Hookworm Infections and Sociodemographic Factors Associated With Female Reproductive Tract Infections in Rural Areas of the Central Region of Togo

Female reproductive tract infections (FRTIs) have a huge impact on women’s health including their reproductive health in rural areas. Immunomodulation by helminth infections could influence the occurrence of FRTIs. This study aimed to investigate the association between FRTIs, hookworm infections, and sociodemographic factors in six rural areas of the central region of Togo. A semi-structured questionnaire was used to collect sociodemographical information, and parasitological assessments were used to diagnose helminth infections. Moreover, cytobacteriological examination of vaginal swabs was performed for the diagnosis of candidiasis and bacterial vaginosis (BV), and real-time PCR method was used to determine sexually transmitted infections (STIs). Finally, a logistic regression analysis was performed to assess the relationship and association of these factors to FRTIs. The prevalence of FRTIs was 82.3% including STIs (74.38%), BV (31.79%), and vulvovaginal candidiasis (9.85%). In detail, FRTIs were caused by bacteria such as Ureaplasma parvum (50%), Ureaplasma urealyticum (26.5%), and Mycoplasma hominis (17.5%) and viruses such us cytomegalovirus (5%) and human papilloma virus (HPV) (20%). No cases of Haemophilus ducreyi, Treponema pallidum, or varicella-zoster virus (VZV) were observed. Interestingly, women who had hookworm infections were at high risk of HPV. The use of condoms was a protective factor [adjusted odds ratio (aOR) = 0.23; 95% CI [0.11–0.51)], while the use of contraceptive methods was a risk factor [aOR = 2.49; 95% CI (1.19–5.19)] for STIs. The risk of BV was lower among participants who had more than four pregnancies [aOR = 0.27; 95% CI (0.11–0.65)]. Furthermore, women who had ever been paid for sexual intercourse were at high probability risk of vulvovaginal candidiasis [aOR = 16.92; 95% CI (1.46–196.48)]. This study highlighted risk factors associated with FRTIs, the control of which would help to reduce the incidence of these diseases. Health-care professionals could develop education and sensitization strategies based on these risk factors, and anti-hookworm treatment concepts may be taken into consideration to minimize the risk of HPV infections.

Female reproductive tract infections (FRTIs) have a huge impact on women's health including their reproductive health in rural areas. Immunomodulation by helminth infections could influence the occurrence of FRTIs. This study aimed to investigate the association between FRTIs, hookworm infections, and sociodemographic factors in six rural areas of the central region of Togo. A semi-structured questionnaire was used to collect sociodemographical information, and parasitological assessments were used to diagnose helminth infections. Moreover, cytobacteriological examination of vaginal swabs was performed for the diagnosis of candidiasis and bacterial vaginosis (BV), and real-time PCR method was used to determine sexually transmitted infections (STIs). Finally, a logistic regression analysis was performed to assess the relationship and association of these factors to FRTIs. The prevalence of FRTIs was 82.3% including STIs (74.38%), BV (31.79%), and vulvovaginal candidiasis (9.85%). In detail, FRTIs were caused by bacteria such as Ureaplasma parvum (50%), Ureaplasma urealyticum (26.5%), and Mycoplasma hominis (17.5%) and viruses such us cytomegalovirus (5%)

