High prevalence of hepatitis B virus dual infection with genotypes A and G in HIV-1 infected men in Amsterdam, the Netherlands, during 2000-2011

A total of 1674 patients tested HBsAg positive in the AMC (Amsterdam, the Netherlands) during 2000-2011, of which 1008 were also tested for HIV-1 infection (HBV tests requested from outside the AMC, but performed here were not included in the numbers). Of those 1008 HBV positive patients, 211 were found to be also HIV-positive. Patients receiving anti-HBV treatment in whom therapy failure was observed and drug resistance was thus suspected (N = 343), HBV genomic fragments were amplified, sequenced, genotyped and analysed for drug-resistance mutations. For this study, we selected AMC patients from the database of the AMC Laboratory of Clinical Virology (Department of Medical Microbiology) that previously were found to be infected with HBV genotype A or G. HBV genotyping was done with an in-house sequencing assay. The Laboratory of Clinical Virology participates in the Quality Control for Molecular Diagnostics Hepatitis B virus Genotype EQA Programme [33] to ensure adequate performance of this genotyping assay. A total of 96 patient entries met these criteria and had sample availability; 91 had been diagnosed as HBV genotype A and 5 as HBV genotype G. Twenty-seven were HIV-positive (all males), 54 were HIV-negative and 15 had an unknown HIV status. In total, sixty-nine patients were male, and 27 were female. Of the 69 included men, 25 originated from the Netherlands; the others were from Suriname (N = 11), Africa (N = 8), other European countries (N = 5), or of unknown or other origin (N = 20). Of the 27 female patients, 6 were from the Netherlands, 15 from Suriname, 4 from Africa, and for 2 the country of origin was unknown.

Viral DNA was isolated from blood plasma samples with the QIAamp UltraSens Virus Kit (QIAGEN, the Netherlands). Single-tube real-time PCR reactions that specifically quantify HBV genotype A or G DNA were developed in the HBV X-protein gene and analysed on an ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the primers and probes shown in Table 1. Primers and probes were highly specific for the targeted HBV genotype (either A or G). Cross-reactivity was not detected as the primers and probes were able to amplify 5 copies of the targeted genotype in a background of up to 10⁸ copies of the non-targeted genotype. All subgenotypes of HBV-A can be quantified with this assay. The lower limit of detection is 5 copies per reaction (= 1000 copies/ml).

HBV genotype G genome fragments were amplified from viral DNA isolated from plasma samples and directly sequenced with the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Electrophoresis and data collection were performed on an ABI PRISM 3100 genetic analyser (also from Applied Biosystems). Sequences were assembled with CodonCode Aligner [34], and aligned with reference HBV sequences from the NCBI nucleotide database [35] using ClustalW implemented in BioEdit Sequence Alignment Editor version 7.0.9 [36]. GenBank accession numbers for the full-length genotype G sequences are: KF767450, KF767451, and KF767452.

No specific approval by the Institutional Review Board (IRB) of the Academic Medical Center (AMC) of the University of Amsterdam was needed for this study in the Netherlands, because the body materials for this study were collected under medical treatment, and can be used for further scientific research when the patients have given consent and samples are analysed anonymously, as was the case here (Code of Conduct as implemented by the Committee on Regulation of Research COREON of the Netherlands Epidemiological Society and the Dutch Federation of Biomedical Scientific Societies that is followed by the IRB of the AMC).

The prevalence of HBV genotypes as determined by the Laboratory of Clinical Virology in HBV-infected patients visiting the AMC is shown in Figure 1. Genotypes D (29%) and A (27%) were the most prevalent, followed by genotypes E (16%), C (15%) and B (12%). Genotype F or G infections are uncommon (≤ 1%).

A total of 96 patients with HBV infection genotype A or G infection for whom samples were available were assessed for the presence of single (A or G) or A/G dual HBV infection with a single-tube real-time PCR assay that specifically quantifies these genotypes. Patient characteristics and results are summarized in Table 2. All female patients (N = 27) were found to be singly infected with HBV-A. Of the 69 male patients, 55 were infected with HBV-A only, but 10 were dually infected with both HBV-A and -G. According to the clinical database all 10 dual infected patients were infected with the HBV subgenotype A2. In four male patients, the HBV pVL was below the detection limit of the assay. None of the patients exhibited a single infection with HBV-G; all 5 patients previously diagnosed as HBV genotype G also carried genotype A. Five patients previously diagnosed as genotype A also carried genotype G. Strikingly, all ten dually HBV-A/G infected patients were also infected with HIV-1. In contrast, none of the HIV-1 negative patients (42 males and 27 females) were dually HBV infected. So, HBV-A/G dual infection is significantly associated with HIV-1 infection (two-tailed Fisher’s exact test, p < 0.0001). In total, 37% of HBV/HIV-1 infected patients harboured two HBV genotypes. All ten dually HBV infected patients were HBeAg positive at the time of testing with the AxSYM® HBe Assay (Abbott Diagnostics, Lake Forest, Ill, USA), suggestive of infection with a precore protein producing strain [19], such as HBV-A [21]. The majority of HIV-1 infected males were of Dutch ethnicity (13/23), with an equal distribution between those singly (7/13) or dually HBV (6/10) infected. For the other HIV-1 infected patients the origin was either unknown (4/23) or varied widely (6/23, from the Dominican Republic, Ecuador, Dutch Antilles, Ethiopia, and Germany). Of the HBV-A/G dually infected patients, five had unknown risk factors for infection, 1 reported heterosexual contacts as a risk factor, and four were labelled as MSM in the database.

