A Serological Survey of Selected Papua New Guinea Blood Donors for Hepatitis B and Related Co-Infections

Hepatitis B virus (HBV) infection is a serious problem and earlier studies in Papua New Guinea have reported a high prevalence of hepatitis B virus infection. These studies were undertaken using insensitive tests and before an expanded immunization program. The current HBV status is therefore uncertain. A retrospective study to investigate the HBV status was carried out using blood donor data at Nonga General Hospital, East New Britain Province, Papua New Guinea, from January 2003 to December 2018. Additional data for Human Immunodeficiency Virus, syphilis and hepatitis C virus were also collected. Data were analysed using NCSS statistical software. The mean hepatitis B antigen (HBsAg) sero-prevalence was 21% for the period of study and showed a downward trend over the period of the study, which may reflect the effect of the extended immunization program. HBsAg prevalence in male donors (23%) was significantly higher than females (16%). Donors living in Pomio district had a significantly lower proportion of sero-positive HBsAg donors (7%) than Gazelle (22%), Kokopo (22%) and Rabaul (20%), which was attributed to this district’s geographical isolation. Ethnically, Pomios donors (8%) had significantly lower HBsAg prevalence than the Taulils, (29%), Bainings (21%) and Tolais (21%). Fifteen to nineteen year olds (23%) were the predominant age group affected, and vertical or perinatal transmission was probably the primary transmission route. Our findings call for greater awareness on the part of public policy makers and should be considered when planning future public health campaigns.


Introduction
Hepatitis B (HBV) infection is a serious problem both globally and nationally. Earlier studies in certain areas of Papua New Guinea (PNG) reported a high prevalence of Hepatitis B virus infection, especially in the highlands of PNG [1][2][3], the Sepik province [4] and in the Autonomous Island of Bougainville [5]. These studies were undertaken in the general population using insensitive tests and before an expanded immunization program. The HBsAg prevalence among blood donors in Papua New Guinea was reported in Port Moresby Blood transfusion services as 22.7% [6], and in the whole country in 1996 as 15% [7]. In about the same period, 24% of blood donors were reported positive for the before an expanded immunization program. The HBsAg prevalence among blood donors in Papua New Guinea was reported in Port Moresby Blood transfusion services as 22.7% [6], and in the whole country in 1996 as 15% [7]. In about the same period, 24% of blood donors were reported positive for the HBsAg in Bougainville [8]. A study undertaken in 1998 that reviewed the HBsAg sero-positivity status in blood donors of Pacific countries stated the prevalence of HBV in PNG was 16.7%, second highest to the Solomon Islands (19.5%) [9]. Despite the high prevalence of HBV antibodies found in populations in Papua New Guinea [1-9], a study in 1987 looking at the aetiology of jaundice in a medical ward of one of the Hospitals in PNG failed to detect viral hepatitis in any of the clinical cases with jaundice [6]. This is in contrast to an earlier study published in 1976, which had examined 17 clinical cases, eight of which were strongly suggestive of viral hepatitis clinically [10]. Since the introduction of neonatal hepatitis B Immunoglobulin (hep B Birth Dose) immunization in 1989 and subsequent addition of the national three-dose infant hepatitis B (hep B3) vaccination program in 1992 [11], little work to establish the prevalence of HBV has been conducted in blood donors in Papua New Guinea. A recent comparative retrospective study in blood donors in Port Moresby General Hospital found a hepatitis B antigen (HBsAg) prevalence of 22.9% in blood donors in 2016 [12]. Coinfections were also indicated in the latter study at 7.3% (20/275) and were mostly seen in Family replacement donors. Co-infections were mainly with Human Immunodeficiency Virus (HIV) and syphilis [12], owing to shared means of transmission [13].
