Prevalence, intensity and associated risk factors of soil-transmitted helminth infections among individuals living in Bata district, Equatorial Guinea

Background Soil transmitted Helminths (STH) infections remain a public health concern worldwide, particularly in tropical and subtropical areas where these diseases are highly endemic. Knowing the prevalence and risk factors of the disease is crucial for efficient STH control strategies in endemic areas. The scarcity of epidemiological data on STH for Equatorial Guinea has motivated the decision to perform the present study. Methods A cluster-based cross-sectional study was carried out in Bata district from November 2020 to January 2021. Stool samples were collected for the diagnostic of STH infections using Kato-Katz technique. Descriptive statistics was performed for determination of STH prevalence and intensity, while logistic regression models were used to assess the risk factors associated with STH infections. Results A total of 340 participants were included in the study with a mean age of 24 years (SD = 23.7) and 1.2 female-to-male sex-ratio. The overall prevalence of any STH was 60% (95%CI: 55–65). The most prevalent species were Ascaris lumbricoides (43%, 95%CI: 37–48) and Trichuris trichiura (40%, 95%CI: 35–46). Intensity of infection were mainly light to moderate. A trend of association was observed between age and any STH infection (overall p-value = 0.07), with a significant difference observed between children aged 5–14 years as compared to those aged 1–4 (aOR 2.12; 95%CI: 1.02–4.43, p-value = 0.04), while locality was significantly associated with STH infection (overall p-value<0.001) with a higher odds observed for peri-urban area as compared to urban area (aOR 4.57; 95%CI: 2.27–9.60, p-value<0.001). Conclusion Bata district is a high STH transmission area, where school-aged children and peri-urban areas are associated with a higher risk of any STH infection. This situation calls for a full implementation of the WHO recommendations for STH control; mass drug administration of anthelminthic twice a year to the whole population with great attention to school age children, and prioritizing peri-urban areas where safe water, improve sanitation, and hygiene education should be implemented to achieve a better control.

