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Is caused by the release of terminal-spine parasite eggs from female S. haematobium. FGS is a serious and chronic gynecological condition causing substantial morbidity among affected women [3,4]. When the eggs are deposited in the tissues of the cervix and lower female genital tract, the combination of the presence of the eggs with host inflammation and increased vascularity in the cervicovaginal mucosa produces typical intravaginal lesions that result in genital itching, pain, bleeding, and dyspareunia [5,6,7]. In addition, eggs deposited in the uterus and fallopian tubes can result in infertility [8]. It causes vaginal discharge, blood in urine, and abdominal and pelvic pain. It is a chronic gynecological condition that, if left untreated, can lead to a range of complications for women living with FGS, including infertility [8], abortion, genital ulcers, modified immunological responses to HPV and HIV [9], and increased risks of contracting HPV [10] and HIV [11,12].According to the World Health Organization (WHO), many young women in sub-Saharan Africa are at risk of acquiring FGS, HIV infection, and cervical cancer [13]. Furthermore, FGS remains largely overlooked within the national health systems and Neglected Tropical Disease (NTD) programs, and its prevalence is underestimated [14]. In some lower- and middle-income countries (LMICs) like Zambia, where schistosomiasis is endemic, knowledge and understanding of FGS among community members and healthcare workers (HCWs) within affected communities is often incomplete and confused with other Sexually Transmitted Diseases (STDs). In addition, there is a paucity of scholarly work exploring the knowledge, perceptions, and practices of women of reproductive age and in HCWs’ knowledge about FGS. To sustainably control and prevent FGS, an understanding of community and HCWs’ perceptions and attitudes towards people with FGS and their knowledge and practices may play an important role. In addition, a lack of knowledge may exacerbate levels of misconceptions [15] that may, in turn, create stigma among women misdiagnosed with STDs instead of FGS.The absence of definitive policies for FGS may, in turn, have caused the following: (i) a lack of treatment and diagnostic guidelines, (ii) limited investment, (iii) a lack of awareness among multiple stakeholders, and (iv) poor FGS surveillance among women of reproductive age. When treatment and diagnostic guidelines are lacking, there is an increased risk of misdiagnosis, inappropriate treatment, and missed opportunities for developing innovative solutions to control FGS in endemic communities. Hence, having the right knowledge, attitude, and improved perception of FGS is essential in

PLoS ONE 2014, 9, e91144. [Google Scholar] [CrossRef] [PubMed] Figure 1. The model of FGS data analysis determines the prevalence and burden in different geographic locations over multiple years. Figure 1. The model of FGS data analysis determines the prevalence and burden in different geographic locations over multiple years. Table 1. Prevalence of Female Genital Schistosomiasis (FGS) among the total samples collected in Ghana, Zambia, and Tanzania. Table 1. Prevalence of Female Genital Schistosomiasis (FGS) among the total samples collected in Ghana, Zambia, and Tanzania. Data SourceTotal SamplesTotal FemalesPositive Females (S. haematobium Prevalence *)Negative Females (S. haematobium Prevalence *)Ghana 20139039 (43%)31 (79.5%)8 (20.5%)Zambia 201613380 (60%)46 (57.5%)34 (42.5%)Zambia 201711060 (54.5%)45 (75%)15 (25%)Tanzania 201810470 (67.3%)43 (61.4%)27 (38.6%) Table 2. Prevalence of FGS based on the DNA concentrations of PCR-positive individuals in Ghana, Zambia, and Tanzania. Table 2. Prevalence of FGS based on the DNA concentrations of PCR-positive individuals in Ghana, Zambia, and Tanzania. DNA Concentration (ng/μL)DNA Concentration Total Females in Ghana 2013Total Females in Zambia 2016Total Females in Zambia 2017Total Females in Tanzania 2018Gr. A (0.5–3)Low9 (29.04%)42 (91.30%)40 (88.89%)41 (95.35%)Gr. B (4–10)Medium 16 (51.61%)4 (8.70%)3 (6.67%)0 (0%)Gr. C (10–above)High6 (19.35%)0 (0%)2 (4.44%)2 (4.65%) Table 3. Infection prevalence of FGS based on hematuria, urine filtration, and the PCR diagnostic test. -- = absence of positive or negative samples. Table 3. Infection prevalence of FGS based on hematuria, urine filtration, and the PCR diagnostic test. -- = absence of positive or negative samples. Data SourceHematuriaUrine