was set up: questionnaires are sent monthly to institutions with c. difficile associated diarrhoea (cdad) outbreaks to obtain information on incidence, c. difficile testing strategies, antibiotics use and control measures taken.measures taken in hospitals dealing with an outbreak of type 027 include: treatment of cdad with vancomycin in stead of metronidazole, emphasis on frequent and thorough cleaning and disinfection, isolation of all patients with diarrhoea until tested negative for c. difficile toxin, cohort isolation of cdadcases if individual isolation capacity is exceeded and strong restriction of certain antibiotics, including fluorochinolones. results: until november 1st, 2005, 224 samples from 23 institutions have been sent in for typing, resulting in 74 type 027 positives. epidemic spread of type 027 has been detected in 7 hospitals and one nursing home. furthermore, in retrospective studies in four hospitals isolated cases of type 027 were detected. it became clear that in one region with three hospitals, the cdad incidence had already risen in 2002, 2003 and 2004, respectively . unfortunately, no samples from that period were available for typing. in the hospitals with epidemic spread of type 027, a wide range in the monthly incidence of cdad was observed, from 50 to 114 per 10,000 admissions during the outbreaks. the incidence in the pre-epidemic period varied from 3 to 7 (see figure) . conclusions: the outbreaks in hospitals are difficult to control: most hospitals continue to have new cases for a long period, although the incidence is decreasing in several hospitals. fortunately, once a c. difficile 027 outbreak in a hospital is recognised, spread to other hospitals has not been observed. objectives: c. difficile is a major cause of antibiotic associated diarrhoea (aad) and colitis (c). the aim of this study was to determine the incidence of these infections in our hospital (450 beds), during a period of 6 months (march-october 2005) . methods: a number of 182 liquid stools from equal adult patients (mean age 61 y, m:110, f: 72) receiving broad spectrum antibiotics (especially cephalosporins) were plated in ccfa (oxoid) and anaerobic brucella agar (ba), after alcohol shock procedure. if the culture was positive, an immunochromatographic test was performed for toxin a (colorpactm toxin a, bd, usa). if the last test was negative, a rapid enzyme immunoassay was performed for toxins a+b (immunocard, meridian bioscience inc. cincinatti, ohio). results: c. difficile was isolated in 23/182 (12.6%) samples. seventeen men (pathological -p, pneumological -pn, surgical -s, urologic -u, outpatients -o, 7, 6, 2, 1, 1 respectivly) and 6 women (p:4, pn:1, o:1) harbored c. difficile in their intestin. twelve out of 23 strains (52%) produced toxin a, while the remaining (48%) produced toxin b. eleven patients had severe diarrhoea (7-10 days). one patient got endoscopic examination, which confirmed colitis findings. the two outpatients received oral cefuroxime in the preceding week of the positive culture. conlusions: (1) the incidence of c. difficile infections in this study is among these reported in international bibliography (12.6%). (2) since toxigenic b c. difficile strains were demonstrated in half cases, the use of the tests detecting both toxins a and b by clinical laboratories is recommended.(3) molecular technics application (e.g. pfge and ribotyping) will offer a better knowledge of c. difficile spread in our hospital. assay of the cytotoxicity of stool samples to cells in tissue culture is commonly considered the 'gold standard' for detection of c. difficile toxin. however the method is slow and therefore its use can result in delayed patient treatment and implementation of infection control measures. we undertook a comparison of two microtitre plate-based elisa kits (techlab c. difficile tox a/b ii and meridian premier toxins a & b) and three rapid immunoassay card kits (remel xpect clostridium difficile toxin a/b, meridian immunocard toxins a & b and techlab tox a/b quik chek) with an in-house cytotoxin assay. all samples tested had been referred for routine microbiological examination. toxin tests were done on unformed samples from adult hospital in-patients and bone marrow transplant recipients and on samples where c. difficile toxin testing was requested by the referring clinician. all kits were used according to manufacturers' instructions. three hundred and thirty three specimens were tested using all five kits and cytotoxin assay. sensitivities and specificities were calculated both (a) assuming the cytotoxin assay to be the 'gold standard' (universally correct) test and (b) taking a concensus view that any sample with at least two tests positive is truly positive. data are shown in the table below. overall, the microtitre plate-based elisa kits were more sensitive than the rapid immunoassay card kits. the cytotoxin assay was negative for seven samples that were positive by at least two other tests. thus the plate-based elisa kits were also more sensitive (but less specific) than the cytotoxin assay if consensus data was used to judge true positivity. we conclude that some immunoassay kits offer an acceptable alternative to cytotoxin assays for the detection of c. difficile toxin, allowing more rapid diagnosis. location of the enterotoxin gene in strains of clostridium perfringens associated with gastroenteritis objectives: clostridium perfringens type a is a common cause of food poisoning and is also associated with non-food borne gastroenteritis including antibiotic associated, infectious and sporadic diarrhoea. the disease symptoms are due to an enterotoxin produced when the organism sporulates in the human small intestine. the c. perfringens entertoxin gene (cpe) has been shown to be located either on the chromosome or on one of two large plasmids and it is generally accepted that c. perfringens strains associated with food poisoning have a chromosomal cpe gene whilst strains isolated from non-food borne diarrhoea have a plasmid encoded cpe gene. spores from strains possessing a chromosomal cpe gene have been found to be far more heat resistant than spores from strains with a plasmid encoded cpe gene. heat resistant spores are more able to survive the cooking process and go on to cause food poisoning, thus explaining why most food poisoning strains have been found to have chromosomally located cpe genes. the purpose of this study was to determine the location of the cpe gene in a range of c. perfringens strains from the uk, including those from both food borne and non-food borne illness. method: a multiplex pcr assay described by miyamoto et al., (2004) was used to determine the location of the cpe gene in 107 strains of c. perfringens isolates associated with food borne illness and 16 strains associated with non-food borne illness. results: by multiplex pcr assay 35% of c. perfringens strains associated with food borne outbreaks in the uk were found to have a plasmid encoded cpe gene. these findings have not been described before. all strains associated with non-food borne illness had the cpe gene located on one of two plasmids, as anticipated. conclusions: a significant number of food borne outbreaks of c. perfringens food poisoning were found to be caused by strains of c. perfringens carrying a plasmid encoded cpe gene. since strains of c. perfringens with a chromosomal cpe and plasmid cpe genes have different physiological characteristics this may have a profound impact on their mode of transmission. references miyamoto, k., wen, q. and mcclane, b. a. (2004) multiplex pcr genotyping assay that distinguishes between isolates of clostridium perfringens type a carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an is1470-like sequence, or a plasmid cpe locus with an is1151 sequence. journal of clinical microbiology 42, 1552-1558. novel multiplex-pcr method for simultaneous detection of clostridium difficile toxin a and toxin b and the binary toxin (cdta/cdtb) genes applied on a danish cohort k.e.p. olsen, s. persson (copenhagen, dk) objectives: a new multiplex pcr method was developed for the detection of the clostridium difficile toxin genes: tcda, tcdb, cdta and cdtb. this method was applied on 210 clostridium difficile strains isolated from 175 danish hospitalised patients with diarrhoea in the period from april to october 2005, in order to investigate the present toxin profiles and their correlation to sex and age. method: a 5-gene multiplex pcr method was developed for the simultaneous amplification of the four clostridium difficile toxin genes tcda, tcdb, cdta, cdtb and 16s rdna as an internal positive control. template dna was prepared from plate grown bacterial colonies by a simple boiling procedure, and amplicons were visualized by standard gel electrophoresis. results: three different toxin profiles were detected in the danish cohort: 43 tcda+, tcdb+, cdta+/cdtb+; 107 tcda+, tcdb+, cdta-/cdtb-and 24 non-toxigenic tcda-, tcdb-, cdta-/ cdtb-. the prevalence of the binary toxin genes in this study was 25% of the clinical isolates.more than half of the strains (62%) were isolated from the elderly part of the population (>59 years), and 74% of these strains displayed the tcda+, tcdb+, cdta+/cdtb+ profile. of the non-toxigenic strains, 83% of the patients were females. one fourth of the strains isolated from children under 2 years of age were non-toxigenic. in four patients, two different toxin profiles were obtained from independent faecal samples. conclusion: this method offers a one-step, rapid and specific identification of clostridium difficile toxin genes. this specific toxin profiling allows an evaluation of the pathogenic potential of the isolated clostridium difficile and surveillance of emerging toxin profiles. further studies of the isolated toxigenic clostridium difficile strains will include gene deletion analyses of the tcda and the tcdc (toxin regulating gene) which independently have been observed to cause enhanced pathogenicity. prevalence of clostridium difficile-associated diarrhoea in hospitalised patients with nosocomial diarrhoea in university of medical sciences hospitals, tehran, iran objectives: this study was aimed at determining the prevalence of clostridium difficile associated diarrhoea in hospitalized patients with nosocomial diarrhoea at three university hospitals in tehran from december 2002 to august 2004. methods: during the study period, the stool samples of 1500 hospitalized patients with nosocomial diarrhoea were cultured and tested by stool cytotoxin assay, toxigenic culture and also 650 of the samples were examined by enzyme immunoassay. results: in 159 (10.9%) of samples c. difficile grew and 108 stool samples (prevalence: 7.2%) were toxin positive by stool cytotoxin assay, enzyme immunoassay or toxigenic culture. there were no significant relationships between c. difficileassociated diarrhoea and sex and age of patients. the results of the present study showed that among requested samples the highest percentage of c. difficileassociated diarrhoea was observed from the transplantation department (13.9%), followed by icu and paediatric section. objectives: the prevalence of toxigenic clostridium difficile (c. difficile) has been reported about 70-80% in korea. toxin a(-)/ toxin b(+) variant c. difficile strain is also important in nosocomial c. difficile infection. however, characterization of clostridial toxin (toxin a, toxin b) had not been studied. methods: we used pcr for toxin a and toxin b genes in 260 c. difficile isolates from patients admitted in three tertiary hospitals during january to december, 2004. primers for toxin a genes were nk1-nk2, nk2-nk3 and nk9-nk11 and toxin b gene was nk104-nk105. results: toxin a and toxin b positive rates using nk1-nk2, nk3-nk2 and nk9-nk11 were concordant and ranged from 62.1% to 90.9% in 3 hospitals. the proportions of non-toxigenic strains were 10-40%. however, we could differentiate toxin a(-)/toxin b(+) variants using nk9-nk 11 primers. the proportion of toxin a(-)/toxin b(+) c. difficile variants were 43.0%, 9.7% and 36.4% in 3 hospitals respectively. objective: administration of antibiotic drugs has long been known to cause alterations in the gut ecosystem. in some patients, these alterations may create a niche that allows the overgrowth of some pathogens such as clostridium difficile, the main causative agent in nosocomial infectious diarrhoea. a predictive tool to assess the risk of development of clostridium difficile, would be of utmost clinical relevance. it remains to be determined whether specific patterns in pre-existing gut microbiota can predict the risk of onset of clostridium difficile, upon initiation of antibiotic treatment. using samples from 87 subjects enrolled in a previously published clinical study on antibiotic-associated diarrhoea (aad), we investigated the potential relationship between their dominant faecal microbiota and the subsequent development of clostridium difficile when subjects received antibiotics. methods: temporal temperature gradient gel electrophoresis (ttge) was used to assess dominant species distribution in gut microbiota. each electrophoregram was digitised from the migration distances and a regression model [partial least square-discriminant analysis (pls)] was built to investigate the correlation between pre-treatment dominant faecal microbiota and the acquisition of clostridium difficile during antimicrobial chemotherapy. results: this pls model could explain 46% of the subsequent onset of clostridium difficile. this result supports the concept of ''permissive'' flora with preliminary data focusing on clostridium coccoides-phylogenetic group. conclusion: to our knowledge it is the first time that dominant faecal microbiota is found to heighten susceptibility to the subsequent onset of clostridium difficile upon initiation of antibiotic treatment. these findings insinuate that strategies reinforcing the control of dominant faecal microbiota at homeostasis would be of clinical relevance. this study has been partially financed by biocodex laboratories. objectives: selective therapy of c. difficile diarrhea (cdd) requires the reduction of pathogen counts in the colon, but spare the normal flora. to determine if par 101 is selective for cdd, serial stool samples were collected at study entry, at day 10, and weekly x5 during the conduct of a phase 2a study of cdd treatment. methods: patients (n = 32) were randomized to receive 50, 100 or 200 mg twice daily of par 101 for 10 days. no prior therapy was given to 24 patients; 8 receive 1 or 2 doses of standard therapy. as treatment controls, 7 additional patients were treated with vancomycin 125 mg qid for 10 days. five well persons donated stools as normal flora controls. fresh stool samples were cultured 10-2, 4, 6, 8 for c. difficile vegetative and spore forms; faecal filtrates were tested for cytotoxin b by cell assay. strains were characterized by tcda/b, ermb, cdta/b pcr and by ribotyping. at study entry and day 10, aerobic and anaerobic faecal flora cultures, diluted 10-3, 5, 7, 9, were examined for major floral shifts. since bacteroides group organisms are ubiquitously present and cultivable, this genera was selected as a indicator of the integrity of the microbial flora. results: at study entry, mean log 10 cfu + sd vegetative counts of c. difficile (all par 101 patients) were 6.8 + 3.6, range 2-10.95; at day 10, with the exception of one patient receiving 50 mg, all other patients had c. difficile quantitative counts reduced < 2 log10/gm faeces. vancomycin was similarly effective. at study entry, bacteroides group counts were < 3, 3-8, & 8.5-10 log10 cfu/gm in~1/3 each of patients. all normal stools showed complex, multi-genera in high counts, with 4-5 bacteroides group species > 11 log10 cfu/g. mean + sd of log10 cfu of bacteroides group counts/g feces wet weight at study entry and day 10 for 50 mg/day (n = 10) were 6.6 + 2.8/ 8.2 + 2.6 (p = 0.11, wilcoxon matched pairs signed-ranks test, 2 tailed); for 100 mg/day (n = 8) were 6.6 + 2.8/6.3 + 2.5 (p = 0.44); for 200 mg/day (n = 11) were 7.0 + 2.9/7.3 + 3.1 (p = 0.56); and for vancomycin (n = 7) 7.4 + 2.7/3.6 + 1.9 (p = 0.03). conclusion: patients with cdd have variably impaired normal flora. par 101 was effective in all dosages in eradicating c. difficile. a dose-dependent reduction in bacteroides counts was not observed. vancomycin significantly reduces bacteroides counts during cdd treatment. par 101 is effective against c. difficile in-vivo, and is relatively sparing of the normal flora. results: the three rt pcr assays were able to detect all enterovirus strains in cell culture supernatants. however the detection limit of the mgb rt pcr was 1 to 2 log10 more sensitive in 14 out of 16 dilutions assays of vc supernatants compared to the rab and ver rt pcr. all ver and mgb negative csf were vc negative. thirty-two csf specimens from 68 patients suspected of viral meningitis were positive by all rt pcr (47.1%), whereas only 8 were found positive by vc (11.8%). the rab rt pcr failed to detect 4 csf confirmed positive by vc (1 echo 6 and 3 non typable ev). among samples positive by rt pcr, sensitivity of ver, mgb and rab was respectively 100%, 100% and 71.9%. conclusion: in our laboratory, mgb rt pcr has a good correlation with ver rt pcr whereas rab rt pcr is less sensitive especially for the detection of echovirus 6. the mgb rt pcr seems to be the most sensitive of the 3 rt pcr. further studies, including more ev strains should help to precise the sensitivity of this assay. a. dalwai, s. ahmad, e. hussein, a. pacsa, w. al-nakib (kuwait, kw) objectives: enteroviruses generally share tissue tropism and present with overlapping disease spectrum, however certain enteroviruses may be over represented in certain diseases than others. coxsackievirus a7 though has been reported to cause several diseases such as febrile illness, herpangina, aseptic meningitis and acute flaccid paralysis, the frequency was very low. the study aimed to determine the prevalent enteroviruses causing non-specific febrile illness, aseptic meningitis, encephalitis, neonatal disease and myositis, in kuwait. it also aimed to study the association between a certain enterovirus and a particular disease and its severity. methods: diagnosis of enteroviral infection was based on detection of enteroviral rna by semi-nested rt-pcr of a portion of the 5'utr of the enteroviral genome followed by southern hybridization with an enterovirus specific probe to confirm the results. the enterovirus was genotyped by sequencing of the 5'utr, the vp4 and a portion of the vp2 encoding regions, and the sequence was analysed by blast analysis, clustalw alignment and phylip phylogenetic analysis package. results: enteroviruses were the only etiological agents detected in 24% (217) of 920 disease cases investigated. coxsackievirus a7 was identified to be the second most predominant enterovirus (24%; 44 of 187 cases genotyped) associated with disease, after only echovirus 9 (39%; 70/187). although identified in all the diseases investigated, coxsackievirus a7 occurred less frequently in cns disease cases (17%; 25/147) than in febrile illness cases (43%; 15/34). in a preliminary study, it was also predominantly detected in 66% (4/6) of myositis cases. the 5'utr of this virus showed 96% homology with that of coxsackievirus a7 prototype strain (parker strain) whereas the vp4 and the adjoining region showed greater homology to human enterovirus b genotype sequence. conclusions: coxsackievirus a7 was determined to be an emerging enterovirus associated with different diseases in kuwait. it was frequently represented in mild febrile illness and myositis cases than in cns disease suggesting that the isolate might be less neurovirulent. molecular analysis suggests that the isolate might have emerged due to recombination between coding and non-coding segments of coxsackievirus a7 and human enterovirus b group genomes. acknowledgement: supported by research administration project grants mi 04/01, ym 03/02 and college of graduate studies, kuwait university. the new proposed enterovirus type 75 is causing meningitis in spain introduction: several new proposed enteroviruses (ev) have been recently described, including the named ev75 [1] . a total of 8 isolates of this serotype were identified from 1974 to 2000 in america, africa or asia associated mainly with acute flaccid paralysis or unspecified disease. objective: to determine if this new serotype circulates in spain and what type of disease produces. methods: a total of 300 ev isolates coming in 2005 to the spanish enterovirus reference laboratory were studied both by micro neutralization assays and by typing pcr [2] . in the isolates in which ev75 was suspected by the mentioned methods complete vp1 gene was amplified and sequenced with specific designed primers. results: four isolates from two different regions of spain were identified as ev75 (more than 80% of homology with the published sequences). three of them corresponded to aseptic meningitis in children and were isolated from csf. discussion: the present work demonstrates that this new proposed virus circulates also by europe and is associated to aseptic meningitis. till the moment it seems that is represented in a minor proportion (4/300 studied), however the possibility of spreading of this viral infection should be considered, as evs may behave in that way, as previously have been demonstrated [3] . objectives: rotavirus is the most important cause of severe gastroenteritis in infants and young children through the world and is responsible of 500,000 deaths annually, mostly in developing countries. therefore, development of rotavirus vaccine is a high priority. rotavirus strains with g1 types account for the majority of the diarrhoea episodes. recently, a monovalent g1 attenuated rotavirus vaccine was licensed in mexico. in view of a hypothetical introduction of such vaccine in europe, we investigated the variability over time of vp7 antigenic genes of g1 rotavirus strains in our area. methods: fifty strains were selected from a total of 780 g1 strains obtained from children of less than 5 years of age hospitalised with acute gastroenteritis at the ''g. di cristina'' children's hospital of palermo in the period 1986-2004. the selected strains were genotyped by rt-pcr and 35 of them were submitted to vp7 gene sequence analysis. results: all but one of the 50 strains were genotyped as g1p(8). the vp7 sequences of 35 of them were distributed into lineages i and ii. lineage i included 14 strains from 7 different years in the range 1986-2004. lineage ii included 21 strains from 9 different years in the range 1989-2004. the degree of similarity among the nucleotide sequences of italian strains in each lineage were comprised between 94% and 100%. an alignment of the deduced amino acid sequences showed major lineage specific amino acid changes in the variable antigenic regions with respect to the reference wa strain. conclusions: sequence analysis indicated that in palermo there was co-circulation of g1 strains belonging to two different lineages. overall, the g1 strains showed a high degree of similarity inside each lineage and shared specific amino acid modifications. the antigenic differences between circulating strains might permit them to escape neutralization and persist in the infantile population. our results suggest that rotavirus strains belonging to the two g1 lineages should be both included in a rotavirus vaccine preparation. epidemic spread of recombinant noroviruses with four capsid types in hungary objectives: noroviruses (''winter vomiting diseases'') are the predominant etiological agent in hungary and common pathogen worldwide in outbreaks of gastro-enteritis in humans. noroviruses are genetically diverse group of viruses with multiple genogroups (gg) and genotypes. more recently, naturally occurring recombinant noroviruses were identified. these viruses had a distinct polymerase gene sequence (orf1, designated ggiib/hilversum) and were disseminated through waterborne and food-borne transmission in europe. our aim was to characterize these emerging recombinant noroviruses causing outbreaks of gastro-enteritis in hungary. methods: stool and rna samples -from norovirus outbreaks between january 2001 and may 2005 -containing ''ggiib/ hilversum polymerase'' (ggiib-pol) were selected for analysis of the viral capsid region (orf2) by reverse transcriptionpolymerase chain reaction (rt-pcr) followed by sequence-and phylogenetic analysis. results: forty (12.6%) of 318 confirmed norovirus outbreaks were caused by the new-variant lineage with the ggiib-pol. viral capsid region was successfully characterized in 24 ggiibpol outbreaks. four different recombinants were detected with capsids of hu/nlv/ggii-3/mexico/1989 (n = 10, 41.7%), hu/ nlv/ggii-2/snow mountain/1976 (n = 9, 37.5%), hu/nlv-1/ggii/hawaii/1971 (n = 4, 16.6%) and hu/nlv/ggii-4/ lordsdale/1993 (n = 1, 4.2%). interestingly, outbreaks caused by recombinant ggiib-pol strains mostly associated with outbreaks among children (47.5%) and had non-winter seasonality. conclusions: epidemic spread of emerging multiple recombinant norovirus strain ggiib-pol were detected in hungary that became the second most common norovirus variants -next to the endemic ggii-4/lordsdale virus -causing epidemics of gastroenteritis in the last 4.5 years. the respiratory infections are the most common diseases in the world being the origin of a great morbidity and mortality especially in infants and elderly. (1) human metapneumovirus (hmpv) was first described in dutch children with acute respiratory tract infections (artis) in june 2001. (2) very limited studies data are available from tropical and developing countries. we sought to determine the role of hmpv in upper and lower respiratory tract infections in cuban patients and correlated the presence of virus with clinical characteristics of the disease. between 1 october 2002 to 30 september 2003 clinical samples received from the national surveillance program of artis at the national reference laboratory of respiratory viruses, for virological study, were used to detect hmpv by rt-nested pcr, amplifying a conserved fragment of 170 nucleotides in the polymerase gene. we found rna hmpv in 9.6% of samples from the patients with artis. 44.4% of individuals who tested positive for hmpv were under 12 months of age. patients with evidence of hmpv had symptoms consistent with either upper or lower respiratory tract disease or both. 66.6% of hmpv positive individuals were detected during august-october (table1). the results of this preliminary study shows that hmpv is present among cuban patients with arti. constitute the first report of the frequency of hmpv infection in a non-preselected group of cuban patients with ages ranged from 3 months to 60 years old. it should be noted that this is the first report of hmpv infection in central america and in the caribbean region, further confirming the worldwide distribution of the virus (3) (4) (5) . detection of human metapneumovirus in paediatric nasopharyngeal aspirates by a taqman minor groove binder probe assay: a one-year prospective study in belgium w. verstrepen, p. bruynseels, a. de smet, a. mertens (antwerp, be) objective: human metapneumovirus (hmpv) has a relative high incidence in acute respiratory infections in children but is difficult to isolate in culture. the aim of the study was to decrease the number of undiagnosed viral respiratory infections in our hospital by means of a taqman minor groove binder (mgb) probe assay. methods: from october 2004 to september 2005 a total of 1387 nasopharyngeal aspirates from children presenting at our paediatric facility were analysed. rna extracts from specimens negative for rsv, parainfluenzavirus and influenzavirus with an (in) direct immunofluorescence assay (ifa) were subjected to a taqman mgb probe assay in parallel with a previously published taqman assay. results: of the 1387 specimens, 371 (27%) were positive by ifa for either rsv (226), parainfluenzavirus (47), influenzavirus a (83) or influenzavirus b (15). hmpv was detected in 83 (8.4%) of the remaining 988 specimens subjected to the newly developed pcr. of the patients with a positive hmpv assay, 46/52 (88.5%) presented with respiratory symptoms. 72% of the positive specimens were from children less than 2 year as compared to only 6% from children older than 5 years. viral load was highest in children less than 1 year. a prominent seasonal variation was noted since more than half of the positive specimens occurred during the months march and april. there was no significant difference in the proportion nor viral load of positive specimens from ambulatory patients, patients admitted to a general ward or patients requiring intensive care. as compared to the published taqman assay, diagnostic sensitivity and specificity were 97.6% and 99.9% respectively, whereas ppv and npv were 98.8% and 99.8%. method comparison (nccls guideline ep-9a) failed to demonstrate a significant difference between both assays when the threshold cycle (ct) was between 22 and 41. strongly positive specimens (ct < 22) were associated with a lower ct using the published taqman assay. however, the new taqman mgb probe assay appeared to be more sensitive for weakly positive specimens (ct > 41). conclusion: the number of viral respiratory infections confirmed in our hospital was substantially increased by means of the hmpv taqman mgb probe assay. the new assay is a reliable alternative to the previously published taqman assay for detection of hmpv in nasopharyngeal aspirates. nucleic acid sequence based amplification and molecular beacon detection for the real-time identification of respiratory syncytial virus in paediatric respiratory specimens r. manji, f. zhang, c. ginocchio (lake success, us) background: respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract infection in infants and young children, with bronchiolitis and pneumonia being the major clinical manifestations. the rapid diagnosis of rsv infections is of central importance for individual patient management (rational use of antibiotics and antiviral agents), hospital infection control and monitoring epidemiological disease patterns. this study included a technical validation and a retrospective clinical evaluation of a real time nasba assay for the detection of rsv a and rsv b in paediatric respiratory samples. methods: samples tested included: dilution panels of in vitro transcribed rna, local rsv isolates, isolates of common respiratory pathogens, and frozen respiratory specimens (nasopharyngeal aspirates, washes or swabs) from 231 children (age range: 5 d to 2 yr) who were evaluated in the paediatric emergency department for respiratory disease. nucleic acid (na) isolation, amplification and detection were performed using the nuclisens easyq basic kit and nuclisens easyq rsv a+b reagents (biomérieux). specimen nas and a rsv specific internal rna control (ic) were co-extracted using nuclisens magnetic extraction reagents and the nuclisens minimag instrument (biomérieux) and co-amplified using a single rsv specific primer pair. included in the reaction were a rsv specific molecular beacon (5'-fam) and an ic specific molecular beacon (5'-rox). target amplification and continuous monitoring of emitted fluorescence were performed using a nuclisens easyq analyzer (biomérieux). results were compared to direct immunofluorescence (dfa) and/or viral culture using r-mix cells (diagnostic hybrids, oh). results: the limit of detection for rsv was 2 rna copies/rxn and the 100% detection rate was 5 copies/rxn. the assay was 100% specific for rsv with no cross reactivity to other respiratory pathogens. the nasba assay detected 10% more positive specimens than dfa and 44% more positive samples than vc. the npvs of the assays were: nasba 94.6%, dfa 90.8% and vc 78.0%. the nuclisens easyq rsv assay demonstrated superior sensitivity to both dfa and viral culture for the detection of rsv a and b from respiratory specimens. the assay was easy to use, required minimal hands on time (1 hr) and a faster time to results as compared to rapid culture (4 hr vs. 24-72 hr). respiratory syncytial virus (rsv) is a major cause of acute lower respiratory tract infection in infants and young children. it has previously been shown that hrsv isolates can be divided into two antigens groups a and b. the g protein is the most divergent both between and within the two subgroups and appears to accumulate amino acid changes with time, suggesting evolution under selective pressure. our knowledge of the molecular epidemiology of rsv has so far been based mainly on studies done in the developed world with e temperate climate. very limited epidemiological data are available from tropical and developing countries, where rsv infections may follow a different pattern. in this report we examine the molecular epidemiology and evolutionary pattern of the g protein of both subgroups a and b rsv through consecutive epidemics in cuba. sixthly four nasopharyngeal swabs were collected from children under 1 years of age with respiratory disease to different hospitals in cuba between 1994 and 2000, to examine the molecular epidemiology and evolutionary pattern of the g protein of rsv. all samples collected from 1994 to 1999 were rsv subgroup a; however both subgroups co-circulate during 2000. the cuban isolated from 1994 to 1996 showed a great homogeneity between them and were resemble to an ancient strain (long) with only five nucleotide differences, this also occur in 1998 and 2000 with two strain. furthermore was detected different size of g protein (297 or 298 for rsv a and 295 for rsv b) due to change in stop codon used he genetic homogeneity of the cuban isolates (1994) (1995) (1996) and their resemble to an ancient strain such as long was an unusual finding in our country. in both subgroups was observed the predominance of strains with almost similar sequences. phylogenetic analysis for subgroup a strains showed that strains were cluster in different genotypes with virus isolated in different geographic regions. both subgroups co-circulated during 2000 and clustered whit south african strains that circulate during at the same time. point mutations in respiratory syncytial virus detected by lightcycler pcr and melting curve analysis u. germer, l. nielsen, k. boye, h. westh (copenhagen, dk) objective: the objective was to analyse rsv real-time pcrpositive isolates from clinical samples, which appeared to belong to three different groups according to melting temperature (tm) of the amplicons. the analysis was done according to genotypic and phenotypic difference and related to geographical distribution. materials and methods: 322 nasopharyngeal aspirates were collected from children with respiratory distress in the city of copenhagen. viral rna extracted using the magnapure lc automated extraction system was amplified in a real-time rt-pcr previously described (1) . five samples from each of the three groups with different tm's were selected for bidirectional dna sequencing using the rsv primers. sequences were analysed using chromas lite version 2.3. results: a total of 322 clinical samples were analysed. 170 (53%) of the 322 samples were positive and 152 (47%) were negative for rsv. three distinctive groups with different tm's could be identified from the melting curve analysis. group 1 (n = 91) had a tm with a median of 60.9°c, group 2 (n = 59) and 3 (n = 20) had lower tm's with a median tm of 58.7°c and 56.8°c respectively. sequence analysis of amplicons showed that the difference in tm was due to differences in genotype between the three groups. genotype 1 and 3 were closely related, differing only in two nucleic acids in position 12525 (c to t) and 12564 (a to t). both were silent mutations. only position 12564 is targeted by the probe. genotype 1 and 3 were both blasted to a complete genome sequence of respiratory syncytial virus subgroup a (genbank rsu39661) with the highest identity score for genotype 1. genotype 2 sequences were blasted to human respiratory syncytial virus mutant cp52 subgroup b (genbank af013255). geographical analysis showed a higher prevalence of the mutant strain (genotype 3) in the northern areas of the greater copenhagen area compared to central, southern and western areas (p = 0.01). conclusion: we found three genotypes of rsv according to the tm of the pcr product. two of the genotypes were closely related with only two point mutations and the same phenotype. genotype 3 was mainly found in clinical isolates from the northern part of copenhagen, suggesting a local epidemic spread. objectives: biomerieux is developing a real-time nasba assay to detect influenza a and b rna in different kind of respiratory clinical samples, by using the nuclisens ò easyq basic kit in combination with specific primers and molecular beacons. methods: 89 nasal/throat swabs in transport medium from hospitalised children (0-16 yrs from edouard herriot hospital, lyon, france) were used for this evaluation. influenza rna is isolated using the nuclisens ò minimag extraction. an internal control is added to the sample prior to nucleic acid extraction. the assay is designed to detect in a single tube, using a three-label approach, the internal control and both influenza a and influenza b rna. amplification reactions were performed in a nuclisens ò easyq analyser allowing real-time detection. the results of the clinical samples were compared to cell culture results. results: among 89 swabs tested, real-time nasba detected 10 (11.2%) samples for influenza a and 2 (2.2%) samples for influenza b. comparatively, by cell culture only 5 (5.6%) samples were identified as influenza a and non as influenza b. interestingly, 1 influenza a positive sample identified by cell culture was found negative in real-time nasba. conclusions: the data showed that nuclisens ò easyq influenza a/b assay detected 50% more influenza a virus than cell culture method. moreover, real-time nasba detected 2 influenza positive samples, which were not detected by cell culture. with this assay a qualitative detection of influenza a and influenza b viruses in a single reaction can be done within 3 hours. it provides a valuable alternative to cell culture method for the clinical management of patients with influenza infections. results: 299 patients have developed mumps meningitis and 17 patients were diagnosed with mumps meningoencephalitis. age limits were from 3 to 17 years and sex ratio m/f was 7/ 4.clinical manifestations involved fever (95%), stiff neck (92%), nausea and vomiting (89%), headaches (86%), photophobia (73%) and neurological manifestations such as: equilibrium disorders and drowsiness (6%), convulsions (0.17%), cerebellum syndrome (0.12%). meningeal symptoms have occurred shortly after parotiditis in 94% of cases and before parotiditis in 3% of cases; the other cases have evolved without parotid swelling. other localizations of the mumps infection were: parotiditis (97%), pancreatitis (61%), submaxillitis (40%) and orchitis (4%). lumbar puncture yields csf containing between 130 and 2830 wbc/mm3. the predominating cells were usually lymphocytes, but 12% of the patients have polymorphonuclear leukocyte predominance at the first puncture. protein levels are normal to mildly elevated in all cases and hypoglycorrachia was founded in 10% of the patients. therapy for mumps meningitis was symptom-based (analgesics and antipyretics) in 67% of cases and glucocorticoid therapy in 33% of cases. conclusions: (1) neurological involvement in mumps occurred in 13.32% of cases; (2) men are afflicted two times more often as women, but the age distribution is the same as for uncomplicated mumps; (3) mumps meningitis was the only localization of the mumps infection in 3% of cases. mumps is acute generalized infection occurs primarily in schoolaged children and adolescents. most prominent manifestation of mumps is swelling and tenderness of salivary glands especially parotid gland. uveitis is a rare manifestation of mumps. here we present a mumps case complicating with uveitis. 26 years old paediatric nurse was admitted to our emergency department because of headache and malaise. on physical examination bilateral parotid enlargement was noticed. opthalmology consulatation revealed anterior uveitis. local prednisolon and cyclopentholate treatment were prescribed. lumbar puncture revealed lymphocytic pleocytosis without hypoglychorachea and elevated protein levels. mumps igm was found positive. differential diagnosis made with other viral infections and sarcoidosis. her headache diminished day after the hospitalisation. uveitis responded very well to local therapy and patient got well in 2 weeks. clinical and epidemiological aspects of a measles epidemic, bucharest, romania results: there were 108 cases; sex ratio m/f:58/50. the mainly affected age group is under 13 months (38.9%) followed by 13 months-2 years (23.15%), 2-7 years (22.22%), > 16 years (10.18%) and 7-16 years (5.55%). 70.37% cases were hospitalacquired (mostly in paediatric clinics), 3.7% were communityacquired; in 25.9% cases the source was unknown. the most common clinical features were fever (100%), rash (100%), conjunctival hiperemia (78.7%), cough (70.37%), micropoliadenopatia (64.8%), diarrhoea (39.81%). pulmonary complications were described in 62.3% of cases; 22.38% of them were bacterial pneumonia, 77.62% were viral pneumonia. in 20.37% of cases we diagnosed acute stomatitis, in 12.96% bacterial conjunctivitis; in 14.81% of cases -otitis; in 1.85% of cases -pharingitis, and in one case (0.92%) -urinary tract infection. 7.4% of the patients were previously diagnosed and treated for pulmonary tb. all cases were confirmed serologically through detection of specific igm antibodies. 25 patients (23.15%) had severe clinical forms of measels. the evolution was good in all cases. conclusion: 1. this year in the south-east part of the country, evolves a measels epidemic with different features comparing to the previous one (1997) (1998) we investigated the recombinant proteins np and hn to develop new antigen with useful properties for applied in elisa test systems. methods: significant antigenic epitopes of nucleoprotein (np) and haemagglutinin (hn) measles virus strain edmonston were generated by computer analysis. using standard geneengineering techniques was evaluated two fusion peptides np and hn consist from only linear t-cell antigenic determinants. the virus-neutralization activity of hyperimmune serum on recombinant proteins was determined by plaque reduction neutralization test (prn). the level of specific igg in serum to genotypes a, d4, and d6 of measles virus was determined by enzyme-linked immunosorbent assay (elisa). we used recombinant proteins np and hn as antigens for elisa. results: hyperimmune serum was collected from mice after immunization by np and hn recombinant proteins. the level of neutralize activity was measured in the prn assay with strain edmonston. the titre reached up to 1:13.5 and 1:22.9 for np and hn recombinant proteins, respectively. interestingly that, hyperimmune serum on recombinant protein np in elisa reacted both with np (titre 1:6400) and with hn (titre 1:3200), and in turn serum on recombinant protein hn reacted only with hn (titre 1:12800). the estimation immunological properties of proteins with use of the panel of serum (50 samples) collected from patients. the diagnosis of measles infection was confirmed in laboratory (by rt-pcr). the nucleotide sequences of rt-pcr products used for genotyping of mv. selective interaction of antibodies in elisa with recombinant proteins in relation to various genotypes is revealed. the interaction with genotypes a and d6 was expressed with high level of correlation whereas with genotype d4 any serum did not react authentically (as the control was used recombinant protein n of sars virus). conclusion: we have shown that neutralize antibodies formed hot only on superficial proteins such as hn, f and sh but also on core proteins such as np. our data demonstrate that the recombinant proteins np and hn could be a cost-effective alternative to current whole virus based elisas for surveillance for immunity to measles and could more efficient in detecting susceptibility to measles in relation to genotypes a and d6. episode 1: a pregnant woman with thirty-eight week of gestation was hospitalized in obstetrics clinic with the complaints of fever, malaise, and severe vaginal bleeding. on admission, white blood cell count was 6700/mm 3 , haemoglobin was 11.5 g/l, platelet count was 8 000/mm 3 . the level of ast was 591 iu, alt 163 iu, lactate dehydrogenase 1079 iu, and creatinin phosphokinase 2132 iu. the baby was delivered by cesarean section. in serum cchf igm was positive by elisa, and per oral ribavirin was administered after delivery. at the first day of delivery, the clinical and laboratory of findings of the baby were found to be normal. however, on his 5th day, he died because of massive bleeding. his cchf igm was found to be negative. episode 2: a pregnant woman with 19 week of gestation was admitted to the hospital. her complaints were fever, malaise, headache, myalgia, nausea, vomiting, diarrhoea, and subconjuntival bleeding. in her laboratory investigation, white blood cell count was 3400/mm 3 , haemoglobin level was 10.4 g/l and platelet count was 53000/mm 3 . the level of ast was 813 iu, alt 539 iu, and ldh 741 iu. in her serological analysis cchf igm and cchf virus -pcr was found to be positive. at the twenty six week of gestation in obstetric ultrasound, fetal intraabdominal fluid was visualized and amniocentesis was performed. in serological analysis of amniotic fluid cchfv-pcr was found to be negative. intraabdominal fluid had increased and scrotal edema was visualized at thirty eighth weeks of the gestation. after her vaginal delivery, baby was severely ill and was operated with the diagnosis of necrotizing enterocolitis. his laboratory findings were normal except high white blood cell count. on his fifth day, thrombocytopenia occurred and he died because of massive bleeding. his cchf igm and pcr were negative. conclusion: to our knowledge, these are the first episodes of intrauterine cchf infection. these episodes show that cchfv can transmit through placenta. obstetricians in endemic countries should consider cchf infection among the patients with massive bleeding and thrombocytopenia. objective: to detect the asymptomatic crimean congo hemorrhagic fever virus (cchfv) infections in an endemic area, and calculate the attack and the infection rate. methods: the study was performed in a cchf endemic region. the household members of the index cases were screened for cchfv igg and igm by elisa. the data related to risk exposure were obtained by a structured form. results: eleven index cases were admitted to the clinic, 45 household members of these cases were screened. all the index patients had positive igm or pcr for cchfv. among the household members, three individuals had igg positivity (%), and only one patient had igm positivity. none of the screened individuals had symptoms. the mean age was 28 (sd 17), and 52% of the subjects were female. tick bite was detected a risk factor (p = 0.040) for cchv infection, whereas patient care and contact with body fluids of the patients were not (p > 0.05). eighteen patients had the history of tick bite, and 8 became infected (44%), and five (27%) became ill. among the infected eight individuals, five became ill (63%). conclusion: although we consider that some of the patients do not notice tick bite, we can still suggest that the infection rate of the virus is rather high compared to similar diseases. tick bite is the major risk factor, in comparison to exposure to blood and body fluids of the infected cases. results: 457 children were included in our study. distribution according sex was: 57.6% female and 42.4% male. median of age was 13 years (iqr = 6). during the follow-up study we recorded 2 years when the number of cases increased. the distribution of cases among the study was: 8.4% in 2000, 33.9% in 2001, 20.9% in 2002, 6.6% in 2003, 7.0% in 2004 and 23.1% in 2005 . the proportion of paediatric patients also varied from; 11.9% in 2000, 9.6% in 2001, 12.4% in 2002, 9.7% in 003, 7.8% in 2004 and 4.4% in 2005 . in panama city we recorded 65.3% of the infants. we detected an increase in the number of patients in the rain season, from may till november. the mean of days between the onset of symptoms and the first blood sample was 6.3 days (ds: 6.7) a second sample was obtained in 23.6% of our infants with an average time of 10.1 days (ds: 7.4). the frequency of classical symptoms related to dengue virus infection was: fever (95.2%), severe headache (74.2%), chill (65.9%) rash (63.5%), myalgia (51.9%), retro-orbital pain (51.6%), arthralgia (43.3%), gastrointestinal symptoms (37.4%), inflamed pharynx (26.7%), cough (26.5%), mild respiratory symptoms (18.6%) and diarrhoea (10.7%). in our infants the symptoms which were detected first were; fever, severe headache, chill, myalgia, retroorbital pain, arthralgia, mild respiratory symptoms, cough and inflamed pharynx. we did not observed differences on clinical features between girls and boys. however, we detected detected significative differences among symptoms when we compared infants who were £5 years old with those who were older (p < 0.005). four of our patients died because of dengue hemorrhagic fever. conclusion: dengue is endemic in panama as in most tropical countries and is one of the world s major emerging infectious disease. more data about this illness are needed to elaborate sanitary programmes which contribute to control this infection. diagnosis of dengue infection by enzyme-linked immunosorbent assay and reverse transcriptionpolymerase chain reaction from oral specimens dengue. salivary elisa has been shown by various investigators to be useful for dengue diagnosis. we sought to perform a pilot evaluation of diagnostic value of elisa and pcr of oral brushes and saliva for dengue diagnosis in adults. methods: adults with acute fever and suspected of dengue infection admitted to our university hospital were enrolled. dengue diagnosis was made by standard elisa using serum or plasma. patients with negative elisa served as controls. buccal mucosal cells were collected for rt-pcr and saliva for both rt-pcr and elisa at least twice, 7-14 days apart. our elisa criteria for saliva were single igm > 20 units or single igg > 60 units or 2-fold increase in igg titre with the second titre >40 units for secondary dengue infection. criteria for primary dengue infection were the same as secondary infection plus igm:igg ratio of over 1.8. results: 23 cases and 16 controls were enrolled. our country is endemic for dengue and thus there was no primary dengue adult case in this study. as the study was performed in hospitalized patients, most of the first samples were collected one day before or on the day of defervescence. the specificities of either methods and the sensitivity of elisa method for saliva were 100%. sensitivities were approximately 32-33% for rt-pcr using buccal cells or saliva specimens. however, a combination of rt-pcr results for both types of oral specimens gave a sensitivity of 52%. the results are summarised in the table. conclusions: collection of oral specimens is less invasive and may be more acceptable in certain situations. a single, acute specimen is adequate for diagnosis by rt-pcr. our specimens, however, were collected late in the course of illness which affected the sensitivity of rt-pcr's. earlier specimens may give a better yield. a study in paediatric patients is needed to assess the value of these methods for primary dengue infection. objective: the aim of this study was to assess the proportion of seropositives against hantaviruses among healthy blood donors. methods: 1607 volunteer donors were recruited by the institute of transfusion medicine, representing the demographic situation in the tyrol regarding gender and residence. sera were tested for igg with a commercially available elisa. positive samples were confirmed by a commercially available dot blot which was also used for identification of the serovar. setting: the study area comprises north tyrol (austria, north of the main ridge of the alps), south tyrol (italy) and east tyrol (austria, both south of the main ridge of the alps). south tyrol belongs to the catchment area of the etsch river, which drains into the adriatic, while north-and east tyrol are part of the catchment area of the danube, which drains to the black sea. results: none of 617 samples from the italian part of the study area yielded a positive result, wherein 7 of 988 donors of the austrian part turned out to be seropositive. two patients were positive for hantaan, 3 patients were positive for puumala, one patient was positive for dobrava and one patient had antibodies against hantaan and dobrava. only one of those patients reported extensive travelling abroad. conclusions: evidence was found for the occurrence of hantaviruses in the austrian part of the region covering the catchment area of the danube, but not in the italian part of the study area covering the catchment area of the etsch river. seropositivity to hantaviruses differs by hydrogeographic areas. objectives: canine coronavirus (ccov) is an enveloped, singlestranded rna virus, belonging to group i coronaviruses within the family coronaviridae. two different ccov genotypes have been recognised, that are designated ccovs type i and type ii on the basis of their genetic relatedness to feline coronaviruses (fcovs) type i and type ii, respectively. ccov is usually responsible for mild, self-limiting infections restricted to the enteric tract. we report the molecular characterisation of a pantropic variant of ccov that caused fatal disease in pups. methods: ccov type ii strain cb/05 was isolated from an outbreak of fatal disease affecting seven dogs housed in a pet shop in apulia region, italy and characterised by fever, lethargy, inappetance, vomiting, haemorrhagic diarrhoea, neurological signs, and severe lesions in the parenchymatous organs. in all tissues, ccov antigen was detected by immunoistochemistry and ccov type ii rna was identified by genotype-specific realtime rt-pcr. the 3' end of the genome of strain cb/05 was determined by amplification of seven partially overlapping fragments. the pcr-amplified products were subjected to direct sequencing and the obtained nucleotide (nt) sequences were assembled and analysed using the bioedit software package and the ncbi's and embl's analysis tools. genbank accession number dq112226 was assigned to the sequenced 8.7-kb fragment. the inferred amino acid sequences (aa) were compared to the analogous proteins available in the online databases. results: the structural proteins s, e, m, n of strain cb/05 displayed a high degree of aa identity to the cognate orfs of ccov type ii, although the s protein showed the highest identity to type ii fcovs. while the nonstructural protein (nsp) 3a had the same length of known ccovs, the nsp3b was 49-aa shorter than expected due to the presence of a 38-nt deletion at position 4704 and to a frame shift in the sequence downstream the deletion that introduced an early stop codon. conclusions: association of strain cb/05 to a severe, fatal disease of dogs, together with virus isolation from organs with remarkable lesions, strongly suggests that this virus has changed the tropism, acquiring the ability to spread from the enteric tract to the internal organs. by sequence analysis of the viral genome, the only striking change was the truncated form of nsp3b, but the role of the deletion in the orf3b in determining the patho-biological change deserves more in-depth investigation. objectives: to perform a surveillance study for sars coronavirus (sars-cov)-like virus in non-caged wild animals from the wild of hong kong special administrative region (hksar). methods: from summer 2004 to spring 2005, 127 bats, 60 rodents and 20 monkeys from 11 locations in hksar were captured. nasopharyngeal and anal swabs and blood samples were collected and tested for sars-cov-like virus rna by rt-pcr using conserved primers targeted to a 440-bp fragment of the rna-dependent rna polymerase (pol) gene. the complete genome of the sars-cov-like virus from bats (bat-sars-cov) was sequenced using rna extracted from three anal swabs of three bats as template. phylogenetic tree construction was performed using neighbor-joining method with growtree using jukes-cantor correction. prediction of signal peptides and cleavage sites was performed using signalp, transmembrane domains using tmpred and tmhmm, potential n-glycosylation sites using scanprosite and protein family analysis using pfam and interproscan. antibodies were detected using a recombinant bat-sars-cov nucleocapsid protein enzyme immunoassay and neutralization assay for human sars-cov. results: we identified a coronavirus closely related to sars-cov (bat-sars-cov) from 23 (39%) of 59 anal swabs of wild chinese horseshoe bats by rt-pcr. sequencing and analysis of three bat-sars-cov genomes from samples collected at different dates showed that bat-sars-cov is closely related to sars-cov from humans and civets. phylogenetic analysis showed that bat-sars-cov formed a distinct cluster with sars-cov as group 2b coronaviruses, distantly related to known group 2 coronaviruses. most differences between the bat-sars-cov and sars-cov genomes were observed in the spike gene, orf 3 and orf 8, which are the regions where most variations were also observed between human and civet sars-cov genomes. in addition, the presence of a 29-bp insertion in orf 8 of bat-sars-cov genome, not in most human sars-cov genomes, suggests that it has a common ancestor with civet sars-cov. antibody against recombinant bat-sars-cov nucleocapsid protein was detected in 84% of chinese horseshoe bats using an enzyme immunoassay. neutralizing antibody to human sars-cov was also detected in those with lower viral loads. conclusion: our data support the existence of sars-cov-like virus in chinese horseshoe bats in hksar. noroviruses are genetically heterogeneous and form at least 27 genotypes within 5 genogroups, gi, gii, giii, giv, and gv, based on the capsid genes. human novs cause an estimated 23 million cases of illness annually in the united states alone and >90% of nonbacterial epidemic gastroenteritis worldwide. porcine calicivirus have been found to be genetically similar to human gii novs or to sapoviruses but calicivirus rna has been detected at low frequency by rt-pcr in adults or fattening pigs. the close genetic relationships between human and porcine novs raise public health concerns regarding their potential for zoonotic transmission and as a potential source of new epidemic human strains. methods: a total of 46 faecal samples of nursing and weaning piglets with enteritis were collected during 2003-2005 in porcine herds in italy. an additional 44 samples were include in the analysis, that had been collected during a rotavirus (rv) surveillance study in 2003-2005, all which tested positive to rv by electron microscopy and by rt-pcr. viral rna was extracted by the guanidine thiocyanate/glass milk method to eliminate enzyme inhibitors. primer pair con2-con3, targeted to the vp4 outer capsid protein, was used for rv detection. a degenerated version of primer pair 289/290 was used for nv detection, that targets a conserved region in the rnapolymerase. results: nov rna was detected in 20/46 of the screened samples, while rvs were detected in 24/46 samples. mixed infections nov+rv were found in 10 samples. screening of the 44 rv positive samples allowed detection of 15 mixed infections with novs. conclusions: in previous investigations novs were detected in 4 of 1,017 normal slaughtered pigs in japan, in 2 of 100 pooled pig