East New Britain province (ENBP) is one of the five island provinces of PNG that lies between four and six degrees south of the equator (Figure 1). It has a total population of 327,355 people with a high literacy rate (86.6%) among the four island provinces and second in the country to the National Capital District (92.4%) [14]. It is also known as the fastest growing province in the country economically [15]. It has four districts inhabited by four distinct ethnic groups, namely the Tolais, Pomios, Taulils and the Bainings, which are all Melanesians (Figure 1). The Kokopo and the Rabaul districts are semi-urban areas and are inhabited predominantly by the Tolais, the Gazelle district is inhabited by the Tolais, the Taulils and the Bainings, while the Pomio district is inhabited predominantly by the Pomios. The current HBV status in ENBP is uncertain, and therefore this study was undertaken to establish the prevalence of HBV and related co-infections in blood donors to help with policy making in the province.

Materials and Methods
A retrospective observational cross-sectional study to investigate the HBV, HIV, hepatitis C virus (HCV) and Syphilis status of blood donors and related epidemiological risk factors in ENBP was carried out using blood donor data at Nonga General Hospital (NGH), ENBP, PNG from January 2003 to December 2018. Mandatory pre-screening for health and social status are done on all prospective blood donors presenting for donation at the blood Transfusion Service. This is in concordance with the PNG National Health Department Blood Policy, in partnership with the World Health Organization [16].
Epidemiological variables included in the data analysis were age, gender, district of origin, ethnicity, infection status and diagnostic tests. Ethnicity was based on the village that was recorded in the database for that patients place of residence, with the assumption that people from a particular village would all be of the same ethnicity, since tribal clustering is still relevant to PNG. Infectious status was determined in the hospital as part of their diagnostic service using commercially available kits and relevant demographic data were recorded at the time. The following kits were used by the hospital for diagnostic purposes (note that this study only looked at the results of the tests and the authors were not involved in the diagnostic process). The Gelatin Particle Agglutination assay using Serodia ® -HBs PA (specificity 100%; sensitivity 99.0%. Fujiebio Inc., Tokyo, Japan) was used to detect HBsAg before 2010. This test had a reported minimum detection limit of 0.5 ng/mL HBsAg. After 2010, immunochromatographic assays were used for HBsAg detection, which included DetermineTM HBsAg (sensitivity 100%; specificity >98%. Abbott Laboratories, Dainabot Co. Ltd., Tokyo, Japan). Alternatively, the Hepa S-Ag test kit using reverse passive hemagglutination (specificity 100%; sensitivity 99%. Green Cross Medical Science, Yongin, South Korea) or the Chembio Diagnostic Systems kit (specificity 100.0%; sensitivity 99.0%. Medford USA) was used, depending on availability. Test kits used were in keeping with World Health Organization (WHO) recommended assay required for EIAs (sensitivity 100%; specificity ≥98%) and RDTs (sensitivity ≥99%; specificity ≥98%) [17]. WHO recommended limit of detection for both EIAs and RDTs is ≤0.13 IU/mL.
Two tests were used for the screening of Syphilis; the Syphilis TPHA liquid Hemagglutination test for qualitative and quantitative detection of T. pallidum antibodies in serum specimens (sensitivity 98.5%; specificity 100%. Hexagon, Germany) or the Syphilis RPR Rapid test for qualitative and semi-quantitative detection of antibodies, associated with syphilis, in serum or plasma (specificity and sensitivity not given by manufacturer. HUMAN Gesellschaft für Biochemica und Diagnostica mbH Max-Planck-Ring 21 65205 Wiesbaden, Germany). In addition, the SD BIOLINE Syphilis 3.0 test was used to test for Syphilis, which is a solid-phase immunochromatographic assay for the qualitative detection of antibodies of all isotypes (IgG, IgM, IgA) against Treponema pallidum (TP) (sensitivity 99.3%; specificity 99.5%. Standard Diagnostic, Korea,). The SD BIOLINE HCV (02FK10, 02FK16, 02FK17) chromatographic testing kit was used to test for the HCV in 2018 blood donors (sensitivity 100%; specificity 100%. SD Standard Diagnostics, Inc., Korea,). This test was introduced to all blood bank centres in PNG in 2018, and therefore only HCV test results for 2018 are reported for our study. Other tests used for the diagnosis of active hep B and C infections were not performed in this setting because of financial constraints. Positive donors for HBsAg were excluded from repeat donation, however, donors were not always informed of their results unless they came back for their results or came back for a second donation. Additionally, all tests positive for HBsAg were not repeated.
