Prevalence and factors associated with human Taenia solium taeniosis and cysticercosis in twelve remote villages of Ranomafana rainforest, Madagascar.

BACKGROUND: Infections with the tapeworm Taenia solium (taeniosis and cysticercosis) are Neglected Tropical Diseases (NTD) highly endemic in Madagascar. These infections are however underdiagnosed, underreported and their burden at the community level remains unknown especially in rural remote settings. This study aims at assessing the prevalence of T. solium infections and associated risk factors in twelve remote villages surrounding Ranomafana National Park (RNP), Ifanadiana District, Madagascar.
METHODOLOGY: A community based cross-sectional survey was conducted in June 2016. Stool and serum samples were collected from participants. Tapeworm carriers were identified by stool examination. Taenia species and T. solium genotypes were characterised by PCR and sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Detection of specific anti-cysticercal antibodies (IgG) or circulating cysticercal antigens was performed by ELISA or EITB/Western blot assays. PRINCIPAL
FINDINGS: Of the 459 participants with paired stool and blood samples included ten participants from seven distinct villages harbored Taenia spp. eggs in their stools samples DNA sequencing of the cox1 gene revealed a majority of T. solium Asian genotype (9/10) carriage. The overall seroprevalences of anti-cysticercal IgGs detected by ELISA and EITB were quite similar (27.5% and 29.8% respectively). A prevalence rate of 12.4% of circulating cysticercal antigens was observed reflecting cysticercosis with viable cysts. Open defecation (Odds Ratio, OR = 1.5, 95% CI: 1.0-2.3) and promiscuity with households of more than 4 people (OR = 1.9, 95% CI: 1.1-3.1) seem to be the main risk factors associated with anticysticercal antibodies detection. Being over 15 years of age would be a risk factor associated with an active cysticercosis (OR = 1.6, 95% CI: 1.0-2.7). Females (OR = 0.5, 95% CI: 0.3-0.9) and use of river as house water source (OR = 0.3, 95% CI: 0.1-1.5) were less likely to have cysticercosis with viable cysts.
CONCLUSIONS/SIGNIFICANCE: This study indicates a high exposure of the investigated population to T. solium infections with a high prevalence of cysticercosis with viable cysts. These data can be useful to strengthen public health interventions in these remote settings.

Introduction

Taenia solium taeniosis/cysticercosis are neglected parasitic tropical diseases mainly affecting people living in Central and South America, South-East Asia, Indian Subcontinent, and Sub-Saharan Africa [1]. Also referred to as poverty-related diseases, these infections are associated with poor sanitary and hygiene conditions, open defecation, free-range pig-husbandry and lack of meat inspection [2]. Despite having been declared potentially eradicable, T. solium infections remain a serious health and economic problem affecting around 50 million people worldwide [3]. T. solium was identified as a leading cause of death from food-borne diseases, accounting for 2.8 million disability-adjusted life-years (DALYs) annually [4].

T. solium taeniosis and cysticercosis are zoonosis with a biological cycle maintained in the environment between humans and pigs [5]. In the natural life cycle of the parasite, humans are the only definitive hosts developing the intestinal adult form (causing taeniosis). Cysticercosis in pigs and in human (respectively the natural and the accidental intermediary host) is caused by the metacestode larval stage of T. solium (called cysticerci). Humans can therefore develop cysticercosis in muscles, eyes or brain. Neurocysticercosis (NCC) is the most severe presentation of this pathology and the most important parasitic disease affecting the central nervous system [6-9]. NCC may count for almost one-third of seizure disorders with an estimated 2 million people affected [10,11].

A few community-based studies were carried out in Africa to estimate the prevalence of T. solium infections using different diagnostic tools [12-14]. Human cysticercosis prevalence varied greatly across African countries, 0.68% to 21.63% of analyzed samples were found positive for circulating antigens while the seropositivity associated with T. solium antibodies ranged from 7.6% to 34.5%. The prevalence of taeniosis reported in Africa was between 0.37 and 13.8% [12]. Nevertheless, there are still little data available regarding the prevalence of T. solium infections and their associated risk factors for the African Continent especially in rural areas [12,15-17].

In Madagascar, the proportion of the population living below the international poverty line was estimated at 75% in 2019 [18]. Around 35% of the population do not have access to basic toilets [19] and 52% of the population (only 35% in rural areas) have access to drinking water [20]. Agriculture, mainly mixed with livestock, is still very dominant with more than seven out of ten employed individuals. In rural areas, about two-thirds of households are traditional farmers where pig breeding (mainly raised in free roaming) holds the third place [18]. Given the persistence of factors determining the spread of T. solium infections such as traditional pig farming, open defecation and poor meat hygiene requirements, Madagascar is among the most infested countries in the world.

Madagascar is a vast island divided into 114 Districts where taeniosis and cysticercosis epidemiological data are scarce [21]. However, the few data available underline the significant burden of T. solium infections in the Malagasy population. At the national level, the only and most recent sources of data on taeniosis were provided by mapping surveys (Based on Kato-Katz tests) performed through integrated national program of the Ministry of Public Health targeting some Neglected Tropical Diseases (i.e. lymphatic filariasis, schistosomiasis and soil-transmitted helminthiasis). These surveys conducted between 2011 and 2015 identified 54/114 co-endemic Districts for schistosomiasis and taeniosis of which 7 districts were endemic only to T. solium [22]. In 2015, an overall prevalence of 0.7% (ranging from 0.3% to 7.3%) was reported affecting 14 of the 24 Districts surveyed. Regarding cysticercosis, very little data are available in the literature and the data collected by the Ministry of Health are mainly based on clinical suspicions rarely confirmed with neuro-imaging or serological diagnosis (detection of T. solium cysticercal antigens by ELISA and/or anti-cysticercal antibodies by ELISA and/or Western blot/ElectroImmunoTransferBlot-EITB). Indeed, since the existence of human cysticercosis in Madagascar which was first documented by Andrianjafy [23], subsequent data have shown that approx. 20–25% of late-onset epilepsy were related to cysticercosis [24,25]. The seroprevalence of the human cysticercosis in the island ranged from 7% in Coastal regions (Mahajanga and Toamasina) to 14–21% in Central Highlands (Ambositra, Ihosy, Mahasolo and Antsirabe) following the geographical importance of pig farming and pig cysticercosis [26-31]. These studies are unlikely to reflect the true situation as they did not cover the whole country and the remote rural areas.

The Ifanadiana District (13 townships) is one of the rural Districts of Madagascar heavily burdened by poverty and reported to have a high rate of intestinal parasites [32-34]. In 2015 however, the national mapping program reported 0% of taeniosis prevalence in 14 fokontany (the smallest administrative subdivision of Madagascar) including 21 rural villages (comprising one of the villages targeted by this study) [35]. The main objective of the current study was to determine by community based cross-sectional survey the prevalence of human Taenia solium taeniosis/cysticercosisis and the risk factors associated in 12 remote villages of Ranomafana and Kelilalina townships (Ifanadiana District, Madagascar).

Methods

Ethics statement

The study was reviewed and approved by the Ethics Committee for Biomedical Research at the Ministry of Public Health of Madagascar (N° 142-MSANP/CE), and by the Institutional Review Board (IRB) of the Stony Brook University (IRB#874952). The field study was conducted under the supervision of the medical team of ValBio Centre and followed ethical principles according to the Helsinki Declaration. All participants received an explanation of the study goals and procedures prior to enrolment. Written informed consent was obtained from adult participants or parent/legal guardians for the children. Participants detected positive for taeniosis were treated by local medical officers with praziquantel at 10 mg/kg in one dose according to the national guidelines. Before this treatment, in order to recover the whole worm properly and avoid dissemination of eggs in the environment, all carriers of Taenia were cured with niclosamide (2 g orally single dose) and with oral purgative (lactulose, 2 tablespoons) one day before and 2 hours after niclosamide. During the 3 days following the niclosamide treatment, stool samples, collected in large plastic bags, were obtained from all Teania carriers.

Study design and population

This is a cross-sectional prevalence study. Overall, 164 households were randomly selected using an electronic random number generator on the village census. Household members older than 5 years and living in the study area (> 50% of their time) during the last 3 months before the beginning of the study were invited to participate. The study was conducted in the District of Ifanadiana (Vatovavy-Fitovinany Region) Southeastern Madagascar. The investigation was carried out in two rural townships: Ranomafana (21°15’36” S and 47°27’17" E, elevation 613m) and Kelilalina (21°17’8” S and 47°33’17" E, elevation 613m). The targeted villages were situated close to the ValBio Centre (CVB), a Stony Brook University Research Centre in the Ranomafana National Park. The study population was enrolled in five Fokontany, namely Vohimarina, Tsaramandroso, Mandrivany, Ampitambe and Kianjanomby. In these fokontany, twelve rural and remote villages (Ambinanindranofotaka, Mangevo, Marojano, Sahavanana, Sahavoemba, Kianjanomby, Mandrivany, Ankazotsara, Ampitambe, Bevoahazo, Torotosy, Ampitavanana) were targeted (Fig 1). These villages were chosen as they constitute the rural villages receiving biannual medical consultations by the medical team of ValBio Centre with a deworming of the entire population.

Fig 1

Map of the study sites.

Map of Ranomafana and Kelilalina townships (Ifanadiana District) showing the twelve villages where sampling was carried out. The figure was created using the humanitarian Data Exchange (HDX) accessible from OCHA (United Nations Office for the Coordination of Humanitarian Affairs) services and using QGIS 2.8.1. https://data.humdata.org/dataset/madagascar-administrative-level-0-4-boundaries. Details on the corresponding license can be accessed via the link below: https://data.humdata.org/about/license.