Introduction Soil transmitted-helminth (STH) infections are the most prevalent among the neglected tropical diseases (NTDs) [1][2][3][4], with 1,500 million people infected each year in 112 affected countries [5]. Four main species are responsible for the disease in humans; Ascaris lumbricoides with an estimation of 807 to 1.121 million people affected globally; Trichuris trichiura affecting 604 to 795 million people globally and hookworms (Ancylostoma duodenale and Necator americanus) infections affecting 576 to 740 million of people globally [6]. These parasites prevail in tropical and subtropical areas where ecological, socioeconomic, and environmental factors, together with the deficiency of hygiene enhance their transmission. At regional level, sub-Saharan Africa represents one of the most endemic regions in the world, supporting 40% of the worldwide burden [7,8].
STHs particularly affect children, hampering their physical growth and cognitive development. In this population, STH infections are known to cause malnutrition, micronutrient deficiencies, poor cognitive function with as consequence school absenteeism and low academic performance and wage earning potential [2,9]. Furthermore, STH infections has been reported to contribute to the host vulnerability to other endemic infectious diseases [10], such as human immunodeficiency viruses [11], malaria [12,13] and tuberculosis [14].
For the control of disease burden, the world health organization (WHO) recommends the implementation of some measures for population living in endemic areas, which include periodic mass drug administration (MDA) with albendazole (ABZ) or mebendazole (MBZ). The frequency of MDAs is determined by the baseline prevalence of STH infection in the community, particularly among school aged children (SAC). Treatment should be administered once a year if the prevalence is between 20-50% and twice a year if prevalence is 50% or above [15][16][17][18]. This highlights the importance of determining the STH prevalence of each endemic area. In addition to MDA and in order to control disease transmission, the WHO recommends the WASH (Water, Sanitation and Hygiene) strategy, which consists of providing safe water to atrisk population, adequate sanitation and hygiene education [15,19]. MDA campaigns are considered fundamental since they are cost effective targeting pre-school-(1-4 years) and school-(5-14 years) -aged children, and women of reproductive age (15 to 45 years old) all considered as more vulnerable [20,21]. However, in high-risk communities, infected adults and untreated children have been reported to act as reservoir of infection, raising the importance to take into account these population during MDA campaigns in such communities [2,9,20]. This strategy was supported in a mathematical model which demonstrate that the exclusion of adults in MDA campaigns in endemic areas contributes to the spread and re-infection of treated children [9].
Equatorial Guinea is a country located in central Africa region known to be endemic for STH [1,22]. The country is bordered to the north by the Republic of Cameroon, to the south and east by Republic of Gabon and to the west by the Equatorial Atlantic Ocean. With an area of 28,051.5 km 2 , Equatorial Guinea is constituted by two regions: the insular region and the continental region which comprises a large part of the country's surface area. Administratively, both regions together are composed by eight provinces, 19 districts (18 districts before 2017) and 37 municipalities. Although the country is located in region known to be endemic for STHs, epidemiological data on STH infection are scarce for the country and those available are particularly old; about nine years and more [23][24][25][26][27]. Indeed, the main data was reported in 2008 through the national NTDs survey conducted by the Ministry of Health and Social Welfare (MoHSW) in all districts of the country, where the overall prevalence for any STH was 96%, and T. trichiura the most prevalent species with 91% prevalence followed by A. lumbricoides and Hookworms with 72% and 24% of prevalence, respectively [24]. On the STH control strategies, the government of Equatorial Guinea through the MoHSW and based on the first national epidemiological survey conducted in 2008, published in 2018 a strategic plan to control NTDs at national level in which the main STH strategies retained were MDA campaigns targeting children aged 5 to 15 years, provide safe water, and health education [28]. However, as indicated by the 2020 Expanded Special Project for Elimination of Neglected Tropical Diseases (ESPEN) report, no MDA have been implemented in the country until 2019 [29] mainly due to lack of funding [7]. Just passive strategies consisting of, deworming of young children during vaccination campaigns and systematic treatment of children consulting at hospital for helminth-like symptoms are being implemented. However, in a context of global development of the country and where the literacy rate among population older than 15 years is 94% [30], the 2011 Demographic and Health survey reported that 55% of the population has improved water sources with for instance, 21% of the population having public taps, 2% having taps at home and 11% using water wells. On sanitary facilities as well, 40% of the population reported having unshared toilets in improved conditions but only 8% discharged into sewers, while 12% of the population used shared sanitary facilities and only 1% of them discharged into sewers [31]. In that context, establishing current epidemiological data on STH infections in the country and more importantly identifying the main risk factors associated with infection could thus serve to develop a tailored guide for implementation of control strategies and to strengthen advocacy of a rapid scale up of interventions at national level. The objective of the present work was therefore to determine the current prevalence, intensity and risk factors associated with STH infections in Bata district, the largest district of Equatorial Guinea.

Ethics statements
The study protocol was approved by the Equatorial Guinea National Ethical Committee in Malabo (CENGE; Nr Reg-2019-028). Administratively, the study received credentials from the public health direction of the ministry of health and social welfare (reference number: 56-150), the regional delegation of Bata of the ministry of health and social welfare (reference number: 2018), and the government delegation of Bata-Littoral from the Ministry of Interiors and Local Corporations (reference number 937). As study inclusion procedure, eligible participants filled out and signed the informed consent form and then were included. For illiterate adults who agree to participate, the informed consent form was filled out and signed by an impartial witness before the participant him/herself signed or thumb-printed the form. For participants less than 18 years of age who orally agree to participate, signed informed consent was obtained from parents or legal guardians before inclusion. The study was conducted in line with Good Clinical Practice principles of the International Conference on Harmonization [32] and the Declaration of Helsinki [33].