FiltrationPCR PositiveNegativePositiveNegativePositiveNegativeGhana 20138 (18.6%)35 (81.4%)----31 (79.5%)8 (20.5%)Zambia 20163 (3.7%)79 (96.3%)082 (100%)46 (57.5%)34 (42.5%)Zambia 20177 (11.7%)53 (88.3%)3 (5%)57 (95%)45 (75%)15 (25%)Tanzania 2018--------43 (61.4%)27 (38.6%) Table 4. Comparison among females and males detected as positive for S. haematobium, S. mansoni, and dual infections. Male = M, Female = F. Table 4. Comparison among females and males detected as positive for S. haematobium, S. mansoni, and dual infections. Male = M, Female = F. LocationTotal Female Total MaleFemaleMaleS. haematobiumS. mansoniCo-OccurrenceS. haematobiumS. mansoniCo-OccurrenceGhana 2013394731 (36%)33 (38%)27 (69%)39 (45%)41 (47.7%)35 (74.5%)Zambia 2016805146 (35%)61 (46.6%)33 (41.3%)32 (24.4%)34 (26%)20(39%)Zambia 2017605045 (41%)47 (42.8%)47 (78%)37 (33.6%)38 (34.6%)31 (62%)Tanzania 2018703443 (41.4%)54 (52%)37 (52.9%)24 (23.1%)28 (27%)23 (67.7%) Table 5. Prevalence of Female Genital Schistosomiasis (FGS) among different female age groups. -- = absence of positive or negative samples. Table 5. Prevalence of Female Genital Schistosomiasis (FGS) among different female age groups. -- = absence of positive or negative samples. Location Total FemalesS. haematobium PositiveGr. A (0–10 years) Gr. B (11–20

Reducing cases of stigma and modifying risk behaviors to reduce the odds of re-infection at both the individual and community levels.This proposed pilot study will provide substantial evidence of the presence, prevalence (infection intensity), geographic distribution, age group, and gender-specific infection prevalence for FGS in females from a database of extracted DNA from field-collected urine samples from Ghana, Zambia, and Tanzania, over multiple years. The project will determine and explore the burden of FGS to develop strategies to improve the control of FGS among endemic communities in sub-Saharan Africa. 2. Materials and Methods 2.1. Data SourceThe data used for this study were preexisting. All the collected data came from analyzing the field-acquired filtered urine samples from previous studies, which were acquired from three different geographical locations over four years. The existing data sources were from previously individually published and unpublished studies conducted in Ghana in 2013, Zambia in 2016 and 2017, and Tanzania in 2018. The existing data were extensively analyzed (Figure 1) to determine the FGS presence, prevalence, distribution, and burden across age groups. 2.2. Model of Data Analysis 2.2.1. Overall FGS PrevalenceThe total FGS-positive samples for each year were determined based on the polymerase chain reaction (PCR) testing. All the duplicates and missing samples were discarded before analysis. The total number of positive and negative results for S. haematobium among females was determined based on three countries (Ghana, Zambia, and Tanzania). The disease prevalence was calculated based on the proportion of positive infections from each test out of the total number of samples evaluated. For S. mansoni, PCR was carried out by amplifying a highly repeated 121 bp Sm1-7 fragment (GenBank: M61098.1) [16]. For S. haematobium, 121 bp Dra, one repeat fragment (GenBank: DQ157698.1) was amplified [17,18]. For both schistosome species, the repeat fragments that makeup 12–16% of each parasite genome (~600,000 copies per cell) are species-specific and occur in different regions of the genomes of these two schistosome parasites, so there is no chance of cross-amplification. 2.2.2. FGS Prevalence Based on Parasite DNA Concentration in UrineThe overall infection prevalence was assessed for each year across three countries: Ghana (2013), Zambia (2016 and 2017), and Tanzania (2018). The infection prevalence was defined as the number of positive samples out of the total number of samples evaluated each year. The extracted DNA from filtered urine samples (used to assess FGS prevalence) for each year was categorized into three groups,

