Clinical evaluation of IntelliPlexTM HCV genotyping kit for hepatitis C virus genotyping.

Genotyping of the hepatitis C virus (HCV) is crucial for determining the most efficient anti-viral therapy. The clinical sensitivity and specificity of the IntelliPlexTM HCV Genotyping Kit was determined by comparing the assay results of 307 specimens with the results obtained by Sanger sequencing. Out of 202 HCV-positive specimens tested, 8 samples yielded discrepant results between the IntelliPlex HCV Genotyping Kit and Sanger sequencing. For 5 of these discrepant samples, the IntelliPlex HCV Genotyping Kit classified the correct genotype but failed to show the same single or dual infected status as determined by Sanger sequencing. A total of 105 samples which tested negative for HCV by In-Vitro-Diagnostics (IVD)-approved viral load assay tested negative for HCV by the IntelliPlex HCV Genotyping Kit. The IntelliPlex HCV Genotyping Kit has a clinical specificity of 100% and a clinical sensitivity of 96.9% and is suited to be used in clinical laboratories to genotype HCV.

Genotype distribution in relation to viral load in a large cohort of Indian patients with chronic hepatitis C virus infection: A retrospective analysis.

INTRODUCTION: Hepatitis C virus (HCV) displays high genetic diversity, characterized by regional variations in the prevalence of genotype posing challenges to the development of vaccines and definitive treatment. Very few reports exist on the distribution and frequency change of HCV genotypes in India. In the present retrospective study, we aimed to understand the distribution pattern of HCV genotypes and viral load among HCV-infected patients attending the Asian Institute of Gastroenterology, Hyderabad, India, a tertiary care hospital. METHODS: Patients referred to the Hepatology Department from January 2009 to December 2015 were screened for this study. Eight hundred and sixty-two chronic HCV patients were included in this study. Genotyping was performed using type-specific probe-based hybridization assay and viral load was estimated by real-time polymerase chain reaction. RESULTS: Out of 862 patients, genotype 1 was detected predominantly in 392 (45.5%), followed by genotype 3 in 344 (39.9%) patients; genotypes 4, 6, and 2 were detected in 115 (13.3%), 8 (0.9%), and 3 (0.3%) patients, respectively. The number of patients having genotype 1 increased in frequency while genotype 3 became less from the year 2009 to 2015. Patients having genotype 1 had significantly (p < 0.0001) higher viral load compared with the patients infected with other genotypes. CONCLUSION: Our study results demonstrate a change in HCV genotypic distribution pattern from genotypes 3 to 1 during the span of 7 years in patients referred to our hospital. In the light of the reported difference in the pathogenic potential of various HCV genotypes, detection of HCV genotype appears to be still essential for better patient management.

Performance of 6 HCV genotyping 9G test for HCV genotyping in clinical samples.

BACKGROUND: A treatment of HCV infection depends on the genotype and sub-genotype. Therefore, accurate HCV genotyping is critical for selecting the appropriate treatment regimen. METHOD: This study included 280 plasma samples to evaluate the performance of 6 HCV Genotyping 9G test. The performance of 6 HCV Genotyping 9G test for accurate detection of HCV 1a, 1b, 2, 3, 4, and 6 genotypes was evaluated by comparing it with LiPA 2.0 assay and sequencing. RESULTS: 6 HCV Genotyping 9G test and LiPA 2.0 assay demonstrated 83.9% (n = 235) agreement. 39/45 samples that showed discrepant results between the two tests were analyzed by sequencing. Sequencing genotyped 39 discrepant samples as 0 (HCV 1a), 24 (HCV 1b), 1 (HCV 6f), 12 (HCV 6i), and 2 (HCV-negative). Results of 6 HCV Genotyping 9G test were very similar to the sequencing as it detected 1, 23, 1, 12, and 2 samples as HCV 1a, 1b, 3 & 6a or 6f, 6i or 6n, and negative, respectively. However, LiPA 2.0 assay showed complete disagreement with sequencing, as it did not detect any of these 39 samples correctly. These results indicate that LiPA 2.0 assay has limitations in identifying HCV genotypes 1b, and 6. The sensitivity, specificity, PPV, and NPV of 6 HCV Genotyping 9G test were 99.5, 98.8, 99.5, and 98.8%, respectively. It is important to note that HCV Genotyping 9G test showed 98.3 and 100% sensitivity for HCV 1b and 6 genotyping, respectively. However, LiPA 2.0 assay demonstrated 57.9 and 71.7% sensitivity for these genotypes. CONCLUSIONS: 6 HCV Genotyping 9G test identifies HCV 1a, 1b, 2, 3, and 6 with good agreement with sequencing. Hence, 6 HCV Genotyping 9G test has a high clinical value because it can provide critical information to physicians and assist them to use the correct drug for efficient hepatitis C treatment.