INTRODUCTION
Female reproductive tract infections (FRTIs) have a high incidence among various gynecological diseases (Yan et al., 2018). FRTIs comprise (i) endogenous infection such as bacterial vaginosis (BV) and vulvovaginal candidiasis; (ii) sexually transmitted infections (STIs) such as syphilis, gonorrhea, and chlamydia; and (iii) iatrogenic infections (Moragianni et al., 2019). Worldwide, FRTIs are a major public health problem and affect predominantly young women (Torondel et al., 2018). It has been observed that the prevalence of these infections varies a lot between countries and even among regions belonging to the same country (Rabiu et al., 2010). Sequelae of untreated FRTIs affect the well-being of women, such as infertility, cervical cancer, ectopic pregnancy, stillbirth, and spontaneous abortion (Diadhiou et al., 2019). Interestingly, FRTIs seriously impact women in the Sub-Saharan African region causing around 85% infertility (Hussen et al., 2018). Helminth infections could expose the person to various infections, protect from auto-immune diseases, or decrease disease severity and influence vaccine efficacity (Moreau and Chauvin, 2010;Hartmann et al., 2019). Helminthic infections which are predominantly prevalent in Africa could therefore contribute to the establishment of infectious diseases (Arndts et al., 2015;Katawa et al., 2015;Ritter et al., 2018Ritter et al., , 2019. The impact of helminthiasis on female reproductive health and associated diseases is widely documented. For instance, (i) Wuchereria bancrofti and Schistosoma haematobium infections were associated with an increased risk of HIV infection, and (ii) an increased prevalence of human papilloma virus (HPV) infection has been observed in the soil-transmitted helminthsendemic area of Peru (Chetty et al., 2020).
Both FRTIs and helminthic infections are co-endemic and have been shown to be highest in low-and middle-income countries (Chetty et al., 2020). Thus, this study was designed to investigate the association between FRTIs, hookworm infections, and sociodemographic factors in the central region of Togo.

Study Population and Ethics
This was a cross sectional study conducted in October 2019 in six villages of the central region of Togo, namely, Sakalaoudè, Tseve, Fazao, Sagbadai, Alheridè, and Kikimini. The sample size (367) was calculated using Schwartz formula n = Z 2 P (1 -P)/d 2 where Z, the accepted risk error, is 1.96; d, the precision, is 0.05; P, the prevalence. In the central region of Togo, the prevalence of STIs is estimated at 25% according to the sociodemographic survey of 2013-2014 (Enquête Démographique et de Santé du Togo 2013-2014, available online) (World Bank, 2017). So using these factors and the prevalence of 25%, the minimum sample size is 288. Therefore, 367 women, sexually active and premenopausal, aged between 18 and 56 years old, were enrolled. Pregnant and/or women positive for HIV were not included in this study. All participants gave their written consent to participate. This study was ethically approved by the "Comité de Bioéthique pour la Recherche en Santé (CBRS)" of the Togo Ministry of Health (N • 26/2017/CBRS) and the Ethical Review Board of the University Hospital Bonn (Lfd. Nr. 273/16).

Variables
Sociodemographic and helminth infections were assessed as risk factors.
For sociodemographic risk factors, a pre-tested and semistructured questionnaire was used to collect sociodemographical data. The questionnaire was validated during a pilot study (Katawa et al., 2021) and administered by a trained medical laboratory technician in French or in a local language. Then, the participants who agreed moved freely to the sampling sites with a ticket proving their participation in the study. The probable associated risk factors chosen for analysis included helminth status, age, marital situation, duration of relationship with current partner, level of education, monthly income, average length of menstruation, vaginal hygiene methods, use of objects for intimate cleansing, introduction of products in vagina, having sex during menstruation, number of pregnancies, outcome of the last pregnancy, use of contraception methods, alcohol consumption, tobacco consumption, having sexual relationships during last 6 months, use of condoms, number of sexual partners during lifetime, use of condoms during last sexual intercourse, partner's HIV positivity, having multiple sexual partners, having a sexual partner who has other sexual partners, and having paid intercourses/sexual relationships. Parasitological assessment was performed for the diagnosis of helminth infections. The confidentiality was maintained by an anonymization number that was attributed to each participant.

Parasitological Assessment
For the diagnosis of helminth infections, stool, urine, and skin snips were collected. Using Kato Katz and urine sedimentation methods, helminths and S. haematobium eggs were determined in the stool and urine, respectively. In addition, two skin snips of 1-2 mm were taken from the left and right iliac crests using biopsy forceps. They were then incubated in 100 µl of NaCl (0.9%) in 96well plates for 18-24 h at room temperature to detect the presence of Onchocerca volvulus microfilariae using microscopy.

