Syphilis and HIV Infection among Pregnant Women Previously Screened Negative during Their First Antenetal Care Visit (ANC) at Some Selected Health Facilities in the Buea Health District, Cameroon

Syphilis and HIV are amongst the world’s most widespread diseases, particularly in low-income countries. Syphilis and HIV infections during pregnancy have been associated with numerous adverse pregnancy outcomes. Of concern now are the rising rates of congenital syphilis and HIV in Cameroon. Cameroon only mandates testing pregnant women for syphilis and HIV during their first ANC visit. This study was aimed at determining the incidence of new syphilis and HIV infections and factors associated with pregnant women who previously tested negative during their first ANC visit. A cohort design was used, where 335 pregnant women were followed up for a period from December 2019 to August 2020. A blood sample was drawn and the serum was analyzed using the WANTAI ELISA and AIDTM HIV 1 + 2 Ag/Ab ELISAPlus test methods for syphilis and HIV respectively at three intervals. A questionnaire was used


Introduction
Sexually transmitted infections (STIs) are an emerging public health concern and are amongst the world's most widespread diseases, particularly in low-income countries [1]. The bacteria Treponema pallidum is the organism that causes the systemic disease syphilis. The infection can be classified as congenital or acquired, transmitted through unsafe sexual intercourse or blood transfusion [2].
The last estimation of WHO showed that approximately, 18 million women worldwide are infected with syphilis and those with pregnancy have approximately 305,000 fetal and neonatal deaths every year and leaving 215,000 infants at increased risk of dying from prematurity, low-birth-weight or congenital disease [3] [4]. Infection with the Human Immunodeficiency Virus (HIV) is also an important public health problem that affects many people around the world. The Global HIV/AIDS statistics estimated 37.9 million, people living with HIV in 2018 [5]. Sub-Saharan Africa remains the most affected area with 70% of all cases [6]. Studies have shown that syphilis infection causes genital ulcers, creating a site for HIV entry due to disruption of the natural mucosal and epithelial barrier integrity [5] [6]. In Africa, it was observed that the overall syphilis prevalence in pregnant women ranges from 4% to 15%, and early untreated syphilis will result in a stillbirth in 25% of pregnancies and death in 14% of newborns [7]. A study across 43 sub-Saharan African countries has shown that adverse outcomes such as stillbirth, neonatal death, low birth weight, and congenital syphilis occur in an estimated 206,000 pregnancies each year [8]. Pregnant women infected with HIV have a higher risk of adverse pregnancy outcomes [9]. An estimated 24% of  [10]. Reports from CDC have recommended testing for syphilis and HIV during pregnancy at the first prenatal visit, and to repeat the testing for mothers at risk during the third trimester (preferably 28 -32 weeks). Increased testing for HIV during pregnancy has helped in drastically reducing vertical, or mother-to-child, transmission of HIV.
Many health facilities [11] require testing pregnant women for HIV during their first and third trimesters, leading to a public health victory in preventing perinatal cases of HIV. However, in most low-income countries most of these rec-

Study Area and Population
This study was carried out among pregnant women who were diagnosed negative for syphilis and or HIV infection during their first antenatal visit at some stratified selected health facilities. We targeted settings in Buea Health District (Rural and Urban) where the potential for benefit would be the greatest and the study would be the most feasible. The three participating ANC clinics are Regional Hospital Buea, CMA Muea, and St Veronica Polyclinic (Biaka). These sites were selected because they are found around the epicentres of high venereal diseases as they are surrounded by many Universities and higher institutions.
The selected clinics comprise a Unit specialized in HIV counselling.

Study Design
A hospital-based cohort study was carried out among 335 pregnant women who

Sampling Technique
A consecutive sampling technique and review of medical records were used to recruit 335 pregnant women who tested negative for syphilis and HIV during their first ANC visit. Participants were recruited over two weeks through a two-stage process. First, the service providers sensitized women coming for the first ANC visits at their respective centres about the study, giving only basic information about the study. This was to give all clients undergoing the screening test for HIV and syphilis an opportunity to choose to participate while avoiding a potentially biased approach by study staff. Subsequently, those who were willing to participate in the study were referred (using a study referral notepad) to the researcher sitting in another room closely. It was considered that those who presented to the researcher were truly willing to participate in the study.

Ethical Consideration
The researcher conducted consenting to every pregnant woman individually by providing in-depth information about the study and answering questions they posed.
Those willing to participate in the study provided written consent. Only pregnant women who had previously tested negative for Syphilis and HIV consented and enrolled. During consenting, everyone was assured of confidentiality and privacy; informed that their information will be kept confidential and that they should not use their names when filling out the forms. Each was assigned a unique number code.
Participation in this study had a risk of mild discomfort, bruising, bleeding, and blood clot at the needle puncture site during the collection of blood. However, in case of a blood clot (hematoma), the needle was removed and pressure applies at the site for two minutes. For discomfort, the patients were talked to and given assurance. In case of excessive bleeding, dry cotton was applied at the site until there is a complete stoppage. Participants that tested positive in the course of the follow-up, were referred to see the counsellor, who disclosed their results and were further sent to see the physician for management. The study was conducted after obtaining clearance from the Institutional Review Board During data collection, all pregnant women eligible for the study was asked to sign the informed consent form before blood collection. For pregnant women aged less than 21 years, assent was obtained from parents, guardians or husbands.