Genotyping of hepatitis C virus by nucleotide sequencing: A robust method for a diagnostic laboratory.

Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases, and it has infected over 170 million people worldwide. HCV is a diverse group of RNA viruses currently divided into genotypes 1-7 as well as subtypes. HCV infection can be treated with antiviral drugs, but the HCV genotype has to be determined for optimal selection of treatment strategy. The aim of this study was to set up a sequencing-based HCV genotyping method suitable for the workflow of a diagnostic laboratory. The established method is robust and stable, and it utilizes a one-step reverse transcription and PCR amplification of the 5' untranslated region (5'UTR) and partial Core region of the HCV genome. Amplification products are sequenced using the standard Sanger method, and the genotype is determined by using a freely accessible web-based genotyping tool. The method was validated at the Helsinki University Hospital Laboratory using 238 previously genotyped serum samples. •A new one-step RT-PCR method for the amplification of the 5' untranslated region and partial Core region of hepatitis C virus was established.•HCV genotype is determined using Sanger sequencing and a freely accessible, easy-to-use web-based genotyping tool.•The method is robust, reproducible and suitable for diagnostic laboratory workflow, and it requires no costly instrumentation or specialized sequence analysis skills.

Evaluation of dried blood spot as an alternative sample collection method for hepatitis C virus RNA quantitation and genotyping using a commercial system.

Dried blood spot (DBS) is a minimally invasive sampling method suitable for sample collection, storage and transportation in resource limited areas. Aim of this study was to compare the diagnostic utility of DBS with plasma sample for HCV RNA quantitation and genotyping using commercial systems. Plasma and DBS card spotted samples were collected from 95 HCV seropositive patients. Both types of samples were subjected to HCV RNA by real-time PCR (Abbott m2000rt, USA). Genotyping was performed using Abbott HCV genotype II kit (Abbott diagnostics, USA) in samples with viral load > 3 log10 IU/ml. In both plasma and DBS, 14 (14.7%) samples were negative and 81 (85.3%) were positive for HCV RNA. Median viral load in plasma (3.78; range 0-7.43) log10 IU/ml was comparable to DBS (3.93; range 0-7.24) log10 IU/ml. DBS demonstrated sensitivity and specificity of 97.5 and 85.7% respectively, with positive predictive value (PPV) of 97.5% and negative predictive value (NPV) of 85.7%. DBS showed good correlation (r2 = 0.866) and agreement (93.5%) with plasma. Genotyping in 20 patients showed 100% concordance between DBS and plasma samples. DBS showed good sensitivity and specificity as a sampling method for HCV RNA quantitation and genotyping.

Next-Generation Sequencing of Hepatitis C Virus (HCV) Mixed-Genotype Infections in Anti-HCV-Negative Blood Donors.

The infection with more than one hepatitis C virus (HCV) genotype especially in subjects with a high risk of multiple HCV exposures has been demonstrated. The role of HCV mixed-genotype infection in viral persistence and treatment effect is not fully understood. The prevalence of such infection varies greatly depending on the technique used for genotype determination and studied population. Next-generation sequencing (NGS) which is suitable for extensive analysis of complex viral populations is a method of choice for studying mixed infections. The aim of the present study was to determine the prevalence of mixed-genotype HCV infections in the Polish seronegative, HCV-RNA-positive blood donors (n = 76). Two-step PCR was used for amplification of 5'-UTR of HCV. Using pyrosequencing altogether, 381,063 reads were obtained. The raw reads were trimmed and subjected to similarity analysis against the entire unfiltered NCBI nt database. Results obtained from NGS were compared with the standard genotyping. One (1.3%) mixed-genotype [3a, 2989 reads (94.8%); 1b, 164 reads (5.2%)] infection was found in a sample diagnosed as genotype 3a only by routine testing. Two samples were identified with different genotypes, compared to routine testing. In conclusion, NGS is a sensitive method for HCV genotyping. The prevalence of mixed-genotype HCV infections in blood donors is low.

Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype-specific PCR and deep sequencing.

