Study area

Mainland region of Equatorial Guinea comprises 4 provinces and 13 districts in a total area of 26,017 km², and is bordered on the north by Cameroon, on the east and south by Gabon, and on the west by the Atlantic Ocean. It has a population of 882,747 inhabitants according to the 2015 census, 72.2% of the whole country’s population (information from the National Onchocerciasis and Other Filariasis Control Programme). All the mainland districts are ivermectin-naïve and defined as O. volvulus and LF co-endemic [5, 8, 10].

Sample size and sampling strategy

From September to December 2019, a cross-sectional study was carried out to estimate the seroprevalence of onchocerciasis and LF in the continental of Equatorial Guinea.

The districts constituted the evaluation unit of the study. A minimum sample of 300 individuals per district was calculated based on WHO protocols [11]. The sampling frame was all communities within each district. The study purposefully sampled 3–5 first line communities in each district. All age groups were targeted. Within the selected districts, the location of the original first-line villages in relation to known breeding sites and distances between other first-line villages were verified. For the selection of communities to survey, we prioritized sites based on the following criteria:

Communities identified with the presence of the aquatic stages of S. damnosum vector during the entomological survey conducted in February–March 2019 (supplementary file 1).

To meet the quota of communities per district (n = 5), we included sentinel communities where a prevalence of nodules/skin snips greater than 10% was recorded in rapid mapping activities by APOC in 2008, 2013, and 2015 (personal communication with the Expanded Special Project for Elimination of Neglected Tropical Diseases, ESPEN).

Should additional communities be needed, we first considered those with a prevalence below 10%, by rapid epidemiological mapping of onchocerciasis (REMO) or skin snip, selecting them in descending order of prevalence. Following this, communities identified with other Simulium species during the 2019 entomological study were selected.

If more communities were still needed, we selected those located within 5 km of a risk zone, defined as areas adjacent to both sides of a river.

Within each community, a randomization system was established to cover approximately 60 members per community based on the community lists provided by the local authorities. Details on strategy sampling and community selection are provided in the Standard Operational Procedure SOP_01 (see supplementary file 2).

Data and sample collection and management

Prior to starting the study, a comprehensive field training program was provided along with training on the proper use of sampling (see additional files 3, 4 and 5). The participation questionnaire was pre-tested one week before the beginning of the project.

All consenting individuals were asked for basic information about gender, age, the number of years lived in this village, travel history, and exposure to ivermectin and/or albendazole. Each participant received a unique, anonymized code assigned to a thick blood smear (TBS), filter paper disk for collecting dried blood spots (DBS) and to their questionnaires, ensuring confidentiality before sample collection.

Data and sample collections were carried out in a health post or a traditional house known as “house of the word” (see supplementary file 6). To this end, the members of the selected households were notified in advance, ensuring that everyone meeting the inclusion criteria was present on the day of the visit. The village or community chief took on the responsibility of summoning the participants.

The survey team also recorded basic information on each selected village including the associated district, village population, village global positioning system (GPS) coordinates, and identification of black flies by community leaders. Data were collected on smartphones and uploaded regularly to the electronic platform tailored by ESPEN Collect using a secure data kit software (SDK) [12]. Data were stored on a secure server. Field staff was previously trained in using these tools for data collection (see supplementary files 7 and 8). All data from questionnaires and samples were documented in notebooks and then input into a secure database by specialized data entry personnel. After the study concluded, DBS were dissociated from personal identifiers to ensure confidentiality.

For logistical reasons, all samples were taken during the day. Blood sampling was performed by fingerstick method collected onto Tropbio Filter Paper Disk and onto microscope slides. Following collection, filter paper disks were stored and transported to Madrid, Spain, for processing. Further details on sampling, sample collection, and transport are provided in the supplementary files 9, 10 and 11.

A follow-up visit was scheduled to collect samples from individuals who tested positive or had undetermined results for onchocerciasis. However, due to the COVID-19 pandemic, this visit was postponed to July 2021. During this visit, skin snip biopsies (see supplementary file 12) were taken from each iliac crest for PCR analysis, and peripheral blood samples were collected in EDTA tubes for serological retesting. Unfortunately, for lymphatic filariasis (LF), it was not possible to obtain a second nocturnal blood sample from individuals with positive or undetermined initial results.

Microscopy laboratory testing

TBS were stained with 3% Giemsa solution for 30 min. TBS were used to calculate the W. bancrofti microfilaremia (microfilariae per milliliter of blood) considering that each TBS contained approximately 20 µl. Morphological identification was performed by two different expert microscopists. Every field on a slide was thoroughly examined before a negative result was determined.

Serological laboratory testing

The blood samples collected on Tropbio Filter Paper Disk underwent analysis using two ELISA tests to detect specific IgG4 antibodies against the recombinant antigens Ov16 and Wb123 from O. volvulus and W. bancrofti respectively (Ov16-ELISA & Wb123-ELISA). Both recombinant antigens were obtained as previously described in Hernández-González et al. [13] and Herrador et al. [14].

The procedures for ELISA tests, including the controls used (including humanized monoclonal IgG4 against Ov16 antigen) [15], the calculation of the serological index (SI) and the interpretation of results, are detailed in the supplementary file 13.

Molecular laboratory testing

Skin snip biopsies and DBS were incubated at 56 °C overnight in ATL buffer previous to DNA extraction following the QIAamp® DNA Mini Kit (QIAGEN GMBH, Germany) instructions. The presence of O. volvulus in the DNA from skin snip biopsies and W. bancrofti in the DNA isolated from blood samples were assayed using the F-RT-PCR described by Formenti et al. [16].

Statistical analysis

Individual data and laboratory results were analyzed to obtain the frequencies of each variable. The prevalence of onchocerciasis and LF by district, along with 95% confidence intervals (CI) was determined using the method recommended by Brown et al. for interval estimation of a binomial proportion [17]. The software GeoDa was used for map representation.