Apparent prevalence (AP) and its corresponding 95% confidence interval (CI) were calculated using proportions of donors at NGH, testing positive on one or more of the diagnostic tests described above. The Chi-Square test was used to look for significant associations between the proportion of HBsAg seropositive donors and demographic variables. The level of significance was set at p ≤ 0.05%. Odds Ratios were calculated to determine the strength of association between the proportion of seropositive HBsAg donors and demographic variables.
For the multivariable logistic regression models, survey data were entered into Microsoft Excel (2007) and the computer software program NCSS (NCSS Inc., Kaysville, UT, USA) for analysis. The associations between seropositive HBsAg donors and various risk factors were analysed using Chi-square tests (i.e., univariate analysis). Risk factors with a p-value < 0.20 on univariate analysis were selected to be included in the multivariable logistic regression models [18]. A hierarchical stepwise forward elimination process with switching of variables based on log likelihood values was used to determine the best fitting logistic regression model. NCSS has a built-in algorithm that does the selection process and several iterations were run before the final model was selected, which contained the best fitting log likelihood value and only the variables with a p-value < 0.05 on the Wald test [19,20]. Due to collinearity between the District and Ethnic groups, two separate models were run with the inclusion of only one of these variables in each model. Ethical Clearance was granted by the Nonga General Hospital Management on 6 February 2017 and the University of Papua New Guinea School of Medicine & Health Sciences Research Ethics Committee (UPNG SMHS REC) approved the research on 10 October 2016. All records collected were de-identified and only data required for analysing trends was collected.

Results
A total of 24,478 donor records from 2003-2018 were analysed. Of these, 41.4% (n = 10,128) were excluded because of missing variables of interest. Of the remaining 58.6% (n = 14350), 67.5% (n = 9686) were males and the rest were females (32.5%, n = 4664). The average number of donors per year and standard deviation over the period studied was 897 ± 497 and ranged from 129 in 2017 to 1832 in 2003 (Appendix A).
The mean ages and standard deviations for males and females were 31 ± 11.6 and 31.8 ± 11.3 years, respectively. Though there was no statistical difference observed between the mean age of males and females, the age range was 74 and 58 years, respectively. In both genders, 20-year olds donated more frequently than the other ages. More males continued to donate annually throughout the study period.
Two logistic regression models were done to identify risk factors in the districts and among ethnic groups since district and ethnic groups showed collinearity. Model one (Table 3), which includes district, uses Gazelle, which has the highest prevalence of HBsAg, as a reference and shows that Rabaul  Model 2 ( Table 4) that includes ethnicity shows that previous donors are less likely to be HBsAg positive (OR = 0.8). Pomio donors are less likely to be HBsAg positive than the Bainings (OR = 0.57) and no difference in risk can be shown between the Taulils and the Bainings or between Tolais and Bainings. Unlike, Model 1, donors positive for HCV were shown in this model to have less chance of being HBsAg positive (OR = 0.39), which means district was probably confounding this relationship. HCV exposure may therefore be associated with ethnicity. Male donors were confirmed to be more at risk of having HBsAg sero-positivity than females (OR1.
