Validation of a modified algorithm for the identification of yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

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.

Evaluation of a real-time polymerase chain reaction assay for the diagnosis of pneumococcal and meningococcal meningitis in a tertiary care hospital.

This study evaluated the performance of a real-time polymerase chain reaction (PCR) assay in comparison with Gram staining and culture of cerebrospinal fluid for the diagnosis of meningococcal and pneumococcal meningitis in patients with suspected bacterial meningitis. The sensitivity for detection of Neisseria meningitidis in cerebrospinal fluid was 87% (20/23) for the PCR assay, 27% (6/22) for Gram staining, and 17% (4/23) for culture. The sensitivity for detection of Streptococcus pneumoniae in cerebrospinal fluid was 100% (14/14) for the PCR assay, 62% (8/13) for Gram staining, and 36% (5/14) for culture. Therefore, we recommend that real-time PCR of cerebrospinal fluid for detection of N. meningitidis and S. pneumoniae become a part of the routine diagnostic procedure for patients with suspected bacterial meningitis.

Evaluation of a rapid real-time RT-PCR assay for detection of enterovirus RNA in cerebrospinal fluid specimens.

BACKGROUND: We previously described the characteristics of a single-tube real-time enterovirus reverse transcriptase polymerase chain reaction (RT-PCR) assay based on a fluorogenic probe and primers directed to highly conserved sequences in the 5'-untranslated region (UTR) of the enterovirus genome. OBJECTIVES: To evaluate the performance of the assay on a larger number of cerebrospinal fluid (CSF) specimens from patients suspected of having viral meningitis. STUDY DESIGN: Real-time enterovirus RT-PCR and viral culture were performed on CSF specimens received from March 2000 to November 2001. Patient records were retrospectively reviewed for final clinical diagnosis. RESULTS: From the 186 CSF specimens tested, culture was positive for enterovirus in 31 cases, whereas real-time RT-PCR detected enterovirus RNA in 45 CSF specimens. The sensitivity of real-time RT-PCR in relation to the clinical diagnosis of viral meningitis was 72.6%, whereas the sensitivity of viral culture reached only 57.4%. Enterovirus RNA was also found in a number of specimens with low leukocyte counts. CONCLUSIONS: We confirm that the real-time enterovirus RT-PCR assay for CSF specimens is significantly more sensitive than viral culture.

Diphtheria immunity in Flanders.

A serological survey to determine the immunity to diphtheria in the Flemish population was conducted according to the recommendations of the World Health Organization. Immunity to diphtheria was determined on a randomised, stratified sample (1679 serum samples) from an existing serum bank (4058 serum samples) representative of the Flemish population. All age groups between 0 and 100 years were included. A tissue (Vero cell) culture toxin neutralisation assay was used to measure serum diph-theria antitoxin concentrations. The results showed that 43% of the Flemish population was protected against diphtheria (antitoxin titre, > or = 0.1 IU/ml), while 32% was susceptible (antitoxin titre, < 0.01 IU/ml); for 25%, protection was of limited duration (antitoxin titre, > or = 0.01 IU/ml and < 0.1 IU/ml). The proportion of susceptible subjects showed a significant age-related increase, with the highest values in the 35 to 44 and 45 to 54 age groups (57.9% and 55.5%, respectively). These results emphasise the need for booster immunization of adults.