A real-time Taqman method for hepatitis C virus genotyping and methods for further subtyping of isolates.

The HCV genome is highly heterogeneous; more and more genotypes, each with several distinct subtypes, are being identified around the world. Knowledge of genotype is important for planning of treatment regimes, whereas subtype identification is useful in epidemiological studies and outbreak investigation. We describe HCV genotyping and subtyping assays, based on real-time PCR, that are sensitive, specific, and reliable. These assays provide fast, accurate, and convenient methods for HCV genotyping/subtyping to support clinical practice.

Establishment of a UK National Influenza H5 Laboratory Network.

Avian (H5N1) influenza continues to pose a significant threat to human health, although it remains a zoonotic infection. Sensitive and robust surveillance measures are required to detect any evidence that the virus has acquired the ability to transmit between humans and emerge as the next pandemic strain. An integral part of the pandemic planning response in the UK was the creation in 2005 of the UK National H5 Laboratory Network, capable of rapidly and accurately identifying potential human H5N1 infections in all regions of the UK, and the Republic of Ireland. This review details the challenges that designing molecular detection methods for a rapidly evolving virus present, and the strategic decisions and choices required to ensure successful establishment of a functional national laboratory network, providing round the clock testing for H5N1. Laboratory partnerships have delivered improved real-time one-step multiplex PCR methodologies to ensure streamlined testing capable of not only detecting H5 but also a differential diagnosis of seasonal influenza A/B. A range of fully validated real-time PCR H5 confirmatory assays have been developed to run in parallel with a universal first-screening assay. Regular proficiency panels together with weekly surveillance runs, intermittent on-call testing for suspect cases of avian flu in returning travellers, and several outbreaks of avian influenza outbreaks in poultry that have occurred since 2005 in the UK have fully tested the network and the current diagnostic strategies for avian influenza. The network has clearly demonstrated its capability of delivering a confirmed H5N1 diagnosis within 3-4 h of receipt of a sample, an essential prerequisite for administration of the appropriate antiviral therapy, effective clinical management, disease containment and implementation of infection control measures. A functional network is an important means of enhancing laboratory capability and building diagnostic capacity for a newly emerging pandemic of influenza, and is an essential part of pandemic preparedness.

A real-time Taqman method for hepatitis C virus genotyping.

BACKGROUND: There is the need for a rapid, inexpensive method for genotyping hepatitis C virus (HCV) to support clinical practice. OBJECTIVES: To develop a real-time (Rotor-Gene 3000) Taqman assay for HCV genotyping in a single tube. STUDY DESIGN: Seven type-specific probes, two for genotypes 1-3 and one for genotype 4 were designed around genotype-specific motifs in the 5' non-coding (NC) region to create two panels of probes. The first panel included two probes for genotype 1 detection and a single probe each for genotypes 2 and 3. The second panel had two probes for confirmation of genotypes 2 and 3 and a first line probe for genotype 4 detection. A comparative analysis of the Taqman assay against our in-house sequence-based method using 154 consecutive clinical samples, from HCV carriers in Cambridge, and four samples from the Quality Control for Molecular Diagnostics (QCMD) System was undertaken. RESULTS: 158 samples were analysed by conventional sequencing: 49% (n=78) were genotype 1, 11% (n=18) genotype 2, 30% (n=47) genotype 3 and 6% (n=10) genotype 4. For two samples, the sequence data was heterogeneous and difficult to analyse, suggesting mixed infection and for three samples, the viral load was insufficient for sequencing. Concordant results were obtained with the novel Taqman assay for 77/78 (99%) of genotype 1 isolates (positive with both genotype 1 probes), 17/18 (94%) of genotype 2 isolates, 43/47 (91%) of genotype 3 isolates and 10/10 (100%) genotype 4 isolates. One isolate, untypeable with sequencing was genotyped with the Taqman assay. CONCLUSIONS: The Taqman assay was sensitive, specific and reliable over a wide range of viral loads and could identify mixed infections. These results highlight the potential of the Taqman assay as a fast, accurate and convenient method for routine HCV genotyping.