Reproductive Tract Infections Diagnosis
FRTIs were defined base on the Nugent score (>6) and the vaginal pH (>5).

Candidiasis and Bacterial Vaginosis Diagnosis
Vaginal swabs were collected by physicians for cytobacteriological examination using fresh vaginal swab smears for yeast screening and gram staining for evaluation of bacterial flora by the criteria of Nugent (BV screening).

Diagnosis of Female Reproductive Tract Infections
One vaginal swab and one cervical swab were collected by physicians from each participant and stored in 1 ml of eNAT R medium (COPAN Italia S.P.A., Brescia, Italy). DNA was extracted using the Seegene Microlab Nimbus IVD automation (Seegene Inc., Seoul, South Korea) according to manufacturer's instructions. Real-time PCR for 27 germs implicated in FRTIs and STIs was performed. Amplification was done on Bio-Rad CFX96 Real-time PCR System (Biorad) using the Allplex TM STI Essential Assay kit that allows the simultaneous detection and identification of seven pathogens: Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Trichomonas vaginalis, Ureaplasma parvum, and Ureaplasma urealyticum; the Allplex Genital Ulcer Assay kit that allows the detection of six pathogens: Cytomegalovirus (CMV), Haemophilus ducreyi, herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), Treponema pallidum, and varicellazoster virus (VZV); and the Anyplex II HPV HR detection kit that allows the detection of HPV infection. All reagents used for DNA extraction and FRTIs diagnosis were manufactured by Seegene Inc. (Seoul, Republic of Korea).

Statistical Analysis
SPSS software (IBM SPSS Statistics 21; Armonk, NY) and GraphPad PRISM software 5.2 for Windows (GraphPap software, Inc., La Jolla, United States) were used to perform statistical analysis. Chi-square test was used to assess the prevalence differences (p-value threshold 0.05). Univariate and multivariate logistic regression analyses were used in order to investigate the risk factors associated with STIs, HPV, BV, and candidiasis including the calculation of odds ratios (OR) with 80% confidence intervals (CI) and adjusted odds ratios (aOR) with 95% CI. A threshold of p-value < 0.2 and < 0.05 were considered for univariate and multivariate analyses, respectively, and we declared the significance of OR/aOR if the value "1" is not included in the CI.

Prevalence of Female Reproductive Tract Infections
The overall prevalence of FRTIs among women of the Central region of Togo was 82.3% (Figure 1). Considering the prevalence per village, we found a high prevalence at Sakalahoudè (64.29%) and Tcheve (50%). Among all FRTIs screened, STIs were more prevalent (74.38%) than BV (31.79%) and candidiasis (9.85%) (Figure 1). The most infections implicated in STIs were caused by U. parvum (50% of women infected), followed by U. urealyticum (26.5%) and M. hominis (17.5%) (Figure 2). Viral infections detected included: HPV (20%) and CMV (5%). There were no cases of H. ducreyi, T. pallidum, or VZV. The prevalence of FRTIs in our study area was elevated with the predominance of hookworm infections. Therefore, we investigated the association between FRTIs and hookworm infections.
Hookworms were the most frequent helminthic infection (95.34%); Schistosoma mansoni and Hymenolepis nana were found, each on 2.33% of parasites that infected women. Therefore, univariable logistic regression analysis was performed. We could not observe any association between hookworm infection and STIs

Sociodemographic Factors Associated With Female Reproductive Tract Infections
Here we aimed to investigate the association between sociodemographic factors with FRTIs. Univariate logistic regression analysis revealed the independent risk factors associated with STIs ( Table 2) After controlling for confounding parameters through multivariable logistic regression analysis, adjusting independent factors linked to STIs, we found that the use of condoms was a  Following the multivariate logistic regression analysis, the use of condoms [aOR = 0.13; 95% CI (0.03-0.49)] appeared to be a protective factor against HPV infections. while having more than one partner during the lifetime [aOR = 2.79; 95% CI (1.32-5.95)] appeared as a risk factor for HPV infections.
In Table 4, the univariate regression analysis revealed that the risk of BV infections was low among participants who had more than four pregnancies [OR = 0.49