Our study demonstrated a downward trend in HBsAg sero-prevalence from 2003-2018 ( Figure 2, Table 1). This is in accordance with studies done in the Solomon Islands, which demonstrated a decline in HBsAg prevalence from 32% in 1977 [21] to 25.1% in 1999 [25] to 21.5% in 2007 [26]. Similarly, Fiji has seen a decline in prevalence from 17.9% in 1982 [22] to 0% in children, 5.6% in adolescents and 3.2% in adults in 2009 [27]. The decline in donor HBsAg prevalence seen in our study, from a high of 35% in 2003 to 5% in 2018 in ENB province, can probably be attributed to the expanded program for immunization in the province and increased awareness of the disease within communities as a result of these campaigns. The fluctuating decline in donor HBsAg prevalence infections may have been indirectly affected by fluctuating hepatitis B immunization coverage rates, which varied from 42% in 2001 to 61% in 2002, 68% in 2003 and a decline to 61% in 2004. In 2005, coverage increased again to 67% and 75% in 2006. In 2007 and 2008, a decline in coverage was again recorded from 66-65%, respectively [28]. Furthermore, the estimation of hep B Birth dose (hep BB) and hep B3 (triple dose) immunization coverage rates in PNG showed declining rates from 26% in 2009 to 25% in 2018, and 66% in 2009 to 61% in 2018, respectively [29].

HBV Infection among the Districts and Ethnic Groups of This Study
The Gazelle, Kokopo and Rabaul districts had the highest prevalences of HBsAg at 22%, 22%, and 20% respectively, which were twice the Pomio district (Table 1). The Rabaul and the Kokopo districts are both semi-urban areas, while the Pomio and Gazelle districts are mostly rural. Though both the Pomio and the Gazelle districts are rural settings, HBsAg seropositivity in the Pomio district was lower than the Gazelle district (Table 2). In fact, Pomio district has lower odds (OR = 0.3) of having HBsAg infection than Gazelle (Table 3). A possible factor that may explain this difference in HBsAg prevalence between Gazelle and Pomio districts could, therefore, be the difference in the ethnicity of donors in the two areas, with most donors in Gazelle being predominantly Tolais and Bainings and some Taulils, while the Pomio district is populated almost exclusively by Pomios. In other studies [30][31][32], familial clustering is a common risk factor among rural inhabitants, or intra-familial transmission [33].
Inhabitants of the two semi-urban districts of Rabaul and Kokopo are either located within the vicinity of a hospital or have easy access to a hospital and blood donation services due to good road networks, unlike most areas of the Gazelle and Pomio districts. It is not clear from our results, however, if this had an influence on exposure rates. Although both Rabaul and Kokopo districts are semi-urban, Rabaul has slightly lower odds of having donors positive for HBsAg than Gazelle, but no difference in risk could be shown between Kokopo and Gazelle. The people in Rabaul and Kokopo districts are predominantly Tolais as is the majority of Gazelle, which could explain the relatively small difference in prevalence of HBsAg between these districts rather than socioeconomic factors. The fact that Bainings were clustered together with Taulils and Tolais in Gazelle and were not living in other districts could explain why no difference in HBsAg prevalence could be shown between these ethnic groups. It is difficult to explain variations between these ethnic groups, but it could mean that accessibility to basic health services is not a key factor. Alternatively, isolation of the Pomios, who are mostly accessible only via air and water transport, may have mitigated against spread to this population. Lifestyle was seen to be a factor in two rural populations of New Caledonia [34], yet a study in Fiji showed high prevalence that appeared not to have been affected by location and lifestyle [21]. A study in Tonga showed increased new cases of HBV infections in rural dwellers compared to urban and attributed this to a lifestyle in rural settings that predisposed them to increased infection [35]. While this may hold true for the Taulils, Bainings and Tolais of the Gazelle district, it is in contrast to the Pomios in this current study, who live mostly in rural areas and yet have the lowest HBsAg prevalence.
The reason why HBV is so prevalent in Tolais is complex but one reason could be that when the Tolais migrated from nearby New Island Province (NIP) to settle in ENBP, they could have carried the virus along with them and introduced it to those who were already there. There is a possibility, therefore, that genetic predisposition could be playing a role amongst the ethnic groups in this study as well.
The overall literacy rate in the province is high [14] and, in other studies, high literacy rates have been found to be associated with decreasing HBV infection among rural populations [36,37]. This is in contrast to this study where the Pomio district has a lower literacy rate as compared to the rest of the districts in the province and yet has the lowest HBsAg prevalence and odds of being HBsAg positive, while the Gazelle district has a high literacy rate [38] and yet has a significantly higher HBsAg prevalence than Pomio.