The incidence of mixed genotype hepatitis C virus (HCV) infections in the UK is largely unknown. As the efficacy of direct-acting antivirals is variable across different genotypes, treatment regimens are tailored to the infecting genotype, which may pose issues for the treatment of underlying genotypes within undiagnosed mixed genotype HCV infections. There is therefore a need to accurately diagnose mixed genotype infections prior to treatment. PCR-based diagnostic tools were developed to screen for the occurrence of mixed genotype infections caused by the most common UK genotypes, 1a and 3, in a cohort of 506 individuals diagnosed with either of these genotypes. The overall prevalence rate of mixed infection was 3.8%; however, this rate was unevenly distributed, with 6.7% of individuals diagnosed with genotype 3 harbouring genotype 1a strains and only 0.8% of samples from genotype 1a patients harbouring genotype 3 (P < .05). Mixed infection samples consisted of a major and a minor genotype, with the latter constituting less than 21% of the total viral load and, in 67% of cases, less than 1% of the viral load. Analysis of a subset of the cohort by Illumina PCR next-generation sequencing resulted in a much greater incidence rate than obtained by PCR. This may have occurred due to the nonquantitative nature of the technique and despite the designation of false-positive thresholds based on negative controls.

A novel next generation sequencing assay as an alternative to currently available methods for hepatitis C virus genotyping.

Chronic HCV infection is one of the leading causes of liver-related death and in many countries it is a primary reason for having a liver transplant. HCV genotype identification has long been used in the clinical practice, since different genotypes have different response rates and required different doses and durations of IFN/RBV treatment; moreover both the frequency and the pattern of resistance to different Direct-Acting Antivirals (DAAs) classes are subtype specific. Hence the necessity to make an accurate HCV subtyping becomes a fundamental tool to optimize current and future clinical management of HCV infected subjects. In the present study the performance of a next generation sequencing (NGS: based on the Ion Torrent Platform-Vela Sentosa SQ 301 sequencer) HCV genotyping assay has been evaluated. The current method targets a region of the NS5B gene and it is the unique NGS based market CE-IVD assay. As a comparative method a commercial method based on the detection via reverse hybridization of 5'UTR and core regions (Versant HCV Genotype 2.0 Assay, LiPA, Siemens) was selected. A total 207 plasma samples from HCV infected individuals were used. No selection was made for these samples that were submitted for routine HCV genotyping. The results show Vela NGS assay assigns major number of HCV subtypes with respect LiPA. Concerning genotype 1 and 3, the discrepancy of assigned subtypes for LiPA with respect to Vela NGS assay is not relevant (1.8% and 2%, respectively); in contrast, the difference of assigned subtypes for genotypes 2 and 4 is very high (96.6% and 100%, respectively). The resistance mutations data, except for 1a and 1b subtypes, remain scarce; the future relevant challenge will be to identify subtypes-specific drug resistance mutations, which are essential to create highly personalized therapeutic pathways.

Clinical evaluation of a newly developed automated massively parallel sequencing assay for hepatitis C virus genotyping and detection of resistance-association variants. Comparison with a line probe assay.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. Recently, HCV was classified into 6 major genotypes (GTs) and 67 subtypes (STs). Efficient genotyping has become an essential tool for prognosis and indicating suitable treatment, prior to starting therapy in all HCV-infected individuals. The widely used genotyping assays have limitation with regard to genotype accuracy. This study was a comparative evaluation of exact HCV genotyping in a newly developed automated-massively parallel sequencing (MPS) system, versus the established Line probe assay 2.0 (LiPA), substantiated by Sanger sequencing, using 120 previously identified-HCV RNA positive specimens. LiPA gave identical genotypes in the majority of samples tested with MPS. However, as much as 25% of LiPA did not identify subtypes, whereas MPS did, and 0.83% of results were incompatible. Interestingly, only MPS could identify mixed infections in the remaining cases (1.67%). In addition, MPS could detect Resistance-Associated Variants (RAVs) simultaneously in GT1 in 56.82% of the specimens, which were known to affect drug resistance in the HCV NS3/NS4A and NS5A genomic regions. MPS can thus be deemed beneficial for guiding decisions on HCV therapy and saving costs in the long term when compared to traditional methods.

