Evaluation of the INNO-LiPA HBV Genotyping Assay for Determination of Hepatitis B Virus Genotype

Abstract

Specific genotypes of hepatitis B virus (HBV) are increasingly recognized for their clinical significance and association with particular viral mutations. Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. A newly available line probe assay (INNO-LiPA HBV Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens. All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%). LiPA was able to detect 19 mixed genotype infections not detected by amplicon sequencing, which for the most part were confirmed by cloning and sequencing of the pre-S1/pre-S2 amplicon. Four specimens had discrepant results between the two methods, and five specimens had indeterminate results by LiPA. The HBV DNA in four specimens was unable to be amplified by the nested INNO-LiPA HBV DR amplification primers; however, the HBV DNA in six specimens unable to be genotyped by sequencing was clearly genotyped by LiPA. The INNO-LiPA HBV Genotyping assay appears to be useful for the rapid genotyping of HBV, particularly for the sensitive detection of mixed genotype infections.It is estimated that more than 350 million individuals worldwide are chronically infected with hepatitis B virus (HBV), of which approximately 20 to 30% risk death from HBV-related liver failure or hepatocellular carcinoma (7, 18). In the past several years, the clinical significance of different HBV genotypes has become increasingly recognized in patients with both acute and chronic HBV infections. For example, genotype C has been more closely associated with a poor prognosis and a more aggressive clinical phenotype (8, 26), while genotype B has been associated with earlier HBeAg seroconversion (2), possibly leading to a lower prevalence of HBV-related cirrhosis. Core promoter and lamivudine resistance mutations were found to be more common in genotypes C and A, while precore stop mutations have been observed more frequently in genotypes B and D (6, 9, 30, 34, 35). Therefore, knowledge of the genotype infecting a patient may assist a physician in making clinical and therapeutic decisions.Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. There are seven genotypes of HBV, based on nucleotide differences of 8% or greater along the entire length of the HBV genome or 4% or greater within the small S gene (HBsAg) (23). Recently, an assay based on the line probe assay (LiPA; INNO-LiPA HBV Genotyping assay, Innogenetics N.V., Ghent, Belgium) was released for research purposes. This method is based on the reverse hybridization principle, such that biotinylated amplicons hybridize to specific oligonucleotide probes that are immobi...

Hepatitis B virus genotype G monoinfection and its transmission by blood components

“…Independent investigations demonstrated a high correlation between the LiPA assay and other genotyping methods combined with the potential to detect very sensitive mixed-genotype infections. 17 Data from the genotypespecific PCR approaches and from the restriction pattern analysis support the absence of further HBV strains in the donor and recipient samples. In all genotype G cases reported so far, the patients were chronically HBV infected.…”

“…Moreover, LiPA has been shown to identify mixed-genotype infections, which may remain undetected by amplicon sequencing. 17,18 As shown in Fig. 2, the samples of the donor and the two recipients (R3 and R4) were positive for genotype G-specific line 16 and for genotype A-specific line 4, which was expected to cross-hybridize with the genotype G sequences.…”

“…The INNO-LiPA HBV Genotyping assay (Innogenetics N.V., Ghent, Belgium) is a line probe assay designed to differentiate among HBV genotypes A-G by hybridizing type-specific sequences in P gene domains B-C. 17 The new genotype H can also be identified. 18 The assay was performed according to the manufacturer's instructions.…”

Hepatitis B virus taxonomy and hepatitis B virus genotypes

“…Using different methods for genotyping, several reports described high rates of double infection with two different HBV genotypes in all parts of the world. Using these methods double infections have been found in 4.4% [56] , 10.9% [57] , 12.5% [58] , 14.1% (Kirschberg et al, unpublished results), 17.3% [59] and 17.5% [60] of HBV infected patients. Even triple infections with HBV of genotype A, B and C have been described in 0.9% of HBV infected intravenous drug users [60] .…”

Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China

“…Since Okamoto et al [18] established the first method to classify HBV, several methods for HBV genotyping have been reported except sequence analysis, such as multiplex PCR [19] , restriction fragment length polymorphism (RFLP) [20] , serological assay [21] and reverse hybridization [22] . These other genotyping methods have a number of advantages over sequence analysis as they are inexpensive and easy to perform.…”