Evaluation of a broadly reactive nucleic acid sequence based amplification assay for the detection of noroviruses in faecal material.

A recently described nucleic acid sequence based amplification (NASBA) assay for the detection of genogroup I (GI) and genogroup II (GII) norovirus RNA in faecal samples was evaluated against a reverse transcription polymerase chain reaction (RT-PCR). Both assays were used to screen a panel of 38 faecal samples known to contain 17 different norovirus strains and 131 clinical samples collected from 60 gastroenteritis outbreaks of unknown aetiology. The NASBA assay detected 13 out of the 17 strains of norovirus in the characterised panel, failing to detect a single GII strain and three GI strains. There was 90% agreement between the two assays used to detect norovirus in clinical samples from outbreaks. NASBA detected norovirus RNA in all 64 samples positive by RT-PCR and also detected norovirus RNA in additional 13 samples that were negative by RT-PCR. The sensitivity and specificity of NASBA was 100% and 80%, respectively, compared to RT-PCR results. The norovirus NASBA assay was shown to be highly sensitive and specific, and its ease of use and rapid turnaround time makes it a favourable alternative to RT-PCR for the investigation of norovirus outbreaks.

HBV DNA levels and transmission of hepatitis B by health care workers.

BACKGROUND: Laboratory-based study funded by the Research and Development Division of the Department of Health to inform the decision making on guidelines for the conduct of exposure prone procedures (EPPs) by health care workers who are hepatitis B carriers. OBJECTIVES: Define the quantity and nature of hepatitis B virus (HBV) DNA in hepatitis carriers whose serum does not contain hepatitis B e antigen (HBeAg) and in surgeons previously cleared to conduct EPPs who have transmitted HBV to their patients. STUDY DESIGN: Cross-sectional survey using HBV DNA quantification, genotyping and sequencing comparing transmitting surgeons and asymptomatic carriers. RESULTS: HBV DNA could be detected and quantified in 64.5% (136 of 211) of carriers whose serum did not contain HBeAg with a median level 3.6 log(10) copies/ml (range of 5.7 log(10) copies). Pre-core mutation appeared not to affect the HBV DNA level, however, all surgeons carried codon 28 variants and transmitted these variants to their patients. The lowest HBV DNA level in a transmitting surgeon was 4 x 10(4) copies/ml. CONCLUSIONS: Pre-core mutations are common in carriers whose serum does not contain HBeAg and do not specifically identify carriers whose HBV DNA levels are high. It was possible to define a level of virus above which transmission of hepatitis B during conduct of EPPs could not be excluded.

Comparison of commercial assays for the quantification of HBV DNA load in health care workers: calibration differences.

Until recently, carriers of hepatitis B virus (HBV) were allowed to undertake exposure prone procedures providing their serum did not contain HBeAg. However, the recent description of hepatitis B transmission events occurring from HBV-infected health care workers who conduct exposure prone procedures demonstrated that the then current Department of Health guidelines needed to be revised. As part of a series of studies carried out to determine if viral load measurements are a more secure means of assessing the conduct of exposure prone procedures, the suitability of commercially available assays for HBV DNA detection and quantification were investigated. This study describes a comparative analysis on the performances of three assays each based on a different methodology. The assays included the QUANTIPLEX HBV DNA Assay (bDNA), (Chiron Diagnostics Ltd.), the AMPLICOR HBV Monitor Test, (Roche Diagnostics Systems) and the Digene Hybrid Capture System HBV DNA Assay (Digene Corporation). Calibration curves from experiments using the Eurohep ad and ay HBV DNA standard controls indicated a close correlation between the three assays over the dynamic ranges claimed by the manufacturers, although the Quantiplex assay did appear to be over-reporting. This became more apparent when testing patients undergoing anti-viral therapy where the Quantiplex assay consistently over-reported by 0.5 log(10) when compared with the Amplicor assay. The results of this study indicate that based on its dynamic range, the Amplicor HBV Monitor test is the most appropriate assay for the routine investigation of anti-HBe carriers, which will have lower levels of HBV DNA. The investigation also highlights the need for using accepted standard HBV DNA control sera. This will be essential when using an assay to establish whether health care workers who are hepatitis B carriers can be allowed to perform exposure prone procedures under the new guidelines of the UK Department of Health.

Screening for Chlamydia trachomatis infection using the BDProbeTec ET Chlamydia trachomatis amplified DNA assay on urine in a GUM clinic setting: a simple, fast and cost-effective alternative.

This study compared the BDProbeTec ET Chlamydia trachomatis amplified DNA assay on urine specimens with culture of genital swabs for the detection of C. trachomatis in patients attending the Department of Genitourinary Medicine (GUM), Cardiff Royal Infirmary. Almost twice as many patients tested positive by BDProbeTec ET than by culture. A similar difference was found for both males and females. The case notes of those patients positive by BDProbeTec ET alone were analysed and a significantly greater number were found to have risk indicators for C. trachomatis infection when compared with age and sex comparable controls, providing clinical validation of our findings. The BDProbeTec ET assay was easy to use, more importantly, the test format features an internal control integral with every sample. The cost per true positive was calculated as comparable with culture. We conclude that the BDProbeTec ET assay is a superior alternative to culture for identifying patients infected with C. trachomatis in the GUM clinic setting.