6 HCV Genotyping 9G test for HCV 1a, 1b, 2, 3, 4 and 6 (6a, 6f, 6i and 6n) with high accuracy.

According to EASL guidelines and WHO recommendations, the accurate detection of HCV genotypes such as HCV 1a, HCV1b, HCV 2, HCV 3, HCV 4, and HCV 6 (6a, 6f, 6i, 6n) is crucial for the efficient treatment of hepatitis C. HCV Genotyping 9G test allows simultaneous genotyping of HCV 1a, 1b, 2, 3, 4, and 6 (6a, 6f, 6i, and 6n) in clinical samples in 30min. The performance of the test was evaluated by comparison with sequence analysis. Serum samples (n=152) from HCV-infected patients (n=110) and healthy individuals (n=42) were processed under blinded codes. The k coefficient (kappa) values indicated high agreement between the HCV Genotyping 9G test and sequencing. The sensitivity and specificity of the test were 99.1% and 99.7%, respectively. The results indicate that HCV Genotyping 9G test is rapid, reliable, sensitive, and accurate for screening and genotyping of HCV in the clinical specimens.

Profile of Roche's cobas® HCV tests.

INTRODUCTION: Molecular assays for detection and accurate quantitation of hepatitis C virus (HCV) RNA have been important for identification and management of the hepatitis C. Furthermore, the HCV genotype should be assessed prior to treatment initiation. Recently, Roche developed the cobas® HCV tests for use on the cobas® 6800/8800 Systems and the cobas® 4800 System and the cobas® HCV genotyping (GT) test for use on the cobas® 4800 System. Areas covered: The analytic and clinical performance of the newly-developed tests is described according to the currently existing literature. Both tests for detection and quantitation of HCV RNA have been shown to be sensitive and linear, and correlate well with established Roche tests used in the routine diagnostic laboratory. The cobas® HCV GT test shows a good performance and is suitable for identification of HCV genotypes 1 to 6 and genotype 1 subtypes a and b in clinical specimens from individuals with chronic HCV infection. Expert commentary: The new tests are effective in screening for hepatitis C infection and in the management of patients with chronic HCV infection ensuring full HCV genotype coverage. They will replace the established Roche tests within the next few years.

6 HCV genotyping 9G test and its comparison with VERSANT HCV genotype 2.0 assay (LiPA) for the hepatitis C virus genotyping.

In this article, we describe the 6 HCV Genotyping 9G test and its evaluation by using clinical samples and plasmid DNA standards. In tests with 981 plasmid DNA standards, the 6 HCV Genotyping 9G test showed higher than 92.5% sensitivity and 99.4% specificity. The 6 HCV Genotyping 9G test was compared with the VERSANT HCV Genotype 2.0 assay (LiPA 2.0) for detection and discrimination of HCV genotypes in clinical samples. The results of both tests were verified by genomic sequencing. The 6 HCV Genotyping 9G test demonstrated a 100% agreement with the sequencing results, which was higher than LiPA 2.0. These results indicate that the 6 HCV Genotyping 9G test can be a reliable, sensitive, and accurate diagnostic tool for the correct identification of HCV genotypes in clinical specimens. 6 HCV Genotyping 9G test can genotype six HCV types in 1 PCR in 30min after PCR amplification. The 6 HCV Genotyping 9G test, thus provide critical information to physicians and assist them to apply accurate drug regimen for the effective hepatitis C treatment.

Ultra-Deep Sequencing Characterization of HCV Samples with Equivocal Typing Results Determined with a Commercial Assay.

Hepatitis C virus (HCV) is classified into seven phylogenetically distinct genotypes, which are further subdivided into related subtypes. Accurate assignment of genotype/subtype is mandatory in the era of directly acting antivirals. Several molecular methods are available for HCV genotyping; however, a relevant number of samples with indeterminate, mixed, or unspecified subtype results, or even with misclassified genotypes, may occur. Using NS5B direct (DS) and ultra-deep pyrosequencing (UDPS), we have tested 43 samples, which resulted in genotype 1 unsubtyped (n = 17), mixed infection (n = 17), or indeterminate (n = 9) with the Abbott RealTime HCV Genotype II assay. Genotype 1 was confirmed in 14/17 samples (82%): eight resulted in subtype 1b, and five resulted in subtype 1a with both DS and UDPS, while one was classified as subtype 1e by DS and mixed infection (1e + 1a) by UDPS. Three of seventeen genotype 1 samples resulted in genotype 3h with both sequencing approaches. Only one mixed infection was confirmed by UDPS (4d + 1a), while in 88% of cases a single component of the mixture was detected (five genotype 1a, four genotype 1b, two genotype 3a, two genotype 4m, and two genotype 4d); 44% of indeterminate samples resulted genotype 2c by both DS and UDPS, 22% resulted genotype 3a; one indeterminate sample by Abbott resulted in genotype 4d, one resulted in genotype 6n, and one was classified as subtype 3a by DS, and resulted mixed infection (3a + 3h) by UDPS. The concordance between DS and UDPS was 94%, 88%, and 89% for genotype 1, co-infection, and indeterminate results, respectively. UDPS should be considered very useful to resolve ambiguous HCV genotyping results.