The villages included in each fokontany and the geographic coordinates (obtained using a handheld Garmin global positioning system, GPS) of the twelve villages investigated are listed in supporting S1 Table. The geo-reference data collected were used to map the study sites using QGIS 2.14.19-Essen software (http://www.qgis.org). All twelve villages, situated around Ranomafana Rainforest Park, are only accessible after 2–7 hours of walking with no roads or paved paths. Investigated villages were found to be traditional, rural, and heavily reliant on subsistence agriculture. Village sanitation was poor, with lack of water infrastructure, common practice of open defecation and free-range pig-husbandry.

Socio-demographic, behavioural and knowledge data collection

A community-based survey was conducted over a two-month period (June to August 2016). Results on the prevalence of soil-transmitted helminthiasis and risk factors associated were presented in another manuscript and are not further discussed here [34].

Socio-demographic features, hygiene practices and behavioural data (water source, use of soap, hand-washing, pork-eating, defecation place), knowledge about worms, some clinical manifestations suggestive of NCC (headache and seizure) and antiparasitic drug-taking were obtained using an individual questionnaire in Malagasy. Heads of households were also interviewed about the size of the household, water and sanitation conditions and pig breeding. All participants were informed on the risks associated with T. solium infections.

Stool sampling and Taenia spp. detection by microscopy

After signing the consent form, stool samples were collected from each participant in a sealed 50 mL cup for parasitological analysis. Approximately 100–250 mg of stools was conserved at room temperature in a 2.5% potassium dichromate solution (VWR chemicals, Ref. 26784.231) until PCR analysis. Fresh stool samples were analyzed for Taenia spp. eggs using two parasitological techniques, Kato-Katz method (KK) and the Spontaneous Sedimentation Technique (SS) [36,37]. Preliminary diagnoses were performed by medical students (Stony Brook University, USA) under the supervision of a trained parasitologist from Stony Brook University and were confirmed by trained parasitologists from Institut Pasteur de Madagascar.

Taenia species identification

Taenia species identification (T. solium, T. saginata and T. asiatica) from stool samples of tapeworm carriers was performed by sequencing the mitochondrial cytochrome C oxidase subunit 1 (cox1) gene following PCR amplification. Copro-DNAs were extracted from approx. 150 mg of the eleven stools positive for Taenia eggs conserved in potassium dichromate. Briefly, after three washes with PBS (DPBS 1X, phosphate buffer saline, Invitrogen), genomic DNA extraction was subsequently realized using the QIAamp DNA Mini Stool kit (Qiagen, Germany, Ref. 51504) according to the manufacturer’s instructions. DNA was eluted in 200μl of buffer and stored at -20°C until use.

Briefly, cox1 gene sequences from geographically different areas and representative of T. asiatica (GenBank accession No. AB107235.1); T. saginata (GenBank accession No. AB107246.1); T. solium Asian genotype (GenBank accession No. AB066488); and T. solium African/American genotype (GenBank accession No. AB066492.1) were obtained from EMBL/Genbank databases and aligned using the Clustalw software [38]. A region of 627 base pairs (starting at the position 472 of the completed T. solium cox1 gene, GenBank accession No. AB491986.1), totally similar within the same species and containing enough polymorphisms to differentiate between Taenia species and T. solium genotypes, was selected to design common primers (Forward: 5’-GACTAATATATTTTCTCGTAC-3’ and reverse: 5- GACATAACATAATGAAAATG-3’). The details of the polymorphic region with the primer delimitation are shown in supporting S1 Fig.

In a first step, primer pairs described by Yamasaki et al. [39] were used to perform Taenia spp. specific PCR. However, PCR amplification performed with these primers used either in multiplex or simplex assays showed slight cross-amplifications: T. asiatica primers amplified both T. saginata and T. solium DNAs and T. solium Asian genotype primers amplified T. asiatica DNA. Therefore, common primer pairs able to amplify the three Taenia species were designed, and PCR product were directly sequenced. PCR reactions using these common primers were performed in a total reaction volume of 20 μL containing 0.5 μM of each primer (Sigma Aldrich, Germany), 0.05U of Phusion HF DNA polymerase (New England Biolabs, M0530S), 1X Phusion HF Buffer including 200 μM dNTPs (New England Biolabs, B0518S) and 5 μl of genomic DNA template. PCR cycling condition was carried out according to the following program: initial denaturation step at 98°C, 30 sec followed by 35 amplification cycles (denaturation at 98°C, 10 sec; annealing at 58°C, 30 sec and elongation at 72°C, 30 sec) and a final elongation step at 72°C, 10min. All PCR reactions were run on GeneAmp PCR System 9700 Applied Biosystem thermal cycler. Parasitic DNA extracted and purified from T. solium cysticerci isolated from pig was used as intra-run positive control and distilled water as negative control.

T. solium cox1 gene sequencing

The amplicons obtained after cox1 gene amplification by PCR (627 bp) were sequenced in both directions by Genewiz (France). Sequence chromatograms were analyzed using BioEdit software. By removing the beginnings and ends of sequences that were illegible after sequencing, a region of 474 base pairs (starting at the position 601 of the completed cox1 gene sequence) for 10 out of 11 Taenia carriers was finally analyzed for Taenia species identification. The sequences were compared with the 627 base pairs reference regions of each species used beforehand to design the primers.

All nucleotide sequences obtained in the present study were made openly accessible under the following accession numbers: MT947371 (IFAI_Mangevo_051), MT947372 IFAI_Kianjanomby_332), MT947373 (IFAI_Kianjanomby_365), MT947374 (IFAI_Ampitambe_378), MT947375 (IFAI_Ankazotsara_452), MT947376 (IFAI_Torotosy_564), MT947377 (IFAI_Torotosy_573), MT947378 (IFAI_Ampitavanana_641), MT947379 (IFAI_Sahavoemba_182), MT947380 (IFAI_Sahavoemba_190).

T. solium genotypes identification and phylogenic analysis

For the T. solium genotypes and phylogenic analysis, the ten sequences obtained in this study were also compared with T. solium Asian and African/American genotype sequences available in EMBL/Genbank databases. Their accession numbers and the corresponding native countries are as followed: Madagascar: AB781355 to AB781361[40]; FM958305 to FM958317 [41]; Japan: AB516957, AB494702.1; India: KC709810.1; Thailand: AB066487; Indonesia: AB631045.1; Nepal: AB491986; AB524780.1; China: AB066486.1; Korea: DQ089663.1; Mexico: AB066490, FN995657; Cameroon: FN995666.1; Brazil: AB066492.1; Ecuador: AB066491.1; and Tanzania: AB066493. The evolutionary analyses were conducted and a tree was constructed using the Maximum Likelihood method and Jukes-Cantor model [42] using the MEGA X software [43] with 100 bootstrap replicates for reliability tests.

Blood collection

After signing the consent form, blood sampling was performed for each participant in order to carry out serological diagnosis of T. solium infections according to the flow diagram represented in Fig 2. Finger prick blood samples (300–500 μL) were collected in microtubes (Sarstedt, 20.1344). The serum was obtained after 10 minutes of centrifugation at 12 000 rpm, transported at 4°C (cool boxes) within 24 hours at the CVB where they were stored at -20°C. At the end of the survey, all serum samples were transferred and stored to the Institut Pasteur de Madagascar (IPM) at -20°C until use for the serological tests.

Fig 2

Flow diagram for taeniosis (coprology and molecular analysis) and cysticercosis (serological assays) performed during this study.

T. solium glycoprotein antigen preparations

An in-house T. solium glycoprotein antigen suspension was prepared for anti-cysticercal antibody (IgG) detection by ELISA and EITB assays. T. solium cysticerci were harvested from infected pork meat obtained in slaughterhouse (Antananarivo) and stored at -80°C in PBS buffer (DBPS 1X, Sigma Aldrich D8537) until use. Lentil lectin T. solium metacestode glycoproteins (LLGP) antigens were extracted and purified according to the method initially described by Tsang et al., 1989 with slight modifications [44]. All extraction and purification steps have not been changed except for the foamy lipid residues above the pellet that was removed carefully with a spatula without using Freon (CHFCl2) and the urea solubilization of the pellet was not carried out.

Anti-cysticercal antibody (IgG) detection by enzyme-linked immunosorbent assay (Ab-ELISA)

In-house Ab-ELISA assay was performed as previously reported [27]. Briefly, ELISA 96-well microplates (Nunc 3455) were coated with 100 μL/well of previously prepared T. solium glycoprotein antigens extract at 0.1 μg. mL-1 in DPBS 1X and incubated overnight at 4°C. After three washing steps in washing solution (1X DPBS, 0,05% v/v Tween 20), non-specific absorption was blocked by adding 150 μL/well of blocking buffer (DPBS 1X, 0.05% Tween 20, 1% casein) and plates were incubated for 2h at 37°C. After three additional washing steps, 100 μL of serum samples, tested in duplicate and diluted 1:200 in blocking solution, were added per wells and incubated at 37°C for 2 h. Well characterized serum samples of patients from the neuropsychiatry department of Befelatanana Hospital (tested by EITB assay as described below and by neuroimaging) and serum samples from voluntary donors (tested by EITB assay) as positive and negative controls respectively were systematically included in each plate. Plates were then washed five times and incubated at 37°C for 1h30 with 100 μL/well of Human antibody anti-IgG conjugated with peroxidase (Sigma Aldrich A8419) diluted 1:10 000 in blocking solution. Plates were then washed five times before a 20 min incubation at 37°C with 100 μL/well of substrate [(4.5 mM OPD, 3.6% H2O2) in 0.2 M citric acid buffer, pH 5.5, Sigma Aldrich, Ref. P9029 and 251275]. The enzymatic reaction was stopped by addition of 100μL/well of 2.5N H2SO4 acid. The optical density (OD) was determined at 492 nm using microplate reader (Labsystem multiscan Plus). The negative cut-off value (0.3 OD) was calculated as the mean optical density (OD) of 38 sera (obtained from European healthy subjects living in T. solium infections non-endemic areas, France) plus 3 standard deviations. A previous study done in Madagascar has reported a specificity of 97.4% and a sensitivity of 96.3% of this in-house Ab-ELISA assay [27].