faecal samples of 3-to 9-month-old fattening pigs in the netherlands and in 6 out of 274 healthy adult and finisher pigs in the united states. interestingly, in this study a high rate of positivity to novs (35/90) was found in nursing and weaning piglets with diarrhoea, a finding that may suggest a higher frequency of infection by nov in young pigs or an association between nov infection and occurrence of enteric disease. altogether, these findings demonstrate that novs are common in porcine herds in italy and provide new insights into the ecology of novs. detection of calicivirus genome in calves using ni/e3 primers m. mahzounieh, t. zahraeisalehi, e. moghtadaei khorasgani (shahrekord, tehran, ir) caliciviruses may cause a wide spectrum of disease in animals and are important etiological agent of viral gastroenteritis in humans. members of the family caliciviridae are small nonenveloped viruses 27 to 35 nm in diameter. they possess a single stranded poly adenylated rna genome. caliciviruses have been isolated from mink, dog, cattle and non-human primates. "norwalk-like viruses" (nlvs) are the most common cause of acute non-bacterial gastroenteritis in humans. cattle may be a reservoir of nlvs although never bovine nlvs have been found in humans. in this study, we try to detect enteric caliciviruses genome from 50 faecal samples of 6 dairy cattle herds in shahrekord area using reverse transcriptase polymerase chain reaction (rt-pcr) assays specific for nlvs found in humans. the primers used for pcr amplification were ni and e3, which amplify a 113-bp product for the detection of both genogroups i and ii srsv rna in fecal material. our results showed that nine specimens (18%) were positive. these findings suggest that calicivirus infection is endemic in dairy herds in shahrekord, iran and may be have an important role in calf diarrhea. objectives: reoviruses are non-enveloped, 10-segmented dsrna viruses. in humans and mammalians three distinct serotypes exist, whose prototypes are strains lang (t1), jones (t2) and dearing (t3). although reoviruses have been isolated both from the enteric and respiratory tract, no diseases has been clearly associated to reovirus infection in humans. the potential association with extra-hepatic biliary atresia, myocarditis, and, above all, neurological and cutaneous diseases require further investigations. reoviruses are ubiquitous and scarcely speciesspecific. reovirus identification is usually based on electronic microscopy or gel electrophoresis and reovirus incidence seems to be very low in humans and most mammalians. in this study, we investigated the presence of reoviruses in dogs by means of molecular methods. methods: one hundred ninety-two rectal samples from dogs with diarrhoea, 12 ocular swabs, 19 nasal swabs and 27 oropharyngeal swabs from dogs with ocular/nasal discharge were subjected to an rt-pcr assay targeting a conserved region of viral genome segment l1 (primers l1-rv5/l1-rv6). positive samples were characterised by polyacrylamide gel electrophoresis (page), serotype-specific rt-pcr assays targeting segment s1 and sequence analysis. to increase the sensitivity, a nested pcr using primers l1-rv7/l1-rv8 was performed on samples tested rt-pcr negative. results: only 4 faecal swabs (2.1%) were found positive (rt-pcr product of 416 bp). by using a serotype-specific rt-pcr assay and/or sequence analysis, two strains was characterised as type 3 and the other ones as type 1. page of viral dsrna confirmed the genetic characterisation. unexpectedly, in secondround pcr 50 faecal samples (26%), 9 ocular swabs and 10 nasal swabs yielded a 344 bp product, while no oropharyngeal swab was positive. conclusions: these data suggest a wider distribution of reoviruses in dogs than previously thought, even if most reovirus infections were detected only by nested pcr. the ability of reoviruses to induce disease in dogs, alone or in synergism with other pathogens, is still unclear, since attempts to reproduce a specific disease in germ-free dogs have given contradictory results. due to their poor species-specificity, reoviruses may be easily transmitted from animals to humans (and vice-versa). further studies are required to understand reovirus ecology and their potential zoonotic impact. objectives: parvovirus b19 is a member of a family parvoviridae. on the basis of genetic distances and evolutionary relationships, human parvoviruses are divided into three genotypes: genotype i corresponding to b19-related isolates, genotype ii to lali-related isolates, and genotype iii to v9-related isolates. parvovirus b19 causes a common exanthematous disease in childhood or adult age, arthropathy, hydrops fetalis, various haematological disorders and myocarditis. up to now, we have had no data of the prevalence of b19 virus in slovenian population. consequently, we also lack information on the genotypes of parvovirus b19 that are involved in the patients who suffer from the infection. methods: to gather information of the genetic variants of b19 virus present in slovenia, we extracted dna from 48 serum samples that were sent for serologic diagnostic of parvovirus b19 infection and were positive for specific igm in the period from january 1997 to june 2005. nearly half of all 48 patients were children and young adults up to 25 years. the ns1 region of parvovirus b19 was amplified by the nested pcr (primers pb19f1, r1 and pb19f2, r2). all pcr products were directly sequenced. the results of our study show that dna of parvovirus b19 was present in all 48 samples that tested positive for specific igm antibodies. after the first round of pcr reaction, 17 samples were positive, and after the second reaction, all 48 samples were positive. altogether 12 unique genotype variants of parvovirus b19 were identified and all were clustered in the genotype i group of b19-related isolates. most of the distinct 12 genetic variants differed in 1% to 2% from the sequences deposited in gene bank. the majority of sequences obtained from the b19 virus epidemic in 1998 represents a single variant of genotype i with the gene bank acc. no. aj781038. we also found that different genetic variants of parvovirus b19 were circulating in 2002 and were 100% or 99% identical to the genotype i variant with the gene bank acc. no. z68146. in our study, we were not able to identify any variants of other rare genotypes (lali or v9). conclusion: parvovirus b19 dna was successfully amplified from all igm positive serum samples of the patients. the genotype i of parvovirus b19 is dominating in infections with parvovirus b19 in slovenia. objectives: great britain has been free of animal brucellosis since 1985 (european commission decision 93/52). the main source of infection for uk residents is through contact with infected material in foreign countries. the objective of this study is to type human samples received in the uk since 1999 using variable number tandem repeats (vntr) molecular typing to confirm results obtained by classical typing and relate these results to the suspected source of infection. methods: classical typing is traditionally used and is based on the phenotypic attributes of each strain and biovar. vntr typing is a recently developed molecular method, which is based on short repeats contained in the dna that can be amplified to give a banding pattern specific to each strain. results: results found using both methods are consistent. the results show geographical differences, consistent with observations of strain genotype distribution found in animal brucellosis. conclusion: patient history has been gathered where possible giving information on recent travels. along with results found by classical typing and confirmed by vntr typing we can draw a picture of the sources of infection. these results illustrate the potential of vntr typing as a tool to aid conventional approaches to epidemiological traceback that, in the presence of a suitably comprehensive database of strain genotypes, could help identify the source of an infection. is711-fingerprinting of brucella isolates from humans e.stubberfield (surrey, uk) objective: brucellosis is a zoonotic disease usually associated with cattle, sheep, goats and pigs. human infection has been attributed to b. melitensis, b. abortus, b. suis, b. canis and b. maris. although the uk is officially bucellosis free there are a number of human cases due to travel and occupation that are submitted to our laboratory for diagnosis. definitive diagnosis of brucella is by bacteriological culture and microbial tests (classical typing), however these require skilled personnel and the results can be subjective. there are a number of molecular tests that have been developed to assist with diagnosis more rapidly and in some cases to strain level less subjectively. is711-fingerpinting is a molecular technique that has proved useful for the identification of brucella isolates to species and in some cases stain level. is711-fingerprinting relies on the variable number and location of the is711 mobile genetic element found in all brucella isolates. method: 73 brucella isolates from humans have been tested. genomic dna was extracted, digested using restriction endonuclease ecor1, and electrophoresed. southern blotting was performed, hybridising with a dig-labelled is711 probe. results: the number of brucella is711 copies range from 5 to more than 20. brucella melitensis remains the most commonly acquired brucella species of travellers, while occupational infections have included b. abortus isolated from cattle farmers and b. suis associated with pig butchers. two marine brucella strains have been isolated originating from an occupational perspective (a laboratory worker) and a natural setting from an unknown source. 7 unusual patterns have been observed, 5 of which are unique. one of the new patterns has been observed only in isolates originating in east african countries. conclusion: although the diagnosis of brucella to species and strain level is not essential for the treatment of human brucellosis, it is useful for epidemiological studies. is711fingerprinting is able to identify the three biovars of b. melitensis, many other techniques do not offer this capability, because of this it may be a useful test in epidemiological studies. this method remains an important diagnostic tool for brucella identification. rapid diagnosis of brucellar epididymo-orchitis by real-time pcr assay in urine samples objectives: to study the diagnostic yield of a real-time pcr assay in urine samples for the rapid diagnosis of brucellar epididymo-orchitis, in comparison with conventional microbiological techniques. methods: ten consecutive patients with brucellar epididymoorchitis were included in the study. the diagnosis of brucellosis was established according to one of the following criteria: first, isolation of brucella spp in blood or any other body fluid or tissue sample or, second, the presence of a compatible clinical picture together with the demonstration of specific antibodies at significant titers or seroconversion. epididymo-orchitis was diagnosed in patients with scrotal enlargement, swelling and pain not due to other causes.for dna amplification we used a sybr green i lightcycler-based real-time pcr assay. the assay amplifies a 223 bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein (bcsp31). the pair of 21 nucleotide primers b4 (5' tgg ctc ggt tgc caa tat caa 3') and b5 (5' cgc gct tgc ctt tca ggt ctg 3) were used in the amplification process. after dna amplication, we performed melting curve analysis to verify the specificity of the pcr products. in order to study the specificity of the technique, all the samples from the patients with brucellosis were paired with an equal number of samples from controls with urinary tract infection. (e. coli four cases, k. pneumoniae two cases, p mirabilis two cases, and c. freundii and p. aeruginosa one of each). results: the mean age was 38.4 years (range 21-69). the duration of the symptoms prior to diagnosis was 30.2 ± 16.2 days (range: 5-51). b. melitensis was isolated from blood cultures in nine cases (90%). wright's seroagglutination was negative or inconclusive in 30% of cases. brucella was isolated from urine in only one case whereas real-time pcr assay in urine was positive in nine (90%) cases and the results were available in four hours, whereas the mean time to availability of the final blood culture results was 5.8 days (range 4.5-7 days). real-time pcr was negative for all the control samples from patients with urinary tract infections. conclusion: sybr green i lightcycler-based real-time pcr assay in urine samples is highly sensitive and specific, easy to perform and could provide the clinician with the results in under five hours. the technique could be a practical and useful tool for the rapid diagnosis of genitourinary complications of human brucellosis. objectives: although the united kingdom remains brucellosisfree, there are more than 500,000 new cases of human brucellosis reported each year according to the world health organisation. uk residents returning from worldwide travel may have encountered exposure through contact with infected animals and animal products such as dairy produce and meat. phenotypic characterisation or classical methods remain the definitive diagnosis though require skilled personnel and have their limitations. the increasing range of molecular techniques can aid the rapid detection and characterisation of brucella species and their biovars and may have significance in epidemiological studies. methods: a study of brucella reference and field strains of mainly human isolates from different geographic locations were analysed for diversity of their genes encoding the outer membrane proteins (omps) 25, 2a and 2b. pcr products of the three genes digested with seven restriction enzymes were analysed for polymorphisms. results: a re-occurring unique pattern profile seen only in human isolates was observed originating in some european countries and beyond. a growing database of strain types giving a recent overview of brucella infection of humans of many countries. conclusions: molecular typing methods may have an advantage over classical typing concerning brucella melitensis, the most common brucella infection of humans. the characterisation of human brucella isolates may be useful in epidemiological studies for a variety of purposes. objectives: a study to demonstrate the rapid detection and speciation of campylobacter jejuni and of campylobacter coli isolates directly from enrichment broth using a taqman ò assay. single nucleotide polymorphism analysis of mapa positive strains was used for rapid identification of c. jejuni clonal complexes. methods: thermotolerant campylobacter species were initially confirmed by culture according to the modified draft iso 17995 method, where water samples were filtered through 0.2 mm pore size nylon membrane. the filters were transferred to selective enrichment in preston broth to improve their recovery and therefore detection of any campylobacter cells present. dna was extracted directly from the enrichment broth culture for real-time detection of c. jejuni and c. coli using the taqman ò . samples, which were map a positive were, further characterise by single nucleotide polymorphism profiling for rapid recognition of c. jejuni clonal complexes. results: environmental samples, which were confirmed by culture were also map a positive by taqman ò . snp profiling of mapa positive isolates identified clonal complexes, which are predominantly contained in isolates of human disease and chicken. conclusions: this study has demonstrated the feasibility of rapid detection and identification of c. jejuni and c. coli following short enrichment incubation using a taqman ò assay. a rapid turnaround time of between 3-4 h per batch of 96 samples was achieved. snp profiling offers important epidemiological grouping at strain level, enabling accurate and phylogenetically valid strain identification for c. jejuni, which may have important host associations for tracing sources of infection and consequently improve public health responses. objectives: campylobacter jejuni and c. coli are recognized as the most common causes of acute bacterial gastroenteritis in humans, c. jejuni being the predominant species in most developed countries. the hippurate hydrolysis test is widely used to differentiate c. jejuni from other campylobacter species. about 10% of c. jejuni isolates fail to hydrolyze hippurate under laboratory conditions. molecular methods represent an alternative to the phenotype-based methods. we tested two multiplex pcr assays for species identification of human campylobacter strains and compared the results with the hippurate hydrolysis test. methods: campylobacter strains isolated from patients were tested for hippurate hydrolysis with rosco diagnostic tablets. 50 hippurate-negative and 15 hippurate-positive strains were selected for two multiplex pcr assays. one pcr-method was based on distinctive ceue-genes of c. jejuni and c. coli, the other pcr-method detected genes from five major clinically relevant campylobacter species: hipo from c. jejuni, glya from c. coli, c. lari and c. upsaliensis, sapb2 from c. fetus subsp. fetus, and 23s rrna gene from campylobacter spp. as an internal validation control. results: the c. jejuni hipo gene was detected in all of the 15 hippurate-positive strains and 10 of the 50 hippurate-negative strains. the c. coli glya was detected in 39 of the hippuratenegative strains. in one hippurate-negative strain, sapb2 from c. fetus subsp. fetus was detected. species-specific genes were detected in 53 of the 65 strains with the ceue-based pcr assay. c. jejuni ceue was detected in 12 hippurate-positive and 5 hippurate-negative strains. c. coli ceue was detected in 36 hippurate-negative strains. conclusion: all hippurate-positive strains were identified as c. jejuni. of the hippurate-negative strains, 78% were identified as c. coli, whereas 20% were identified as c. jejuni and one strain as c. fetus subsp. fetus. the results of the two pcr assays were concordant, although some strains could not be identified with the ceue-based pcr assay. the results suggest that molecular species identification should be performed on hippuratenegative strains after the hippurate hydrolysis test for accurate species identification. multiplex-pcr is quick and easy to perform. using the pcr assay that simultaneously detects five campylobacter species also diminishes the need for further phenotypic testing. phenotypic typing of cryptosporidium species isolated from children in kuwait: a role in unique transmission j. iqbal, p. hira (safat, kw) background: cryptosporidiosis is recognized worldwide as a significant cause of diarrhoeal diseases in both adults and children especially in children less than 2 years of age. objective: cryptosporidium spp. isolated from young children in kuwait were characterized at the molecular level to understand the transmission of infection. the study was approved by the ethical committee, faculty of medicine, kuwait. methodology: over a period of 2 years, faecal specimens from 97 kuwaiti children with persistent diarrhoea found to be positive for cryptosporidium spp. by microscopy were genotyped and sub-typed with a small subunit rrna-based pcr-restriction fragment length polymorphism analysis. informed consent was taken from all individuals included in the study. results: the median age of infected children was 4.9 years, and the majority of the infections (>70%) occurred during the cooler months january-april, indicating a marked seasonal variation. more than 85% of the children with cryptosporidiosis had only cryptosporidium infection. socio-demographic information did not reveal any particular mode of transmission of infection. genotyping of the organisms isolated showed that ninety-two (95%) of the children had c. parvum, 4 (4%) had c. hominis, and 1 (1%) had both c. parvum and c. hominis. altogether, 9 subtypes of c. parvum and c. hominis were observed. objectives: the intracellular respiratory pathogen chlamydophila pneumoniae (cp) might be involved in the pathogenesis of atherosclerosis. several studies have demonstrated a serological association between cp and cardiovascular disease and dna from the bacteria has been found in various atheromatous vessels. after infection in the respiratory tract, cp is believed to be disseminated systemically within alveolar macrophages. the prevalence of cp within peripheral blood mononuclear cells (pbmc) has in some studies been shown to be higher in patients suffering from cardiovascular disease than in control patients. we investigated the presence of cp dna in aortic heart valves and pmbc in 55 patients (37 men; 18 women; mean age 69 years) undergoing aortic valve replacement because of aortic stenosis. also, the presence of cp mrna was investigated in the sclerotic aortic heart valves as a marker of viable bacteria. methods: dna was extracted from aortic valve biopsies and pbmc using the qiaamp dna mini kit (qiagen). mrna and dna were extracted from another piece of the same biopsy using trizol (invitrogen). real-time pcr directed against the chlamydia momp gene was used to detect cp-specific dna and mrna. patient sera were tested for cp-specific igm, igg and iga antibodies by the microimmunofluorescence technique. results: cp dna was found in aortic heart valves from 20% (11/55) of the patients and in pbmc from 5% (3/55) of the patients. in one patient cp dna was found in both pbmc and heart valve. no patient had cp-specific igm antibodies. in patients that were pcr-positive for cp dna in the aortic heart valves, 73% had igg ‡1:64 and 45% had iga ‡1:32. in patients that were pcr-negative in the aortic heart valves, 59% had igg ‡1:64 and 23% had iga ‡1:32. cp-specific mrna in aortic heart valves will be presented on the poster. conclusion: cp-specific dna was found in sclerotic aortic heart valves from 20% of patients undergoing aortic valve replacement. this confirms previous investigations supporting a role for cp in the pathogenesis of aortic valve sclerosis. the prevalence of cp in pbmc was 5% which is comparable to that reported in healthy blood donors and lower than that recorded in patients suffering from other cardiovascular diseases. if the bacteria are involved in the pathogenesis of aortic sclerosis they have likely been spread to the aortic valve long before the patient is in need of surgery because of the stenotic valve. introduction: diarrhoea is one of the most common causes of morbidity and mortality among young children in developing countries. diarrheagenic e. coli strains include several emerging pathogens of worldwide public health. six important categories are entero-aggrigative e. coli (eaec), entropathogenic e. coli (epec), enterotoxigenic e. coli (etec), enterohemorrahgic e. coli (ehec), entroinvasive (eiec) and shigatoxin-producing e. coli (stec). this study investigated the role of different diarrheagenic e. coli in iranian children with acute diarrhoea by molecular methods and the antibiotic susceptibility of isolated strains. methods: from april 2003 to january 2005, one thousand eighty five children with acute diarrhoea in tehran hospitals in were enrolled in the study. the fecal samples were cultured on macconkey for conventional bacterial pathogen and sorbitol macconkey agar for non sorbitol fermenting phenotype, than they were incubated in 37ordm;c. the primary stool cultures were subjected to six different pcr reactions targeting stx1 and stx2 gene, heat-labile enterotoxin (lt) producing gene, heatstable enterotoxin (st) producing gene, eae gene and pcvd432 plasmid. the kirby -bauer disc diffusion method was used for antibiogram of 110 isolated strains from different diarrheagenic e. coli by 12 different antibiotics. results: two hundred seventy one diarrheagenic e. coli strains were detected. stec was the most prevalence with 125 (46.1%). the frequency of other strains was 28.7%, 16.3%and 8.8% for etec, eaec and epec, respectively. out of 120 stec isolated 86 strains (% 68.8) had stx1 or stx2 gene, and 4 strains had stx1 and stx2 gene. the eae gene was found in 13 (10.4) stec strain. out of 110 tested strains, 102(92.5%) were resistance to ampicllin and cefalotin, and 99 (90%) to streptomycin. conclusion: in this study stec was the most frequent associated with diarrhoea. the strong association between use of antibiotics and colonization with antibiotic resistant e. coli, suggest a major role for selection of resistant strains while using antibiotics. the existence of other unknown intestinal adherence factors has been suggested by the isolation of stec strains that lack the eae gene but are still associated with bloody diarrhoea or hemolytic ureamic syndrome (hus). since there is no specific treatment, there is an urgent need for effective preventive measures based on detailed understanding of the epidemiology of stec infections. identification of shiga toxin-producing escherichia coli in raw beef using dna hybridization with digoxigenin-labelled probes and multiplex pcrs m. weiner, j. osek (pulawy, pl) shiga toxin-producing escherichia coli (stec) is an important cause of bloody diarrhoea, haemorrhagic colitis, haemolytic uremic syndrome and thrombotic thrombocytopenic purpura. transmission of stec occurs through consumption of contaminated food, especially meat, dairy products and water. objectives: to develop a three-steps procedure based on two multiplex pcrs and dna hybridization with digoxigeninlabelled probes for identification of stec in raw beef. methods: beef samples inoculated with different number of e. coli o157:h7 cells were incubated in tsb medium at 37°c for 6 h. the cultures were then transferred to tsb with mitomycin c and incubated for another 18 h. the resulted cultures were used as a source of dna template. the mpcr-1 was established to identify shiga toxins genes (conserved sequence). the positive culture samples were subjected to dna hybridization with dig-labelled probes as follows: the culture was diluted and inoculated onto agar plates supplemented with tergitol ò and incubated at 37°c for 18 h. then, the nylon membranes were put on agar plates, carefully removed and incubated in denaturation, neutralisation and equilibration solutions following incubation with the stx-specific dig-labelled probes, anti-dig conjugates and finally developed with enzyme substrates (bcip and nbt). dark spots visible on the membranes were compared with the respective bacterial colonies on the original agar plates. the corresponding bacterial colonies were isolated and characterized using the mpcr-2 test which allows amplification of stx1 (shiga toxin type 1), stx2 (shiga toxin type 2), rfbo157 (e. coli o157) and flich7 gene (h7 antigen). an internal control of amplification (e. coli 16s rrna gene) was also included in both mpcr tests. results: the first mpcr resulted in two amplification products: 230 bp for stx and 401 bp for 16s rrna genes. the positive meat samples were further tested with dna probes and positive colonies were then characterized with the second test (mpcr-2), generating the amplicons either of 348 bp (stx1), 584 bp (stx2), 420 bp (rfbo157), 247 bp (flich7) or 798 bp (16s rrna). the specificity of this procedure was confirmed by testing e. coli o157:h7, o157:h-and non-stec bacteria. the sensitivity of the method was estimated as 4 cfu/g of meat. conclusion: the obtained results demonstrated the high specificity of the procedure developed and the possibility of using it for identification of shiga toxin-producing e. coli in raw beef. correlation between virulence pattern, phylogenetic group and extended spectrum betalactamases genes in escherichia coli strains isolated from blood cultures m. damian, c. usein, d. tatu-chitoiu, s. ciontea, d. jardan, a. palade (bucharest, ro) e. coli, heterogeneous species consisting of commensal and pathogenic strains, is causing a broad spectrum of human diseases, including extra intestinal and enteric infections.the strains isolated from invasive infections were documented to be carriers of a large number of genetic structures coding for virulence, as well as for resistance to antimicrobial agents. the aim of this study was to evaluate the virulence of strains in comparison with the presence of esbl genes and their distribution among the different phylogenetic groups. a total of 51 e. coli strains, isolated from blood cultures, in hospitalised patients, adults and children, were screened for virulence factors-encoding genes (pap, sfa/foc, afa, hly, cnf, aer and fimh), for genes encoding resistance to extended spectrum betalactam antibiotics (bla shv and bla tem genes) and the appurtenance to one of the main four phylogenetic group based on presence or absence of markers chua, yjaa and tspe4.c2 three strains, negative for all virulence genes, were included in the phylogenetic group a. ten strains, which were positive for five or six virulence genes, were identified as b2 group. no matter the phylogenetic grouping, the remaining strains possessed at least one virulence gene. no strain was pcr positive for all seven virulence genes targeted. among the 16 strains which were positive in the double disk test, 12 strains exhibited both bla shv and bla tem genes and 4 strains only bla tem gene. restriction with pst i and dde i and sequencing of the amplicons were performed in order to identify the type of esbl gene expression product. taking into account the link between phylogenetic group and virulence, we obtained a good correlation for the bacteremic e. coli strains analysed, but there was no relationship with the production of esbls. isolation of shiga-toxin producing escherichia coli from meat samples, phenotypic and genotypic characterisation of isolated strains f. baghbani-arani, f. jafari, m.r. zali, s. salmanzadeh-ahrabi (tehran, ir) objective: shiga-toxin producing escherichia coli (stec) is an emerging foodborne pathogen of worldwide public health importance. this bacterium has been reported as an etiological agent of many outbreaks and sporadic cases. definition of the diversity and antimicrobial susceptibility of (stec) may be helpful in the management of sporadic cases and outbreaks. studies in different countries show that food items maybe contaminated by this pathogen. the present study was carried out to determine the frequency of contamination of meat samples by stec collected in tehran as well as defining genotypes, serotypes, antibiogram susceptibility patterns and molecular diversity of isolated bacteria. methods: from july 2004 to june 2005, 250 beef samples were collected from different part of tehran. a 25 grams of each samples was enriched in ec broth and subculture on mac-conkey agar. dna was extracted from a loop full of bacteria taken from primary first streaking area of mac-conkey agar and was subjected to three different pcr reactions targeting stx1, stx2 and eae genes. as much as colonies required were tested for finding the colony responsible for positive results in the first pcr. antibiogram susceptibility patterns of isolated strains were determined by standard disk diffusing method. the 17 antimicrobial agents were used at this study. all isolates were serotyped by slide agglutination test using standard antisera (mast groups) subtyping of strains was done with rapd-pcr by 1283primer. results: among 250 samples, 47 (19%) samples were positive and their genotypes were as follow: 1(0.4%) stx1+, stx2-, eae-, 35(14%) stx1-, stx2+, eae-, 6(2.4%) stx1-, stx2+ and eae+.4 (1.6%) stx1+, stx2+, eae-, 1(0.4%) stx1+, stx2+, eae+. among these positive samples 30 strains were isolated. according to the antibiotic susceptibility tests, all isolates were resistance to erythromycin (e) and oleandomycin (ol), and were sensitive to imipenem (i); gentamicin (g) norofloxazin (nx) enterofloxazin (ex) ciprofloxazin (cf) and ceftazidim (ca). in otyping and htyping the most frequency were o112ac and h2 serotypes. analysis of isolates by rapd-pcr yielded 17different patterns. conclusion: our results show that contamination of meat samples by stec is a life-threatening health problem. combinational analysis of antibiogram susceptibility patterns and serotypes with rapd-pcr patterns can aid to survey the characteristics of stec strains. factors affecting the conjugative transfer of plasmid pip501 in enterococcus faecalis a.m. al-qurashi (dammam, sa) objectives: factors which are known to influence plasmid transfer were studies using the conjugative plasmid pi501, which encodes erythromycin resistance, in enterococcus faecalis. methods: the donors strains streptococcus a agalactiae v666 (group b) is resistant to in rifampicin and fusidic acid, non hemolytic and b-lactamase-negative. it contains the broad host range plasmid pi501, which confers resistance to erythromycin and chloramphenicol. the recipient is enterococcus faecalis strain jh2-2 group d. results: transfer of pip501 occured on a agar, on filters and in broth cultures at relatively high densities (106-108 bacteria/ml). transfer frequency was largely unaffected over a wide range of temperatures (23-42°c). the ph of the medium, in the range ph 5-9 had little effect on the transfer frequency. log phase cultures and donor: recipient ratios of 1:1000-100:1 were required for optimal for plasmid transfer. conclusion: factors which modified the transfer efficiency of the conjugative plasmid pip501 were mating media, solid or liquid environment, mating time, mating temperature, selection temperature, growth temperature of donor and recipient of prior to mating, ph culture age, and donor/recipient cells ratio, to obtain a better understanding of this plasmid and its transfer process will help understand what role they may have in the dissemination resistance among streptococcal and enterococcal populations. enterococcus faecium blood-culture isolates collected during a five-year period h. billströ m, å . sullivan, b. lund (stockholm, se) background: enterococcus faecalis and enterococcus faecium have during the last years become a significant nosocomial problem. this could be due to the enterococcus hardy nature combined with intrinsic and acquired antibiotic resistance. since most individuals harbour enterococci in their normal intestinal microflora there has been a discussion regarding the origin of these isolates. during the last ten years the isolation ratio between e. faecalis and e. faecium have shifted from 10:1 to 3:1. this could be because of increasing antibiotic resistance among infectious e. faecium isolates compared to infectious e. faecalis ones. it is possible that this increase also depends upon different virulence genes such as enterococcal surface protein (esp), hyaluronidase variant gene (hylefm) and e. faecalis antigen a variant (efafm). objectives: the objectives in this study were to determine the presence and frequencies of seven different enterococcal virulence genes in infectious isolates. further objectives were to see if the number of virulence genes in these isolates vary or increase over time. methods: a total of 257 strains isolated from bacteraemia patients during year 2000-2005 at the karolinska university hospital, huddinge were used. all isolates were screened for seven different virulence genes using a multiplex pcr. these seven virulence genes were aggregation substance (asa), cytolysin (cyt), collagen binding protein (ace), e. faecalis antigen a variant (efafm), enterococcal surface protein (esp), gelatinase (gel) and hyaluronidase (hyl). results: according to the results about half of all isolates were esp-positive. the prevalence of the other virulence genes asa, efafm, gel and hyl were detected, but in low frequencies (<5%). conclusion: it seems like the esp gene is the most dominant virulence gene in e. faecium isolates. the occurrence of virulence traits in these isolates further indicates that the potential to cause infection is potentiated among this enterococcal population the data from this investigation supports the hypotheses that enterococci causing infection in hospitalized patients are probably of nosocomial origin rather than endogenous. objectives: the ability of l. monocytogenes to tolerate alkaline stress is of particular importance, as this pathogen is often exposed to such stress in food processing environments cleaned with alkaline detergents or in the mildly alkaline ph values which prevail within engulfing phagolysosomes. this study aims to investigate the alkaline tolerance response (altr) in listeria monocytogenes 10403s using dna microarray technology. knowledge of the alkaline-induced stress response will be useful in understanding how this pathogen tolerates alkaline stress. methods: transcription profiling of l. monocytogenes 10403s was carried out at 15, 30 and 60 min at high ph in order to capture an early, an intermediate and a prolonged expression response to alkaline stress using oligo arrays from the pathogen functional genomic resource centre. to verify the microarray results the regulation of some ph stress response genes were confirmed by real time quantitative polymerase chain reaction (rt-pcr). results: about 1584 genes were upregulated and 1754 genes (of 6347 open reading frames represented on the arrays) were down regulated at least 1.5 fold upon alkaline shock. many of the repressed genes encode enzymes that are involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating a metabolic adjustment of the cells to the high ph. notably, the strongest alkaline-inducible genes were involved in the membrane transport systems. conclusion: the analysis of the data revealed that cells sense and respond to alkaline stress with an extensive program of changes in gene expression. interestingly, there is a strong correlation between the altr and virulence gene expression. comparison to various microarray data already in the literature revealed similarity between the response to alkaline stress and the transcriptional response to stresses such as osmotic shock. engineering improved listerial stress tolerance "with a twist"! r. sleator, c. hill (cork, ie) objectives: to engineer listeria monocytogenes strains with a significantly improved ability to tolerate stresses encountered in the external environment and during gastrointestinal transit, thus, improving listeria's efficacy as a potential vaccine and drug delivery platform. methods and results: using a directed evolution approach, based on a random mutagenesis strategy involving the e. coli xl1-red mutator strain, we generated a mutant variant of the listerial betl gene (designated betl*), encoding a secondary betaine uptake system. the mutant betl* promotes a dramatic increase in resistance to a number of biologically relevant stresses when expressed in a variety of different surrogate hosts. using a luciferase (lux) reporter system in combination with the ivis imager system (xenogen corporation, alameda, ca), we tracked betl* expression, in real time, both in vitro under various environmental stresses and in vivo in animal models of infection. in each case strains expressing betl* demonstrated a marked improvement over those expressing wild type betl, both in terms of gene expression and bacterial growth. sequence analysis of the mutated gene revealed a single nucleotide deletion in the spacer region between the -10 and )35 promoter elements upstream of the betl coding region. this deletion presumably introduces a conformational 'twist' in the putative promoter, thereby increasing its transcriptional output. furthermore, the betl* mutation appears to counter the heretofore unreported 'twisted' cell morphology observed using scanning electron microscopy of l. monocytogenes grown at elevated osmolarities. conclusions: it is possible to selectively improve genes required for bacterial stress survival both inside and outside the host. such mutated genes systems may ultimately be used for the construction of more physiologically robust bacterial based vaccine and drug delivery platforms. a.r. samarbaf-zadeh, s. tajbakhsh, s.m. moosavian (ahwaz, ir) introduction: peptic ulceration following infection of stomach with h. pylori is a common disease. accurate and rapid detection of the bacteria can lead to implementation of appropriate treatment and recovery. this research was undertaken to evaluate the sensivity and specificity of fluorescent in-situ hybridization (fish) in the detection of h. pylori in patients who were suffering from dyspepsia. methods: for this purpose, one hundred gastric biopsy samples taken from antrum and corpus of stomach by endoscopy were tested by fish and compared with conventional culture method complemented with biochemical tests. results: fish detected h. pylori in 48 clinical samples while conventional method detected 42 samples. the sensivity and specificity of fish for detection of h. pylori were calculated as 98% and 100% respectively. conclusion: the findings of this study suggest that fish is a highly suitable and rapid method for diagnosis of h. pylori, especially when the samples are taken from the antrum and the corpus of the stomach this technique potentially can be applied routinely for detection of this bacterium in clinical samples. objective: numerous studies have demonstrated that h. pylori is ubiquitous; approximately 50% of the world's population is infected with the organism. gastroduodenal diseases associated with h. pylori infection are manifested principally in adults. however, it's usually during chilhood that the infection is acquired, and it is possibile that mucosal and humoral responses at this time may determine, at least in part, the course of the natural infection. our study will describe the prevalence of the h. pylori oral carriage in children resident in bari, south of italy, using the pcr method. methods: the evaluation was performed in 404 children, with ages ranging from 6 to 11 years, from primary school district of local health unit of bari, italy (ausl ba/4). the school and the class have been selected using the cluster sampling method. a standardized questionnaire was used to verify socio-economic standard, hygiene and history of previous gastrointestinal disorder. a standard full-mouth examination was made to detect periodontal diseases, then dental plaque and saliva collected from children were placed in pbs and transported in laboratory. h. pylori infection status was checked by pcr method. dna was extracted from oral samples by the boiling method and evaluated for the presence of h. pylori caga and urea genes using commercial kit (ab analitica, padova). results: a total of 404 children (212 females and 192 males) partecipated to the study. the presence of gene coding for caga was found in 57 children (14%), but gene urea was detected only in 22 (5%). the bacteria was detected in saliva, supragingival and subgingival plaque, suggested that these sites may be considered reservoirs for h. pylori in ureasi-positive patients. there was statistically significant relationship between who didn't wash their hands frequently and the presence of urea gene (o.r. 1.71). conclusions: current knowledge implies that acquisition of h. pylori seems to occur predominantly in childhood and that once acquired the infection persists life-long in most infected subjects. it has been reported at a worldwide level that h. pylori infection prevalence in children varies between 10% and 80% and increases with low socio-economic and educational levels and age. the results of this study suggest that oral carriage of h. pylori may play a role in the transmission of infection and that the hand may be instrumental in transmission. the role of helicobacter pylori in otitis media with effusion t. yilmaz, m. ceylan, y. akyon, o. ozcakir, b. gursel (ankara, tr) objectives: otitis media with effusion (ome) is such a common disease of childhood and its pathogenesis still remains unsettled. pepsinogen and pepsin has been shown in the middle ear fluid of patients with ome, indicating that gastric juice could reach as far as middle ear. if gastric juice could enter the middle ear, helicobacter pylori, a common inhabitant of gastric juice and mucosa, would also be expected to be found in the middle ear of patients with ome. the objective of this study was to evaluate possible role of helicobacter pylori in pathogenesis of otitis media with effusion. methods: the study group consisted of 22 children who are to undergo bilateral ventilation tube insertion, adenoidectomy, tonsillectomy with a diagnosis of ome, adenoid hypertrophy and chronic tonsillitis. the control group consisted of 20 children who are to undergo adenoidectomy, tonsillectomy with a diagnosis of adenoid hypertrophy and chronic tonsillitis. for the study group, middle ear fluid was aspirated and a small biopsy was taken from the promontorium mucosa. for the control group, myringotomy was done and a small biopsy was taken from the promontorium mucosa. for both groups, 5 mm deep tissue specimens were obtained from tonsil and adenoid. for all the specimens taken from the patients, culture and a nested-pcr were performed to show helicobacter pylori. results: middle ear fluid culture was positive for h. pylori in 2 patients and mucosa culture was positive in 1 patient only. in the control group middle ear mucosa cultures were always negative. when culture and pcr results were combined together; the middle ear was positive for h. pylori in 10 patients in the study group and in 2 patients in the control group. this difference was statistically significant. h. pylori presence in the tonsillar and adenoid tissues by culture and pcr was also significantly more frequent in the study group compared to the control group. conclusion: this study is the first to grow h. pylori in the middle ear in ome. significantly increased colonization by h. pylori of the middle ear, tonsillar and adenoid tissue in patients with ome indicates that the bacteria reaching the middle ear through gastroesophageal reflux might be involved in the pathogenesis of ome. for ome cases resistant to medical treatment it may meaningful to evaluate the patient for gastroesophageal reflux and h. pylori. distribution of the serine-aspartate repeat protein-encoding sdr genes among nasal carriage and invasive staphylococcus aureus strains objectives: this study was designed to examine the distribution of the sdr genes among nasal carriage and invasive staphylococcus aureus strains as well as methicillinsensitive s. aureus (mssa) and methicillin-resistant s aureus (mrsa). methods: the presence or absence of the sdr genes using dna from 497 s. aureus strains was determined by a novel triplex pcr procedure. the two-tailed fisher's exact test was used to analyse the distribution of the sdr genes among s. aureus strains originating from different hosts. p values less than 0.05 were considered a statistically significant difference. results: the sdr locus was found in all 497 investigated s. aureus strains although in 29 strains it contained only the sdrc gene (sdrd -sdre-). the sdrc + sdrd -sdre-gene profile was exclusive to mssa strains (fisher's exact test; p = 0.0005) and was not found in the strains collected from bone infections (p = 0.0019). we also found a strong association between the presence of the sdrd gene and mrsa strains (p < 0.0001). conclusion: our findings suggest that mssa strains with the newly uncovered sdrc + sdrd -sdre-gene profile have a substantially decreased potential to establish bone infection. sequencing of luks-pv and lukf-pv in methicillin-sensitive and methicillin-resistant staphylococcus aureus of diverse genetic backgrounds in a swedish county c. berglund, b. sö derquist (ö rebro, se) objectives: community-aquired methicillin-resistant staphylococcus aureus (ca-mrsa) have been reported to carry the loci for panton-valentine leukocidin (pvl) in high frequency. the aim of this study was to describe variations within the pvl genes (luks-pv and lukf-pv) in methicillinsensitive and methicillin-resistant s. aureus of diverse genetic backgrounds. methods: twelve pvl-positive s. aureus were characterised by multilocus sequence typing (mlst) and mrsa also by staphylococcal cassette chromosome mec (sccmec) typing. ten of these were isolated between 1990-2005 in ö rebro county, sweden. oligonucleotide primers were designed to yield a product size of~2500 bp including luks-pv and lukf-pv and flanking regions by pcr amplification. cyclic sequencing was performed with several sets of primers to overlap the sequences on both strands and was separated on abi prism ò 3100 genetic analyzer (applied biosystems). the nucleotide sequences were analysed using abi prism ò autoassembler tm dna sequence assembly 1.4.0 software and compared using bioedit 7.0.1. results: analysis with mlst differentiated the pvl-positive ca-mrsa into six different sequence types (st8, 36, 80, 152, 154 and 256) with either sccmec type iv, iv c, v or unknown types. six additional sts (st5, 22, 25, 30, 88 and new) were detected among the pvl-positive methicillin-sensitive s. aureus. sequencing luks-pv and lukf-pv revealed eight point mutations among these isolates with twelve different origins. five substitutions had occurred in luks-pv and three in lukf-pv. only one substitution was nonsynonymous (histidine fi arginine). conclusion: the pvl-genes were well conserved despite the different genetic origins of the isolates analysed. the pvl is an extracellular product and the genes are not subject to any selective forces and thereby diversify very slowly. additional nonsynonymous mutations might result in a non-functional toxin. the first case of staphylococcus pseudintermedius in humans isolated from an icd lead l. van hoovels, a. vankeerberghen, k. van vaerenbergh, a. boel, h. de beenhouwer (aalst, be) introduction: staphylococcus pseudintermedius is recently described as a new coagulase-positive species from animals (devriese et al., 2005) . the pathogenic significance of this novel species remains unclear and to our knowledge no human infection due to s. pseudintermedius has been reported to date. here, we present the first isolation of s. pseudintermedius in humans with important clinical significance. patient and methods: a 60-year old male patient was referred to our centre for an ischemic cardiomyopathy and ventricle tachycardia for which he recieved an implantable cardioverterdefibrillator (icd) in january 2004. in august 2005 he presented with complaints of migration of the icd device. clinical examination revealed perforation of the icd pocket. infection was suspected and confirmed by the presence of pus in the pocket. the infected icd was completely removed and several samples (ventricular lead, pus and a tissue sample from the pocket) were sent for culture.bacteria obtained by routine culture were further characterised by phenotypical identification, pastorex ò staph-plus (biorad), api staph ò (biomérieux) and phoenix ò (bd). for molecular analysis, pcrs were performed targeting the nuclease (nuc) and coagulase (coag) genes of s. aureus. additionally, sequencing of the 16s rrna gene was performed and further analysed using blast. results: staphylococci with identical phenotypical appearance were isolated from 2 of the 3 icd samples (lead and pus). colonies were beta-hemolytic on sheep blood agar, dnase and coagulase positive but clumping factor, mannitol and pastorex ò negative. biochemical identification by api staph ò and phoenix ò gave a presumptive identification of s. aureus with a confidence value of respectively 88,5% and 97%.the pcrs for the nuc and coag genes were both negative. 16s rrna gene sequencing resulted in the identification of s. pseudintermedius based on a 100% sequence similarity with a previous reported sequence by devriese et al. conclusion: this case report describes the first identification of s. pseudintermedius as a significant pathogen in human. growth characteristics and commercial identification systems misidentify the organism as s. aureus. when confronted with an inconsistent phenotypical identification pattern, clinical labs should consider the use of 16s rrna gene sequencing for final confirmation. characterisation of staphylococcus aureus isolates recovered from dairy sheep farms (agr group, adherence, slime, resistance to antibiotics) e. vautor, m. sabah, g. mancini, m. pepin, h. carsenti-dellamonica (sophia-antipolis, nice, fr) objectives: the purpose of this study was to investigate 46 staphylococcus aureus natural isolates associated with dairy sheep mastitis for epidemiological key features (agr group, adherence, slime production and antibiotics resistance). methods: the s. aureus isolates (n = 46) were recovered from a field study in the southeast of france in 2001-2004 (28 from subclinical mastitis, 10 from clinical mastitis, 8 from the environment of the dairy sheep farm). a total of thirteen dairy sheep farms, producing cheeses manufactured with raw ewe's milk, were involved. the agr group were determined by multiplex and real-time pcr. the evaluation of adherence and slime production were assessed with methods previously described by christensen et al. (1982) . the susceptibility patterns to 11 antibiotics were determined using the discdiffusion method on mueller-hinton agar plates. oxacillin susceptibility testing was performed on all the isolates. the 10 others antibiotics susceptibility was only studied on the 28 isolates recovered from subclinical mastitis as they represent the major source of cheese contamination. results: 80% (37/46) of the isolates belonged to agr group 3, regardless of clinical findings. 39% (18/46) were adherent, strongly adherent or with maximal adherence (biofilm producers). 