Hepatitis B virus sero-profiles and genotypes in HIV-1 infected and uninfected injection and Non-injection drug users from coastal Kenya

Background Information about HBV sero-markers, infection stages and genotypes in HIV-1 infected and uninfected injection and non-injection drug users (IDUs) in Kenya remains elusive. Methods A cross-sectional study examining HBV sero-marker, infection stages and genotypes was conducted among HIV-1 infected and uninfected, respectively, IDUs (n = 157 and n = 214) and non-IDUs (n = 139 and n = 48), and HIV-1 uninfected non-drug using controls (n = 194) from coastal, Kenya. HBV sero-marker and infection stages were based on HBV 5-panel rapid test plasma sero-reactivity. DNA was extracted from acute and chronic plasma samples and genotypes established by nested-PCR and direct sequencing. Results HBsAg positivity was higher in HIV-1 infected IDUs (9.6 %) relative to HIV-1 uninfected IDUs (2.3 %), HIV-1 infected non-IDUs (3.6 %), HIV-1 uninfected non-IDUs (0.0 %) and non-drug users (2.6 %; P = 0.002). Contrastingly, HBsAb positivity was higher in HIV-1 uninfected IDUs (14.6 %) and non-IDUs (16.8) in comparison to HIV-1 infected IDUs (8.3 %), and non-IDUs (8.6 %), and non-drug users (8.2 %; P = 0.023). HBcAb positivity was higher in HIV-1 infected IDUs (10.2 %) compared to HIV-1 uninfected IDUs (3.3 %), HIV-1 infected non-IDUs (6.5 %), HIV-1 uninfected non-IDUs (2.1 %) and non-drug users (4.6 %; P = 0.038). Acute (5.7 %, 1.4 %, 0.0 %, 0.0 % and 1.5 %) and chronic (5.1 %, 0.9 %, 3.6 %, 0.0 % and 1.5 %) stages were higher in HIV-1 infected IDUs, compared to HIV-1 uninfected IDUs, HIV-1 infected and uninfected non-IDUs and non-drug users, respectively. However, vaccine type response stage was higher in HIV-1 uninfected IDUs (15.4 %) relative to HIV-1 infected IDUs (6.4 %), and HIV-1 infected (6.5 %), and uninfected (10.4 %) non-IDUs, and non-drug users (5.7 %; P = 0.003). Higher resolved infection rates were also recorded in HIV-1 uninfected IDUs (11.2 %) compared to HIV-1 infected IDUs (8.3 %), and HIV-1 infected (7.2 %), uninfected (6.3 %) non-IDUs, and non-drug users (6.7 %; P = 0.479), respectively. Only A1 genotype showing minimal diversity was detected among the study participants. Conclusion HBV sero-markers and infection staging are valuable in diagnosis and genotyping of HBV infections. Among IDUs, higher HBsAg and HBcAb positivity in HIV-1 infected and higher HBsAb positivity in HIV-1 negative IDUs suggests frequent exposure. Additionally, HBV genotype A is the dominant circulating genotype in both high and low risk populations of Kenya.


Background
Despite existence of an effective vaccine, about 2 billion people have been exposed to hepatitis B virus (HBV) leading to at least 300 million chronic HBV infections and 0.6 million deaths worldwide [1]. The problem is further compounded by the spreading co-endemicity with HIV-1 leading to at least three million co-infections in the world [2]. The higher rates of HBV and HIV-1 coinfections are driven by the shared portals of entry such as parenteral, perinatal and sexual routes [3].
Hepatitis B virus sero-prevalence is determined by examining for the presence of hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), hepatitis B pre-core antigen (HBeAg), hepatitis B pre-core antibody (HBeAb) and hepatitis B core antibody (HBcAb) sero-marker reactivity [4]. HBsAg represent active acute or chronic infection, HBeAg indicate high viral replication and persistence while HBsAb and HBeAb signify hepatitis B resolution [4,5]. HBcAb is a non-protective total antibody of the classes IgM and IgG denoting acute-window and past infection, respectively [4]. To our knowledge, no study has estimated HBV seroprevalence by concurrent testing of the five hepatitis B virus sero-markers in Kenya.
The incubation period following HBV exposure varies from one to six months and correlates with magnitude of the inoculum [6]. Resolution and outcomes of HBV infection are governed by host immunity, age, gender, infection route and genotype [7]. While immune competent individuals successfully resolve HBV infection [8], immune-suppressed subjects such as HIV-1 infected patients rapidly progress from acute to chronic stages [9]. Although most individuals recover from acute or mild infections, occult infection persists in the liver with reactivation largely occurring in immune-suppressed individuals [10]. In Sub-Saharan Africa, HBV and HIV-1 are co-endemic [11], but information about HBV infection stages in HIV-1 infected and uninfected injection drug users (IDUs) and non-IDUs is unknown.
To date, ten genotypes (A-J) of HBV have been reported with Africa having mainly genotype A and to a varying extend genotypes C, D and E [12]. Although genotype A is the most common in blood donors, commercial sex workers and HIV-1 infected patients on antiretroviral treatment (ART) in Kenya, genotypes D and E occur less frequently [13][14][15][16][17]. HBV genotypes influence disease severity and response to antiviral treatment, and cluster with risk-population [10,18]. However, circulating HBV genotypes in most-at-risk populations (MARP) in Kenya particularly among IDUs and non-IDUs remains elusive. This study, therefore, examined hepatitis B virus sero-markers, infection stages and genotype distribution in HIV-1 infected and uninfected IDUs and non-IDUs residing at Coastal Kenya.