Enhanced clinical utility of the NucliSens EasyQ RSV A+B Assay for rapid detection of respiratory syncytial virus in clinical samples.

The aim of the present study was to compare traditional methods for the detection of respiratory syncytial virus with a newly developed commercial assay based on real-time nucleic acid sequence based amplification. Respiratory syncytial virus is a major cause of severe respiratory infection in infants and in certain groups of older children and adults. Treatment options are limited, but a rapid diagnosis improves patient management and infection control. The rapid diagnosis of respiratory syncytial virus currently relies on antigen detection assays. These tests are limited to use in certain good-quality types of samples, which are rarely obtained from adult patients. Molecular-based assays for the detection of respiratory syncytial virus are shown to be highly sensitive, specific, and more rapid than cell culture techniques. This retrospective study compared traditional laboratory techniques for the detection of respiratory syncytial virus in 508 respiratory samples collected during the winter months of 2003-2004 against the recently developed, commercially available NucliSens EasyQ Respiratory Syncytial Virus A+B assay (bioMérieux, Marcy l'Etoile, France), which is based on real-time nucleic acid sequence based amplification using molecular beacons and an internal control. Using traditional techniques, the prevalence of respiratory syncytial virus in the samples tested was found to be 21%. Using the real-time nucleic acid sequence-based amplification assay, an additional 41 samples from patients with a clinically diagnosed respiratory illness were found to be positive for respiratory syncytial virus. The NucliSens EasyQ assay was shown to be sensitive and specific for the detection of respiratory syncytial virus A+B in different types of respiratory samples. Moreover, the time required to complete the assay was <4 h, so results could be obtained on the same day as sample receipt in the laboratory.

Rapid identification of methicillin-resistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR.

Methicillin-resistant Staphylococcus aureus septicemia is associated with significant morbidity and mortality and requires treatment with intravenous glycopeptides. For blood cultures positive for gram-positive cocci, 24 to 48 h is required for the detection of S. aureus bacteremia and the provision of antibiotic susceptibility testing results. We describe a molecular biology-based assay that requires 2 h from the time of initial positivity of blood cultures. The assay correctly detected 96% of the S. aureus isolates including all methicillin-resistant S. aureus isolates. Clinical data collected during the study suggest that 28% of patients with S. aureus bacteremia do not receive early and appropriate treatment and that 10% of patients may initially be receiving inappropriate glycopeptide treatment.

The integration of HPV-18 DNA in cervical carcinoma.

AIMS: Little information is available on the patterns of integration into the host chromosomal DNA of cervical carcinomas of human papillomavirus type 18 (HPV-18) DNA, which is associated with up to 20% of these carcinomas. Because integration of the viral genome may be extremely important in the pathogenesis of cervical carcinoma, the aim of this study was to investigate which regions of HPV-18 DNA are integrated into the cellular DNA of cervical carcinomas. METHODS: Southern analysis using four subgenomic probes covering the entire HPV-18 genome was used to map viral DNA integrated within cellular DNA. The polymerase chain reaction (PCR) was used to confirm the presence of specific regions of the viral genome. RESULTS: In all 11 carcinomas there was a single major HPV-18 DNA integrant, retaining approximately 4000 bp of HPV-18 DNA, indicating that approximately half of the virus genome had been lost upon integration. Southern analysis suggested strongly that the viral breakpoint was within the E1/E2 gene boundary, with concomitant loss of part or all of the E2 ORF (open reading frame), all of the E4, E5, and L2 ORFs and part of the L1 ORF. These data were supported by the PCR results, which confirmed that the region of integrated HPV-18 DNA from nucleotides 6558 to 162 was present in all the carcinoma samples studied. Assuming that no genomic rearrangements, deletions, or insertions had occurred, 4131 bp of integrated HPV-18 DNA could be accounted for in eight cervical carcinoma samples. The results of Southern analysis also suggested that integration of HPV-18 DNA may have occurred at a specific host chromosomal site. CONCLUSIONS: Broadly, the viral sequences retained upon HPV-18 integration resemble those found when HPV-16 is integrated. However, it appears that the HPV-18 E2 region is more consistently deleted.

The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering.

The dominant late elongated hypocotyl (lhy) mutation of Arabidopsis disrupted circadian clock regulation of gene expression and leaf movements and caused flowering to occur independently of photoperiod. LHY was shown to encode a MYB DNA-binding protein. In wild-type plants, the LHY mRNA showed a circadian pattern of expression with a peak around dawn but in the mutant was expressed constantly at high levels. Increased LHY expression from a transgene caused the endogenous gene to be expressed at a constant level, suggesting that LHY was part of a feedback circuit that regulated its own expression. Thus, constant expression of LHY disrupts several distinct circadian rhythms in Arabidopsis, and LHY may be closely associated with the central oscillator of the circadian clock.