HBV Subgenotypes D1, D2, D-del! Are ‘Old‘ Genotyping Methods Interpreted Correctly?

Implication for health policy/practice/research/medical education: Molecular virology methods are developing rapidly and allow new insights into molecular epidemiology of viral infections. Despite the recent introduction of advanced methodology to genotype hepatitis B virus, using old but rapid and inexpensive genotyping tools like restriction fragment length poly-morphism have been utilized widely for large population studies. Although the usage of this technique might have some advantages, misinterpretation of old methods may result in wrong conclusions and may negatively affect patient's health. Infections with the hepatitis B virus (HBV) are one of the major global public health problems. HBV has been categorized into different genotypes and subgenotypes that are distributed distinctively around the world. These classifications provide important information as the genotypes differ with respect to the clinical course of disease as well as in their response to antiviral therapy, and subgenotyping allows relevant conclusions about transmission routes, global or local spreading of infections or phylogenetic relations between viral strains (1). Several molecular virology methods are utilized to (sub) genotype HBV including direct sequencing (as gold standard), restriction fragment length polymorphism (RFLP), restriction fragment mass polymorphism (RFMP), oli-gonucleotide microarray chip (DNA Chip), INNO-LiPA, and a range of (real-time)-PCRs with distinct advantages and limitations (2). Among these methods for (sub) ge-notyping, RFLP is widely considered as one of the most favorable methods among scientists, since it is technically simple, robust, inexpensive and can be established in virtually any laboratory with basic molecular biology facilities. As a consequence, RFLP has been frequently and successfully employed for many studies worldwide including large cohorts of HBV-infected patients (3-5). Despite its clear benefits, this assay also has a number of pitfalls that may impede to assess the correct HBV genotype (4). For instance, any variation within HBV genome that affects the assay's enzyme restriction site(s) negatively impacts the outcome of the method. HBV genomic variations regularly occur in patients due to natural viral evolution or endogenous and/or exogenous pressures such as immune responses, antiviral therapeutic regimes and/ or vaccination (6). However, the mal-interpretation of results obtained with the RFLP assay is also an important trap that needs to be considered by researchers using this assay. In this editorial, we would like to illustrate our concern by commenting on several studies applying an established RFLP-based HBV genotyping method, in which the results were (to our opinion) interpreted incorrectly (Table 1) (7-12). The latest of these studies (published in 2012) …