Data Collection
Data was collected using a pretested questionnaire. This questionnaire was used to explore information on socio-demographic characteristics and clinical data, risk factors for infection with syphilis (such as level of education, number of sexual partners, marital status, partner's status) and past medical history.
Blood samples were collected into dry tubes, centrifuged and the serum was preserved.

Laboratory Analysis
5 mL of Blood samples were collected through the venous puncture into dry tubes. These blood samples were centrifuged and the serum was obtained and transferred to cryo tubes which were frozen at −40 c. These samples were later used to test for syphilis using the WANTAI anti-TP ELISA test methods meanwhile the HIV was analyzed using the AIDTM HIV 1 + 2 Ag/Ab ELISAPlus test methods.

Data Analysis
Data was input into a Microsoft Excel sheet for easy access while the questionnaire was properly preserved. All source documents and laboratory reports were reviewed and ensured that they are accurate and complete. Data were analyzed using the SPSS version 23.0 software for statistical analysis. The Chi-square test was used to access association and the multinomial logistic regression was used to access risk factors at a 95% confidence interval (95% CI). A P-value < 0.05 was considered significant.

Socio-Demographic Characteristics of Pregnant Women in the Buea Health District
Out of the 335 pregnant women who were enrolled in this study, the majority originating from the North West region was 203 (60.6%

Prevalence of Syphilis and HIV Infections Post First ANC Visit in the Buea Health District, Cameroon
Out of the 335 pregnant women who previously tested negative for syphilis and HIV during their first ANC visit who participated in this study, the majority 49 (14.6%) were later diagnosed positive for syphilis, followed by 54 (16.1%) positive for HIV and 10 (3%) co-infected with Syphilis and HIV (as shown in Figure 1).

Prevalence of Syphilis and HIV Infections among Pregnant Women Follow-Up at Two Months Post 1 st ANC, 2 nd and 3 rd Trimester Visit at the Buea Health District Cameroon
Concerning trimester distribution with regard to the incidence of new cases of HIV and syphilis within the 9-month follow-up, it was observed that the majority of the pregnant women became diagnosed positive for syphilis in the second trimester 32 (9.4%) as compared to the third trimester 17 (5.1%) as seen in Figure 2.

Co-Infection of Syphilis and HIV among Pregnant Women Occurring at the 2 nd and 3 rd Trimester of Previously Tested Negative Participants
It was observed that new cases of syphilis and HIV co-infections occurred mostly in the second trimester 8 (2.3%) and a few 2 (0.5%) occurred in the 3 rd trimester as seen in Figure 4.

Incidence of Syphilis and HIV Infections with Respect to Age among Pregnant Women Previously Tested Negative during Their First-Trimester Visit at the Buea Health District, Cameroon
The incident of syphilis during pregnancy was higher at 8% in the older age  Figure 5.

Prevalence of Syphilis and HIV Infections with Respect to Educational Level among Pregnant Women Previously Tested Negative during Their First-Trimester Visit at the Buea Health District, Cameroon
The prevalence of syphilis was higher in pregnant women who ended at the   and tertiary level 2.8%. The prevalence of HIV was higher in pregnant women who ended at the Primary level 18.4% followed by those who ended at the Secondary level 5.6% and the least being those who ended at the tertiary level 1.9%.
Co-infections were higher in those who ended at the primary and secondary levels with a prevalence rate of 4.1% and 1.5% respectively. There was no co-infection prevalence at the tertiary level likewise those with no formal education.
Those with no formal education also had no Syphilis or HIV prevalence. The distribution of new cases of HIV and syphilis during pregnancy as per educational level can be seen in Figure 6.

Risk Factors Associated with Contracting New Syphilis Infections among Pregnant Women Previously Tested Negative during Their First ANC Visit at the Buea Health District, Cameroon
The findings of this study revealed that pregnant women between the age group of 16 -29 years had a higher risk aOR (1.302, 95% CI) of contracting syphilis infection after their first semester ANC screening negative test results. It was also observed that pregnant women resident in Buea station were three times more likely to contract a new infection of syphilis aOR (3.158, 95% CI). Pregnant women with no formal education and primary education levels were more likely to contract the new infection and were statistically significant with aOR of (9.055, 95% CI) (P = 0.001) and (6.764, 95% CI) (P = 0.006) respectively. Pregnant women in the second trimester also showed a high risk of acquiring new infections of syphilis aOR (2.018, 95% CI). Pregnant women who had inadequate knowledge of syphilis were 2 times more likely to acquire a new infection of syphilis during pregnancy aOR (2.176, 95% CI) and those who were not aware of their partner's syphilis status also had a high risk at 3 times more likely of acquiring new syphilis infection aOR (3.190, 95% CI) as seen in Table 2.

