Performance of the VITEK®2 advanced expert system™ for the validation of antimicrobial susceptibility testing results.

Time to reporting antimicrobial susceptibility testing (AST) results to physicians plays an essential role in antibiotic stewardship programs. Expert software has been developed for facilitating the microbiologists' AST review process. The reliability of the VITEK®2 Advanced Expert™ software to effectively alert the microbiologist in detection of atypical and inconsistent AST results was assessed at the Labor Berlin-Charité Vivantes services. The results demonstrated a confidence rate of 99.3% in reporting fully consistent AST results to physicians.

MCR: modern colistin resistance.

Recently, plasmid-mediated and, therefore, transferable bacterial polymyxin resistance was discovered in strains from both humans and animals. Such a trait may widely spread geographically, while simultaneously crossing microbial species barriers. This may ultimately render the "last resort" polymyxin antibiotics therapeutically useless. Colistin is currently used to treat infections caused by Gram-negative carbapenemase producers and colistin resistance may lead to practical pan-antibiotic resistance. We here analyzed the medical and diagnostic consequences of (emerging) colistin resistance and propose pathways toward adequate diagnostics for timely detection of both asymptomatic carriage and infection. Culture-based testing using chromogenic and selective media for screening clinical (and veterinary) specimens may constitute key tools for that purpose. Relevant molecular tests are also discussed.

Digital antimicrobial susceptibility testing using the MilliDrop technology.

We present the MilliDrop Analyzer (MDA), a droplet-based millifluidic system for digital antimicrobial susceptibility testing (D-AST), which enables us to determine minimum inhibitory concentrations (MICs) precisely and accurately. The MilliDrop technology was validated by using resazurin for fluorescence readout, for comparison with standard methodology, and for conducting reproducibility studies. In this first assessment, the susceptibility of a reference Gram-negative strain Escherichia coli ATCC 25922 to gentamicin, chloramphenicol, and nalidixic acid were tested by the MDA, VITEK®2, and broth microdilution as a reference standard. We measured the susceptibility of clinically relevant Gram-positive strains of Staphylococcus aureus to vancomycin, including vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-susceptible S. aureus (VSSA) strains. The MDA provided results which were much more accurate than those of VITEK®2 and standard broth microdilution. The enhanced accuracy enabled us to reliably discriminate between VSSA and hVISA strains.

Performance of chromID® CARBA medium for carbapenemases-producing Enterobacteriaceae detection during rectal screening.

Chromogenic chromID® CARBA medium was compared with CDC method and MacConkey agar with imipenem for its performance in detecting carbapenemase-producing Enterobacteriaceae (CPE) during a faecal screening surveillance program. Double rectal swabs were collected from patients hospitalized in the ICU. One swab was inoculated onto the solid media chromID® CARBA and MacConkey agar with imipenem, while the other was tested according to CDC protocol. Suspected colonies from all procedures were identified to species level and tested for their susceptibility to carbapenems by phenotypic tests. All carbapenem non-susceptible isolates were tested by the modified Hodge test (MHT) and synergy tests. Positive results were confirmed by PCR testing for carbapenemase gene detection. Performance of all three procedures applied was statistically analyzed as compared to MHT and PCR results for the presence of carbapenemase-encoding genes. Out of 177 rectal samples tested, 86 samples were found to contain one or more CPE verified by molecular detection of carbapenemase encoding genes among isolated Enterobacteriaceae. Sensitivity of chromID® CARBA was 96.5 % in clinical samples. Specificity was 91.2 % at the reading level and 100.0 % after Gram staining. chromID® CARBA performed with high accuracy among the phenotypic methods applied, giving early results.

International dissemination of Escherichia coli strains with discrepant behaviour in phenotypic antimicrobial susceptibility tests.

Preventing the dissemination of antimicrobial resistance depends on appropriate antibiotic stewardship and accurate antimicrobial susceptibility testing (AST). We report the international dissemination of Escherichia coli strains, showing discrepancies between reference methods when phenotypically tested for susceptibility to piperacillin/tazobactam (TZP). We demonstrate that these related strains are predisposed to problematic TZP AST interpretations.

Laboratory diagnosis of oxacillin resistance in Staphylococcus aureus by a multiplex-polymerase chain reaction assay.