T. solium cysticerci antigens detection by ELISA (Ag-ELISA)

The serum samples were tested for the presence of excretory secretory circulating antigens of the metacestode of T. solium using the commercially available cysticercosis Antigen-ELISA Kit (ApDia Ltd., Turnhout, Belgium) based on the B158/B60 monoclonal antibodies enzyme-linked immunosorbent assay (ELISA) [45]. The ELISA was also used and carried out according to the manufacturer’s instructions and the OD was read at 450nm. The Ag-index was calculated following the recommended cut-offs: an Ag-index less than 0.8 classified as negative result, an Ag-index greater than or equal to 1.3 considered as positive and values in between classified as doubtful. The reported sensitivity and specificity of the Ag-ELISA for detecting active cysticercosis in humans were 90% (95% CI: [80–99%]) and 98% (95% CI: [97–99%]), respectively [45,46].

Enzyme-linked Immunoelectro-Transfer Blot (EITB) assay

The EITB assay was performed as previously described by Tsang et al., 1989 [44]. The specificity and sensitivity of the EITB for detecting exposure to the parasite was reported ranging from 97% to 100% and from 97% to 98% respectively [44,46]. The usual concentration for LLGP-antigens in-house EITB assay was about 30ng/μL per mm of membrane. Briefly, each strip membrane was incubated for 2h at 37°C with human diluted (1:10) sera. The strips were thereafter incubated for 1h at 37°C with diluted (1:6000) alkaline phosphatase-conjugated goat anti-human IgG (Sigma Aldrich, A1543). Immune reactivity was detected using BCIP/NBT blue substrate solution (Sigma Aldrich, B3804). Among the seven glycoprotein (GP) bands commonly recognized by antibodies from sera of patients with cysticercosis, only the immune reactivity against one or several GP < 50 kDa (i.e. GP42-39, GP24, GP21, GP18, GP14, and GP13) were considered as positive. As a precaution, tests giving a single 50kDa band were not considered as a positive result in this study. Indeed some studies have shown the lack of specificity of a single positive 50kDa band in EITB assay for cysticercosis [47-49].

Statistical analysis

Descriptive analyses on the study population were first conducted. Prevalence of Taenia infections was estimated as the number of samples tested positive divided by the total number of participants provided samples. Data were analyzed with STATA 15 software. Due to the lack of the specificity of Ab-ELISA [47,50-53], only the prevalence obtained with EITB and Ag-ELISA was used for the association analysis using STATA software. Variables showing more than 25% of missing data were not included into the statistical analysis. A univariate analysis using logistic regression was performed to determine associations between the prevalence and each risk factor. A p value ≤ 0.2 was included in a model (Initial model) for multivariable logistic stepwise regression analysis. A backward deletion method was used to eliminate the factor one at a time considering a p ≤ 0.05 as statistically significant (Final model). A Confidence Interval of 95% (95% CI) and a significance level of 0.05 were performed for the association analysis and the odds ratio calculation.

Results

Participant characteristics

People of all age and both genders, living in the targeted villages and willing to participate to the study were enrolled. A total of 574 willing participants (≥ 5 years old) from 12 villages belonging to 164 households were included in this study. Stool and blood samples were obtained from 543/574 (92.7%) participants following the exclusion of thirty-one participants with missing basic data (age or gender) and missing biological samples (stool or serum). Seventy-three and eleven participants were further excluded for the analysis respectively due to the insufficient volume of sera to perform all three serological tests used for cysticercosis diagnosis (i.e., Ab-ELISA, ETIB and Ag-ELISA tests) and due to the completion of the epidemiological data (<25%) for the statistical analysis. The final study population consisted of 459 participants surveyed having all epidemiological and serological data available. Fig 2 shows the flowchart describing the enrolment and analysis performed.

Socio-demographic characteristics and health/hygiene conditions of the study population are described in Table 1. Genders were equally represented (235 males and 224 females, sex ratio 1.05). The median age was 23.9 years, ranging from 5 to 82 years old with a high proportion of adults over 15 (56%) and just over a quarter (27.7%) were children between 5 and 10 years old. The households were composed mainly of large families (52.1% of households with 4 to 6 people, median 6.4 people) and the adult population was mostly composed of farmers (54.5%). The highest education was the elementary level (68.6%), of which 38.3% were adults. Thirteen-point five percent of adults ≥ 15 years old never attended school. More than two-thirds of participants lived 6 km or more from the main national road (72.1%) and used river stream as house water source (73.2%).

Table 1

Characteristics, behaviours, health and hygiene conditions of the study population (n = 459).

	Number	% (95% CI)		Number	% (95% CI)
Gender			Habit of washing veggies
Male	235	51.2 (46.6–55.8)	Yes	272	59.3 (54.7–63.7)
Female	224	48.8 (44.2–53.4)	No	80	17.4 (14.2–21.2)
Sex ratio	1.05		Missing	107	23.3 (19.7–27.4)
Age groups (in years)			Pigs nearby
Extreme (Median)	5 – 82y (23.9y)		Yes	186	40.5 (36.1–45.1)
5–10 y	127	27.7 (23.8–32.0)	No	237	51.6 (47.1–56.2)
11–15 y	75	16.3 (13.2–20.0)	Missing	36	7.8 (5.7–10.7)
> 15 y	257	56.0 (51.4–60.5)	Pork consumption
Number of household members			Yes	435	94.8 (92.3–96.5)
1	16	3.5 (2.1–5.6)	No	14	3.1 (1.8–5.1)
2 to 3	98	21.4 (17.8–25.4)	Missing	30	6.5 (4.6–9.2)
4 to 6	239	52.1 (47.5–56.6)	Meat source (Pork)
7 to 9	94	20.5 (17.0–24.4)	Local/Home slaughter	63	13.7 (10.9–17.2)
≥10	12	2.6 (1.5–4.6)	District slaughter	297	64.7 (60.2–68.9)
Profession (> 15 y)			Both	69	15.0 (12.1–18.6)
Government official (Teacher)	1	0.2 (0.0–1.5)	Missing	10	2.2 (1.2–4.0)
Farmers	250	54.5 (49.9–59.0)	Concept of disease spreading within households
Students	145	31.6 (27.5–36.0)	Yes	42	9.2 (6.8–12.2)
Missing	21	4.6 (3.0–6.9)	No	318	69.3 (64.9–73.3)
Education			Missing	99	21.6 (18.0–25.6)
None (> 15y.)	62	13.5 (10.7–17.0)	Cysticercosis knowledge
NA (Children ≤ 6y)	42	9.5 (6.8–12.2)	Yes	12	2.6 (1.5–4.5)
Elementary school	315	68.6 (64.2–72.7)	No	276	60.1 (55.6–64.5)
High school	22	4.8 (3.2–7.2)	Missing	171	37.7 (33.0–41.8)
College	1	0.2 (0.0–1.5)	Age of first seizure (year of old)
Missing	17	3.7 (2.3–5.9)	<2y.	10	2.2 (1.2–4.0)
House distance to the national main road			2-4y.	15	3.3 (2.0–5.3)
< 6 km	128	27.9 (24.0–32.2)	5-10y.	11	2.4 (1.3–4.2)
≥ 6 km	331	72.1 (67.8–76.0)	11-15y.	2	0.4 (0.1–1.6)
House water source			16-19y.	3	0.7 (0.2–1.9)
Stand pipe	8	1.7 (0.9–3.4)	20-37y.	5	1.1 (0.5–2.5)
Stream (river)	336	73.2 (69.0–77.0)	No	63	13.7 (17.2–10.9)
Missing	115	25.1 (21.3–29.2)	Missing	350	76.3 (72.2–79.9)
Latrine use			Headache frequency
Yes	201	43.8 (39.3–48.4)	1 every 3 months or so (Rare)	56	12.2 (9.5–15.5)
No	239	52.1 (45.8–59.1)	> 1 every 3 months (Frequent)	49	10.7 (8.2–13.9)
Missing	19	4.1 (2.7–6.4)	Never	257	56.0 (51.4–60.5)
Opportunity for washing hands			Missing	97	21.1 (17.6–25.1)
Yes	444	96.7 (94.6–98.0)	Deworming frequency
No	1	0.2 (0.0–1.5)	Only when prescribed	2	0.4 (0.1–1.7)
Missing	14	3.1 (1.8–5.1)	1/year	60	13.1 (10.3–16.5)
Soap use for washing hands			2/year	362	78.9 (74.9–82.4)
Yes	276	60.1 (55.6–64.5)	Never	8	1.7 (0.9–3.5)
No	127	27.7 (23.8–31.9)	Missing	27	5.9 (4.1–8.5)
Missing	56	12.2 (9.5–15.5)

Percentage (%) and 95% CI (95% Confidence Interval) are based on the total number of participants. NA = Not applicable.

Hygiene conditions were poor: 52.1% of participants did not have access to latrines and practiced open defecation. Surprisingly, a large part of the study population however declared having the opportunity to wash their hands (96.7%), using soap for washing hands (60.1%) and the habit of washing fruits or vegetables (59.3%) before eating/cooking.