DISCUSSION
This study revealed that STIs were predominantly prevalent (74.38%) in six rural villages of the central region of Togo. Serious complications of STIs are ectopic pregnancy, pelvic inflammatory disease, preterm labor, miscarriage, stillbirth, and congenital infection, and they may lead to chronic disability (such as infertility and genital cancer) and death. Here, we diagnosed 27 germs incriminated in STIs. In detail, U. parvum was the most prevalent affecting 50% of women, followed by U. urealyticum (26.5%) and M. hominis (17.5%). Those three species belong to the gender Mycoplasma, a gender of bacteria frequently isolated from the genital tract of both men and women (Kokkayil and Dhawan, 2015). In Italy, Leli et al. (2018) found that U. parvum was the most isolated mycoplasma among women with a prevalence of 38.3%, and a study in South Africa revealed that U. parvum was isolated from 72.4% vaginal samples of pregnant women (Redelinghuys et al., 2013). In general, U. parvum is associated with intrauterine inflammation (Kasper et al., 2010). Among viruses found in the cohort, human CMV was predominately prevalent (5%). In Africa, CMV is a neglected pathogen, but its burden is important with a seroprevalence close to 100% (Bates and Brantsaeter, 2016). CMV is a member of Herpesviridae family and is classified as an emerging STI (Kent, 2017) that is transmitted through direct contact with multiple fluids of the body like saliva, urine, milk, and genital secretions. In addition, it can also be transmitted from an infected mother to her fetus (CDC, 2020b). The virus can stay latent in infected persons until the immune system weakening allows the virus to reactivate (Grinde, 2013).
In regards to risk factors, this study revealed that the use of contraceptive methods appeared as a high risk of STIs.
Contraceptives are used for birth control and have a socioeconomic impact. According to Deese et al. (2018) some contraceptive methods have related to STIs, but robust studies should be conducted to provide accurate information and facilitate contraceptive choice by women. Moreover, the presence of STIs was also associated with alcohol consumption, confirming other studies showing that increased risk of STIs was influenced by alcohol drinking across a wide variety of populations (Arasteh et al., 2008;Hutton et al., 2008). That is justified by the fact that alcohol drinking can lead to immune deficiency and increase sensitivity to infectious diseases (Zitkute and Bumbuliene, 2016). Another independent factor associated with STIs was the type of relationship and sexual behavior, especially the number of sexual partners during the lifetime, confirming a study in Brazil among rural women, which revealed that a higher number of partners in life was a risk factor for STIs (Oliveira et al., 2008). In addition, having had more than one partner during the lifetime appeared as a high-risk factor for HPV infections, confirming a previous study from Molano et al. (2002) who found that having had more than one regular sexual partner increased HPV infection risk (OR 1.9). In general, HPV plays an important role as a leading cause of most cervical cancers. More than 100 types of HPV have been identified and at least 14 of them are associated as risk factors for cancer and especially 2 types (HPV 16 and HPV 18) cause 70% of cervical cancers and pre-cancerous cervical lesions (WHO, 2019). Nevertheless, in our cohort, HPV 16 and HPV 18 were less prevalent (respectively, 1.54 and 1.03%), but associations to cancer diseases need to be investigated in future studies.
Interestingly, the impact of helminth immune regulation on susceptibility to STIs is known. Given the global incidence of helminths and their detrimental impact on public health (WHO, 2017), the geographical overlap between helminth exposure and STIs might be a result of parasite-induced changes on female reproductive health (Gopinath et al., 2000;Wolday et al., 2002) and helminth-induced immunomodulation (Brown et al., 2005). Indeed, here we showed that women who had hookworm infections had a 2.22 times risk of HPV infection than those who were not infected by hookworm. Gravitt et al. (2015) found that women infected with soil-transmitted helminths (STHs) had 60% higher prevalence of HPV, compared with those without STHs infection with a prevalence ratio of 1.6. The helminth-induced Th2 response in vagina could explain the plausibility of high HPV occurrence (Gravitt et al., 2015).
Concerning endogenous infections, 31.79% of women were affected by BV and 9.85% had candidiasis. In Sokodé, the principal city of the central region of Togo, Tchelougou et al. (2013) found on pregnant women a prevalence of 55.31% of Gardnerella vaginalis and 50.77% of candidiasis, which is caused by Candida species and has been shown to be responsible for inflammatory changes in the vaginal and vulvar epithelium (Jeanmonod and Jeanmonod, 2020). However, the differences between the studies could be due to pregnancy status with unbalance vaginal flora.
In addition, BV, which is the most common vaginal condition in women aged between 15 and 44 years (CDC, 2020a), is known to increase predisposition to STIs (Lata et al., 2010; Bautista et al., 2017). In detail, it is characterized by an altered vaginal microflora including an increase in the vaginal pH, reduced lactobacilli species, and increased hydrogen peroxide producing species and facultative and anaerobic bacteria numbers and/or types (Eschenbach et al., 2000). Interestingly, the risk probability of BV was high among those who were not married or not in a relationship and those who had multiple partners. Ranjit et al. (2018) found that 100% of unmarried women were more prone to BV, and indeed, another study revealed that BV has been associated with having three or more male sexual partners in the past 12 months (OR = 1.60, 95% CI: 1.19-2.04) (Smart et al., 2004). Moreover, the number of pregnancies more than 4 was a protective factor against BV infections. This was in line with a study conducted in Ghana where Konadu et al. (2019) found that the fact of having more than four pregnancies was protective for BV.
In a logistic regression model with monthly income and practicing paid sexual intercourse, we found that women who practiced paid sexual intercourse were at high probability risk of candidiasis. Unlike our results, Konadu et al. (2019) did not find any possible risk factor associated with candidiasis, although they did not include the practice of paid sexual intercourse as a possible risk factor. We can notice that the study population of Konadu et al. (2019) was pregnant women attending antenatal clinic, whereas our study population was exclusively non-pregnant women.
Finally, the use of condoms was a protective factor against STIs, confirming a previous study (Ginindza et al., 2017). Moreover, several studies revealed that consistent condom use was also associated with significantly decreased risk of gonorrhea, chlamydia, genital ulcer disease, BV, HPV, and pelvic inflammatory disease but did not have an impact on candidiasis (Baeten et al., 2001).
The present study had some limitations including the final samples size and one stool sample per participant.