Study area
This study was conducted in the municipalities of Bata and Rio Campo, in Bata district. Bata is the capital city of Littoral province, one of the eight provinces of Equatorial Guinea (Fig 1). Littoral province has the largest population in the country and is composed of three districts: Bata with 25% of country's total population, Cogo, and Mbini. Bata district has three municipalities namely; Bata, the most populous; Machinda and Rio Campo [34]. Municipalities in Bata district constitute of urban, peri-urban, and rural areas. Urban areas are characterized by the presence of basic infrastructure available such as pipe water, internet, health, and education systems with easy access. In those areas, industrial economic activities are predominated, especially those of the secondary (manufacturing) and tertiary (services) sectors. Peri-urban areas are intermediate areas between urban and rural areas and were mainly located in peripheral regions of large cities. They are mainly made up of populations migrating from rural to urban areas in search of better living conditions and are therefore characterized by populations with low economic resources. Poor housing, unimproved latrines; precarious hygienic-environmental conditions are mainly predominant in peri-urban area while basic infrastructures such as sanitation systems for sewage, availability of safe drinking water, internet connection, health system, and school facilities are absent or scarce. Rural areas are areas located at great distances from urban centers and where predominate productive activities are of primary sector such as agriculture, livestock, and fishing. However, rural houses are better organized than in periurban areas. Bata district is known to be endemic for STH [7,8]. Indeed, a 94% overall prevalence of any STH was reported in the district in 2008, with T. trichiura as the most prevalent species [24].

Study design and study population
We conducted a cross-sectional survey between November 2020 and January 2021. Participants of both sexes, from one year of age and above, residing in the study area at least 3 months before the start of the survey were eligible for the study.

Sample size calculation
The sample size of the present work was calculated to determine the overall STH prevalence in the district of Bata where according to the 4 th population and housing census of 2015 [34], where 309,345 inhabitants are living. A previous survey conducted in the country in 2008 reported a 94% overall prevalence of STH in the same area [24]. Using the formula described elsewhere for sample size calculation for cross-sectional studies [35], 87 or more participants were needed to have a confidence level of 95% that the real prevalence is within ±5% of our measured value. As we planned to include our study population from the three main areas of the district of Bata; urban, peri-urban, and rural areas, we considered to include from each of those areas the calculated number of participants approximately equally distributed between neighborhood communities or village councils within each area. This gave us a total of 261 minimum participants to surveyed over the study area.

Sampling procedure
A multi-stage cluster sampling technique was used for study sample selection. Bata district was our study area. At the first stage, we selected two (Bata and Rio campo municipalities) out of the three municipalities constituting the district of Bata. Bata municipality is composed by urban areas divided in neighborhood communities and peri-urban areas divided in village councils while Rio Campo municipality is composed by rural areas only divided in village councils. At the second stage of sample selection, five neighborhood communities in the urban areas and nine villages councils (five in the peri-urban areas and four in rural areas) were randomly selected using the list of neighborhood communities and villages provided by the Bata Littoral Government Delegation of the Ministry of Interior and Local Corporations. At the third stage, households were selected by convenience and from each household, a maximum of two eligible participants among those who were willing to participate were randomly selected and included in the study.

Study procedures
After arriving at a selected house, we requested permission from the person in charge of the household. When permission was granted, the study objectives and participations conditions were explained to the house inhabitants in Spanish and when it was necessary in the community local language, and subject's participation was requested. Among those who agreed to participate, eligible participants were included in the study. An identification code was assigned for each participant for confidentiality reasons. Socio-demographic data were recorded, and a short-standardized questionnaire was administered to all participants to collect data on the household conditions. For children aged less than nine years old, the head of family respond to their questionnaire as they were less likely to provide accurate answer. After administering the questionnaire, a clearly labeled plastic container for stool sample collection was provided to the participant who was invited to provide a morning stool sample the next day, or if not possible, the day after. Instructions on how to collect specimen were given to the participants prior to specimen collection. Fresh stool sample was collected in the morning, preserved in a thermal box and transported to Bata regional hospital laboratory in the following two hours of collection for analysis. Results were given back to all participants and to those found infected, MBZ 100 mg twice a day for three consecutive days was given free of charge.

Stool sample processing
All stool samples collected were analyzed under a microscope using a Kato-Katz technique with a 41.7 mg mold, following the WHO standard procedure [18,36,37]. For each stool sample, two slides were prepared, and the two separate slides were read by two technicians. Preparation was examined under microscopy (10x and 40x power objective) after 30 minutes of slide's preparation in a systematic manner. The eggs of each species counted was reported on a laboratory case report form. Participants was considered positive if at least one egg was found on any of the duplicate Kato-Katz thick smear slides [38]. STH infection intensity were calculated by multiplying the species specific average of egg counted from duplicate Kato Katz by factor of 24 [38].