Based on the number of positive cases found by each diagnostic test against the total number of samples evaluated. The data and analysis were processed through Microsoft Excel and JMP 12 (JMP® v12, SAS Institute Inc., Cary, NC, USA) and converted to numerical values (1 = positive and 0 = negative) for statistical analysis. Excel and JMP analyses were performed blindly and then compared for accuracy. 3. Results 3.1. Overall FGS PrevalenceFor Ghana in 2013, the prevalence of FGS was 79.5% (31 out of 39 females were positive). For Zambia in 2016 and 2017, the figures were 57.5% (46 out of 80 females were positive) and 75% (45 out of 60 females were positive), whereas, in Tanzania in 2018, the prevalence was 61.4% (43 out of 70 females were positive, Table 1).Out of the total samples, all the duplicates and missing samples were discarded. Positive samples are based on PCR testing. 3.2. FGS Prevalence Based on Parasite DNA ConcentrationData for each year were categorized as follows based on the concentration of extracted DNA collected from filtered urine samples of PCR-positive individuals to assess FGS prevalence: high DNA load (>10 ng/μL), medium DNA load (4–10 ng/μL), and low DNA load (0.5–3 ng/μL). The highest positivity rate was found in the low DNA load over three years, as follows: in Zambia (2016), 42 (91.30%) females were positive; in Zambia (2017), 40 (88.89%) females were positive; and in Tanzania (2018), 41 (95.35%) females were positive. Based on a medium DNA load, the positivity rate ranged between 0 and 16 (0–51.61%). For a high DNA load, the positivity rate ranged between 2 and 6 (4.44–19.35%, Table 2). 3.3. FGS Geographical Distribution Based on Diagnostic TestingThe FGS prevalence based on PCR detection ranged from 57.5% (Zambia in 2016) upwards to 79.5% (Ghana in 2013) and others in between (Table 3). PCR consistently detected S. haematobium-specific DNA (4–15 times) in extracted DNA from urine samples collected from different endemic countries compared to traditional diagnostic tests, such as hematuria and urine filtration. 3.4. FGS Distribution Comparison Based on GenderS. haematobium (the causative agent of FGS) infections were comparatively higher among females than in males in Zambia (2016: 35% vs. 24.4%, and 2017: 41% vs. 33.6%) and Tanzania (2018: 41.4% vs. 23.1%) (Table 4). Similarly, females had higher S. mansoni infections over three years (Zambia 2016 and 2017, and Tanzania 2018) than males with S. mansoni infections. Co-occurrence

(S. haematobium and S. mansoni) among females and males remained somewhat similar throughout the years in the three countries (Table 4). 3.5. FGS Distribution Based on Age GroupThe FGS infection level had been consistently higher among pre-teens to young adults (11–20 years) for three years (2013, 2016, and 2017) in two countries (Ghana and Zambia) (Table 5). The infection level was higher for mid-ages (>31 years) for only Tanzania in 2018 (Table 5). The second highest infection level was found among children and juveniles (1–10 years) (Table 5). 4. DiscussionThe outcome of this study highlights that FGS has been predominant in females in different endemic countries over the years, with a high overall prevalence in certain age groups and when compared to males. The findings are crucial as awareness and knowledge of FGS are largely absent in communities and healthcare workforces in endemic countries [2]. The analytical outcome highlights that FGS is predominant in females among the evaluated urine samples in Ghana (2013), Zambia (2016 and 2017), and Tanzania (2018), and the prevalence ranges from 57.5% to 79.5% (Table 1). This is a prelude to underestimating the true prevalence of FGS as it is currently based on a small number of studies [5,20].Cell-free S. haematobium-specific DNA detection via Polymerase Chain Reaction (PCR) consistently detected more infections (4–15 times) than traditional diagnostic tests, such as hematuria and urine filtration for urine samples collected from different endemic countries over the years (Table 3). The presence of S. haematobium-specific DNA in urine is an indication of an active infection that clears within two weeks of Mass Drug Administration (MDA [21]). The level of DNA extracted from the urine of PCR-positive individuals is reflective of the infection level and mirrors the high (10 and above ng/µL), medium (4–10 ng/µL), and low infection (0.5–3 ng/µL) levels. PCR has shown its effectiveness as most of the positive infections detected occurred in low-level infections (Table 2). Sensitive and effective diagnostics are paramount for gaining a true sense of the prevalence and extent of FGS in endemic areas, as it is currently substantially under-reported [5,22,23].Infections related to S. haematobium were relatively higher among females compared to males in three years for two different countries, except for Ghana (Table 4). Co-occurrence along with S. mansoni was higher only for Zambia across two years and lower for Ghana and Tanzania when compared to males (Table 4). FGS infection was consistently