CONCLUSION
This study depicted a high prevalence of FRTIs in six villages of the central region of Togo. Among them, STIs were predominately prevalent, and factors such as helminth infection, the use of contraceptives, the number of pregnancies, the number of partners, the monthly income, the methods of cleansing, the marital status, and alcohol drinking were associated with the occurrence of FRTIs. Local healthcare authorities must focus on women education and sensitization strategies based on these risk factors to optimize prevention and control measures against FRTIs.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Comité de Bioéthique pour la Recherche en Santé (CBRS) du ministère de la santé du Togo. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
GK, AHA, CNT, PT, CV, AN'd, SA, HK, BM, and OA carried out the survey and the field work. CV, MR, GK, AHA, MO, CNT, and PT analyzed the samples. GK, MR, LEL, AH, WH, MK, YA, and SK designed the study protocol, analyzed, interpreted, and validated all the data. GK, WH, MR, CNT, and LEL wrote the manuscript which was then read and approved by all other authors. All authors contributed to the article and approved the submitted version.

FUNDING
This study was funded by the German Research Foundation (DFG; Grant LA 2746/2-1) within the "German-African Cooperation Projects in Infectiology" through grants awarded to GK, WH, and LEL. Additionally, AH was financially supported by the Federal Ministry of Education and Research (BMBF; initiative Research Networks for Health Innovations in sub-Saharan Africa: TAKeOFF) and was a member of the Excellence Cluster Immunosensation (DFG, EXC 1023). LEL and AH were also members of the German Center of Infectious Disease (DZIF).