26% (12/46) were slime producers (moderate or strong producers). all the isolates (n = 46), but seven, were susceptible to all the antibiotics tested. two isolates recovered from subclinical mastitis were resistant to oxacillin and partly resistant to ampicillin and penicillin-g. the five other isolates were found: partly resistant to erythromycin (n = 1), cefoperazone and penicillin-g (n = 1), erythromycin (n = 1), neomycin (n = 1) or resistant to enrofloxaxin and partly resistant to ampicillin and penicillin (n = 1). conclusions: s. aureus isolates recovered from sheep mastitis in the southeast of france are mainly related to agr group 3 suggesting a role for agr-regulated proteins in the persistence of this bacteria in the sheep udders. biofilm and slime production may also be an important aspect for intracellular survival of s. aureus which could promote the development of persistent intramammary infections. finally, ewe's milk does not appear to represent a source of resistant s. aureus and specially methicillin (oxacillin)-resistant s. aureus (mrsa) for human health. detection of virulence genes in staphylococus aureus isolates from dairy sheep, goats and cows mastitis, using single-dye dna microarray e. vautor, v. magnone, g. rios, m. pepin, p. barbry (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy farms animals. although many putative virulence factors have been identified in s. aureus genomes (kuroda et al., 2001) , the differences in pathogenic potential between naturally occurring isolates remain largely unaddressed. the relative importance of host (tissue) factors versus bacterial virulence determinants in disease pathogenesis is not well known, but it is widely accepted that bacterial factors including toxins, cell wall-associated adhesions, and secreted exoproteins are involved in the process. in this study, we use a single-dye dna microarray assay to investigate the presence or absence of 196 putatives virulence genes in 75 s. aureus isolates recovered from cases of ovine, caprine and bovine mastitis. methods: mastitis s. aureus isolates: sheep (n = 25), goats (n= 22), cows (n = 20).dna microarray: the arrays were spotted with long oligonucleotides (65-mer) representing 192 known virulence genes and new candidates identified in mu 50 genome (a human strain) and other s. aureus genomes. each gene were spotted four time. dna extracted from the strains were labelled with fluorescent cy5 using the bioprime ò array cgh (invitrogen). control strains with known genetic and phenotypic characteristics were used to normalize the data. results: (i) the majority of the virulence gene was detected in all the isolates (e.g. coa, ica adbc operon, htra, hysa, nuc, sbi, sdre, ssp, feob, fnb, sib, spa). (ii) genes were not detected in the majority of the isolates (e.g. cna, edin, lukf-pv, sav1999,…). (iii) genes were not found in isolates, depending on the herd (e.g. aur or sav1038 absent in isolates from some dairy sheep farm), on the isolates whatever the species (i.g. bsap, caph, entk, eta, fnbb, hsds, lpl2, lukd, …) . but we found gene mainly related to species (e.g. agriii, sav2496,…) comprehensive results will be given in the poster. conclusions: the present study indicated that the prevalence of virulence genes among s. aureus isolates recovered from dairy farm species depends on the gene. these observations suggest a common occurrence of host-adapted (or tissueadapted) s. aureus strains in which particular virulence genes may play a significant role. when taken with complementary methods such as pcr or/and southern hybridisation, singledye dna microarrays may provide a powerful tool to type s. aureus strains for epidemiological and possibly pathogenesis studies. detection of dna sequences distinguishing two closely related genomes of staphylococcus aureus from subclinical versus gangrenous ewe mastitis strains n. chevalier, c. huard, r. thiery, e. vautor (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy sheep. the severity of mastitis ranges from subclinical to gangrenous forms. subclinical mastitis is an inflammation that is not readily detected clinically whereas gangrenous form is an acute necrotizing mastitis. with the ain to find genetic markers or virulence factors that are only present in gangrenous strains a suppression substractive hybridisation (ssh) method was used in the present study to compare two strains of s. aureus respectively recovered from subclinical or gangrenous mastitis in the same dairy sheep herd. methods: 42 ewes were held in the investigated farm. the subclinical strain was recovered in january 2002 from the milk of 6 ewes. the gangrenous strain was recovered in december 2002 from an primipare dairy sheep that subsequently died from this acute mastitis. dna extracted from the strains were first compared by pulsed field gel electrophoresis (pfge). then, ssh was performed by using dna from the subclinical strain (driver), as described in a commercial kit (clontech pcr-select bacterial genome substraction kit). results: using pfge, four band differences were found between the two strains. two dna fragments, presumably specific from the gangrenous strain were detected by ssh and sequenced: (i) a 262 bp (98% of homology with the sulfide quinone reductase contained in orf 11 pathogenicity island of the mrsa 252 strain) (ii) 280 bp (98% homology with a gene coding a bacteriophage holine contained in the s. aureus n315 genome). control pcr tests using primers designed from these specific gene candidates confirmed that they were only present in the s. aureus gangrenous strain. conclusions: according to tenover et al. (1995) , a 4 band difference using pfge indicates that the strains may possibly be related genetically. although genes classically involved in the virulence of s. aureus were not detected in the present study, two putative virulence factors were detected. the sulfide quinone reductase allows s. aureus to growth on sulfide (found in animal manure). the holine protein breaks the internal membrane of s. aureus to release daughter phages suggesting that a mechanism of horizontal gene transfer could have been mediated by bacteriophages and could explain the acquisition of virulence factors. antimicrobial clinical trials p1702 outpatient treatment of acute pyelonephritis in pregnancy after 24 weeks. a randomised controlled trial z. ahmadinejad, s. hantooshzadeh (tehran, ir) objectives: the purpose of this study was to compare the safety and efficacy of outpatient and inpatient treatment of acute pyelonephritis in pregnancy. methods: this was a randomized controlled, clinical trial. one hundred twenty eight gravidas past 24 weeks' gestation admitted in imam khomeini hospital, tehran & sahid dr bahonar hospital, kerman, divided by random blocks to outpatient or inpatient therapy, received two 1-g doses of intramuscular ceftriaxone at 24-hour intervals while hospitalized, then were discharged and reevaluated within 48-72 hours or remained hospitalized until afebrile for 48 hours. all patients completed a 14-day course of oral cephalexin. we performed urine cultures on admission and 10-14 days after therapy. results: the two groups were similar with respect to age, parity, temperature, estimated gestational age, initial white blood cell count, and incidence of bacteremia. there were not any significant differences between two groups about the clinical improvement after 48-72 hours, bacteriuria 10-14 days after treatment, relapse of pyelonephritis, requirement to change in antibiotic, date of pregnancy at delivery and preterm labor. the relapse of bacteriuria and preterm labor in inpatients were significantly more than outpatients (pv = 0.0077 and 0.030 respectively). the birth weight of neonate in outpatients were significantly more than inpatients (pv = 0.013). conclusion: outpatient antibiotic therapy is effective and safe in selected pregnant women with pyelonephritis. however in this study, the neonatal outcomes were better in outpatients and the maternal outcomes in inpatients. experience with daptomycin in patients with renal insufficiency investigators collected demographic, disease state, clinical and microbiological data; outcomes were defined using standard definitions. patients nonevaluable for outcome were excluded. core data were divided and data on cohorts of pts with a creatinine clearance (crcl) ‡30 or <30 ml/min were examined. results: of the 1160 pts enrolled, 770 (66%) had evaluable pt outcomes and either crcl ‡30 ml/min (nml, n = 598) or crcl <30 ml/min not yet requiring renal replacement therapy (ri, n = 172). the distribution of males and females was equal in both groups. ri pts were older (45% ‡66 yrs vs 29%, p < 0.01). the groups did not differ in the percent coming from the community setting prior to starting dap (nml 46%, ri 49%). nml had more frequent history of fractures/orthopaedic procedures (11 vs 4%, p < 0.01) and haematological cancers (6 vs 1%, p < 0.02) while ri had higher rates of any renal disease (4 vs 17%, p < 0.01), chf (6 vs 12%, p < 0.05) and other immunologic/ inflammatory disease (2 vs 11%, p < 0.01). ri had higher rates of skin infections (60 vs 69%, p < 0.05) and endocarditis (3 vs 6%, p < 0.05). infections that were frequently reported for nml and ri were bacteremia, non-catheter-related (10 vs 10%), bacteremia, catheter-related (10 vs 4%), osteomyelitis (14 vs 7%), and foreign body-orthopaedic (6 vs 5%), all p > 0.05. methicillin-resistant staphylococcus aureus was the most common pathogen; nml 41%, ri 38%. ri had higher rates of coagulase-negative staphylococci (9 vs 15%, p < 0.02) and viridans streptococci (0.3 vs 1.7%, p < 0.05). there was no difference in the percentage receiving antibiotics prior to dap; nml 75%, ri 68%. the mean dap dose and duration were similar; nml 4.7 mg/kg for 18 d, ri 4.6 mg/kg for 16 d. the most frequent dose was 4 mg/kg; nml 58%, ri 39%. ri initial dap dosing was more frequent than recommended (q 48 h) in 76%. the mean time to clinical response was similar; nml 5.2 d, ri 5.4 d. more pts in nml received concomitant antibiotics with dap; 53 vs 35%, p < 0.01). the clinical success (cure and improved) rates were; nml 95%, ri 96%. conclusion: dap shows favourable clinical success rates in pts regardless of the presence of renal insufficiency. in vitro activity of second line antibiotics against helicobacter pylori infection objective: the aim of our study was to determine the in vitro activity of levofloxacin, ciprofloxacin and rifampicin in clinical strains of h. pylori. material and methods: 40 isolates of h. pylori from biopsies of dyspeptic patients were obtained following standard methodology. in vitro activity of metronidazole, clarithromycin, levofloxacin, ciprofloxacin and rifampicin was determined by e-test using 5% sheep blood agar and incubated at 37ordm;c during 3-5 days in a co2 atmosphere. mic was determined as the point of complete inhibition of growth. breakpoint of the nccls for other microorganisms were considered for fluorquinolones: resistant if mic > 4 mg/ l. for rifampicin we considered the strain susceptible if mic <32 mg/ l, as same studies reported. results: 42.5% of the strains were resistant to metronidazole and 50% to clarithromycin. mic50, mic90 and range (mg/l) was: 0.064, 0.125 and 0.012 fi 32 for levofloxacin, 0.064, 0.25 and 0.006 fi 32 for ciprofloxacin and 0.75, 1.5, and <0.002-4 for rifampicin. all the strains were susceptible to rifampicin and only 2% of them were resistant to fluorquinolones. conclusions: the fluorquinolones tested and rifampicin showed an excellent in vitro activity against h. pylori, despite the high resistance rate to metronidazole and clarithromycin. however, in vitro susceptibility test should be done before the use in clinical practice. vibrio antibodies in serum and breast milk samples of parturient women in calabar, nigeria objectives: serum and breast milk samples from 335 parturient women and serum from non-parturient controls were analysed for prevalence and titres of vibrio antibodies. methods: v. cholerae agglutinins and vibriocidal antibodies in serum samples were analysed by direct agglutination and immune bacteriolysis techniques respectively, using 96 well microtitre plates. the protective value of breast milk was evaluated by haemagglutination inhibition and rabbit intestinal mucosal attachment of v. cholerae cells. results: vibrio agglutinins were detected in serum samples of 191(57.0%) parturient and 78 (33.9%) non-parturient subjects (p < 0.05). high prevalence rates of 68.9% and 39.1% occurred among parturient and control subjects of 21-25 years of age respectively. at 1:160 cut off titre to evaluate vibrio cholerae specific bacteriocidal antibodies, activity was detected in samples of 31 (57.1%) and 9 (56.3%) parturients and controls respectively aged 26-30 years. breast milk from 67 (20.0%) parturients contained vibrio agglutinins with titres ranging between 1:20 and 1:320, while milk samples from 32 subjects showed haemagglutination inhibition (hi) activity titres of p 1:40. of the 19 hi positive milk samples 17 (89.5%) showed inhibition of v. cholerae adherence to rabbit intestinal mucosa at titres p 1:80, and 53-92% reductions in cell attachment. conclusion: our study confirms that parturient women in calabar may benefit from significant serum titres of v. cholerae antibodies and provide immune protection for their babies through breast milk secretions. moxifloxacin vs clarithromycin for treatment of community-acquired pneumonia associated with common respiratory pathogens: a pooled analysis objectives: streptococcus pneumoniae and haemophilus influenzae are pathogens commonly associated with community-acquired pneumonia (cap). this study compared the clinical and bacteriologic efficacy of moxifloxacin (mxf) to clarithromycin (clar) in cap patients with these pathogens. patients and methods: data were pooled from three doubleblind, multicenter, phase iii trials comparing oral mxf 400 mg qd to clar 500 mg bid for 10 days. all patients included had mild-to-moderate cap. clinical and bacteriologic success rates were identified for s. pneumoniae and h. influenzae isolated from these studies. data for the efficacy-valid population was recorded at the test-of-cure (toc) visit (7-35 days post-therapy). results: 1437 patients were entered, of which 356 were microbiologically evaluable. infection with s. pneumoniae and/or h. influenzae was documented in 149 (42%) of patients (5 mxf and 2 clar patients had mixed infection). within this cohort, the two treatment groups were well balanced based on demographic/baseline medical characteristics (55% male, mean age 50 yrs, 41% smokers, 8% recent antimicrobial therapy). clinical success and bacteriologic eradication rates (one response per patient) at toc are presented in the table. conclusions: in cap associated with s. pneumoniae and h. influenzae there was a trend towards greater bacterial eradication for mxf vs clar. clinical success rates were significantly higher for mxf monotherapy vs clar. variability of creatinine clearance measurements in inpatients with community-acquired pneumonia r. grossman, s. choudhri, d. haverstock (mississauga, ca; west haven, us) objectives: moxifloxacin, levofloxacin and gatifloxacin have been recommended as empiric therapies for patients with community-acquired pneumonia (cap). levofloxacin and gatifloxacin require dose-adjustment for renal insufficiency while no dose adjustment is required for moxifloxacin. this study was designed to determine the frequency and underlying variability of renal insufficiency in patients with cap. methods: a pooled analysis of data from 2211 patients with mild to moderate or severe cap entered into one of six randomized, controlled clinical trials was undertaken. renal function (calculated creatinine clearance; crcl) was assessed in each patient prior to treatment with mxf and then again during and post-treatment. results: baseline crcl levels in this pooled population of patients with cap were: <25 ml/min in 47 (2.1%) of patients, 25-49.9 ml/min in 436 (19.7%) and ‡50 ml/min in 1728 (78.2%) patients. after the pre-treatment crcl measurement 88 patients (4%) were lost to follow-up, so there was no during or post treatment value. in patients with cap the crcl improved from baseline in many patients during or post-treatment, while some patients experienced a worsening of renal function (see table) . conclusions: renal function (crcl) is highly variable in cap patients with baseline evidence of renal insufficiency. renal function should be monitored closely to permit appropriate dose adjustments if levofloxacin or gatifloxacin is used in this patient population. moxifloxacin may be a better empiric choice in this setting as it does not require dose adjustment in patients with renal insufficiency or renal failure. a prospective, controlled, randomised, nonblind, comparative study of the efficacy and safety of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice-daily ceftazidime plus amikacin in the empirical therapy of febrile neutropenic patients objective: empirical antibiotic treatment for febrile neutropenia is well established. the best regimen is still controversial. the purpose of this study was to evaluate the efficacy, safety and cost of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice daily ceftazidime plus amikacin in neutropenic febrile patients. patients and methods: ninety-five patients with febrile neutropenia were included in a prospective, controlled, randomized, non-blind, comparative study. patients were randomly assigned to either treatment group (63 in the ceftriaxone/ciprofloxacin group and 32 in the ceftazidime/ amikacin group) and evaluated as successes or failures according to defined criteria. daily assessments were made on all patients all adverse events were record. results: the overall incidence of documented infections was 45.9%: 24/47 (51.1%) in the ceftriaxone/ciprofloxacin group and 10/27 (37%) in the ceftazidime/amikacin group. there was significant difference in clinical efficacy between groups (p = 0.011) at the end of therapy. ceftriaxone/ciprofloxacin group had an overall incidence of resolution and improvement of 95,7% in comparison to the 75% of the ceftazidime/amikacin group. thirty-nine organisms were isolated, 26 (66.67%) gramnegative and 13 (33,33%) gram-positive. there was low incidence of adverse events in both groups. conclusion: the combination of high dose single daily ceftriaxone plus ciprofloxacin was more effective than the standard combination of thrice daily ceftazidime plus amikacn with no significant adverse events in either group. objective: in past studies of azithromycin in children, a posttreatment (pt) benefit was observed at day 28. in 5 recent phase 3 trials in adults, single-dose zmax was at least as effective as standard comparators for treatment of respiratory tract infections (rtis), including cap. our objective is to demonstrate a pt benefit in this adult population. methods: post-hoc analyses, including respiratory adverse event burden (raeb), were conducted on the all treated population (n = 2596; 1292 az-m, 1304 comparators) in the 5 phase 3 studies. the raeb is the sum of duration, in days, of all respiratory adverse events, divided by total number of observation days of all patients, normalized to 1 year. the overall and per study day raeb were calculated for zmax and the pooled comparators for the 5 studies combined. results: raeb, in days/patient year, was 15.5 for az-m patients vs 20.2 for comparator patients (p = 0.08). the difference in raeb consistently and progressively favoured zmax, beginning at day 11 and achieving statistical significance between days 29 and 35, when the upper limits of the 95% cis around the differences were below zero (figure). faropenem medoxomil (fm) is an oral penem with potent activity against streptococcus pneumoniae and haemophilus influenzae. this integrated analysis was conducted to summarize the efficacy of 10 days of 300 mg bid of fm compared with other beta lactams in the management of community acquired pneumonia (cap). methods: efficacy was determined in three multicenter randomized double-blind controlled trials (rct) and a single uncontrolled study of faropenem medoxomil. comparators were 14 days of cefpodoxime (c), 10 days of amoxicillinclavulanate (ac), or 10 days of amoxicillin (a). the analysis allowed examination of treatment effects by age, race, gender and study site subgroups. results: a total of 2267 subjects were studied. studies 1 and 4 were conducted in n. america, studies 2 and 3 in europe, latin america, israel, and s. africa. n. american vs. other studies included subjects at least 12 (vs. at least 18) years of age and only out patient (vs. outpatient and hospitalized) subjects. the clinical responses for fm in both per protocol and intention-to-treat populations were non-inferior to comparator for each study and for the three trials combined. no differences were found in treatment effect by age, race, gender, or country. recovery of an etiologic agent from initial respiratory or blood culture varied between 9.4 and 17.5% of cases in the 4 studies for a total of 236 microbiologically evaluable subjects. s. pneumoniae was eradicated or presumed eradicated in 82/90 (91.1%) and 49/52 (94.2%), h. influenzae in 49/61 (80.3%) and 30/32 (93.8%), s. aureus in 13/14 (93%) and 8/10 (80%), h. parainfluenzae in 6/8 (75%) and 0/2 (0%), and m. catarrhalis in 6/7 (85.7%) and 4/4(100%) fm and comparator recipients, respectively. clinical response for s. pneumoniae bacteremic patients was 18/21 (85.7%) for fm. conclusions: fm efficacy was consistent across studies, within subgroups, and non-inferior to comparators. it is efficacious against the most common bacterial pathogens and in the most severe form (bacteremic) disease. fm is a good option for the treatment of cap. propionibacterium acnes strains isolated from acne vulgaris and severe infections c. oprica, c.e. nord (stockholm, se) propionibacterium acnes is a member of the resident flora of the skin and is an important factor involved in inflammatory reactions in acne patients. during the last years the prevalence of different severe infections due to p. acnes has increased. objectives: 1) to detect the prevalence of resistant p. acnes strains isolated from acne patients in stockholm and different severe infections in europe; 2) to identify the mechanisms of resistance and the genetic diversity among resistant strains. methods: p. acnes strains isolated from acne vulgaris and severe infections were tested against clindamycin, erythromycin, linezolid and tetracycline and pulsed-field gel electrophoresis was used for further characterization. pcr and sequencing of the genes encoding domain v of 23s rrna for clindamycin and erythromycin resistant strains and 16s rrna for tetracycline resistant strains were performed. results: i) antibiotic-resistant strains were more often isolated from antibiotic treated patients with moderate to severe acne area than from non-antibiotic treated acne patients. an individual might harbor different pulsotypes of p. acnes with various degrees of resistance. ii) among the 304 clinical isolates from 13 european countries were found resistant strains to tetracycline, clindamycin, and erythromycin. overall, in the southern europe a higher prevalence of erythromycin-resistant strains was noticed and in southern and eastern europe a higher prevalence of resistance to clindamycin. it was noticed a high genomic diversity and the geographical spread of some clones in related areas but also in geographically distant countries. most clindamycin or erythromycin resistant p. acnes isolates, were found to be members of a single clone that has spread in different geographically countries. iii) p. acnes clindamycin and erythromycin resistant strains carrying one of the described mutations within the 23s rrna were predominantly isolated from swedish acne patients compared to strains from other infections. forty-four per cent of tetracycline resistant strains were found to carry a mutation in the 16s rrna. these strains were isolated from swedish acne patients, were highly resistant and were clustered in one pulsotype. conclusion: surveillance of both the prevalence of resistant p. acnes strains and associated resistance mechanisms is important due to the rapid variation in resistance patterns, both in acne patients and other severe infections. antimicrobial activity of unisepta quick and deconex solarsept on the surface contamination and dental instrument in dental clinics in iran f. shahcheraghi (tehran, ir) objectives: quaternary ammonium compounds (qacs) are amphoteric surfactants that are widely used for the control of bacterial growth in clinical and industrial invironment.unisepta quick and deconex solarsept are new generation of qacs is widely used as adjuncts in iran to hygine in dental clinics.the aim of present study was to investigate clinical efficiency of these substances on the surface and instruments in dental clinics. material and methods: the following bacteria and fungi on the base of aoac standard were used.pseudomonas aeruginosa (atcc 27853), staphylococcus aureus (atcc 25923) bacillus. subtilis atcc (6051), mycobacterium bovis atcc (35443) and wild types of trichophyton mentagraphit, p. aeruginosa and salmonella typhimorium (a common fungi in iran). a stock solution of deconex solarsept (borer chemie) and unisepta quick (micro 10 unident) was prepared as recommended by the manufacturer. the concentration of bacterial suspention was 0.5 macfarland and the results were reported on the base of decreasing in (cfu) colony forming unit from 107 to 102. results: the results shows that both of these disinfectants have bactericidal and fungicidal activity on the standard p. aeruginosa, s. aureus, s. typhimurium and trichophyton mentagraphit, the number of bacteria decreased significantly (p < 0.05), but no significant difference was seen with b. subtilis, wild type of p. aeruginosa and m. bovis. conclusion: the results confirm that these qacs are not able to sterilize or disinfect medical and dental instruments, and they can not be used lonely, and it must be used with the other methods for sterilization of surface and dental instruments. macrolide as long-term treatment in patients with bronchiectasis colonised by p. aeruginosa background: a certain efficacy of macrolide against p. aeruginosa has been described in vitro, mainly through mechanisms such disruption of quorum sensing and suppression of inflammation. aim: to evaluate the efficacy of macrolide in patients with bronchiectasis colonised by p. aeruginosa. methods: the study prospectively included patients with bronchiectasis and p. aeruginosa isolated in sputum in stable state. all subjects received either azithromycin 250 mg · 3 days/week or clarithromycin 500 mg daily on long term and completed daily diary cards for symptoms and pef values until the end of therapy. follow-up period was 1 year. results: 26 patients with bronchiectasis and p. aeruginosa evidence in sputum were included (17 men, mean age 55.0 ± 12.1 yrs.). 12 patients received azithromycin and 14 patients clarithromycin, with a mean duration of 4.5 ± 1.7 months. five (19.2%) patients discontinued treatment after less than 2 weeks because of adverse events. at the end of therapy, 12 (46.1%) patients showed no evidence of p. aeruginosa in sputum while 9 (34.6%) patients still had ps. aeruginosa in sputum. an improvement in the following parameters could be observed in all patients: sputum volume (75.5 ml/day before therapy versus 31.6 ml/day after therapy, p = 0.04); pef (311.2±77.1 l/min before therapy versus 476.2 ± 30.2 l/min after therapy, p = 0.03); number of exacerbations/year (2.8 in the previous year versus 1.7 in the follow-up year, p = 0.01). conclusion: the study shows that macrolide may be an effective therapy in patients with bronchiectasis colonised by p. aeruginosa. independently of the microbial eradication, an improvement of the clinical symptoms and a reduction of exacerbations were observed in all patients. fungal pathogens from haematoncology patients and their susceptibility to new and old antifungal drugs the expanding population of immunocompromised hosts has been infected with many established and emerging opportunistic fungi. most pathogens can be treated empirically whereas for an increasing number of species proper treatment starts once the mic becomes available. though invasive aspergillosis remains the principle life threatening complication in the haematoncology patients (hop) other pathogens cannot be ignored as selection and resistance during prophylaxis increases the risk of treatment failure.in order to understand the frequency of rare fungal pathogens, selection and emergence of resistance in our trust all fungi from hop were identified using standard mycological techniques and the mics to amphotericin b (amb), flucytosine (5fc), fluconazole (fcz), itraconazole (itz), voriconazole (vcz) and caspofungin (cfg) were determined using the nccls method. 513 specimens were processed, 53% respiratory, 17.5% blood, 12.8% oral, 8.4% other sterile (bile, csf, drains, lines and tissue biopsies) and 8.3% nonsterile sites. yeasts accounted for 85% and filamentous fungi (ff) for 15%, representing 12 candida sp, 7 other types of yeast, 6 aspergillus sp and 8 other types of ff. c. albicans represented 35.7%, c. glabrata 29.3%, c. krusei 8.6%, a. fumigatus 10% and other aspergillus sp 2% of all isolates. the mic 90s for all isolates were amb 0.5, 5fc 2, fcz >64, itz 1, vcz 1 and cfg 0.06 mg/ l. with the exception of acremonium sp, a. versicolor, a. terreus and scedosporium apiospermum all isolates including the 15 isolates of c. lusitaniae were sensitive to amb. most but not all ff and only one isolate of c. albicans from the yeasts were resistant to 5fc. all ff, rhodotorula sp, c. albicans 3%, c. glabrata 53% and c. krusei 41% were resistant to fcz. only absidia corymbifera, acremonium sp 25%, c. albicans 1%, c glabrata 33% and saccharomyces cerevisiae 4% were resistant to itz. for vcz a. corymbifera, acremonium sp 25%, c. albicans 2%, c. glabrata 31%, c. krusei 2%, c. tropicalis 10%, rhodotorula sp 66.6% and p. aecilomyces variotii 50% had an mic ‡2 mg/l. with cfg the effective concentration was ‡0.5 mg/l for a. corymbifera, fusarium solani, geotrichum capitatum, sporobolomyces salmonicolor, acremonium sp 50% and c. parapsilosis 25%.the data show that hop are exposed to many different fungal pathogens some of which are resistant to the old and the new antifungals and that amb is still the drug with the broader spectrum and less developed resistance for both yeasts and ff. faropenem medoxomil in the treatment of acute bacterial sinusitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial agent with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute bacterial sinusitis (abs) was evaluated in 3 phase iii trials; 100288, 10186, and 100287. study 100288 was conducted in n. america, study 10186 was conducted in europe and israel and study 100287 was conducted in the us and argentina. 100288 and 10186 were prospective, randomized, double-blind, active comparator trials and 100287 was an open-label ''sinus tap'' trial. the dose of fm was 300 mg bid in all 3 studies. the comparator in 100288 and 10186 was cefuroxime axetil (cfx) 250 mg bid. the duration of fm treatment in 100288 was 7 days and 10 days vs cfx for 10 days. in 10186, fm or cfx were given for 7 days. in 100287, fm was administered for 7 days. the primary efficacy variable in all 3 studies was clinical response at the test-of-cure (toc). microbiologic response at the toc was a secondary efficacy variable in 10186 (sinus puncture and endoscopic collection) and 100287 (sinus puncture and aspiration). non-inferiority was defined as the difference in cure rates (fm minus comparator) where the lower boundary of the 95% ci was greater than -10%. results: the cure rates at the toc are shown in the table for the valid per protocol (vpp) and the intent-to-treat (itt) populations. the frequency of isolation of key pathogens and the rate of eradication in samples obtained by endoscopicallyguided swab and in samples obtained by tap were consistent across studies. the eradication rates for s. pneumoniae, h. influenzae, and m. catarrhalis were 94.8% vs. 96.3% (fm 7/ 10 d vs. cfx 7/10 d), 82.4% vs. 90.5% (fm vs. cfx) and 96.2% vs. 83.3% (fm vs. cfx), respectively. conclusions: fm 300 mg bid x 7 days was shown to be noninferior to cfx in clinical efficacy in two prospective, doubleblind, comparative trials. a third, open-label trial, demonstrated similar efficacy in microbiologically documented abs caused by key pathogens. longer (10 d treatment) with fm provided no additional efficacy. faropenem medoxomil in the treatment of acute exacerbation of chronic bronchitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute exacerbation of chronic bronchitis (aecb) was evaluated in 2 phase iii trials. study 10187 was conducted in europe, israel, mexico, and south africa. study 100291 was conducted in the us and argentina. both were prospective, randomized, double-blind, active comparator trials. the dose of fm was 300 mg bid for 5 days in both studies. the comparators were clarithromycin (clr) 500 mg bid for 7 days and azithromycin (azi) qd for 5 days (500 mg on day 1 and 250 mg on . the primary efficacy variable was clinical response at the test-of-cure (toc). microbiologic response at the toc, in subjects with a baseline pathogen was a secondary variable. non-inferiority was defined as the difference in cure rate (fm minus comparator) where the lower boundary of the 95% ci was greater than )10%. results: the cure rates are shown below for the valid per protocol (vpp), intent-to-treat (itt) and modified itt populations (all itt subjects who met inclusion/exclusion criteria). in both the individual studies and the pooled analyses, for all populations, treatment with fm was not less effective than either comparator. 20% of treated subjects in 10187 and 28% of subjects in 100291 were evaluable for microbiological response. in 10187, the eradication rates for the microbiologically evaluable population was higher in the clr group (77.1%) compared with the fm group (68.6%) (95% ci )23. 4, 6.3) . in contrast, the eradication rate in 100291 was similar in the fm (80.0%) and azi (78.9%) groups (95% ci -9.7, 11.8). when the data were pooled across studies, the response rates were similar with fm (75.8%) and combined comparator (78.2%) groups (95% ci -11.0, 6.3). the combined eradication/presumed eradication rates in the pooled fm and comparator groups were 81.8% vs. 86.6%, respectively for s. pneumoniae and 76.0% vs. 77.6%, respectively, for h. influenzae. conclusions: fm was shown to be non-inferior to either azi or clr in clinical efficacy in two adequate and well-controlled trials. pooled analysis further strengthened the clinical noninferiority conclusion. the difference in eradication rates observed in study 10187 (clr) was not supported by study 100291 (azi). an integrated safety analysis of faropenem medoxomil: results of 5,023 subjects from phase ii/iii clinical trials r. echols, r. tosiello (milford, us) objective: to evaluate the safety profile of faropenem medoxomil (fm), a novel oral penem antibiotic. methods: 5,023 subjects from 3 phase ii and 14 phase iii clinical trials received fm, 300 mg bid for 3-10 days for treatment of acute bacterial infections. randomized controlled trials (rcts) included 4,223 fm and 3795 comparator treated subjects. analyses were conducted to identify possible disparate adverse event (ae) reporting based on type of infection, subject age (12-17, 18-45, 4664, 65-75, >75) and gender, duration of treatment (3/5 d v. 7/10 d), geography (na, eu, row), study design (open label v rct), relationship to treatment. comparisons were made to control treatment based on antibiotic class (b-lactam v. other), and individual antibiotic treatments. results: fm compared favourably to penicillins, cephalosporins and macrolides. fm was better tolerated than tmp/smx and co-amoxiclav. open labeled trials had higher aes reported v. rcts. aes reporting na = row > eu except serious aes and deaths where row = eu>na. aes for fm 3/5 d = fm 7/10 d. underlying infection did influence ae reporting. female gender had higher ae reporting than male gender. fm was tolerated equally well across age ranges, although deaths and saes were more common in >75 age group. common aes (>1%/related from rcts) were diarrhoea, nausea, fungal vaginosis and headache and were generally less frequent with fm than control rx. no evidence of neuro or cardio toxicity was identified. laboratory tests identified no hepatic, renal or hematopoietic signals. conclusion: faropenem medoxomil, a novel oral penem antibiotic, has the safety profile expected of a b-lactam but is better tolerated than co-amoxiclav with approximately one-third the gi side effects. the efficacy of non-surgical and systemic antibiotic treatment regimens in smoking and non-smoking patients e. pähkla, k. lõ ivukene, p. naaber, m. saag (tartu, ee) periodontitis is a chronic infectious disease, which leads to the destruction of periodontal ligament fibres and alveolar bone until tooth loss. the objective of this study was to compare the longitudinal effect of combination of non-surgical periodontal therapy with systemic antibiotics in smoking (s) and nonsmoking (ns) patients. methods: there were total of 28 patients with severe generalized chronic periodontitis involved in this study (14 s, 14 ns), who did not respond well to previous mechanical periodontal treatment. the clinical examination included recordings of visible plaque index (vpi), modified gingival index (mgi), bleeding on probing (bop) and suppuration after probing (sup), probing pocket depths (pd) and clinical attachment levels (cal). the non-surgical periodontal therapy was performed within 4 weeks. clinical parameters were recorded at baseline, 2-3 weeks after the first mechanical treatment and 14 months after combined treatment, during a regular check-up visit. as the patients did not respond to the conventional periodontal therapy, the microbiological analyses were taken and a combination of systemic amoxicillin 500 mg · 3 and metronidazole 250 mg · 2 for 7 days, was prescribed. results: the results suggested that the combined systemic antibiotic therapy is effective in case of severe generalized chronic periodontitis, as vpi, bop, sup, cal, and mgi improved significantly after the treatment. in the ns group all parameters, except cal, improved significantly after the treatment. the s showed markedly smaller reduction in sup, mgi, and cal.after instrumentation, no periodontal pathogens were isolated in 11 (39%) patients, while 17 patients (61%) were infected with one to three different pathogens. among the pathogens, prevotella intermedia/nigrescens (10 patients) and actinobacillus actinomycetemcomitans (8 patients) were dominating. the total level of microbial load (log10 cfu/ml) as well as the spectrum of pathogens in s and ns patients remained similar. conclusions: despite of positive treatment effect in general, there were insignificant improvements in any clinical parameters in the smoking group. smoking has adverse effect on periodontal therapy; therefore the dentist should cooperate with patients in counselling of smoking cessation to achieve better results in the treatment of periodontitis. objectives: laminin (ln), which is a large multidomain glycoprotein of the extra cellular matrix, has attracted much attention because of its importance in many cellular functions, including induction of cell adhesion, growth promotion and mediation of cell communication. the target of this study was to find out whether there is any relation between the levels of serum ln and the inflammatory activity of a microbial infection. patients/methods: from june to october 2005, 48 immunocompetent adults, with confirmed bacterial infection were admitted to our hospital (17 with pneumonia, 24 with pyelonephritis and 8 with cholecystitis) (group 1). at the same time 50 hospitalised patients for non-infectious causes (stroke, gastrointestinal bleeding, anaemia) were also studied (group 2). the levels of serum ln and crp were measured on the day of admission in both groups. the levels of ln were measured using an enzyme immunoassay kit (takara laminin eia kit) and 100 healthy volunteers were used to determine its normal limits (130-520 ng/ml). plasma crp concentration was assessed by immunoturbidometric method (using randox, uk kits). normal values were considered those below 10. results: the mean serum ln levels of patients of group 1 were 800.90 ± 249.29 (much higher that the normal limits), while the mean crp value was 117.53 ± 87.78. the mean corresponding values in group 2 were 475.79 ± 239.25 for ln (within normal limits) and 24.15 ± 32.77 for crp. there is a statistically significant difference between the mean ln levels of the two groups (p < 0.001). additionally, there is a statistically significant correlation between the levels of ln and crp (a well studied serum inflammatory marker) in patients with bacterial infection (group 1) (pearson correlation coefficient r = 0.565, p = 0.01). conclusions: the definition of the ln levels could constitute a new reliable, simple, direct serum marker for the confirmation of an active bacterial infection. additionally, as the crp levels are above normal in group 2 too (patients without infection) while ln lies within normal limits, maybe ln is even more specific than crp. more studies are required in the future, with more patients included, in order to confirm the outcome of this study. performance and clinical significance of a direct tube coagulase test using serum separator tubes for rapid identification of staphylococcus aureus from blood culture broth d. kwa, t. schü lin-casonato, p. sturm (nijmegen, nl) objective: blood cultures are important in the diagnosis of serious infections. early administration of effective antibiotics is associated with improved patient outcome. the performance of the direct tube coagulase (dtc) using serum separator tubes (ssts) for rapid identification of s. aureus from blood culture broth (bcb) was investigated. the clinical significance of rapid identification was assessed. methods: consecutive blood cultures with gram-positive cocci in clusters were tested. bcb was collected in ssts using a subculture-venting unit. after centrifugation, the supernatant was discarded and 1 ml rabbit plasma was added to the remaining pellet of bacteria. coagulation was evaluated after 2 and 4 hours incubation at 37°c, and after overnight incubation at room temperature. in parallel, a direct tube coagulase test was performed using a 1:10 saline dilution of bcb as described previously. isolates were identified by standard microbiology procedures. clinical significance was measured by comparison of antimicrobial prescription based on gram stain results, direct coagulase results, and culture results. results: over a 6-week period, 90 bcbs from 46 patients were tested. s. aureus was present in 22 bcbs. using the serum separator tube method and the saline dilution method, the sensitivity of the dtc after 2 hours incubation was 91% and 41%, and after 4 hours 100% and 88%, respectively. the specificity of both methods was 100%. rapid identification of s. aureus resulted in initiation (n = 1) or streamlining (n = 4) of antimicrobial therapy in 5 of 13 patients with s. aureus bacteremia. rapid identification of coagulase-negative staphylococci resulted in changes in antimicrobial therapy in 1 of 33 patients. conclusion: the dtc using ssts for bacterial enrichment is a very reliable, rapid, cheap and easy to perform method for identification of s. aureus from bcb. implementation of this test can improve antimicrobial therapy. evaluation of the results of the spanish seimc external quality control program for the diagnosis of enterococcus faecalis and klebsiella pneumoniae infections r. guna, j.l. pérez, n. orta, c. gimeno on behalf of seimc objectives: to evaluate the results obtained from four shipments of two different strains by the participants in the seimc external quality control program (eqcp). these controls were intended to analyse the percentages of correct species identification and the ability of the participants in detecting some special features of the control strains: vanb phenotype in the case of e. faecalis, and the production of extended spectrum betalactamase (esbl) in k. pneumoniae. methods: the same strain of each microorganism was sent in two different shipments. the vanb e. faecalis strain was sent both in a control of year 2002 as well as in other of 2004, while the esbl-producing k. pneumoniae was sent in 1999 and in 2005 to an average of 200 laboratories. the results obtained were compared with those of a reference laboratory that certified both the species identification and the resistance features. results: in the 2002 control, 92.6% of participants identified correctly e. faecalis, while 97.8% did it in 2004. as for the glycopeptide resistance pattern of the enterococcal strain, 39.9% and 53.3% of participants detected the vanb phenotype in 2002 and 2004, respectively. overall, the k. pneumoniae strain was correctly identified in both separate controls by most of the participants (97.1% and 98.6%, respectively). interestingly, the percentage of laboratories that detected the presence of the esbl in the k. pneumoniae strain sharply increased from 55.0% in 1999 to 89.4% in 2005. the overall percentages of correct species identification were high for the two microorganisms and for both control points. most important, the ability of the spanish clinical laboratories in detecting the special resistance features of these strains clearly improved along the study period. these data confirm the importance of implement a continuous surveillance of the diagnostic training in the clinical laboratory, as well as the possible positive intervention of the seimc external quality control program in such improvement, since the analysis of results is accompanied of updated reviews on the subject of each control. a. bonnet-pierroz, a. resenterra, o. péter (sion, ch) objective: to evaluate 2 new elisa ridascreen ò borrelia igg and igm for antibody response in patients with confirmed lyme borreliosis and to compare to the results of vidas lyme (igg-igm) and in-house immunoblots (b. garinii igg and igm for early cases or b. burgdorferi sensu stricto, b. afzelii, b. garinii, b. valaisiana igg for late cases). methods: elisa ridascreen ò borrelia igg and igm was used to screen sera from patients with clinically confirmed erythema migrans em (n = 30). patients with confirmed neuroborreliosis by intrathecal antibody synthesis (n = 45) were evaluated for igg antibodies to borrelia. sera from patients with acrodermatitis chronica atrophicans aca (n = 30) and sera and synovial fluids from patients with lyme arthritis (n = 30) were also evaluated for igg antibodies. patients with syphilis (n = 10) and infectious mononucleosis (n = 10) were screened for igg and igm antibodies to borrelia in order to estimate the specificity. conclusion: the elisa ridascreen ò borrelia igg and igm have shown a good sensitivity for the serological diagnosis of lyme borreliosis. the short evaluation for the specificity of the igg test revealed a good assay with few false positive reactions, whereas the igm assay was, as expected more prompt to give false positive results with sera from patients with infectious mononucleosis. so far any equivocal or positive tests should be confirmed by immunoblots. is it necessary to incubate the bact/alert blood culture bottles more than 3 days? objective: to assess the incubation time reduction of the aerobic and anaerobic bact/alert system bottles from 5 to 3 days. methods: from 1996 to 2004 we processed 94.303 blood culture sets and detected 9.432 (10%) positive blood cultures with clinical significance. we retrospectively examined the detection time of positive bottles and assessed the clinical significance of the bottles that were positive between the fourth and fifth day. results: out of 9432 positive blood cultures with clinical significance, 9238 (97.9%) were detected within the first 3 days of incubation. out of the 194 positive blood cultures detected between the fourth and fifth incubation days, 105 were recovered in concurrent cultures within the first 3 days. chart reviews were conducted from 78 patients with the remaining 89 isolates. only in 24 patients (0.3% positive blood cultures) changes in antimicrobial therapy based upon the positive blood culture results on day 4 to 5 were made, in the other patients the empirical treatment was adequate. the isolated microorganisms in those 24 patients were: 7 gram-positive cocci (3 staphylococcus spp. not s. aureus, 1 staphylococcus aureus, 2 streptococcus viridans and 1 streptococcus pyogenes), 5 anaerobes, 3 enterobacteriaceae, 2 pseudomonas aeruginosa, 2 campylobacter spp., 2 candida spp. 1 cryptococcus neoformans, 1 brucella spp. and 1 haemophilus influenzae. conclusions: incubation of bact/alert blood cultures bottles only for 3 days would have represented a detection loss of 0.3% of the clinically significant isolates, which led to antimicrobial therapy changes. although we keep employing a 5-day incubation for routine blood cultures, we could reduce the incubation time to 3 days depending on current instrument capacity. an enzyme immunoassay for anti-diphtheria antibodies: a practical alternative to the vero cell assay r. budd, e. harley, r. george, a. efstratiou, k. broughton, a. bradwell (birmingham, london, uk) introduction: in this extended study, results from an anti-diphtheria toxoid enzyme immunoassay (eia), specifically designed to detect higher affinity antibodies, were compared with those from a vero cell assay (vca). methods and results: 154 serum samples with antibody concentrations ranging from 0.008-8.000 iu/ml on the vca from the respiratory and systemic infectious laboratory (rsil) were assayed by eia (the binding site ltd, uk). a further 100 samples from rsil selected on the basis of being close to the protective level, were assayed to confirm the performance of the eia. the eia was calibrated, against the nibsc reference material 00/496 and the assay measuring range was 0.004-3.0 iu/ml results were compared using the who guidelines of 0.01-0.1 iu/ml as minimum protective level, and > 0.1 iu/ml as protective. relative agreement, sensitivity and specificity for the first 154 samples were: 91.6%, 98.8% and 83.3% respectively, for the second set of 100 samples performance was: 90.0%, 84.5% and 97.6%, and for the combined 254 samples results were: 91.3%, 92.9% and 89.4% respectively. roc analysis of the total 254 samples confirmed the highest sensitivity 92.9% and specificity 89.4% occurred at a cut-off of precisely 0.1 iu/ml for the elisa assay. conclusion: of the total 22 discrepant samples, 14 had vca and eia values < 0.149 iu/ml, therefore we suggest the possibility of establishing an equivocal zone for the interpretation of the eia results. if the test is part of a general immune status assessment a grey zone is not required. if undertaken to determine the requirement for immunization, the use of the equivocal zone is recommended. by applying these criteria in the eia, only one sample would have suggested inappropriate immunization, as indicated by a vca result > 0.149 iu/ml. because of the > 90% agreement between the two assays, significant advantages of cost and speed, ease of use and the potential for automation, the eia could therefore be considered as an alternative to the vca. evaluation of accuracy limits of countable colony-forming units on agar plates j. arbique, a. rendell, k. forward (halifax, ca) objectives: accurate colony counts are an essential component of many microbiology research projects and clinical laboratory processes. the suggested range of accuracy of colony-forming units (cfu) extends from 30 to 300 (standard methods for the examination of water and wastewater). this recommendation dates to 1907, and fails to adequately address the numerous sources of inter-and intra-variability. without more detailed analysis it is difficult to estimate the sample size and number of replicates necessary to ensure accurate results. the purpose of this study was to determine the validity of accuracy limits for quantifying cfus on agar plates. methods: escherichia coli (atcc 25922) and staphylococcus epidermidis (atcc 12228) were used to prepare series of four organism densities ranging from approximately 40-500 cfu, on three different days. on each day, each of the 4 densities for both organisms was plated on sba and viable organisms were counted following incubation. an average of the margins of error obtained over the 3 days of testing was used to determine the reproducibility of agar plate counts, and to estimate the optimum number of replicate plates (sample size) required for each organism at each concentration. results: margins of error for both organisms were greatest with suspensions yielding approximately 40 cfu, and lowest for suspensions yielding 300 and 500 cfu. nine replicate plates were required for a suspension of s. epidermidis yielding 40 cfu to achieve the same margin of error as obtained with 3 replicate plates at concentrations yielding 100-300 cfu. seven replicates plates were required for a suspension of e. coli yielding 40 and 100 cfu to achieve similar margins of error to those obtained with 4 replicate plates at concentrations yielding 300 cfu, and 3 replicate plates at concentrations yielding 500 cfu. conclusion: we found that the greater the concentration (300 and 500 cfu), the fewer replicate plates necessary to reliably estimate organism concentrations. the lower the organism density (40 cfu), the more plates necessary to reliably estimate cfus. contrary to the recommendations described in standard methods for the examination of water and wastewater, cfu of 500 were reliably reproducible. for greatest accuracy, experiments should be conducted so as to assure that colony counts are in the range of 300-500. direct microscopy: a valuable instrument for diagnosis and prognosis of periodontal disease objective: to appreciate the composition of micro flora from periodontal pockets, using light microscopy and to compare it with clinical status. introduction: it is generally accepted that periodontal disease occurs when anaerobic gram-negative flora increase in number with the subsequent decrease of facultative anaerobe grampositive bacteria. in other words, the switch from gram-positive to gram-negative of sub-gingival flora has a pathologic significance and could be observed using direct microscopy. materials and methods: 30 specimens sampled with sterile paper points from periodontal pockets and 10 samples from clinical healthy persons were included in this study. each sample was diluted in 0.5 ml saline solution and, with a calibrated loop, was taken 10 ll aliquots in order to prepare a smear for microscopic examination and for inoculation on solid media (columbia with 5% sheep blood). the smears were gram stained and the culture plates were incubated in anaerobic conditions (72 h, 37°c) and in air (24 h, 37°c). results: in 95% of samples from patients with periodontal disease, easily notable, high number of gram-negative bacteria at direct microscopy, associated with abundant growth in anaerobic condition and poor growth in air. in 9 from 10 healthy patients, the gram-negative flora was almost absent and gram-positive bacteria were in high number, correlated with the absence of bacterial growth in anaerobiosis and some growth in air.the presence of treponema spp. at direct microscopy was associated with deep and bleeding periodontal pockets. after few days of proper therapy, the good clinical status was well correlated with an increasing number of gram-positive bacteria. conclusions: 1) using a diluted sample for microscopic examination, the value of the method increase, offering important information about the composition of sub-gingival flora.2) the good correlation between the clinical status and microscopic finding recommend it as an easy to use diagnostic method in dentistry. identification of species and glycopeptide resistance among enterococcal isolates by bd phoenix objectives: vancomycin resistant enterococci are emerging in europe necessitating their fast and accurate identification by the laboratory. there was an attempt to evaluate the performance of the bd phoenix automated microbiology system (bd diagnostic systems, sparks, md.) for the correct identification of species and glycopeptide resistance in comparison to the gold standard of diagnosis, pcr, using a large collection of clinical strains. methods: a total of 232 enterococcal isolates were tested by the bd phoenix sytem. these strains were isolated from 145 faecal, 55 urine, 18 pus, 5 blood and 9 samples from other body sites cultures. a multiplex pcr was applied using 8 different pairs of primers, specific for the identification of e. faecium, e. faecalis and the vana, vanb, vanc, vand, vang, vane glycopeptide resistance genotypes. susceptibility to the glycopeptides was also confirmed by the etest (ab biodisk, solna, sweden). results: according to the pcr, there were 101 e. faecium (including 85 vana-positive strains), 72 e. faecalis (including 1 vana-positive and 1 vanb-positive strains) and 58 e. cass/gall isolates. two strains were not identified and were excluded from the analysis. discrepant results between the multiplex pcr and the phoenix system were obtained for 23/232 isolates (10%) with similar rates amongst faecal (15/145, 10.4%) and the rest of the isolates (8/87, 9.4%). the most common discrepancies were the misidentification of 11 e. faecium vana strains and 7 e. faecalis strains as e. cass/gall by phoenix. two e. faecalis strains were incorrectly characterized as vancomycin resistant, two e. faecium strains were misidentified as e. hirae and e.cass/gall, respectively, and one e.cass/gall strain was reported as e. faecium resistant to both glycopeptides. thus, the sensitivity and specificity for the identification of e.cass/gall by phoenix were 98.3% (57/58 strains) and 88.4% (145/164 strains), respectively, while 12.8% of vana strains (11/86 strains) were not recognized by this system. conclusion: this study demonstrates that the new identification system, phoenix, similarly to other automated or manual systems, presents with problems regarding correct identifica-tion of enterococcal species and glycopeptide resistance. specifically, laboratories should be aware that clinically significant isolates identified as e.cass/gall should be confirmed by another method. an audit of sputum requisition practices p. lal, i. balakrishnan (london, uk) objectives: to analyse the indications and rationale for the processing of sputum specimens in a london teaching hospital. methods: sputum samples received from 01/12/2004 -28/02/ 2005 were included in this study. data were obtained from the patient requisition forms and the winpath systems and were analysed further as per the objectives. results: a total of 1309 specimens were received during this period. 1202 (92%) from hospital in-patients and 107 (8%) from general practitioners. out of the total of 1202 samples received from hospital-in patients 167 (13.9%) had > 25 epithelial cells/ lpf. no clinical details were mentioned in 258 (21.4%) and 388 (32.2%) were from patients already on antibiotics. repeat specimens within one week were sent in 340 (28.2%) cases and 375 (33%) had atypical serology also sent. out of the 1202 hospital-in patient samples 372 (30.9%) had a significant isolate and 488 (40.5%) had normal respiratory tract flora isolated. others were reported as: ''gross oral contamination'' [168 (13.9%)], ''no growth'' [29 (2.4%)] and ''no significant growth'' [37 (3.07%)]. there were a few specimens reported as ''inappropriate specimen -two days old'' [35 (2.9%)] and ''leaking'' or ''saliva only' ' [22 (1.8%) ]. out of a total of 107 samples received from gp patients 14 (13.1%) of samples had > 25 epithelial cells/lpf, 25 (23.3%) had no clinical details provided, 5 (4.6%) samples were sent while patients were on antibiotics and 9 (8.4%) samples were repeated within one week. only 4 (3.9%) had atypical serology also sent. conclusions: -less than one-third of specimens yielded a significant pathogen.-adequate clinical details were lacking in about one-fifth of specimens.-nearly one-third of specimens were repeated within one week, without a clear indication.-about 16% of specimens were of poor quality.