Hepatitis C virus in Vietnam: high prevalence of infection in dialysis and multi-transfused patients involving diverse and novel virus variants

Abstract

Hepatitis C virus (HCV) is a genetically diverse pathogen infecting approximately 2-3% of the world's population. Herein, we describe results of a large, multicentre serological and molecular epidemiological study cataloguing the prevalence and genetic diversity of HCV in five regions of Vietnam; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. Individuals (n=8654) with varying risk factors for infection were analysed for the presence of HCV Ab/Ag and, in a subset of positive specimens, for HCV RNA levels (n=475) and genotype (n=282). In lower risk individuals, including voluntary blood donors, military recruits and pregnant women, the prevalence of infection was 0.5% (n=26/5250). Prevalence rates were significantly higher (p<0.001) in intravenous drug users (IDUs; 55.6%, n=556/1000), dialysis patients (26.6%, n=153/575) commercial sex workers (CSWs; 8.7%, n=87/1000), and recipients of multiple blood transfusions (6.0%, n=32/529). The prevalence of HCV in dialysis patients varied but remained high in all regions (11-43%) and was associated with the receipt of blood transfusions [OR: 2.08 (1.85-2.34), p=0.001], time from first transfusion [OR: 1.07 (1.01-1.13), p=0.023], duration of dialysis [OR: 1.31 (1.19-1.43), p<0.001] and male gender [OR: 1.60 (1.06-2.41), p=0.026]. Phylogenetic analysis revealed high genetic diversity, particularly amongst dialysis and multi-transfused patients, identifying subtypes 1a (33%), 1b (27%), 2a (0.4%), 3a (0.7%), 3b (1.1%), 6a (18.8%), 6e (6.0%), 6h (4.6%), 6l (6.4%) and 2 clusters of novel genotype 6 variants (2.1%). HCV genotype 1 predominated in Vietnam (60%, n=169/282) but the proportion of infections attributable to genotype 1 varied between regions and risk groups and, in the Southern part of Vietnam, genotype 6 viruses dominated in dialysis and multi-transfused patients (73.9%). This study confirms a high prevalence of HCV infection in Vietnamese IDUs and, notably, reveals high levels of HCV infection associated with dialysis and blood transfusion.

Figure 1. Seroepidemiology of HCV in 8 Different Vietnamese Risk Groups, n = 8654.

The total percentage of HCV Ab/Ag positives in each population group is shown at the end of each bar. The number of samples tested from each group is listed below. 59.7% of CSWs testing positive for HCV also reported intravenous drug use.

Figure 2. Seroepidemiology of HCV in Dialysis Patients.

This figure depicts HCV prevalence in relation to duration of dialysis in years. Increased duration of dialysis is strongly associated with an increase in HCV infection (p<0.001).

Figure 3. Phylogenetic Analysis of the HCV NS5B Gene.

Vietnamese HCV gene sequences from the present study (n = 259 NS5B) are presented with reference sequences (n = 74) downloaded from the Los Alamos database. Analysis was based on a 329-bp of the HCV NS5B gene (nucleotides 8282–8610 relative to H77 NC004102). “A” depicts a midpoint rooted radial phylogenetic tree constructed using the neighbour joining distance method under a Kimura-2-paramter model of evolution. Bootstrap values >70% were obtained for all major nodes separating the confirmed genotypes (not shown). The scale bar indicates an evolutionary distance of 0.08 nucleotide substitutions per site. Branches and annotations are colour coded for all HCV subtypes identified in this study, with reference sequences shown in black. Sequences are annotated by the study cohort from which they were obtained, namely: IDU, intravenous drug user; SW, commercial sex worker; DIA, dialysis patient; MT, multi-transfused patient; ES, elective surgery patient; MR, military recruit. Reference sequences are annotated by subtype name. “B” represents all obtained HCV genotypes/subtypes in the varying risk groups. In total, genotypes were identified for 282 specimens - 201 based on both the NS5B and core/E1 regions, 58 from the NS5B region alone and 23 from the core/E1 region only. Genbank accession numbers are JX102664–JX103137.

Figure 4. Molecular Characterisation of HCV Genotypes in Vietnamese Dialysis and Multi-transfused Patients.

Analysis of HCV patient (n = 113) and reference sequences was performed on a 329-bp fragment of the HCV NS5B gene (nucleotides 8282–8610, numbering based on H77). A neighbour joining tree was constructed using the Kimura-2-parameter model of evolution with gamma distribution and a proportion of invariable sites. Bootstrap values over 70% are shown at the respective nodes. The scale bar indicates an evolutionary distance of 0.1 nucleotide substitutions per site. Reference sequences are annotated with their confirmed genotype and subtype, country of isolation where known and Genbank accession number. Sequences from dialysis and multi-transfused patients are represented in colour, differing by location - red for Ha Noi, orange for Hai Phong, purple for Da Nang, blue for Khanh Hoa and green for Can Tho. Open triangles are used to denote sequences from dialysis patients, open circles are used to denote sequences from multi-transfused patients and filled squares are used where a patient fits both of these criteria. Brackets select the subtypes in which sequences were identified in this study. Two unassigned groups of sequences were identified – a group of sequences obtained from four dialysis patients in Can Tho and sequences obtained from two multi transfused patients in Khanh Hoa and these are labelled as unclassified groups 1 and 2. For 6 additional specimens, 6a genotypes (5 Ha Noi and 1 Can Tho) were identified by core/E1 sequencing.

Figure 5. Maximum Likelihood Phylogenetic Analysis of Novel HCV Genotype 6 Sequences in both NS5B and core/E1 Genes.

HCV sequences identified in this study (coloured and marked by brackets) and reference sequences for all currently recognised subtypes of HCV genotype 6 are analysed with A) a 329-bp fragment of the HCV NS5B gene corresponding to nucleotides 8282 to 8610 of the H77 strain and B) a 446-bp fragment of the HCV core and E1 genes corresponding to nucleotides 860 to 1305 of the H77 strain. Rooted trees were constructed using submodels of the general time reversible (GTR) model, a gamma distribution and a proportion of invariant sites. Bootstrap values over 70% are shown on the corresponding branches. Scale bar indicates an evolutionary distance of 0.2 substitutions per site. “Unassigned group 1” sequences were obtained from 4 dialysis patients in Can Tho and the sequences in unassigned group 2 were from multi-transfused patients in Khanh Hoa.