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.

FIG. 1

Principle and procedure of S-PSMEA. (A) Amplification of 5′ UR of the HCV genome by PCR. (B) PSMEA reaction with an incomplete set of dNTPs (dCTP and dGTP) and a Cy 5.5 dye-labeled primer. (B-1) Primer extension cannot initiate if a mispair(s) is present at and/or within the 3′ end of the primer. (B-2) Single mispair formation and extension may occur at certain nucleotide positions that are more than one nucleotide away from the initiation site of primer extension. (B-3) Two consecutive mispairs or a single mispair closely followed by more than one mispair can completely terminate primer extension. (C) Genotype-specific profiles of the PSMEA reaction determined by FLPA after electrophoresis of PSMEA reaction products. (Top profile) No extension, showing a single peak for an unextended primer, due to a mispair that exists at the nucleotide position immediately adjacent to the 3′ end of the primer (see B-1). (Bottom two profiles) Primer extended with the addition of a different number of bases, showing a genotype-specific pattern (also see B-2 and B-3). d, c, and a indicate the position of each detected peak.

FIG. 2

Evaluation of the sensitivity of S-PSMEA for detection of low levels of HCV mixed-genotype infections with a defined mixture of genotypes 1b and 2a in different proportions. (A) FLPA graphs showing the genotype-specific profiles and proportion of primer extension in the PSMEA reactions with different proportions of genotypes in the mixtures. The first peak from the left is the unextended primer, and the other two peaks represent extended primer to which different numbers of bases were added. See the report by Hu et al. (11) for details. (B) Quantification analysis of primer extension with different percentages of genotype 2a in the mixture with FLPA software. The proportion of primer extension in agreement with the percentage of the template (genotype 2a) in the mixtures was shown to be 100%. The formula for the calculation was as follows: the percentage of each genotype in the mixture is equal to the [percent primer extension of target genotype/(percent primer extension of target genotype + percent primer extension for each other genotype in the mix)] × 100.

FIG. 3

Detection of mixed-genotype infections (genotypes 1a and 1b) by S-PSMEA. (A) Genotypic profiles of primer extension of genotypes 1a and 1b by PSMEA reactions with universal primer 1BR in presence of different sets of dNTPs. (A-1) No extension with dTTP and dGTP in the presence of genotype 1b alone; (A-2) primer extension profile with dCTP and dGTP in the presence of genotypes 1a and 1b in a mixture; (A-3) no extension with dCTP and dGTP in the presence of genotype 1a alone; (A-4) primer extension profile with dTTP and dGTP in the presence of genotypes 1a and 1b in a mixture. (B) Nucleotide sequences of the templates (genotypes 1a and 1b), primers, and extension products illustrated in panel A. The capital letters CGCGGGGC and GT represent the bases that were extended. The majority of primer extension products ended at a pair with a single mismatch: g in genotype 1b or a in genotype 1a (B-2 and B-4). A small proportion of primers extended with several more bases (gcgccc or ggggg) and ended at a pair with a double mismatch (xx) (B-2 and B-4). The percentage of genotypes 1a and 1b in a mixture is calculated as follows: [percent primer extension of genotype 1a or 1b/percent total primer extension of genotype 1a + genotype 1b (i.e., percent primer extension with G and T + percent primer extension with C and G)] × 100.