Risk Factors Associated with Contracting New HIV Infections among Pregnant Women Previously Tested Negative during Their First ANC Visit at the Buea Health District, Cameroon
The findings of this study showed that pregnant women between the age group of 16 -29 years had a higher risk aOR (1.302, 95% CI) of contracting HIV infection. Those who had primary and secondary level of education was eleven and three times more likely to contract new HIV infection which was statistically significant with aOR of (11.912, 95% CI) (P = 0.001) and (3.144, 95% CI) (P = 0.002) respectively. Pregnant women two months post 1 st ANC and second trimester also showed a high risk with aOR (1.174, 95% CI) and (1.081, 95% CI) respectively of contracting new HIV infection. Pregnant women who had more than one sexual partner were seven times at risk of contracting the new HIV infection aOR (7.342, 95% CI) while those who had inadequate knowledge were two times at risk of acquiring this infection aOR (2.866, 95% CI) as shown in Table 3.  With the high rate of pregnancy in the Fako health district, the Cameroon health care system should improve surveillance system for HIV and Syphilis.

Discussion
The prevalence of syphilis infections in pregnant women post-first ANC visit was high (13%) considering the fact that these women had been screened for this disease during the first-trimester visit and all tested negative. This study corroborate with a study that indicate that syphilis is fast becoming a growing burden in the population, Kengne-Nde et al., 2020 [14]. However, the findings of our study were contrary to the study conducted by Halle-Ekane et al., 2018 [12] in Buea which had a prevalence of 6.3% and far lest obtained by Befekadu B et al., 2022 [15]. The difference might be due to the fact that their study was a crosssectional study wherein data was collected just at one point in time. Our findings were also higher than the 3.92% gotten by Roch et al., 2017 [16]  than that of this study with a prevalence rate of 1.2% in one of its sentinel study sites of Impfondo. The differences recorded in most of these studies might be related to their studies using cross-sectional studies. Using a cross sectional study design might lead to under reporting of most cases. Since new cases are not easily captured. Risk factors associated with contracting new HIV and Syphilis infections among pregnant women who previously tested negative during their first ANC visit at the Buea Health District, Cameroon associated with the younger age group, leaving in an urban area, having lower level and having no formal education, inadequate knowledge on the diseases, many sexual partners and pregnant women unaware of their partner status.
Following this study, the age group 16 -29 years which is the younger age group were at increased risk of contracting both syphilis and HIV. Another study carried out by Roch et al., 2017 [16] in Congo showed that younger pregnant women had a low risk of contracting Syphilis and HIV but rather older pregnant women were at increased risk of contracting these diseases. This could be due to the fact that the older pregnant women in that study area were more exposed to these infections. This study also showed that participants living in Buea station were at increased risk of contracting both syphilis and HIV. Other studies such as Jodie et al., 2016 [19] rather compared their study areas in terms of rural and urban settings and the urban areas showed significant risks. Preg-nant women with no formal education, primary and secondary levels of education showed high risks of contracting syphilis and HIV infections. Pregnant women with no formal education and those with primary levels of education showed significant risk with P values of 0.001 and 0.006 for syphilis respectively and a P value of 0.001 and 0.002 for HIV respectively. Roch et al., 2017 [16] showed no relationship between educational status and syphilis or HIV. This could be due to an increased awareness in the study population. There are several risk factors that were shown to be associated with contracting new HIV and Syphilis infections among pregnant women who previously tested negative during their first ANC visit at the Buea Health District, Cameroon, There is a need for constant education on these diseases during every visit. Persons in charge of carrying out the education session should highlight these identifiable factors when talking to pregnant women during their ANC visits despite the outcome of their previous or current test results.

Conclusions
There are increased new cases of syphilis and HIV infection during pregnancy among women who previously tested negative at their first ANC visit.

Risk factors associated with contracting new HIV and syphilis infections
among pregnant women who previously tested negative during their first ANC visit at the Buea Health District, Cameroon, were associated with a younger age group, residents of an urban area, lower level and no formal education, inadequate knowledge on the diseases, many sexual partners and pregnant women unaware of their partner status.

Consent
Participants were issued consent/assent forms to seek for their/guardian's approval. Participants were recruited only after they or their guardians signed the consent/assent forms indicating their acceptance to participate in this study.

Ethical Approval
The study was conducted after obtaining clearance from the Institutional Review Board (IRB) of the Faculty of Health Science, University Of Buea and also an administrative authorization was obtained from the delegation of public health in Buea. An additional clearance was obtained from the Regional Hospital Buea, Muea Integrated Health Center and St Veronica Polyclinic where study participants were recruited.

Data Availability Statement
Data is available at the University of Buea library.

Author's Contribution
This work was carried out in collaboration among all authors. Authors MGM