HIV , syphilis and hepatitis B coinfections in Mkushi , Zambia : a cross-sectional study

Human immunodeficiency virus, syphilis and hepatitis B virus Background: (HBV) are major global public health problems. They are sexually transmitted diseases with overlapping modes of transmission and affected populations. The aim of this study is to assess the seroprevalence of HIV 1, hepatitis B virus and syphilis coinfections among newly diagnosed HIV individuals aged 16 to 65 years, initiating on antiretroviral therapy, in Mkushi, Zambia. A total number of 126 sera were collected from HIV 1 infected Methods: patients attending Mkushi district hospital/ART clinic for antiretroviral therapy initiation. Hepatitis B surface antigen test and serologic test for syphilis were conducted between March and May 2018. Of the 126 participants, hepatitis B surface antigen (HBsAg) was Results: detected with a prevalence of 9.5% among newly diagnosed HIV infected patients, while that of syphilis was as high as 40.5% in this same population group. Three patients recorded HIV coinfections with both syphilis and hepatitis B virus (2.4%) at the same time. After analysis, the results indicate that there was no significant association between gender for both dependent variables: HIV/syphilis or HIV/hepatitis B virus coinfections (alpha significance level > 0.05). Those who had a history of syphilis infection in the past were more likely than those who had none to be HIV-syphilis coinfected (53.6% vs 34%, respectively; odd ratio [OR] 2.236; 95% confidence interval [CI] 1.045 – 4.782). The high prevalence rates for HIV, HBV, and syphilis Conclusion: coinfections strongly indicate the need for HBV and syphilis screening for HIV infected individuals. Furthermore, the high number of patients previously treated for syphilis who retest positive for syphilis in this study calls for use of the Venereal Disease Research Laboratory test to identify true syphilis infection (titers ≥ 1:8 dilutions, strongly suggestive).

, they are transmitted through sex (Sexual transmitted diseases, STDs) 2-5 and cross over affected populations 1 . The World Health Organization (WHO) reported an estimated 36.9 million people were living with HIV in 2016 6,7 . It was also reported that 248 million had chronic Hepatitis B virus (HBV) infection (persistent HBs Ag ≥ six months) 8 .
It is estimated that about a million sexually transmitted diseases are acquired daily throughout the world 5 . Moreover, STDs have impact on neonatal, sexual and reproductive health. They can also cause severe complications if not treated. Additionally, STDs pose major a socioeconomic challenge in the form of treatment costs 5,9,10 . Many studies have demonstrated a two-sided relationship between HIV and a number of STDs, including syphilis and HBV 4 . Syphilis and HBV has the potential to increase genital and plasma HIV 1 RNA levels. This increase can increase the likelihood of transmission of HIV 1 to others. In the same way, HIV 1 infection to impact the clinical manifestation, the therapeutic outcome and the HBV/syphilis disease progression 4 .
HIV is associated with a higher prevalence of both HBV and Hepatitis C Virus (HCV) in Sub-Saharan Africa 11-13 in this region, many people living with HIV are HBV or HCV coinfected. About 8% of individuals infected with HIV were HBV coinfected in another study conducted in this region 14 . In a trial conducted in Sub-Saharan Africa 15 , a number of factors were associated with high rates of HIV and syphilis coinfection, including: young age, divorce, widowhood, or separation. Although it is helpful to recognize syphilis risk factors, many women without the risk factor characteristics were syphilis-seroreactive 15 . Also, a high proportion of prevalent rapid plasminogen reagin (RPR)-positive infections remain serofast despite treatment 16 .
In a number of studies conducted in Zambia 17-29 , it has been observed that HIV and chronic hepatitis B (CHB) coinfection was common in both children and adults. It was also observed in these patients that many risk factors such as liver complications and impaired immunologic recovery increased morbidity and mortality. In these settings, syphilis infection remains prevalent in HIV infected adults 30 . As HIV, syphilis and HBV coinfections are clinically consequential, there is great need to screen for syphilis and HBV in people living with HIV/AIDS. The aim of this study was to determine the prevalence of HIV1, HBV and Treponema pallidum serological markers, and therefore their coinfection prevalence among newly diagnosed HIV individuals attending ART clinic in Mkushi rural district of Zambia.

