Rapid Quantification of Hepatitis B Virus DNA by Automated Sample Preparation and Real-Time PCR

Monitoring of hepatitis B virus (HBV) DNA in serum by molecular methods has become the standard for assessment of the replicative activity of HBV. Several molecular assays for the detection and quantification of HBV DNA have been described. However, they usually lack automated sample preparation. Moreover, those assays, which are based on PCR, are limited by a short dynamic range (2 to 3 log units). In the present study, the use of RealArt HBV LC PCR Reagents in conjunction with automated extraction on the COBAS AMPLIPREP analyzer was evaluated. Members of an HBV proficiency program panel were tested; linearity, interassay, and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was evaluated with a total of 117 clinical specimens. When members of the HBV proficiency program panel were tested by the new molecular assay, the results were found to be within ±0.5 log unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve over more than 6 log units. The interassay variation of the RealArt HBV LC PCR Reagents by use of the automated sample preparation protocol ranged from 16 to 73%, and the intra-assay variation ranged from 9 to 40%. When clinical samples were tested by the new assay with the automated sample preparation protocol and the results were compared with those obtained by the COBAS AMPLICOR HBV MONITOR Test with manual sample preparation, the results for 76% of all samples with positive results by both tests were found to be within ±0.5 log unit and the results for another 18% were found to be within between 0.5 and 1.0 log unit. In conclusion, the real-time PCR assay with automated sample preparation proved to be suitable for the routine molecular laboratory and required less hands-on time

The Polypore Mushroom Irpex lacteus, a New Causative Agent of Fungal Infections

Irpex lacteus, a wood-decaying basidiomycete, was isolated from a pulmonary abscess of an immunosuppressed child. This medical strain was compared morphologically and by sequencing of the ribosomal intergenic spacers with specimens from both culture collections and herbarium desiccated material. The patient was treated successfully with amphotericin B

Detection of Methicillin-Resistant Staphylococcus aureus and Simultaneous Confirmation by Automated Nucleic Acid Extraction and Real-Time PCR

A molecular assay for the simultaneous detection of a Staphylococcus aureus-specific gene and the mecA gene, responsible for the resistance to methicillin in staphylococci, was evaluated. The assay included an automated DNA extraction protocol conducted with a MagNA Pure instrument and real-time PCR conducted with a LightCycler instrument. The performance and robustness of the assay were evaluated for a suspension of methicillin-resistant S. aureus (MRSA) strain with a turbidity equivalent to a McFarland standard of 0.5, which was found to be the ideal working concentration. The specificity of the new molecular assay was tested with a panel of 30 gram-negative and gram-positive bacterial strains other than MRSA. No cross-reactivity was observed. In a clinical study, 109 isolates of MRSA were investigated. All clinical MRSA isolates gave positive results for the S. aureus-specific genomic target, and all but one were positive for the mecA gene. In conclusion, the new molecular assay was found to be quick, robust, and laborsaving, and it proved to be suitable for a routine molecular diagnostic laboratory

Evaluation of Automated Sample Preparation and Quantitative PCR LCx Assay for Determination of Human Immunodeficiency Virus Type 1 RNA

Efforts have been made to achieve full automation of molecular assays for quantitative detection of human immunodeficiency virus type 1 (HIV-1). In the present study, the Abbott LCx HIV RNA Quantitative assay was evaluated in conjunction with automated HIV-1 RNA extraction on the MagNA Pure LC instrument and compared to the conventional LCx HIV RNA Quantitative assay, which uses a manual nucleic acid extraction protocol. Accuracy, linearity, and interassay and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was tested with a total of 105 clinical specimens. When the accuracy of the LCx HIV RNA Quantitative assay with the automated sample preparation protocol was tested, all results were found to be within ±0.5 log unit of the expected results. Determination of linearity resulted in a quasilinear curve over 3.5 log units. For determination of interassay variation, coefficients of variation were found to be between 21 and 66% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 10 and 69% for the LCx HIV RNA Quantitative assay with manual sample preparation. For determination of intra-assay variation, coefficients of variation were found to be between 7 and 25% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 7 and 19% for the LCx HIV RNA Quantitative assay with manual sample preparation. When clinical samples were tested by the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and the results were compared with those of the LCx HIV RNA Quantitative assay with manual sample preparation, 95% of all positive results were found to be within ±0.5 log unit. In conclusion, the assay with automated sample preparation proved to be suitable for use in the routine diagnostic laboratory and required significantly less hands-on time

High Prevalence of VanA-Type Vancomycin-Resistant Enterococci in Austrian Poultry

Fecal samples from humans and food-producing animals were analyzed for the presence of vancomycin-resistant enterococci (VRE). The VRE carriage rate in humans was 6%, and there was a predominance of VanC-type resistance. Enterococcus faecium with vanA-mediated resistance was frequent in broiler chickens (42%) but rare in cattle and pig samples

Road and rail traffic noise induce comparable extra-aural effects as revealed during a short-term memory test

To examine extraaural effects as induced by 20 min of road (ROAD) and 20 min of rail (RAIL) traffic noise with same loudness (75 dBA), a laboratory study was carried out. The study (N = 54) consisted of 28 high and 26 low-annoyed healthy individuals as determined by a traffic annoyance test. To control attention, all individuals performed a nonauditory short-term memory test during the noise exposures. A within-subject design, with phases of ROAD, RAIL, and CALM (memory test only), alternated by phases of rest, was defined. Heart rate (HR), systolic blood pressure (sBP), total peripheral resistance (TPR), as well as three autonomic variables, preejection period (PEP), 0.15–0.4 Hz high-frequency component of HR variability (HF), and salivary stress biomarker alpha amylase (sAA) were measured. In relation to CALM, HR increased (RAIL +2.1%, ROAD +2.5%), sBP tended to increase against the end of noise exposure, PEP decreased (RAIL −0.7%, ROAD −0.8%), HF decreased (RAIL −3.4%, ROAD −2.9%), and sAA increased (RAIL +78%, ROAD +69%). No differences were found between RAIL and ROAD, indicating that both noise stressors induced comparable extraaural effects. Factor annoyance showed significant during CALM. Here a reduced sympathetic drive (higher PEP values) combined with an increased vascular tone (higher TPR values) was found at the high-annoyed subgroup

Emergence of Enterobacteriaceae Isolates Producing CTX-M Extended-Spectrum β-Lactamase in Austria

Among 149 extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates collected from patients in southeast Austria from 1998 to 2004, 38 Escherichia coli isolates and 11 Klebsiella spp. were CTX-M producers. The proportion of CTX-M-producers among all ESBL producers rose from 0% in 1998 to 58% in 2004. In general, CTX-M-producers had heterogeneous pulsed-field gel electrophoresis patterns, but one E. coli isolate was identical to a United Kingdom epidemic CTX-M-15-producing strain, although no epidemiological link with the United Kingdom was apparent