-atypical serology was only performed in 3.9% of outpatients, as compared with 33% of in-patients.this audit brings forth the fact that the clinical indications for which sputa are being sent for culture need to be clearly defined and an educational campaign instituted amongst relevant healthcare professionals. sputum collection techniques need to be rigorously applied if good-quality specimens are to be obtained. indications for performing atypical serology need to be defined and reinforced, particularly in primary care. a new approach to laboratory diagnostic of infectious gastroenteritis -a follow-up objectives: in order to optimize use of laboratory facilities and ensure flexibility in relation to current epidemiology, a new approach to laboratory diagnosis of infectious gastroenteritis was applied: from an algorithm the decision of which organisms to test for was defined by the demographic, clinical and epidemiological information submitted to the laboratory on paper/electronic request forms. methods: from april 1, 2004 -june 30, 2005 , hospitals and general practitioners submitted a request form with the following information together with the stool sample (s): (1) acute or persistent diarrhoea (duration > 2 weeks); (2) bloody stools; (3) recent history of foreign travel; (4) > 2 patients within same epidemiological setting; and (5) nosocomial infection. provision of data is mandatory when submitting electronically. based on these data, analyses were performed according to an algorithm. examination for salmonella, shigella, yersinia, campylobacter, and clostridium difficile was done by culturing. verotoxin producing e. coli (vtec), enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec) and enteroinvasive e. coli (eiec) were identified by pcr for virulence genes and serotyping. rota and adenovirus were detected by antigen tests and parasites by microscopy. results: in total we examined 8,628 samples from 4,088 patients. a pathogen was isolated in 19% of patients. in 673 cases (17%) clinical/epidemiological data were missing. • 1,205 patients had diarrhoea < 2 weeks: 13% with campylobacter, 5% with salmonella, 4% with etec, 3% with giardia, and 1% each with eiec and vtec • 1,502 patients had diarrhoea > 2 weeks: 3% with campylobacter, 2% with giardia, 1% each with epec and vtec • 740 patients had a history of foreign travel: 7% with campylobacter, 6% with etec, 4% with salmonella, and 4% with giardia • 214 patients had bloody stools: 18% with campylobacter, 3% with salmonella, and 2% with vtec • 896 patients were < 7 years: 4% with campylobacter, 3% with epec, 2% each with giardia, vtec and salmonella, and 1% with etec conclusions: campylobacter was the most common bacterial pathogens in all groups and rotavirus was the most common pathogen in children < 7 years. the new approach had a number of advantages: more relevant microbiological analysis, collection of data on defined patient groups, and flexibility regarding adaptation to current epidemiological knowledge. increasing use of electronic submission of request forms will optimize the approach used. objectives: small colony variants (scvs) are an emerging infectious disease problem, presenting as a naturally occurring, slow-growing subpopulation of staphylococcus aureus that are characterized by tiny colonies on solid media. studies on scvs recovered from patients with persistent infections are hampered due to their frequent unstable phenotype. in particular, scvs are not easily distinguishable from the normal phenotype in broth media and a reversion of scvs into the normal phenotype is not traceable. methods: a set of isogenic s. aureus isolates comprising the (i) normal and the (ii) scv phenotype (isogenic to the isolate with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding mutants mimicking the scv phenotype (knock-out of hemb), and (iv) their complemented mutants were used to investigate the feasibility of fourier-transform infrared (ftir) spectroscopy to trace the expressed phenotype in broth media. the respective isolates cultured on solid media served as controls. in addition, all isolates were genotyped by pulsed-field gel electrophoresis and spa typing. results: using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information in three different spectral ranges resulted in a dendrogram that showed a clear discrimination between both staphylococcal phenotypes. distinct clusters comprising the clinical and mutant scv phenotype on one hand and the normal phenotype (isolate with normal phenotype and complemented mutant) on the other hand were found. thus, scvs from different clonal lineages gave spectra that were more similar to one another than to their normal growth parent. ftir was also shown to be able to trace the switch of the phenotypes in broth when the medium was supplemented. conclusion: ftir spectroscopy allows a rapid, reproducible and clear discrimination of different phenotypes of s. aureus in fluid media for diagnostic and research purposes. in contrast to genotyping approaches, ftir staphylococcal fingerprinting is only reliable for typing purposes if the isolates exhibit the same phenotype. in future studies, this technique may also provide an approach for tracing the scv phenotype in infected tissues. objectives: triggering receptor expressed on myeloid cells-1 (trem-1) is a recently discovered cell surface molecule whose expression on phagocytes is up regulated by exposure to bacteria or fungi. a soluble form of trem-1 (strem-1) can be measured in various body fluids. we studied whether strem-1 in cerebrospinal fluid (csf) could serve as a biomarker for the presence and outcome in patients with bacterial meningitis. methods: in this retrospective study on diagnostic accuracy we used an elisa to determine levels of strem-1 in csf from 46 adults with bacterial meningitis, confirmed by csf culture, who participated in the prospective dutch meningitis cohort study; 8 patients with viral meningitis, confirmed by polymerase chain reaction of csf; and 9 healthy control subjects, who underwent lumbar puncture to exclude the diagnosis of subarachnoid haemorrhage. the mann-whitney u test and the chi-square test were used to identify differences between groups. a receiveroperating-characteristic curve (roc) was constructed to illustrate various cut-off csf levels of strem-1 in differentiating between the presence and absence of bacterial meningitis and diagnostic accuracy was quantified by 95% confidence intervals (95% ci). results: levels of strem-1 in csf were higher in patients with bacterial meningitis as compared to those with viral meningitis [median, 159 pg/ml (range, 0 to 988 pg/ml) versus 0.5 pg/ml (range, 0 to 48 pg/ml); p = 0.001] and controls [0 pg/ml (range, 0 to 36 pg/ml); p < 0.001; fig]. patients with viral meningitis and controls had similar csf strem-1 levels. the area under the roc curve for discriminating between patients with and without bacterial meningitis was 0.88 (95% ci, 0.80 to 0.96; p < 0.001). at a cut-off level of 40 pg/ml, strem-1 yielded a sensitivity of 0.80 (95% ci, 0.69 to 0.88) and a specificity of 0.94 (95% ci, 0.77 to 0.99). in patients with bacterial meningitis, csf strem-1 levels were associated with mortality [survivors versus nonsurvivors: median 99 pg/ml (range, 0 to 365 pg/ml) versus 214 pg/ml (range, 0 to 988 pg/ml); p = 0.02]. conclusions: measuring strem-1 in csf may be a valuable new approach to accurately diagnose bacterial meningitis and identify patients at high risk for adverse outcome. therefore, a prospective study on strem-1 as biomarker in bacterial meningitis is needed. systematic review of rapid diagnostic tests for enterohaemorrhagic e. coli i. abubakar, l. irvine, l. shepstone, s. schelenz, c. aldus, p. hunter (norwich, uk) objective: a variety of rapid tests for the detection of enterohaemorrhagic escherichia coli (ehec) have recently emerged. culture on sorbitol macconkey (smac) agar and biochemical identification, while easy to use and inexpensive, is slow and lacks sensitivity in the detection of non o157:h7 serotypes. this study sought to determine the accuracy of rapid serological or polymerase chain reaction (pcr) assays which have been evaluated for the detection of all ehec serotypes compared to culture. methods: a systematic review and meta-analysis of 146 articles, identified via searches of electronic databases, hand searching of selected journals, and through contact with experts and commercial test manufacturers. the majority of these needed to be excluded due to low quality or lack of accuracy data. sensitivity and specificity of each method was calculated using full biochemical identification as the reference standard. twenty-one studies met the inclusion criteria, of which 7 used pcr methods and 10 used serological assays and 4 were based on culture. a summary receiver operator curve (sroc) was constructed from these data and the area under the curve (auc) calculated (using the trapezium rule). results: serological tests had individual sensitivities ranging from 0.82 to 1.00 and specificities ranging from 0.67 to 1.00. pcr tests had individual sensitivities ranging from 0.94 to 1.00 and specificities ranging from 0.92 to 1.00. additional analysis comparing smac agar culture with toxin detection methods showed poor sensitivity compared to pcr and serological tests (ranging from 0.24 to 0.38) yet the specificity was very good (1.00 for all 4 studies considered). our results suggest that both molecular and serological tests may have a potential role in detecting ehec infection. whilst there is very little difference in the effectiveness of these techniques, both are faster and have improved sensitivity when compared to traditional culture methods. fast, reliable diagnosis could lead to more informed treatment choices and improved outbreak control measures. however, given the substantial extra cost of these assays, an assessment of economic feasibility is necessary prior to use in everyday practice. antibodies against bordetella pertussis detected by slow agglutination test and elisa in two agerelated groups of vaccinated people suspected of acute pertussis: a comparative study objective: the aim of presented study was to describe differences between results of two tests used for detection of antibodies against bordetella pertussis (slow agglutination and elisa) in two age-related groups of patients suffering from respiratory infection. each of the people has undergone vaccination against b. pertussis. methods: paired sera obtained from two age-related groups of patients [(1). age 0-6 years, n = 49; (2). age above 6 years, n = 455] suffering from acute respiratory infection were tested. the first group comprised the children who were vaccinated earlier than one year before testing; the second group was determined by longer interval between the vaccination and the testing. the criterion of positivity of the slow agglutination was based on quadruple increase/decrease of the titer of specific antibodies; the criterion of serodiagnosis of the illness was the same. each of the patients was tested by elisa iga,igg,igm (virotech) during the same period, positive results of each of class of immunoglobulins were evaluated as positive elisa. the differences of results obtained by the two tests were assessed inside and between the groups. results: there were found 12.2% (respective 16.9%) concordant positive and 36.7% (respective 19.6%) concordant negative results between the tests in the first (respective second) group. there were found the following discrepancies in the frame of non equal results: agglutination positive/elisa negative sera were present in 8.2% (respective 13.2%) persons and agglutination negative/elisa positive samples were present in 42.9% (respective 62.2%) persons. conclusion: (1) . the frequency of serologically confirmed infection based on results of slow agglutination is higher in the group of people older six; the interpretation of the results in the younger group is limited by the influence of actual vaccination. (2) . the elisa evaluated as described above shows extremely high frequency of positivity in both groups, thus, the usefulness for diagnostics of acute infection seems to be low. (3) . the study will be continued to asses relationships between the positive results detected by slow agglutination and the positive ones detected by elisa in separate classes of specific immunglobulins. accuracy of the microscan walkaway system to identify coagulase-negative staphylococci a. sáez, b. ruiz, l. martínez-martínez (santander, es) objective: to determine the reliability of the identification of coagulase-negative staphylococci (cons) with the microscan walkaway 96 (wa, dade behring) system at species level when a > = 90% probability is obtained, considering as a reference the results of molecular identification. methods: one hundred and sixty-eight isolates of cons from clinical samples (october 2003 -may 2005 for which the identification with the wa system was ‡ 90%, and 9 atcc type strains were evaluated. bacteria were identified with the wa system using pos combo 1s panels. absence of coagulase was determined with a latex assay (pastorex ò staph-plus, bio-rad). reference identification was established by sequencing of the 16s rrna; when identification with wa and 16s rrna disagree, definitive identification was defined after sequencing of the soda and tuf genes, as previously described (drancourt et al. jcm 2000; 38: 3623-30 and heikens el al. jcm 2005; 43: 2286-90) . for identification, the sequences of 16s rrna, soda and tuf were compared with those in genebank. homologies values above 97% were considered reliable. results: all 9 type strains were correctly identified by 16s rrna sequencing as named by the atcc. among the 168 clinical isolates, the molecular method identified the following species (number): s. hominis (39), s. haemolyticus (35), s. saprophyticus (30), s. epidermidis (27), s. lugdunensis (12), s. schleiferi (7), s. capitis (7), s. simulans (4), s. pasteuri (2), s. warneri (2), s. intermedius (2) and s. equorum (1). the wa system correctly identified 8 out of the 9 atcc strains. s. pasteuri is not included in the wa database, and the corresponding atcc strain was misidentified as s. warneri. one hundred and fiftyseven out of the 168 (93.4%) clinical isolates were correctly identified by the wa. five s. haemolyticus were identified by wa as s. auricularis (2), s. simulans (2) and s. warneri (1) . other errors corresponded to: two s. pasteuri misidentified as s. warneri, one s. epidermidis as s. hominis, one s. lugdunensis as s. schleiferi, one s. hominis as s. haemolyticus and one s. equorum as s. cohnii. all isolates of s. saprophyticus, s. schleiferi, s. capitis, s. simulans, s. warneri and s. intermedius were correctly identified by the wa system. conclusions: the microscan walkaway 96 is reliable to identify cons at species level when a probability of > = 90% is obtained. s. pasteuri should be incorporated to the wa database in order to improve its performance. objectives: the aim of this study was to analyse the results of proficiency testing obtained by polish microbiology laboratories participating in polmicro. haemophilus influenzae is an important pathogen causing a variety of community-acquired respiratory tract infections, acute otitis media and purulent meningitis. two mechanisms of ampicillin (amp) resistance in this organism are described. one is mediated by the production of beta-lactamases tem-1 and rob-1; these amp-resistant strains are termed beta-lactamase-producing, amp-resistant (blpar). the second mechanism involves development of altered penicillin-binding proteins (pbp) with decreased affinity to amp and other beta-lectam agents. strains with resistance mechanisms mediated by pbp alterations are termed beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae. methods: four hundred seventy eight laboratories participated in this part of the scheme. each participating laboratory received haemophilus influenzae (pm-63)-beta-lactamase negative, ampicillin-resistant strain (blnar). the laboratories were asked to provide identification to the species level and of the susceptibility results and interpretation. results: correct identification to the species level of this strain was reported by 454 laboratories (97.2%) of the 467 labs involved. thirteen laboratories reported the analysed strain as haemophilus parainfluenzae. three hundred ninety eight laboratories (88.1%) of 452 correctly detected the mechanism of resistance to beta-lactams. only three laboratories incorrectly reported the organism as beta-lactamase producer. the greatest dispersion of inhibition zone was observed in the susceptibility of h. influenzae to ampicillin, amoxicillin-clavulanic acid and clarithromycin. conclusions: over 90% of the laboratories correctly identified and interpreted beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae strain. purpose and methods.the architect syphilis tp assay is a chemiluminescent eia that employs three recombinant antigens of treponema pallidum on the solid phase and an anti-human igm and igg conjugate. we evaluated this assay in comparison with a conventional eia (diesse enzywell syphilis screen recombinant) on unselected routine serum samples and on repository specimens for whom the results for specific igg and igm and of the rapid plasma reagin (rpr) assay were already known. in both instances an immunoblot (ib: inno-liatm syphilis score, innogenetics) has been employed on discordant specimens as a confirmatory assay. the precision and robustness of the architect assay were also evaluated.results.on 1.165 routine samples (975 from volunteer blood donors and 190 from in and outpatients) the concordance between architect and eia was high (1.155 samples, or 99.1%; 7 positives, 1,148 negatives). one of the discordant, positive by architect and negative by eia, was confirmed by ib. the specificity of the architect assay was 99.4% (95% confidence limits: 98.9-99.8). the 177 repository samples assayed belonged to three groups: 1) 9 biological false positives from 6 subjects: all negative by architect; 2) 134 true positives, all positive by architect, with a significantly stronger signal (average s/co: 26.19 vs. 14.53) on the 23 igm positive samples, all of whom were also positive by rpr; 3) 34 samples positive by rpr and negative by eia igg: 32 of them were negative by architect as well and for igm, while two specimens were strongly positive by architect and positive also for specific igm and with three specific bands by inno-lia, suggesting a pattern of recent infection. the reproducibility of the architect assay was good, with cvs of 7.78%, 5.71% and 5.51% on 28 replicates over 4 weeks of the assay's negative and positive control and of an internal control; finally, the s/co distribution of negative specimens confirmed the robustness of the assay, with a mean of 0.09, a median of 0.06, 10 standard deviations between the mean and the cut-off value and the 99th percentile at a s/co value of 0.53.conclusion.the automated assay for anti-treponema pallidum antibodies on the architect system has an excellent sensitivity and a good specificity. the analytical performances, coupled with the elevated throughput and minimal samples handling, make this method a first-choice option for syphilis screening and diagnosis in medium and large volume laboratories. objective: quantitative urine culture is the gold standard for defining the diagnosis of urinary tract infection (uti), because it allows identification of the uropathogenic species. however, this method is time consuming and expensive. approximately, up to 70% of urine cultures are negative with high cost for unnecessary testing. thus, we have evaluated the usefulness of two automated analysers for uti screening to quickly identify the negative samples that can be prompt reported to the clinicians, improving in the quality of patient care and allowing the laboratory to direct more effort into positive samples. methods: 1.165 of midstream urine samples submitted for microbiological examination were analysed by conventional urine culture plates (mcconkey agar + trypticase soy agar + bile esculine azide), sysmex uf-100 (sysmex, japan) and coral uti screen (coral biotechnology, ca, usa) automated analysers. uti was defined positive as follows: one or two strains of bacteria with at least 10 5 ufc/ml for the culture plates, more than 5.000 bacteria/ll and more than 15 wbc/ll for the uf-100 and/or an rlu value grater than 2% of the calibrator value for the coral. when more than two strains of bacteria were found, the culture was classified as contamined. results: the diagnostic performance of sysmex uf-100 and coral uti screen are shown in table 1. the sensitivity (95.8%) and negative predictive value (96.4%) confirm that sysmex uf-100 and coral uti screen are an excellent screening for uti. after this evaluation, we decide the use of the sysmex uf-100 and coral uti screen on our routine workflow for uti screening. the results of both the analysers are sent to a software system (labfinity dasit, italy) connected to the lis. if the results are lower than the cut-off values, uti can be excluded and directly reported to the physician. positive results are submitted to microbiological culture and reported within 24 or 48 hours depending on negative or positive bacterial growth. in our experience, evaluated on further 2.955 samples, this means that 78% of samples are immediately reported within very few hours. of the 22% of positive samples, 222 (35%) were confirmed by culture and reported within 48 hours, 418 (65%) were not confirmed and reported within 24 hours. comparison of the blood and bone marrow culture positivity rates for the diagnosis of brucellosis objectives: brucellosis is a common disease, seen worldwide as well as in our country. the diagnosis of brucellosis is made with certainly when brucellae are recovered from blood, bone marrow. in our study, we aimed to compare the blood and bone marrow culture positivity rates in patient with brucellosis. methods: this study was performed in the infectious diseases and clinical microbiology department of ankara research and training hospital between 2002 and 2004. the diagnosis of brucellosis was made on the history, physical findings, serologic findings and the isolation of the organism. the number of patients with brucellosis included to the study was 102. blood and bone marrow samples were taken from all of the patients on admission and cultured by using the bactec 9050 system. results: blood culture positivity for brucellosis was 48% (49/ 102), while bone marrow culture positivity was 34% (35/102). the difference between those positivity rates was found to be statistically significant (p < 0.05). the isolation ratio from blood cultures among acute cases was 66% (40/61) while it was 31% (9/29) among subacute cases. brucella isolation from blood was not detected in 12 chronic cases. the isolation rates of the microorganism from bone marrow of acute, subacute and chronic cases were 45.9%, 20.7%, 0.9% respectively. among our patients, 32 had history of medical therapy for brucellosis before admission and 27 of them was treated inadequately. of those 27 cases, the organism was isolated in 7 (25%) from blood and in 9 (33%) from bone marrow.in the cases with high standard tube agglutination titers, the rate of positivity was also high both in blood and bone marrow cultures. however when compared with low standard tube agglutination titers, that difference was not statistically significant.the mean growing time for the positivity of cultures was 4.2 days for bone marrow and was 5.8 days for blood cultures. the difference between the mean growing times of two culture types was found statistically significant (t-test. p < 0.05). conclusion: premedication, subacute and especially chronic phases decrease the possibility of isolation of the microorganism from blood culture. therefore we suggest taking bone marrow culture only for these kinds of patients as it. is a traumatic process. serological findings in blood sera of patients with yersinia-triggered arthritis e. golkocheva, r. stoilov, h. najdenski (sofia, bg) objectives: immunoblot analysis of iga and igg antibody response of blood sera from patient with yersinia triggered reactive arthritis and with undifferentiated arthritis were made. patients and methods: serum samples were obtained from patients admitted to clinic of rheumatology at medical university, sofia, bulgaria with suspicion of yersinia triggered reactive arthritis, based on diagnostic criteria. a total of 20 blood serum samples were analysed by immunoblot analysis with specific antigens-yops (yersinia outermembrane proteins). when y. enterocolitica is cultivated at 37 o c under calcium restriction (0.2 mm ca 2+ ), large amounts of yops are secreted into medium. these proteins were separated by 2d-sdselectrophoresis. results: immunoblot analysis of iga and igg antibody response against yops in 20 blood sera from patients with arthralgias and polyarthropathies was carried out. yersinia enterocolitica, serotype o: 8, was used as source for yop. seven strong bands of the molecular weights 26 kda-yope, 33 kda-yopn, 36 kda-yopd, 41 kda -v-ag, 43 kda-yopb, 46 kda-yopm and 51 kda-yoph were visualized. for immunoblot assay the optimal concentration of antigen was established by analytical electrophoresis. of the 10 blood sera from the patients with yersinia triggered reactive arthritis igg antibodies were detected against yoph, yopm, yopb, yopd, yopn and yope. iga antibodies were established against yopm, yopb, yopd, yopn and yope. all 5 sera from the patients with other rheumatic diseases were negative for the presence of anti-yersinia iga antibodies and two of them were positive for igg against yopd. antibodies from two classes were not detected in 5 sera samples from healthy people. conclusions: yops are borne by the virulence plasmid, which mean that they are clearly associated with virulence properties of pathogenic strains. moreover, yops is not restricted to single serotype and this made them a specific antigen in diagnosis of different yersinia infections. conventional techniques such as culture and demonstration of serum agglutinins prove to be insufficient to demonstrate invasive or chronic yersiniosis in contrast with the determination of specific serum iga and igg antibodies by immunoblot analysis and antigen detection. the detection of anti-yops igg and iga antibodies by immunoblot can be used for diagnosis of yersinia triggered arthritis. acknowledgements: this work was sponsored by natoreintegration grant 981256. objectives: to evaluate the identification and susceptibility results by using suspensions obtained directly from positive blood cultures. methods: during the period between 1st august and 31st october 2005 we selected all positive cultures grown in bact/ alert ò sa and sn bottles (biomérieux) from gram-negative bacilli. only the first culture positive from each patient was included. we inoculated 5 ml fluid from a positive bottle into a serum separator tube (bd vacutainer systems, plymouth, united kingdom) and centrifuged at 1500 x g for 10 minutes and the supernatant was carefully aspirated. using a cotton swab the bacteria were removed from the top of the separator layer to be suspended in 0.45% saline solution to get 0.6 mcfarland. the suspension was processed according to standard inoculation procedure for gn and ast-n020 vitek ò 2 cards. positive bact/alert 3d bottles were also sub cultured and after an overnight incubation several colonies were used to make a 0.63 mcfarland suspension in 0.45% saline. the suspension was processed according to standard vitek ò 2 inoculation procedure for gn and ast-n020 cards. results: identification: a total 56 gram-negative bacillus from positive blood cultures were investigated. fifty (89.2%) strains were correctly identified to the species level, four (7.1%) strains were not identified and two (3.6%) strains were misidentified. antimicrobial susceptibility testing: in all, 1040 mics were determined for 52 isolated by both methods. the unidentified strains (4) were excluded. the overall mic agreement between direct and standard inoculation was 93.9%. all individual antimicrobial agents scored > 90%. the overall minor error rate was 2.8% (30 of 1040). the overall major error rate was 1.05% (11 of 1040). the overall very major error rate was 2.1% (22 of 1040). the highest rate of mic agreement was for amikacin, norfloxacin (100%), meropenem (98%), gentamicin and ofloxacin (96.1%). conclusion: the direct method from positive bact/ alert&#61650; cultures cannot totally replace the approved methods of identification and susceptibility but in some cases provides earlier information which allows a better patient management and also reduce cost in patient care. investigation of listeria monocytogenes "o" antibodies in maternal and cord sera with the agglutination test e. us, a.t. cengiz, o. gelisen (ankara, tr) objectives: listeria monocytogenes is a gram-positive food borne pathogen that is responsible for listeriosis, a human infection with a mortality rate of 30%, which could cause severe motherto-child infections. this serious pathogen in pregnancy could be treated if diagnosed, but there is no routine screening test for susceptibility to listeriosis during pregnancy. therefore, we investigate different l monocytogenes serotype o antibodies for diagnosis of listeriosis in 275 maternal sera with agglutination test. 69 of them had spontaneous abortion, premature labour or stillbirth (group i), while 206 had no obstetric patology (group ii) in their previous pregnancies. cord bloods were also obtained at the delivery and tested. methods: all sera were being tested against antigens with the o formulation of serotype 1/2c, 3b, 4ab, 4c and 4d. the antigens were prepared by the method of osebold, and larsen et all. the bacterial suspensions were trypsinized for 15 min at 37°c to prevent cross-reactions and contaminations. sera were diluted by doubling serially in saline followed by addition of an equal volume of antigen. a positive titre of greater than or equal to 1:320 was chosen as positive test result to maximize the sensitivity and specificity. results: 4.36% of cases have ingested raw milk and diary products, 1.81% ready-to-eat foods, and 5.81% developed nonspecific febrile illness (nfi) during their pregnancies. 20% of group i were found positive (28.5% developed nfi) while at group ii 22% had positive (26.6% developed nfi) agglutination titres to one ore more serotypes. all the cord blood sera of group i were found negative, whereas two in group ii (all 4ab) were positive, with the positive maternal sera of the same serotype. it's evaluated as transmission of the antibody from mother to foetus. at group i the frequent serotypes were 1/2c = 4ab, at group ii 4ab, 1/2c, respectively. the newborns showed no symptoms or signs of listerial foeto-maternal infection. conclusion: the women encountered the antigens of l monocytogenes in any period of their life time (most 21-25 years of age) and produce antibodies against this pathogen. there is a relationship between nfi and positive titres. if the disease is recognized, it is possible to treat the mother and allow the birth of a healthy infant. we propose the less time consuming and easy to perform agglutination test as a routine screening test for susceptibility to listeriosis during pregnancy to prevent bad pregnancy outcomes. objectives: to evaluate the performance of a real time pcr assay (with a fluorogenic target-specific probe), mrsa-idi (geneohm sciences) for mrsa detection directly from mucocutaneous swabs in hospitalized patients. methods: clinical swabs (1 to 14 samples with a median of 2.1 samples per patient) from nares (n = 330) and skin (n = 326) were prospectively collected for mrsa screening from 321 patients admitted to a 858-bed teaching hospital. swabs were inoculated onto selective mrsa agar (mrsa-id, biomérieux), into the buffer extraction solution for idi-mrsa pcr assay and into enrichment broth (bhi with 7.5% nacl). after 24h, bhi broths were subcultured onto mrsa-id agar. selective agars were incubated for 48 h at 35ordm;c and examinated daily. suspected colonies were identified by coagulase testing; oxacillin resistance was tested by cefoxitin disk diffusion according to clsi recommendations. the pcr assay was performed according to the manufacturer's instructions. pcr results were compared with phenotypic identification test results. in case of discordant results, the assay was repeated, but only results from first testing were considered for calculating test performance. results: mrsa was detected by culture in 74 specimen (11.3%) from 38 patients. the sensitivity and specificity of the pcr compared with culture was 82.4% and 96.9%, respectively. positive predictive value and negative predictive value were 77.2% and 97.7%, respectively. the sensitivity of pcr (92%) was higher on nasal swabs than on swabs from other sites (77.5%, p < 0.001). the pcr assay detected mrsa in 34 patients (89.5%). the pcr assay provided results in 2 to 12 versus 48 to 72 hours for conventional method. conclusion: in our hospital, the id-mrsa pcr assay detected 89.5% mrsa carriers in less than 12 hours when performed on multiple specimen. the assay appeared more sensitive in testing nasal swabs than other clinical specimens. prospective studies are needed to evaluate the impact of this assay for rapid implementation of infection control procedures and its global costs and benefits. the purpose of this study was to establish a rapid and sensitive real-time polymerase chain reaction (pcr) method for detection of methicillin-resistant staphylococcus aureus (mrsa) from blood culture bottle. as a result of over use of broad-spectrum antibiotics after the 1960s in whole the world, an outbreak of mrsa infection has been seen. severe nosocomial infections with mrsa such as bacteraemia and sepsis may lead to multiple organ failure and high mortality in the hospital. although standard method took at least 48 hours to identify mrsa by the blood culture method, the presence of meca and nuc genes which is specific for methicillin resistance and s. aureus was determined by real-time pcr method within only 2 hours after blood culture signal positivity. nineteen s. aureus and 33 coagulase negative staphylococci positive blood culture bottles were studied retrospectively for detection of s. aureus and methicillin resistance. staphylococci were identified with classical methods and mics of oxacillin were determined by etest (ab biodisk) on mueller-hinton agar supplemented with 2% nacl. real-time pcr was performed to all positive blood culture samples for s. aureus and methicillin resistance determination. nineteen (100%) s. aureus were determined correctly by real-time pcr method. forty-four methicillin resistant and 8 methicillin sensitive staphylococci were detected by etest. using the real-time pcr method, the meca gene was detected in 47 staphylococci except 3. when compared with etest and realtime pcr method gave sensitivity, specificity, and positive and negative predictive values of 100%, 63%, 94%, 100% for both positive and negative tests, respectively. agreements between two methods were high (94%); there were 3 discrepant results among the 52 strains were tested. detection of mrsa bacteraemia and methicillin resistance with real-time pcr definitely is useful for reducing mortality and morbidity of this type infection. in conclusion, this method, as many as sensitive and specific for detection of mrsa bacteraemia and clinically should be beneficial for prevention of unnecessary antibiotic use and determination of appropriate antibiotic treatments of mrsa infection. pcr detection of class b, c and d betalactamases in environmental and clinical aeromonas strains t. fosse, c. giraud-morin, f. la louze (nice, fr) objectives: aeromonas spp. strains are waterborne opportunistic pathogens. they are able to produce different types of beta-lactamases (class b, c and d). the determination of beta-lactamase content is not easy by phenotypic methods. we have developed a pcr tool to study diversity and distribution of class b, c and d beta-lactamases in a set of representative clinical and environmental aeromonas species. method: a total of 22 references, 20 environmental and 80 clinical strains were tested. identification was realized by conventional tests and gyrb sequence analysis. beta-lactam antibiotic susceptibility was determined by diffusion agar and micro broth dilution methods. three sets of specific primers were defined for the pcr amplification of the internal region of class b beta-lactamase (mei1 and mei2, 297 bp size), class c beta-lactamase (aercp1 and aercp2, 840 bp) and class d betalactamase (aerd1 and aerd2, 514 bp). all pcr products were sequenced. results: class d pcr was positive with most strains except a. trota, a ticarcillin susceptible species (3 strains) . class c pcr was positive with most cephalothin resistant strains (mic >16 mg/l; 61/67 strains, 91%) including a. hydrophila and a. caviae phenospecies. class b pcr was positive with most strains of a. hydrophila and a. veronii phenospecies (33/38; 87%) including three imipenem susceptible strains (mic < 5 mg/l). beta-lactamase type distribution was species related and was particularly useful to better characterize environmental species such as a. bestiarum, a. popoffii and a. allosaccharophila. partial beta-lactamase gene sequence analysis allowed phylogenic studies. some cephalosporinase gene from environmental species was probable progenitor of ampc plasmidic beta-lactamase. conclusion: pcr with specific primers was a good method to detect class b, c and d beta-lactamase in aeromonas species. beta-lactamase type distribution and sequence analysis phylogeny were largely species related and could be helpful for molecular diagnostic and taxonomic purpose. objectives: the aim of this study was to develop a convenient dna extraction method and to optimise a pcr reaction in order to detect enterotoxin b producing s. aureus strains directly from milk. methods: we applied a chemical extraction method of bacterial dna from milk samples artificially inoculated with s. aureus. a pcr based method was used for the detection of seb gene (coding for enterotoxin b) and nuc gene (coding for termonuclease). a protocol for the multiplex pcr was developed and optimized. the sensitivity of the reaction was checked by determining the minimum number of organismsaeml -1 , which can be detected in the multiplex pcr and in each single pcr reaction. amplification specificity of the seb gene was verified by amplicon digestion with restriction endonucleases. results: the specific bands for both genes in the multiplex pcr were detected in samples containing a dna quantity corresponding to 5000 organismsaeml )1 . in the same reaction, the amplicon for nuc gene was visible for as little as the dna concentration corresponding to 1000 organismsaeml )1 . the sensitivity of each single pcr reaction was similar with those of multiplex pcr reaction. conclusion: the applied dna extraction method allowed us to obtain a good quality dna and can be used for a direct milk extraction. multiplex pcr reaction is a simple, rapid and reliable method for detecting enterotoxin b producing s. aureus strains from milk. objective: to detect the resistance to fluoroquinolones in 30 acinetobacter baumannii strains by a pcr-rflp assay. methods: thirty a. baumannii clinical isolates were obtained from different specimens (bronchial aspirates, blood-cultures, catheters, etc.) . the mics (minimal inhibitory concentrations) for ofloxacin were determined by agar dilution following standard methodology.a pcr-rflp method using one primer pair for amplification of a 344 bp fragment related to gyra gene (which codifies subunity a of dna-gyrase) and using one restriction enzyme hinf i was developed to study the resistance to ofloxacin in the different a. baumannii strains. when an a. baumannii strain is resistant to fluoroquinolones, a mutation in the position ser 83 of the dna-gyrase has been detected, decreasing the affinity for the antimicrobial. agarosa gel was used to determine the dna pattern: 2 fragments of 289 bp and 55 bp when there is not mutation and 1 fragment of 344 bp when the ser 83 to leu 83 mutation is present. results: the relationship between the pcr-rflp pattern and the mic to ofloxacin is shown in the table 1. the results of pcr-rflp analysis of most strains were in agreement with the results of mic. one isolate was susceptible to ofloxacin by agar dilution (mic = 0.25 mg/l) whereas by pcr-rflp this isolate seems to be resistant because it presents the mutation in gyra gene. two isolates with intermediate mic (4 mg/l) showed mutation in gyra. the genotypic study by pcr-rflp proved that ofloxacin resistant a. baumannii strains showed a punctual mutation in gyra gene, in the same position inside the sequence of gene. evaluation of a rapid amplification-detection assay for the identification of vancomycinresistant enterococci j. fuller, l. turnbull, s. shokoples, b. lui, l. rosmus, r. rennie (edmonton, ca) objective: the routine identification of vancomycin-resistant enterococci (vre) in clinical laboratories often yields a lengthy turn-around-time that may impede infection control efforts, particularly in an outbreak situation. in search of an improved vre test, we evaluated the genotype ò enterococcus assay (hain lifescience, germany), which provides both species and van gene identification for vre, and compared the results to conventional methods. methods: forty clinical enterococcal strains isolated on vrescreen agar media were selected for study. lactococcus and pediococcus were used as negative controls. conventional testing involved basic culture and identification tests, e-test susceptibility testing for vancomycin and teichoplanin, and pcr for vana, b, and c genes. the genotype ò enterococcus assay involved multiplex dna amplification and reverse hybridization of amplified product on an immobilized dna strip-blot containing probes for e. faecium, e. faecalis, e. casseliflavus, e. gallinarum, vana, vanb, vanc1, and vanc2/3. the genotype ò enterococcus assay produced correct species and van gene identification for all 40 (100%) vre isolates, including 7 e. faecalis vanb, 12 e. faecium vana, 12 e. faecium vanb, 6 e. gallinarum vanc1, 1 e. gallinarum vana-vanc1, and 2 e. casseliflavus vanc2/3. the only minor discrepancy was an e. casseliflavus that hybridized very weakly with the vanc1 probe in addition to the expected vanc2/c3 probe. the costs per specimen were comparable for each test method. however, the genotype ò enterococcus assay could be completed within a normal working day in contrast to conventional testing, which required a minimum of two days from the point of isolation on the vancomycin-screen media. conclusion: from this preliminary evaluation, the genotype ò enterococcus amplification-detection assay provides vre species and van genotype identification in a rapid and costeffective manner, superior to conventional culture methods. although further study is required, this kit may have clinical utility during a vre outbreak. application of minimal sequence quality values prevents misidentification of blashv type in single bacterial isolates carrying different shv extended-spectrum beta-lactamase genes background: detection of extended spectrum beta-lactamase (esbl) genes by pcr and sequence analysis is the gold standard for detection of shv-type beta lactamases. usually, quality values of sequence analyses are not reported. during a study on esbl epidemiology, three strains for which the default sequence assembly showed an shv)2 or shv-5 gene, showed low quality values at certain positions in individual sequence traces. we investigated the reason for these lower values. methods: shv genes were amplified by pcr from three isolates (escherichia coli, enterobacter cloacae and pseudomonas aeruginosa). individual sequence traces were analysed with the computer programs phred and codon code. pcr products were ligated in vector pcr2.1 and transformed to e. coli. sequence analysis was performed on eight individual clones from each transformation. results: visual inspection of the low quality positions in the sequence traces showed signals for two different nucleotides at three positions in the shv sequence: a or t at position 92, a or g at position 402 and a or g at position 703. the polymorphisms at positions 92 and 703 lead to aminoacid substitutions, the four different combinations would give shv types 2, 2a, 5 or 12. the double signals suggested that two or more blashv alleles were amplified. pcr amplicons were cloned in e. coli, in the sequences of individual clones only two combinations of the three polymorphisms were present: a92g402a703 and t92a402g703. these two combinations correspond to shv-2 and shv-12, respectively. conclusions: (i) in isolates of three different species, two different shv genes were present: shv-2 and shv-12. (ii) genotypic detection with default sequence assembly parameters may lead to misidentification of the number and type of shv genes carried by a single strain. (iii) careful interpretation of sequence data of shv genes, including analysis of low quality positions, may further improve our understanding of the epidemiology and evolution of these esbl genes. antimicrobial susceptibilities and epidemiological analysis of salmonella typhimurium human isolates in slovakia by phage typing and pulsed-field gel electrophoresis v. majtán, l. majtánova, m. szabó ová (bratislava, sk) objectives: salmonella typhimurium is a common cause of salmonellosis among humans and animals in many countries. in the last few decades the incidence of multidrug-resistant s. typhimurium infections appears to pose a particular health risk. the objectives of this study were analysis by antibiotic susceptibility, phage typing and pulsed-field gel electrophoresis (pfge) of s. typhimurium human isolates. methods: a total of 145 strains isolated during 1997-september 2005 were analysed. the susceptibility of isolates to ten antibiotics was evaluated by a disk diffusion method. the phage types were identified according to anderson et al. (1977) in the national reference center for phage typing of salmonellae. pfge was used to resolve xbai macro restriction fragments from all strains. results: of human isolates 88 (60.7%) were resistant to more than two antibiotics. sixty-three of isolates (43.4%) showed a classic dt104 resistance profile to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline (acssut). among this resistance type 37.2% were dt104, 7.6% were dt120 and one strain was dt20a. isolates encompassed 18 phage types. the majority of isolates was found to be definitive phage type dt104, representing 62.8% of all isolates. other phage types were mainly dt120, dt41 and dt20a. nine pulsotypes and 18 subpulsotypes were obtained using xbai restriction enzyme, but pattern x1 with its subtypes predominated (86.9%). a major pulsotype x1 was represented by 51.7% of dt104 isolates and was also found among dt120 isolates. conclusion: results indicated the spread of different clones of the multidrug-resistant s. typhimurium in the slovakia, but with predominance of one clone represented mainly by dt104 isolates. the phage typing as well as pfge may offer an improved level of discrimination for the epidemiological investigation of s. typhimurium human strains. novel reverse hybridisation assay to identify ctx-m genotype in cephalosporin-resistant isolates from uk and india to validate the assay results by dna sequencing. methods: isolate collection 1: 110 enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in london and south-east england. these isolates were known to carry phylogenetic group 1 blactx-m, but precise genotypes had not been determined. isolate collection 2: 130 enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in aligarh, north india. resistance determinants had not been investigated previously. a novel multiplex pcr was used to amplify blactx-m. reverse hybridisation was carried out using biotinylated pcr amplicon and sequence-specific oligonucleotides designed to identify members of ctx-m phylogenetic group 1. hybridisation results were validated by dna sequencing for 20 representative isolates from each collection. results: 109/110 london and se england isolates known to carry group 1 blactx-m gave a consistent profile, corresponding to that for ctx-m-15 and ctx-m-28; 1/110 gave a profile corresponding to ctx-m-3 and ctx-m-22. 82/130 indian isolates had blactx-m genes, all of which belonged to group 1, and all these gave a hybridisation profile corresponding to ctx-m-15 or ctx-m-28. ctx-m-28 and ctx-m-22 are rare variants, suggesting that the enzymes present were more likely to be ctx-m-15 and ctx-m-3, and this was confirmed by dna sequencing. conclusions: this is the first reported application of this novel reverse hybridisation assay to the analysis of large numbers of cephalosporin-resistant enterobacteriaceae. results were validated by dna sequencing. the assay is cheap and convenient, enables reasonable throughput, provides results within one day and can be used in place of dna sequencing. we believe it will be valuable for monitoring the prevalence and genotypes of blactx-m genes in enterobacteriaceae. detection of mexa and mexx efflux genes in p. aeruginosa: correlation between qc-rt-pcr and real-time pcr objectives: efflux systems are rarely identified as such in clinical microbiology laboratories. yet, over expression of transporters such as mexab-oprm and mexxy-oprm are likely to cause antibiotic multi-and cross-resistance in pseudomonas aeruginosa, leading to potential clinical treatment failures because of their inducible character. we have previously developed and validated with reference strains a qc-rt-pcr method to quantify mexa and mexx expression levels (eccmid 2005 . in the present study, we have developed a real-time-pcr assay and present here the correlation between both methods using control strains and clinical isolates. methods: expression levels of mexa and mexx were measured by both techniques in (i) 4 reference strains expressing only one of these efflux mechanisms [mexa (2) or mexx (2) ]; and (ii) 8 clinical isolates, in comparison with the wild-type strain pao1 (basal mexa and mexx expression levels). results: real-time pcr showed an inter-day reproducibility of 95 ± 5.3% (triplicates of 10 strains). among the clinical strains, 5 over expressed mexa and 3 mexx. the table shows (i) the mean level of overexpression of mexa and mexx in comparison with the wild type strain pao1 (set at 1), as detected by real-time pcr for all strains; (ii) the ratio of these values to those observed by qc-rt-pcr for the corresponding transporters. conclusions: both qc-rt-pcr and real-time-pcr are potentially useful in clinical laboratories as sensitive and rapid diagnostic tools to quantify the expression level of mexa and mexx in p. aeruginosa. combined with phenotypic characterization, this approach may help in a better understanding of the resistance mechanisms and epidemiology of resistance in this difficult-to-treat nosocomial pathogen. molecular detection of penicillin resistance in streptococcus pneumomiae n.g. rizk, n.a. abo khadr, s.m. abdel salam, n.m. gamil, m. hassan (alexandria, eg) objectives: the aim of the study was to detect penicillin resistant streptococcus pneumoniae by using seminested polymerase chain reaction (pcr) and to compare it with minimum inhibitory concentration (mic) of penicillin g. methods: fifty clinical isolates of streptococcus pneumoniae where isolated from patients admitted to alexandria main university hospital in egypt and were recovered from sputum (36 strains), throat swabs (11 strains), and pleural effusion (3 strains) . two species-specific primers 1a-1 and 1a-2, which amplified 1043 bp region of the pbp1a penicillin-binding gene, were used for pneumococcal detection. two resistance primers, 1a-r1 and 1a-r2, were used to bind to altered areas of pbp1a gene which, together with the down stream primer 1a-2, amplify dna sequences of 224 bp and 569 bp from isolates with penicillin mic > 0. objective: lipopolysaccharide-binding protein (lbp) is an acute phase protein produced in the liver. the objective of our study was to evaluate lbp as a marker of severity and prognosis in patients with bacteraemia. methods: 42 adult patients with community-acquired bacteraemia were included in a prospective manner. daily blood sampling for lbp and interleukin-6 (il-6) was performed. the patients were classified according to the systemic inflammatory response syndrome (sirs) criteria. demographic data, co-morbidity, microbiological aaetiology, routine biochemical parameters, focus of infection, severity score and mortality on day 28 were recorded. lbp and il-6 levels were analysed on plasma samples with a chemiluminescent immunometric assay (immulite-1000 ò ). results: the median age was 71 yrs. the mortality rate on day 28 was 16.6%. 5 patients had bacteraemia without sirs, 17 patients had sepsis and 20 patients had severe sepsis. lbp concentrations are presented as medians and range: 32.2 lg/ml (28. 2-34.1) in patients without sirs, ) in patients with sepsis and 50.9 lg/ml (22.9-96.5) in patients with severe sepsis (p < 0.05). lbp levels correlated to levels of il-6 (rs 0.52), c-reactive protein (rs 0.68), leukocytes (rs 0.44) and neutrophils (rs 0.46) (p < 0.01). lbp did not predict the outcome of the patients with bacteraemia. conclusion: lbp levels increased with the severity of sepsis in patients with bacteraemia. lbp correlated to il-6, c-reactive protein, leukocytes and neutrophils. lbp did not predict the outcome of the patients in this small cohort. pyrosequencing of the gra6 gene to discriminate type i, ii and iii toxoplasma gondii in clinical samples b. edvinsson, b. evengård on behalf of the esgt objectives: infection with toxoplasma gondii in immunocompromised transplant recipients is rare but often fatal. to increase our knowledge about the significance of the genotype of the parasite during infection, methods suitable for routine use need to be developed. pyrosequencing is a rapid sequencing-bysynthesis method performed in real-time. it is developed for detection of short nucleotide polymorphisms (snps), and is suitable for molecular genotyping of microorganisms. we here present a pyrosequencing assay for rapid and reliable discrimination of toxoplasma gondii type i, ii and iii in clinical samples. methods: twenty-two isolates of t. gondii were used for pyrosequencing analysis of the gra6 gene. real-time pcr was performed using a lightcycler 2.0 instrument to amplify a 176 bp fragment of the gra6 gene. pyrosequencing analysis of two different snps contained within a 10 bp fragment of the amplified product was preformed to identify t. gondii type i, by detection of nucleotides g and a at these respective positions. type ii was g and g, and type iii was a and a. to test the assay in a clinical context, blood samples and lung tissue from an immunocompromised patient was analysed. results: the detection limit of the assay is 10 parasitic genomes in a sample. reproducibility (r) was calculated as r = nr/n (nr = the number of isolates assigned the same type on repeat testing and n = the number of isolates tested). r was determined using three independent runs, and was 1, suggesting clearly interpretable results with little variation. typeablility (t) of the assay was calculated as t = nt/n (nt = the number of typeable strains and n = the number of isolates tested). t was determined using three independent runs, each including four atypical isolates. t was 0.82, suggesting that the assay discriminates correctly between the three main genotypes of t. gondii, but does not detect atypical strains. analysis of the clinical samples revealed type ii t. gondii in blood samples and lung tissue. conclusion: when preceded by real-time pcr, pyrosequencing is a rapid process with a high reproducibility and throughput. this makes it a good candidate for routine use. the method does, however, not detect atypical or recombinant strains. more than one gene may have to be analysed for that purpose. acknowledgement: in particular, we want to thank marie-laure dardé and hervé pelloux for provision of the t. gondii isolates. virulence genes in escherichia coli isolates from calves in shahrekord area, iran shiga toxin-producing escherichia coli (stec) strains, also called verotoxin-producing e. coli (vtec) strains, represent the most important recently emerged group of food-borne pathogens around the world. members of this group are a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (hc) or the hemolytic uremic syndrome (hus), which is the main cause of acute renal failure in children. domestic ruminants, mainly cattle, sheep, and goats, have been implicated as the principal reservoir. transmission occurs through consumption of undercooked meat, unpasteurized dairy products and vegetables, or water contaminated by feces of carriers because stec strains are found as part of the normal intestinal floras of the animals.we studied the prevalence of shiga toxinproducing escherichia coli (stec) in stool specimens of calves with diarrhoea or other gastrointestinal alterations from 10 dairy cattle farms of shahrekord city (central of iran). the virulence genes, stx1, stx2, eae, intimin hly, enterohemolysin, st, lt, were detected by multiplex pcr method. stec strains were detected in 7 (11.5%) of 61 e. coli from 180 cases investigated. stec o157 was isolated in 7 cases (11.5%), whereas non-o157 stec strains were isolated from 4 animals (6%). stec strains were the most frequently recovered enteropathogenic bacteria. pcr showed that 5 (8.2%) isolates carried st gene. none of isolates carried an ehxa, eae, and lt (labile toxin) genes. our results suggest that stec strains are a significant cause of calf infections in this area and confirm that, infections caused by stec non-o157 strains are more common than those caused by o157:h7 isolates. the high prevalence of stec strains (both o157 and non-o157 strains) also found in human patients by other investigators, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of stec in spanish clinical microbiology laboratories. objectives: shiga toxins are a-b holotoxin including one enzymatically active a subunit associated non-covalently to five identical receptor binding b subunits. each subunit can cause different signalling pathways in different cells. to assess the effect of each single subunit the specific clones for expressing the single subunit was designed. periplasmic expression yielded native ab5 holotoxin or b5 pentamer. methods: o157 was used as bacterial strain for pcr amplification of shiga toxin gene. each subunit was amplified by specific primers and the amplified genes were cloned in pbad expression vector. the expression of the cloned genes was induced and optimized by different concentration of arabinose. the expressed proteins was assessed on sds-page and detected by elisa and western blotting. the expressed recombinant ab5 holotoxin and b subunit were purified and assessed for its biological activity on cells. cell cytotoxicity was shown by the expressed (ab5) holotoxin. moreover inhibition was observed by b subunit and antibody against it. results: e. coli clones expressing recombinant shiga toxin a and recombinant shiga toxin b subunits were established to release the toxin to periplasmic space. expressed toxin was examined by sds-page to visualize two subunits. the whole structure of these expressed subunits was checked in native gel. active ab5 structure expressed in periplasmic space was extracted by polymyxine b. the biological activity of the constructed recombinant shiga toxin showed both vero cell cytotoxicity and inhibition of in vitro protein synthesis. conclusion: in this study it was shown that for b subunit assembly and secretion to periplasmic space as b5 pentamer homologous leader sequence is not needed. although for biological active holotoxin (ab5) secretion to periplasmic space the presence of homologous leader sequence of gene is essential. these subunits can be used for studying on cell cytotoxicity and also as a vector for antigen presentation in immunotherapeutic approaches. characterisation of gram-positive anaerobic cocci by biochemical tests and partial 16s rrna sequencing a. bryk, a. kanervo-nordstrom, m. hyvonen, e. kononen (helsinki, fi) objective: gram-positive anaerobic cocci, which are common findings in various infections, are difficult to identify in clinical microbiology laboratories, where identification is based only on few phenotypic tests. in recent years, this group of organisms (traditionally known as peptostreptococci) has encountered several taxonomic changes. the aim of the present study was to compare the characterization made by a selection of key phenotypic tests to that by partial sequencing of the 16s rrna gene. methods: fifty-nine clinical isolates sent to our laboratory as gram-positive anaerobic cocci were examined for their colony and cell morphologies and biochemically characterized using spot catalase and indole reaction, 8 enzyme reactions by individual diagnostic tablets (rosco), sodium polyanethol sulphate susceptibility, glucose fermentation, and determination of metabolic end products. in addition, commercial identification test kit (rapid id 32a) patterns were performed. the sequencing of the 16s rrna gene of the 59 clinical isolates and 4 reference strains comprised of about 470 bp, and the sequences obtained were compared to those in genbank database by using the multisequence advanced blast comparison software from the national center of biotechnology information. results: the biochemical characteristics of the isolates were consistent with those of peptostreptococcus anaerobius (n = 8), peptostreptococcus (micromonas) micros (n = 6), finegoldia magna (n = 25), peptoniphilus asaccharolyticus (n = 8), peptoniphilus sp. (n = 8) and anaerococcus sp. (n = 2), whereas 2 isolates remained as unidentified gram-positive anaerobic cocci. biochemical identification correlated with that obtained by partial 16s rrna sequencing in 56/59 (95%) isolates at genus level and in 40/59 (68%) isolates at species level. the agreement of the biochemical and sequence-based identification was 100% for p. micros and f. magna. of 8 isolates biochemically identified as p. asaccharolyticus, 2 isolates were identified as peptoniphilus harei and 6 remained as peptoniphilus sp. by sequencing. according to the sequence data, the 2 unidentified isolates were peptoniphilus ivorii. conclusion: most isolates from human infections proved to be f. magna. a relatively good agreement of identification was obtained using biochemical testing and partial 16s rrna sequencing. objectives: molecular methods for identification of infectious agents in patients with clinical infectious disease are increasingly being used. especially in cases where antibiotics have been given prior to sampling or when fastidious bacteria difficult to grow are the aaetiology of the infection. infectious arthritis is a serious disease where identification of the etiological agent is mandatory for optimal antibiotic treatment as well as indication of the primary focus if not the joint it self. methods: in the present prospective study, 227 synovial fluids taken from patients in elucidation of affected joints and sent to a clinical microbiological laboratory in the copenhagen area, denmark, were examined by conventional (culture, phenotypic tests) and molecular methods (pcr/sequencing of 23s ribosomal genes). conventional methods included gramstaining and microscopy, aerobic and anaerobic culture and identification. pcr/sequencing included dna extraction, pcr assay which produced a 700 bp fragment of 23s rdna, and sequencing of both dna strands of the amplicons. sequencing data were edited and a blast search in the ncbi database was done. results: overall a microorganism was identified in 24 of the 227 synovial fluids (10.6%). in 14 synovial fluids from nine patients bacteria were identified by either methods [staphylococcus aureus (n = 8), streptococcus pneumoniae (n = 3), streptococcus dysgalactiae (n = 2), citrobacter freundii (n = 1)]. six synovial fluids were only culture positive; in four of those six specimens coagulase negative staphylococci were isolated. in three of the 227 synovial fluids a microorganism was identified by 23s pcr only. in two synovial fluids 23s pcr identified only one microorganism, whereas culturing resulted in two isolates. conclusion: the present study indicates a significant contribution by molecular methods (pcr/sequencing of 23s ribosomal genes) in recognizing and identification of microorganisms from foci normally considered sterile like synovial fluids. continued suspicion of infected arthritis despite of negative cultures should result in use of molecular diagnostics. direct detection of cardiobacterium hominis by broad-range 16s rrna pcr and sequencing in the serum of a patient with infective endocarditis e. malli, d. klapsa, a. vasdeki, m. morava, m. pitsitaki, e. petinaki, a. maniatis (larissa, gr) objectives: to describe the detection of cardiobacterium hominis directly in the serum of a patient with infective endocarditis, by employment of broad-range 16s rrna pcr followed by sequencing. methods: a series of blood cultures were taken from the patient before starting empirical treatment. in addition, 10 ml whole blood was collected in rubber sealed pyrogen-free tubes for direct detection of bacterial dna. bacterial dna was detected by a broad range pcr reaction and sequencing process allowed identification of bacteria species. results: cardiobacterium hominis was identified as the causative agent of infective endocarditis, on two days after the serum collection. blood cultures, simultaneously obtained with the serum sample, remained negative after 5 days of routine incubation; however, after a prolonged incubation of twelve days a gram negative bacterium was isolated from the aerobic bottles, that was identified as c. hominis species, by the usual phenotypic studies (catalase, oxidase reaction, indole, nitrate, etc) which are time-consuming. conclusions: to our knowledge this is the first report of direct detection of c. hominis in the serum using molecular methods, emphasizing the need for the establishment of such methods especially for infections caused by fastidious organisms. identification of dangerous bacterial pathogens by 16s ribosomal rna gene sequence analysis w. ruppitsch, a. stoeger, a. indra, d. schmid, k. grif, c. schabereiter-gurtner, a. hirschl, f. allerberger (vienna, at) to assess the usefulness of partial 16s rrna sequence analysis for identification of dangerous bacterial pathogens, a total of 28 isolates comprising bacillus anthracis, brucella melitensis, biovars melitensis, suis, abortus and bovis, burkholderia mallei, burkholderia pseudomallei, francisella tularensis, yersinia pestis, and 9 genus-related and unrelated control strains were sequenced and analysed using the genbank database (blast 2.2.10, national institute of health, u.s.a), the microseq500 database (version 1.4.1 and v1.0, applied biosystems, foster city, u.s.a.) , the ribosomal database project-ii database (rdp-ii, release 9, update 26, michigan state university, u.s.a), and the ribosomal differentiation of medical microorganisms database (ridom, university of wuerzburg, germany). on genus level all isolates were identified using genbank, rdp-ii, and microseq v1.0. the older microseq 1.4.1 database identified 92% of the tested samples correctly on genus level. the ridom database did not include sequence data of the tested species even on genus level, the ridom database none (''there seems to be, at least currently, no close relative available''). genbank and rdp-ii identified all dangerous pathogens correctly. the microseq v 1.0 database identified four of the six species of dangerous pathogens. on species level none of the dangerous pathogens was correctly identified using microseq 1.4.1 or ridom. as previously noted by various other authors, the most important reason for failure of databases in identifying a bacterium is a lack of the 16 s rrna gene sequence of the particular bacterium in the database rather than misidentification because of poor sequence quality. one must also be aware that the following bacterial species or subspecies have the same 16s rrna gene sequence, which makes differentiation by sequence analysis impossible: b. anthracis and b. cereus, y. pestis and y. pseudotuberculosis, all brucella subspecies, and francisella tularensis ssp. holarctica and mediasiatica. in addition to 16s rrna gene analysis complementary methods are essential to discriminate between these bacteria on species or subspecies level. identification of nontuberculous mycobacteria by sequence analysis of the 16s ribosomal rna, the heat-shock protein 65 and the rna polymerase beta-subunit genes s. shin, j.h. yoon, e.c. kim (seoul, kr) objectives: the diagnosis of diseases caused by nontuberculous mycobacteria (ntm) is difficult because ntm are prevalent in the environment such as soil and water and because they have fastidious properties. in this study, we investigated the distribution pattern of ntm clinical isolates and the identification to the species level. methods: among the presumptive ntm clinical isolates, cultured in a third referral hospital from 21-jan-2003 to 20-jan-2004 in seoul, south korea, which were negative by probe hybridization method for mycobacterium tuberculosis complex, we selected those of more than 10 colonies or those cultured more than twice in a same patient. a total of 120 isolates were studied for the distribution of ntm including 97 isolates recruited for species identification by direct sequencing of 16s rrna, hsp65 and rpob gene segments. (2.5%) were also identified in the presumptive ntm isolates. the identification rate by sequencing of 16s rrna, rpob, and hsp65 were 65%, 82% and 87%, respectively. hsp65 or rpob gene was more efficient than 16s rrna in identification of ntm by sequencing. conclusions: some ntm are considered to be the causative organisms of clinical diseases even in the countries with intermediate burden of tuberculosis, so accurate identification method by direct sequencing can be adapted to clinical laboratories. evaluation of the genotype mtbdr assay for the simultaneous detection of resistance to rifampicin and isoniazid of mycobacterium tuberculosis clinical strains f. brossier, c. truffot-pernot, n. veziris, v. jarlier, w. sougakoff (paris, fr) objectives: the rapid determination of drug resistance in mycobacterium tuberculosis is an important challenge to ensure a rapid effective chemotherapy. the genotype mtbdr test is a commercially available dna strip assay enabling the molecular genetic identification of the m. tuberculosis complex and its resistance to rifampicin (rif-r) and isoniazid (inh-r) by detecting the most commonly found mutations in the genes rpob (asp516val, his526tyr, his526asp, ser531leu) and katg (ser315thr). here, we report the evaluation of the genotype mtbdr assay from a set of 106 clinical isolates of m. tuberculosis. methods: 106 clinical isolates were collected in france over a 2 years period (2003) (2004) and were included in the study: 77 were rif-r, 96 were inh-r (of which 73 were also rif-r) and 6 were susceptible to both drugs. the susceptibility tests were carried out by the standard proportion method. the mutations involved in rif-r and inh-r in rpob, katg, inha and his promoter region, were characterized by dna sequencing. results: the genotype mtbdr assay identified 100% of the 77 rif-r strains harbouring mutations in the rpob gene, of which 37 (48%) showed a ser531leu mutation and 14 (18%) a his526asp or tyr mutation. 