Ethical considerations
This study was approved by Mkushi District Health Office. Participants were informed and provided verbal consent prior to enrollment in the study. Samples were collected as part of routine recommended national baseline tests before initiating ART, therefore only verbal consent was sought.

Study design
We performed a cross-sectional study to assess the seroprevalence of HIV 1, HBV and syphilis coinfections among newly diagnosed HIV individuals aged 16 to 65 years, initiating on cART, in Mkushi, Zambia. Sociodemographic and clinical data were collected from study subjects including age, sex, number of years of education, marital status, residence, occupation, household income, HIV transmission route, history of tuberculosis, past syphilis infection, and WHO clinical staging. A data collection sheet (Extended data 31 ) was used to collect information in addition to client' files.
Inclusion and exclusion criteria: this study included a patient population starting ART between March and May 2018, aged 16 to 65 years old, confirmed HIV 1 infection, being ART naïve (never taken ART for their HIV infection), regardless of WHO clinical staging. Subjects previously exposed to ART, children below the age of 16, and clients with HIV 2 or HIV 1 & 2 coinfections were excluded. A total number of 126 subjects were included.
Diagnosis: All tests were performed at Mkushi hospital laboratory. Four milliliters of venous blood were collected from each client using aseptic technic. Following centrifugation, sera were separated and tested immediately for HIV antibodies. HIV serologic test was conducted using Determine screening test (ALERE) (CHASE BUFFER REF 7D2243, ALERE Medical Co. Ltd. Japan); followed by SD Bioline confirmatory test, discriminating between HIV 1 & HIV 2. (SD HIV1/2 3.0) (Assay diluent Lot 03ADDC013, SD Standard diagnostics, Inc. Hepatitis B virus infection was assessed by an immunochromatographic test (one step rapid test, Lot No. HBPDR00117, NeckLife, Punjab), following the manufacturer's instructions. Syphilis serology was examined using rapid plasma reagin (RPR) (one Step Rapid Test, Lot No. SYS-02-18Q, Ensure Biotech, Hyderabad-500 076), following the manufacturer's instructions.
Statistical analysis: The collected data were analyzed using the IBM SPSS statistics software (trial version 20). The Chi-Square test was used for analysis of the differences between proportions and p < 0.05 was considered as significant. The statistical analysis of frequencies was also conducted and the confidence interval was set at 95%.
A total number of 126 blood samples were collected from HIV 1 infected patients attending Mkushi district hospital/ART clinic for antiretroviral therapy initiation (see underlying data 24 ). HIV 1, HBV, and Syphilis coinfections were recorded together in 2.4% of cases (3/126). Table 1 shows the prevalence of syphilis coinfection with HIV by age group.
The below table shows the hepatitis B virus and syphilis coinfections results by gender among HIV infected subjects (Table 2).
Hepatitis B surface antigen (HBsAg) was detected with a prevalence of 9.5% (12/126) among newly diagnosed HIV infected patients, while that of syphilis was as high as 40.5% (51/126) in this same population group. The above data was analyzed and the results indicate that there was no significant association between gender and syphilis (Chi square value = 0.373, p = 0.542). This study also shows that HBV presence was independent of gender (alpha significance level > 0.05).
A past medical history was taken from each individual to obtain information on past syphilis infection. The results of past and current syphilis are shown in the table below (Table 3).
Finally, those HIV clients who had a history of syphilis infection in the past were more likely than those who had none to be HIV-syphilis coinfected (53.6% vs 34%, respectively; OR 2.236; 95% CI 1.045 -4.782). The relative Odds of HIV-Syphilis coinfection is calculated at 1.573 (95% CI: 1.044 -2.370).