More than half (51.6%) reported not having pigs near where they lived and pork consumption was reported by a high proportion (94.8%) of participants principally from district slaughter (64.7%). Most of the participants (78.9%) declared having a history of deworming drugs with a treatment taken at least twice a year. The survey data also showed that 69.3% of the study population was not aware of the concept of disease spreading within households. Nevertheless 60.1% of the people had knowledge about cysticercosis. Questionnaire analysis also showed that 56.0% of the participants declared never having headaches versus 10.7% declared having frequent headaches (more than 1 headache every 3 months). 10.1% of the participants reported having had their first seizure of which 1.1% (5 participants) were between 20–49 years old. However, a large proportion of the participants (76.3%) lacked data on their first seizure including 154 participants (31.6%) aged between 20–49 years old.

Taeniosis prevalence

The estimated overall prevalence of taeniosis screened by stool microscopy for Taenia spp. carriage was 2.4% (95% CI: 1.3–4.3) corresponding to eleven tapeworm carriers living in eight distinct villages. Sedimentation technique detected 10 positive tapeworm carriers whereas Kato-Katz technique detected 9. Taenia carriage seems to be more prevalent in women (n = 7, 3.1%, 95% CI: 1.5–6.4) than men (n = 4, 1.7%, 95% CI: 0.6–4.5), and in adults (n = 7, 2.7%, 95% CI: 1.3–5.6) than children under 15 years old (n = 4, 2.0%, 95% CI: 0.7–5.2) but these differences were not statistically significant (P > 0.05). All age classes were affected: average age of the tapeworm carriers was 20.1 years, ranging from 6 to 51 years old. The number of detected tapeworm carriers varied slightly across villages: two for each of the villages of Kianjanomby, Sahavoemba, Torotosy and one for each of the villages of Marojano, Mangevo, Ampitavanana, Ankazotsara, Ampitambe; No tapeworm carrier was detected in 4 out of the 12 villages where the sampling was carried out (Ambinanindranofotaka, Bevoahazo, Mandrivany and Sahavanana) (Table 2).

Table 2

Prevalence of taeniosis by villages investigated.

Results obtained after stool analysis by Kato-Katz (KK) or sedimentation assays (SS).

	Tapeworm carriers
Villages investigated (stools analyzed/villagers >5y. old in the selected HH/total villagers >5y. old#, n)	Positive samples by KK (n)	Positive samples by SS (n)	Positive samples per villages n (%, 95% CI)
Kianjanomby (50/89/721)	1	2	2 (4, 1.0–14.9)
Sahavoemba (35/41/363)	1	1	2* (5.7, 1.4–20.6)
Torotosy (52/80/383)	2	2	2 (3.8, 0.9–14.3)
Marojano (23/50/303)	1	1	1 (4.3, 0.6–26.2)
Mangevo (31/37/186)	1	1	1 (3.2, 0.4–20.3)
Ampitavanana (40/65/432)	1	1	1 (2.5, 0.3–16.1)
Ankazotsara (45/89/294)	1	1	1 (2.2, 0.3–14.5)
Ampitambe (41/65/369)	1	1	1 (2.4, 0.3–15.8)
Ambinanindranofotaka (40/48/253)	0	0	0
Bevoahazo (10/12/403)	0	0	0
Mandrivany (68/114/805)	0	0	0
Sahavanana (24/31/255)	0	0	0
Total	9	10	11

n: number of villagers and participants, total and per village or positively detected by each test, prevalence (%) and 95% CI (Confidence Interval).

HH: household

#excluding 121 participants with unspecified age (not knowing their age and without birth certificate)

*Sahavoemba, two (2) different tapeworm carriers were detected either by KK or sedimentation coprology tests.

Taenia species and T. solium genotypes

Of the 11 positive stools in microscopy, cox1 PCR product was obtained from extracted copro-DNAs. Their sequencing analysis amplified cox1 DNA fragments and showed that ten belonged to T. solium species and one was an unspecific amplification. Two T. solium genotypes were identified: T. solium Asian genotype and T. solium African-American genotype. The majority of the sequences (9/10) belonged to the Asian genotype and was detected in stool samples from the villages of Ampitavanana, Torotosy, Ankazotsara, Kianjanomby, Mangevo, Ampitavanana and Sahavoemba. Only one cox1 sequence matched with the African-American genotype and was identified from one of the tapeworm carriers living in the village of Sahavoemba (Fig 3).

Fig 3

Phylogenic tree showing the Asian and the African-American Taenia solium genotypes of the specimens obtained in this study and in databases.

Cox1 sequences obtained in this study (from human stool with detected Taenia eggs) are underlined in blue. The sequences obtained from EMBL/Genbank databases (from cysticerci of pig) are indicated with their accession numbers followed by the name of each District/township for genes described in Madagascar and by “FOR” for specimens collected in foreign country. The percentage (%) of bootstrap replicates for the reliability tests of the associated taxa clustered together is shown next to the branch (up and down for the genomic and coding forms respectively).

The phylogenic tree analysis performed with cox1 sequences, obtained from human stools, and the cox1 Asian genotypes sequences (obtained from pig cisticerci) deposited in EMBL/GenBank databases revealed that the majority (8/9) of the sequences obtained in this study are absolutely conserved. These T. solium Asian genotypes clustered together within the corresponding genotype sequences isolated from pig cisticerci collected in Central, South and Western parts of Madagascar [40,41] and from other countries (India, Nepal, Japan and Thailand). All these cox1 sequences belonged to the Asian T. solium genotype, cluster I (Fig 3). This first cluster was less close to the cox1 DNA specimen collected from the eastern part of Madagascar (Toamasina, cluster II) and distant to those found in Indonesia (Asian T. solium genotype Cluster II) and in other countries such as Korea and China (Asian T. solium genotype Cluster IV). The last cox1 T. solium Asian genotype sequence from Sahavoemba formed a cluster apart (Asian T. solium genotype Cluster V).

The only African-American genotype found in this study shared 100% similarity to the sequences already identified in the Southern part of Madagascar (Toliara, from pig cysticerci) and in other African/American countries (Tanzania and Mexico) forming a cluster (African-American T. solium genotype cluster III) which was less close to those found in Ecuador (African-American T. solium genotype cluster II) and more distant to those obtained in Mexico, Cameroon and Brazil (African-American T. solium genotype the Asian T. solium genotype cluster I). The variation of nucleotides and the corresponding amino acids for each T. solium genotype cluster are shown in the supporting S2 Fig.

Exposure to T. solium parasite: seroprevalence of anti-T solium cysticercus antibodies (IgG)

The presence of anti-T. solium IgGs against metacestode glycoproteins in serum was estimated by ELISA (Ab-ELISA) and EITB methods, which given a fairly similar seroprevalence: 27.5% (95% CI: 23.5–31.7) and 29.9% (95% CI: 25.8–34.2) respectively (Table 3). Females seem to be more exposed to T. solium parasite using Ab-ELISA test (Sex ratio: 0.7) while no differential exposure was observed using the EITB assay (Sex ratio: 1.01). The average age of Ab-ELISA seropositive individuals was 22 years, (ranging from 5 to 79 years old) whereas the average age of EITB seropositive individuals was 24.4 years (ranging from 5 and 74 years old). Using both methods, anti-cysticercal IgG seroprevalence was quite similar between the three defined age-groups. Depending on the village investigated, the exposure rate found by Ab-ELISA and IgG Western Blot assays ranged between 13% - 50% and between 16% - 70% respectively (Table 3). At household level, the overall seroprevalence of IgG detected by Ab-ELISA and EITB within the same household in which a tapeworm carrier has been detected was 46% and 34% including 5/11 and 3/11 Taenia carriers respectively with co-infection (taeniosis and cysticercosis).

Table 3

Seroprevalence of T. solium anti-cysticercal antibodies (Ab-ELISA and EITB assays, IgG) and cysticercal Antigens (Ag-ELISA) for all participants investigated and within the Taenia carrier households.