61 of the 96 inh-r strains (63%) harboured a ser315thr mutation in katg, all identified by the genotype mtbdr assay. 58 of this 61 strains displayed a high level of inh-r. among the other inh-r strains, 3 showed a katg mutation at the level of the 315 regions, which was different from ser315thr (2 of which showing a low level of inh-r), and one harboured a deletion in katg (with a high level of inh-r). these 4 mutations were also detected by the strip. finally, among the 31 remaining inh-r strains not detected by the mtbdr assay, 13 were characterized by a mutation in position -15 of the promoter region for the maba-inha regulon (10 with a low level of inh-r), 7 by a ser94ala mutation in inha (all with a low level of resistance) and 11 by other mutations. conclusions: the mtbdr assay, which can readily be included in a routine laboratory workflow, identified 100% and 68% of the strains resistant to rif and inh, respectively. interestingly, 60 of the 68 inh-r strains showing a high level of resistance (88%), but only 5 of the 28 inh-r strains with a low level of resistance (18%), were detected by the mtbdr assay, indicating that complementary tests are necessary for detection of the m. tuberculosis strains having a low level of resistance to inh. variation in the streptococcal 16s rdna detected by pyrosequencing m. haanperä, p. huovinen, j. jalava (turku, fi) originally the aim of this study was to identify alpha-haemolytic streptococcal isolates to the species level by pyrosequencing the v1 and v2 regions of the 16s rdna and comparing the results to the sequences of type strains that have been determined earlier. however, the isolates could not be unambiguously identified due to sequence variations detected in the alpha-haemolytic isolates. materials and methods: invasive s. pneumoniae isolates (n = 17), alpha-haemolytic streptococcal blood culture isolates (n = 16) and alpha-haemolytic streptococcal isolates from the normal pharyngeal microbiota (n = 34) of six elderly persons were analysed by pyrosequencing the v1 and v2 regions. results: varying degree of genetic variation was found in different types of streptococcal isolates. in the pneumococcal isolates, no sequence variation was detected as all the isolates contained the sequence specific for s. pneumoniae in both regions. also the sequences of the alpha-haemolytic blood culture isolates were well in agreement with the sequences of the streptococcal type strains. however, most of these isolates could not be unambiguously identified, as they contained sequences belonging to different species in the v1 and v2 region. consequently, only five of the isolates could be unequivocally identified as s. gallolyticus (n = 2), s. anginosus (n = 1), s. mitis (n = 1) and s. sanguinis (n = 1). the commensal streptococci contained numerous sequences to which an identical type sequence could not be found. also sequences identical to type strains were found; but similarly to the blood culture isolates, the results enabled the identification of only four isolates: s. mitis (n = 2), s. parasanguinis (n = 1), and s. salivarius or s. vestibularis (n = 1). moreover, the pyrograms of three blood culture isolates and ten pharyngeal isolates indicated heterogeneous 16s rdna alleles. one such pyrogram of the v1 region is presented in the figure. interestingly, four of the eight different nonheterogeneous v1 and v2 sequence combinations of the blood culture isolates were also present among the pharyngeal isolates. the results of this study indicate that the variation in commensal streptococci is greater than that of the streptococcal type strains and pathogenic isolates. the presence of identical sequence combinations among the blood culture and pharyngeal isolates supports the assumption that potentially pathogenic isolates are present in the normal microbiota. evaluation of partial 16s rrna gene sequencing for identification of clinical isolates of nocardia species m. marín, m. sánchez, m. del rosal, e. cercenado, p. martín-rabadán, e. bouza (madrid, es) new species of nocardia are being described. conventional identification based on biochemical characteristics and pcrrestriction enzyme analysis is frequently unable to distinguish them. partial sequencing of 16s rrna gene has proven useful in the identification of bacteria. objective: to evaluate the utility of 5'end 16s rrna gene pcr and sequencing in the identification of clinical isolates of nocardia sp. compared with conventional methods and pcr-rflp of hsp65. methods: 48 clinical isolates of nocardia sp. were characterized by biochemical reactions and disk diffusion susceptibility testing. molecular identification was performed by hsp65 pcr-rflp and pcr of 5'end of 16s rrna gene followed by sequencing. the sequences obtained were compared with those included in genebank. only alignments with similarities higher than 99% were considered. a comparison of sequences of our nocardia isolates with those deposited in genebank and well characterized phenotypically was performed using clustal x 1.8 software. results: distribution of species after pcr-rflp of hsp65 was n. asteroides vi (22), n. farcinica (11), n. nova (6), n. asteroides i (4), n. otitidiscaviarum (4) and n. asteroides iv (1) . partial sequence analysis of 16s rrna revealed a great heterogeneity between the isolates of n. asteroides vi, as follows: n. cyriacigeorgica (15 isolates), n. abscessus (3 isolates) and n. carnea (1 isolate). for 3 isolates, no genebank sequence was found with more than 99% similarity. all n. farcinica isolates had the same sequence and showed 100% similarity with those deposited in genebank. n. nova, n. asteroides i and n. otitidiscaviarum also showed sequence heterogeneity. three n. nova isolates matched with the recently described n. veterana and 3 with n. nova. n. asteroides i isolates were identified as n. abscessus (1) and n. beijingensis (3) . all n. otitidiscaviarum were identified properly. the isolate of n. asteroides iv was identified as n. transvalensis. conclusions: sequencing of 5'end 16s rrna gene is a useful and rapid molecular tool for the identification of nocardia clinical isolates. this method could provide more accurate results than the conventional ones used routinely in our laboratory. sequence analysis of the 5'end 16s rrna has enabled us to recognize great diversity and new species among our nocardia isolates. several species would have gone unnoticed using non-sequencing-based methods. antibacterial susceptibility studies-iii p1765 anaerobic bacteraemia due to fusobacterium necrophorum and clostiridium cadaveris: a case report m. panopoulou, e. alepopoulou, e. chrisafidou, a. tsaroucha, c. simopoulos, s. kartali (alexandroupolis, gr) introduction: anaerobic bacteremia is uncommon accounting 0.5-9% of bacteremias and it is associated with a high mortality rate, which is strongly and independently associated with underlying liver disease. case report: a 62 year-old man presented to our hospital with a 2-day fever and rigor. he had a history of cancer of the extrahepatic biliary tree, which was found incidentally during an operation for the treatment of echinococcal cyst of the liver. physical examination reveals high fever (39 c) and tachycardia. blood tests showed the following results: hb: 9.9 gr/dl, wbc: 22.500 /ul, plt: 296.000 /ul, tprot: 5.9 each colony type subcultured to blood agar plates and incubated aerobically and anaerobically (aerotolerance test). after 24 hours of incubation the two organisms grew only in anaerobic conditions. they identified by the api 20a system (bio-merieux-france) as fusobacterium necrophorum and clostiridium cadaveris. the patient's treatment started with metronidazole, amikacin and ceftriaxone and followed by metronidazole and imipenem. he was discharged after 3 weeks in a good condition. conclusions: although anaerobic bacteremia is rare, there is value in performing separate anaerobic blood cultures. the early recognition of anaerobic bacteremia and administration of the appropriate antimicrobial therapy play a major role in preventing mortality especially in patients with underlying disease. fluoroquinolone resistance among enterobacteriaceae strains isolated from urinary tract infections v. skandami-epitropaki, p. fostira, a. tsiringa, a. xanthaki, k. zampitha, m. toutouza (athens, gr) objectives: to study the frequency and antibiotic susceptibility of quinolone resistant bacterial stains isolated from patients with community-aquired bacteriuria and compare it with urinary pathogens from hospitalized patients. methods: during a 12-month period (october 2004 -october 2005 a total of 772 bacterial strains were isolated out of 8369 urine samples submitted for culture in our hospital laboratory from the community and from hospitalized patients with urinary tract infection symptoms. cultures and bacterial identification were obtained by conventional methods. antibiotic susceptibility testing was done by kirby-bauer disk diffusion method according nccls criteria. results: of the 772 bactrial strains studied (escherichia coli 604, klebsiella pneumoniae 97, proteus mirabilis 71), 14.6% of them were found to be quinolone resistant. the percentage of quinolone resistance was 19.9% for hospitalized patients (hp) and 5.7% for community patients (cp). the quinolone resistance for e. coli was 11.3% (16.5% for hp and 4.3% for cp), for k. pneumoniae 24.7% (28.0% for hp and 6.7% for cp) and for p. mirabilis 29.6% (29.6% for hp, 29.4% for cp). susceptibility pattern of the 113 quinolone resistant isolates to other antimicrobial agents was for hospitalized patients and community patients respectively as following: for e. coli ampicillin (am) 7%-9.1%, amoxicillinclavulanate (amc) 45.6%-54.5%, piperacillin-tazobactam (tzp) 69.4%-81.8%, cefuroxime (cxm) 64.9%-72.7%, trimethoprimsulfamethoxazole (sxt) 15.8%-18.2%, ceftazidime (caz) 66.7%-72.7%, cefepime (fep) 66.7%-81.8%, gentamicin (gm) 80.7%-72.7%. for k. pneumoniae am 0%-0%, amc 25%-0%, tzp 68.4%-100%, cxm 37.5%-100%, sxt 15.8%-100%, caz 37.5%-100%, fep 41.7%-100%, gm 79.2%-100%. for p. mirabilis am 9.5%-20%, amc 23.8%-40%, tzp 90.5%-100%, cxm 25%-40%, sxt 4.8%-0%, caz 23.8%-40%, fep 100%-100%, gm 56.3%-60%. seven strains of k. pneumoniae (29.2%) were carbapenem resistant and metallo-beta lactamase producing. conclusions: high resistance rates to fluoroquinolones were observed in uropathogen bacteria isolated not only from hospitalized patients but also from patients with communityacquired urinary tract infections in greece. increasing resistance rates to the rest antibiotic agents make the treatment of urinary tract infections a very difficult problem. susceptibility of pseudomonas aeruginosa isolated from the mystic programme to the carbapenems: meropenem and imipenem p.j. turner (macclesfield, uk) objectives: the meropenem yearly susceptibility test information collection programme (mystic) was initiated in 1997 in order to track the susceptibility of organisms in centres that were prescribing meropenem. this poster seeks to examine the susceptibility of pseudomonas aeruginosa isolates over this period to the carbapenems; meropenem and imipenem and, in particular, records the susceptibility of imipenem-resistant isolates to meropenem and vice versa. methods: pseudomonas aeruginosa isolates were speciated by the methods in current use at the participating centres. minimum inhibitory concentrations of meropenem and imipenem were determined using reference methods described by clsi. results: a total of 15709 isolates of pseudomonas aeruginosa have been tested globally, of these 78.2% were susceptible to meropenem at the breakpoint of < 4 mg/l and 70.1% to imipenem. globally, susceptibility to the two carbapenems has remained stable over the period 1997-2005, however when imipenem-resistant isolates were examined (n = 4625) 32.7% proved to be susceptible to meropenem, conversely of the 3359 meropenem-resistant isolates only 7.8% proved to be susceptible to imipenem. a similar pattern was seen when isolates were separated into global regions:usa 425 imipenem-resistant isolates, 25.4% susceptible to meropenemusa 346 meropenemresults: bacteroides fragilis group (bafg) accounted for 53% of the isolates, fusobacterium spp. for 7%, other gram negative bacilli (ognb) for 11%, clostridia (clos) for 14%, nonsporeforming gram-positive bacilli (nsfgpb) for 7% and cocci for 8%. beta-lactamases (bl) were detected in 61% of isolates. most bl + strains belonged to bafg (98%) and ognb (70%). at nccls-recommended breakpoints, more than 95% of isolates were susceptible to tzp, mtz, chl and mem, 92% to amc but only 77%, 70%, 62% and 33% to fox, ctt, cli and pen respectively. no nccls-breakpoints for anaerobes are available for mxf, lzd and tig. mic 50 and mic 90 for mxf were 1 and 64 mg/l, for lzd 2 and 4 mg/l and for tig 0.5-8 mg/l. in comparison with similar surveys conducted in 1987 and 1993 -1994 susceptibility of bafg to clindamycin decreased from 83% in 1987 , to 66% in 1993 -1994 and 48% in 2004 in bafg 92% of b. fragilis and 78% of non-b. fragilis were susceptible to amc in this study; in 1993-1994 susceptibility in these groups was 95% and 89% and in 1987-97% and 94% respectively. all isolates, except 6 bafg and 1 clos, were susceptible to mem. 98% of the isolates were susceptible to chl. susceptibility to mtz remains stable and is high in all groups except nsfgpb where mtz is active on merely 35% of the isolates. conclusions: tzp, mem and mtz remain very potent antimicrobial agents in the treatment of anaerobic infections. although still rare, resistant organisms were detected to each of them. therefore susceptibility testing of anaerobic isolates is indicated in severe infections to confirm appropriateness of antimicrobial therapy. further monitoring of background susceptibility is necessary to guide empiric treatment. comparative in vitro activity of levofloxacin against escherichia coli isolated from acute pyelonephritis in france in 2005 c.j. soussy, c. lascols, c. dib-smahi and the multicenter group study. objectives: the objective of this study was to evaluate the in vitro activity of levofloxacin (lvx) comparatively to other antibiotics against escherichia coli strains isolated from acute pyelonephritis in women consulting emerging rooms by 23 french hospitals in 2005. methods: mics of lvx, ofloxacin (ofx), ciprofloxacin (cip), nalidixic acid (nal), amoxicillin-clavulanic acid (amc), ceftriaxone (cro), cefixime (cfm), amikacin (an), gentamicin (gm) and cotrimoxazole (sxt) were determined by agar dilution according to the eucast breakpoints approved by 2005 recommendations of the comité de l'antibiogramme de la société française de microbiologie. quality control was performed with e. coli strain atcc 25922. results: a total of 231 strains were collected. 46.3% of strains were isolated from urinary samples, 10.8 % from blood culture and 42.9% from the two specimens. mics 50/90 (mg/l), the range of mics and the percentage of susceptibility (%) are presented in the following table: concerning the fluoroquinolones, mics50/90 of lvx were one/two dilution lower than those of ofx and two/one dilution higher than those of cip. for the other antibiotics, a higher percentage of susceptibility was observed with cro and an, when a lower percentage of susceptibility was observed with amc and sxt. conclusions: levofloxacin exhibited good in vitro activity against e. coli strains isolated from acute pyelonephritis with 93.1% of susceptible strains. in vitro activity of double and triple combinations of colistin, imipenem, rifampicin and linezolid against epidemic strains of multidrug-resistant acinetobacter baumannii producing oxa carbapenamases d.w. wareham, d.c. bean (london, uk) objectives: a. baumannii has emerged as an important cause of nosocomial infection in critically ill patients worldwide. in the uk three strains in particular exhibiting multi-drug resistance and producing oxa carbapenamases have been responsible for ongoing outbreaks. treatment options for infection with these organisms are limited as only colistin and tigecycline retaining significant activity in vitro. animal models and in vitro studies using other multi-resistant strains suggest that drugs in combination with colistin may be effective. we assessed the activity of colistin in combinations including imipenem, rifampicin and linezolid against epidemic strains from a recent uk outbreak. methods: isolates of a. baumannii exhibiting resistance to carbapenems were recovered from patients at barts and the london nhs. isolates were referred to the health protection agency and confirmed as belonging to clones producing oxa carbapenemases. activities of polymyxin, imipenem, rifampicin and linezolid alone and in double and triple combinations were determined using standard chequerboard assays with increasing concentrations of drug 1 on the x axis, drug 2 on the y axis and drug three in multiple replicate plates. after incubation at 24 hours wells were examined for growth and mic's determined for each combination. synergy between agents was defined as a fixed inhibitory concentration index (fici) of < 0.5. results: the isolates tested belonged to the oxa-23 clone 1, oxa-23 clone 2 and the south east clone, as confired by the hpa. colistin was the most active agent alone with mics from 1-2 mg/l. imipenem mic's varied from 4-32 mg/l. the most active combinations were colistin plus rifampicin (fici = 0.38) and colistin, rifampicin and imipenem (fici = 0.39). synergy was not seen with colistin in combination with imipenem alone. linezolid in combination with colistin (fici = 0.37), or imipenem (fici = 0.38) was synergistic but at therapeutically unobtainable linezolid concentrations (64 mg/l). conclusion: multidrug resistant strains of a. baumannii from the uk producing oxa carbapenemases remain susceptible to polymyxin in vitro. polymyxin exerts its effect on the bacterial cell wall; theoretically assisting other antibiotics to reach their respective targets, and seems a logical choice for inclusion in combination therapy. we have shown that rifampicin is synergistic with polymyxin against these isolates in vitro and may be effective in treating severe a. baumannii infections in man. a comparative in vitro evaluation of resistance development after exposure to teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin in staphylococcus spp. and enterococcus spp. mssa, mrse, msse, e. faecium and e. faecalis strains was determined on agar plates containing each antibiotic at clsi resistance breakpoints and at peak blood concentrations. after incubation at 37°c for 48 h colonies were counted and compared to the inoculum to calculate frequency of mutation. colonies grown in plates containing antibiotics were sampled for determination of mic values. results: frequency of mutation was less than 10-9 for all the tested antibiotics at peak blood concentrations. same results were obtained when breakpoint concentrations for each drug were used. conclusion: this one-step in vitro study demonstrated the ability of teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin to prevent growth of resistant mutants of staphylococci and enterococci, thus suggesting no occurrence of mutational events leading to resistance when bacteria are exposed to blood concentrations of these drugs. in order to establish the development of resistance after in vitro serial exposure to the same antibiotics simulating different in vivo concentrations, further studies are needed and are now in progress (multi step induction of resistance). in vitro activity of antimicrobial agents against legionella obtained from hotel water systems in turkey objectives: the aim of this study was to evaluate the in vitro activity of colistin against endemic pan-resistant acinetobacter baumannii (including resistance to imipenem) isolated during a 4 year period in a university hospital. methods: 150 imipenem-resistant acinetobacter spp. isolates were collected between january 2001 and october 2004, from a variety of clinical specimens of different patients attending distinct wards in a university teaching hospital. isolates were identified by api32gn and by sequencing the 16s rrna gene. mics of colistin were determined by agar dilution method, according to nccls susceptible breakpoint (£ 2 mg/l). pfge (apai restriction enzyme) was performed. results: 141 of 150 a. baumannii isolates (94%) were susceptible to colistin. colistin resistance (mic ‡ 4 mg/l) was observed in 9 isolates (5 isolates with a mic of ‡ 16 mg/l and 4 isolates with a mic of 4 mg/l) recovered from different patients in distinct wards. among these imipenem-and colistin-resistant isolates, 3 distinct pfge patterns were identified (clones a, b, and c). resistance to almost all beta-lactams (including carbapenems) and variable susceptibility to aztreonam, amikacin and tobramycin was a common feature of clone a. isolates belonging to clone b showed resistance to imipenem, amoxicillin and its association with clavulanic acid (amc), ureidopenicillins and their associations; susceptibility to ceftazidime; and variable behaviour to meropenem, cefepime, cefpirome and aztreonam. the susceptibility profile to aminoglycosides was variable, differing from clone a in its susceptibility to netilmicin and minocycline. clone c was resistant to imipenem, amoxicillin, amc, piperacillin, piperacillin + tazobactam, ticarcillin and ticarcillin + clavulanic acid, but remained susceptible to meropenem, aztreonam, cefpirome, ceftazidime and cefepime. conclusion: only colistin, one of the few effective drugs available against multi-drug-resistant acinetobacter infections, showed in vitro activity against the majority of acinetobacter spp. strains isolated within the sampled hospital. the observed 6% a. baumannii resistance to the recently re-introduced colistin seems like the first chapter of a novel repeatedly told for several antibiotics. emergence of high-level gentamicin resistance in clinical enterococcal isolates of companion animals in portugal objectives: to characterize in vitro gentamicin susceptibility among enterococci causing infections in cats and dogs, in order to evaluate the impact of high-level gentamicin resistance in small animal therapeutics. methods: the samples were collected at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. from january 1998 until november 2005, a total of 43 enterococci were isolated from dogs and cats with urinary tract infection (uti), otitis externa (oe) and pioderma. bbl crystal gram positive id system was used for identification at the species level. minimal inhibitory concentrations (mic) were determined by the microdilution method according to nccls (1) . the bifuntional enzyme gene that confers high-level gentamicin resistance (hlgr) was detected using pcr (2) . results: enterococcus faecalis was the predominant isolate (n = 35), followed in frequency by enterococcus faecium (n = 6). mic cumulative data analysis showed that mic50 values were 8 lg/ ml and mic90 1024 lg/ml. six (14%) hlgr clinical enterococcal isolates were detected, with mic ranges between 1024-2048 lg/ml. four of these enterococci were isolated from uti and 2 from oe. four of the phenotypically high-level gentamicin resistant isolates carried the aac(6')-ie-aph(2'')-ia gene. conclusions: the importance of enterococcal infection in small animal clinical samples has increased over the last years. mic cumulative data points out low-level gentamicin resistance among clinical enterococci isolates of veterinary origin and the emergence of high-level isolates, as previously detected (2). this fact compromises cell-wall active agents (such as ampicillin or vancomicin) and aminoglicoside in vivo synergy. the aac (6')-ieaph(2'')-ia gene carriage is of concern because its expression confers resistance also to tobramicin, netilmicin, amikacin and kanamicin. our findings are of critical importance, as they may have a direct impact in therapeutic decision in the management of companion animal's infections by enterococci. furthermore, transfer of resistance genes and resistance strains between animals and owners/caretakers by direct contact is a concerning probability. references: (1) results: interpretative criteria were used according to nccls 2002.during the study period penicillin resistant strains of s. pneumoniae was noted as follows: 67% in sputum or ta,19% in blood,12%in csf,and 75% in others,against cefuroxim resistant strains:15% in sputum or ta,10% in blood,4% in csf.regarding the susceptibility to ofx,penicillin resistant s. pneumoniae strains from sputum or ta revealed 97.9%.the penicillin resistant strains coming from sputum or ta showed resistance as follows; 48% to em and 78% to sxt,against strains isolated from others: 58% to em and 49% to sxt.no resistant strain to va was found. conclusion: the percentage of the penicillin resistant s. pneumoniae isolates from the lower respiratory tract, middle ear fluid, eye fluid and sinus was markedly higher than that of the isolates from blood and csf. the most efficient drugs against penicillin resistant pneumococci were cefuroxim and ofloxacin. these results from romania also underline the previous observations regarding the higher emerging rates of resistance in s. pneumoniae worldwide. penicillin resistance in streptococcus agalactiae objectives: streptococcus agalactiae has become recognized as a cause of serious illness in newborns, pregnant women, and adults with chronic medical conditions. heavy colonization of the genital tract with streptococcus agalactiae also increases the risk that a woman will deliver a preterm low-birthweight infant. early-onset infections (occurring at < 7 days of age) are associated with much lower fatality than when they were first described, and their incidence is finally decreasing as the use of preventive antibiotics during childbirth increases among women at risk. penicillin or ampicillin remains the drug of choice for intrapartum antibiotic prophylaxis for streptococcus agalactiae colonization in pregnant women. erythromycin and clindamycin are the drugs of choice for women with serious penicillin allergy who are colonized with streptococcus agalactiae. the objective of this study is to estimate the insorgence of penicillin resistance among streptococcus agalactiae. methods: all streptococcus agalactiae were tested against penicillin by agar dilution method according to clinical and laboratory standards institute 2005 (clsi); breakpoints for resistance were those recommended by the clsi. antimicrobial agents were obtained from their manufacture as laboratory grade powder. results and discussion: four hundred seven (407) clinical isolated were analysed during 2005. streptococcus agalactiae resulted resistant to penicillin in 1 case; and about 2% resulted borderlines.the present findings indicate a probable evolution in s. agalactiae toward penicillin resistance this finding suggest the need a continuous national and international surveillance programs to provide timely data on the evolution of incidence of penicillin resistance in this pathogen. ciprofloxacin susceptibility of the most common isolates at bacterial conuctivitis conclusion: according to the average numerals we concluded that all the isolated strains are highly susceptible at ciprofloxacin. its application in the conuctivial saccus is especially important in curing the conuctivial infections with resistent strains like pseudomonas aeruginosa. we successfully cure the bacteria chronic conuctivitis with the adequately used therapy according to antibiogram. antimicrobial resistance patterns of acinetobacter baumannii in clinical isolates g.t. tsilika, v.p. pliatsika, m.t. tsivitanidou, d.s. sofianou (thessaloniki, gr) objectives: a. baumannii is a nosocomial pathogen, commonly isolated from critically ill and immunocompromised patients. the aim of the present study was to evaluate the antimicrobial resistance of a. baumannii strains isolated in a tertiary care hospital througout a three-year period. methods: a total of 1311 a. baumannii strains were selected from january 2002 to december 2004.the specimens were obtained from inpatients hospitalized in intensive care unit (icu) and pediatric intensive care unit (picu) and other departments of our hospital.the identification and the antimicrobial susceptibility testing were performed using the vitek 2 automated system(biomerieux,france conclusions: the emergance and rapid spread of multidrug resistant a. baumannii isolates are of a great concern worldwide.imipenem was one of the most potent agents for treatment of those infections caused by multiresistant strains.the increasing prevalence of imipenem resistance limits therapeutic options and leads to outbreaks of carbapenems resistant strains. tigecycline in vivo studies objectives: antibacterial agents disrupt the ecological balance of the normal human microflora. disturbances may lead to the emergence of antibiotic resistance and/or to infections by potentially pathogenic bacteria. tigecycline, a member of a new class of antibiotics (glycylcyclines), has been shown to have a potent expanded broad-spectrum activity against most grampositive and gram-negative aerobic and anaerobic bacteria. the aim of the study was to investigate the ecological effects of tigecycline on the normal oropharyngeal and intestinal microflora in healthy subjects. methods: thirteen (13) white subjects (6 women, 7 men) aged 20 to 31 years, received 100 mg of tigecycline in the morning on day 1 as a 30-minute intravenous (iv) infusion, followed by 50mg doses of tigecycline given every 12 hours as a 30-minute infusion for 10 days. one (1) subject was withdrawn on day 2 because of an adverse event. serum, saliva, and faecal samples were collected before, during, and after administration for microbiologic cultivation and for assays of tigecycline. all new colonizing bacteria were tested for susceptibility (resistance >8 mg/l) during the investigation period. results: the serum concentrations on day 9, 12 hours after dosing, were 0.2 to 2.1 mg/l (mean value 0.4 mg/l, median value 0.2 mg/l, and sd 0.5 mg/l). the faecal concentrations on day 8 were 3.0 to 14.1 mg/kg (mean value 6.0 mg/kg, median value 5.6 mg/kg, and sd 2.9 mg/l). saliva concentrations were generally low, with highest mean value 0.18 mg/l, median value 0.22 mg/l, on day 10, 3 hours after dosing. a minor effect on the oropharyngeal microflora was observed. the numbers of enterococci and escherichia coli in the intestinal microflora were reduced at day 8, while other enterobacteria and yeasts increased. there was a marked reduction of lactobacilli and bifidobacteria but no impact on bacteroides. no clostridium difficile strains were isolated. two (2) klebsiella strains and 5 enterobacter strains resistant to tigecycline were found. conclusion: tigecycline had a minor effect on the oropharyngeal microflora. tigecycline's effect on the intestinal microflora was due to its spectrum of antibacterial activity and intestinal concentrations. objectives: to examine and report the use of tigecycline (wyeth) in the treatment of multidrug resistant acinetobacter (mdra) culture positive sepsis in 11 patients requiring mutiorgan support. methods: all patients were managed within the liver intensive care unit. physiological data was collected prospectively and entered onto a specialist database. patients received standard intensive care management; antibiotic and antifungal therapy administered as indicated by microbiological cultures. systemic inflammatory response (sirs) features initiated blood cultures (vascular lines and peripheral), drain fluid culture and broncoalveolar lavage (bal). screening swabs were undertaken weekly and samples sent for culture at laparotomy. mdra positive cultures from blood, bal, drain fluid or samples taken at laparotomy in the context of sirs resulted in the initiation of tigecycline treatment. results: 11 patients received tigecycline treatment for mdra infections. the underlying disease states were necrotizing pancreatitis (1), post hepatectomy (1), polytrauma (1), all with postive intra-abdominal cultures. acute and acute on chronic liver failure (4), mdra +ive broncho-alveolar lavage ± blood cultures and 4 post liver transplant patients (necrotising pancreatitis in one, 2 with recurrent small bowel perforation and 1 with retroperitoneal haemorrhage) all with positive blood cultures and in 3 positive intra-abdominal tissue/clot. mean time from admission to treatment for mdra was 25 days. mean duration of treatment was 10 days (range 4-15). mean apache ii score at initiation of therapy was 18 (range 13-26); 4/11 patients survived to intensive care discharge and 3/11 to hospital discharge. microbiological clearance of mdra was observed in 8/11 cases. in those who did not achieve microbiological clearance cause of death was intra-abdominal haemorrhage, recalcitrant organ failure with recurrent small bowel perforation and vasopressor resistant shock. in these patients one remained culture positive for intraabdominal sepsis despite full treatment (small bowel perforation x5). the drug was well tolerated with the only side effect being that of hypercalcaemia observed in 5/12 patients, mean corrected calcium 2.59 mmol/l, range 2.32-2.81. in all cases this resolved on drug discontinuation. conclusion: tigecycline appears to be an efficacious agent in the treatment of deep seated mdra infections. objectives: nausea (n) and vomiting (v) have been reported with tigecycline, a new glycylcycline with expanded broad spectrum activity. exposure-response relationships and patient covariates predictive of the first n and v occurrence were evaluated in patients with complicated intra-abdominal infections (ciai). methods: data from patients from 3 ciai trials (one phase 2 and two phase 3), receiving 100 mg loading dose and 50 mg every 12 hours, were pooled for analysis. n and v (definitely, possibly, or probably related to tigecycline) reported from the start of infusion until 24 hours after the last dose were included. individual exposure measures [auc0-12 and cmax] were calculated using a previously developed population pk model. logistic regression was used to evaluate predictors of first n and v occurrence. covariates included age, weight, sex, region of treatment, and baseline n and v. results: the dataset included 928 patients (218 with pk). mean (sd) age and weight were 46 (18) years and 73 (16) kg. 64% of patients were men and 24%, 37%, and 18% were enrolled in north america, europe, and latin america, respectively. baseline nausea or vomiting was reported in 47% and 35%. overall, n and v occurred in 18% and 13% of patients receiving tigecycline, however most (62%; 67%) of first n and v events were mild in nature. women had more n and v (23%; 17%) than men (15%; 11%). n and v were lower in europe (10%; 6%) than in other regions. auc0-12 and cmax were not predictive. the final nausea model included weight, sex, region, baseline nausea, and the interaction of weight/region as predictors of the first nausea occurrence (p = 0.671, 0.0006, 0.205, 0.033, & 0.023, respectively objective: because hospitalisation for community-acquired pneumonia (cap) is associated with substantial morbidity and health resource utilisation, we evaluated the predictors of prolonged hospital length of stay (los) and treatment duration. methods: we conducted a retrospective analysis of data from a double-blind, randomised, multicentre clinical study that compared the efficacy and safety of tigecycline with that of levofloxacin in the treatment of patients with cap requiring hospitalisation. patients were stratified by the fine pneumonia severity index and randomly assigned to receive tigecycline or levofloxacin via iv administration for at least 7 days. treatment duration and hospital discharge were based on physician assessment of signs and symptoms of infection and patient condition. we used cox proportional hazards modelling with stepwise selection to identify statistically significant predictors (p < 0.05) of treatment duration and hospital los. results: among 426 patients with cap in the clinical intent-totreat population with complete hospitalisation data, mean age was 49.8 years (range 17-92) and 22.8% of patients were aged ‡65 years. diabetes (11.5%), chronic obstructive pulmonary disease (7.8%), and congestive heart failure (7.0%) were leading co-morbidities. about 37.1% of patients were smokers and 5.6% were characterised by alcohol abuse. median fine pneumonia severity index score was 3; 21.1% of patients had a score >4. . there were no significant differences between the 2 groups in treatment duration or los. conclusions: tigecycline, a first-in-class glycylcycline, was associated with treatment duration and los similar to that of levofloxacin, adjusting for several identified risk factors. tigecycline effective in treating patients with intra-abdominal or skin/skin structure infections who have bacteraemia e.j. ellis-grosse, r. maroko (collegeville, us) objectives: the treatment of bacteraemia, which is a potentially fatal complication of infections originating at other body sites, is complicated by increasing resistance. tigecycline, a first-in-class glycylcycline, has an expanded spectrum of activity against gram-positive, gram-negative, anaerobic, and atypical bacteria including resistant strains. tigecycline is safe and effective in treating complicated skin and skin structure (csssi) and intraabdominal infections (ciai). this analysis examines tigecycline clinical trial experience in patients with ciai or csssi who had bacteraemia (presence of bacteria in blood) at baseline. objectives: treatment of complicated intra-abdominal infections (ciai) is challenging due to diverse bacteriology and bacterial resistance. the efficacy and safety of tigecycline (tgc), a first-in-class glycylcycline approved in mexico, brazil, peru, colombia and usa for treating ciai and complicated skin and skin structure infections, was compared with imipenem/cilastatin (imi/cis) in adult hospitalised patients with ciai in two double-blind, phase 3 multinational trials. this analysis evaluated tgc efficacy and safety in the european region of the integrated results of these two trials. methods: one study was conducted in 96 centres (17 countries) and the other study was conducted in 94 centres (27 countries). patients were stratified by disease severity (apache ii score £15 vs >15 but £30), and randomly assigned to iv tgc (100 mg loading, then 500 mg q12h) or iv imi/cis (500/500 mg q6h) for 5-14 days. clinical response at test-of-cure (toc, 12-44 days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) were co-primary efficacy endpoints where cure/failure responses were determined. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, 703 patients were mitt (received ‡1 dose), 556 m-mitt (283 tgc, 273 imi/cis) and 460 me (237 tgc, 223 imi/cis). treatment groups were balanced with respect to demographics. patients were mostly white (98.2%) men (58%) with a mean age of 50 years. for me, clinical cure rates at toc were 92.4% (219/237) for tgc vs 88.8% (198/ 223) for imi/cis (95% ci = )2. 2, 9.4 ; test for non-inferiority p < 0.0001). clinical cure rates for m-mitt were 87.3% (247/283) for tgc vs 83.5% (228/273) for imi/cis (95% ci = )2.5, 10.0; test for non-inferiority p < 0.0001). most commonly reported treatment emergent aes (teaes, mitt) for tgc and imi/cis were nausea (14.7% and 11.8%, p = 0.267) and vomiting (10.7% and 7.3%, p = 0.146). the imi/cis group had significantly higher teaes of fever (7.3% imi/cis vs 3.2% tgc, p = 0.017), hyperglycaemia (1.7% imi/cis vs 0 tgc, p = 0.031) and dyspnoea (2.8% imi/cis vs 0.3% tgc, p = 0.011) where tgc had significantly higher amylase increase (3.2% tgc vs 0.6% imi/cis, p = 0.011) and bun increase (2.3% tgc vs 0 imi/cis, p = 0.003). conclusions: similar to the overall integrated analysis of the two phase 3 trails, in the european analysis, tgc was safe and effective in the treatment of hospitalised patients with ciai in comparison with imi/cis. tigecycline is safe and effective in the treatment of complicated skin and skin structure infections: european experience of two double-blind phase 3 comparison studies with vancomycin/aztreonam r. maroko, n. dartois, d. sarkozy, j. goodrich, e.j. ellis-grosse on behalf of the tigecycline 300 and 305 study groups objectives: tigecycline (tgc) a first-in-class expanded spectrum glycylcycline, has been approved in mexico, brazil, peru, colombia and usa for treating complicated skin and skin structure infections (csssi) and complicated intraabdominal infections. two phase 3, randomised, double-blind studies were conducted in hospitalised men and women with csssi to determine tgc safety and efficacy compared with vancomycin/aztreonam (v/a). the objective of this analysis was to evaluate the efficacy and safety seen in the european population of the integrated analysis of these 2 phase 3 trials. methods: one study was conducted in 53 centres in 8 countries while the other study was conducted in 65 centres in 21 countries. patients were randomly assigned (1:1) to receive either tgc (100 mg, followed by 50 mg iv twice daily) or vancomycin (1 g iv twice daily) plus aztreonam (2 g iv twice daily) for up to 14 days. clinical response at test-of-cure (toc, 12-92 days after therapy) for clinically evaluable (ce) and clinical modified intent-to-treat (c-mitt) populations were coprimary efficacy endpoints in which cure/failure responses were determined. secondary objectives included determination of in vitro susceptibility to tgc of a range of bacteria that cause csssi and microbiological efficacy. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, 385 patients comprise mitt (received ‡1 dose of study drug), 326 comprised ce (167 tgc, 159 v/a/cis) and 376 comprised c-mitt (189 tgc, 187 v/a/ cis). treatment groups were balanced with respect to demographics. patients were mostly white (99.7%) men (63.6%) with a mean age of 50 years. in the european region, clinical responses to tgc and v/a at test-of-cure were similar: c-mitt, 84.7% (160/ 189) versus 86.6% (162/187), difference tgc-v/a was -2.0% (95% ci -9.5, 5.6). similar results were noted in the ce population with tgc curing 89.8% (150/167) and v/a curing 95.0% (151/ 159), difference tgc-v/a was -5.1% (95% ci -11.6, 1.2).most commonly reported treatment emergent aes (teaes, mitt) for tgc and v/a were nausea (17.3% and 3.2%, p < 0.001) and vomiting (6.1% and 1.6%, p = 0.032). the v/a group had significantly higher teaes of sgpt increase (6.9% v/a vs 2.0% tgc, p = 0.025) and rash (2.6% v/a vs 0 tgc, p = 0.028). conclusion: in the european analysis of the integrated phase 3 worldwide clinical studies, tgc monotherapy is as safe and efficacious as the combination of v/a in the treatment of patients with csssi. safety and tolerability of tigecycline r. maroko, n. dartois, g. rose, e.j. ellis-grosse (collegeville, us; paris, fr) objectives: tigecycline (tgc), a glycylcycline, is a first-in-class, extended, broad-spectrum iv antibiotic that has demonstrated clinical activity in patients with complicated intra-abdominal infections (ciai) and complicated skin and skin-structure infections (csssi). the safety of tigecycline was evaluated in four phase iii trials. methods: a total of 1415 hospitalized patients from these trials were pooled and evaluable for safety analysis. in the ciai trial, patients received tgc 50 mg q 12 hrs (following a 100-mg loading dose) or imipenem 500 mg and cilastin 500 mg q 6 hrs. those in the csssi study were treated with either tgc (same dose/schedule) or vancomycin 1 gm with or without aztreonam 2 gm q 12 hrs. results: the most frequently reported adverse events (aes) in both tgc-treated groups were nausea (n) and vomiting (v). the incidence of n was 29.5% while v was approximately 19.5%; these were generally mild to moderate in severity. infection-related serious aes were slightly more frequent with tgc versus comparators (6.7% vs 4.6%). discontinuations due to treatment-emergent aes (including n/v) occurred at similar rates with tgc and comparators (5.0% vs 4.7%). six patients (0.36%) treated with tgc presented with intestinal perforations and developed sepsis/septic shock compared with 2 (0.12%) for imipenem/cilastatin, with higher baseline apache ii scores in the tgc group; the relationship to treatment could not be determined. in the overall efficacy analysis, subjects with ''perforation of the intestines'' were balanced between the two groups, and overall efficacy was not statistically different. no clinically significant renal, hepatic, cardiac (qtc), bone marrow, or cns toxicities were noted with tgc. conclusion: tgc appears to be safe and tolerable for patients with ciai and csssi. n/v were generally mild to moderate in severity, self-limiting, and did not result in increased overall drug discontinuation. there did not appear to be clinically significant renal, hepatic, cardiac, bone marrow, or neurological toxicities related to tgc treatment. all-cause mortality rates did not statistically differ between those treated with tgc and the comparators. its demonstrated efficacy and favourable toxicity profile make tgc a good monotherapy option for selected serious infections. tigecycline as effective as imipenem/cilastatin in the treatment of complicated intra-abdominal infections: experience in india objective: due to diverse bacteriology and bacterial resistance, treatment of complicated intra-abdominal infections (ciai) is a challenge. in a double-blind, phase 3, multinational trial, the efficacy of tigecycline, a first-in-class glycylcycline, was compared with imipenem/cilastatin (imi/cis) in hospitalised patients with ciai. this subanalysis evaluated tigecycline safety and efficacy from 12 investigational sites in india. methods: patients were stratified by disease severity (apache ii score £15 vs >15 but <31), and randomly assigned to iv tigecycline (100 mg loading, 50 mg q12h) or iv imi/cis adjusted for body weight (500/500 mg q6h for ‡70 kg) for 5-14 days. clinical response at test-of-cure (toc, 12-44 days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) populations were co-primary efficacy endpoints where cure/failure responses were determined. safety evaluations included vital signs, laboratory tests and record of adverse events (aes). results: in india, 167 patients received at least 1 dose (mitt, 84 tigecycline, 83 imi/cis), 149 patients were clinically evaluable (ce), 85 were me, 120 were m-mitt. treatment groups were balanced with respect to demographic/baseline medical characteristics. primary diagnoses (mitt) were complicated appendicitis (31%), gastric/duodenal perforation (26%), perforation of intestine (18%), cholecystitis (12%), peritonitis (6%), and intraabdominal abscess (5%). cure rates at toc in me in india were 36/41 (87.8%) tigecycline and 40/44 (90.9%) imi/ cis, which are consistent with overall me results [80.6% (199/ 247) tigecycline vs 82.4% (210/255) imi/cis (95% ci = )8.4, 5.1; non-inferiority p < 0.001)]. in india m-mitt, cure rates at toc were 47/59 (79.7%) tigecycline and 54/61 (88.5%) imi/cis, similar to the overall m-mitt results [73.5% (227/309) tigecycline vs 78.2% (244/312) imi/cis (95% ci = )11.0, 2.5; non-inferiority p < 0.001)]. noninferiority of tigecycline among india patients could not be statistically demonstrated because of insufficient sample sizes, however, magnitude of response to study drugs in patients treated in india was comparable to that in overall patients. in india, treatment aes were similar with significantly higher incidence of dyspnoea in tigecycline (9.5%) vs imi/cis (0.0%), p = 0.007. conclusions: efficacy results in india are consistent with findings from the overall study and results at other centres, suggesting tigecycline is noninferior to comparator in treating ciai. nosocomial infection: control of environment, viral infections p1788 bacterial flora contamination of blood pressure cuffs in use on hospital wards n. walker, r. gupta, j. cheesbrough (preston, uk) blood pressure cuffs are a plausbile vehicle for the transmission of nosocomial infection between patients. despite this, few studies have examined the level of bacterial contamination and tested for the presence of common nosocomial pathogens on their surface. we swabbed 24 cuffs currently in use on hospital wards. using sterile gloves, a disposable template measuring 10 · 10 cms was placed onto the cuff and a moistened sterile swab was rubbed onto the defined area for 1 minute and then transported in 10 mls of buffer medium. from each sample, 0.5 mls of the buffer was plated onto 6 different media which included a non-selective agar medium for total viable count (tvc) and selective media for s. aureus, mrsa, c. difficile, coliforms and vancomycin resistant enterococci (vre.) bacterial growth was recovered from all 24 cuffs. pathogenic organisms were isolated from 14 cuffs (58%). mssa from 5, mrsa from 1 and c. difficile from 5. the remaining three cuffs grew more than one pathogenic organism; mssa + mrsa + c. difficile from one and mssa + c. difficile from 2 cuffs. colifroms and vre were not isolated from any of the cuffs. the range of total viable counts recovered per 100 cm 2 area of the cuff varied from 1000 > 20000 cfu and the cuffs with the highest counts tended to have more pathogens present. mssa and c. difficile were isolated from 33% of the cuffs sampled and mrsa from 8%. while the actual importance of this potential route of transmission for nosocomial pathogens remains unclear, it can not be dismissed. the impracticality of decontaminating blood pressure cuffs between patients suggests that single patient use cuffs or a barrier between cuff and skin would be a more viable option on a busy general ward. needlestick and sharp injuries of health care personnel in a newly founded tertiary hospital: a prospective study m. falagas, i. karydis, g. georgoulias, p. hatzopoulou, d. nikita, i. kostogiannou (athens, gr) objectives: needlestick and sharp injuries of health care workers are a major cause of anxiety and may expose susceptible employees to the risk of infectious diseases. however, the incidence of such injuries has not been examined in a newly founded hospital while preventive programmes are taking place. methods: we prospectively studied the needlestick and sharp injuries of employees in a newly founded tertiary hospital in athens, greece while a vaccination program against hepatitis b virus as well as educational activities for avoidance of injuries were taking place. serologic studies for hepatitis b and c virus as well as human immunodeficiency virus (hiv) were performed in all injured employees and the source patients (when known). results: sixty-eight needlestick, 8 sharp injuries, and 3 splashes were reported during the study period (01/10/2002 to 28/02/ 2005) in 71 nurses, 2 housekeepers, 3 technicians, and 3 ambulance workers. the overall incidence (percutaneous injuries and splashes) per 100full-time employment-years (100 fteys) was 2.4% whereas the incidence of percutaneous injuries alone per 100 fteys was 2.3%. a higher incidence of injuries was noted during the first than the second half of the study period (3.8% versus 1.8%, p = 0.003). no source patient was found positive for hepatitis c or hiv. the use of high-titre immunoglobulin after adjustment for the incidence of injuries was higher in the first than the second half of the study period (22.6% vs 3.8%, p = 0.05). conclusion: although we did not adjust for possible confounders, our data show that educational and vaccination preventive programs for needlestick and sharp injuries led to a statistically significant decrease in the incidence of such injuries and use of high-titre immunoglobulin. epidemiology of occupational needlestick and sharps injury among healthcare-workers in turkey s. hosoglu on behalf of the occupational infections study group, turkey background: health care workers (hcws) are frequently exposed to the danger of infectious agents through needle stick and sharps injury (nssi) in their occupational efforts. in turkey, the hepatitis b and c viruses cause an essential threat to the hcws because of their prevalence rate (2%-4% and 0.5%-1%, respectively). a cross-sectional countrywide survey study was performed on the epidemiology of nssi among hcws at 30 hospitals in 19 cities throughout the country. data relating to the epidemiology of nssis were collected using a standard questionnaire in 2004. results: totally 5048 hcws completed the questionnaire forms. nurses are the leading group (2016 persons) that joined into the study were followed by doctors (1452 persons) andlaboratory technicians (475). totally 2498 of them (49.5%) declared an occupational exposure or nssi in the last 12 months related their job. needle stick injury was reported in 1698 of them (33.6%), splash into the eye in 1132 (22.4%), sharp injury in 737 (14.6%), and the other injuries in 419 (7.0%). the hepatitis positivity was reported in 567 cases (2.27%) objectives: to assess the microbiological status of reprocessed single-use devices for interventional cardiology by testing bioburden, sterility and pyrogenic load. methods: a total amount of 154 electrophysiology non-lumen catheters (ep) were collected after the first clinical use on patient. 20 devices were contaminated with bacteria spiked human blood and underwent four different pre-sterilization protocols including chlorine, polyphenol, and enzymatic agents. treated samples were assayed by cultural quantitative methods (cqm) for bactericidal properties and electron microscopy (em) for biologic residuals. 