Development and validation of a real-time, reverse transcription PCR assay for rapid and low-cost genotyping of hepatitis C virus genotypes 1a, 1b, 2, and 3a.

Hepatitis C virus (HCV) infection affects millions of people and leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Treatment regimen selection requires HCV genotype (Gt) and Gt 1 subtype determination. Use of a laboratory developed, reverse transcription (RT)-PCR assay was explored as a low-cost, high-throughput screening approach for the major HCV genotypes and subtypes in North America. A commercial line probe assay (LiPA) was used for comparison. Sequencing and/or an alternative PCR assay were used for discordant analyses. Testing of 155 clinical samples revealed that a paired, duplex real-time RT-PCR assay that targets Gts 1a and 3a in one reaction and Gts 1b and 2 in another had 95% overall sensitivity and individual Gt sensitivity and specificity of 98-100% and 85-98%, respectively. The RT-PCR assay detected mixed HCV Gts in clinical and spiked samples and no false-positive reactions occurred with rare Gts 3b, 4, 5, or 6. Implementation of the RT-PCR assay, with some reflex LiPA testing, would cost only a small portion of the cost of using LiPA alone, and can also save 1.5h of hands-on time. The use of a laboratory developed RT-PCR assay for HCV genotyping has the potential to reduce cost and labour burdens in high-volume testing settings.

Personalized genetic testing and norovirus susceptibility.

BACKGROUND: The availability of direct-to-consumer personalized genetic testing has enabled the public to access and interpret their own genetic information. Various genetic traits can be determined including resistance to norovirus through a nonsense mutation (G428A) in the FUT2 gene. Although this trait is believed to confer resistance to the most dominant norovirus genotype (GII.4), the spectrum of resistance to other norovirus strains is unknown. The present report describes a cluster of symptomatic norovirus GI.6 infection in a family identified to have norovirus resistance through personalized genetic testing. CASE PRESENTATION: In January 2013, four members of a family determined by a direct-to-consumer genetic test to be homozygous for the norovirus resistance trait (A/A genotype for single nucleotide polymorphism rs601338) developed symptoms consistent with acute viral gastroenteritis. Stool and vomitus samples were submitted for enteric viral pathogen testing. Samples were positive for norovirus GI.6 in three of the four cases. CONCLUSIONS: The present report is the first to describe norovirus GI.6 infection in patients with the G428A nonsense mutation in FUT2; this cluster of cases suggests that the G428A mutation in FUT2 may not confer resistance to norovirus GI.6. Direct-to-consumer genetic testing is empowering members of the public to identify novel associations with their genetic traits. Expert consultation is important for the interpretation of personalized genetic test results, and follow-up laboratory testing can confirm any potentially novel associations.

HISTORIQUE: Les tests génétiques personnalisés destinés directement aux consommateurs permettent au public d'accéder eux-mêmes à l'information génétique et à l'interpréter. Il est ainsi possible de déterminer divers traits génétiques, y compris la résistance au norovirus par une mutation nonsens (G428A) dans le gène FUT2. Même si on pense que ce trait confère une résistance au génotype du norovirus le plus dominant (GII.4), on n'en connaît pas le spectre de résistance à d'autres souches de norovirus. Le présent rapport décrit une grappe d'infection symptomatique au norovirus GI.6 au sein d'une famille dont la résistance au norovirus avait été établie au moyen de tests génétiques personnalisés. PRÉSENTATION DU CAS: En janvier 2013, quatre membres d'une famille qui, d'après un test génétique destiné directement au consommateur, étaient homozygotes au trait de résistance au norovirus (génotype A/A du polymorphisme de nucléotide simple rs601338) ont présenté des symptômes évocateurs d'une gastroentérite virale aiguë. Des coprocultures et des prélèvements de vomissures ont été soumis à un test pour déceler un virus entéropathogène. Dans trois des quatre cas, les prélèvements étaient positifs au norovirus GI.6. CONCLUSIONS: Le présent rapport est le premier à décrire l'infection à norovirus GI.6 chez des patients présentant la mutation nonsens G428A dans le gène FUT2. Ce groupe de cas laisse croire que la mutation G428A dans le gène FUT2 ne confère pas de résistance au norovirus GI.6. Les tests génétiques destinés directement aux consommateurs permettent aux membres du public d'établir de nouvelles associations avec leurs traits génétiques. Il est important de consulter un expert pour en interpréter les résultats, et des tests de laboratoire effectués en suivi peuvent confirmer ces associations potentielles.

Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories.

BACKGROUND: Noroviruses (NoVs) are the leading cause of infectious gastroenteritis worldwide. Real-time reverse transcription PCR (real-time RT-PCR) is the preferred method of NoV detection for the majority of testing laboratories. Although the accepted target region for molecular detection assays is the conserved ORF1/ORF2 junction, multiple variations have been published with differences in primers, probes, reagents, multiplexing, etc. OBJECTIVES: We assessed the detection limit for GII.4 NoV real-time RT-PCR assays as well as the ability to detect the non-GII.4 NoV genotypes in each participating laboratory. STUDY DESIGN: A panel of 25 RNA samples was circulated to 18 testing laboratories for comparison of their real-time RT-PCR procedures for NoV detection. RESULTS: Multiple protocols with slight differences in reagents or conditions successfully detected 10 genome equivalents or fewer of NoV per reaction. Multiplex procedures were significantly associated (p=0.04) with false negative results, particularly for a GI.2 strain. Sensitive detection was associated with false positive results (p=0.03). CONCLUSIONS: Overall, the data indicate that comparable results are produced under slightly different assay conditions.

Multicenter comparison of two norovirus ORF2-based genotyping protocols.

Point source norovirus outbreaks can be difficult to track due to high background levels of the virus in the environment and the limited strain variation in some genotyping regions. However, rapid and accurate source identification can limit the spread of a foodborne outbreak and reduce the number of cases. Harmonization of genotyping assays is critical for enabling the rapid exchange of sequence data nationally and internationally. Several regions of the genome have been proposed for this purpose, but no consensus has been reached. In the present study, two standardized genotyping protocols (region C and region D) were evaluated by nine laboratories in Canada and the United States, using a coded panel of 96 fecal specimens representing 22 different norovirus genotypes. Overall, region C typing had a success rate of 78% compared to 52% for region D; however, region D provides greater nucleotide sequence diversity for identifying new GII.4 variant strains. Significant differences in the genotyping success rate were observed among the nine participating laboratories (10% to 100%) and among the different genotypes (6% to 100%). For several genogroup II strains, reduced region D amplification correlated directly with mismatches between primer sequences and the template. Based on overall performance, we recommend the region C protocol for routine genotyping of noroviruses, while the region D protocol may be useful for identifying new GII.4 variants. Standardized genotyping protocols will enable rapid exchange of outbreak and sequence data through electronic norovirus surveillance networks.