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

ABSTRACT Background: Human Immunodeficiency Virus, syphilis and Hepatitis B Virus are major global public health problems, they are sexually transmitted infections with overlapping modes of transmission and affected populations. Objective: 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. Methods: A total number of 126 sera were collected from HIV 1 infected 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. Results: Of the 126 participants (out of 131 enrollments), Hepatitis B surface antigen (HBsAg) was 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). Conclusion: The high prevalence rates for HIV, HBV, and syphilis 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).


Introduction
Hepatitis B virus, together with syphilis and human immunodeficiency virus (HIV), are important public health challenges worldwide 1 , they are transmitted through sex (Sexual transmitted infections, STIs) 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 infections 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, STIs pose major socioeconomic challenge on treatment costs 5,9,10 . Many studies have demonstrated a twosided relationship between HIV and a number of STIs, including syphilis and HBV 4 . Syphilis and HBV has the potential to increase genital and plasma HIV 1 RNA levels. This increase can raise the likelihood of transmission of HIV 1 to others. In the same way, HIV 1 infection has the potential 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 location 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 these 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 and the region 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. The lack of current prevalence estimates of HIV/HBV and/or syphilis co-infections in Mkushi provides the rationale for this project. Also, laboratory based diagnostic tests for the three infections are not routinely conducted together in much of Zambia.

Ethical considerations
This study was conducted after obtaining permission from the Mkushi District Health Office. The waiver for ethical clearance was sought and obtained from the ERES Converge Zambian Institutional Review Board (IRB); the authority to publish was sought and obtained from the Zambian National Health Research Authority (NHRA). Participants were informed and provided verbal consent prior to enrollment in the study.

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. The study was conducted by a team of trained medical officers, nurses and laboratory technicians.
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

Amendments from Version 1
This new version of the study has addressed issues raised by our reviewers. The total number of patients who were seen during this period has also been included to the abstract as the enrollment. The relevance and need for the study have been included to the introductory notes of the current version. The term STI has been used instead of STD as it appears that many of these infections were sub-clinical. The statement on ethical clearance has been completely revised: "The waiver for ethical clearance was sought and obtained from the ERES Converge Zambian Institutional Review Board (IRB), the authority to publish was sought and obtained from the Zambian National Health Research Authority (NHRA)". The number of participants informed of the study and how/ who conducted the study have been described. The study citations have been checked and corrected accordingly (the studies conducted in Zambia and the region have been mentioned). All grammatical errors have been corrected, the sentence referring to the national guidelines has been revised. Limitation of screening diagnostic tests have been described. No data errors were observed. The limitations of the research have also been discussed. Disclose competing interests: the authors have no conflicts of interest to disclose.
Any further responses from the reviewers can be found at the end of the article REVISED 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

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.
Limitations: The difference in performance observed with various hepatitis, syphilis, and HIV diagnostic test kits available in the market might influence the comparison of results with other studies. Moreover, this facility based survey may limit the generalizability of findings to all people living with HIV due to possible selection and information bias.
However, as shown earlier, the occurrence of these coinfections is clinically consequential. The information provided in this study will therefore assist policy makers re-direct resources for concomitant screenings/diagnosis of HIV, HBV, and syphilis.
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 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. Thank you for the opportunity to review this manuscript. Overall the manuscript is sound but there are several grammatical errors and some sentences that do not make sense eg the reference to the National Guidelines. Additionally, there does not seem to be ethical approval. My understanding is that Mkushi District Health Office is the administrative body that falls under the Ministry of Health and manages the public health services in that area. Unless they have a special mandate, of which I am not aware, they are not authorized to provide Ethical Clearance as they are not considered a regulatory body. They are, however, required to give permission for research to occur in their facilities. Given that this study involves human subjects, I recommend getting retrospective ethics approval if not yet obtained or, if it has been obtained, including this in the manuscript from a recognised regulatory authority. Also, please include the total number of patients who were seen during the same period as the enrolment (ie the denominator), it is not clear what proportion of patients who were seen, enrolled in the study and had a specimen drawn. Please also check citations-the paragraph that starts with ... in Zambia (17-29) includes references that are regional but not necessarily in Zambia. Lastly, I suggest using STI instead of STD as it appears many of these infections were subclinical.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?
pertinent resources needed to curtail the infections while identifying gaps in the drive to end the HIV epidemic. However, the manuscript is not written to best advantage and will require significant revisions to be impactful. Within this context, the authors may wish to consider the following concerns.

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?
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.

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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?
The relevance and need for the study have been included. The number of participants informed of the study and how/ who conducted the study have been described. The 4 Ml venous blood draws were the routine blood draws for ART clients base line tests as recommended by the national guidelines. Limitation of screening diagnostic tests have been described. Data errors have been corrected. The total number of patients who were seen during this period has also been included as the enrollment. Citations have been checked and corrected accordingly.
The waiver for ethical clearance was sought and obtained from the ERES Converge Zambian Institutional Review Board (IRB), the authority to publish was sought and obtained from the Zambian National Health Research Authority (NHRA). This will also appear in the new version. The limitations of the research have also been discussed.