73 ep were tested for sterility. by the repetition of simulated-use (bacteria spiked blood) and regeneration (enzymatic and chlorine treatment, gas plasma sterilization) we obtained 54, 39, 26, 28, 36, 22 samples respectively reprocessed 1, 2, 3, 4, 5, 6 times. devices were cultured for 28 days in trypticase soy broth. the pyrogenic status of 61 ep was monitored after clinical use, after decontamination-cleaning treatments and after complete reprocessing by lal test. results: high-resolution em and cqm confirmed the superior properties of chlorine releasing agent added to enzymatic detergent for devices treatment before sterilization. hypochlorous acid based protocols were more biocide (>3.1 log cfu reduction) than polyphenolic (3.2-1.9 log cfu reduction). sterility tests showed no positive sample to inoculated strain until the fourth cycle of reprocessing. catheters showed the growth of the inoculated strain, bacillus subtilis in 1/35 and 1/22 samples after five cycles and six cycles respectively. every reprocessed device was non-pyrogenic (<20 eu/catheter). in addition, tests conducted on in-vitro spiked catheters showed that pyrogenic loads of 200 eu/device were reduced to less than 11 eu/device. conclusions: reprocessing procedures following the adopted regeneration protocol were able to satisfy the fundamental microbiological requirements until five in-vitro reuses. sterility tests showed that devices' sterility was not guaranteed after five reuses. pre-sterilization treatments including enzymatic solutions and chlorine revealed high cleaning properties with effective bioburden reduction. storage intervals among reprocessing steps longer than 24 hours should be avoided in order to limit contamination and pyrogenic load. technical considerations suggest to consider the introduction of reprocessing procedure only in hospitals with a considerable workload. room disinfection in the hospital setting using akacid plus ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: akacid plus ò , a novel polymeric guanidine with broad antimicrobial activity also against multi-resistant bacterial strains, was used in the present study as room disinfectant. methods: disinfection of closed rooms experimentally contaminated with antibiotic-susceptible and multi-resistant staphylococcus aureus (mrsa), pseudomonas aeruginosa and escherichia coli was performed using akacid plus ò at concentrations of 0.1%, 0.25% and 0.5% for 100 minutes. bacterial suspensions were distributed on stainless steel plates and placed in a test and control room. recovery of the test bacteria was determinedbefore nebulizing, 60 and 100 minutes after the beginning and 4 hours after the end of room disinfection by a modified simple swab-rinse technique. for the detection of mrsa in isolation units, surface samples were collected by direct swab and enrichment culture. results: the swab-rinse method demonstrated a dose-and time-dependent effectiveness of akacid plus ò in eradicating s. aureus, e. coli and p. aeruginosa on stainless steel plates. nebulizing of 0.5% akacid plus was successful in eliminating all hospital pathogens in 340 min contact time, while mrsa was still detectable after use of 0.25% akacid plus ò . 0.1% akacid plus ò achieved a reduction >100 cfu of s. aureus and p. aeruginosa, but was only able to eradicate e. coli during the observation time. the results suggest that nebulized akacid plus ò at a concentration of 0.5% is a potent substance for eradication of pathogenic organisms in the hospital setting. study on the antiviral efficacy of citrofresh ò , a flavonoid based organic acid complex sanitizer z. nack (north-geelong, au) objective: determine the antiviral efficacy of this organic sanitizer against enveloped and non-enveloped viruses using a carrier based method. seeking registration for citrofresh ò in australia and in the eu as a hospital grade antiviral sanitizer. methods: the study was performed according to the american society of testing and materials (astm) designation (e 1053-85) recommended by the australian therapeutic goods administration (tga) to determine the efficacy of a disinfectant intended to use on inanimate, environmental surfaces. we tested citrofresh ò (diluted in standard hard water) in three different concentrations: 1%, 2% and 4% on adherent cell lines (pk-15, mrc-5, mdck, a549, l929) in four replicates against five different viruses including: porcine parvovirus (non-enveloped, high resistant against sanitizer); human rhinovirus-16 (non-enveloped, high resistant against sanitizer); human adenovirus-4 (non-enveloped, moderate resistant against sanitizer); human influenza type a (h3n2) virus (enveloped, moderate resistant against sanitizer); human herpes simplex virus type 1 (enveloped, low resistant against sanitizers). prior to the viral testings, acute toxicity assay was carried out to determine the adherent cells viability against citrofresh ò . results: cell lines exhibited >80% viability after exposure to all three concentration. herpes simplex type 1, human influenza type a and human adenovirus-4 exhibited the most significant viral log reduction of log10 4 to 5 at 4% concentration of citrofresh ò followed by the human rhinovirus-16 and porcine parvovirus log10 4 reduction at 4% concentration. the reduction of viable virus load was exhibited after 1 minute exposure time to citrofresh ò , which means no time-dependant activity. citrofresh ò clearly exhibited concentration and ph dependent viral load reduction activity against influenza type a and the human adenovirus -4 and human herpes simplex type 1 virus. the reduction in viral titre for porcine parvovirus and human rhinovirus-16 is probably ph dependent (the ph of 1% citrofresh ò is 6.5, 2% is 4.5 and 4% is 3.5). conclusion: our investigation shows that citrofresh ò is an effective disinfectant on environmental surfaces, eliminating enveloped and non-enveloped viruses and sufficient to achieve the minimum 4-log reduction with complete viral inactivation which is prerequisite for registration. rapid environmental recontamination of an intensive care unit after decontamination with hydrogen peroxide vapour objectives: to evaluate the effectiveness of hydrogen peroxide vapour (hpv) to reduce the levels of total bacterial and methicillin resistant staphylococcus aureus (mrsa) environmental contamination on an intensive care unit (icu), and to establish the rate of environmental recontamination. methods: the study took place on a 9 bed open plan icu. on each environmental screen 3 sites in each bed space (under the bed, the workstation and the monitor) were examined using broth enrichment for the detection of mrsa. in addition total bacterial counts were determined for under the bed and workstation using rodac plates. environmental screening was carried out monthly for the 3 months preceding the usage of hpv, increasing to weekly for the 4 weeks prior to usage. additional sampling was carried out immediately before patients were discharged from icu, following the subsequent terminal clean and then immediately after hpv use. after readmission of patients sampling was carried out at 24 h, 48 h and then weekly for a period of 8 weeks. patients were screened for mrsa on admission and then weekly. results: sampling of the environment prior to the usage of hpv revealed contamination of the environment with mrsa on 6/7 occasions, with mrsa colonised patients being present on only 3/7 occasions. after discharge of the patients and terminal cleaning of the environment, mrsa was isolated from 5 (13%) environmental sites. after the use of hpv, mrsa was not isolated from any environmental sites upon immediate sampling, but 24 h after patients were readmitted, including 2 patients known to be colonised with mrsa, mrsa was isolated from 5 sites. these sites were not clustered around the colonised patients but were widespread across the icu. in the 8 weeks post hpv usage mrsa has been isolated every week. the mean total bacterial counts prior to the use of hpv were 22.0/10 cm 2 underneath the beds and 3.8/10 cm 2 on the workstations, this was reduced after hpv to 0.1/10 cm 2 and 0.2/10 cm 2 respectively. after patients readmission the counts were 4.1/ 10 cm 2 underneath the beds and 1.2/10 cm 2 on the workstations after 48 h and returned to pre-hpv levels of 16.9/10 cm 2 and 4.8/10 cm 2 at each site respectively after 1 week. conclusion: hydrogen peroxide vapour is effective in eliminating bacteria from the environment. the rapid rate of recontamination of the environment suggests that the use of hpv is not an effective means of maintaining low levels of environmental contamination on an open plan icu. objectives: the nosocomial infections are more serious and dangerous than community acquired infections since they have high rate of morbidity and mortality as well as they increase the cost of therapy. recently many precautions have been taken to prevent these infections. one of these applications is that covering of the floor of the wards, clinics, intensive care units and operating rooms of the hospitals with vinyl flooring material, which is believed to be cleaned easily and effectively. in this study it was aimed to determine the duration of survive of the staphylococcus aureus, enterococcus feacalis, escherichia coli and pseudomonas aeruginosa, which were most common encountered as nosocomial infection agents, on the surface of flooring materials such as vinyl flooring, ceramic laminated wood and galvanized sheet at room temperature. methods: four kinds of flooring materials were prepared approximately in 4-6 cm 2 coupons and sterilized. separate bacterial suspensions equal to mc farland 1 turbidity were swapped to the surface of each flooring materials by sterile cotton swabs. all contaminated test materials were put in sterile petri dishes with cover and kept at room temperature without subjecting to the direct sunlight. on the third day, culture samples were taken from the surface of each material by sterile cotton swaps soaked with sterile saline and streaked on the blood agar surface. culturing procedure was repeated every other day until no growth detected. in case of three consequently, negative culture results obtained culturing was ended. results: overall results of the study were presented on table 1. conclusions: among the four flooring materials, galvanised sheet seemed to be the most unsuitable one for the bacteria to survive long period. in other words this material should be preferred as to laminated wood for covering benches and laboratory tables. as for the flooring of the floors the vinyl flooring material is better than ceramic. covering the complete cmv ie-1 and pp65 proteins.results: cmv seropositive transplant recipients had significantly hightened ie-1 and pp65 specific t cell frequencies compared to seronegative individuals. patients withevidence of cmv antigenemia or dnaemia could not be discriminated based on cmv-and donor-reactive t cells or serum creatinine. however, recipients of seropositive grafts with low ie1 response showed a tendency towards more frequent cmv infection. cmv disease was observed in only 3/64 individuals. 2 had no detectable ie1or pp65-t cell response, the third presented with a dominant pp65 response. interestingly, ie1-specific t cells correlated inversely with early post-tx donor-reactive t cell frequencies during weeks 1-2 post-tx. most importantly, ie1-specific t cell frequencies correlated inversely with serum creatinine at 6 and 12 months at several times post-tx. in patients without acute rejection, even pre-transplant ie-1 specific t cells correlated inversely with 6 and 12 months creatinine. conclusion: these data suggest subclinical control of cmv infection by ie-1 specific t cells and subsequently less graft injury by (cmv-induced) alloimmunity. universal precautions: knowledge, attitude and practice of healthcare workers regarding hiv, hepatitis b and c v. gupta, s. bhoi, a. goel, p. aggarwal (new delhi, in) objectives: increasing incidence of hiv, hepatitis b (hbv) and hepatitis c (hcv) in the patients expose the healthcare professionals of acquiring these infections during occupational exposure. we studied the knowledge, attitudes and practices of healthcare workers regarding hiv, hbv, hcv and the risk of occupational transmission of these diseases. methods: an interview survey was conducted among all the health care workers (hcw) using a standardised questionnaire comprising of 34 items in english and local language, as suitable, by an expert in the emergency ward of a tertiary care teaching hospital of a developing nation. data analysis (bivariate and multivariate analysis) was done using spss version 10. results: 170 (response rate: 80%) hcw participated in the study. the mean age was 33 ± 6 years, 54 were females. the study population comprised of 40% doctors, 23% nurses, 25% lab technicians and 12% support staff. respondents had adequate knowledge about causative (65%) usual transmission (63%), symptoms (69%) of aids but poor knowledge about hbv and hcv (43%, 41% and 30% respectively). inadequate knowledge was also revealed about the infectious bodyfluids (31%), disinfection of equipments (32%), pregnancy in hcw as a susceptibility factor (22%), post exposure prophylaxis (34%) and comparative infectivity of hiv and hepatitis (38%). 70% of hcw became anxious while treating these patients. poor compliance with universal precautions was noticed. high compliance was reported for wearing masks (72%) and wearing gloves (53%). doctors were more likely to suffer needlestick injury (p = 0.04) occupational exposures was found to be high (48%) with poor declaration rate (10%). guidelines adherence was influenced by profession (p < 0.001), availability or adequacy of protective equipments but not by work experience as hcw (p = 0.8). all of the respondents urged for an interactive information session. conclusions: results from this study reveal that there is a fair level of knowledge about hiv/aids but hepatitis b and c have not generated adequate concern among the hcw. incongruity between perceived knowledge and reported practice suggests that there is a need for an interactive awareness course about the universal precautions. the educational programmes need to consider attitudes in conjunction with empirical knowledge. objectives: the sero-prevalence of hepatitis a (hav) antibodies are known to be low in young adults in korea. recently, seventeen cases of hepatitis a have been reported in health-care workers (hcw) of icu in a university hospital from may 2005 to july 2005. we performed surveillance, and determined molecular identification of outbreaks. methods: 1. we checked the hav igm from all the patients of sicu with elevated ast/alt retrospectively and screened ast/alt level from all the nurses and the doctors in contact with suspicious index case. 2. when we determined the existence of outbreak, the molecular subtypes of hav from a blood of hcw were determined to provide the data for epidemiologic study. we determined the index case, a transmission route and the intervention for control an outbreak were planned. results: 1. seventeen hcw including 13 nurses and 4 doctors who are 22 to 32 years old, suffered from acute hav over 7 weeks period. 2. the possible transmission of hav was fecaloral route from the bed-ridden patients with diarrhea to the exposed hcw. 3. seventeen hcw were identified with a positive anti-hav igm. the eight hcw had a positive hav rna. analysis of the vp1-p2a region of each isolate showed genotype 1a in five strains and co-circulation of 1a and 1b in others. conclusions: the occurrence of hav outbreak highlights the importance of standard precaution in a hospital. the hav vaccination is considered in young aged-hcw. the genotype identification of blood would be useful for the epidemiologic study of suspicious hav outbreak in a hospital. management of a norovirus-associated gastroenteritis outbreak on two psychiatric wards a. buehling, u. arnold (magdeburg, de) objectives: we report a norovirus-associated outbreak of gastroenteritis on a closed psychiatric and a gerontopsychiatric wards from december 2004 to february 2005. during this time 46 patients and 25 healthcare workers (hcws) were affected. introduction and results of hygiene measures based on published guidelines on psychiatric wards are described. methods: effective and adapted measures had to be implemented to stop the outbreak and to prevent the spread of disease to other areas of our hospital. isolation or cohorting of the psychiatric patients was excluded for therapeutic reasons. regular hand disinfection in patient rooms was impossible because of the high risk of abuse. the following measures have been introduced: use of gowns, masks and gloves by hcws during care of infected patients-frequent hand disinfections with alcohol-based disinfectants by hcws using ''pocket bottles''; recommendation for all persons entering the station to use gowns, gloves and masks and to disinfect their hands frequently, distribution of handouts describing the measures; hand disinfection by all patients after using toilet, before and after taking meals (distribution of disinfectants by hcw); increased frequency of routine surface disinfection (3 times daily) instead of routine cleaning once daily; routine disinfection of door handles, handrails, wash-basins and -fittings and light switches 3-4 times a shift; avoidance of patient transfer via hospital; visitor restriction during outbreak time; daily evaluation of recommended measures and adaptation to the current situation; exclusion of affected staff from the ward until 48 h symptom free. results: the hygienic measures have been explained to the local hcws in daily meetings. they have been fully accepted only after a severe staff shortage in the fifth week of outbreak because of 3 new cases of gastroenteritis during hcws and 14 newly infected patients. because of the restrictive application of the adapted guidelines for these special wards the outbreak has been stopped within 4 further weeks. conclusion: in case of norovirus-based gastroenteritis outbreaks on closed psychiatric wards hygienic measures which are adapted to the concrete situation are necessary. especially in these cases the compliance with guidelines can be increased by daily meetings and daily evaluation of recommendations. staff shortage during the outbreak forced the strict compliance with the recommended measures. regional spread of antibiotic resistance methods: we performed surveillance of patients, healthcare stuff and icu environment and we registered the infections of ab during 3 periods of 21 days each one. the interval between 1st-2nd period was 6 months and 2nd-3rd period was 1 year. rectal, oropharyngeal swabs tracheal aspirates from patients, handswabs from stuff and samples from environment were taken weekly. the identification of ab was performed using vitek ii system the susceptibility was tested by kirby-bauer and mic methods and the <<dna fingerprints>>obtained by pulsed field gel electrophoresis (pfge). results: during the 1st 2nd and 3rd period, 19 patients (14 men, 5 women), 13 patients (7 men, 6 women) and 12 patients (8 men, 4 women) were hospitalized in icu respectively. ab was isolated in 74 from 250 samples (29.6%) at the 1st period, 59 from 336 (17.5%) at the 2nd and 62 from 260 (24%) at the 3rd period. totally ab was isolated in 195 from 846 specimens (23%) at the 1st 2nd and 3rd period among the patients carrying ab, 7/14 (50%), 7/10 (70%) and 5/8 (62%) were infected respectively. the infections observed during the study period were: sepsis (8), urinary tract infection (1), pneumonia (11), meningitis (1), thrombophlebitis (2) . all the isolated ab strains were multiresistant to antimicrobial agents. molecular analysis of 169 isolated strains by pfge distinguished the following types: a (64, subtypes a1-a7), b (10) at the 1st period a(6), c(7), d(22), e(2), f(1), g(1), h(2), i(1). j(1) at the 2nd period a(25), b (16), d(1), h(3), k(2). l(5) at the 3rd period. infections were caused mainly by a and d types while the same types were isolated from the environment and the hands of the icu stuff. conclusion: there was a high rate of colonization and infection of icu patients by multiresistant clones of ab. the persistence of clone a of a. baumannii and the appearance of b type at the 3rd period after its disappearance at the 2nd period despite the application hygiene measures, indicates the need for more strict reinforced infection control in icu. the transmission via the hands of stuff to patients has become the most important contributor factor in patient colonization and/or infection. objectives: the antibiotic resistance and its mechanism of group a streptococci (gas) varies according to nations or study period. we have investigated antibiotic resistance and mechanism of macrolide resistance for the strains isolated from korean children and compared to the previous (2002) results. methods: throat cultures were taken from 2351 elementary school children in jinju, korea from october to december, 2004 to isolate gas. antibiotic susceptibility test to erythromycin (em), clindamycin (cc), and tetracycline (tc) was performed by disk diffusion method. macrolide resistance phenotype and genotype as well as emm genotype were studied. results: isolation rate of gas was 14.0% (328/2351). resistance rates of em, cc, and tc were 9.8%, 8.8%, and 18.3% respectively, which were dramatically decreased from 51%, 34%, and 30% in 2002 at the same area. emm44/61 was prevalent (29%), while emm12 was the most common type (34%) in 2002. cmlsb, m, and imlsb were observed in 87.5%, 9.4%, and 3.1% respectively, compared to 64%, 34%, and 2% in 2002. the strains with cmlsb and imlsb had ermb gene and the ones with m phenotype were positive with mefa gene. conclusion: the resistance rates to em and cc were dramatically decreased compared to the past (2002). education to the public and physicians, decreased consumption of antibiotics, acquisition of immunity to the resistant strains, or change of prevalent emm types could be considered to explain the reason of decrease of antibiotic resistance. although antibiotic resistance rate was decreased, cmlsb type which has high mic was prevalent suggesting treatment failure for those children carrying these resistant strains in jinju, korea. analysis of skin and soft tissue infections in european medical centres: report from the sentry antimicrobial surveillance program (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) g. moet, p. strabala, h. sader, t. fritsche, r. jones (north liberty, us) objective: to analyse the skin and soft tissue infections (ssti) or wound infections in hospitalized patients in the sentry program for pathogen prevalence and resistance (r) variations in european (eu) medical centres for the years 1998 to 2005 (8 years). this program also included north america (na) and latin america (la) for the same years, except 2003. methods: 50 consecutively isolated pathogens/site were collected from each centre per year and varied in number of sites each year in eu: 1998 eu: (23), 1999 eu: (6), 2000 eu: (16), 2002 eu: (3), 2003 eu: (25), 2004 eu: (23), and 2005 . susceptibility testing was determined by clsi (formerly the nccls) broth microdilution methods and interpreted by current (2005) breakpoints. results: table of all years total of ssti pathogens. (see table) sa was the predominant pathogen in eu ranging from 35.7% of ssti isolates in 1998 to 47.5% in 2005. the top 5 most prevalent organisms accounted for 80.0% of isolates in all years with psa and ec ranking second and third, respectively with 22.5% combined, and enc and ent ranked fourth and fifth with 10.7% of the total isolates. compared to the americas, mrsa and vre isolation was at a lower occurrence rate in the eu; but between the rates of other monitored continents for ctz-r psa, cipro-r ec and ctz-r ent (ampc). vre increased in eu over the 8 year. conclusions: pathogen prevalence in ssti for eu has been consistent over the 8 monitored years although sa (with mrsa) appears to be increasing. eu is not a world leader in any key r marker compared to the americas. however, the r rates are evolving which suggests continued need for surveillance programs at regular intervals to detect mobile genetic r elements. objectives: carbapenems play an important role in the therapy of pseudomonas aeruginosa infections. the aim of our study was to characterize the molecular alteration responsible for changed susceptibility towards carbapenems in multiresistant p. aeruginosa strains from germany. methods: 19 multiresistant p. aeruginosa strains from 9 cystic fibrosis and 9 non cystic fibrosis patients were collected in 3 german hospitals in 2004. the strains showed reduced susceptibility (intermediate or resistant; din guidelines) to imipenem, piperacillin, ciprofloxacin and gentamicin. clonality was tested using pfge. a pcr screening for vim and imp was carried out. effluxpump overexpression was detected using an effluxpump inhibitor (epi) test. oprd and for strains with positive results in the epi test the effluxpump repressorgenes mexr and nfxb were sequenced. results: pfge patterns revealed no clonal relationship among the multiresistant strains. neither vim nor imp was detected. the geno-and phenotypes found are depicted in table 1. defective oprd genes caused by premature stopcodons or frameshifts were found in 17 strains. among those 11 had no mutations in mexr or nfxb and showed the highest mics found ranging from 8 to >32 and 4 to 32 mg/l of imipenem and meropenem, respectively. 3 additionally had defective mexr genes, but intact nfxb genes, 2 also had modifications in mexr and nfxb, and 1 showed only in nfxb additional alterations. for 2 strains no alterations in oprd but in mexr were proven. conclusions: the predominating mechanism of carbapenem resistance in multiresistant p. aeruginosa strains from germany was the loss of oprd. accessory overexpression of mexaboprm due to modifications in mexr did not result in significantly elevated mics of meropenem. moreover, the additional overexpression of mexcdoprj did not lower the mic of imipenem. in 2 strains with modifications only in mexr only elevated mics of imipenem indicate a reduced expression of oprd accompanied by overexpression of mexefoprn as conferred by nfxc-type mutants. objective: mart (study for monitoring antimicrobial resistance trends) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intraabdominal infections (iai). the aim of this sub-analysis was to assess antimicrobial susceptibility patterns among gramnegative bacilli from 5 different regions of the world during 2004. methods: 6pa total of 81 major medical centres in north america, latin america, europe, middle east/africa, & asia/ pacific tested the in vitro activity of antimicrobial agents commonly used to treat iai against consecutive unique aerobic and facultative gram-negative bacilli from iai using microdilution techniques according to clsi guidelines & breakpoints. results: enterobacteriaceae were recovered from 4905 (88%) & non-enterobacteriaceae were recovered from 826 (15%) of the 5596 patients in the study worldwide, constituting 5317 (86%) & 839 (14%) of the 6156 total isolates, respectively. e. coli (n = 2979; 56%) and klebsiella spp. (n = 978; 18%) were the most commonly isolated enterobacteriaceae. pseudomonas spp. (n = 605; 72%) and acinetobacter spp. (n = 110; 13%) were the most commonly isolated non-enterobacteriaceae. isolates from asia/pacific and latin america were generally more resistant. 2267 (43%) of the enterobacteriaceae & 272 (32%) of the non-enterobacteriaceae were recovered <48 hours after hospitalization. the % susceptible isolates are reported below: conclusion: in this study, enterobacteriaceae were the predominant intraabdominal isolates recovered both <48 h and >48 h after hospitalization. carbapenems were overall the most active agents against enterobacteriaceae worldwide. resistance rates varied among geographic regions, with the asia/pacific and latin america regions generally having the most resistance. characterisation of streptogramin resistance genes among enterococcus faecium isolates from austrian animal husbandry a. eisner, g. gorkiewicz, g. feierl, f. dieber, e. marth, j. kö fer (graz, at) objectives: the streptogramin virginiamycin has been widely used as a growth promoter in animal husbandry in the european union but was banned in 1998 because of concerns about evolving cross-resistance to the streptogramin quinupristin-dalfopristin used in human medicine. the aim of the present study was to investigate the prevalence of streptogramin resistance genes of enterococcus faecium recovered from animal faecal specimens collected in southeast austria. methods: we analysed 300 e. faecium isolates of cattle (n = 100), pig (n = 100), and poultry (n = 100) for the presence of streptogramin resistance genes. we used selective enterococcal broth for isolation. species identification was done on basis of gram stain, catalase and pyrrolidonyl arylamidase activity, motility, lancefield group d antigen typing, and by the 20 strep apitest (biomérieux). detection of the resistance genes vat(e), vat(d), and erm(b) was done by pcr. results: the erm(b) gene encoding macrolide, lincosamine, and streptogramin b (mlsb) resistance was found in each 2 e. faecium isolates recovered from pig and cattle, none of the isolates from these animals carried genes coding for streptogramin a resistance. on the contrary, 14 e. faecium isolates from broiler specimens contained the vat(e) gene and one isolate contained the vat(d) gene. all of these isolates also contained the erm(b) gene. conclusion: our data indicate that the use of the meanwhile banned antimicrobial feed additive virginiamycin has created a reservoir of streptogramin-resistant e. faecium in southeast austrian poultry. characterisation of macrolide-resistant streptococcus pneumoniae isolates from russia objectives: streptococcus pneumoniae (spn) resistance to macrolide antibiotics continues to be of major concern. the aim of present study was to analyse phenotypic and genotypic characteristics of macrolide resistant spn isolates. methods: eighty one macrolide resistant spn isolates were collected in moscow, 2003 moscow, -2005 . the susceptibility testing was performed according to the clsi guidelines. macrolide resistance phenotypes were characterized by triple-disk diffusion test, using erythromycin, clindamycin and rokitamycin disks. detection of genes, coding resistance to macrolides, was done by rt-pcr. sequencing for qrdr mutations was performed on levofloxacin resistant spn isolates. selected isolates were analysed by mlst. results: by the triple-disk test, 21 isolates were assigned to the m phenotype, 20 of them were carrying mefa gene, and one was negative. twenty eight isolates were cmlsb-phenotype, 25 of them were carrying both ermb and mefa genes, in two isolates only ermb was detected, and one isolate was negative for both genes. imclsb phenotype was demonstrated by 29 isolates, both ermb and mefa genes were detected in 6 of them, and only ermb in 23. three isolates didn't demonstrated blunting of zone of inhibition around rokitamycin disk. associated resistance to penicillin g, tetracycline, chloramphenicol and co-trimoxazole was observed in 67.9%, 86.4%, 42.0% and 75.3% of spn isolates respectively. nine multidrug resistant isolates, harbouring both mefa and ermb genes, were subjected to mlst. among them one isolate was found to share the allelic profile st 81 (spain23f-1 clone), and four isolates were single-allele variants of st 81. in four isolates new allelic profiles were detected. three isolates were resistant to levofloxacin (mic ‡4 mg/l), in two of them with levofloxacin mic > 32 mg/l (st81 single-allele variants) e85k, s79f and i460v substitutions were detected in gyra, parc and pare, respectively. d83n and i460v substitutions were detected in parc and pare of one isolate with new allelic profile. conclusion: high prevalence of macrolide resistant spn, harboring both ermb and mefa genes is observed in moscow, macrolide resistance is associated with resistance to other groups of antibacterials. some multidrug resistant isolates are highly related to internationally disseminated multiresistant clone spain23f-1. strains with fluoroquinolone resistance in moscow were all single locus variants of the spain23f-1 clone. occurrence of tet(w) gene in a clostridium difficile clinical isolate p. mastrantonio, f. barbanti, p. spigaglia (rome, it) objectives: to investigate the presence of tet(w), a tetracycline resistance gene recently identified in anaerobic commensal bacteria from animals and humans, in c. difficile clinical isolates. methods: several c. difficile clinical isolates from different italian hospitals were analysed for the presence of a tet(w) gene by pcr assays. the primers used were designed on the tet(w) sequences available in genbank. pcr fragments obtained by these amplifications were sequenced. tet(w) dna flanking regions were also examined with a set of pcrs constructed on the sequence of the conjugative transposon tnb1230 of butyrivibrio fibrisolvens 1.230, that is the only element carrying a tet(w) partially characterized so far. tet(w) positive isolates were also examined for the tet(m) gene and for the presence of int and tndx, markers for the tn916 and the tn5397like elements, respectively. the tn916-like elements were further characterized by pcrs designed on enteroccus faecalis tn916 sequence. tetracycline mic values were determined by the e-test method. tetracycline resistance gene transfer was evaluated by filter mating experiments, using c. difficile p881r strain as recipient. results: a tet(w) gene was found in only one isolate, c. difficile cd5, also positive for the tet(m) gene. this isolate was resistant to tetracycline with a mic of 8 mg/l. sequence analysis of the tet(w) pcr fragment (about 1860 bp) showed that this gene had an identity of 99% with the genes found in clostridium spp strain k10, mitsuokella multacida and butyrivibrio fibrisolvens. no amplifications were obtained with the primers designed on tnb1230, indicating the presence of a different genetic support for tet(w) in c. difficile. tet(m) gene of c. difficile cd5 was carried by a tn916-like element that showed nucleotide sequence mutations in the region containing orf 17-20 compared to the element of e. faecalis. conjugative transfer of tet(w) was not observed, whereas the tet(m) gene was transferred to the recipient strain. c. difficile transconjugants were resistant to tetracycline with a mic of 8 mg/l. conclusion: the results obtained in this study demonstrate for the first time the presence of a tet(w) gene in a clinical isolate of c. difficile, providing further evidence of the spread of this resistance determinant among gastrointestinal bacteria. macrolide resistance determinants are prevalent and readily selected for in viridans group streptococci among healthy norwegian adults background: norway has a low prevalence of antimicrobialresistant bacteria including macrolide resistant (mr) respiratory tract pathogens. we have observed an increase in macrolide consumption in norway and there is a lack of knowledge on the reservoir of macrolide resistance determinants among viridans group of streptococci (vgs) in the pharyngeal flora. objectives: examine the occurrence, selection and persistence of macrolide resistance determinants in vgs pharyngeal flora in healthy norwegian adults before and after treatment with azithromycin. methods: throat samples were collected before (day 1), after treatment (day 7) and after 3 months (day 90) from 20 healthy volunteers. the samples were plated directly as a lawn on pdmii agar plates with 5% defibrinated blood with an erythromycin etest strip. photos were used as quantitative comparisons. up to 10 morphological different colonies with erythromycin etest mic ‡1lg/ml from each specimen were collected; day1 (n = 59), day 7 (n = 157) and day 90 (n = 76). in total 86 representatives mr, vgs-isolates were selected for further studies: (i) mics of erythromycin, tetracycline and penicillin were determined by etest. (ii) pcr's for erm(b), erm(tr), and mef(a/e), and subsequent sequence-typing of mef. species identification was performed by soda sequencing. results: a total of 17/20 persons carried a low number (<5) of mr vgs in day1 specimens, while 20/20 had a significant higher number (>100) of mr strains in day2 specimens. in day90 specimens, 20/20 carried a low number of mr, resembling day1. reduced susceptibility to penicillin was observed in 32/86 (34%) isolates. tetracycline resistance was found in 43/86 (57%), and mainly in erm(b)-positive strains. mef(a/e)-positive dominated day1 (53%) and erm(b) day2 specimens 52%. sequence typing revealed mef(e) (n = 41) and mef(a) (n = 3). soda sequence; s. mitis (n = 42), s. oralis (n = 9), s. parasanguinis (n = 12), s. salivarius (n = 22), and s. sanguinis (n = 1). conclusion: there is a pool of vgs carrying macrolide resistance determinants in the normal pharyngeal flora of healthy adults that are readily selected for during azithromycin exposure. the mef(e) and erm(b) were the most prevalent resistance genes and co-resistance to tetracycline was frequently observed, resembling the findings in norwegian clinical isolates of s. pneumoniae. these vgs may provide a pool of resistant bacteria that may transfer resistance determinants to more pathogenic organisms. relationships in genotype, phenotype, t type and pfge type among macrolide-resistant streptococcus pyogenes strains isolated in the czech republic v. jakubù , p. urbášková, l. straková (prague, cz) objectives: to determine relationships between phenotypic and genotypic methods among erythromycin-resistant s. pyogenes strains. methods: a total of 1331 clinical isolates of s. pyogenes resistant to erythromycin were collected in 39 microbiology laboratories during 2001-2003. erythromycin susceptibility was tested by the disk diffusion method. strains with an inhibition zone <21 mm around the erythromycin disk (15 lg) were sent to the national reference laboratory for antibiotics (nrl). presences of mlsb resistance genes (ermtr, ermb and mefa) were tested by pcr. t serotypes were determined in randomly selected representatives of each phenotype (n = 370). pfge type were determined in strains from year 2003 only (n = 68). results: the rate of the most prevalent phenotype (constitutive mlsb resistance) was 63%, 55% in the year 2001 and 2003, respectively. the major prevalent t types among the analysed strains were serotype t 28 (58%), t 12 (10%), t 4 (8%) and t b3264 (8%). gene ermb was the most frequent (54%). the results of pcr method was highly congruent with observed phenotype of resistance. pfge patterns of strains with constitutive mlsb resistance were highly identical. conclusion: m phenotypes, constitutive and inducible resistance to mlsb antibiotics were found and ermtr, ermb and mefa genes were detected among the analysed strains. the t serotype 28 was identified the mainly prevalent in our collection. the majority of strains harbouring t serotype 28 were constitutively resistant to macrolides. the study showed close relationships among genotypes, t types, specific resistotypes (phenotype) and pfge types. objectives: since recognition of transferable clindamycin and tetracycline resistance in bacteroides, we have undertaken a us national survey on the susceptibility of b. fragilis group to analyse emergence of resistance and trends, since these species are not routinely tested for susceptibility in hospital clinical laboratories. methods: agar dilution mics were determined for 5225 isolates from 1997-2004 for b. fragilis and related species from 9 geographically diverse centers in the us. antibiotics included 3 carbapenems, 3 b-lactam/b-lactamase inhibitors, 2 quinolones, a tetracycline, clindamycin, metronidazole, chloramphenicol, a glycylcycline and linezolid. isolate identity was confirmed by api 20a. results: analysis of resistance trends from 1997-2004 showed a decrease in geometric mean mic's (geomic) for imipenem (0.80 mcg/ml to 0.32 mcg/ml, p < 0.001) and meropenem (0.52 mcg/ml-0.42 mcg/ml, p = 0.003) for the bacteroides species. ertapenem geomic remained unchanged (0.99 mcg/ ml). for the b-lactamase inhibitors, piperacillin-tazobactam geomic declined from 2.56 mcg/ml to 2.22 mcg/ml (p < 0.001). ampicillin-sulbactam geomic did not change. few isolates were resistant to any carbapenem or b-lactamase inhibitor combination. clindamycin resistance increased, especially for b. fragilis, b. ovatus and b. thetaiotaomicron (all p < 0.001). among quinolones, resistance of bacteroides to moxifloxacin increased (geomic went from 2 mcg/ml to 3.5 mcg/ml, p < 0.001). b. fragilis remains the most sensitive bacteroides species to moxifloxacin, although approximately 45% of stains have mic's ‡to 4 mcg/ml in 2004. tigecycline susceptibility, tested over 5 years, did not change. the first confirmed metronidazole-resistant isolate (mic = 64 mcg/ml) obtained in the us was noted in 2002 but none were noted in 2003 or 2004. conclusion: improved susceptibility of bacteroides species to some carbapenems and the b-lactamase inhibitor combinations is unexplained but significant. clindamycin resistance continues to increase, especially for b. fragilis. moxifloxacin susceptibility for the non fragilis species shows that the majority of strains are resistant. the first metronidazole resistant isolate has been reported from the us. since resistance trends are associated with species, the differentiation within the species is of extreme importance, since it may impact the choice of antimicrobial agent for the treatment of infections caused by this group of anaerobes. observed duration of nasopharyngeal carriage of penicillin-resistant pneumococci: relations to age and serogroup p. geli, l. hö gberg, h. ringberg, e. melander, m. lipsitch, k. ekdahl (solna, malmö, lund, stockholm, se; boston, us) background and objectives: knowledge of how the duration of pneumococcal carriage varies with age and serogroup is essential to understanding how immunity to carriage arises throughout the course of life, and designing appropriate models for the effects of vaccination or other public health initiatives aiming to reduce the pneumococcal transmission in the community. using data from an ongoing swedish intervention project, the duration of nasopharyngeal carriage of penicillinresistant pneumococci (mic pcg >0.5 mg/l) stratified by both serogroup and age of the carrier were estimated. methods: the mean duration and corresponding 95% confidence interval was estimated by fitting a gamma distribution to the observed duration of carriage for each serogroup and age stratum. results: the mean duration of carriage for all cases was 37 days (95% ci 35-38). children below the age of 5 years carried prp for significantly longer periods (43 days, 95% ci 41-45) compared with older individuals (25 days, 95% ci 24-27). there were also differences within the group of cases below the age of 5 years, as the duration of carriage became significantly shorter for each year older the cases were. serogroup 9 and 14 were carried for significantly shorter periods compared with serogroup 6. serogroup 9 also had significantly shorter carriage duration compared with serogroups 19 and 23 for cases 0-4 years. for cases 5 years or older, no significant difference in carriage duration for different ages or serogroups could be noted. conclusions: even though the estimate does not cover any correction for the censored carriage duration and therefore not yield an estimate of the total length of carriage, the results highlight the importance to take both serogroup and age of the p1823 exploring the molecular basis for differences in phenotype of salmonella enteritidis typing phage n. delappe, d. morris, m. cormican (galway, ie) objectives: the salmonella enteritidis phage tying scheme of the laboratory of enteric pathogens, health protection agency, uk, is a widely used method for subtyping this important pathogen. the method is rapid and highly discriminatory. interpretation of results can be subjective and the typing phage which are central to the method have not been well characterised. complete sequence data is available for the salmonella typhimurium podovirus phage p22. methods: the 16 typing phage were propagated on s. enteritidis pt1b (pb406). phage were visualised by electron microscopy. phage dna was extracted and digested with hindiii. consensus pcr primers were designed based on sequences of p22 and other s. typhimurium phage. additional primers were designed based on the sequence of the s. enterititidis typing phage 3 (a siphovirus). amplification, sequencing and dna probe hybridisation of various phage genes were performed using standard techniques. results: on em the 16 typing phage comprise 6 podoviridae (phage 1, 8, 10, 14, 15 and 16) , 6 siphoviridae (phage 3, 5, 7, 11, 12 and 13) and 4 myoviridae (phage 2, 4, 6 and 9). digestion with hindiii subdivided each morphotype into 3 groups. the podoviridae contained genes homologous to p22 while the siphoviridae contained genes homologous to the sequenced s. enteritidis typing phage 3. some sequence variation was detected in podovirus and siphovirus genes however in some cases phage, which differ in their phenotype had no difference detected in hindiii digestion pattern or partial sequence. conclusions: the s. enteritidis typing phage set comprise 3 distinct phage morphotypes. in some instances distinct phage that contribute to differentiation between s. enteritidis phage types had no dna sequence variation detected. variations in phage typing reactions may in part be due to epigenetic difference in typing phage, e.g. due to methylation of phage dna. salmonella enteritidis typing phage biology could provide a model for developing approaches to phage therapy. tularemia is a zoonotic bacterial disease. the causative agent, francisella tularensis, is spread to humans by direct contact with infected rodents, inhalation, ingestion of contaminated water or by arthropod bites. in some endemic regions, outbreaks occur frequently, whereas nearby rural parts may be completely free. we presented two cases of tularemia in non endemic region of the turkey. case 1: a 46 year old female patient referred to tertiary hospital due to swollen on the neck for 2 months. before admission beta lactam antibiotics had been prescribed to her for tonsilopharyngitidis. but her complaints had been continued. so she admitted to our hospital. she had been suffered fever sore throat and neck pain. she had a palpable and painfull cervical lymphadenopathy which was not suppurated. leukocytosis and elevated c reactive protein were predominant. at screening there were not any lymphadenopathy detected elsewhere. she had been examined about cytomegalovirus epstein barr virus and brucellosis. they were negative. fine needle aspiration from neck was negative considered as malignancy. cultures were negative for routine bacteriologic examination. microagglutination test for tularemia was 1/320 positive. then we decided to treat her with gentamycin for 10 days. after treatment cervical lymphadenopathy became small. leukocyte count and c reactive protein levels were reach normal range. case 2: a 29 year old female patient referred to university hospital due to cervical lymphadenopathy and fever and sore throat. before admission beta lactam antibiotics were prescribed to her for 2 weeks. but no apparent benefits had been detected. there was a palpable and fistulated cervical lymphdenopathy. drainage was examined microscopically and cultured for bacteria, mycobacteria and fungi. on routine cultures no microorganisms were grown. fine needle aspiration was done. it was reported that suppurative granulamatous lympadenitis. so we were examined for tularemia, cat scratch disease. microaggltunation test for tularemia was 1/320 positive. then streptomycin had been given for 10 days and excision of lymphadenopathy had been done. no complications or recurrence occur. results: both patients were applied to us from non endemic and different regions of the turkey. they had no known insect bite history. both of them were diagnosed by serological tests. conclusions: in the differential diagnosis of tonsillopharyngitidis, tularemia also must be considered in the non endemic regions. tularaemia presenting with tonsillopharyngitis and cervical lymphadenitis: two case reports b. kandemir, i. erayman, m. bitirgen, e. turk aribas, a.c. inkaya, s. guler (konya, tr) tularemia is a zoonotic disease caused by francisella tularensis. francisella tularensis is transmitted to humans by direct contact or ingestion of infected animal tissues, through the bite of infected arthropods, by consumption of contaminated food or water, or from inhalation of aerosolized bacteria. in this report we describe two cases of oropharyngeal tularemia who presented with tonsillopharyngitis and cervical lymphadenitis. case i: a 43 years old woman with multiple cervical lymphadenitis has been admitted to our clinic. her complaints started 2 months ago with signs and symptoms of tonsillopharyngitis. she had received non specific treatment (ampicillin+sulbactam) and ten days later cervical lymph nodes appeared. the diagnosis was made serologically. the antimicrobial therapy (streptomycin 1 · 1 g im) was given for fourteen days. the patient recovered completely. case ii: a 16 years old girl with multiple cervical lymphadenitis was admitted to hospital. her complaints started 3 months ago with throat ache after which multiple cervical lymphadenitis appeared. she was admitted to our out patient clinics and diagnosed to have tularemia. anti-microbial therapy (streptomycin 1 · 1 g im+doxycyciline 2 · 100 mg) was given for four weeks but no clinical response was achieved. patient was admitted to the hospital and surgical drainage was performed. treatment against tularemia was prolonged. patient was finally recovered at the end of nine weeks of therapy. it can be concluded that early diagnosis and treatment of tularemia are important. some patients may benefit from surgical drainage and prolonged therapy. a case of nonclostridial crepitant cellulitis which is due to escherichia coli c. ayaz, m. ulug, m.k. celen, m.f. geyik, s. hosoglu (diyarbakir, tr) objectives: this condition is caused by gas forming bacteria that involve the skin, either or as an extension from deeper structures. the origin of infection is an abdominal wound, perianal disease, or operative incisions that have become secondarily infected. tracking of gas-forming organisms from deeper sites of infection may also present as crepitant cellulitis without a break in the skin. diabetics are more likely to acquire such infections, especially in the lower extremites. among the bacteria isolated are anaerobic organisms such as bacteriodes or anaerobic streptococci, or coliform bacteria, especially escherichia coli and klebsiella. because of this reason we reported a case of a nonclostridial crepitant cellulitis which is due to escherichia coli. case: a 40 year old man who was previously healthy, has come with fever, pain, oedema, erythema, crepitant and limitation of movement at the right lower extremity. in his history he had no complaint until 2 weeks ago. perianal abscess has developed at this time and it has drainged spontaneously 3 days later. than his complaints has comprised 3 day duration. on physical examination, the temperature was 38.9°c, pulse rate 116/ minute, respiratory rate 42/minute and blood pressure was 90/50 mmhg. laboratory evaluation showed a haemoglobin 8.1 g/dl, leucocyte count of 32700/mm 3 (neutrophils 92%). serum electrolytes, renal and liver function tests were within normal limits. c reactive protein was elevated up to 354 mg/dl, esr was 77 mm/h. escherichia coli was isolated from wound and blood cultures. he was treated initially with ampicilinsulbactam (6 g/day) and required attempt. even with optimal surgical and medical therapy, he dies at the third day of the treatment from septic shock. conclusion: the onset is generally gradual, and there is usually mild local pain and systemic toxicity, allowing clinical differentiation from the more fulminant clostridial myonecrosis. the surgical approach should be aggressive, but tailored specifically to the underlying cause of infection. antibiotic therapy is directed at a mixed aerobic-anaerobic flora, until culture reports are available. a case of iliopsoas abscess which is due to pseudomonas aeruginosa objectives: pyogenic psoas abscess, a rare but life-threatening infection, results from primary suppuration or is secondary to the spread of infection from an adjacent structure. primary iliopsoas abscess occurs probably as a result of hematogenous spread of an infectious process from an occult source in the body. primary iliopsoas abscess can occur in diabetus mellitus, intravenous drug abuse, aids, renal failure and immunosupression. ultrasound is diagnostic in only 60% of the cases. computed tomography should be done for definitive diagnosis and is considered the gold standard. stapylococcus aureus is the causative organism in patients with primary iliopsoas abscess, but pyogenic psoas abscess caused by pseudomonas aeruginosa is uncommon. because of this reason we reported this case. case: a previously well 67 year old woman presented with a month history of right loin to groin pain, limping or limitation of hip movement, fever and nausea. she was a diabetus mellitus patient for 7 years. on her physical examination, the temperature was 38.1°c, pulse rate 104/minute, respiratory rate 28/minute and blood pressure was 140/90 mmhg. examination of the respiratory system, cardiovascular system and abdomen were found to be normal. laboratory investigations revealed total leucocyte count of 17800/mm 3 (polymorphs 88%), c reactive protein was elevated up to 168 mg/dl, esr was 77 mm/h. serum electrolytes, renal and liver function tests were within normal limits, but serum glucose level was elevated to 351 mg/dl. her blood cultures were sterile, but abscess culture yielded pseudomonas aeruginosa which was taken during the surgery. she was treated imipenem (2 g/ day) + amicasin (1.5 g/day) and required surgical drainage. she was treated and followed up 21 days, and discharged at the end of the treatment. conclusion: in these patients treatment involves the use of appropriate antibiotics along with drainage of the abscess. an adequate knowledge of the causative organisms should guide the initial choice of antibiotics. depending on the results of the abscess fluid culture and sensitivity, adjustments should be made. percutaneous drainage or surgical drainage may be done in them. in conclusion early recognition, empiric antimicrobial coverage and aggressive drainage or debriment are indicated in these patients. cervical lymphadenitis in a diabetic woman f. ç okça, a. azap, s. gö çmen, h. erdi sanli, s. gü l (ankara, kirikkale, tr) objective: rhodococcus equi infections are commonly seen in immunocompromised patients. exposure to domestic animals, such as horses and pigs may play a role in some cases. two thirds of the r. equi infections in immunocompromised were reported in hiv infected patients, and the rest divided between transplant recipients, immunosupressive medications and other kinds of immunosupression. the clinical picture presents with pulmonary infection in 80% of patients. here, we report a rare case of cervical lymphadenitis in a diabetic women due to r. equi. case: a sixty-year-old diabetic woman was admitted with the complaints of fever, right cervical erythematous swelling with tenderness and warmth. on physical examination; inflammation beginning from the right submandibular region and descending to the upper chest was detected. a tender mass of 8 · 5 · cm. was palpated on the right cervical region. ampicillin/sulbactam 4 g/day was given emprically for a week with no improvement. the ct scan of the neck showed conglomerated lymphadenopathy extending from the submandibular area to the supraclaviculary region with 10.5 · 3 cm in size. the mass began to fluctuate and 500 cc abscess material was drained surgically. gram's stain of the purulent material showed polymorphonuclear leukocytes with pleomorphic gram positive coccobacilli. the cultures of the material grew r. equi. therapy was changed to teicoplanin and ciprofloxacin combination and surgical care of the wound with antiseptics was performed. after a month, intrevenous medical therapy was changed to oral route with roxythromycin and ciprofloxacin and was continued to 2 months with complete resolution. conclusion: increased awareness and improved laboratory techniques help for the early diagnosis of rhodococcal infections. timely diagnosis is important because the microorganism is usually resistant to penicillin g, oxacillin, ampicillin, carbenicillin and cefazolin. the use of at least one antibiotic with intracellular activity is necessary in the treatment of r. equi infections. empirical two drug regimens with erythromycin, rifampin and/or ciprofloxacin are recommended. objectives: to analysed the features of spondylodiscitis (sd), their clinical presentation, the commonest diagnostic methods and the kind of treatment applied according to the different groups of the study. methods: a retrospective and descriptive study taking place amongst the patients diagnosed as having sd from 1998 till 2003. in each case we studied the presence of underlying disease, primary infectious sources in the prior 6 months, the way symptoms started, location, diagnostic methods, treatment and evolution, comparing between different aetiologies. results: 67 patients with sd were studied.53 of them had pyogenic sd,(35 had spontaneous sd and 18 had an sd after spinal surgery) and 14 patients had tuberculous sd. 45 were men (16 to 84 years; mean 51.8). patients with postoperative sd were the youngest (mean 42.17 y, p = 0.001). underlying diseases were found in 75% of patients, mainly in postoperative sd (50% of cases) (p = 0.009). an episode of previous bacteremia or infectious source was found in 40% and 63% respectively of patients with spontaneous pyogenic sd, significantly higher than in surgical sd (6% had bacteremia and 11% other infectious source, p < 0.01). the most common presenting symptoms were back pain (98.5%) and neurological deficits (54%). frank fever occurred in 29% of cases, being more frequent in spontaneous sd (49%) than in postoperative sd (6%) or tuberculous sd (7%), p £ 0.002. leukocytosis was found only in 28% of patients. postoperative sd presented the lowest levels of esr (p = 0.008). s. aureus was the most frequent bacteria isolated (31%) in pyogenic spontaneous sd, as coagulase negative staphylococci was in surgical sd. lumbosacral localization was detected in 45% of spontaneous pyogenic sd and in 95% of postoperative sd. tuberculous sd predominate in dorsolumbar region. paravertebral abscess formation was observed in 39% of pyogenic sd and in 93% of tuberculous sd (p = 0.001). surgical treatment was required in 46.2% of tuberculous sd and in 9% of pyogenic sd (p = 0.005). outcome of patients with spontaneous sd was worse (sequelae in 62%), than in patients with surgical sd (38.2%) or tuberculous sd (14%) (p = 0.015). conclusions: 1) spontaneous sd was the most frequent and it occurred mainly in patients suffering from underlying diseases; 2) nearly all patients had pain but only in 1/3 of them was accompanied by fever; 3) the lumbar zone was the most frequent location; 4) the majority of patients had a complete resolution of their symptoms only with medical treatment. background: the ethiopathogenesis of cns abscess includes a broad spectrum of pathogens and predisposing conditions, so that a polymicrobial flora is a quite frequent event. capnocytophaga spp. includes fastidious gram-negative organisms, usually underestimated in the common clinical practice, and poorly tested in vitro for antimicrobial susceptibility. surprisingly, also agents usually active on gram-positive pathogens demonstrated some efficacy against capnocytophaga spp. (i.e. erythromycin, rifampin, tetracyclines, cotrimoxazole, chloramphenicol, and glycopeptides), which is usually responsible of anecdotal episodes of cns infection (meningitis, brain abscess, and subdural empyema). methods and results: the fourth case report of capnocytophaga spp. brain abscess is herewith reported. a probable origin from a recent cat bite and a mandibular granuloma is suspected. due to the lack of clinical and neuroradiological response to neurosurgical debridement and an association therapy including imipenem, amikacin, clindamycin and fluconazole, empiric administration of linezolid (1200 mg/day) was attempted, and a rapidly favorable clinical, microbiological, and neuroradiological response was achieved. notwithstanding the identification of capnocytophaga spp. as the sole microorganism yielded by purulent drainage of a cns abscess, patients with multiple risk factors and recent surgery are expected to suffer from a polymicrobial cns infection. due to its favourable cns penetration and its dual mode of administration (both i.v. and oral), linezolid may represent an alternative option in the event of cns diseases borne by numerous risk factors and a suspected polymicrobial origin, especially when a lack of response to first therapeutic attempts is of concern. in the management of a cns abscess where the role of microorganisms with an unpredictable sensitivity profile remains of concern, chemotherapy should be directed also against potentially multiresistant organisms. considering also the relevant limitations given by the often poor cns penetration, the activity of glycopeptide agents is limited, compared with that of linezolid. aetiologies and antimicrobial resistance profiles of purulent meningitis study carried out in a hospital of infectious diseases, algiers objectives: bacterial meningitis is a serious clinical and medicolegal consequences if management is incorrect. meningitis protocols have recently been published by the british infection society/meningitis research foundation and are widely disseminated in our institution. local guidelines are also available on the hospital intranet and in the emergency department and acute medical admissions wards. this study investigated the level of understanding about meningitis and knowledge of the guidelines in medical staff of different grades working in the emergency department and the acute medical admissions unit in a large teaching and emergency hospital. methods: 90 medical staff were interviewed faced to face and asked a series of questions on the management of meningitis. results were stored on a database and responses were analysed. results: general knowledge about meningitis was variable. although 96% knew that bacterial meningitis was a notifiable disease only 40% knew the procedure for informing the health protection agency and only 43% would notify viral meningitis. only 30% of responders were aware that guidelines could be viewed on the hospital intranet. only 41% correctly identified the indications and cautions for lumbar puncture. although the majority recognised the need for urgent administration of antibiotics 18% would omit antibiotics until further assessment and lumbar puncture results. only 30% were aware of the need to consider adding ampicillin to cover listeria in patients over 55 years of age and there was uncertainty about the management of patients with penicillin resistance. conclusions: although protocols and guidelines for meningitis have been produced and are easily accessible the majority of medical staff were uncertain how to access and utilise this information. the level of knowledge and expertise in managing meningitis amongst medical staff working in a and e and the acute medical unit was poor and there is a need for further education to improve patient management. guidelines are of no value if they are not disseminated to front-line medical staff. objectives: the aim of this study was to evaluate the prevalence of penicillin resistant and multi-drug resistant pneumococci isolates in streptococcus pneumoniae meningitis. methods: a retrospective study was carried out on 46 clinical records between january 2001 and october 2005. among the 46 csf samples the pneumococcal ethiology was confirmed by 55% positive cultures and 70% latex agglutination. antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. isolates of pneumococci with oxacillin zone sides of >20 mm are susceptible (mic < 0.06 microg/ml) to penicillin, while at those of <19 mm the mic has to be determined (by e -test). results: isolates from 12 patients (26%) were found with penicillin-resistance (prp) -of which 67% were multi-drug resistant-and 34 (84%) with penicillin susceptibility -of which 35% were resistant to other drugs. an abrupt onset of disease was found in 58% prp patients and 79% from non-prp ones. chest x ray pulmonary determinations were found in 50% prp patients and 35% non-prp ones. sixty-six per cent of prp patients and 29% of non-prp ones had a prior hospitalization. only 8% of non prp patients had a positive blood culture. antibiotic switch was made in 41% cases with prp isolates and 29% cases with non prp ones. the overall rate of mortality was 8%, with 16% for prp patients and 6% for non-prp ones. conclusions: non-prp isolates were the prevalent ethiology of s. pneumoniae meningitis. 