Background
Viral hepatitis infection including hepatitis B virus (HBV) is still challenging as the public health concern, having global prevalence of 3.5% and 1.34 million deaths in 2015 [
1]. The prevalence of HBV infection might differ in each World Health Organization (WHO) region [
2] and the high prevalence of 6.1 and 6.2% were found in Africa and Western Pacific region respectively [
1].
Although the gross decrement of HBV prevalence was reported in the developed countries after discovery of effective hepatitis B vaccine (HepB) since 1981 [
3], the prevalence is still high in developing countries. Mother-to-child transmission (MTCT), also known as vertical transmission, still ranks as the main route of HBV transmission in intermediate and high endemic countries. Cambodia, one of the developing countries in WHO Western Pacific Region, has been reported high hepatitis B surface antigen (HBsAg) prevalence ranging from 7.7 to 13% [
4,
5]. In 2005, Cambodia started phasing-in HepB vaccine to National Immunization programme (NIP) and the coverage was achieved over 90% since 2008 [
6]. After introduction of HepB vaccine in the whole Cambodia, HBsAg prevalence among ≤5 years old children markedly reduced to 3.5% in 2006 [
7] and then dropped to 0.33–3.45% in three provinces of Cambodia in 2011 [
5]. Recent nationwide study on HBsAg prevalence among mother-child pairs in 2017 revealed the positive rate of 0.56% among children and 4.39% among their mothers [
8]. Very low HBsAg positive rate in children with its reciprocal high positive rate in mothers indicates the needs for further study on HBV in Cambodia. Moreover, the clinical outcomes of chronic HBV infection rely on HBV genotypes and sub-genotype as viral factor. Understanding HBV genotypes and sub-genotypes can predict not only liver disease progression but also the response to antiviral treatment [
9]. Although there were only a few reports about HBV genotype distribution in Cambodia [
4,
10], the nationwide distribution pattern of HBV genotypes was still unknown.
Additionally, the widespread use of HepB vaccine in combating HBV infection potentially threatens the emergence of mutant strains at hepatitis B surface gene. The mutation in S gene causes the amino acid substitution either single or multiple mutations in HBsAg especially
a determinant region between amino acid 120 and 147 and mutation in this region reduces the sensitivity to diagnostic test, failure of response to both HepB vaccine and HBIG [
11]. It is later denoted as vaccine escapes mutation and is abundantly occurred in those children who had received plasma-derived vaccines (0.3%) rather than recombinant vaccines (0.06%) [
12]. The emergence of vaccine escapes mutants threatens the efficacy of HepB vaccine among infants and now raising as the public health concern in elimination pathway of HBV. Although Cambodia has a long track of using HepB vaccine over a decade, there is no study on S gene mutation of HBV meanwhile.
Therefore, this study aimed to detect HBV genome sequences and their potential mutant strains specifically mutation at S gene of HBV as of nationwide scale using dried blood spot (DBS) samples and then to provide the up-to-date reference data for consideration of prevention, control and surveillance of HBV infection in Cambodia.
Discussion
This study is the first report to present HBV DNA positive rate, its amplification rate, genotype distribution and existence of potential HBV variants among the strains isolated from mother-child pairs in Cambodia as of its nationwide scale.
The overall HBV DNA positive rate in children was 0.48% which definitely reflects the well-established vaccination program in Cambodia. But, MTCT rate was 9.47% (9/95) which is higher than the previously reported rate among vaccinated Asian (2–3%) [
26]. The homology between HBV strains isolated from these mother-child pairs was 99.62–100% which strongly indicated that the transmission was vertical.
The genome sequences revealed the genotype distribution pattern of HBV in the whole Cambodia. HBV genotype C was abundantly found in almost all provinces of Cambodia except Kandal, Kampong Cham, Tbong Khmum and Svay Rieng provinces where HBV genotype B was predominant. Some studies also reported that genotype C is the predominant genotype in Cambodia and then followed by genotype B [
10,
27]. Cambodia is bordered by Thailand to the northwest, Laos to the northeast, and Vietnam to the east and the Gulf of Thailand to the southwest. The genotype distribution pattern is linked to the neighboring countries of Cambodia. In our study, HBV genotype B was exclusively circulated in Svay Rieng, Kampot and Tbong Khmum provinces; the border regions to the Vietnam where HBV genotype B is predominant (72.6%); particularly sub-genotype B4 (87.8%) [
28]. HBV genotype C was abundantly found in the Stung Treng, Ratanak Kiri and Preah Vihear provinces, the northeast part of Cambodia and border region to Laos, where HBV genotype C (55.4%) is also predominant [
29]. Meanwhile, in Otdar Meanchey, Pursat and Battambang provinces; the west and northwest regions of Cambodia bordering to Thailand, HBV genotype C was exclusively found where 73 to 87.5% of the detected HBV strains were genotype C [
30,
31]. In fact, HBV genotypes B and C are the most prevalent types in Asia and the genotype C has more pathogenicity in compared with genotype B [
32]. By this study, it is supposed to have the historical relation of HBV genotype between Cambodia and its neighboring countries. Therefore, this nationwide genotype distribution pattern raises two important issues for the infection control of HBV in Cambodia. Firstly, the health sector should be aware of geographic variation of HBV genotypes and its historical relation among neighboring countries and should consider for implementation of effective HBV preventive strategies among migrants from both sides. Secondly, the reported predominant genotypes and sub-genotypes of our study can be the clue for better understanding of viral factors on liver disease progression in chronic hepatitis B carriers in Cambodia.