Potential risk of misclassification HCV 2k/1b strains as HCV 2a/2c using VERSANT HCV Genotype 2.0 assay.

The performance of a hepatitis C virus (HCV) NS5B sequencing method described by Murphy et al. (Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences. J Clin Microbiol 2007;45(4):1102-12.) was compared with the VERSANT HCV Genotype 2.0 assay. The sequencing strategy led to detection of HCV recombinant genotype 2k/1b, previously identified as genotype 2a/2c, which reveals the importance of exact HCV genotyping and subtyping.

Fast, reliable and low cost user-developed protocol for detection, quantification and genotyping of hepatitis C virus.

Early detection and genotyping of HCV infection is important for disease management. It is important to develop fast and cost-effective semi-automated techniques allowing an accurate and reproducible detection, quantification and genotyping of HCV. The proposed protocol includes a real-time RT-PCR assay for HCV detection/quantification and a type-specific one-tube RT-PCR assay for genotyping. Both assays detect genotypes 1-4 as intended. The limit of detection was 112IU/ml for the real-time assay and 600±278IU/ml (mean±SD) for the genotyping assay. Concordance between the real-time assay and AMPLICOR HCV v2.0 test was 100%. The real-time assay has wide linear dynamic range of detection and quantification and excellent reproducibility with 2% and 0.75% coefficients of variations, for inter- and intra-assays, respectively. The observed correlation with AMPLICOR HCV Monitor v2.0 kit was linear with the correlation coefficient of 0.988. The diagnostic specificity and sensitivity of the genotyping assay, tested on 102 samples, was 100% and 95%, respectively. The overall procedure of HCV diagnosis is completed within 6h in a closed system with minor contamination risk. In addition to being fast and cost-effective, this approach is reproducible and avoids post-PCR enzymatic and hybridization steps while detecting and genotyping HCV with high clinical sensitivity.

Nucleotide polymorphisms in the 5'-UTR region of HCV can affect the ability of two widely used assays to assign an HCV genotype.

Determination of hepatitis C virus genotype is crucial for establishing the duration of antiviral therapy and predicts response to treatment. In this study, consecutive serum samples collected from two patients with chronic hepatitis C infection were tested by two assays used widely, the Abbott RealTime HCV Genotype II and the Versant HCV Genotype 2.0 assays, in order to assign a genotype to the virus. The obtained results were verified by phylogenetic analysis of the NS5B region and sequencing of the 5'-UTR of the viral genome. Testing of the serum samples from both patients gave an indeterminate result with the Abbott assay. By contrast, the Versant assay gave an indeterminate result for one patient and identified an HCV-2b subtype in the other patient. Phylogenetic analysis of the NS5B region confirmed the presence of HCV-2b in this latter patient and disclosed the presence of HCV-3h in the other patient. Sequencing of the 5'-UTR revealed the presence of nucleotide changes at positions -166 and -119 of HCV-2b, and at positions -138, -108 and -99 of HCV-3h. Nucleotide mutations located in the 5'-untraslated region of hepatitis C virus may impair the ability of commercial assays to assign an HCV genotype.

Study of the hepatitis C virus in the republic of macedonia.

Hepatitis C virus (HCV) is a major public health problem. It is a leading cause of chronic liver disease and the most common indication for liver transplantation. The therapy for eradication of HCV infection is successful in only 50.0-80.0% of patients and is highly dependent on the HCV genotype. Molecular detection and characterization of HCV in the Republic of Macedonia started in 1990. Since then, more than 4000 samples have been analyzed at the Research Centre for Genetic Engineering and Biotechnology (RCGEB) "Georgi D. Efremov," Skopje, Republic of Macedonia. The prevalence of HCV infections in the healthy population of the Republic of Macedonia was found to be 0.4%, while it varies between 23.0 and 43.0% in different at-risk groups of patients. The prevalence of HCV genotypes, according to associated risk factors in HCV infected patients from the Republic of Macedonia, was analyzed. We found genotype 1 to be predominant in a group of hemodialysis patients, while genotype 3 was predominant in intravenous (IV) drug users. Association of six polymorphisms in the Oligoadenylate synthetase (OASL)-like interferon-stimulated gene with a sustained virological response was also analyzed. Our preliminary results suggest that non ancestral alleles in four of the six studies polymorphisms in OASL gene are associated with sustained virological response among HCV infected patients in R. Macedonia.