Discussion and conclusion
The correlates of HIV, syphilis and hepatitis B coinfections were age (between 20 and 39 years), primary education, and of course sexual activity. This may be influenced by the rural settings where our study was conducted (especially low level of education). The HBV/HIV coinfections prevalence of 9.5% in this study is in line with the prevalence of 9.9% documented in Zambia by Kapembwa and colleagues 27 , and also the 12.2 reported by Vinikoor et al. 19 . The later also demonstrated that HIV coinfected adult males were more likely to be coinfected with HBV than their female counterparts. This contrast to our findings may be due to our small sample size. In our study, Pearson Chi-square analysis showed no statistically significant difference between gender for both HIV/HBV and HIV/Syphilis coinfections. Our findings demonstrate that HIV infected clients are more likely to be infected with other STIs, especially syphilis. The coinfection prevalence of 40.5% for HIV and syphilis agrees with previous study conducted in Zambia (43%) by Odom and colleagues 16 . We found past syphilis infection to have positive association with two-fold increase risk of HIV/syphilis coinfections. CONCLUSION: The high prevalence rates for HIV, HBV, and syphilis coinfections strongly indicate the need for HBV and syphilis screening for HIV infected individuals. The 2018 Zambian consolidated guidelines for prevention and treatment of HIV infection to address the management of these coinfections 32 . However, there is urgent need to monitor the implementation of HBV and syphilis testing among HIV infected subject to close the gap between policy and practice. Furthermore, in previous study conducted in Zambia, Odom and colleagues 16 demonstrated that a high proportion of RPR positive infections remain serofast despite treatment. The high number of patients previously treated for syphilis who retest positive for syphilis in this study calls for use of the Venereal Disease Research Laboratory test to identify true syphilis infection (titers ≥ 1:8 dilutions, strongly suggestive) 33 . Both government and Non-governmental organizations need to up their efforts to support these health care service needs appropriately. This project contains the following extended data: Data collection sheet.docx (data collection sheet)

Grant information
The author(s) declared that no grants were involved in supporting this work.

INTRODUCTION
This has accurate information but fails to specifically indicate relevance and need for study. What are the current prevalence estimates for HIV/HBV and/or syphilis co-infections in Mkushi? in Zambia? Is this information lacking and, therefore, partly formed the basis for the research study?
Are laboratory-based screening and/or diagnostic tests for the three infections routinely conducted together in much of Zambia, or this is lacking and provided another impetus for the study? METHODS Clarify if the "Mkushi District Health Office" has formal authority to independently review and give ethical approval to conduct clinical research studies consistent with the norm. Presented data are scanty and lack details. The authors are encouraged to expand this section and give additional information as well as correct errors in counts. Consider reporting socioeconomic and demographic characteristics of participants in Table format. Give values as mean ± STD here and elsewhere, accordingly.
Stated syphilis seroprevalence is 51 of 126 cases. This is then subdivided into age groups, i.e., 1 for below 20 years; 24 for 20-29 years; and 15 for 30 -39 years. But this yields a total of 40 and not 51 cases. What happened to the missing 11 patients?
Like with syphilis, should also report "seroprevalence" data for HIV and HBV both by age and gender. Authors need to be consistent accordingly.
Need clarification on the reliability of the collection of "past syphilis infection" history. How exactly was this done?
More rigorous statistical analyses needed to properly interpret data and show significance as applicable. The significance of findings are unconvincing (and lost) as reported.

DISCUSSION
Interpretation and significance of findings are unconvincing. It is difficult to make an independent assessment since presented data lack much detail.
Need to explain what gaps in knowledge have been filled by the study. For example, how will this information assist with patient care and/or re-direction of resources for concomitant screenings/diagnosis of HIV, HBV, and syphilis?
Are the above services readily available in all health centers in Zambia, or there is a disparity between urban and rural health centers? Explain.
Need to discuss the limitations of the research study.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results?