2.1 Description of study area
Benishangul-Gumuz region, western Ethiopia, is located 34° 10’N and 37° 40’E and 09° 17’N and 12° 06’ N. The region is predominantly (75%) lowland. The total population is around 784,345 with an estimated density of 15.91 people per kilometer square (BGRoHB, 2019).The study encompassed six VL high risk districts (Figure 1) as per the environmental factor based risk model by Tsegaw et al (2013)[4]; Dangur, Guba and Pawi from Metekel zone, and Banbasi, Kumruk and Sherkole from Assosa Zone. The region is one of the development corridors with large scale agricultural, mining and Dam projects which entailed change in settlement pattern and deforestations, and large influx of peoples for temporary work and/or permanent settlement.
2.2. Study design and sample size determination
A cross sectional survey was done from 2018 to 2020 to assess the epidemiology, explore if any zoonotic significance of VL and risk factors associated with exposure to Leishmania infection. Samples were selected using a multi-staged sampling technique. At primary sampling unit among the three administrative zones of the region, overlaying theenvironmental factor based risk map [4] two zones namely Assosa and Metekel were selected because they had large areas under VL high risk. Similarly, within the selected zones districts with high risk area were selected. Then an operation map was prepared overlaying the risk map of selected districts and the kebele level shapefile to identify high risk kebeles.Subsequently,study households were randomly selected from each of the kebeles targeting up to 5% of their total population, with overall sample size of 1342 individuals for LST testing.
Following LST reactive individuals as a focal point dogs were sampled for serological tests, rK39 and DAT. Also, 253 human blood samples, 67 purposively from LST reactive and 185 randomly from non-reactive individuals were tested by DAT and rK39.
After explaining the purpose of the study, a written informed consent was obtained from each participant; parents or guardians for minors. Similarly, informed assent was obtained from dog owners to sample dogs. Blood sample were aseptically collected using 5mL disposable syringe or plain vacutainer tube from cephalic/saphenous vein of both human and dogs. Of the collected blood, 20 µL was used to prepare dried blood spot (DBS) on 3MM Whatman paper (Whatman, Maidstone, UK) allowed to fully air dry without exposing to direct sunlight. Sera from both dog and human were used for rK39 ICT and DAT testing.
2.2.1. Leishmanin skin test (LST)
Prior to LST, socio-demographic information was documented from the study participants using pre-tested semi-structured questionnaire. Then, intradermal injection of 0.1 mL leishmanin antigen (Pasteur Institute of Iran, Tehran, prepared from L. major) was made at the volar surface of the arm. After 48–72 hours delayed hyper sensitivity reaction was measured with the ballpoint techniques; 5.0 mm and above of the average of the two diameters of an induration was considered positive (Fig 2).
2.2.2. rk-39 immunochromatographic test (rK39 ICT)
The rK39 ICT (DiaMed- ITLEISH; Bio-Rad Laboratories, Marnes-la-Coquette, France) was done following the supplier’s recommendation. In brief, 20μL serum sample was added to the absorbent pad well with a 150μL (2-3 drop) of chase buffer provided with the kit. Results were read after 10–20 minutes and recorded as follows: positive when both control and test lines appear; negative when only control line appears or invalid when no control line appears (in such cases tests were repeated).
2.2.1. Direct Agglutination Test (DAT)
Sera were transported to Benishangul Gumuz Regional laboratory with ice box and stored at −20°C. Then samples were transported to AHRI under cold chain and stored at -20 OC until processed. Direct agglutination test was performed according to the manufacturer’s instructions (Institute of Tropical medicine, Antwerp, Belgium). The presence of antileishmanial antibodies below or at cutoff of 1:3200 titers was used to decide as negative. Both negative and positive controls were run for every batch of kit used. In brief, sera were diluted serially from 1:200 to 1:204800by transferring 50 μL of diluted serum and discarding the same amount from the last dilution. The diluent used was a solution containing 0.24 mL of 2-ME (2-Mercaptoethanol) to 1 vial (30 mL) of DAT-DILUENT. After serial dilution, 50μL DAT antigens diluted with DAT-BUFFER was added to the sera in a microwell plate containing 50μL of diluted serum, the plate was sealed with plate sealer and shacked clockwise and anticlockwise to settle splashing. After 18hours of incubation at room temperature, the DAT result was read over white board (Fig 4).
2.2.3. Data analysis
STATA version 13 data software (College Station, Texas, USA) was used for data analysis. Descriptive statistics were employed to summarize in terms of frequencies and percentages. Univariate and multivariate logistic regression were used to determine the association of Leishmania infection with the risk factors and expressed as odds ratio and 95% confidence interval. For all analysis a P < 0.05 was considered for significant difference.
2.2.4 Ethical consideration
The protocol was approved by AHRI/ALERT ethical review committee (AH01275/0012/18, 19/12/18). Informed consent was obtained from all participants or guardian/parents of minors. For participants between 11 and 18, verbal assent was sought in addition to the parental/guardian consent. Similarly, for dogs informed assent was obtained from the owners.