Thus, HBV-G infection in Dutch patients is strongly associated with HBV-A and HIV-1 co-infection, which likely explains the male-specificity in this analysis, as the majority of HIV-1 infected patients in the Netherlands are male (80% in 2011), of which 73% belong to the MSM risk group [37]. These results suggest that HBV-G is primarily circulating among MSM in the Netherlands.

To assess the possibility that both HBV-G and HIV-1 circulate in a certain subgroup of patients, as well as investigating a potential simultaneous transmission of both viruses we analysed HIV-1 polymerase sequences that were available for 6 out 10 HIV-1/HBV-A/G infected patients and 10 HIV-1/HBV-A2 infected patients that were not infected with HBV-G. A phylogenetic tree was generated of these 16 sequences plus reference sequences of HIV-1 subtypes A-K with the neighbour-joining option in MEGA5 based upon a Kimura 2-parameter distance matrix [38]. The NJ tree suggested no significant relationship between the HIV-1 polymerase sequences of the 6 HBV-A/G dual infected patients (not shown) suggesting that transmission of HIV-1 and HBV-G are independent events.

To investigate whether HBV-G strains circulating in the Netherlands are similar to global HBV-G strains, the DNA genome of three virus isolates (from patients 7, 23 and 27, all of Dutch ethnicity) were amplified and completely sequenced. The sequences demonstrated the characteristic 36-nucleotide insertion at the 5′ end of the core protein gene and two stopcodons in the precore region that preclude HBeAg expression. Also, a fourth, but incompletely sequenced HBV-G genome from patient 26 (originating from the Dominican Republic, but infected in the Netherlands, where he presented with an acute HBV infection) showed high sequence similarity with the other Dutch strains (not shown). HBV-G strains of patients 7 and 27 were closely related to each other and to an HBV-G strain originating from a Dutch blood donor with a HBV-G mono-infection [14]. The virus isolates of both patients showed resistance mutations against 3TC in the RT region (L180M and M204V).

All three completely sequenced Dutch HBV-G strains clustered with HBV-G reference sequences in a phylogenetic analysis (Figure 2). Genetic distances between the Dutch HBV-G genomes and reference HBV-G sequences were extremely low, and ranged between 0.002-0.008 for the full-length sequences. This is in line with the low genetic diversity of less than 0.5% detected for all HBV-G strains isolated worldwide [39].

For the other 7 patients, a 359 nucleotide fragment spanning the precore/core region of HBV-G was amplified and sequenced. All 7 isolates contained the typical 36-nucleotide insertion as well as the two stopcodons, and clustered with HBV-G sequences.

For three HBV-A/G dually infected patients (nos. 7, 12 and 23), serial blood plasma samples were available for a longitudinal analysis. In all three patients, both genotypes were already present in the first available sample, so that the order of infection could not be established. A longitudinal analysis of the pVL of each HBV genotype was performed with the real-time PCR assay (Figure 3). All three patients were also HIV-1 infected, and treated with antiretroviral therapy, including lamivudine (3TC) and tenofovir (TDF) that target both the HIV-1 and the HBV RT enzymes. In general, the pVL of HBV-A and HBV-G follow a similar course in these patients, increasing or decreasing in parallel.

In patient 7, the HBV-G pVL exceeded the HBV-A pVL considerably (>10⁵ difference) at all time-points over a 4-year period (Figure 3A). The HBV-A and HIV-1 pVL decreased significantly after the start of an ART regimen containing 3TC in 2001, and remained close to or below the detection limit of the assay afterwards for HBV-A (Figure 3A). 3TC drug resistance occurred in the HBV-G strain, and its pVL rebounded after an initial drop. Subsequent initiation of TDF treatment (in addition to 3TC) resulted in a significant decrease in the HBV-G pVL.

In patient 12, the HBV-G pVL exceeded the HBV-A pVL at least tenfold over a 42 month period, except in the first sample measured (Figure 3B). A period without ART resulted in an increase in HBV pVL of both genotypes that decreased again with the restart of ART that included TDF (Figure 3B).

In patient 23, the pVL of the two genotypes was comparable over an 8-year period, despite dramatic fluctuations due to therapy changes (Figure 3C). The HBV-A and -G pVL also decreased sharply with the start of an ART regimen that contained both 3TC and TDF. However, mutations associated with 3TC resistance (L180M and M204V) developed three years later in both genotypes. A regimen containing TDF was subsequently installed, which lowered both HBV pVLs to just above the detection limit of the assay.