Hepatitis C virus (HCV) genotyping is a tool used to optimize antiviral treatment regimens. The newly developed Versant HCV genotype assay (LiPA) 2.0 uses sequence information from both the 5' ...untranslated region and the core region, allowing distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1. HCV-positive samples were genotyped manually using the Versant HCV genotype assay (LiPA) 2.0 system according to the manufacturer's instructions. For the comparison study, Versant HCV genotype assay (LiPA) 1.0 was used. In this study, 99.7% of the samples could be amplified, the genotype of 96.0% of samples could be determined, and the agreement with the reference method was 99.4% when a genotype was determined. The reproducibility study showed no significant differences in performance across sites (P = 0.43) or across lots (P = 0.88). In the comparison study, 13 samples that were uninterpretable or incorrectly genotyped with Versant HCV genotype assay (LiPA) 1.0 were correctly genotyped by Versant HCV genotype assay (LiPA) 2.0. Versant HCV genotype assay (LiPA) 2.0 is a sensitive, accurate, and reliable assay for HCV genotyping. The inclusion of the core region probes in Versant HCV genotype assay (LiPA) 2.0 results in a genotyping success rate higher than that of the current Versant HCV genotype assay (LiPA) 1.0.
Abstract Background Hepatitis B virus (HBV) is primarily transmitted from mother to child, by sexual contact, intravenous drug abuse, or unsafe health care-related injection practices. Despite ...increased safety efforts, nosocomial acquired hepatitis B infection remains problematic. Objectives A large HBV outbreak was investigated comprising 36 patients with acute HBV infection in a primary care physician's practice. Study design In a retrospective study (2003–2008), 36 serum samples from patients with acute HBV infection were collected. They had received several injections by the same physician at least 3 months before the onset of clinical symptoms. As a control group, sera were collected from HBV patients from other physicians from the same province. Full-length HBV genomes were amplified and were phylogenetically analysed. Results HBV complete genomes of 32 patients were successfully amplified and sequenced, and clustered together with the reference genotype A, subgenotype A2 strains. We also analysed 26 control HBV genotype A samples. All 32 HBV strains from the patient group clustered in a monophyletic branch with a bootstrap value of 100, whereas the control samples branched separately in another clade. The genetic distance value showed small differences within the patients group, whereas the rate within the control group was seven times higher. These observations confirm that the source of transmission was clearly different in both groups. Conclusion Maximum likelihood analysis and genetic distance calculations based on the full-length genomes of HBV strains isolated from patients and controls provided strong evidence for a common nosocomial source of infection for all 32 patient cases.
Since auditory disability causes serious problems in the development of speech and in the total development of a child, it is crucial to diagnose possible hearing impairment as soon as possible after ...birth. This study evaluates the neonatal hearing screening program in Flanders, Belgium. The auditory ability of 118,438 babies was tested using the automated auditory brainstem response. We selected 194 babies with indicative hearing impairment and 332 matched controls to investigate the association between the presence of human cytomegalovirus (HCMV) in urine samples and sensorineural hearing loss and to analyze the sensibility and specificity of a cell culture assay and a quantitative PCR detection method. Our results indicate that significantly more babies with confirmed hearing impairment were HCMV positive after birth. Further, based on the results of our study, babies with HCMV viral loads above 4.5 log copies/ml urine seem to be 1.4 times more likely to have confirmed hearing impairment. Our follow-up study suggests that the hearing impairment of children infected with HCMV after birth is less likely to improve than that of HCMV-negative infants. Our results confirm that the presence of HCMV before or shortly after birth influences the outcome of hearing impairment.
Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses.
In our study, 102 volunteers experiencing upper airway infection ...were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.
Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs.
Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections.
The prior diagnosis of fatal astrocytoma in a 60-year-old man with Crohn's disease treated with natalizumab, a monoclonal antibody against alpha4 integrins, was reclassified as JC virus-related ...progressive multifocal leukoencephalopathy (PML). Analysis of frozen serum samples showed that JC virus DNA had appeared in the serum three months after the initiation of open-label natalizumab monotherapy and two months before the appearance of symptomatic PML. There was staining of the brain lesion for polyomavirus. This case report, along with two others, suggests that anti-alpha4-integrin therapy can result in JC virus-induced PML.
To validate a commercially available line probe assay for samples containing the infrequently found hepatitis C virus (HCV) genotype 5a, we sequenced a 511-nucleotide fragment of the NS4b region of ...Belgian and South African HCV genotype 5a samples. Phylogenetic analysis of the sequence data was performed. For the 77 HCV genotype 5a samples collected, there was 100% concordance between the genotype assignment by the line probe assay and the genotyping based on nucleotide sequencing, despite sequence heterogeneity in the probe binding sites of some samples.
•Direct antivirals can help HCV patients, but select for drug-resistant variants.•A full-genome sequencing strategy could detect these resistant mutants.•Genotyping is performed by PCR amplification ...and Sanger sequencing or NGS.•Three partial overlapping PCR fragments span almost the whole HCV genome.•Due to the high genetic variability, sensitivity is lower than for HIV genotyping.
A near-full genome genotypic assay for HCV1b was developed, which may prove useful to investigate antiviral drug resistance, given new combination therapies for HCV1 infection. The assay consists of three partially overlapping PCRs followed by Sanger population or Illumina next-generation sequencing. Seventy-seven therapy-naïve samples, spanning the entire diversity range of currently known HCV1b, were used for optimization of PCRs, of which ten were sequenced using Sanger and of these ten, four using Illumina. The median detection limits for the three regions, 5′UTR-NS2, E2-NS5A and NS4B–NS5B, were 570, 5670 and 56,670IU/ml respectively. The number of Illumina reads mapped varied according to the software used, Segminator II being the best performing (81%). Consensus Illumina and Sanger sequencing results accord largely (0.013% major discordances). Differences were due almost exclusively to a larger number of ambiguities (presumably minority variants) scored by Illumina (1.50% minor discordances). The assay is easy to perform in an equipped laboratory; nevertheless, it was difficult to reach high sensitivity and reproducibility, due to the high genetic viral variability. This assay proved to be suitable for detecting drug resistance mutations and can also be used for epidemiological research, even though only a limited set of samples was used for validation.
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