The logistic regression models generated in this study only had a correct classification rate of 51 to 57%, indicating that numerous other risk factors not included in the models were likely playing a role. Other factors, such as poor knowledge and attitude about HBV infection, the importance of vaccination against it, parents being older, and families with low monthly income have all been identified as having an association with higher HBV prevalence rates [37,39,40].

HBV Infection among Each Gender and Sub-Age Groups
The disease is more prevalent in males than in females in our study (Table 2). Males are also more likely to be positive for HBsAg than females (OR = 1.3). This is in contrast to Nkrumak et al. [41], whose study demonstrated higher HBsAg prevalence in females than males (21.4%, 95%CI 11.6-34.4 vs. 13.2%, ). More males than females were presenting for blood donation in this study, which is consistent with other similar studies [42,43], and this may be confounding the prevalence of HBsAg in males. However, the higher prevalence of positive males could also be due to increased male susceptibility to infection [21] and a gender tendency to chronicity [4]. In this study, HBsAg infection is lower in the Pomio district among both genders. One possible reason that Pomios males have less infection could be due to male circumcision practiced in this ethnic group. This practice reduces the risk of transmission sexually transmitted diseases, as seen in another study [42]. A reason why females are less at risk, as given by Mazzur and Jones [21], is that females do not venture out of the home to socialize as often as males.
HBV exposure was seen in our results to be highest among adolescents and young adults in both genders (Table 1). This is similar to a study done in Bougainville [5] but in contrast to an earlier long-term study in the Sepik district, where HBV infections increased with age in all populations studied [3]. Though both Hawkes et al. and Wong et al. [4,5] also employed other markers of past infections such as the anti-HBc, which increased their detection rates, the general scenario was that, for both genders, evidence of HBV infection increased with age, although the positivity rate of increase varied. This indicated vertical and horizontal acquisition from an early age [21,34,[43][44][45][46]. In this study, high levels of HBsAg were seen among teenagers and young adults, the 15-29-year-olds. The highest prevalence of HBV infection was observed in 2003 among 15-29-year-olds. This may be because donors aged 16-29 years during this time would not have been vaccinated against the virus, as the vaccine was first introduced into the country in 1989. This suggests that acquisition was mostly vertical or perinatal. This is further supported by the fact that IV drug use is rare in these communities.
It could not be concluded in this study if the HBsAg positive donors were chronic carriers as repeat donors positive for HBsAg were not followed up.

HBV Co-and Triple Infection with Syphilis, HIV and HCV
According to the World Health Organization [47], about 2.7 million (1%) or 7.4% of people with HBV are HBV/HIV-co-infected. Our study found that only 0.7% of HBV positive donors co-infected with HIV. However, donors infected with HIV were more likely to be positive for HBsAg (OR = 4.8), no significant association between donors positive for HCV and HBsAg was seen. This means that donors positive for HCV are less likely to be infected with HBV (OR = 0.39).
Many studies have demonstrated continuous occurrence of HBsAg co-infections with HIV apart from syphilis, especially among asymptomatic MSM [48][49][50][51][52][53][54][55][56], sex workers [49,50], high-risk sexual behaviour individuals [48,54] and among the immuno-compromised population [54][55][56][57]. Furthermore, it has been found that the risk of developing HBV chronicity is 5-6 times higher in individuals positive for HIV than in those negative for it [52]. HIV infection has also been indicated to be increasing in ENBP [58] and in the country as a whole at an exponential rate [59] and is most common among the young adults of 20-29 years of age [60]. In our study and others [21,43], this is also the sub-age group in which HBsAg is significantly prevalent, which is a cause for concern. Although HBV/HIV co-infection seems to be relatively low in this study as compared to others [61,62], it is imperative for blood banks in PNG to take note that escape mutants in donors can escape being detected using conventional assays, posing a greater risk to recipients.