Study area, design and population
A cross-sectional study of drug users was conducted at Bomu Hospital, Mombasa, coastal Kenya. A description of the study site and recruitment procedure is presented elsewhere [19]. Injection drug users were defined as individuals exhibiting needle-scars and reporting use of any illicit injection drug from the United Nations Office on Drugs and Crime (UNODC) report [20] for at least once in the previous month. Non-injection drug users were defined as persons who had never injected drugs but have used at least one non-injection drugs listed in the UNODC report [20]. The study participants were stratified into HIV-1 infected and uninfected IDUs, HIV-1 infected and uninfected non-IDUs, and HIV-1uninfected non-drug using controls (from the general population). About, 3.5 mL venipuncture blood was collected in EDTA BD vacutainer TM anticoagulant tubes (BD, Franklin Lakes, USA) for HIV-1 serological testing, CD4+ T cell enumeration and plasma harvesting as described elsewhere [19].

HBV sero-marker testing
The five HBV serological markers (HBsAg, HBsAb, HBeAg, HBeAb and HBcAb-IgM) reactivity of plasma samples was determined using the one-step HBV-5 panel rapid diagnostic cassette (Healthaw Medical limited, Hangzhou, China) according to the manufacturer's instructions. Briefly, 5 μL of the test plasma was placed into each of the five sample wells corresponding to the sero-markers. Two drops of buffer were added to each well and HBV sero-marker reactivities recorded after 20 min.

Statistical analysis
Statistical analyses were performed using SPSS version 19.0 (IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp.). Age and CD4+ T cell counts were compared among groups using Kruskal Wallis test followed by Dunn's post-hoc correction for multiple comparisons. Differences in the distribution of proportions of gender, HBV sero-markers, infection stages and genotypes were compared across the study groups using the Chi-square tests. All tests were 2-tailed with statistical significance set at a critical alpha value of 5 %.

Ethical considerations
The study was approved by Kenyatta University Ethical Review Committee and was conducted according to the Helsinki Declarations [24]. Written informed consent was obtained from each participant before enrolment and confidentiality was ensured in the course of the study. All the study participants were provided with free health education on sexually transmitted infections (STIs) including HIV, hepatitis B and C, tuberculosis, hygiene and nutrition. Participants testing positive for HIV were referred to the comprehensive care centres at Bomu Hospital or the Coast General Referral Hospital for treatment, care and support.

Baseline characteristics
The baseline characteristics of the study participants are summarized in Table 1. A total of 752 adults were recruited into the study. The study participants were categorized into the following five study groups

Hepatitis B viral marker distribution
Hepatitis B sero-marker reactivities in the study participants are shown in Table 2