A polymerase chain reaction (PCR) test was developed in which the mecA gene responsible for the intrinsic resistance to oxacillin in Staphylococcus aureus and the gyrA gene, always present in this species, were amplified in one operation. Among the 468 clinical isolates tested, the results obtained for 454 of the isolates (97%) were consistent with those of MIC determination. Discrepant results were noted for strains with low-level oxacillin resistance (MICs, 4-8 micrograms/ml) and mecA gene negative. For these strains, susceptibility to oxacillin was restored in the presence of a beta-lactamase inhibitor, which suggested a resistance by penicillinase hyperproduction. In contrast, all of the high-level resistant strains (MICs, > 8 micrograms/ml) carried the mecA gene. The presence of this gene has frequently been associated with resistance to gentamicin, tetracycline, erythromycin, lincomycin, and pefloxacin. The PCR assay described in this study can be accomplished with ease and total confidence in the clinical microbiologic laboratory for a rapid and effective establishment of antistaphylococcal chemotherapy.

Clinical impact of rapid oxacillin susceptibility testing using a PCR assay in Staphylococcus aureus bactaeremia.

OBJECTIVES: The aim of this work was to establish the clinical impact of rapid oxacillin susceptibility testing in nosocomial Staphylococcus aureus bacteraemia. METHODS: This study was performed in 145 critically ill patients infected by S. aureus. Patients were randomly assigned to one of two groups: patients for whom susceptibility testing was performed using a rapid same day multiplex PCR assay for detection of the staphylococcal mecA (mean delay of response: 6 h) and those for whom testing was accomplished using traditional overnight techniques (21 h). RESULTS: The results of this study showed no significant difference between the two groups in terms of age, Simplified Acute Physiologic Score, severity of infection, severity of underlying disease and clinical outcome (control vs. PCR): unfavourable outcome of infection, 12.32 vs. 12.5%; 95% CI for the difference = -11.49 to 11.09 (P = 0.975); unfavourable general outcome, 16.43 vs. 20.83%; 95% CI for the difference = -17.35 to 8.50 (P = 0.497). For the oxacillin-susceptible S. aureus bactaeraemia, results were: unfavourable outcome of infection = 13.04 vs. 11.11%; 95% CI for the difference = -11.38 to 16.18 (P = 0.767); unfavourable general outcome = 13.04 vs. 20.37%; 95% CI for the difference = -22.12 to 8.07 (P = 0.331). CONCLUSION: This study seemed to demonstrate that rapid oxacillin susceptibility testing using a PCR assay did not have a major impact on the care and outcome of patients with S. aureus bactaeremia.

Nosocomial infection with Candida albicans in a pancreatic transplant recipient investigated by means of restriction enzyme analysis.

Restriction enzyme analysis (REA) of total DNA was used in order to investigate the possible transmission of Candida albicans from the grafted pancreas in a woman with a kidney-pancreas transplant. A strain of Candida albicans was recovered from the pancreas-transplant preservation medium cultured routinely before transplantation. Four infecting isolates and one vulval isolate were recovered from the recipient in the early post-operative stage. In addition, 12 unrelated control strains were studied for comparison. By means of EcoRI and HinfI, restriction patterns of the isolates recovered from the preservation medium and from the patient were found to be identical (100% similitude according to Jaccard's coefficient) apart from that of the strain isolated from the vulva. HinfI gave two characteristic fragments of 5.9 and 4.6 kb. In contrast, the 12 control strains generated 12 different patterns. The percentage of similarity between the patterns of the infecting strains and those of the control and the vulval strains was less than 60%. These findings provide evidence of transmission of the infecting strain via the pancreas transplant and the nosocomial nature of infection.

Comparison of three primer sets for the detection of Mycobacterium tuberculosis in clinical samples by polymerase chain reaction.

A number of studies have underlined the interest of the polymerase chain reaction (PCR) in the detection of Mycobacterium tuberculosis in clinical samples. Among the different parameters to be carefully studied the choice of target gene and primers is essential. The amplification of nucleotidic sequences localised on three different target genes (groEL, IS6110, Pab) was examined in 196 clinical samples from patients with suspected tuberculosis or receiving antituberculous therapy. The results obtained after hybridization with non-radioactive labelled probes were compared with the culture data. None of the primer sets studied showed a satisfactory sensitivity (79% to 84%) suitable for it to be used alone. The false-negative specimens with the PCR tests usually corresponded to those that contained few mycobacteria. With the methods described in this study, the use of two or three primer sets located on different target genes allowed to improve the positivity rate compared to the culture and sensitivity of the test (90-98%), particularly for paucibacillary samples. On the other hand, the interpretation was easier when concordant results were obtained.