The prevalence of HBsAg/Syphilis and HBsAg/HIV co-infections in this study was significantly higher than with HCV and triple infections. More males were co-infected with syphilis than females, while females were more commonly co-infected with HIV than with syphilis and HCV. Triple infection was more common among males than females (Table 2). This is in contrast to Butsashvili et al. [53], who did not find any association between being positive for HBsAg and having antibodies against Treponema pallidum among blood donors. Triple infections with HIV and syphilis have also been observed in other studies in asymptomatic high-risk individuals such as MSM and sex workers and is attributed to people not knowing if they are infected [54,55]. A lack of knowledge about having been infected has been found to result in 3.5 times higher risk than those who know their serological status [63]. The feedback of results to patients is therefore important in combating these sexually transmitted diseases.

Conclusions
Hepatitis B virus was still prevalent in the overall population in ENBP in 2018 despite existing control measures, which call for greater awareness on the part of public policy makers in the province, since it is possible that low levels of escape mutants will remain undetectable in blood donor blood and may pose a threat to the recipients as has been demonstrated in other studies [59,60]. Transmission of HBV in this study is likely to be perinatal or vertical, as demonstrated by annual high levels of HBsAg prevalence in young adults (15-29 years) from 2003-2018 and supported by the fluctuating hep B immunization coverage over the years [28]. Male donors are at a greater risk of being infected with HBV than females, which may be attributed to lifestyle. The Pomios people living in the Pomio district had a significantly lower prevalence of HBV than Kokopo, Rabaul and Gazelle districts or the Tolais, Taulils and Bainings people, which was attributed to their remoteness and isolation from other ethnic groups. Co-infections with HIV in females and syphilis in males is of concern and efforts to make donors aware of their infective state should be a priority at hospitals testing for these diseases.
These findings should be considered when planning public health campaigns and high-risk groups need to be targeted for intervention and existing control measure reviewed.

Limitations
Syphilis testing only started in 2007 and therefore an accurate conclusion on reactive syphilis cases could not be drawn from this study. Within the period that syphilis was being tested for, some analysis was not done periodically due to reasons such as a shortage of reagents or inconsistencies on the part of the laboratory personnel responsible for the analysis. A total of 32 (0.35%) of the total population studied had an initial reactive test for HIV and had to undergo confirmatory testing but were never confirmed, and therefore the percentage of positive cases of HIV in blood donors in this study could have been higher. Risk factors for the possible ethnicity of blood donors were identified based on the location (villages) in the districts where their blood was drawn during blood collection and therefore, the likelihood of misidentifying someone as belonging to that ethnic group is possible but would constitute a small portion of the sample due to clustering of people along tribal lines in PNG. Another limitation of this study is the inability to trace repeat donors, and therefore it may have been possible that some donors had donated more than once but were treated as new donors. Additionally, seroconversions were not identified and therefore the same people may have been counted more than once.
Although the specificity and sensitivity of assay kits employed were indicated to be at WHO-approved levels, limits of detection were not indicated, and therefore this could have impacted on the results obtained over the years. Consistency in the use of a single assay needs to be improved, and existing criteria for follow-up of donors positive for HBsAg need to be reviewed.
Author Contributions: F.V. and B.G. designed the study F.V. acquired and processed the raw data; F.V. and B.G. analysed the data and F.V. wrote the paper under supervision of B.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Acknowledgments:
The Authors thank the Australian Aid (AUSAID) for providing the financial means during data collection. This research received no external funding. We also thank Jeffery Warner for his role in the conception of this study and the Nonga General Hospital management, the Officers In-charge of the Laboratory and the Blood Transfusion Services for allowing us to extract these data from their records. We are also grateful to Catherine Warpit, Germaine George, Lawrencia Blasius, Camila Blasius, Daniel Wahawe, David Charles, Ileso Waram and Beverly Warigu for entering some of the data.

Conflicts of Interest:
The authors declare no conflict of interest.