	Ab-ELISA		EITB		Ag-ELISA
	Positive (n)	% (95% CI)	Positive (n)	% (95% CI)	Positive (n)	% (95% CI)	Doubtful n (%)
Total, N = 459	126	27.5 (23.5–31.7)	137	29.9 (25.8–34.2)	57	12.4 (9.7–15.8)	30 (6.5)
Gender
Male	53	22.6 (17.6–28.4)	69	29.4 (23.9–35.5)	36	15.3 (11.2–20.5)	18 (7.7)
Female	73	32.6 (26.7–39.0)	68	30.4 (24.7–36.7)	21	9.4 (6.2–14.0)	12 (5.4)
Sex ratio	0.7		1.01		1.7		1.5
Age groups (in years)
Extreme (Median)	5–79y (22y)		5–74y (24.4y)		5–74y (29.1y)		5–79 y (25.9)
5–10y	37	29.1 (21.9–37.7)	34	26.8 (19.8–35.2)	11	8.7 (4.8–15.0)	7 (5.5)
11–15y	21	28.0 (19.0–39.3)	25	33.3 (23.5–44.8)	7	9.3 (4.5–18.4)	5 (6.7)
> 15y	68	26.5 (21.4–32.2)	78	30.4 (25.0–36.3)	39	15.2 (11.3–20.1)	18 (7.0)
Villages for all participants (serum analyzed/villagers >5y. old in the selected HH/total villagers >5y. old#, n)
Kianjanomby (50/89/721)	13	25 (15–39)	23	44 (31–58)	5	9.6 (4–21)	0
Sahavoemba (35/41/363)	12	34 (20–52)	10	29 (16–46)	7	20.0 (10–37)	4 (11)
Torotosy (52/80/383)	10	19 (11–32)	17	33 (21–47)	11	21 (12–35)	6 (12)
Marojano (23/50/303)	5	22 (9–43)	16	70 (48–85)	2	9 (2–30)	2 (9)
Mangevo (31/37/186)	11	36 (21–54)	9	29 (16–47)	5	16 (7–34)	4 (13)
Ampitavanana (40/65/432)	14	35 (22–51)	13	33 (20–4)	8	20 (10–35)	4 (10)
Ankazotsara (45/89/294)	13	29 (17–44)	8	178 (9–32)	6	13 (6–27)	2 (4)
Ampitambe (41/65/369)	16	41 (27–57)	9	23 (12–39)	5	13 (5–28)	2 (5)
Ambinanindranofotaka (40/48/253)*	8	20 (10–35)	10	25 (14–41)	3	8 (2–21)	1 (3)
Bevoahazo (10/12/403)*	5	50 (21–79)	2	20 (5–56)	1	10 (1–50)	2 (20)
Mandrivany (68/114/805)*	16	24 (15–35)	11	16 (9–27)	2	3 (1–11)	1 (2)
Sahavanana (24/31/255)*	3	13 (4–33)	9	38 (21–58)	2	8 (2–29)	2 (8)
Village per Taenia carrier and per household (mHH included/Total mHH)
Total, N = 41	19	46 (32–61)	14	34 (22–50)	8	20 (10–34)
Kianjanomby (4/7)	2¤	50 (9–91)	2¤	50 (9–91)	2¤	50 (9–91)	0
Kianjanomby (2/4)	0		0		0		0
Sahavoemba (6/8)	2	33 (6–70)	2	33 (6–70)	1¤	17 (1–56)	0
Sahavoemba (6/7)	5¤	83 (44–99)	1	16 (1–56)	1¤	17 (1–56)	0
Torotosy (2/4)	1¤	50 (3–97)	2¤	100 (18–100)	1	50 (9–91)	0
Torotosy (4/9)	1	25 (1–70)	1	25 (1–70)	1	25 (1–70)	0
Marojano (3/6)	1¤	33 (2–88)	1¤	33 (2–88)	0		0
Mangevo (4/5)	2	50 (9–91)	2	50 (9–91)	0		1 (25)
Ampitavanana (4/6)	2	50 (9–91)	2	50 (9–91)	2	50 (9–91)	0
Ankazotsara (3/3)	0		0		0		0
Ampitambe (3/6)	3¤	100 (44–100)	1	33 (2–88)	0		1 (33)

Ab-ELISA: LLGP antigens-based enzyme-linked immunosorbent assay.

EITB: Enzyme-linked Immuno-electroTransfer Blot detecting specific antibodies (IgG) against T. solium cysticercus glycoproteins (GP39/42-GP24-GP21-GP18-GP14/13 kiloDaltons, LLGP antigens) [44].

Ag-ELISA: B158/B60 monoclonal antibodies-based enzyme-linked immunosorbent assay. An Ag-index less than 0.8 was classified as negative result, an Ag-index greater than or equal to 1.3 was considered as positive and values in between classified as doubtful.

n: number of villagers and participants, total and per village or detected positively by each serological test respectively

HH or mHH: household or member of household including participant tested positively

#excluding 121 villagers with unspecified age (those not knowing their age and without birth certificates)

*Villages where no Taenia eggs carriers were detected.

¤Taenia carrier tested positively for each serological test

Prevalence of current cysticercosis infection with viable cysts

Current cysticercosis was detected by measuring the excretory/secretory circulating T. solium antigens based on B158/B60 enzyme-linked immunosorbent assay (Ag-ELISA) [45]. Circulating antigens were detected in 57/459 participants giving an overall seroprevalence of 12.4% (95% CI: 9.7–15.8). Cysticercosis affected all age classes, ranging from 5 to 74 years old with an average age of 29.1 years (Table 3). T. solium antigen seroprevalences were higher in males (15.3%, sex ratio: 1.7) and in adults over 15 years old (15.2%) but did not reach statistical significance (p = 0.07 and p = 0.32 respectively). The seroprevalence of T. solium antigens varied between 3% and 21% according to the village investigated. Doubtful cases (n = 30, 6.5%, 95% CI: 4.6–9.2), with an Ag-index < 0.8, were also detected in the study population. These doubtful cases were higher in men (7.7%, sex ratio: 1.5) and quite similar between the 3 age groups. The rate of these cases, which should be confirmed by a second blood sampling, varied from 0% to 20% according to the village investigated (Table 3). Considering the participants within the same household, the overall prevalence of circulating T. solium antigens was 20% comprising 3/11 Taenia carriers.

Risk factors associated with T. solium infections: univariate analysis

Several epidemiological and behavioral factors were evaluated for their association with T. solium infections and summarized in Table 4. For both serological techniques used (EITB and Ag-ELISA), sixteen variables were firstly assessed using univariate analysis. Univariate analysis suggests that a high number of householders (≥ 4 people, OR = 2.0, p = 0.001) and open defection behaviors (OR = 1.6, p = 0.02) could be risk factors associated with the presence of T. solium anti-cysticercal antibodies (EITB). With p value ≤ 0.2, housing more than 6 km from the national main road (OR = 1.5), pork consummation (OR = 3.1), district slaughter as meat (pork) source (OR = 0.6) and frequent headache (OR = 0.6) were also included in the multivariate analysis for EITB test (Table 4). Contrariwise, regarding T. solium circulating antigen positivity (Ag-ELISA seropositivity), being male (OR = 0.6 for female, p<0.001) and no use of latrine (OR = 1.6, p = 0.049) could be associated with a higher risk of developing active cysticercosis. Multivariable analysis was completed for Ag-ELISA test with the following factors (with p value ≤ 0.2): adulthood (over 15 years, OR = 1.7), use of stand pipe for house water source (OR = 0.3 for stream river), use of soap for washing hands (OR = 1.5), and no habit of washing veggies (OR = 0.7 for habit of washing veggies).

Table 4

Univariate analysis on risk factors and T. solium infections (positive EITB and Ag-ELISA results).

	Positive EITB		Positive Ag-ELISA
Variables	OR (95% CI)	p-value	OR (95% CI)	p-value
Age groups (in years)
5–10ya	1		1
11–15y	1.4 (0.7–2.5)	0.323	1.1 (0.5–2.6)	0.700
> 15y	1.2 (0.7–1.9)	0.468	1.7 (1.0–3.1)	0.100*
Gender
Malea	1		1
Female	1.0 (0.7–1.6)	0.816	0.6 (0.4–0.9)	0.000*
Number of household members
≤3 personsa	1		1
≥ 4 persons	2.0 (1.2–3.2)	0.001*	1.0 (0.6–1.8)	0.900
Education
No schoolinga	1		1
Elementary and high school levels	1.4 (0.8–2.2)	0.220	0.8 (0.5–1.4)	0.516
House distance to the national main road
< 6 kma	1		1
≥ 6 km	1.5 (1.0–2.3)	0.070*	1.0 (0.6–1.7)	0.940
House water source
Stand pipea	1		1
Stream (river)	1.2 (0.3–6.2)	0.800	0.3 (0.1–1.2)	0.090*
Washing hands after toilet, before eating
Yesa	1		1
No	1.1 (0.6–2.1)	0.744	0.6 (0.3–1.5)	0.260
Soap use for washing hands
Noa	1		1
Yes	0.9 (0.6–1.4)	0.633	1.5 (0.8–2.6)	0.179*
Habit of washing veggies
Noa	1		1
Yes	1.1 (0.8–1.7)	0.559	0.7 (0.5–1.2)	0.179*
Pigs nearby
Noa	1		1
Yes	0.9 (0.6–1.4)	0.753	1.1 (0.7–1.8)	0.672
Pork consumption
No, without responsea	1		1
Yes	3.1 (0.9–10.6)	0.070*	0.9 (0.3–2.4)	0.809
Meat source (Pork)
Local/Home slaughter or botha	1		1
District slaughter	0.6 (0.3–1.1)	0.070*	0.8 (0.5–1.3)	0.412
Latrine use
Yesa	1		1
No	1.6 (1.1–2.4)	0.020*	1.6 (1–2.6)	0.049*
Disease spreading concept within households
No/without responsea	1		1
Yes	1.2 (0.6–2.4)	0.610	0.8 (0.4–2.0)	0.692
Frequent headache (> 1 every 3 months)
Noa	1		1
Yes	0.6 (0.3–1.2)	0.131*	2.0 (0.5–2.1)	0.912
Deworming frequency (2/year)
Noa	1		1
Yes	1.3 (0.8–2.1)	0.324	1.5 (0.8–2.8)	0.203

a Reference variable

*Risk factors with a p value ≤ 0.2 were included in multivariable analysis.

Risk factors associated with T. solium infections: multivariate analysis

In the multiple regression analysis, the risk factors that were significantly associated with anti-cysticercal Antibodies (EITB) or cysticercal Antigens (Ag-ELISA) positivity (p-value ≤ 0.05) are presented in Tables 5 and 6 respectively. A high number of householders (≥ 4 people, OR = 1.9, 95% CI: 1.1–3.1, p = 0.017) and open defecation behaviors (OR = 1.5, 95% CI: 1.0–2.3, p = 0.038) were found to be the main risk factors of exposure to T. solium infections (seropositivity for T. solium antibodies) [see Table 5]. On the other hand, females (OR = 0.5, 95% CI: 0.3–0.9, p = 0.010) and house water source from river (OR = 0.4, 95% CI: 0.2–0.7, p<0.001) were less at risk of developing active cysticercosis with the presence of circulating T. solium antigens (Table 6). At the opposite, adults (over 15 years old, OR = 1.6, 95% CI: 1.0–2.7, p = 0.049) had an increased risk of developing cysticercosis with detectable T. solium circulating Ag (Table 6).

