Evaluation of in-house and commercial genotyping assays for molecular typing of hepatitis C virus in Hong Kong.

This study aims to evaluate genotyping assays for hepatitis C virus (HCV). An in-house nucleic acid sequencing method is performed in parallel with the Roche Linear Array HCV genotyping test on 73 HCV-positive (66 clinical samples and seven proficiency testing quality control samples) and 12 HCV-negative samples (11 clinical samples and one proficiency testing sample). The performance of the in-house method was comparable with that of the Roche assay (concordance rate: 89.4%). Discordant results included four mixed infections missed by the in-house method, two false-negatives with the Roche assay, and three discrepant results. The in-house method exhibited a higher resolution (subtype vs. genotype level) at a lower running cost (25% of the commercial assay). The in-house method was also used to genotype 375 HCV clinical isolates to determine the genotypic distribution of HCV in Hong Kong between 2005 and 2008. A total of 441 (52.8%) clinical isolates proved to be genotype 1, which shows a poorer response to interferon therapy. Genotype 6 was the next most common (32.0%). Prevalence of genotypes 2 and 3 was 7.7% and 6.6%, respectively, and prevalence of genotypes 4 and 5 was 0.9% and 0%, respectively. Although the in-house nucleic acid sequencing method failed to detect a few cases of mixed HCV infection, its high resolution and low running cost make it suitable for surveillance and outbreak investigation.

Differential fadE28 expression associated with phenotypic virulence of Mycobacterium tuberculosis.

Ability to persist in human macrophages is central to the virulence of Mycobacterium tuberculosis and is not invariable among various strains. Differential gene expression that is associated with phenotypic virulence may provide additional information of virulent genes involved in the pathogenesis of M. tuberculosis, which is not fully elucidated. Three hypervirulent strains of M. tuberculosis isolated from patients suffering with tuberculous meningitis were shown to grow more rapidly inside human macrophages in a previous study. In the current investigation, expression of 7 mycobacterial genes (fadE28, mce1A, mymA, acr, sigA, sugC, and Rv3723) of these strains during ex vivo macrophage challenge and in vitro acid shock was quantified by real-time PCR. Using rrs gene as a normalisation gene, fadE28 gene exhibited differential gene expression that is associated with phenotypic virulence, whereas the other 6 genes showed indistinguishable expression patterns. Up-regulation of fadE28 gene in the hypervirulent strains may account for virulence by increasing the efficiency of beta-oxidation, which is important for the persistence in macrophages as M. tuberculosis uses fatty acids preferably inside phagosome of macrophages. The fadE28 gene, together with its adjacent genes may also be critical in the process of lipid modification that could facilitate parasitism in human macrophages.