Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.

Internal quality assurance in diagnostic microbiology: A simple approach for insightful data.

Given the importance of microbiology results on patient care, high quality standards are expected. Internal quality assurance (IQA) could mitigate the limitations of internal quality control, competency assessment and external quality assurance, adding a longitudinal insight, including pre- and post-analytical steps. Here, we implemented an IQA program in our clinical microbiology facilities with blind resubmission of routine samples during 22 months. One-hundred-and-twenty-one out of 123 (98.4%) serological analyses and 112 out of 122 (91.8%) molecular analyses were concordant. Among the discordances in molecular biology analyses, 6 results were low positive samples that turned out negative, likely due to stochastic repartition of nucleic acids. Moreover, one identified retranscription error led us to implement automated results transmission from the Applied Biosystems instruments to the laboratory information system (LIS). Regarding Gram stain microscopy, 560 out of 745 (75.2%) of compared parameters were concordant. As many as 67 out of 84 (79.8%) pairs of culture results were similar, including 16 sterile pairs, 27 having identical identification or description and semi-quantification and 24 only showing variations in semi-quantification with identical description or identification of colonies. Seventeen pairs had diverging identification or description of colonies. Culture was twice only done for one member of the pairs. Regarding antibiotic susceptibility testing, a major discrepancy was observed in 5 out of 48 results (10.4%). In conclusion, serological tests were highly reproducible. Molecular diagnosis also revealed to be robust except when the amounts of nucleic acids present in the sample were close to the limits of detection. Conventional microbiology was less robust with major discrepancies reaching 39.5% of the samples for microscopy. Similarly, culture and antibiotic susceptibility testing were prone to discrepancies. This work was ground for reconsidering multiples aspects of our practices and demonstrates the importance of IQA to complete the other quality management procedures.

Early detection of extended-spectrum ß-lactamase from blood culture positive for an Enterobacteriaceae using ßLACTA test.

Bacterial pellets from Enterobacteriaceae positive blood cultures prepared using ammonium chloride were tested for rapid detection of ß-lactamase using the commercial ßLACTA test and read after 30 minutes. During 7 months, 137 bacterial pellets were tested prospectively. ßLACTA test exhibited a sensitivity of 75% and a specificity of 100% for the detection of third-generation cephalosporin resistance. False negative tests were mainly observed with hyperproduced chromosomal or plasmid-borne AmpC.

Preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A simple blood-culture bacterial pellet preparation for faster accurate direct bacterial identification and antibiotic susceptibility testing with the VITEK 2 system.

An ammonium chloride procedure was used to prepare a bacterial pellet from positive blood cultures, which was used for direct inoculation of VITEK 2 cards. Correct identification reached 99% for Enterobacteriaceae and 74% for staphylococci. For antibiotic susceptibility testing, very major and major errors were 0.1 and 0.3% for Enterobacteriaceae, and 0.7 and 0.1% for staphylococci, respectively. Thus, bacterial pellets prepared with ammonium chloride allow direct inoculation of VITEK cards with excellent accuracy for Enterobacteriaceae and a lower accuracy for staphylococci.

Asymptomatic oral yeast carriage in HIV-infected patients: frequency and fluconazole susceptibility profile.

OBJECTIVES: Fluconazole-resistant oropharyngeal candidiasis (OPC) is a rapidly growing problem in HIV-infected patients. To better understand the pathogenesis of fluconazole resistance in this setting, asymptomatic candidal carriage was determined by means of oral swabs regularly performed in all patients without clinical signs of OPC seen at our HIV outpatient clinic. Controls were 204 asymptomatic healthcare workers without previous exposure to fluconazole. METHODS: Swabs were plated on three solid media and put in a Sabouraud broth. Phenotypically different colonies were identified to the species level. Susceptibility to fluconazole was determined using a disk diffusion test with 50 microg fluconazole disks on yeast nitrogen agar, with a cut-off value of 25 mm. RESULTS: Swabs were performed in 538 consecutive HIV-positive patients, of whom 216 (40%) had had prior episode(s) of OPC and/or were previously exposed to fluconazole. Yeasts were grown in 418/538 HIV-positive patients (78%), compared to 57/204 controls (28%) (p < 0.05). In HIV-positive patients, yeasts were grown in 189/216 (88%) of those with past fluconazole exposure, and in 229/322 (71%) without exposure (p < 0.05). A total of 589 isolates were grown in the 538 HIV-positive patients (451 C. albicans, 88 C. glabrata, 22 C. tropicalis, 11 C. krusei, and 17 isolates from 12 other species). Resistance to fluconazole was present in 121/589 (21%) Candida species isolates in HIV-positive patients and in 2/59 (3%) in controls. Among C. albicans isolates, there were 18 fluconazole-resistant strains in HIV-positive patients (4%) and none in controls.CONCLUSIONS: Using sensitive culture methods, oral yeast colonization was detected significantly more frequently in HIV-infected patients (78%) than in a control group of HIV-negative persons (28%). In addition, yeast colonization was quantitatively more important in patients with lower CD4+ lymphocyte counts and for those who had been exposed to fluconazole for episode(s) of OPC. Fluconazole-resistant C. albicans isolates were observed only in HIV-positive patients, and all patients (17/18) for whom this information could be ascertained had had prior exposure to fluconazole.

Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species.

Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual "prominent growth reduction" (MIC(2)) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode +/- one dilution range across all 17 laboratories. MIC(2) at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.