Table 5

Multivariable analysis on risk factors for exposure to T. solium infections (positive EITB).

	Positive EITB
	Initial model		Final model
Variables	OR (95% CI)	p-value	OR (95% CI)	p-value
Number of household members
≥ 4 persons	1.7 (1.0–3.0)	0.047*	1.9 (1.1–3.1)	0.017*
Education
Elementary and high school levels	1.6 (0.9–2.6)	0.088
House distance to the national main road
≥ 6 km	1.4 (0.9–2.2)	0.181
Pork consumption
Yes	3.0 (0.8–10.6)	0.097
Meat source (Pork)
District slaughter	0.8 (0.5–1.2)	0.267
Latrine use
No	1.6 (1.0–2.4)	0.038*	1.5 (1.0–2.3)	0.038*

OR: odds ratio; 95% CI: 95% confidence interval.

*Significance level: p ≤ 0.05.

Table 6

Multivariable analysis between risk factors and T. solium infections (positive Ag-ELISA).

	Positive Ag-ELISA
	Initial model		Final model
Variables	OR (95% CI)	p-value	OR (95% CI)	p-value
Age classes
> 15y	1.8 (1.1–3.1)	0.026*	1.6 (1.0–2.7)	0.049*
Gender
Female	0.5 (0.3–0.8)	0.007*	0.5 (0.3–0.9)	0.010*
House water source
Stream (river)	0.3 (0.1–1.5)	0.100	0.4 (0.2–0.7)	0.000 *
Soap use for washing hands
Yes	1.2 (0.7–2.2)	0.467
Habit of washing veggies
Yes	0.7 (0.4–1.1)	0.139
Toilet use
No	1.6 (1.0–2.7)	0.057

OR: odds ratio; 95% CI: 95% confidence interval

Significance level: p ≤ 0.05*

Discussion

There is little information available on taeniosis/cysticercosis epidemiology in Madagascar [21]. To the best of our knowledge, the current study is the first community-based survey reporting the prevalence of human T. solium infections using three different diagnostic tests for cysticercosis and analyzing the associated risk factors in rural and remotely region of Madagascar.

Little data is available regarding the prevalence of adult tapeworm carriers (taeniosis) who are the source of cysticercosis in humans and pigs [54]. The main difficulty for studying taeniosis especially in Africa is the lack of a simple, sensitive and T. solium specific diagnostic tool. Stool microscopy techniques classically used are not able to discriminate eggs of T. solium and T. saginata and have poor sensitivity and specificity [55]. In Madagascar, national surveys did not report any tapeworm carriers in the district of Ifanadiana [35]. Kato Katz and Spontaneous Sedimentation techniques used in this study however allowed estimating a high overall prevalence of taeniosis reaching 2.4% and classifying these rural areas as hyperendemic [56] and at a higher rate when compared to the results reported previously in Madagascar [35,57]. The prevalence of taeniosis found in this study are quite similar to those previously described (0.1%–1.4%) in many African countries (Burundi, Congo, Ethiopia, Togo, Zambia, Cameroon, Kenya and Tanzania) and in some endemic countries of Latin America (Peru and Ecuador) using comparable coprological examination. Conversely these prevalence rates could be low compared to those reported in other African countries (Guinea, Nigeria, Ghana, and Gambia: up to 13%) [12,14,15,54] and those obtained using more sensitive diagnostic tools such as coproantigen ELISA and EITB methods (Zambia, Tanzania, Kenya and Democratic Republic of Congo: up to 23.4% [58-61].

No adult tapeworm carriers were detected in 4 out of the 12 villages investigated where participants presenting antibodies anti-Taenia solium and circulating antigens were however detected. Indeed, studies have shown that microscopy techniques are weakly sensitive missing 60–70% cases of taeniosis [62]. The best diagnostic assay for the intestinal taeniosis and constituting an effective tool for epidemiological studies would be the coproantigen detection ELISA having a sensitivity of about 95% and a specificity over 99% [62]. However these data have never been independently validated. This ELISA assay has been estimated to be two to ten times more sensitive than coprology methods [63]. But neither the corresponding test nor the necessary polyclonal antibodies for the coproantigen ELISA are available on the market and the method remains genus specific. Regarding molecular tools, a triplex Taq-Man probe-based qPCR for the detection and discrimination of T.solium, T. saginata and T. asiatica in human stool [64] and the field application of a Loop-mediated isothermal AMPlification method (LAMP) for rapid identification of human taeniosis were also reported [65]. Using these tests especially the simple and sensitive LAMP technique in the context of Madagascar could be very useful in the field.

Taeniosis cases reported in this study affected all age (mean age: 20.1 years) with 2.7% of prevalence in adults more than 15 years old. This population is not targeted by the national mass treatment programs (mainly treating children schooled between 5 to 15 years old). Thereby these results could guide the national program on mass chemoprevention (as primary intervention strategy against T. solium infections) to treat the entire population of all ages, especially the inhabitants of rural and remote areas.

Species of the Taenia spp. eggs identified in this study were all T. solium, the only species associated with cysticercosis. In order to analyze T. solium genotypes, we used cytochrome c oxidase subunit I which has been widely used for studying polymorphism and for establishing phylogenetic trees of Taenia genus (T. solium, T. saginata and T. asiatica) [41]. Our results confirm the findings of previous molecular analysis of T. solium worldwide, reporting that T. solium could be divided into two genotypes, Asian and African/American genotypes [66-68](Fig 3). Our data strengthen the previous results of Michelet et al. and Yanagida et al. [40,41] showing that the two genotypes geographically disjunct are sympatric in Madagascar. Indeed confirmed by analyses of mtDNA and nuclear DNA, Malagasy people have mixed origins from Southeast Asia and from Southeast Africa [69]. Our results extend the previous findings that were made in Madagascar (on cysticerci from pigs) that the sympatric distribution of Asian and African-American T. solium genotypes is also found in the adult form and in the eggs of T. solium from human stool. The predominance of the Asian genotype (9/10 specimens) corroborated also to these prior results observed at a national level which occurred evenly at a District level of Ifanadiana for this study (Fig 3). Asian genotypes from this study are close to the Asian genotypes from India and Thailand but distant to the Asian genotypes from Indonesia, Korea and China as already reported [40,41], supporting the importance of the Indian influence on the diversity of people and culture in Madagascar [40,69,70]. In this study, one of the two sequences obtained from the village of Sahavoemba resembles the Asian genotype but contains a base at the position 867 that is substituted (adenosine to guanine), as found in the African-American genotypes (Fig 3). Cross-fertilization and hybridization between individual worms possessing different genotypes has already been suggested by analyzing the nuclear genes [40]. Only one specimen from the village of Sahavoemba was an African-American genotype in our study. The sequence is closer to the other specimens isolated in Madagascar (Toliara) and Tanzania but distant to the sequences from Cameroon and Brazil (Fig 3).

The antigens used in this study based on lentil lectin affinity chromatography preparation [44] and Ag-ELISA (B158/B60) method [45] are used worldwide and constitute assays recognized by the WHO, both in sero-surveys for prevalence estimation and also for the diagnosis of individuals [50].

In this study the prevalence of antibodies (IgG) associated with an exposure with T. solium parasite detected by ELISA and by EITB methods using T. solium metacestode glycoproteins was quite similar (27.5% and 29.9% respectively). One study in Zambia has reported a higher seroprevalence (34.5%) than that found in this study using an EITB assay [71]. However this positivity rate is relatively high compared to those found in the general population or among villagers in other African countries using similar methods (ranging from 1.3% to 14.3%) and very high compared to the prevalence obtained in people with epilepsy (0 to 2.8%) [12-15,54]. The detection of cysticercosis-specific antibodies during field-based studies could help to identify areas of disease transmission [50] however they tend to over-estimate the prevalence of cysticercosis in sero-epidemiological surveys. Indeed, as antibodies disappear after 1 to 3 years in only 30–40% of seropositive people in endemic countries [72], some seropositivity will reflect past exposure to the parasite. In addition, infection in the general population could be in any tissue unlike epileptic individuals with NCC with cysts mainly localized in the Central Nervous System. Lower antibodies related to NCC may also be due to the lower sensitivity of EITB in parenchymal localization with few or calcified cysts [73] [74].

The presence of circulating antigens asserting an active cysticercosis case with viable cysts was observed at a prevalence of 12.4%. This prevalence is relatively high compared to those obtained in the following African countries (Cameroon, Zambia, Kenya, Burkina-Faso, Togo, and Senegal: 0.7% to 8.1%) [12-14,16,58] but extensively low compared to those found in Tanzania, Congo, and Togo (16.8% à 38%) [12,13]. However this positivity is similar to the prevalence of circulating Taenia antigens detected in Zambia, 12.5%[71].

Regarding the associated risk factors analysis, the use of soap for hand-washing was surprisingly found associated to active cysticercosis. A study using the Ag-ELISA test reported that participants who declared washing their hands by a dipping method and using the same water were more likely to be seropositive unlike those who used running water [59]. In our study villages, using soap for hand washing as a risk factor may be explained by the fact that people use the same contaminated river water for multiple purposes (washing vegetables, dishes, hands, etc). However other factors would be interesting to consider and could help understand this surprising result. The risk analysis result obtained with the variable “house water source” using standpipe and the probable protective factor when using the river with renewed running water seems to strengthen this previous report. In fact, clean water used directly as a source of water might be a protective factor of cysticercosis in this study.