Hepatitis B infection stages
HBV infection stages are summarized in Table 2

Discussion
Hepatitis B sero-markers, infection stages and genotypes are integral in selecting, initiating and monitoring response to treatment and in sero-and molecular epidemiologic surveillance [8,25]. Although HBV exhibits geographic and risk-group clustering of genotypes [18], distribution of HBV genotypes in HIV-1 mono-and coinfected injection and non-injection drug users from Africa is largely unknown. This study examined HBV seromarker reactivity, infection stages and genotypes among Fig. 1 Phylogenetic tree of HBV isolates. Neighbour-Joining method based on 1000 bootstrap replicates and p-distances were used for generating the phylogenetic tree [23]. HBV genotype sequences from GenBank together with their country of origin and accession numbers are presented. Wooly monkey HBV (AY226578-WMHBV) was used as the out-group. Relevant bootstrap values are indicated. HBV isolates from study participants are indicated by diamond signs. The scale bar represents genetic distance HIV-1 infected and uninfected IDUs and non-IDUs resident at coastal Kenya. The contrasting sero-positivity rates of HBsAg, HBsAb and HBcAb-IgM in HIV-1 infected and uninfected IDUs and non-IDUs reflect frequent HBV exposure to a "competent" (HIV-1 uninfected IDUs) and "incompetent" (HIV-1 infected IDUs) and less exposure to a "competent" (HIV-1 negative non-IDUs) and "incompetent" (HIV-1 positive non-IDUs) immune system. These findings, are in part, consistent with previous studies illustrating contrasting HBsAg, HBsAb and HBcAb positivity in HIV-1 infected and uninfected IDUs [26]. Most importantly, the results also partly mirror HBsAg positivity rates of 7 % in HIV-1 infected female sex workers from Mombasa (the same region as the current study area) [13]. The lower rates of HBsAg positivity in the HIV-1 negative IDUs and non-drug users, and HIV-1 infected non-IDUs may be linked to perinatal and/or early childhood acquisition of HBV infection [27,28]. However, the higher rates of HBsAg in the HIV-1 infected IDUs suggest secondary acquisition of HBV post-HIV infection from increased high risk injection and sexual practices that are prevalent among HIV infected IDUs at Coastal Kenya [29]. While the underlying mechanisms remain undefined, it is possible that depleted CD4+ T cell counts in HIV-1 infected IDUs, cause diminished HBsAg sero-conversion and antibody production [30]. HBeAg and HBeAb rates observed in this study are partly consistent with previous studies among HIV-1 positive Nigerian women and Czech IDUs [31,32]. Altogether, these findings suggest an altered host response to HBV infection in HIV-1 infected and uninfected IDUs and non-IDUs from coastal Kenya.
Although complete serological testing of the five hepatitis B virus sero-markers (HBsAg, HBsAb, HBeAg, HBeAb and HBcAb) is key in guiding accurate diagnosis and infection staging of hepatitis B virus [4,5], to our knowledge, this is the first study to examine utility of the five hepatitis B virus sero-markers in identifying the infection stages of HBV infections in HIV-1 infected and uninfected IDUs and non-IDUs from Kenya. Although the sensitivity and specificity of various HBV rapid diagnostic tests varies [33,34], acute hepatitis B infection rates of 5.7 % in the HIV-1 infected IDUs were detected. This finding corroborates with previous studies showing acute hepatitis B virus infection rates of 5.1 % in IDUs from a high HIV-1 risk region of Scotland [35]. In addition, chronic hepatitis B virus infection rates identified in this study are comparable with rates among HIV-1 infected IDUs in the United States of America [36], and female sex workers from Mombasa (a most-at-risk population from the same study area as the current study) [13]. Taken together, the higher rates of acute and chronic hepatitis B virus infection in HIV-1 infected IDUs suggest that HIV-1 infection indirectly and/or directly increase the risk of acquiring and developing hepatitis B virus infection in IDUs. Our laboratory is currently investigating this hypothesis.
Higher rates of vaccine type responses and resolved infection stages in HIV-1 uninfected IDUs are related to the higher CD4+ T cell counts recorded in these study participants. These observations are similar to previous vaccine-type response and resolved infection rates, respectively, detected in young American and Iranian IDUs [37,38] and HIV-1 uninfected South Africans [39]. The higher CD4+ T cell counts are responsible for increased HBV surface antigen recall responses [40], and resolution of HBV infection [41] in HIV-1 infected and uninfected individuals. Thus, incorporating immune modulators and vaccine booster dose regimens may greatly enhance immunity in co-infected individuals.
Only HBV genotype A sub-genotype A1 were detected in all the study participants, supporting the stability and persistence of this genotype in Kenya. This finding is in line with previous findings in which only genotype A1 was identified among HIV-1 infected adults on antiretroviral therapy from Nairobi [17] and commercial sex-workers from Mombasa [13]. The minimal genetic diversity in the HBV A genotypes found in this study, suggest recent introduction in the HIV-1 infected and uninfected populations of IDUs in Kenya.
Finally, it is important to outline the limitations of this study. This cross-sectional study used HBcAb-IgM to classify acute infection stages even though HBcAb-IgM may persist for several years after acute infection [4]. While the sensitivity and specificity of the various HBV rapid diagnostic tests varies [33,34], nucleic acid testing was performed on plasma samples obtained from study participants presenting with acute and chronic infection stages based on sero-profile. Even though prospective studies would be important in examining HBV seroresponses, genotypes, viral load and occult infections in HIV-1 infected and uninfected IDUs and non-IDUs, this cross-sectional study has delineated the value of the 5panel sero-markers in directing infection staging and genotyping of HBV infections.

Conclusion
HBV 5-panel sero-marker testing is important in guiding infection staging and genotyping of HBV infections. Contrasting sero-reactivities characterized by higher HBsAg and HBcAb positive rates and lower HBsAb positive rates are associated with HIV-1 driven immune suppression. Additionally, it is evident that genotype A is the main circulating HBV infection among IDUs and non-IDUs at coastal Kenya.