Comparison and Application of a Novel Genotyping Method, Semiautomated Primer-Specific and Mispair Extension Analysis, and Four Other Genotyping Assays for Detection of Hepatitis C Virus Mixed-Genotype Infections

Abstract

To date the true prevalence of hepatitis C virus (HCV) mixed-genotype infections has not been established mainly because currently available methods are not suitable for the detection of mixed genotypes in a viral population. A novel semiautomated genotyping method, primer-specific and mispair extension analysis (S-PSMEA), which is more reliable than other genotyping assays was developed for detection of HCV mixed-genotype infections. A genotype present at levels as low as 0.8% in a defined mix of HCV genotypes was detected, showing a 20-fold increase in sensitivity over that of direct DNA sequencing. A total of 434 HCV isolates were genotyped and analyzed for a comparative study of the accuracy between S-PSMEA and four current genotyping methods. The results showed that viruses in approximately 40% of the samples from this group determined to be infected with mixed genotypes by S-PSMEA were undetected by direct DNA sequencing due to its low sensitivity. Type-specific PCR, line probe assay, and restriction fragment length polymorphism analysis performed poorly, being able to identify only 38.5, 16.1, and 15.4% of mixed-genotype infections, respectively, that were detected by direct DNA sequencing. The prevalence of mixed-genotype infections detected by S-PSMEA was 7.9% (12 of 152 donors) among HCV-infected blood donors, 14.3% (15 of 105) among patients with chronic hepatitis C, and 17.1% (6 of 36) among thalassemia patients who had received multiple transfusions. The data lead us to conclude that HCV mixed-genotype infections are more common than previously estimated and that S-PSMEA may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections is required due to its higher level of sensitivity.

Detection of multiple hepatitis C virus genotypes in a cohort of injecting drug users

“…Amplification of virus sequences by type-specific primers to the NS5a region can identify minor species and HMA is able to detect mixtures not apparent from the LiPA. Our conclusion, mirroring those reached by other authors with respect to non-IDU populations [14,15], is that the most common method of genotyping, LiPA, underestimates the true prevalence of mixed infections in IDUs. Mixed infections were detected by HMA in three IDUs selected solely on the basis of their high-risk behaviour, which was similar to that described by more than a dozen other participants.…”

Evaluation of three different hepatitis C virus typing methods for detection of mixed‐genotype infections

“…genotypes in which confounding host factors could be completely eliminated; 12 and (iii) use as a marker for monitoring the presence of risk factors in a population. 13 Though most current genotyping methods are reliable 7 for the general genotyping of HCV, none of these methods is suitable for detecting mixed genotypes. 6 Population-based DNA sequencing is the only reliable method for detecting mixed-genotype infections, however, it is not practical for large cohort studies since it is expensive, time-consuming and requires expertise.…”

The nationwide distribution and trends of hepatitis C virus genotypes in mainland China

“…This review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement issued in 2009. 22…”