Single-step real-time PCR to quantify hepatitis B virus and distinguish genotype D from non-D genotypes.

Hepatitis B virus (HBV) viral load and its genotype play important roles in clinical outcome, management of disease and response to antiviral therapy. In many parts of the world such as Europe or the Middle East, distinguishing HBV genotype D from non-D is most relevant for treatment decisions, because genotype D-infected patients respond poorly to interferon-based therapeutic regimens. Here, we developed an in-house real-time PCR to concordantly assess HBV genotype (D vs non-D) based on melt curve analysis and quantify the viral load. Genotype distinction was established with control plasmids of all HBV genotypes and validated with 57 clinical samples from patients infected with six different HBV genotypes. Our in-house real-time PCR assay could discriminate HBV genotype D from non-D using single-step melt curve analysis with a 2 °C difference in the melt curve temperature in all samples tested. Viral load quantification was calibrated with the WHO HBV international standard, demonstrating an excellent correlation with a commercial kit (r = 0.852; P < 0.0001) in a linear range from 3.2 × 10(2) to 3.2 × 10(10) IU/mL. In conclusion, we developed a rapid, simple and cost-effective method to simultaneously quantify and distinguish HBV genotypes D from non-D with a single-step PCR run and melt curve analysis. This assay should be a useful diagnostic alternative to aid clinical decisions about initiation and choice of antiviral therapy, especially in geographical regions with a high prevalence of HBV genotype D.

Hantavirus nephropathy as a pseudo-import pathology from Ecuador.

We report a case of hantavirus infection (nephropathia epidemica) diagnosed in a Belgian backpacker returning from a trekking expedition in Ecuador, after likely heavy exposure to rodents. Because of epidemiological inconsistency, molecular investigation was performed and revealed a Puumala infection acquired during very limited exposure in Belgium upon return.

JC viral loads in patients with Crohn's disease treated with immunosuppression: can we screen for elevated risk of progressive multifocal leukoencephalopathy?

BACKGROUND AND AIMS: Anti-alpha4 integrin therapy with natalizumab is efficacious in refractory Crohn's disease and in multiple sclerosis, but carries an estimated 1/1000 risk of progressive multifocal leukoencephalopathy (PML) caused by reactivation of latent JC virus infection. Although anti-alpha4 integrin therapies are likely to be introduced in the clinic, screening for the risk of PML has not been developed. METHODS: We prospectively collected urine, serum, plasma and buffy coats from 125 patients with Crohn's disease, 100 control subjects with gastrointestinal (GI) disease, and 106 healthy volunteers. Four to eight weeks after this first sample collection, we additionally collected a set of urine, serum, plasma and buffy coat samples from the 125 patients with Crohn's disease, and a next set of samples was collected 12-16 weeks after the first collection. JC viral loads were determined with quantitative real-time polymerase chain reaction (PCR), and JC virus seroprevalence with a specific enzyme-linked immunosorbant assay (ELISA). RESULTS: The overall JC virus seroprevalence was 65%. JC virus DNA copies were detected in the urine from 29-44% of subjects, both those with Crohn's disease and controls. Median viral loads were significantly higher in patients with Crohn's disease who were immunosuppressed (7.36x10(6) copies/ml) compared to healthy volunteers (2.77x10(5) copies/ml) and compared to GI controls (1.8x10(6) copies/ml). Clearance at any time point occurred in 4/107 (3.7%) subjects only. JC viraemia was found in two patients with Crohn's disease. CONCLUSIONS: The natural history of JC virus in patients with Crohn's disease is still unknown. Our study results show that JC virus latency and urine viral shedding is frequent in immunosuppressed patients with Crohn's disease. More prospective studies are needed in order to agree on possible recommendations concerning the exclusion of patients with JCV viraemia from anti-alpha4 integrin treatment.

Hepatitis delta virus facilitates the selection of hepatitis B virus mutants in vivo and functionally impacts on their replicative capacity in vitro.

To identify molecular interactions between hepatitis B virus (HBV) and hepatitis delta virus (HDV), HBV sequences were analysed in HBV/HDV-infected patients. Characteristic amino acid substitutions were found in cytosolic domains of hepatitis B surface antigen (HBsAg), in contrast to HBV-mono-infected controls. The functional impact of HDV on the replication of wild-type and mutant HBV was assessed in vitro. HDV co-transfection significantly reduced the replication of HBV strains containing precore or basal core promoter mutations, and HBV polymerase or surface antigen mutants affected HDV replication in vitro. Conclusively, our study revealed distinct HBsAg mutational patterns in HBV/HDV-infected patients and novel functional interactions between HBV and HDV.

HBV subgenotype misclassification expands quasi-subgenotype A3.

Recently, we proposed a new classification for 'subgenotype A' of hepatitis B virus (HBV), in which the novel 'quasi-subgenotype A3' group comprising HBV 'subgenotype A3', 'tentative A4', and A5 was introduced. Newly 'Tentative subgenotype A7' strains from Cameroon were introduced by Hubschen et al. However, our meticulous phylogenetic analysis demonstrated that these isolates should also be classified into 'quasi-subgenotype A3'. Such misclassification can be avoided by following established principles for HBV subgenotyping. Moreover, their close evolutionary relationship with A3 highlights our hypothesis that geographical origin may be an important factor in further classification of HBV subgenotypes.