Clinical evaluation of multiplex RT-PCR assays for the detection of influenza A/B and respiratory syncytial virus using a high throughput system.

BACKGROUND: Lower respiratory tract infections are a major threat to public health systems worldwide, with RSV and influenza being the main agents causing hospitalization. In outbreak situations, high-volume respiratory testing is needed. In this study, we evaluated the analytical and clinical performance of a pre-designed primer/probe set for the simultaneous multiplex detection of both viruses on a high-throughput platform, the cobas® 6800, using the "open channel" of the system for integration of lab-developed assays for the detection of influenza and RSV. RESULTS: Using the influenza/RSV qPCR Assay with swabs, LoD (95%) in TCID50/mL for influenza-A was 0.009, influenza-B 0.003, RSV-A 0.202, and RSV-B 0.009. Inter-run variability (3xLoD) was low (<1 Ct for all targets). Of 371 clinical respiratory specimens analyzed, results were concordant for 358 samples. The calculated sensitivity and specificity of the assay were 98.3% and 98.4% for Flu-A, 100% and 98.5% for Flu-B, and 98.6% and 99.7% for RSV. All quality assessment panel specimens (N = 63, including avian influenza strains) were correctly identified. None of the tested microorganisms showed cross-reactivity. CONCLUSION: Compared with CE-IVD assays, the assay evaluated here showed good analytical and clinical sensitivity and specificity with broad coverage of different virus strains. It offers high-throughput capacity with low hands-on time, facilitating the laboratory management of large respiratory outbreaks.

Molecular characterisation of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates from hospital and ambulatory patients in Germany.

The increase of Escherichia coli producing extended-spectrum ß-lactamases (ESBL) in hospitals and their emergence as intestinal colonisers of healthy humans is of concern. Transmission ways and the extent of spread of distinct E. coli clones or ESBL genes among humans and animals via the food chain or the environment is a matter of debate. In this study we determined ESBL genotypes in E. coli isolates (n=233) resistant to 3rd generation cephalosporins from hospitals and medical practices using PCR and sequencing. Bacterial strain typing was performed by PCR-based phylogrouping, multilocus sequence typing (MLST) and a ST131-specific PCR. Results showed that CTX-M-15 (50.4%), CTX-M-1 (28.4%) and CTX-M-14 (5.6%) were the most common ESBL types. Especially, CTX-M-15 was associated with E. coli ST131 of phylogenetic group B2, which was the dominant sequence type among our isolates (35.8%). MLST typing revealed 40 different sequence types (STs), with ST131, ST410, ST10 and ST38 as the most prevalent ones. Our findings give an overview of the current distribution of ESBL-producing E. coli isolates from humans in Germany. E. coli O25b:H4-ST131 was confirmed to be the most common clone, which is known for its successful dissemination worldwide. Although heterogeneity among the isolates was found, several successful clones previously described in animals (ST410, ST10) also occurred in our isolate collection. Further detailed investigations of ESBL-producing isolates from different habitats are needed to evaluate possible transfer ways.

Validation of the FluoroType® MRSA assay for the rapid identification of methicillin-resistant Staphylococcus aureus directly from patient material.

We performed the first evaluation study of the new HyBeacon based FluoroType(®) MRSA assay for the detection of MRSA directly from 617 patient specimens. Using culture as the reference method sensitivity and specificity were higher than 95%. Results were available within 2.5h, including DNA extraction.

Emergence of OXA-48-type carbapenemase-producing Enterobacteriaceae in German hospitals.

Nine carbapenem-resistant Enterobacteriaceae isolates collected from eight patients in five German hospitals were investigated. Six isolates produced the OXA-48 carbapenemase, and three isolates produced OXA-162, which is a point mutant form of OXA-48. Both carbapenemase genes were located on IncL/M-type conjugative plasmids. Insertion sequence IS1999 (truncated or not by IS1R) was located upstream of the bla(OXA-48) and bla(OXA-162) genes in all of the isolates. Pulsed-field gel electrophoresis typing indicated the clonal transmission of an OXA-48-producing Klebsiella pneumoniae strain in two hospitals.

Retrospective evaluation of a PCR based assay for the direct detection of methicillin-resistant Staphylococcus aureus in clinical specimen.

We evaluated the performance of a PCR-based dipstick assay used in our routine laboratory for the direct detection of Methicillin resistant Staphylococcus aureus (MRSA). 2941 clinical swab specimens were evaluated. Sensitivity and specificity were 94.1% and 98.3%, respectively. The PCR assay combines low instrumentation costs and minor hands-on-time with reliable results for MRSA identification.

Performance of a matrix-assisted laser desorption ionization-time-of-flight mass spectrometry system for the identification of bacterial isolates in the clinical routine laboratory.

BACKGROUND: Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has emerged as a new tool for the fast and reliable identification of microorganisms. We evaluated the performance of a MALDI-TOF MS-system for the identification of various clinical isolates in the routine microbiology setting. MATERIAL AND METHODS: For the evaluation study a set of 1116 bacterial isolates were collected in the routine microbiology laboratory. Additonally 108 isolates of strain culture collections (ATTC, DSMZ) were utilized. Identification of the bacterial isolates was perfomed with a Microflex LT mass spectrometer in combination with the MALDI-Biotyper 2.0 software (Bruker Daltonik GmbH, Bremen, Germany). The results of the MALDI-TOF MS were compared to phenotypic bacterial identification systems used in our routine laboratory. Discrepancies were resolved by 16 S rDNA-sequencing. RESULTS: Of the 108 reference strains tested, 101 (93.5%) were correctly identified to species level. Overall, 1062 (95.2%) of the 1116 strains collected in the routine laboratory were correctly identified with the MALDI-Biotyper. Accuracy for the identification of Enterobacteriaceae, non-fermenting gram-negative rods, staphylococci, enterococci and streptococci with the MALDI-Biotyper was 95.5%, 79.7%, 99.5%, 100% and 93.7%, respectively. Results were available in 12 minutes for direct smear and in 20 min with an extraction method. CONCLUSIONS: The MALDI-TOF method proves to be a fast and reliable method for the identification of the most important bacterial isolates in the clinical laboratory.