A single-tube real-time reverse transcription-PCR (RT-PCR) assay for enterovirus detection in cerebrospinal fluid (CSF) was developed based on a fluorogenic probe and primers directed to highly conserved sequences in the 5 untranslated region of the enterovirus genome. Quantitative detection of enterovirus genome was demonstrated in a linear range spanning at least 5 logs. Endpoint titration experiments revealed that the in-tube detection limit of the assay was 11.8 enterovirus genome equivalents (95% detection rate) corresponding in our current extraction protocol to 592 enterovirus genome equivalents per ml of CSF. Twenty CSF specimens not suspected of viral meningitis were all found to be negative, and no cross-reactivity with herpes simplex virus type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza viruses A and B was observed. Nineteen CSF specimens from 70 patients suspected of viral meningitis were determined to be positive by PCR (27.1%), whereas only 17 were found to be positive by viral culture (24.3%). The sensitivity of the assay was 100% and the specificity was 96.2% compared to viral culture. Data from the real-time RT-PCR assay were available within 4 h. Our data suggest that the novel real-time RT-PCR assay may offer a reliable but significantly faster alternative to viral culture. Owing to the elimination of postamplification detection steps, its conduct required considerably less hands-on time and was associated with a substantially reduced carryover risk compared to previously described PCR-based enterovirus detection assays.Enterovirus infections account for a substantial number of aseptic meningitis and encephalitis patients requiring hospitalization in the summer and fall (21). Viral culture is the "gold standard" used to diagnose enteroviral infections in cerebrospinal fluid (CSF) specimens, but it takes several days to be conclusive. A rapid diagnostic test may therefore have a strong impact on the diagnosis and clinical management of viral meningitis (16). During the last decade, different reverse transcription-PCR (RT-PCR) assays were developed as a more convenient alternative to viral culture (1,2,15,18,22,24). Various panenterovirus primer sets directed to highly conserved sequences in the 5Ј untranslated region (5ЈUTR) of the enterovirus genome have been designed (16). Many of these assays require gel electrophoresis to detect the amplified product and are in a (semi-)nested format, which is associated with considerable hands-on time, poses a serious hazard for amplification product carryover, and limits the number of specimens that can be processed simultaneously. Specimen throughput is increased in commercial assays based on reverse hybridization to detect the amplification product in microplate format, but the cross-contamination risk, as well as the laborious and timeconsuming postamplification detection steps, remains (19).In recent years, TaqMan technology combined the 5Ј nuclease activity of Taq DNA polymerase and Förster resonance energy transfer to de...

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Optimising antifungal treatment requires the fast and species-specific identification of yeast isolates. We evaluated a modified protocol for the rapid identification of clinical yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) technology. First, we evaluated a simplified extraction procedure using 54 clinical yeast isolates. Second, we validated a new protocol with this simplified extraction procedure and lower identification threshold by analysing 167 isolates with either MALDI-TOF or conventional identification techniques. MALDI-TOF analysis with both the standard and short extraction procedure yielded identical identification results, although the log-scores were lower with the latter. With the modified protocol, 163/167 (97.6%) isolates showed a correct identification as compared to conventional identification techniques. A total of 135 out of the 163 (82.8%) correct identifications showed log-scores above 1.7, which we considered as the minimum log-score for secure species identification. The rapid identification of clinical yeast isolates is crucial in patient management. The MALDI-TOF technique using a short extraction procedure can be an alternative for the labourious standard procedure, although the log-scores will be lower. The identification of clinical yeast isolates with the modified protocol is a practical and accurate alternative for conventional identification techniques. If the isolate shows a log-score below 1.7, the standard extraction procedure should be used.

OBJECTIVE: To evaluate the incidence of hemolytic uremic syndrome (HUS) in Belgium and to determine the role of verocytotoxin-producing Escherichia coli O157:H7 and other serotypes (non-O157 VTEC). METHODS: Twenty-two centers, including the seven university hospitals, registered prospectively all cases of HUS; they collected clinical samples for isolation of VTEC strains and serum for detection of specific O-lipopolysaccharide antibodies. RESULTS: Forty-seven cases of HUS (including five incomplete cases) were recorded. Three cases were seen in non-residents. The incidence of complete HUS in Belgian residents was 4.3 cases/100 000 in children <5 years old, 1.8 cases/100 000 when all children <15 years were considered, and 0.42/100 000 when patients of all ages were taken into account. By combining bacteriologic and serologic results, evidence of VTEC infection was obtained in 64% of the patients, mainly but not exclusively in children with prodromal diarrhea. The 13 VTEC isolates belonged to serotypes O157:H7 (nine isolates), O26:H11, O121:H---, O145:H--- and O172:H--- (one each) and all produced VT2 (+VT2vh-a in three O157 strains) and were positive for the eaeA gene. CONCLUSIONS: The incidence rate found in this study and the high mortality and morbidity linked with this syndrome warrant further registration of pediatric and post-diarrheic adult HUS cases and also examination of stools for both O157 and non-O157 VTEC strains. For effective prevention of this disease, further study of the serotypes and accessory virulence factors associated with HUS is needed.

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