35% of non -prp strains developed other drug resistance, and 67% prp strains were multi -drug resistant. prp meningites evolved more as a hospital-related pathology, with an abrupt onset, frequently associated with pulmonary determinations and higher mortality rate. background: although vaccination strategies have shifted the age distribution of meningitis to older age groups, few studies have specifically examined bacterial meningitis in the older adult. methods: from october 1998 to april 2002, we prospectively included 696 episodes of community-acquired bacterial meningitis, confirmed by culture of cerebrospinal fluid, which occurred in patients aged >16 years. we dichotomized the cohort with respect to age: patients aged ‡60 years were defined as older adults and patients aged 17-59 years as younger adults. predictors for an unfavourable outcome (defined as score 1-4 on the glasgow outcome scale) were determined by logistic regression. we tested for statistical interaction between age group and potential prognostic factors by adding multiplicative interaction terms to the model. the mann-whitney u test and the chi-square test were used to identify differences between groups. results: 257 of 696 episodes (27%) occurred in older adults and 439 episodes in younger adults (69%). streptococcus pneumoniae was the most common pathogen in older adults (69%). meningitis in younger adults was caused by neisseria meningitidis and s. pneumoniae in 50% and 40% of the episodes, respectively. older adults were more likely to present with the classic triad of bacterial meningitis (fever, neck stiffness and altered mental status) than younger adults (58% versus 36%; p < 0.001). the prognostic value of independent risk factors for unfavourable outcome was similar in both age groups. older adults had more complications during clinical course, resulting in a higher mortality rate than in younger adults (34% versus 13%; p < 0.001). sepsis was the most common cause of death in both age groups (30% in older adults versus 32% in younger adults; fig) . whereas older adults tended to die more often due to cardiorespiratory failure (25% versus 11%; p = 0.06), younger adults more often died due to brain herniation (23% versus 2%; p = 0.004). conclusions: bacterial meningitis in older adults is associated with high morbidity and mortality rates. elderly patients often present with classic symptoms and s. pneumoniae is the most common pathogen within this age group. whereas older adults often die due to cardiorespiratory failure, younger adults more often die due to brain herniation. incidence of serogroups and penicillin susceptibility in neisseria meningitidis isolates (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) objective: the aim of this study was to analyse the serogroup incidence and penicillin susceptibility in n. meningitidis before and after spanish epidemic outbreak in 1997. in this year the public health service decided a massive vaccination in our sanitary area (galicia, north-west of spain, 459.180 inhabitants) and in autum of 2000 the inclusion of vaccine against n. meningitidis serogroup c in vaccination programme. methods: retrospective study of all cases of meningococcal disease confirmed by culture and/or pcr in the health care area of santiago de compostela (galicia) from 1996 to 2004. results: in the period 1996-2004 we identified 151 meningococcal disease episodes by microbiologic diagnosis (56.3%, 41.7% and 2.0% to b, c and w135 respectively). in 1996, serogroup c were the 80% of the isolates. in 1997 and 1998 the serogroup incidence was almost the same (b = 45.7%, c = 48.6%). from 1999 an increase in b serogroup cases were detected, in 1999 (66.6%), in 2000 (88.9%) and in 2003 (76.9%). the most frecuent phenotype has been 2b p1:2.5 in c serogroup. during this period an increase in penicillin susceptibility was observed (in b serogroup 0% in 1996 and 44% in 2004 of the isolates were susceptible and in c serogroup 0% in 1996 and 50% in 2004). conclusions: the b serogroup is the most frecuent isolate during this period except in the years 1996 and 1997. the strain that cause the epidemic outbreak in 1997 (c:2b p1:2.5) was not isolated since 2000. in our health care area, c:2a serotype, was isolated for first time in 2000, and since then, is the unique serotype isolated in c serogroup. incidence rate in c serogroup has changed from 3.34/100.000 in 1997 to 1.09/100.000 in 2004. this decrease was caused by the drop of incidence rate on the youngest groups (<2 years and 2-4 years). the incidence rate in b serogroup during these years was modified from 1.48/100.000 in 1997 to 2.17/100.000 in 2004. a four-year retrospective analysis of infective endocarditis in a belgian university hospital n. de visscher, b. delaere, b. krug, y. glupczynski (yvoir, be) objectives: to establish the epidemiology of infective endocarditis (ie) and determine the prognostic factors for adverse outcome in patients admitted to a university hospital with a cardiovascular surgery department. methods: between 01/00 and 12/04, the clinical and laboratory features of all consecutive adult patients with a definite diagnosis of ie (duke criteria) were evaluated retrospectively by two infectious diseases physicians on the basis of clinical data charts and microbiological laboratory. results: 45 patients (24 men, 21 women) presented with a definite diagnosis of ie. mean age was 64, 6 yrs; 34 cases (76%) were native valve endocarditis (nve) and 11 (24%) were prosthetic valve endocarditis (pve); 30% of patients with nve had underlying valvular abnormalities (3 bicuspidies, 4 mitral prolapsus or regurgitation, 3 others). ten out of 11 cases of pve were late-onset episodes (>1 year after surgery). global mortality was 42% (18/45 pts), including 15 patients (33%) still under antibiotic therapy. a higher mortality rate was observed in pve [7/11; (64%)] than in nve [11/34; (32%)]. overall, 18 pts (42%) underwent surgery (mean: 18 days following admission). valvular replacement was contra-indicated in 11 pts because of critical status and/or major co-morbidities. the distribution of isolated pathogens was: streptococci: 20 cases (44%) including 4 cases of s. bovis, s. aureus: 14 cases (31%, including 2 mrsa), enterococci: 3 cases (7%), miscellaneous: 8 cases. the affected valves were: only aortic: 19 (43%), only mitral: 15 (33%), only tricuspidal: 1, aortic and mitral: 4, mitral and tricuspidal: 2, aortic, mitral and tricuspidal: 2. a high mortality rate was observed in s. aureus ie (9/14 [64%]), especially in the subgroup of patients with a pve (4/5 pts [80%] ). the mortality rate in patients with ie episodes caused by streptococci amounted 21% (5/24 pts). clinical microbiology and infection, volume 12, supplement 4, 2006 related, 20% (n = 40) had previous surgery and 9% (n = 19) were related to urinary or digestive tract procedures. only 2 patients had illegal substance abuse. the most frequent predisposing acquired cardiac condition for native valve endocarditis was degenerative valvular disease in 55% (71/ 129). twelve percentage (n = 24) had prior ie. the most frequent predisposing congenital cardiac condition was a bicuspid aortic valve in 5% (n = 11). in 16% (n = 32), no predisposing heart disease was discernible. causative microorganisms included: staphylococci in 43% (n = 87) with s. aureus in 31% (n = 62), cons in 12% (n = 25), streptococci in 26% (n = 52) with s. viridans in 12% (n = 25), s. bovis in 8% (n = 16), enterococci in 17% (n = 34) and other pathogens in 3% (n = 7). culture negative ie was reported in 11% (n = 23). both in community-acquired and nosocomial ie, s. aureus was the most frequent causative agent. twenty-three percentage (14/62) were methicillin-resistant s. aureus. s. viridans ie was mainly community-acquired while enterococcal ie was nearly equally distributed between community and nosocomial origin. conclusion: compared to older series, we observed a high proportion of nosocomial ie and of prosthetic valve ie. s. aureus and e. faecalis were the most prevalent causative microorganisms. enterococci were nearly equally distributed between community and nosocomial origin, suggesting that nosocomial enterococcemia should be added as a major criterion, as proposed before for s. aureus. the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis: a meta-analysis of comparative trials m. falagas, d. matthaiou, p. papastamataki, i. bliziotis (athens, gr) objectives: the addition of an aminoglycoside to a beta-lactam for the treatment of patients with infective endocarditis has been supported mainly from data from laboratory and animal studies. we sought to review the evidence from the available comparative clinical trials regarding the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis due to gram-positive cocci. methods: the studies for our meta-analysis were retrieved from searches of the pubmed database and from references of relevant articles. included studies were trials that provided comparative data regarding the effectiveness of the treatment and/or mortality in patients receiving monotherapy with a betalactam or beta-lactam/aminoglycoside combination therapy. two independent reviewers performed the literature search, study selection, and extraction of data from relevant studies published in english during the period 01/1975-08/2005. results: no clinical trial comparing beta-lactam monotherapy to beta-lactam/aminoglycoside combination therapy for the treatment of enterococcal endocarditis was found. we performed a meta-analysis of 5 available comparative trials (4 randomized controlled trials and 1 comparative prospective trial) that included 261 patients with bacterial endocarditis in native valves due to staphylococcus. aureus (4 studies) or streptococcus viridans (1 study). there was no statistically significant difference between the compared arms regarding mortality (or 0.59, ci 95% 0.21-1.66), treatment success (or = 1.25, ci 95% 0.49-3.05), treatment success without surgery (or = 1.66, , and relapse of endocarditis (or = 0.79, . nephrotoxicity was less common in the beta-lactam monotherapy arm compared to the beta-lactam/aminoglycoside combination therapy (or = 0.38, ci 95% 0.16-0.88, p = 0.024). conclusion: the limited evidence from the available prospective comparative studies does not offer support for the addition of an aminoglycoside to beta-lactam treatment of patients with endocarditis due to gram-positive cocci. a large multicenter randomized controlled trial may be necessary to reach a definitive conclusion on this issue. outpatient antimicrobial therapy for infective endocarditis. single-centre experience objectives: to evaluate the characteristics and outcome of infective endocarditis (ie) patients included in a outpatient antimicrobial therapy (opat) program. methods: from january 1997 to may 2005 all patients who received opat therapy for an ie were prospectively evaluated. inclusion in opat program require clinical stability and agreement of patients. active drug addiction was contraindicated for inclusion. antibiotic treatment was administered in bolus for once-daily antibiotics regimens. we used cadd-legacy tm plus (deltec, inc. st paul. usa) portable infusion system for either continuous or intermittentprogrammed bolus infusion. results: we included 65 patients, 51 male (78%), mean age 60 years old (sd: 19.3 years). the diagnostic of ie was definite in 45 cases (13 with pathologic diagnosis), 15 probable and 5 possible. mostly of the cases were community-acquired ie (83%). mitral valve ie was the most frequent anatomical site involved (46%), followed by aortic (32%). native-valve ie represent the majority of cases (55%), but 32% were prostheticvalve and 12% were pacemaker lead ie. viridans group streptococci was the most frequent isolate (31 patients, 48%) with 4 cases of s. bovis ie. eleven patients had s. aureus ie (17%). at the time of the diagnosis, 10 patients had valve rupture and 4 patients had periannular abscess. a total of 15 patients required some surgical intervention for the ie [9 valvular replacement (2 of them associated with aortic graft), 5 pacemaker extraction and 1 aortic graft]. the majority of the patients received outpatient monotherapy (65%). the most frequent antibiotic used was ceftriaxone (55% of the cases), followed by cloxacillin 20%, gentamycin 20%, vancomycin 14%, teicoplanin 14%, ampicillin 8% and other antibiotics in 14%. in 60% of the patients the vascular access was a perifericallyinserted venous central catheter and in 26% we used a portable infusion system. twelve patients (18%) had some complication during opat that require hospital readmission, of which 5 could return to opat program. three patients had a fatal outcome (deaths) during admission, not related to ie complications. the mean duration of opat was 18.9 days per patient, and globally supposed 1.230 days of hospital admission savings. conclusion: opat for ie can be a good therapeutic option for ie stable patients. this procedure can represent a considerable amount of hospital admissions savings, improving also patients' well-being, and must be take into account for the treatment of this disease. objectives: botulism, a neuroparalytic illness, is caused by toxin produced by clostridium botulinum. food born botulism, a potentially lethal neuroparalytic disease, is caused by ingestion of preformed toxin. clinical illness is characterised by cranial nerve paralysis, followed by descending flaccid muscle paralysis. in this article we report a case series including a family group of type e botulism after ingestion of an iranian traditional soup. methods: in january 2005, 11 patients of a family group developed clinical manifestations of botulism 1-2 hours following ingestion of a traditional soup. their main clinical presentations were severe weakness (90.9%-10 case) and lethargy (72.8%-8 case). other signs and symptoms were blurred vision, fixed and dilated pupils, diplopia, dry mouth and decreased gag reflex. based on clinical finding, all patients received 3 monovalent antitoxins (a, b, c). stool, gastric fluid and serum samples were sent for toxicological evaluation using the standard mouse bioassay. results: type e toxin was detected in the stool and serum sample of only one patient. all patients recovered and discharged one week after admission. conclusion: this study confirmed that prompt administration of antitoxin can prevent progression of disease based on clinical judgment and also may be life saving. in this case series study, we observed a short incubation period of 1-2 hours only in type e botulism. an outbreak of group g streptococcal pharyngitis among hospital personnel considered to be foodborne n. karabiber, a. gurbuz ertas, m. karahan, e. aykut arca, z.c. karahan, a. tekeli (ankara, tr) introduction: food-born outbreaks of streptococcal pharyngitis are relatively rarely reported,and while group a streptococci are the main causative agents, only a few epidemics caused by group g streptococci have been published. we describe here an outbreak of group g streptococcal pharyngitis occurred among the staff of a teaching hospital in ankara.the outbreak: an explosive outbreak of pharyngitis occured mainly among the staff working in certain departments (i.e. intensive care units, operation rooms) of tü rkiye yü ksek ihtisas teaching hospital, in 29 january 2004. methods: a total of 377 (111 and 266; 3 and 47 from catering firm personel) throat cultures were evaluated in 2 days,and 124 bhs strains were isolated, 65 and 59 on the first and the second days, respectively. presumptive identification by nbacitracin and trimethoprim/sulfametoxazole disk diffusion test showed that 121 strains were non-group a, 3 strains were group a streptococci. in definite grouping by streptococcus grouping kit (avipath-strep,omega),121 strains were found to be lancefield group g,3 strains were found to be group a streptococci (gas). one of the gas strains was isolated from a catering staff on the first day, the other two were isolated from two health care personnels on the second day. during the outbreak, 16 of 50 catering firm personel (32%) were found to positive for group g streptococci. all the bhs tested were found sensitive to penicillin g and erythromycin by agar disc diffusion method. conclusions: the configuration of the epidemic curve suggested a common source of exposure. since respiratory spread of streptococci in such a rapid fashion would be highly unlikely and that 16 of 121 positive throat culture were from the staff of the catering firm that provide all the food services for the hospital, and that most of them were working at the departments in which the outbreak occurred, we considered that the outbreak might be food-borne. prompt treatment with penicillin all the ill personnel and 9-day holiday coming consequently 30 january, terminated the outbreak. all the strains were cryopreserved for further typing studies.we are now typing these strains by pulsed field gel electophoresis (pfge) after digestion with smai restriction endonuclease. our initial results show that these strains are of the same origin. outbreak of acute gastroenteritis in an air force base in western greece e. jelastopulu, t. constantinidis, t. kolokotronis, d. venieri, g. komninou, c. bantias (patras, andravida, gr) objectives: on 20 september 2005, an operative training day at the air force base in western greece, soldiers and staff experienced an outbreak of acute gastroenteritis. the purpose of this study was to determine the causes of the outbreak and develop control measures. methods: following the assessment of descriptive epidemiology, a case-control analytic approach was utilized with 100 randomly selected cases and 66 controls. patients completed a questionnaire pertaining to the presence and severity of gastrointestinal disturbances, date and time of symptoms onset and consumption of food items served in the base on the implied training day. adequate questionnaire was administered to the controls. odds ratios were calculated and statistical significance was determined using x2 test. samples of food items were collected for bacteriological examination. results: the overall attack rate was at least 50% among the approximately 1050 attendees. the outbreak started abruptly in the late afternoon on 20 september, peaked at midnight and ended about 25 hours later. from the interviews and the analysis it was established that the lunch (beef, macaroni, tomato sauce and grated cheese) consumed several hours prior to onset of symptoms by affected military personnel was the likely source of the outbreak with a strong statistical association. there was only one subject who did not eat lunch. among the symptoms the most prominent were watery diarrhoea (96%) and abdominal pain (73%). relatively few indicated vomiting (8%) and nausea (7%). the mean incubation period was 9 h. in the bacteriological examination, staphylococcus aureus was detected in a sample of raw beef and in two samples of grated cheese (rest-cheese from lunch and an unopened package). conclusion: the short incubation period with abrupt onset, the symptomatology and the short, self-limiting nature of the illness, are suggestive of gastroenteritis caused by an enterotoxin-producing bacterium. s. aureus is considered to be the most likely cause. although mortality and longer-term morbidity are uncommon with food poisoning caused by enterotoxin-producing bacteria, this outbreak highlights its capacity to cause short term, moderately-severe illness in a young and healthy population. it underscores the need for proper food handling practices and reinforces the importance of appropriate microbiological specimen collection from cases, as well as the public health importance of timely notification of such outbreaks. occurrence, characterisation and antimicrobial resistance pattern of staphylococcus aureus strains isolated from dairy products in southern italy g. la salandra, e. goffredo, c. pedarra, m.c. nardella, a. parisi, a. dambrosio, n.c. quaglia, g.v. celano, g. normanno (foggia, valenzano, it) objectives: the ingestion of food contaminated by enterotoxins (ses) synthesized by staphylococcus aureus is responsible of one of the most common foodborne diseases (staphylococcal food poisoning-sfp). since s. aureus is often involved in cases of subclinical mastitis of ruminants, milk may results contaminated. infact, the dairy products are frequently related to cases of sfp, expecially in areas characterized by a high level of consumption of these products. consequently an active microbiological surveillance is needed in order to control the risk of sfp and to allow the improvement of the public health standards. s. aureus also show a large antimicrobial resistance pattern. in this work are reported the results of a survey conducted on the occurrence of s. aureus in dairy products from apulia region (southern italy). furthermore, the isolated strains were characterized in order to determine their ability in synthesizing ses and to evaluate their antimicrobial resistance pattern. methods: 250 samples of dairy products (milk, cheese, mozzarella cheese, ricotta cheeses) were analysed for the detection of s. aureus. the isolated strains were tested for the detection of ses, using the reverse passive latex agglutination test (sea to sed) and submitted to pcr to detect enta, entb, entc, entd and ente genes. furthermore, the strains were tested for susceptibility to ampicillin, tetracycline, gentamicin, eritromycin, enrofloxacin, co-trimoxazole, teicoplanin and vancomycin, by the agar diffusion method. results: out of 250 samples analysed, 36 (14.4%) resulted contaminated with s. aureus and, among these, 19 (52.7%) have been recognized as enterotoxigenic strains (10 samples of milk, 5 samples of mozzarella cheese, 3 samples of cheese from ovine milk and 1 sample of cheese). all the strains tested (one per each positive sample) showed antimicrobial resistance properties but none of these was resistant to teicoplanin and vancomicin. conclusions: the results obtained from this survey show that milk and dairy products from southern italy are frequently contaminated by enterotoxigenic strains of s. aureus and highlighted the need to implement strict hygienic control measures along the food chain in order to decrease the risk of spf. furthermore, the presence of antimicrobial-resistant strains of s. aureus in food may be considered a source of communityacquired infections, with the direct risk of transfer of the antimicrobial-resistance to intestinal human microflora. objectives: infection accounts for about one-third of cases of fever of unknown origin (fuo), which remains a major diagnostic challenge. recently, f-18-fluorodeoxyglucose (fdg) positron emission tomography (pet) has entered the field of clinical infectious diseases. fdg accumulates in tissues with a high rate of glycolysis, which is present in malignant cells and in all activated leukocytes. the aim of this prospective multi-centre study was to validate the use of fdg-pet as part of a structured diagnostic protocol in the general patient population with fuo. methods: from december 2003 to july 2005, 70 patients with fuo, defined according to the revised petersdorf criteria, were recruited from one university hospital and five community hospitals. a structured diagnostic protocol was used. fdg-pet was performed after certain obligatory laboratory tests, chest xray and abdominal ultrasound. the final clinical diagnosis was used for comparison with the fdg-pet results. results: a final diagnosis was established in 35 patients (50%): 12 infections, 5 malignancies, 16 non-infectious inflammatory disorders and 2 miscellaneous causes. of the total number of fdg-pet-scans, 33% were helpful. positive predictive value of fdg-pet was 70% and negative predictive value was 95%. fdg-pet was helpful in all patients diagnosed with an infection except for one case of pyelonephritis. contribution of fdg-pet to the final diagnosis did not differ significantly between the university hospital and the community hospitals. fdg-pet was not helpful in any of the patients with normal erythrocyte sedimentation rate (esr) and c-reactive protein (crp). conclusion: in addition to the apparent value of fdg-pet in diagnosing different infectious diseases as described in several case series, fdg-pet is a valuable imaging technique as part of a diagnostic protocol in the general patient population with fuo and a raised esr or crp. based on previous studies comparing gallium-67-citrate or labelled leukocyte scintigraphy and fdg-pet in patients with fuo and resulting from favourable characteristics of fdg-pet, conventional scintigraphic techniques may be replaced by fdg-pet in institutions where pet is available. emergence of clindamycin-resistant streptococcus pyogenes causing cellulitis epidemiology of viral respiratory infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus as a cause of community-acquired respiratory illness seroprevalence of human metapneumovirus in japan 1626-28. carriers into account when studying the dynamics of pneumococcal transmission and modelling the effect of pneumococcal vaccination in young children erythromycin-resistant streptococcus pneumoniae isolated in spain: serotypes, clones and mechanisms of resistance 14 (10%) and 23f (10%) that accounted for 73% of eryr strains. among eryr strains 109 (87%) had mlsb phenotype (73% constitutive and 14% inducible) and 16 (13%) had m phenotype. the genes detected in mlsb isolates were: ermb in 102 isolates, and ermb and mefe genes in 7 isolates. all 96 (88%) mlsb isolates with resistance to tet had tetm gene and 79 of them had int gene (related to tn916-like). seven positive ermb strains susceptible to tet had int gene spain23f-1, spain6b-2, sweden 15a-25 and st89-19f) accounted for 44% of these strains. capsular switching was observed in two clones, spain23f-1 (serotypes 19f and 19a) and sweden15a-25 (serotypes 23a, 23f suggesting the spread of tn916-like elements. the ermb positive strains, related to spain23f-1, spain6b-2, sweden 15a-25 and st89-19f clones, were more frequently isolated in adults us) objective: to characterize changes in the frequency of occurrence of bacterial pathogens responsible for pneumonia in hospitalized patients in europe for the years 1998-2004 and examine select antimicrobial susceptibilities (s) for predominant pathogens. the emergence of resistance (r) among pathogens responsible for pneumonia has resulted in changes to empiric therapy, with increasing reliance upon third-and fourthgeneration cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, carbapenems and fluoroquinolones. methods: participating european medical centres (10-31/year) referred 50 consecutive, non-duplicate pathogens (8419 isolates) from lower respiratory tract sites determined to be significant by local criteria as the probable cause of pneumonia. all identified isolates were tested for s by the broth microdilution method 7 and 49.0%, respectively), increased significantly in 2001 (84.9% s), and returned to near-1998 levels in 2004. esblphenotypes (cro or caz or aztreonam mic ‡2 mg/l) remained essentially unchanged among ec between 1998 and 2004 (8.2% and 8.3%, respectively), whereas among ksp increases were more substantial (16.7% and 26.9%). metallobeta-lactamase-producing pa were identified during the study from italy vim-1) methods: four-hundred consecutive mrsa isolates were collected at 10 centres (max. 40 isolates per centre) as part of a multicentre study conducted throughout germany in 2004. isolates were collected from various sources, including colonization sites as well as infectious foci. only one isolate per patient was included and all isolates were spa-genotyped. cps were determined by slide agglutination with cp-specific antibodies (anti-t5-dt, anti-t8-conjugate, anti-336-repa). the serotypes were confirmed by immunodiffusion using lysostaphin-digested cell lysates. results: in the present study, we serotyped mrsa isolates collected most recently in a german multicentre study. all 400 mrsa isolates evaluated were one of the serotypes tested invasive pneumococcal disease in adults in north-rhine westphalia methods: surveillance for our current study focused on north-rhine westphalia, the largest federal state in germany (18 million inhabitants). 202 (52.2%) acute care hospitals27 microbiological laboratories serving these hospitals agreed to participate. we studied hospitalized patients older than 15 years of age. a case of ipd was identified by the isolation of s. pneumoniae from an otherwise normally sterile site. isolates were verified for species diagnosis by optochin testing and bile solubility, and for serotyping by the neufeld quellung reaction. mics of penicillin g, amoxicillin, cefotaxime, cefpodoxime, cefuroxime, clarithromycin us) objectives: carbapenems are the most reliably active betalactam antibiotics against enterobacteriaceae and are often the treatment of choice for infections caused by multi-drug resistant isolates. while carbapenem resistance has occasionally been reported in enterobacteriaceae, there are limited data on its frequency and distribution. methods: two large ongoing surveillance databases were searched for imipenem (imp) and ertapenem (etp) resistance in enterobacteriaceae: smart (study for monitoring antimicrobial resistance trends), a worldwide program focusing on community-and hospital-acquired intra-abdominal pathogens, and iss (icu surveillance survey), a us program focusing on icu isolates from any sterile body site. results: the overall frequencies of carbapenem-resistant enterobacteriaceae remained <2% in smart and <4% in iss throughout the periods of observation (see table). for 8% of esbl producing k. pneumoniae and e. coli were resistant to etp or imp and rates varied by geographic region. all isolates studied to date have exhibited multiple resistance mechanisms. conclusion: carbapenem resistance was uncommon among clinical isolates of enterobacteriaceae in these surveillance studies. its observed frequency varied by species and geographic region no significant seasonal variability in the prevalence of emergence of streptococcus b-haemolyticus strains in swabs was observed. conclusions: 1. seasonal fluctuations of pharyngeal discussion: with regard to high prevalences of giardiasis and enterobiasis it increase the prevalence that intestinal parasitic infections. it is suggested to decrease the rate of these parasitic infections in the region by strict programes that help to increase the knowledge of students, their parents and teachers about hygen. results: the study included 634 patients, with a mean age of 57.58 ± 19.2. 456 (71.9%) were women. the predisposing factors were: renal lithiasis patients (26.7%), prostatic adenoma (8.2%), vesical structure disease (4.3%), vesical functional disorder (6.6%), chronic kidney failure (4.3%). the underlying diseases included: diabetes mellitus (30.3%), immunosuppression (6.8%), previous urinary tract instrumentation (4.3%), permanent catheter (3.6%). the mean hospital stay was 10.93 ± 8.7 days. the mean duration of symptoms was 5.9 ± 8.2 days the absence of leukocyturia or mictional syndrome does not exclude the presence of complicated upper uti. 3) the high percentage of bacteriemia necessitates blood cultures, with e. coli being the most common pathogen. 4) the associated morbidity and mortality are important in association with sepsis or septic shock gr) objectives: to estimate the incidence of streptococci in community acquired urinary tract infections (uti) and also to carry out the in vitro antibiotic resistance of streptococci in urinary tract infections 3%) were enterococcus avium, 6 (2.0%) were enterococcus gallinarum and 38 (12.6%) were streptococci group b. the in vitro antibiotic resistance of enterococcus faecalis was: penicillin g 38.9%, ampicillin 2.4%, gentamicin500 29%, streptomycin2000 52.7%, nitrofurantoin 0%, ciprofloxacin 44.4%, tetracyclines 54.6%, vancomycin 4.3%, linezolid 0%. the in vitro antibiotic resistance of enterococcus faecium was: penicillin g 79%, ampicillin 69.8% tremolieres for the french aup study group background: short-course therapy for acute uncomplicated pyelonephritis (aup) is the newly suggested standard. talan et al. have demonstrated that oral ciprofloxacin (cip) for 7 days, was associated with greater cure rates than a 14-day trimethoprim-sulfamethxazole regimen. we assessed efficacy and tolerance of a 7-d. regimen of cip (study i), then of levofloxacin (lvx) in study ii results: of 171 (i) and 82 (ii) enrolled pts. 139 aged 31.7 ± 12.9 y., and 69, aged 3.58 ± 15.0 y. were retained for itt analysis; 16.5% and 16.0% had positive blood cultures. escherichia coli was the uropathogen in 99.2% and 100% of cases. finally 122 (i) and 60 (ii) were retained for per protocol (pp) analysis. at v4 bacterial eradication rates were 93.4% and 98.3 %. global cure rates were 89.0 and 92.8% at v4 and 77.3% and 75.0% at v5 with only less than 7% of lost to follow-up between v4 and v5 in both cases. side effects were observed in 12.8% and 27.6% of pts. who received 1 or more fq doses. conclusions: aup treatment with lvx 500 mg hiv-infected patients and drug addicts were excluded. antimicrobial susceptibilities of all s. pyogenes isolates were studied by microdilution method, and macrolide resistance phenotype by double disc test. macrolide resistance genes were detected by pcr. results: over the 10-year study period, there were 523 episodes of cellulitis. the infection was microbiologically 020). of note, all 4 cases of cellulitis due to clindamycin-resistant strains occurred during the last 3 years of the study. five (13%) patients presented with stss and died (1 due to an erythromycin-resistant strain). overall mortality (<30 days) was 33% this resistance might become a problem when treating s. pyogenes infections, especially stss cases. p1864 risk factors for community-acquired bacteraemic gram-negative cellulitis an administrative database was used. then we selected the patients with blood cultures obtained at the time of the cellulitis episode using the microbiology laboratory database. nosocomial cellulitis were excluded. a standardized data collection form was used to review the hospital records. in statistical analyses, student's t test was used for the comparison of mean values and chi square test and fisher's exact test for the comparison of categorical data (two tailed). results: of the 2251 patients with limb cellulitis identified in the study period, 301 patients had blood cultures and were selected for the analysis. bacteremia was detected in 59 of the 301 patients (19.6%), 15 of them due to gram-negative microorganisms hemorrhagic rash was present in 4.14% cases. koplick spot was found in 74.56% cases. measles was associated with streptococcal tonsillitis in 47.92% cases, with oral candidiasis in 20.22% cases and with pulmonary tuberculosis in 8.12% cases. severe forms of evolution were observed in complicated cases with: encephalitis (0.79%) or bronchopneumonia (5.89%), which required intensive care unit survey. we registered only one deceased, in a case of measles encephalitis in gipsy collectivities even it's very difficult, it's necessary to was performed. respiratory samples were tested routinely for twelve common respiratory pathogens. results: over the study period, of 587 samples processed, 40 -six cases were community-acquired and 23 (60%) patients had significant co-morbidities. cough was the predominant reported symptom. chest x-ray was performed in 31 cases, of which 28 showed abnormalities. bronchiolitis (22/ 39) was the commonest initial clinical diagnosis. the majority (69%) of patients received antibiotic therapy, but a convincing bacterial pathogen was isolated in less than half of these cases. thirty patients were admitted for management. more than one virus was identified from 14 patients, with rhinovirus being the predominant co-infection. overall, the average length of stay was 6.6 days. however, where hmpv was the sole pathogen identified, average length of stay was 3.1 days. conclusion: our data suggests that hmpv infections are more common in children with underlying co-morbidities. the rate of radiological imaging was higher than expected and perhaps is a reflection of the patient population or the degree of severity of illness. nosocomial acquisition occurred in 3 cases, which has implications for patient cohorting acknowledgements: the financial support for this study was provided by kuwait university research grant 03/03. evaluation of infection control practices in 31 haematopoietic stem-cell transplant facilities in german-speaking countries: variation of measures reflects lacking evidence s. wenzler-rö ttele, a. conrad, w.v. kern, h. bertz, f.d. daschner, m. dettenkofer (freiburg, de) objective: haematopoietic stem cell transplant (hsct) recipients are highly immunocompromised during pre-and postengraftment. thus, they are cared for in specialised facilities and versatile precautions are practised in order to prevent nosocomial infections. however, there is a lack of evidence whether these interventions are effective. furthermore, most of the measures are cost-intensive and restrict the patients' comfort. for evaluation of precautions, a survey was performed to assess the spectrum of measures commonly practised. methods: a questionnaire was compiled asking in detail for infection control measures differing according to allogeneic and autologous hsct recipients. the questionnaire was sent to 61 hsct facilities in germany, austria and switzerland. results: 31 questionnaires (51%) were filled in and sent back. among the 31 centres, 24 were university hospitals, and 4 teaching hospitals. the overall number of transplantations that were performed by the facilities varied considerably and ranged from 3 to 120/y for auto hscts and from 3 to 110/y for allo hscts. 80% of the institutions performing allo and auto hsct have implemented different precaution standards for each group. some measures regarding allo hsct were routinely adhered to in practically all institutions: accommodation in single rooms (96%), interdiction of plants and opening of windows (100% each) and protection from waterborne bacteria by use of terminal tap water filters (92%). 83% of hsct facilities perform their allo transplantations in hepa-filtered rooms and 54% are providing laminar air-flow for this population. there was a broad spectrum of different measures regarding barrier precautions: gowns when entering the room (required in 43% of centres for allo and 24% for auto hsct) and face masks (83% allo and 66% auto hsct). precautions to be followed by the patient varied among centres, e.g. specification of the face mask/respirator to be worn outside the isolation room (for allo hsct: 41% surgical mask, 5% ffp1, 36% ffp2 and 18% ffp3). conclusion: the broad variety of different preventive measures performed by the different facilities reflects lacking evidence for many infection control precautions that are commonly practised in the care of hsct recipients. this survey provides the basis for further studies within the onko-kiss project (hospital infection surveillance system for patients with haematologic/ oncologic malignancies). objectives: in this study it was our aim to evaluate the microbiological contamination of physiological serum flasks in use in medical day center for wound cleaning and to identify the isolated microorganisms. methods: we have collected 44 saline solutions from 22 health care centres localized in the health sub-region of coimbra. from each centre we have recovered 2 aleatory flasks in current use.the samples were transported at 4ordm;c and maintained at this temperature until its processing. saline solutions were seeded by the pour-plate technique in plate count agar and plates incubated at 28 and 37ordm;c for 48 h. the saline solutions were evenly spread over the surface of blood agar and sabouraud chloramphenicol agar (sab chl-d). the transfers of saline solutions flasks were also tested for microbiological contamination with a sterile cotton swab that was rubbed vigorously, over the transfer surface and directly applied on blood agar media. blood agar plates were incubated at 37°c for 48 h and sab chl-d plates were incubated at 28°c and 35°c and examined daily for a period of 30 days before declared as culture negative. microbial identification was firstly accomplished by employing conventional morphological and biochemical tests. when identification was not possible by these methods, 16s rrna gene sequence determination and phylogenetic analysis were used for bacterial strains and in the case of moulds we performed the amplification and sequencing of internal transcriber spacers region of 5.8s gene. results: from the 44 saline solutions analysed, 54.5% were contaminated. a total of 38 strains were isolated, 66% could be identified to species level using morphological and biochemical tests, the remaining 34% were identified by gene amplification and sequencing. about 69.6% of the identified strains were gram-positive cocci, the second dominant type of strain were gram-positive bacilli (13%), and the third dominant type of strains were gram-negative bacilli and moulds, both with 8.7%. the most frequent contaminants belong to human normal flora (64%), supporting the idea that the source of contamination of saline solutions analysed was human, in contrast with 36% of contamination due to the environment. conclusions: the contamination of the saline solutions is due to inadequate clinical practices. these results claim for more strict hygienic measures and for the replacement of big flasks by single use flasks with an incorporated overture used for wound irrigation. frequencies of cmv-ie specific memory t cells are inversely correlated with alloimmune memory and serum creatinine in kidney transplant patients p. nickel, g. bold, f. presber, c. schö nemann, j. pratschke, d. bitti, f. kern, h.-d. volk, p. reinke (berlin, de) background/aims: cytomegalovirus infection is a significant cause of morbidity in transplant patients and has been associated with allograft rejection. in this study frequencies of ifng-producing t cells following ex-vivo stimulation with protein-spanning peptide pools for cmv proteins pp65 and ie1 as well as donor-reactive t cell frequencies were serially determined during the first 6 months after renal transplantation (tx) to analyse the relation of cmv specific t cells, virus control and alloimmunity. patients: 64 kidney transplant recipients were included. immunosuppression generally consisted of anti-il-2r mab, calcineurin inhibitor, mmf and steroids. 2 presensitized patients received an induction by 2x low dose okt-3, anti-tnf mab, anti-cd20 mab and 5x plasmapheresis. 8 patients received fty-720, cyclosporine and steroids. methods: pbmcs from renal transplant recipients were analysed in a computer-assisted elispot assay before and at multiple times (mean 5) post-transplantationfor ifn-yproducing t cells following in-vitro stimulation for 24 hrs by irradiated donor cells and pools of overlapping peptides conclusions: although temporary r declines were seen among some european pneumonia pathogens, all showed increasing r to most class agents during the study period. the increase in esbl among enterobacteriaceae, and r among pa to most agents except polymyxin b, are especially worrisome. continued longitudinal comparisons of emerging pathogens and changing susceptibility profiles are critical elements in guiding empiric therapies and epidemiologic interventions. week, all participating hospital inpatients were swabbed on three anatomical sites: throat, nose and groin. we investigated the molecular epidemiology of the 46 mrsa isolates collected from 34 patients in 5 hospitals using the pfge method with the smai restriction enzyme. cluster analysis was carried out using bionumerics software. band-based similarity dice coefficients were used for dendrogram construction, which provides a quantitative assessment of strain similarity. samples were defined to belong to a cluster using a similarity coefficient of 75% or higher. pfge profiles were compared with the most similar strains from the harmony iums global mrsa database.results: 21 different restriction profiles were observed among the 46 mrsa isolates and 34 patients. isolates from the same patient but from different anatomical sites had similar pfge profiles. 7 clusters of mrsa strains could be identified with the two largest clusters containing 15 (44%) and 10 (29%) patients, respectively. strains from these 2 major clonal clusters occurred in 5 and 4 out of the 5 hospitals, respectively. isolates from the cluster with 15 patients were most similar to the well-known iberian clone: france a (10 strains), belgium e1 (2 strains), france b, france c and northern germany i (1 strain each). isolates from the next largest cluster of patients correlated with a group of strains previously found in finland and belgium: belgium e3 (2 strains), finland e1 (7 strains) and finland e7 (1 strain). the remaining strains were most closely related to belgium ec2 (3 strains), berlin iv (1 strain), southern germany ii (2 strains) and uk e15 (3 strains). conclusion: two major clonal clusters of mrsa strains were found to be dominant among hospitals inpatients in luxembourg. the molecular diversity of circulating strains was fairly diverse and profiles were very similar to previously described patterns in neighbouring countries and europe. further sequence-based genotyping is warranted to gain a better understanding of the clonal structure and elucidate transmission patterns. enterococci were identified by basic tests and by pcr amplification of ddl genes. susceptibility testing was performed using the icls broth microdilution method. resistance genes were detected by pcr, selected vana, vanb and vanc1 amplicons were sequenced. macrorestriction analysis (smai) resolved by pulsed-field gel electrophoresis (pfge) was performed. results: during the study 31 vre isolates with different phenotypes of resistance to glycopeptides were obtained from 3578 specimens. the prevalence of vre in the gastrointestinal tract was 3.5%. one e. faecalis (isolated from patient arrived from us) and 23 e. faecium isolates, harbouring vana genes, demonstrated mic's of vancomycin (van) and teicoplanin (tec) 256-1024 and 16-128 mg/l respectively. three e. faecium and four e. gallinarum isolates were vanb-positive, with van and tec mic's >32 and 0.25-1 mg/l respectively. all stains were susceptible to linezolide. among e. faecium isolates with vana genes one predominant pegf type was observed, differentiated in nine pegf sub-types. each of three other pegf types detected seemed to be unique. among six vana genes sequenced, four demonstrated similarity to vana gene from e. faecium (genebank af516335) and two to -vana gene from e. faecalis (genebank ay697425). in two sequenced vanb genes from in e. gallinarum nucleotide substitutions, resulting in seven new amino acid substitutions, were detected. conclusions: heterogeneity of glycopeptide-resistance genes, circulating in haematological centre, leads to the conclusion that their spread is not a local phenomenon. spread of vre is an emerging and, possibly, underestimated problem for russia. study of resistance and clonal relatedness of clinical isolates of stenotrophomonas maltophilia from a hospital in northern spain c. valderrey, e. sevillano, f. calvo, l. gallego (bilbao, es) objectives: the aim of this work was to study the antibiotic resistance and genetic relatedness among clinical isolates of s. maltophilia isolated from patients with tract respiratory infections. methods: the study included 36 s. maltophilia isolates obtained in a hospital from bilbao (northern spain) during 2005 (from january to october). susceptibility to antimicrobial agents was determined by the disk diffusion method following the nccls recommendations. the antibiotics tested were imipenem, meropenem, cefotaxime, ceftazidime, cefepime, aztreonam, amikacin, tobramicin, ciprofloxacin, ofloxacin and trimethroprim/ sulfamethoxazole. total dna was used as target for pcrfingerprinting experiments with primers rd1, eric2, ap3, m13 and rnar 1 and 2. to detect class 1 integrons, primers 3cs and 5cs were used in amplification experiments.results: resistance to antibiotics tested was the following: imipenem (100%), meropenem (67%), cefotaxime (92%), ceftazidime (28%), cefepime (64%), aztreonam (86%), amikacin (8%), tobramicin (14%), ciprofloxacin (25%), ofloxacin (25%) and trimethroprim/sulfamethoxazole (0%). pcr-fingerprinting technique was only useful when eric2 primer was used identifying 28 distinct genotypes. the other primers were not able to produce reliable band patterns. patients with several isolates maintained the same clone along time, although there are two patients from which two different genotypes have been isolated, and two clones that have been isolated from more than one patient. class 1 integrons were detected in 56% of isolates ranging in size from of 1500 to 300 bp (8 isolates bored combinations of two structures). conclusions: trimethroprim/sulfamethoxazole and amikacin showed the best activity against the isolates tested. for pcrfingerprinting experiments the best primer was eric2 which produced reliable and reproducible band patterns. there was a high clonal diversity since 28 different genotypes were identified among the 27 patients included in the study. many isolates bored class 1 integrons with sizes similar to those detected in other nonfermenters bacilli from the same environment.methods: -identification: the strains were identified by colonial morphology, haemolysis on blood agar plates, biochemistral and antigenic identification; antibiotic susceptibility testing: all the strains were tested by disk diffusion according to the national committee for clinical laboratory standard methods. mics were determinated by screening test or mic evaluation in solid media. results: our study concerns 252 bacterial strains isolated from january 2000 to june 2005. among the strains isolated, neisseria meningitidis represented the most number of cases with 43.7 %.these were distributed among all different age groups. serogroup a was the most predominant and represented 54.5% of total strains while groups b and c represented 15.5% and 14.5% respectively. streptococcus. pneumoniae represents the second causes of purulent meningitis with 32.5% while haemophilus. influenzae b is the third causative bacterial agent with 23.8%.this last agent is most predominant among infants less than 2 years of age in 80% of cases. neisseria. meningitis is susceptible to all types of antibiotics tested. however, haemophilus. influenzae b produced an inactivating enzyme (penicillinase) in 26.7% of cases. the resistance was associated to cotrimoxazole in 68.7% of cases. the results of mic done on streptococcus. pneumoniae show that 35.4% of strains has an intermediate resistance to penicillin and high level of resistance in 4.9%. the amoxycillin is active in 98.8% of the strains,in the opposite cefotaxim has an intermediate resistance in 6.1% and a high level of resistance in 2.4% of the strains. the resistance to penicillin was associated with resistance to erythromycin, cotrimoxazole or to both in some cases. conclusion: streptococcus. pneumoniae represents the second causative bacterial agent responsible of purulent meningitis and showed an increasing prevalence of resistance profiles to penicillin and cefotaxim in our hospital. this implicates an effective microbiological and epidemiological control.conclusion: as expected in a referral hospital with a cardiac surgery department, the prevalence of s. aureus ie was elevated as well as the attributable mortality rate. the high global mortality rate may be explained by the high frequency of severe co-morbidities and by the late referral of patients to hospital. our data suggest that there is room for improvement in the diagnosis and management of ie in a multidisciplinary collaborative approach. objective: to determine the clinical, epidemiological, diagnostic, and therapeutic characteristics of a series of 111 cases of prosthetic valve endocarditis. methods: we undertook a retrospective, descriptive study of 111 cases of prosthetic valve endocarditis obtained from a series of definite or probable 626 left sided infectious endocarditis from six second-or third-level andalusian hospitals from 1985 to 2005. results: of the 111 cases of prosthetic valve endocarditis, 96 (86.5%) were definite and 15 (13.5%) possible. the mean age was 58 ± 13 years, and they were more common in men (61%). late infection was more common than early involvement (74 vs. 37 cases). the aortic valve was involved in 54 cases (48%) and the mitral valve in 48 cases (43%. most (66%) of the valves were made of metal and prior handling had taken place in 26 cases (23%). clinical characteristics were fever 88%, constitutional syndrome 36%, murmur 41%, vascular events 37%, and immune phenomena 17%. complications included left ventricular failure 54%, kidney failure 22%, peripheral embolism 20%, cns embolisms 18% and heart block 9%. the etiology was as follows: in early prosthetic valve endocarditis the three most common pathogens were s. coagulase-negative (40%), s. aureus (22%) and enterococcus (11%). late prosthetic valve endocarditis involved s. viridans (30%), s. coagulase-negative (17%) and s. aureus (13%). transesophageal echocardiography alone in 14 cases (12%), and transthoracic followed by transesophageal echocardiography in 53 cases (48%). medical therapy was applied in 54 cases (48.6%) and surgery in 57 (51.3%). a cure was achieved in 71 cases (64%), the other 40 (36%) dying. of those who underwent surgery, 38.5% died and 31.4% of those who were treated medically died. the death rate from early prosthetic valve endocarditis was greater than that for late prosthetic valve endocarditis (54% vs. 29%). conclusions: 1) prosthetic valve endocarditis is a very serious infection which is still associated with an excessively high mortality, despite advances in diagnosis and treatment. 