Statistical considerations
The statistical analysis aimed to describe the distribution of STH infections in the study population and the factors associated. Participants were considered as positive for any STH infections if found positive for at least one STH species. STH infection intensity was categorized as light, moderate or heavy based on WHO classification of intensity [39] as presented in S1 Table. Explanatory variables were sociodemographic characteristics, including age, sex, and location. Age was categorized into three groups based on the classification of population at risk of helminths infection in endemic communities as defined by the WHO; 1 to 4 years-old considered as preschool-age children (PSAC), 5 to 14 years-old considered as SAC and participant aged 15 years-old and more [39]. Similarly, we considered women aged 15 to 45 years-old as women of reproductive age (WRA). Education was classified as 'no education', 'primary level', and 'secondary/university level'. As described early, locality was classified as 'urban', 'peri-urban', and 'rural'.
The data was collected in a participant case report form and entered into Excel by a data clerk. A second data clerk double checked digitalized data. The clean database (S1 Dataset) was exported to R software version 4.1.1 for statistical analysis. Continuous variables were described as mean and standard deviation (SD) while categorical variables were described as frequencies or percentages and 95% confidence interval (CI). The difference between proportions was determined under the assumption that nonoverlapping CI indicated statistical significance. Independence between STH infection status and other categorical variables were tested using the Chi-squared test of independence (with Yates' continuity correction for small sample size). Univariate and multivariate logistic regression model were used to identify factors associated with any STH infections. To fit the multivariate model, only indicators significantly related to outcome variable at a 25% level in univariate analysis were included. However, sex was forced in the model. Multicollinearity between variables was assessed using the variance inflation factor. Variable with the highest variance inflation factor was step by step removed from the model. Model with variance inflation factor less than five for all variables was retained as final model (Tables A and B in S2 Table). The significance level of all statistic tests was set at 5%.

Participant's enrollment flow
A total of 286 households were selected for a total of 406 participants evaluated for their eligibility to participate in the study. Of them, 403 participants were eligible and were invited to provide stool sample (Fig 2). Of them, 61 (15%) participants did not provide stool sample, while two (0.5%) samples were rejected because they were not correctly identified. This make a total of 63 participants considered as non-responders of which 54% was male and 63% were under 15 years old. Finally, samples from a total of 340 (84%) participants were retained and included for analysis.

Study population characteristics
The mean age of the 340 study participants was 24 years (SD = 23.7). As presented in Table 1, participant age ranged between one and 86 years, with 42% of them aged from 15 to 86 years old and PSAC and SAC representing 19% (95%CI: 15%-23%) and 39% (95%CI: 34%-44%) of the study population, respectively. A total of 187 (55%) of the study population was female, with 1.2 female-to-male sex ratio. Women of reproductive age represented 21% (39/187, 95% CI: 15-27) of all females. Also, 41%, 35%, and 23% of the study population lived in urban, peri-urban and rural areas, respectively. A majority (55%) of them had primary school education level. Living conditions are presented in Table 2, 96% (326/340) of participants reported not to have sewerage system in their homes. For water source, a majority of 56% (189/340) of participants claimed to use tap water while 26% (89/340) of them used water from wells. The remaining 18% (62/340) use river water. Most study participants (49%, 168/340) were students; all individuals attending classes during the survey, irrespective of the educational level.

Intensity of STH infection
The median (IQR) egg count was 528 (IQR: 72-2256), 192 (IQR: 72-576), and 72 (IQR: 24-72) for A. lumbricoides, T. trichiura and hookworm infection, respectively. Fig 3 displays the proportion of the intensity of STH infections by species. STH infections were basically light intensity, with 100% for Hookworm infection, 90% for A. lumbricoides and 85% for T. trichiura. The prevalence of moderate intensity was 10% and 13% for A. lumbricoides and T. trichiura, respectively. Heavy intensity was observed for T. trichiura only, with 2%, and all of them are from peri-urban area.

