Comparative evaluation of INNO-LiPA HBV assay, direct DNA sequencing and subtractive PCR-RFLP for genotyping of clinical HBV isolates

Abstract

Genotypes (A to H) of hepatitis B virus (HBV) influence liver disease progression and response to antiviral therapy in HBV-infected patients. Several methods have been developed for rapid genotyping of HBV strains. However, some of these methods may not be suitable for developing countries. The performance of INNO-LiPA HBV Genotyping assay (LiPA), direct DNA sequencing and subtractive PCR-RFLP of genotype-specific HBV genome regions were evaluated for accurately determining the HBV genotypes by analyzing sera (n = 80) samples from chronic HBV patients. Both, LiPA and DNA sequencing identified 63, 4 and 13 HBV strains as belonging to genotype D, genotype A and mixed genotype A and D, respectively. On the contrary, the PCR-RFLP-based method correctly identified all 4 genotype A but only 56 of 63 genotype D strains. Seven genotype D strains yielded indeterminate results. DNA sequence comparisons showed that a single nucleotide change in the target region generated an additional restriction site for Nla IV that compromised the accuracy of this method. Furthermore, all the mixed genotype A and D strains were identified only as genotype A strains. The data show that the PCR-RFLP-based method incorrectly identified some genotype D strains and failed to identify mixed genotype infections while LiPA and DNA sequencing yielded accurate results.

Figure 1

Alignment of nucleotide sequences of the HBPol gene product of HBV genotype D. A-to-T nucleotide substitution occurred at nt 204 in all HBV Kuwaiti isolates (KWT1-16). These substitutions represent unique signature sites for local HBV genotype D isolates (shaded). Representative sequences of genotype D are shown (X59795-ITALY, AJ344116-FRANCE, AY161157-INDIA, AB033559-PAPUA, AB078032-JAPAN, AY090453-SWEDEN, AY741796-IRAN, AB126581-RUSSIA, AB104712-EGYPT and AY721605-TURKEY).

Figure 2

Alignment of nucleotide sequences of the S-gene product. Representative sequence of genotypes A (X75669) and D (X65259) are shown at the bottom of the raw. Inherent recognition sites of Nla IV in genotype D are shaded. T-to-C nucleotide substitution occurred at nt 291 (box) in KWT-43 (local HBV isolate). These substitutions resulted in generation of a new recognition site of Nla IV (motif: GGNNCC).