2) early prosthetic valve endocarditis has a worse prognosis than late prosthetic valve endocarditis, due to its distinguishing pathophysiological features. 3) the greater mortality seen in patients who underwent surgery is probably associated with the fact that they had more complications, such as perivalvular abscesses or persistent infection. outcome of infective endocarditis e.e. hill, s. vanderschueren, p. herijgers, m-c. herregods, p. claus, w.e. peetermans (leuven, be)objectives: despite progress in diagnosis and therapy, almost half of patients with infective endocarditis (ie) has at least one complication and overall mortality remains high. the aim of the present 5-year prospective observational study was to define predictors of outcome in patients with ie. methods: from june 2000 through december 2004, all first episodes of definite ie by the modified duke criteria, encountered in a single tertiary-care medical center, were registered and followed-up for 6 months. results: overall, 193 patients suffered 203 ie episodes. sixtyone percentage were males. the median age was 62 years (range 54-73). fifty-five percentage of episodes were referred from another hospital. at least one complication occurred in 79%. surgical intervention was performed in 63% and was mainly indicated because of congestive heart failure. the median time from diagnosis to surgery was 6 days (range 0-92). six-months mortality was 22% (n = 42). in bivariable analyses, factors associated with 6-months mortality were: age, female gender, causative microorganism, nidus of infection and therapeutic policy. six-months mortality was 18% for native valve ie and 31% for prosthetic valve ie; twenty-five% for nosocomial ie and 19% for community-acquired ie. six-months mortality rates for microorganisms were: staphylococci 33% (n = 26) [s. aureus 32% (n = 19) and cons 35% (n = 7)], enterococci 24% (n = 8), streptococci 8% (n = 4) and other microorganisms 14% (n = 4). the 6-months mortality for patients with a contraindication to surgery was 74% (n = 20), for patients conservatively treated without a contraindication 7% (n = 3) and for combined surgical-medical treatment 16% (n = 19). in multivariable logistic regression predictors of 6-months mortality were age (or, 1.05; 95% ci, 1.01-1.1; p = 0.03), causative microorganism (or, 0.74; 95% ci, 0.55-1; p = 0.049) and a contraindication to surgery (or, 32.26; 95% ci, 7.2-145; p < 0.001). conclusion: in the present prospective single centre study of 193 patients with definite ie, 6-months mortality rate was 0.22, and was especially high in patients with preestablished contraindications to surgery, in the elderly and in patients with staphylococcal ie. six-months mortality in patients with combined surgical-medical treatment versus exclusively medical therapy in patients without a contraindication to surgery was not statistically significant. staphylococcal and enterococcal ie had a worse prognosis compared to streptococcal ie. epidemiology and aetiology of infective endocarditis e.e. hill, p. claus, m-c. herregods, p. herijgers, s. vanderschueren, w.e. peetermans (leuven, be)objectives: the epidemiological features of infective endocarditis (ie) have changed. we report the results of a 5-year prospective observational study investigating trends in the epidemiology and etiology of ie. methods: from june 2000 through december 2004, we registered 203 definite ie episodes according to the modified duke criteria in 193 patients older than 16 years, hospitalized in a single tertiary-care center. results: sixty-one% of episodes involved males. the median age was 62 years (range 54-73).fifty-five percentage (n = 112) were referred from another hospital. forty-four percentage (n = 90) were nosocomial. thirty-four percentage (n = 70) involved prosthetic valves and 17% (n = 12) thereof were of early postoperative onset. the mitral valve was most frequently involved. exposure to ie risk factors during the previous 6 months was recorded in 67% (n = 136) of the episodes. twenty-four percentage (n = 49) were intravascular catheterobjective: to determine the eco-epidemiology of cryptosporidiosis in the health services executive -western area (formerly the western health board).concerns about the incidence of cryptosporidiosis in the western area prompted the department of public health to undertake further investigation of potential links between cryptosporidiosis and environment by focusing on farming activity and water supplies in the first instance. background: cryptosporidiosis was not notifiable in the republic of ireland prior to 2004, unless cited as a cause of gastroenteritis in a child less than two years old. as a result the incidence of cryptosporidiosis in the republic of ireland at the time was unknown. nationally it was estimated that up to 8% of cases of gastroenteritis in children less than two years old could be attributed to cryptosporidium. in the western area from 1999 to 2003 the proportion of cases of gastroenteritis in children less than two years old attributable to cryptosporidium ranged from 10.8% to 24.5%. this was cause for concern.many rural locations in the western area are served by voluntarily-operated water schemes. water quality from these schemes is often microbiologically unsatisfactory. the department of public health methods: initial research involved analysis of notification records for cases of cryptosporidiosis received from 1999 to 2003 inclusive. crude incidence rates for cryptosporidiosis in the western area were compared with crude incidence rates in england & wales, northern ireland, and scotland for the same time period. cases of cryptosporidiosis from the western area were geo-coded and mapped to visualize the geographic spread of cases, and are being contrasted with geographic data for farming activity, and also with available data on water supplies. the results of the initial phase of this research indicated the incidence of cryptosporidiosis in the western area may be cause for concern. the geographic spread of cases and potential links to farming practices and water supplies will be presented. objective: the evaluation of epidemiology and seasonal fluctactions of bacterial flora in pharyngeal swabs taken from family doctors' patients. material and methods: a total of 7807 of positive pharyngeal swabs ordered by primary care physicians from silesia were examined during the 2000-2005 period. the microbiological analysis was performed in silesian analytic laboratories. the intake of material, its transport and final identification complied with laboratory standards. results: the most common pathogens were, in order of prevalence: streptococcus viridans (57.4%), moraxella catarrhalis (45.2%), staphylococcus aureus along with mrsa (31.6%) and mrsa alone (1.6%), e. coli (18.8%), klebsiella pneumoniae (6.6%), streptococcus b. haemoliticus (4.7%). candida albicans was identified in 14.2% of positive specimens. considering seasonal fluctuation, the number of positive swabs in each month tended to gradually increase in spring with its culmination in may (10.4%). as for the most common pathogens streptococcus viridans and moraxella catarrhalis mirrored the general tendency and dominating in spring season (up to 11.7 and 12.1%, respectively) and having less stronger impact in automn (up to 9.3 and 9.4%). the frequency of isolation of the other pathogens revealed seasonal fluctuations confined to either spring, as in the case of klebsiella pneumoniae, escherichia coli and staphylococcus aureus strains (up to 17.7, 12.3 clinical microbiology and infection, volume 12, supplement 4, 2006 aim: the aim of this study was to identify the microorganisms isolated from corneal and conjuntival samples, isolated from patients attending the ophtalmology department of a spanish hospital. material and methods: a total of 69 corneal scrapes and 544 conjunctival swabs were obtained since october of 2002 to october of 2005 in an university hospital of madrid. samples were cultured into blood and chocolate agar plates and incubated at 35ordm;c in o 2 and co 2 atmospheres, respectively, for two days (conjunctival swabs) and fifteen days (corneal scrapes). identification and susceptibility tests were performed following standard methodology. results: thirty four (49.3%) out of 69 corneal samples and 96 (17.6%) out of 544 conjunctival swabs yielded positive cultures, respectively. results are summarized in the following table:conclusions : corneal scrapes yielded a higher number of positive cultures than conjunctival swabs. gram-positive microorganisms were more prevalent both from corneal scrapes and conjuntival swabs although the difference was more evident in corneal scrapes. s. aureus was the specie most prevalent in conjunctival samples meanwhile cns were the most prevalent in corneal scrapes. methods: vitreous fluid samples (n = 177) were obtained from 150 patients (94 male, 56 female) undergoing vitrectomy for endophthalmitis between january 2001 and october 2005. specimens of undiluted aqueous and vitreous fluid were cultured for aerobic, anaerobic bacteria and fungi by conventional methods. identification and antibiotic susceptibility were performed by the api system, vitek ii system (biomerieux) and the agar disk diffusion methods according to clsi recommendations. results: ninety one isolates were recovered from the samples. gram stain was positive in 133/177 (75.1%), while cultures were positive in 94/177 (53.2%) samples. gram-positive bacteria were the most common isolates (71/91, 78%), followed by gramnegative bacteria (11/91, 12%) and fungi (9/91, 10%). staphylococci coagulase-negative were isolated in 41/91 (45%). the next most common species isolated among gram-positive bacteria were s. aureus (6.6%), streptococcus spp (9.9%), propionibacterium acnes (9.9%), bacillus spp (3.3%), streptococcus. pneumoniae (1%) and enterococcus faecalis (1%). among gramnegative bacteria eight isolates were enterobacteriaceae, two were non fermenters and one was haemophilus inlfuenzae. two of the fungal isolates were candida albicans, one acremonium spp and six aspergillus fumigatus. polymicrobial growth was observed in six patients with two at least isolates. of staphylococci coagulase-negative 10/41 (24%) were resistant to methicillin. only one strain of staphylococcus aureus was methicillin resistant. all gram positive isolates were susceptible to vancomycin. all isolates were sensitive to amikacine and ceftazidime while resistance was observed in 9/177 (5%) isolates to fluoroquinolones. conclusion: a variety of microorganisms was isolated from the vitreous fluid of patients. the predominant isolates were grampositive bacteria, especially staphylococci coagulase-negative with low resistance rate to methicillin. so, therapy should be based on the isolation and identification of the infecting agent and the in vitro antibiotic susceptibility to the appropriate antibiotics. the prevalence of intestinal parasitic infection in the students of primary schools in nazloo region in urmia during [2004] [2005] k. hazrati tappeh (urmia, ir)background: intestinal parasitic infections are of the most important hygienic and economical problems of millions of people in all over the world, mostly from developing countries. understanding their epidemiological situation and relation to environmental and social factors is necessary for struggling with them in every society. this investigation was designed to study the prevalence of parasitic intestinal infections among primary school attending students in nazloo region of urmia district in 2004. materials and methods: 271 students were chosen randomly from 7 schools upon their population. having their questionnaires filled, two faecal samples were taken from each student and examined with direct wet mount and formalinether sedimentation technique. scotch tape was also applied in order to detect the enterobiasis and taeniasis. 271 students completed the test. all infected persons by e. vemicularis, h. nana were treated by mebandazole and giardia lamblia were treated by metronidazole. results: overall prevalence of parasitic protozoan infections was 29.5%. giardia lamblia was found in 28 cases (10.3%), entamoeba coli in 27 cases (10%) and blastocystis hominis in 2 cases legionella pneumophila as an occupational risk factor for inter-city bus drivers y. polat, ç . ergin, i. kaleli, a. pinar (denizli, ankara, tr)objectives: legionellaceae are ubiquitous aquatic microorganisms that usually isolated from evaporative condensers. various man-made sources such as cooling towers, whirlpools and spas are sources for legionella pneumophila. in hot climate, bus air-conditioning and aircirculating systems are possible sources for the organism. in this study, serologic status of bus drivers and their assistants for legionella infections as well as bus air-conditoner moisture exit samples for legionella species were investigated.methods: serum samples were collected from bus drivers (n = 63) and their assistants (n = 19). samples were tested for anti-legionella antibodies by indirect immunofluorescence technique. 1/320 dilution was accepted as a positive result for anti-legionella pneumophila antibodies. results were analysed according to risk factors based on hot/cold climate route (aegean and mediterranean parts of the turkey were accepted as hot climate region), immundeficiency, chronic diseases and work hours. according to serologic test results, air-conditioners of buses which has been driven by 1/10 dilution seropositive persons, were investigated. air-conditioner moisture exit samples were cultured on bcye-alpha agar supplied with bmpa. same samples were tested by pcr targeting a 108-bp fragment of the 5s rrna gene of legionella. results: anti-legionella pneumophila antibodies were positive in 12 (15.2%) bus-persons. bus drivers' seropositivity was higher than assistants (p < 0.05). in hot climate route, seropositivity was higher than cold climate route (p < 0.05). no positive pcr result was detected. coclusion: in conclusion, higher seropositivity rates in bus drivers were pointed out a newer occupational risk factor for legionellosis. although pcr positivity was not detected for bus air-conditioners, high seropositivity rates show that bus drivers have been somehow exposed to legionella. further legionellosis surveillance studies for bus drivers may help to understand legionella exposure during travel. objective: asymptomatic bacteriuria is an important risk factor contributing to pyelonephritis and renal disfunction in diabetic patients. in this study, the relationship between microalbuminuria and age, body mass index, duration of the disease, the level of glycohemoglobin, glycosuria and glomerular filtration rate is studied prospectively in diabetic patients who have asymptomatic bacteriuria. methods: a hundred and twenty-three type 2 diabetic outpatients who were admitted to baskent university konya medical and research center between january-october 2005 were included in the study. ages of the patients were within the range of 26-80 years. the diagnosis of asymptomatic bacteriuria was established according to the cdc criteria. concurrent samples for urinary culture, glomerular filtration rate, microalbuminuria and glycohemoglobin were obtained. results: twenty-two of 123(17.8%) patients had significant bacteriuria. of these patients 74% were female. although age, body mass index, creatinine clearence and presence of microalbuminuria were similar, there was a significant difference in glycohemoglobin levels, duration of diabetes and glycosuria between the two groups (p < 0.05). e. coli was the most common microorganism obtained from urinary samples. risk factors for asymptomatic bacteriuria were shown in the table. conclusion: the frequency of asymtomatic bacteriuria was found to be similar with the previous studies. high glycohemoglobin levels and long duration of diabetes were found to be the risk factors contributing to asymptomatic bacteriuria in type 2 diabetic patients. descriptive study of complicated pyelonephritis objective: the evaluation of prevalence and contributory factors associated with the development of urinary tract diseases among women with urinary incontinence. material and methods: 124 women aged from 35 to 65 years had their urine culture examination performed. the material was taken from the central stream of first catch urine and transported on uromedium. antibiogram was carried out with the use of becton-dicinson's discs. results: in 14 cases the urine culture tested positively which accounted for 11.3% of subject women. the most common pathogens of urinary tract were, in order of prevalence:e. coli-64.3%, staphylococcus aureus -14.3%,citrobacter diversus-7.13% and klebsiella pneumoniae-7.13%. candida albicans strains were isolated in one patient. e. coli had the highest sensitivity to norfloxacin -100% and cefuroxim -100%, amoxicillin with clavulonian acid -88.8%, ampicillin nitrofurantoin and trimethiprim -sulfamethoxazole -77.7% in each case, cefalothin -66.6%, tetracycline -55.5%, and amikacin -33.3% but only in 11.1% to amoxacillin. staphylococcus aureus proved sensitivity only to gentamicin (100%) and nitrofurantoin (100%). in the case of citrobacter diversus 100% sensivity to norfloxacin, nitrofurantoin, tetracycline, trimethoprim / sulfamethoxazole, ceftazidim and cefotaksym was confirmed.klebsiella pneumoniae also proved sensitivity to amoxicillin with clavulonian acid, cefuroksime, nitrofurantoin, norfloxacine, tetracyclin and trimethoprim / sulfamethoxazole. when considering the sensitivity of pathogens to antibiotics in the family practise setting of higher reliabilty are nitrofurantoina, norfloksacyna.after the administration of guided therapy complete release from symptoms was observed in 10 women (71 %).conclusions: women with urinary incontinence relatively seldom suffer from urinary tract infections. the most common pathogen among women with urinary incontinence was e. coli sensitive to floxacins and cephalosporins but with impaired reaction to amoxycillin. incidence and in vitro antibiotic resistance of streptococci in community-acquired urinary tract infections uncomplicated community-acquired urinary tract infections (ca-utis) and non-pregnant women in london hospital in kuwait over a period of two years. methods: eighty-six pregnant and 90 non-pregnant women with signs of ca-utis were enrolled in the study. the strains isolated from the patients who had significant bacteriuria were included in the microbiological analyses. the identification of the strains was performed using the api 20e system (biomerieux), while their susceptibility was determined by disk diffusion method. the interpretation of the results was realized according to nccls guidelines. quality control was performed using reference strain e. coli atcc 25922. oserotyping was carried out with polyvalent and monovalent antisera. hemolysin production was tested on human blood agar plates. possession of k1 antigen by e. coli was tested with agglutination by murine monoclonal antibodies to the group b meningococcal capsule. results: we found o serogroups o1, o2, o4, o6, o7, o18 and o75 among strains isolated from pregnant and non-pregnant women. hemolysin was presented in 35% and 40% respective. k1 antigen was presented in 40% of strains in studied groups.there are some statistically significant differences in antimicrobial resistance between both groups. amoxicillinclavulanate (amx-clv) resistance was higher among uti haemolytic isolates of e. coli in pregnant women (54%) then in non-pregnant women (38%). similar distinction in cefuroxime resistance was found -15% and 0. amikacin resistance was higher among uti isolates of e. coli in non-pregnant women (47%) then in pregnant women (23%).conclusions: there are no significant differences in expression of virulence factors of e. coli from pregnant and non-pregnant women with ca-utis in london hospital, kuwait. the resistance rates of e. coli from pregnant women to amx-clv and cefuroxime are significantly higher than in non-pregnant women. the penetration of telithromycin in gynaecological tissues and activity in cervicitis patients h. mikamo (gifu, jp)objectives: chlamydia trachomatis and neisseria gonorrhoeae are major causative organisms for sexual transmitted infections in japan. although several oral antimicrobial agents are active against c. trachomatis, few effective oral antimicrobial agents against n. gonorrhoeae exist in japan. two studies were conducted: a clinical pharmacology study examining penetration of telithromycin (tel), an oral ketolide antibiotic, in female genital organ tissues and a clinical study examining tel 600 mg once daily (qd) in cervicitis patients (pts chronic prostatitis (cp) is believed to be an infectious disease in most cases. both aerobic and anaerobic bacteria are involved in the polymicrobial microbiocenosis found in prostate specific specimens. coryneform bacteria form a remarkable part of this community, yet scarce knowledge exists about their clinical significance, species composition and antibiotic susceptibility.our aim was to compare the corynebacteria of the seminal fluid of cp patients and controls and to evaluate their antibiotic susceptibility.material and methods: semen samples from 66 controls and 49 cp patients (nih iiia or iv category) were analysed. corynebacterium seminale was identified by beta-glucuronidase activity, the rest of coryneforms using api coryne (biomerieux). e-test method was used for susceptibility testing.results: coryneforms were found from 78% cp patients and 84% controls (p > 0.05). twelve species and genera were found among 120 strains identified, the most frequent being c. seminale (in 60% cp patients and 58% controls). cp patients harboured significantly more arthrobacter sp. (17% vs 2%, p = 0.03) andcorynebacterium group g (14% vs 2%, p = 0.01), the latter association was especially eminent in case of patients with serious inflammation (>1 wbc/ml): 33% vs 2%, p = 0.0003. all tested 65 strains were susceptible to ampicillin-sulbactam, single strains were resistant to doxycycline (5%) and tmp/smx (5%), however, moderate resistance was common to doxycycline (29%). resistance to clindamycin (38%), benzylpenicillin (28%), nitrofurantoin (17%), erythromycin (16%) and norfloxacin (11%) was observed as well. half of cp-related corynebacterium group g strains showed resistance to nitrofurantoin and benzylpenicillin. in addition, they were often moderately resistant to clindamycin, erythromycin and, finally, norfloxacin frequently used to treat cp. conclusions: most of men have coryneforms in their semen, more than half harbour c. seminale. corynebacterium group g and arthrobacter sp are more frequently found in cp patients than the controls. in the treatment of cp of unknown etiology it is useful to take into consideration the susceptibility profile of corynebacterium group g. objective: to evaluate the role of cmv and listeria monocytogenes in abortion.methods: this descriptive prospective study was done on 450 women, 250 women with spontaneous abortion before 20th weeks of pregnancy as a case group and 200 healthy woman with full term delivery as a control group. serum samples were taken from all patients. elisa test was done for evaluation of cmv (igg and igm) and listeria antibodies in both groups. prevalence of seropositivity was determined. data were analysed by x2 and chi-square test.results: 450 seologic tests were done on samples. average age in case group was 25.6 ± 7.6 and in control group was 25.3 ± 6.5 years. in cases with abortion 89 (35.6%) and in control group 35 (17.5%) were seropositive for listeria monocytogenes. difference in seropositivity between 2 groups is statistically significant (p = 0.0001). cmv igg antibodies were positive in 235 (94%) of case group and in 150 (75%) of control group; the difference is significant statistically (p = 0.0001). cmv igm antibody was positive in 13 (5.2%) of case group and none in control group. difference is significant (p < 0.0001) there was no correlation number of previous abortion and seropositivity for listeria and cmv. conclusion: the present study showed an important role of listeria monocytogenes and cmv infection in abortion. serum and prostatic tissue concentrations of moxifloxacin (400 mg) after a single intravenous infusion in patients with benign prostatic hyperplasia undergoing transurethral resection of the prostate background: the spectrum of bacterial prostatitis comprises gram-negative, gram-positive and atypical pathogens. because of its broad spectrum of activity, moxifloxacin might be a suitable antibiotic for the treatment of bacterial prostatitis. aim: in this study the penetration of moxifloxacin into prostatic tissue after intravenous application of 400 mg as single dose was investigated.methods: in a prospective, multicentric study patients with benign prostatic hyperplasia received a single dose of moxifloxacin 400 mg in an 1 hour lasting infusion (250 ml) for perioperative prophylaxis before undergoing transurethral resection of the prostate (tur-p). serum concentrations were determined in all patients before infusion, at the end of infusion (time point 0), 0.5, 1 and 2 h after the end of infusion. patients were randomized for tissue sampling either 0, 0.5, 1 or 2 h after the end of infusion. at the beginning of tur-p approximately 1 g of tissue was sampled for analysis. concentrations of moxifloxacin in serum and tissue were determined by hplc. results: 39 patients were evaluated in the study. the concentrations (mean, sd, median, 25/75% quantile) are shown in the table. the prostatic tissue concentrations of moxifloxacin were approximately twice as high as in serum. at the end of infusion the tissue and serum concentrations were already equilibrated, because the tissue-serum ratios did not differ significantly from the end of infusion until 2 h after the end of infusion. after an intravenous infusion of 400 mg the serum and prostatic tissue concentrations of moxifloxacin were well above the mic values of the most important prostatic pathogens until 2 h after the end of infusion. therefore, moxifloxacin might be a good alternative for the treatment of bacterial prostatitis and/ or perioperative prophylaxis for tur-p. statistical significant differences were detected between patients with and without bgnc in the proportion of patients older than 65 years (81.3% vs 42.5%), the antecedent of recent animal bite (18.8% vs 0.7%), the presence of immunosuppression (12.5% vs 1.8%), the presence of haematological illness (12.5% vs 0.7%), and the degree of leukocytosis at admission (8989 ± 3736 vs 12690 ± 6028 cel/ll). conclusions: bgnc is frequently detected in our patients. age older than 65 years, the existence of immunosuppression, the existence of haematological illness, and the antecedent of animal bite are more frequent among patients with bgnc. patients with bgnc had a lower degree of leukocytosis at admission. these factors should be borne in mind to select empiric therapy for patients with cellulitis. is erysipelas-associated tinea pedis a site of streptococcal colonisation? objectives: tinea pedis is considered the most frequent portal of entry of erysipelas of legs (sel) but whether it is the site of streptococcal colonisation is unknown. methods: from june to october 2005 we prospectively searched for clinical tinea pedis in patients hospitalised in our infectious diseases ward for sel (acute and unilateral feature with fever were only retained). all patients had bacteriological samples on inter-digital spaces of both feet (sel side and contra lateral side).results: fifteen patients were included. all but one were treated by intra-venous penicillin-g followed by oral amoxicillin. on sel side: tinea pedis was found in 10/15 (67%) and, when present, streptococcal colonisation (c or g streptococcal groups) was found in 7/10 (70%), although streptococcal colonisation was never found (0/5) in its absence. on contra lateral sides : no streptococcal colonisation was found without tinea pedis, which was observed in 7/15, with streptococcal colonisation in 6/7. then there is a strength statistical association between streptococcal colonisation and tinea pedis, on sel side (p = 0.025) as well as on contra lateral side (p = 0.003). in one patient blood-cultures yielded with the same streptococcus than found in foot samples. discussion: streptococcal colonisation of tinea pedis is a common finding on both feet of patients hospitalised for sel. whether inter-digital colonisation is a primary stage of invasive disease remains unproved. in our experience, a strain of streptococcus that colonised inter-digital space was isolated in patient's blood, suggesting this hypothesis may be true in some cases. if confirmed, this concept could lead to a new strategy for secondary prophylaxis of recurrent sel by decontaminating streptococcal colonisation of tinea pedis. among ggs, 11 different emm types were found; stg6, stg480 and stg643 predominated. among gas, 8 types were found, emm81 predominated. one patient had the same ggs isolate in throat and skin. six patients had recurrent infections during the study; two of them with 3 disease episodes. of the 35 culture positive skin samples, 12 were taken from the erysipelas infection focus (42% positive for ggs) and 23 from another site (61% positive for ggs), e.g. wound, intertrigo, between toes or an unknown site.conclusion: a predominance of ggs was seen in the throat of erysipelas patients and their families whereas ggs was not present in control subjects. ggs, instead of gas, also seems to predominate in erysipelas skin lesions. several emm types were present in both groups and there was no clear predominance of a distinct type. the recurrent nature of erysipelas became evident also during this study. the evaluation of fournier's gangrene severity index score in 11 patients m. ulug, m.k. celen, m.f. geyik, c. ayaz, s. girgin (diyarbakir, tr)objectives: fournier's gangrene is synergistic necrotizing fasciitis of the perineum and abdominal wall along with the scrotum and penis in men and the vulva in women. it is rare but life-threatening process. in this study we identify effective factors in the survival of patients with fournier's gangrene and to determine the accuracy of the fournier's gangrene severity index score (fgsis). methods: we evaluate 11 patients with fournier's gangrene who were threated and follewed up from us between january 2005 and september 2005 in the department of general surgery prospectively.results: the results were evaluated in two groups: those who died (n: 3) and those who survived (n: 8). no statiscally significant difference was found between the age of the survivors and those who died. the admission and final laboratory parameters that correlated statiscally signinificant with outcome includes leucocyte count, hematocrit, urea, creatinine, lactate dehydrogenase, bicarbonate and albumin. sites of culture were skin/soft tissue (75, 74 and 92%), respiratory tract (3, 4, and 3%) , blood (4, 7 and 1%), urine (3, 11 and 0%) , and other (3, 2 and 2%) . 30-day mortality was 0% in this population. 50% of patients received antibiotic therapy alone, 5% surgery alone, 30% antibiotics + surgery, 3% other therapy, and 12% no treatment. the most common antimicrobial classes received were vancomycin (27%), beta-lactams (23), fluoroquinolones (11), and cotrimoxazole (10) with 14% of patients receiving multiple agents. median duration of antibiotic therapy was 12, 15, and 10 days, in the ca-mrsa, ha-mrsa and ca-mssa groups respectively. 47, 82, and 57% received adequate antimicrobial therapy (p < 0.001). hospital admission was required in 46, 57, and 17% of patients (p < 0.001). clinical success rates of initial therapy were 61, 63, and 84% (p < 001), and recurrences were more common in the ca-mrsa group, (18, 0, and 6%, p < 001). characteristics associated with outcome are listed in table 1 . in multivariate analysis, presence of mrsa and diabetes were predictive of clinical failure.conclusion: in the community setting, mrsa infections are associated with an adverse impact on outcome compared to mssa infections and patients with ca-mrsa are significantly less likely to receive adequate antibiotic therapy. microbiological analysis of root canals associated with periapical abscesses and the antimicrobial susceptibility of isolated bacteria s. ozbek, a. ozbek, m. koseoglu, s. evcil, a. erdogan, a. ayyildiz (erzurum, tr)objective: the periapical abscess is a collection of pus in the pulp or around the root of teeth. many odontogenic infections can be managed without antimicrobial therapy or bacteriologic investigation. however, when an acute bacterial infection has progressed or antimicrobial therapy might be of benefit to patients, antibiotics are prescribed. we aimed to identify microorganisms in root canals with periapical abscess and the antimicrobial susceptibility profile of them and to revise antimicrobial treatment protocols when antimicrobials is used empirically. methods: 30 patients with odontogenic infections included in this study. the microbiologic investigation was performed under strict aseptic conditions. a standardize routine of root canal therapy was instituted, and in each case a single root canal was sampled. in multirooted teeth only the largest canal was sampled to preserve the identity of a single endodontic/ microbiologic ecosystem. for microbial sampling, two sequential paper points were introduced into the full length of the canal, and kept in place for 1 min. one of the paper points was used for aerobic culture and the other one for anaerobic culture. to identify isolated bacteria, whole bacterial fatty acid profiles were evaluated by using microbial identification system. antimicrobial susceptibility results were obtained by disc diffusion test for aerobics, and e-test for anaerobics. results: totally 156 bacterial strains were isolated. 86 of them were aerobic and 70 of them were anaerobic. 18 or 60% of cultured specimens yielded mixed (aerobic and anaerobic) species. the most prevalent bacteria were staphylococcus spp. as aerobic, peptostreptococcus prevotii and streptococcus morbillorum as anaerobic. conclusion: beta-lactam antibiotics combined with beta-lactam inhibitor (amoxicillin-clavulanic acid) had a quite effect on gram (+) and (-) aerobics. when we take into consideration that beta-lactam antibiotics stimulate production of beta-lactamase, amoxicillin-clavulanic acid combination appears a good first step antimicrobial. clindamycin may be second alternative for that purpose. for anaerobics, cefoxitin and metronidazol had well effect. although imipenem and piperasilin-tazobactam are perfect, they should not be first step of therapy. due to the frequency of mixed infections, a combination of amoxicillinclavulanic acid and metronidazol or a combination of clindamycin and metronidazol considered to have well effect for mixed infections. clinical microbiology and infection, volume 12, supplement 4, 2006 study is to review the spectrum of p. multocida infections in our centre. methods: we studied the medical records of all patients who had positive cultures for p. multocida between 1993 and 2004. demographic, epidemiological, clinical and microbiological data including age, sex, animal exposure, site of infection, underlying diseases, type of therapy and outcome were evaluated. all isolates were identified by standard conventional microbiological methods. antibiotic susceptibility testing was performed by the disk diffusion method onto muller-hinton agar supplemented with 5% sheep blood and the mics of the antibiotics tested were determined by the e-test method. results: thirteen cases of p. multocida infections were diagnosed during this period. the male to female ratio was 10:3 and most patients (62%) were >70 years of age. respiratory tract infections were most commonly encountered (61.5%), followed by soft-tissue infections (30.8%) and septicemia (7.7%). underlying disease was present in 8 (61.5%) patients. among them, 4 presented a kind of malignancy. bullous pemphigoid, mitral valve stenosis, coronary disease, chronic obstructive pulmonary disease, and intracranial haemorrhage served also as predisponding factors. a traumatic animal exposure was reported in only 3 patients and non-traumatic in 2 cases. all isolates were susceptible to beta-lactams (penicillin, amoxicillin, amoxicillin/clavulanic acid, cefepime, cefuroxime, ceftriaxone, imipenem, and meropenem), quinolones (ciprofloxacin, norfloxacin, levofloxacin, and sparfloxacin), chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and 54% were intermediately resistant to aminoglycosides (gentamicin). appropriate antibiotic therapy was administered to all patients and a clinical response was observed in 10 (77%) of them. mortality rate was 23%. conclusions: pasteurella multocida must be considered as a possible etiology for a variety of infections, even without an obvious animal exposure. although this organism is susceptible to a large spectrum of antibiotics, a failure to treatment may be recorded especially in severe infections and in compromised patients. infections caused by nocardia cyriacigeorgici in zaragoza, spain: identification and antibiotic susceptibility c. villuendas, b. moles, v. rodriguez-nava, a. couble, f. laurent, m. revillo, p. boiron (zaragoza, es; lyon, fr)objectives: nocardia species known to date differ in their clinical presentation, antibiotic resistance patterns and geographic distribution. nocardia cyriacigeorgici is a recently described species.the aim of this study is to analyse the identification results, antimicrobial susceptibility together with the clinical data, of 13 n. cyriacigeorgici clinical isolates, recovered from 1998 to 2004 in our laboratory. methods: identification of nocardia spp. isolates was achieved in our laboratory on the basis of the following: visualization of the colony, gram stain and parcial acid-fast positivity by modified acid-fast staining, casein, xanthine and tyrosine hydrolysis, opacification of middlebrook 7h10 agar, production of arylsulphatase after 14 days incubation and antimicrobial susceptibility pattern.identification at species level was achieved by 16s rdna gene sequencing (laboratoire de mycologie. faculté de pharmacie. lyon. france)antimicrobial susceptibility tests included commercial broth microdilution (emiza 9 ef sensititre ò ) and gradient strip agar dilution (e-test ab biodisk ò ). interpretation of results was done according to nccls standard guidelines. in the six years of study, 129 isolates of nocardia spp. were recovered, 13 of them belonging to n. cyriacigeorgici species (10%). n. cyriacigeorgici represents the third species in frecuency in our serie, after n. abscessus and n. farcinica. the 13 strains were recovered from 13 patients, 12 from respiratory specimens and one from blood-culture.pneumonia was the most frequent clinical manifestation, being copd and previous corticosteroid therapy the most common predisposing conditions. all n. cyriacigeorgici isolates showed susceptibility to: amikacin, tobramycin, cefotaxime, imipenem, trimethoprimsulfamethoxazole and linezolid, and resistance to: amoxicillinclavulanic acid and ciprofloxacine. conclusion: n. cyriacigeorgici is not an infrequent cause of nocardiosis in our geographical area. the uniformity showed in the antimicrobial profile can be useful for its identification. in our hospital, patients with copd and receiving corticoid therapy is the most important group of risk for adquiring n. cyriacigeorgici infection. whit the technics available in our laboratory the isolates were identified as nocardia spp. and identification at species level was only possible by phylogenetic analysis using 16 rdna sequencing. high frequency of single-step resistance mutations in nocardia farcinica exposed to quinolones u.s. jensen, j.d. knudsen, k. schønning (hvidovre, dk)objectives: nocardia farcinica infections often require prolonged antibiotic therapy and perorally administered agents are desirable. isolates commonly display in vitro susceptibility to quinolones when tested by disc diffusion methodology. in the present study, we investigated the activity of three different quinolones (ciprofloxacin, levofloxacin and moxifloxacin) against n. farcinica and assessed the robustness of their activity by determining the frequency of single step resistant mutants when exposed to inhibitory concentrations of quinolones. methods: 10 isolates of n. farcinica were used in the study; correct identification to the species level was verified by 16s rdna sequencing. mics of ciprofloxacin, levofloxacin and moxifloxacin against n. farcinica as well as s. aureus atcc 25923 and e. coli ccug 17620 were determined by the agar dilution method using inocula of approximately 10.000 cfu and 48 h of incubation. single step mutation frequencies were determined by heavily inoculating selective agar plates containing quinolone at a concentration of 4x mic and counting resistant colonies after 5 days incubation. inoculum was quantified by seeding a dilution series of the inoculum employed on unselective plates and counting colonies after 48 h of incubation and frequencies were calculated by dividing the number of resistant colonies by the number of cfu present in inoculum. results: when mics were determined by agar dilution method all quinolones displayed roughly the same potency against n. farcinica isolates (mics between 0.25 and 4). as expected moxifloxacin were the most potent quinolone against s. aureus. however, all three quinolones selected for single step resistant mutants, the frequency of which was higher for ciprofloxacin (~10 )6 ) than for levofloxacin (10 )7 -10 )8 ), which again was higher than for moxifloxacin (10 )8 -10 )9 ). however, even for moxifloxacin the frequency against n. farcinica was comparable to the single step mutation frequency of ciprofloxacin against s. aureus (10 -9 ).conclusions: although quinolones may exhibit activity against n. farcinica, n. farcinica is capable of rapid development of resistance. therefore, quinolones should probably be avoided, at least as single agents, in the treatment of nocardia infections. correlation between clinico-laboratory findings and a positive igm elisa test for leptospira: a retrospective study e. mendrinou, p. goudas, a. regli (patra, gr) objective: to correlate a positive elisa test for igm antibodies against leptospira with the clinical and laboratory findings in patients with suspected leptospirosis. method: we retrospectively analysed the history, clinical course and laboratory findings in a total of 420 patients, with suspected leptospirosis. all patients fulfilled the criteria for clinical diagnosis of leptospirosis. from the 420 patients, 21 had to be transferred to the dialysis unit for haemodialysis and 6 patients had to be admitted to the intensive care unit (icu) due to severe pulmonary haemorrhage. serum samples from all patients were tested for igm antibodies against leptospira. results: from the total of 420 patients death occurred to only four, due to respiratory failure from severe pulmonary haemorrhage. the rest of the patients recovered completely. from the total of 420 patients 25 had a positive elisa igm test for leptospirosis (5.95%). however, from the 21 patients that were transferred to the dialysis unit, 8 had a positive leptospirosis test (25%) and from the six patients admitted to the icu, three had a positive test (50%). among other laboratory findings there was a stronger correlation between very low platelet levels (<30.000 mm3) and very high blood bilirubin levels (>20 mg/dl) with a positive test for leptospirosis. all 25 patients with a positive test had less than 30.000 platelets per mm3 and 18 had blood bilirubin over 20 mg/dl. the differential diagnosis of icterohemorrhagic fevers includes a vast number of pathogens, some of which are untraceable with the common laboratory methods. in our study, from the total of 420 patients, only 5.95% had a positive test for leptospirosis. in 92 of the rest 395 patients, many different pathogens were traced, most of them being several kinds of viruses (cmv, ebv), brucella and coxiella. in 303 of the patients no pathogen was traced. conclusions: taking into consideration the high sensitivity of the elisa test we conclude that: 1. icterohemorrhagic leptospirosis comprises only a small subtotal of icterohemorrhagic fevers; 2. there is a correlation between higher levels of bilirubin and/or very low platelet levels with leptospira infection; 3. there seems to be a correlation between leptospira infection and severity of icterohemorrhagic fevers. evaluation of continuous ambulatory peritoneal dialysis-related peritonitis attacks in ankara s. tekin koruk, m.a. yetkin, i. koruk, f.s. erdinc, s. sahan, n. tulek, m. duranay, a.p. demirö z (ankara, konya, samsun, tr) objectives: peritonitis is a common clinical problem that occurs in patients with end stage renal disease treated by peritoneal dialysis. the aims of this study were to assess demographic aspects, rates of peritonitis, causative organisms, clinical outcomes and treatment approach for continuous ambulatory peritoneal dialysis (capd) -related peritonitis cases. methods: seventy cases of peritonitis occurred in 55 patients treated in infectious diseases and clinical microbiology department between may 2003 and april 2004 were enrolled into this study. the mean age of the patients was 48.4 years (range 16-77 years). cloudiness of the peritoneal dialysis fluid and/or abdominal pain were considered suggestive of peritonitis and were confirmed by cell count and culture. baseline cell count, gram stain, and cultures were obtained, and repeated with periodic follow-up. results: the overall incidence of peritonitis was 2.5 ± 2.5 episodes/patient-year. in 52.7% of patients there were only one peritonitis attack, where as in 47.3% of them had two or more attacks. age, gender, education and profession of the patients have not been found as a risk factor in peritonitis attacks.the most common presenting symptoms of the patients were abdominal pain, cloudiness of the peritoneal dialysis fluid, nausea and vomiting. peritoneal dialysate fluid white blood cell count was 1773 ± 1224/mm3 in 70 episodes. cultures were positive in 51 (72.9%) peritonitis episodes; coagulase-negative staphylococci was the most common organism (%22.8), followed by staphylococcus aureus (%21.4), 19 episodes (%27.2) had negative culture results. there was a statistically significant decrease in serum crp and esr levels and at the end of the treatment when compared with the levels on admission.at the end of the study, 61 episodes of 70 peritonitis cases were treated with ip cefazolin and gentamicin protocol. seven of the patients did not respond initiate therapy and the therapy was converted to iv protocol. seven episodes were treated with iv antibiotics on admission for medical reasons (systemic infection and/or concurrent exit-side or tunnel infection). there were two deaths. two catheters were removed and the patients were transferred to haemodialysis programme. conclusion: despite all technical improvements during recent decades peritonitis is still the major complication of capd. for the accurate treatment of complications, causative organisms and their antimicrobial susceptibilities must be known. objective: viruses are a frequent cause of upper respiratory tract infections in children. among the respiratory viruses, influenza viruses are known to cause outbreaks globally. the present study was carried out to identify the influenza virus serotypes causing acute respiratory infection in children attending univesity hospital in konya in turkey. methods: thorat swabs were collected from 117 acute viral upper respiratory infection suspected children attending the out patient clinic of meram medical faculty hospital. two swabs were taken fron each chidren and one of the swabs was used for bacteriological cultures and if these were negative the other one was used for viral diagnosis. totally 100 bacteriological cultures negative swabs were investigated by real-time pcr for the presence of parainfluenza 1, 2 and 3, influenza a and b. results: one or more viral pathogens were detected in 24 children, with parainfluenza 1 9% being the most commonly identified virus. parainfluenza 2 in 7% and parainfluenza 3 in 5%, influenza a were identified in 5% and influenza b in 1%. from the specimens of 3 children more than one virus detected. conclusion: the influenza viruses cause morbidity and mortality among children and elderly. this study analysed the occurrence of influenza and paranfluenza respiratory ifections due to influenza and paranfluenza viruses. molecular methods used directly on clinical material have an important role in the rapid diagnosis and surveillance of influenza viruses and can be applied in clinical practice for correct diagnosis and administration of effective treatment. 1, 2, 5, 6, 7, 8 and 35 . the demographics, clinical presentations and laboratory findings of the 147 patients with serotype 3 were presented. results: the mean age was 4 y7 m, ranging from 5 months to 12 y2 m. seventy percents of children with serotype 3 infection clustered between october 2004 and january 2005. the mean duration of a positive culture result was 8.5 days. the mean duration of fever was 6.0 days, with 4 days before admission. forty (27%) children were treated as outpatients. the mean length of hospital stay was 5.4 days. the 3 most common diagnoses were exudative tonsillitis (25%), pneumonia or bronchopneumonia (24%) and pharyngoconjunctival fever (12%). the 3 most common symptoms and signs were fever (100%), cough (88%) and coryza (74%). neurologic complications were noted in 2 children. eighteen children had documented coinfection (including virus, bacteria and mycoplasma pneumoniae). leukopenia (wbc < 5000/microliter) was noted in two of 111 cases while leukocytosis (wbc > 15000/microliter) in 32 (29%). six (5.5%) of 110 cases had a normal serum c-reactive protein (crp) level (<10 mg/l), while 80% of 110 children had a serum crp greater than 40 mg/l. seventy (47.6%) of 147 children ever received antibiotics therapy. the outcomes were excellent in these cases. conclusion: recognizing that children with adenoviral serotype 3 infection may present with prolonged high fever, leukocytosis and elevated crp, which mimics bacterial infection, the clinician may not prescribe unnecessary antibiotics for these children. the infectious mononucleosis like syndrome (im) is an acute febrile disease of older children and young adults, and is characterized by lymphadenopathy, tonsillitis, splenomegaly, liver dysfunction and by the presence of peripheral lymphocytosis with >10% atypical lymphocytes. epstein -barr virus (ebv) is responsible for over 90% of the cases, cytomegalovirus (cmv) for 5%-7% and toxoplasma gondii <1%.herpes simplex, rubella and adenovirus are rare. the infection is usually characterized by mild symptoms. however in some cases the clinical manifestations may be rather atypical and severe. objective: to determine the prevalence of im like syndrome among patients in a children's hospital and its possible association with etiologic factors, age, major symptoms and atypical manifestations. material and methods: during a one-year period (january to december 2004) a total of 700 samples were examined in our laboratory. the study population was children between 1-15 years old, which either examined in the outpatient's clinic or hospitalized. all serum specimens were examined by1. indirect immunofluorescence for the presence of igg and igm antibodies against the viral capsid antigen (vca) ebv, 2.immuno chemistry luminescence for the detection of igg and igm antibodies to cmv and 3.eia for the detection of igg,igm abs of herpes simplex i and ii and toxoplasma gondii. results: of the 700 children examined 43 (6.1%) were found positive for igg and igm vca antibodies and 28(4%) showed positive specific igg and igm antibodies for cmv. these patients had one or more of the primary following symptoms: fever (87%), lymphadenopathy (80%), pharyngalgia (50%), cough (15%), skin eruption (9%). atypical manifestations as meningoencephalitis were found in two children one aged 20 months (caused by ebv) and the other of 7 years old (caused by hsv i) confirmed by pcr. the laboratory data showed positive serology for ebv and cmv infection, the existence of atypical lymphocyte (76%), ldh, asat and alat were moderately elevated (44 %) and crp increased (18%). conclusion: the frequency of im like syndrome in greece, though it's relatively low, it's not rare. the above results suggested that ebv, cmv, hsv should be considered in any young patient with im and acute neurological illness of uncertain etiology. objectives: enterovirus, parvovirus b19 and human herpes virus type 6 (hhv-6) are a common cause of infection in young infants. the objective of this study was to determine what portion of the infants who received a clinical diagnosis of febrile syndrome have a viral etiology by these three genera of viruses. methods: ninety-six patients were included in the study, all of them were admitted to the pediatric casualty of a tertiary care hospital, and all of them presented a febrile syndrome without a clear focus of infection (urinary tract, lung and meningeal infections were discarded). the assay was carried out in 96 blood samples by real-time pcr. dna was isolated from 200 ll of blood by semi-automated system magna pure lc total nucleic acid isolation kit (roche diagnostics, nederland bv). pcr was performed in a lightcycler instrument (roche molecular biochemicals) by a uniform cycling parameters: 10 min at 95°c for polymerase activation, and 50 cycles of 15 s at 95°c and 60 s at 60°c for amplification of the specific target sequence (5 utr gene for enterovirus, vp2 gene for parvovirus b19 and dna polymerase gene for hhv-6). pcr product formation was detected continuously by the use of taqman probes. results: a viral amplification was detected in 52 (54%) of the 96 patients included in this study. enterovirus was detected in 12 (12.5%) of the patients, parvovirus b19 in 10 (10.4%) and hhv-6 in 35 (36.4%). in five cases two viral amplifications were detected at the same time: 3 parvovirus b19/hhv-6 and 2 enterovirus/hhv-6. the mean age of the patients was 2 years old (range from 24 days to 14 years). in group of infants <6 months old (n = 27) there were 7 enterovirus and 6 hhv-6. in the infants from 7 months to 3 years old (n = 53) there were 3 enterovirus, 3 parvovirus and 24 hhv-6. in the last group of infants >3 years old (n = 16) there were 2 enterovirus and 7 parvovirus b19. conclusions: viral infections are an important cause of sepsis in infants admitted to hospital. enterovirus was the most frequent virus detected in infants <6 months, parvovirus b19 the most frequent in children >3 years old, and the hhv-6 was detected in all age groups. qualitative real-time pcr in blood is a rapid and sensitive method for diagnosis of enterovirus and parvovirus. however, is not the better method for diagnosis of hhv-6, a latent virus, in which this technique is not capable of distinguish between recent and acute infection. objectives: group a rotaviruses are a major cause of acute gastroenteritis in infant and young children worldwide. in this study, the molecular epidemiology and clinical features of rotavirus infection in iranian children was investigated. methods: between february 2004 to january 2005, thirty hundred and seventy two diarrhoea stools from children under 5-years-old with acute diarrhoea that attended the biggest paediatric hospital in tehran (iran), were analysed using elisa, electropherotyping and reverse transcriptionpolymerase chain reaction (rt-pcr). results: ninety-four samples (25.3%) were positive for the presence of rotavirus either by page, elisa, or both. according to page, the predominant electrophoretic pattern detected was the long profile 62 of 67 (92.5%) followed by the short electropherotype five of 67 (7.5%). out of the positive samples, 49 were further characterized by rt-pcr typing assay for identification of g types, resulting in 29 strains of g1 genotype while 20 samples could not be assigned a g type. all of g1 genotypes had a long rna electropherotype. among the patients with rotavirus infection, 23 (24.5%) required hospitalization. watery diarrhoea (92.6%), vomiting (73.4%) and fever (64.9%) were significantly more frequent in children suffering from rotavirus gastroenteritis. seven out of 94 rotavirus-positive patients had severe dehydration (p < 0.0001). rotavirus infection mostly affected children under 2 years of age with a peak incidence of 40% in children 1-2 years of age and it occurs year round with a seasonal pattern: more frequently during winter (46.2%). conclusion: this study revealed that rotavirus is an important etiological agent of acute gastroenteritis in tehran. we found that a major proportion of the specimens were untypeable. improved detection and characterization of incompletely typed strains will help to develop comprehensive strain information that may be required for tailoring effective rotavirus vaccines. serological study of prevalent rotaviruses in tehran e. habibi, s. ghorbani, a. jarollahei, z. habibi (tehran, zanjan, ir)objectives: rotaviruses are icosohedral and non-enveloped viruses that belong to reoviridae family which consist of three layers of protein surrounding 11 segments of dsrna. rotavirus is one of the most important agents of acute gastroenteritis in children. in this survey, the most prevalent serotypes in tehran and seasonal distribution in a year were detected. methods: in this study, a total number of 180 specimens of faecal samples of children and infants with acute gastroenteritis were collected from two children hospitals in tehran. the samples were tested by elisa procedure. serotyping investigation of iranian rotavirus isolates, using 7 serotypes monoclonal antibodies (g1-g2-g3-g4-g6-g8-g9) in elisa tests and immunosorbent electron microscopical studies using trapping and decoration techniques were performed. results: rotavirus type a infection was identified in 66 samples (37%). serotyping investigation in elisa tests proved that serotypes g1 and g4 were the most common serotypes circulating among infected children and infants in tehran. by electron microscopic studies the characteristic of rotavirus particles were observed in the faecal samples of infected children. the maximum incidence of infection was determined to occur among the cold months of the year. conclusion: it was approved that g1 and g4 serotypes are the main rotavirus serotypes present among children in tehran. it was detected that rotavirus diarrhoea was most prevalent among children of under 3 years of age. results: from 447 patients with varicella 19 presented neurological manifestations (sex ratio m/f:10/9). 18 had acut cerebellar ataxia and one had encephalitis. we estabilished the diagnosis on the basis of clinical aspects (including neurological examination), cerebrospinal fluid examination and electroencephalogram. the age interval was between 7 months and 30 years. most cases were diagnosed in children and teenager (17); one case toddlers, and 2 cases in adults. neurological manifestations appeared in most cases among 7 and 10 days after the onset of rash (15 cases). in the order of frequency: gait disorders (15), cerebellar ataxia (11), fever (7), vomiting (5), nistagmus (3), seizures and coma (1) . csf showed limphocytic pleiocytosis and elevated levels of protein (10 cases); in 9 cases csf had normal aspects. electroencephalogram had dominant theta wave with totally or partially suppression of alpha activity in all patients. all cases showed clinical and eeg improvement at the end of the treatment. conclusions: the most frequent neurological manifestation was cerebellar. the evolution was good under treatment, with no sequelae at 1 month of follow up.