Other risk factors tested significantly in this study are common factors classically reported in several previous investigations of T. solium infections in Africa and worldwide, such as open defection [54,71,75-78], pork consumption (especially in preparation and cooking methods) [54,77,79] and increased age (adults >15 year old) [2,12,59,80-83]. Many family members (≥4) in the same household is a high risk insofar as a single T. solium carrier may easily cause cysticercosis to his family members through T. solium eggs shedding [11,84,85]. Among all identified factors, lack of knowledge of the population about soil-transmitted diseases and their risk (68.8% of the participants with elementary level of education) [16,75,86], the poor hygiene (52.1% of participants without latrines and practicing open defecation) and the isolation of these villages (≥ 6km from the main national road) promote the spread of T. solium [17,87]. Therefore, public health education in Madagascar should be a complementary approach for T. solium control. Indeed, these education and communication-based strategies have already been evaluated with success in several places like in rural communities of Burkina Faso [88], in an urban community of Mexico [54], through a computer-based educational health tool on T. solium in Tanzania and in Zambia [89,90], or also through a participatory community education workshop about T. solium in Peru [91]. Some protecting factors have nonetheless been identified as being a female, district versus local slaughter for pork source and habit of washing vegetables. Similar results were also obtained in village-community studies performed in Congo and in Burkina Faso that have reported a positive association between the presence of T. solium circulating antigens and being a male [2,61,92]. Likewise, several publications have reported that in the Districts where official meat inspection could take place, unlike local/home unregistered slaughtering (that has been largely shown to be an important source of transmission of T. solium infections), the prevalence of parasite carriage and associated infections decreased significantly [15,17,93-95].

Our study may present limitations related to the number of samples analyzed. However, the epidemiological data obtained on the geographical distribution of T. solium taeniosis / cysticercosis in humans living in remote areas of Madagascar is important for identifying high-risk populations and applying control measures. This study identified and treated some tapeworm carriers of all age. Indeed, the treatment of taeniosis in humans is one of the basic "quick impact" interventions for strengthening the control/elimination of the parasite transmission and preventing the health burden associated with NCC. During this study, eight cases of active cysticercosis including three co-infections (taeniosis and active cysticercosis probably through self-infestation) were observed and clustered at household level with Taenia carrier. Some suggestive clinical manifestations of health burden associated with NCC (seizure in individuals aged between 20–49 years old [96]) were also recorded without being significantly associated with cysticercosis (Probably due to the high number of missing data: 76.3%) and without confirmation of diagnosis by neuroimaging. Indeed, neuroimaging is an Absolute-Major-Confirmative criteria of NCC. Nevertheless, important criteria of NCC according to the classification of Del Bruto and colleagues in 2017 (such as: specific anticysticercal antibodies/cysticercal antigens detected by immunodiagnostic tests, evidence of a household contact with T. solium infection, and clinical manifestations suggestive of NCC: mainly seizures) were recorded during this study. These data were reported to the local health authorities for the confirmation of the diagnosis of these cases and their eventual management.

Human health interventions in Madagascar remain challenging. Indeed, through a three-year pilot project (2015–2017) in Madagascar, a significant reduction in the prevalence of taeniosis was observed after annual Mass Drug Administration (MDA) targeted adults and children >5 years old. However this reduction could not be maintained [97]. Investigations in pig health and environmental sector as part of a "one health" approach are therefore required to break the cycle of infection [98]. An ongoing project combining pig vaccination, drug treatment in pigs and in human tapeworm carrier will investigate this approach in Madagascar [99].

Conclusions

This is the first large-scale study in Madagascar to examine the prevalence of Taenia solium taeniosis/cysticercosis in humans and the association between potential risk factors measured at the individual-, household-, and village-level. Our results showed high rates of taeniosis cases affecting all ages in both townships investigated, strongly exposing human and pigs to cysticercosis by considering open defecation practices of the participants. Indeed, search of Taenia carrier among household contacts is currently recommended to identify the potential source of infection clustered within household and to reduce further spread/burden of the diseases. This high exposure of participants confirmed by the high rate of active cysticercosis cases should be managed with neuroimaging for a targeted and more efficient treatment. These data would be useful for guiding the authorities and all the entities involved in the programs and in the strategies for combating these diseases especially in rural and remote areas in Madagascar Island. Investigations in pigs and in environment would also be necessary as part of a “one health” approach to complete control strategies for those zoonotic neglected tropical diseases.

Position of the common primers used to amplify cox1 gene and discriminate Taenia species by PCR and sequencing.

Multiple alignments of T. solium Asian or African/American genotypes (T. sol Gen Asia, GeneBank Accession No° AB066488; and T. sol Gen Af/Am, GeneBank Accession No° AB066492.1), T. asiatica (GeneBank Accession No° AB107235.1) and T. saginata (GeneBank Accession No° AB107246.1) were performed. Similarity and variation are marked by a star and space respectively. Common forward and reverse primers are indicated highlighted in bold and with an arrow.

(DOCX)

Click here for additional data file.

Multiple alignment of the partial nucleotide sequence of cox1 showing polymorphism between T. solium genotypes obtained in this study and in databases.

Five T. solium Asian genotypes (T. sol Asian I to V) and three for T. solium African-American genotype (T. sol Af/Am I to III) were aligned. T. solium Asian genotype cluster I sequence is shown as reference. Similarity is indicated with dot. Variant nucleotides are shown in lowercase and in capital letters if affecting amino acid sequence. Comparing to the Asian T. solium genotype cluster I (containing the majority of sequences obtained in this study), the Asian genotype cluster II (from Toamasina, Madagascar) showed one changed base (G instead of A) at the position 934 of the cox 1 completed sequence which changed the corresponding amino acid while the Asian genotype cluster III (from Indonesia) presented 3 substituted nucleotides (C, G and A changed into T, A and G: at the positions 650, 666 and 994 respectively). This last changed base was also the only variation observed in the Asian genotype cluster IV (from Nepal, Japan, China and Korea). The last Asian T. solium genotype obtained in this study, from Sahavoemba (Asian genotype cluster V) was also closely related to the majority of Asian T. solium genotype found in this study (Cluster I) except for one base: A modified in G at the position 867 which is a common substitution in the African-American T. solium genotype. The only African/American genotype found in this study forming the African-American genotype cluster III (with Tanzania, Toliara Madagascar and Mexico) showed six substituted nucleotides (of which 3 modified amino acids) compared to the Asian genotype cluster II. The African-American genotype cluster II (Ecuador) and I (Mexico, Cameroon and Brazil) counted seven and nine substitutions respectively both with four changed amino acids.

(TIF)

Click here for additional data file.

Composition of the Townships and Fokontany (Ifanadiana District) investigated in this study.

The geographic coordinates (GPS) of the 12 villages are indicated.

(DOCX)

Click here for additional data file.

29 Jun 2021

Dear Dr Rahantamalala,

Thank you very much for submitting your manuscript "Prevalence and factors associated with human Taenia solium taeniasis and cysticercosis in twelve remote villages of Ranomafana rainforest, Madagascar" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jessica K Fairley, MD, MPH

Associate Editor

PLOS Neglected Tropical Diseases

Aaron Jex

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The objectives are clearly stated and the study design seems appropriate, but some important details are still missing (see below). In general, the analyses seem appropriate and ethical and regulatory requirements are met.

L157: how exactly were the households randomized? And out of how many households were these households randomly selected (i.e. how many were not selected)? And why 164? A sample size consideration is missing.

L196: please give an indication of the time of conservation (between stool sample collection and extraction/PCR analysis)

L188-193: The questionnaire should be described more clearly. The results of the questions could be very different, depending on how the questions were formulated. For example the question pork consumption (yes/no) is too vague. It could be interpreted as having ever eaten pork in your life, but also as eating pork weakly, which are two completely different things. I would suggest to add the questionnaire in appendix.

Is having more than once headache every 3 months considered as chronic headache? It would be good to add a reference to this definition of chronic headache.

Reviewer #2: The hypothesis is clearly stated as the determination of the prevalence of human taenia solium, taeniasis/cysticercosis and risk factors in remote areas of Madagascar. The human population of the area is clearly described. The study design is appropriated and the sample size seems sufficient to address this hypothesis, statistical analyses do not rise any concern. Ethical and regulatory requirement seem to have been met.

Reviewer #3: The objectives of the study are clearly articulated and the population is also clearly described. The sample size is sufficient to ensure adequate power to address the hypothesis. The statistical analysis support conclusions.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analyses match the analysis plan and the results are compete, but at times the results are over interpreted (especially regarding the results at village level).

The primary aim and secondary/exploratory outcomes could be defined more clearly. I think the sample size is not appropriate for a thorough risk factor analysis. In my opinion, the prevalence estimates seem like the primary aim, and the risk factors are more exploratory. I would suggest to make that more clear.

L348: 574 willing participants, out of how many in these households? Do you have an indication how many people in the selected households were not willing to participate? And L350: not really out of 574 villagers, but out of 574 participants. I assume that the number of villagers is higher than this number because of the exclusion criteria (above 5) and willingness to participate. If available, it would be good to provide this information in Figure 2 and/or the text (how many were excluded because of age or because they were not willing).

L389: this is an over-interpretation of the data; I don’t think you should conclude these villages are “most infected” based on the data (because it’s based on 0, 1 or 2 positive cases per village). This is also clear from the wide CI around the estimates in Table 2. The % positive samples per village should therefore not have digits, because the estimates are very uncertain due to the relatively low sample size per village. It’s OK to present the results per village, but don’t overanalyse them. As mentioned earlier, it would be good to give an indication of the number of people living in each village, because that’s not clear from the manuscript. For example the results from only 10 people from Bevoahazo: how many people actually lived in that village at the time of recruitment?

L460-463: I find this sentence very difficult to follow. And also for the sentence below: the message is not very clear, and as mentioned above, the categorization of villages between presence/absence of taeniosis based on the very few positive samples is likely an overinterpretation of the results due to the large uncertainty around the taeniosis prevalence estimates per village.