Factors associated with STH infections
As presented in Table 5, a significant association was found at crude analysis between STHs infection and age (p-value = 0.005), locality (p-value<0.001). As compared to PSAC, the SAC had 2.29 odds (95%CI: 1.27-4.19) to be found positive for STH infections while no significant difference was observed with the participants aged 15 and above (cOR = 1.04; 95%CI: 0.60-1.80). As compared to participants with no education, those with primary education level had higher odds, to be STH positive (cOR = 1.9; 95%CI: 1.14-3.18) while no difference was observed with those with secondary/university (cOR = 1; 95%CI: 0.52-1.93) education level.
Regarding occupation status, farmer/fisher tend to have a lower odds of being infected with STH, compared to students (cOR = 0.56, 95%CI: 0.31-1.00, p-value = 0.05). The final model for adjusted analysis included age, sex, locality, education, type of toilet, and type of toilet floor. As presented in Fig 4, only a trend of a statistically significant

Discussion
Our results reveal a high prevalence of STH infections, classifying our study area as a high STH transmission area [10]. This finding agrees with the high prevalence previously reported  in the same area through the National NTDs survey in 2008 [24]. However, our results are different to the moderate STH prevalence reported from a study on intestinal parasitism in an elementary school in Bata in 2008 [25]. This difference in result could be explained by the fact that the author conducted the survey in one primary school only. Our result suggests a persistence of infection transmission over time. Indeed, in Cameroon and Gabon, two border countries of Equatorial Guinea, a low STH infections prevalence has been reported after an MDA as compared to before intervention [40,41]. As those countries belong with Equatorial Guinea to the same geographical area, we can therefore assume that the lack of public health control measures implementation observed in our study area during almost the last 20 years [42] could explain the high prevalence reported. This high prevalence therefore expresses an urgent need of implementation of MDA campaigns as recommended by the WHO in order to reduce the STH prevalence in particular and the disease morbidity in general that we can assume to be high in our study area [43]. We investigated the distribution of STH species in our population and found that the most predominant species are A. lumbricoides and T. trichiura with moderate prevalence for each, as compared to the prevalence of hookworm species which was found to be low. This result could indicate a lower transmission of hookworm infection in the study area, compared to A. lumbricoides and T. trichiura. Natural environmental conditions such as tropical climate, soil type mainly sandy typical in Bata district [34], and local temperatures [44] combined with the absence of sewage system, habits of no processing drinking tap water could explain the finding. In our study population for instance, 96% of study participants reported not having a sewage system connected to their houses. In addition, knowing that T. trichiura and A. lumbricoides eggs can resist several years in the soil, and considering that A. lumbricoides and T. trichiura transmission is fecal-oral, in absence of adequate sewage system, feces and sewage containing parasite eggs can persist in the environment, contaminating water, soil and get infective [19,37,45] and favor the transmission of these two species in particular. In the same vein, investigating the STHs co-infection in our study population, the level of prevalence we reported for each species could explain the pattern of co-infection we found. Indeed, A. lumbricoides and T. trichiura were both involved in 96% of co-infections. This pattern of STH distribution corroborates with those published in the central Africa region in 2009 [1] but opposes the finding from Democratic Republic of Congo where the most prevalent species where hookworm, followed by A. lumbricoides and T. trichiura [46]. Our finding could indicate that A. lumbricoides and T. trichiura are the species with highest transmission even in the   reported by some authors in area with either moderate [49] or high [50] STH prevalence and where the intensity of the infections was mainly light to moderate. We hypothesize that the low proportion of heavy infection intensity we observed could be due to the combined effect of deworming young children during vaccination campaigns and the "systematic" administration of anthelminthic treatment to individuals with helminth-like symptoms consulting in hospitals, as it is empirically observed in the country. Indeed, heavy intensity infection should be more frequently associated with clinical symptoms and will therefore lead patients to seek treatment. Treatment of mainly patients presenting symptoms could therefore contribute to the reduction of intensity of infection and morbidity in the community.