S gene mutant strains of HBV were isolated from 17 mothers and 2 children. The overall S gene mutation rate of HBV among HBV DNA positive sera was 23.94% in mothers and 18.18% in children, 24.24% in genotype C and 18.75% in genotype B. This rate was lower than that reported from Singapore (39%) [
33] but is higher than Thailand (22.4%) [
34] and Malaysia (9%) [
35]. By this study, high S gene mutation rate of HBV among mother-child pairs of Cambodia suggested the potential spread of vaccine escapes mutant strains in Cambodia. S gene mutation of HBV specifically
a mutant was occurred most frequently among immunized children and who received plasma derived HepB vaccine [
12] and the similar results were found among immunized children of our study but there was no statistically significance. The vaccine itself driven S gene mutation through immune pressure causing amino acid substitution and point mutation [
36] although we could not exclude MTCT of S gene mutants.
In our study, only 2 out of 17 children born to mothers with S gene mutants of HBV became infected and both of them did not receive HepB-BD. But, no infection was found if the children received HepB-BD. This could be explained by the hypothesis, that the S gene mutant strain of HBV itself has lower replication rate and also has negative effect on replication of wild type HBV in mixed infection through high T cell immune response causing less infectivity and transmissibility of HBV infection [
37]. If the child had received HepB-BD within 24 h, the vaccine totally interrupts MTCT. If the child missed HepB-BD, it causes high possibility of MTCT despite previous study reported on low level of viral replication among mutant strains. Although it was not clear whether S gene mutants of HBV were transmitted vertically or only under immune pressure due to vaccination in our study and the number of isolated mutant strains was quite small to compare, it was revealed that HepB-BD is crucial for preventing MTCT of HBV either wild type or S gene mutants.
S gene mutation of HBV was profoundly occurred in genotype C in our study than genotype B. In fact, genotype B was documented to have high potential for occurrence of amino acid substitution than genotype C [
38]. This discrepancy might be due to difference genotype distribution pattern. But the existence of S gene mutants of HBV in Cambodia alarms the possible breakthrough infection among immunized children which may threaten the long term effect of massive immunization. Despite the successful establishment of HepB vaccination, Cambodia has no specific program and protocol for PMTCT of HBV until now. It is challenging for Cambodia on its pathway to meet WHO’s viral hepatitis elimination goal of by 2030. Therefore, the health sector should develop and disseminate the national guideline, HBV screening, assurance of HepB-BD administration to all newborns within 24 h after delivery and provide specific anti-viral treatment to HBV carrier mothers.
Apart from S gene mutation, preS deletion (22.58%), double (48.39%) and combination mutation (32.26%) were also found in HBV genotype C1 strains. In fact, HBV genotype C can easily mutate [
39] and its mutation is significantly related to the HCC occurrence [
10,
27,
40]. In our study, although we could not correlate the mutant variant with respective liver disease condition, based on recently published study [
40], it indicates the need of proper counseling, early and proper referral to the specialized center, assessment for eligibility to anti-viral therapy and regular follow-up care which should be offered to them even they are currently asymptomatic.
This study used the DBS samples to detect not only the HBV sero-markers but also HBV DNA and consequently both partial and full length genome sequences, which is the critical tool for the advanced molecular epidemiology. According to recent systematic review and meta-analysis report, the pooled estimate of sensitivity and specificity for HBV-DNA using DBS was 95% (95% CI: 83–99) and 99% (95% CI: 53–100), respectively [
14]. Despite the whole blood samples by venipuncture still ranks as the gold standard for biological specimen, this study proved the capable of DBS for HBV full-length genomes sequences and it is useful as alternative blood collection tool for large scale molecular epidemiological study especially in resources limited countries which may accelerate the surveillance of target virus.
The limitations were present in this study. Firstly, our study could not evaluate the S gene mutation rate by type of HepB vaccine used in the children. Secondly, the study is cross-sectional so that the investigation of liver disease stages and their progress is impossible. Based on the previous study, we could only suggest that HBV C1 infected participants of our study have high possibility to HCC occurrence [
40]. At last, even we used DBS samples for detection of partial and full-length HBV genome sequence; we could not compare it with gold standard venous blood samples. Further comparative study on detection of viral genomes in both DBS and venous samples is needed.
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