L466: the highest prevalence in Marojano: the sample size is too low to draw conclusions about which village has the highest prevalence (Marojano is only based on 23 samples)

L485-489: same comment as above

L500: how was this cutoff (4 or more) determined? Same comment for house distance.

Figure 2: first box with Excluded: “missing epidemiological data”: what exactly does this mean? In table 1, it seems there are missing data for some of the 459 participants (but they are not excluded)? It would be better to specify how many are missing due to missing epidemiological data (and what exactly is meant) and how many due to missing biological samples (and which: serum and/or stool). Second box with Excluded: all serological analyses (not analysis)

Reviewer #2: Analyses presented match the analysis plan and the results are clearly presented.

Reviewer #3: The results and figures are clearly and completely presented. The figures are of sufficient quality for clarity.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The discussion clearly addresses the public health relevance and also includes some limitations of the study. The discussion could be slightly more comprehensive.

L540-550: this whole section seems rather introduction than discussion. The results from the current manuscript are currently not mentioned/discussed here.

L582: “more prevalent in adults”: this cannot be concluded from the data with certainty: the difference between adults and children was not significant and actually seemed very minor. The CI are almost completely overlapping. This should be rephrased; perhaps include other studies where they did find significant differences with age.

L600: I don’t understand the “common history” between pigs and human.

L645-647: “water source”: it’s not so clear to which results this is referring to: it seems that river water is protective according to Table 5 (relative to stand pipe). Also, why is water source not included in the initial model in table 6 since p<0.2 according to table 5?

L675: I don’t think any results/analyses at household level are included in the paper?

Reviewer #2: Conclusions are supported by the data presented, but as Taenisasis/Cysticercosis is also a zoonose. This study rise even more question to address this public health problem. Beyond determination of prevalence in remote area, this work focus upon genetic of taenia solium and lack of perspective in order to give valuable data to fight taenia solium in this area and in low and middle income countries in general. Strains of taenia solium have been clearly identified and genotyped, but neither characteristic linked to the intermediate hosts (pork) nor the neurological conditions or pathogenicity (burden) in human participants been evaluated. Moreover, more information upon local habits and environment could help to explain the results of multivariate analyses. As an example, during deworming campaigns, taenia carriers are expected to expel scolex and proglottids, the latter need to be correctly treated in order to be sure to efficiently destroy eggs and avoid a massive contamination of environment and particularly the water used and ingested by both humans and livestock. Inefficient treatment of gravid proglottids following mass treatment can lead to a counter intuitive augmentation of cases in area. Identification of tapeworm carriers remains a challenging problem in community and anti-helminthic treatment could help in such. Authors broach the subject of self-infestation (Cysticercosis) in tapeworms carriers but diagnostic of infections are not presented for the 11 carriers identified in the study nor for the people living in the same household while a higher rate of exposure and infection are expected.

Reviewer #3: The conclusions are supported by the data presented and limitations of analysis are clearly described. Data are public health relevance addressed.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Author summary: I’m not sure if this is understandable enough for the lay public

L86: associated “with”?

L92-93: the life cycle is not maintained among humans. This is confusing and should be rephrased

L139-141: this sentence is not clear to me. Perhaps explain fokontany already here (first use of the word).

L179: common instead of commonly

L194: “spp.” should not be in italics (a species name should be in italics, but the word species not)

L304: use sensitivity (not sensibility)

L341: this sentence is confusing as it reads “p > 0.05 as statistically significant”, whereas values < 0.05 are considered statistically significant. It should be rewritten

L352: diagnosis instead of diagnostic?

Table 1: why not just write “4 to 6” instead of “4 ≤ pers. ≤ 6”?

L405-409: I think this is material and methods (or introduction), but not results

Fig 3 caption: not really “from Taenia eggs of human stool”, but rather DNA extraction of stool samples in which eggs were detected? Based on M&M, it seems that DNA was extracted from the stool sample, not only the eggs? Same for L429

L479: cysticerci (spelling mistake)

L496 “behaviors factors” (grammar)

L497: Table (not tables)

L499: these are methods (not results)

L501: school attendance = education? I would suggest to use the same wording as in table 4. I don’t understand the * for education in Table 4, since the p value is > 0.2. I assume it’s a mistake?

Table 4: there seem to be many more questions compared to Table 1? Why are some of these questions also included in Table 1, but not others?

L508-509: This is confusing, since this seems to be a mixture of both EITB and Ag ELISA results?

L538: grammar

L545: ranging between what? Between villages/districts/…?

L557-565: this is a long and complicated sentence. Please rephrase it. Also, I agree that you should only compare prevalence estimates among studies with a comparable methodology and that microscopy might result in a lower prevalence estimate, but “weak” isn’t the best word for it (if that’s what is meant in the last part of the sentence) .

L565-566: I don’t think the comparison with Honduras is very relevant? Is it just because the point estimate almost the same, or are there other reasons to specifically compare to Honduras? Keep in mind that your 95% CI was 1.3 – 4.3, so it might also be “similar” to other studies above? I also think that this part can be merged with the next paragraph.

L614: I don’t understand the connection between “… Brazil (Figure 3).” and “The description of T. solium…”. Add a sentence to connect both parts or make a new paragraph.

L629: this sentence suggests that antibodies are lower in people with epilepsy compared to the general population. Rephrase and add more references for the seroprevalence in people with epilepsy.

L634: replace “à” by “to”; “weak” is not correct

Reviewer #2: Taeniasis/Cysticercosis by taenia solium are diseases which need a multidisciplinary approach to be fought. Authors would consider their study in a more one-health approach and would discuss additional research to be perform in this specific area with the inherent field limitations and highlight in what their result contribute to eradication.

Reviewer #3: Minor Revision

The manuscript should review by a native english speaker to improve the quality of the written.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This is an important and interesting study that describes the prevalence of taeniosis and cysticercosis in a remote area in Madagascar. The design and methods are appropriate, but certain aspects should still be clarified (such as the randomisation process and (an estimate) of the total population in these villages, in order to be able to assess the representativeness of the analysis set for the total population, and more details about the questionnaire that was used). The authors should also be more nuanced when drawing conclusions from the data.

Reviewer #2: Although this study give some interesting information upon the realization of a survey upon the use of correct diagnostic test and the sharp genetic identification of parasites from a deprived setting, little is discussed on how about these assays and data could be usefully mobilized to give a dedicated response to the burden of tapeworm infection by Taenia solium in remote areas of Madagascar.

Reviewer #3: This is the first large-scale study in Madagascar to examine the association between potential risk factors measured at the individual-, household-, and village-level and the prevalence of Taenia solium taeniasis/cysticercosis in humans. The authors showed high rates of taeniasis cases affecting all ages in both townships investigated, strongly exposing human and pigs to cysticercosis. This study has a sufficient sample size, well-designed analysis and is quite robust. However, a native english speaker review should improve the quality of writing.

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8 Sep 2021

Submitted filename: Response_to_reviewers.docx

Click here for additional data file.

23 Nov 2021

Dear Dr Rahantamalala,

Thank you very much for submitting your manuscript "Prevalence and factors associated with human Taenia solium taeniasis and cysticercosis in twelve remote villages of Ranomafana rainforest, Madagascar" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please address minor comments from Reviewer 1 and then it will be ready for acceptance.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jessica K Fairley, MD, MPH

Associate Editor

PLOS Neglected Tropical Diseases

Aaron Jex

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The changes made by the authors made the methodology more clear.

Reviewer #2: The objectives are clearly stated, the study design is appropriate. The population is well described, the sample size and the statistical analyses do not call for any remarks. The ethical and regulatory procedures appear to have been correctly carried out.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are generally clear, though the analysis at household level is not clear.

Table 3: I don’t fully understand “Village per Taenia carrier and per household (mHH included/Total mHH))”. There were 11 tapeworm carriers, but 41 households in this subgroup, so the subset is not clear to me. Also in L480, the sentence and the subgroup is not clear. It could also be an estimate at household level (if one person is positive, the household is positive because the sentence starts with “At household level”)? This should be clarified.

Reviewer #2: The results, figures and tables are clearly presented.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The authors' conclusions are supported by their results. The public health relevance and perspectives of this work are clearly and appropriately addressed in this revised version of the manuscript.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: L390: write 46 in full; the sentence is not correct/not clear. Rephrase.

Table 3: given the extremely wide CI per village, it’s not appropriate to report decimals for the proportions, because the estimates are very uncertain.

L622-627: I still find the hypothesis of common history of people and pigs very strange. I would suggest to remove it (or clarify).

L657 and further: “In our study villages, washing hands even with soap seems to expose participants to contaminated eggs”: you determined an association; this is not necessarily a causal relation. This should be rephrased. I assume there are likely confounding factors.

L1163: how were these 8 active cases determined? I assume these are the 8 sero Ag people in the unclear subgroup.

English should still be improved.

Reviewer #2: in spite of some somewhat "academic" passages, which could be lightened, I recommend to accept this revised version of the manuscript.

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Summary and General Comments

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Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Reviewer #2: No

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References

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29 Dec 2021

Submitted filename: Database.xls

Click here for additional data file.

17 Feb 2022

Dear Dr Rahantamalala,

We are pleased to inform you that your manuscript 'Prevalence and factors associated with human Taenia solium taeniasis and cysticercosis in twelve remote villages of Ranomafana rainforest, Madagascar' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Aaron Jex

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***********************************************************

31 Mar 2022

Dear Dr Rahantamalala,

We are delighted to inform you that your manuscript, "Prevalence and factors associated with human Taenia solium taeniasis and cysticercosis in twelve remote villages of Ranomafana rainforest, Madagascar," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Paul Brindley

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PLOS Neglected Tropical Diseases