[Biological diagnosis of whooping cough: contribution of gene amplification]. / Diagnostic biologique de la coqueluche: apport de l'amplification génique.

An upsurge in pertussis infections, despite mandatory vaccination in France since 1966, has occurred again in developed countries due to progressive loss of vaccinal immunity and wider circulation of the causal bacterium, Bordetella pertussis. Unfortunately, the classical culture method is insufficiently sensitive and serology can only confirm diagnosis retrospectively. New techniques are needed for rapid diagnosis, and subsequent treatment and preventive measures. One new method, gene amplification using polymerase chain reaction (PCR), has been particularly useful in detecting Bordetella pertussis. PCR is highly specific and more sensitive than culture. It is thus quite useful in case of atypical clinical presentations and in previously treated or vaccinated patients. Less restrictions on sample transportation and preservation make PCR a technique which general practitioners can use for rapid easy diagnosis of pertussis.

[Comparison of different phenotypic methods with polymerase chain reaction (PCR) for the detection of resistance to oxacillin in coagulase negative staphylococci]. / Comparaison de différentes méthodes phénotypiques avec la réaction de polymérisation en chaîne (PCR) pour la détection de la résistance à l'oxacilline chez les staphylocoques à coagulase-négative.

The mecA gene which confers the oxacillin resistance has been searched by PCR in 290 (124 positives, 166 negatives) coagulase-negative staphylococci (CNS) belonging to twelve species. The results were compared with the oxacillin MIC values obtained by agar dilution (4% NaCl) or by the ATB STAPH method (Api-bioMérieux; 0%, 2%, 5% NaCl) and growth inhibitory diameters obtained by agar diffusion with an oxacillin disk placed at 30 degrees C without NaCl, or at 35 degrees C in presence of 2% or 5% NaCl. Sensitivity of oxacillin resistance detection depends upon the salt concentration and the method used. The optimum concentration is 2%. With this concentration, the Api ATB test appears as the more performant (sensitivity: 89.8%). Search for the mecA gene by PCR represents a very interesting method that detects 96.9% of the oxacillin-resistant CNS strains.

Rapid diagnosis of Mycobacterium tuberculosis infections by an ELISA-like detection of polymerase chain reaction products.

A polymerase chain reaction (PCR) assay was developed for the detection in clinical samples of mycobacteria belonging to the Mycobacterium tuberculosis complex. PCR products were detected with a simple and rapid colormetric method. With this method, 50 fg of M. tuberculosis DNA were detectable with the repetitive DNA-sequence-derived primers, corresponding to 10 genome equivalents. Detection of M. tuberculosis in 258 clinical samples by PCR was compared with detection by culture. PCR was positive for 56 of 57 culture-positive and Ziehl-Neelsen-staining-positive (ZN) samples, 11 of 18 culture-positive and ZN-negative samples. The presence of groEL DNA sequences was also investigated by PCR for all the specimens with the same revelation protocol. Three of the eight false-negative samples with the repetitive element-derived primers were found to contain groEL DNA sequences specific for the Mycobacterium genus. Among the 183 culture-negative samples, 30 were positive by PCR. When clinical data were known, the diagnosis of tuberculosis was established for the patients from whom those samples had been obtained. The results show that the rapid and simplified PCR assay described here is slightly more sensitive than culture and can be used in routine clinical practice.

European multicentre evaluation of a commercial system for identification of methicillin-resistant Staphylococcus aureus.

A commercial system for the rapid detection of methicillin-resistant Staphylococcus aureus, the BBL Crystal MRSA test (C-MRSA ID; Becton Dickinson, USA), was evaluated prospectively and compared with a polymerase chain reaction test for the presence of the mecA gene. Ten European centres tested a total of 676 isolates of Staphylococcus aureus from blood cultures. The system correctly identified 661 (97.8%) isolates within 4 h. All but three mecA gene-negative isolates (99.4% specificity) yielded a negative C-MRSA ID reaction, and 158 of 170 mecA gene-positive isolates were accurately detected (92.9% sensitivity). After repeated testing of discrepant results, sensitivity and specificity increased to 99% and 100%, respectively.