Assessing the risk factors of STH infections in our communities, we found locality as a main risk factor. As compared to urban or rural areas, peri-urban areas were found to be more at risk of STH infections. Indeed, we found a significant highest prevalence of A. lumbricoides and T. trichiura in peri-urban area compared to urban and rural areas. Our result corroborates with the finding reported from a georeferenced estimates of STH prevalence worldwide were A. lumbricoides and T. trichiura infections were found most prevalent in peri-urban areas [51]. However, a study conducted in South West Cameroon found rural areas as more at risk locality [52]. We observed that peri-urban areas with inadequate housing conditions, and poor socio-environmental hygiene conditions that increase the risk of STH infection as disease transmission [51]. This finding indicates that the MDA campaigns implemented in the periurban areas must be supported by the full implementation of the WASH strategies in order to be efficient.
We found SAC to be more at risk of infection, compared to other age groups which corroborates with many other studies [10,20,53]. This is mostly due to their playing habits, less hygiene practices, and lack of WASH in schools and at home. Although SAC were more at the risk, we found a high prevalence of STH infections in adults which can have an implication on the control program in the country. Indeed, based on the WHO recommendations, the periodic MDA should target PSAC and SAC, WRA, and adults with at-risk activities such as farming, selling agricultural products at the market, and less hygiene measures practices [54]. Therefore, not taking into account all adult groups in MDA strategies, particularly in endemic areas where less or non STH control strategies were previously developed, can make them to serve as reservoir of infection in the communities [47], and make further control programs ineffective. We therefore advocate inclusion of adults in MDA campaign in our communities where STH prevalence is high if we want the country to control the disease first and then to reach the WHO 2030 targets for STH elimination as public health problem [43,55]. Education of SAC on STH transmission and prevention could boost the prevention of the disease in the community.
We used Kato-Katz technique for the STH infection diagnostic. Although this technique is mostly used in population-based prevalence survey, it is known to be more sensitive for A. lumbricoides and T. trichiura, and less for hookworms [18,56,57]. This could thus contribute to the low prevalence and low intensity for hookworm infection we reported, even if similar prevalence (4%) was already reported in 2012 in Bata but among HIV population [11]. Despite all, Kato Katz technique is considered by the WHO as the gold standard diagnostic method for the detection and quantification of STH intensity [18]. It has been demonstrated that sensitivity of Kato Katz and other microscopic method also depends on the infection intensity. We reported here a moderate or low infection intensity, which could lead to an underestimation of the prevalence we found but not on the conclusion we drawn on the level of disease transmission in the area. Despite the limitation of our diagnostic method technique, the present study offers a picture of the situation of STH infection in Bata district, classified as high STH infection transmission area. Such information can therefore serve as a guide for the orientation and implementation of control strategies [19,53]. In addition, the present study was carried out in only one out of 19 districts. Our conclusion could be therefore limited to the Bata district. However, as Bata district is the most populous district of the country, we could anticipate that our finding could drive the implementation of the STHs control programs in the country. Also, the sample size include in the present study could appears low, particularly to assess the risk factors of the disease and to compare prevalence between modalities of variables. However, on one hand the 95% confidence interval we reported for the prevalence we found may support the consistency of the sample size we included, and on the other hand the statistically significant results we found with the actual sample size could assume a strong association between those risk factors and STH infectious status, particularly when looking at the high STH prevalence reported in the study area. Finally, no information was collected on the use of anthelminthic in the study population months prior the samples collection, could influence the prevalence and intensity we reported in the present study, but cannot affect our conclusion.

Conclusions
The present study reveals that Bata district is a high STH transmission area, where age and locality are the main risk factors of infection in the population. Our results indicate a need of the implementation of two rounds of MDA campaigns per year which should include populations of all age, but with particular attention to SAC and population living in peri-urban areas where WASH concept should be implemented. For a tailored and efficient interventions, future research on knowledge, attitudes, and practices of population regarding STH infections could be contributive.  of the Bata municipality and the municipality of Rio Campo, visited during this research. (Bikuy II, Bomundi-V, Ekuku-II, Ikunde-II, Bongoro, Edjabe Zona única, Nkomakac, Tika, Adjap Cdo, Bicom Cdo, Bindung Cdo, Esun Cdo, San Jose de Alum Cdo). We do not forget parents, children and the tutors who accepted to participate in this research; and in a special way we thank the Baney Laboratory team for their support by providing us with a physical space and in some cases material for the development and successful completion of this research. We would also like to thank Mr. Olivier Nkoghe Mvie for designing the map of the study area.