Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme in Ouagadougou (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (12°25'N, 1°20'W) a site located at 15 km in the north-east of Ouagadougou, the capital city of Burkina Faso (Figure 1). Goden, is a rural village with Sudanian Savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belonging to the Mossi ethnic group, are mostly devoted to agriculture and growing few animals (e.g. pigs, dogs, goats and chickens) within their compounds. LLINs were distributed in 2016 to approximately 90% of the population (N’F S and MWG, Unpublished data). Goden is known for its high density of malaria vectors due to its proximity to the Massili river. The field study was carried out during the rainy season (August to November 2019) corresponding to the high vector density and high malaria transmission period. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches before the tests started.
Human volunteers’ preparation
Healthy adult’s male volunteers aged between 18 and 40 years, were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, nor alcohol 12 hours before the start and throughout the experiments. To establish the amount of repellent required for application in the experiments, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula:
Area= ½ (Cw+ Ce) Dwe
Here Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm and Dwe is the distance in cm between Ce and Cw (21). The amount needed for each volunteer was then determined depending on their forearm or leg length. In addition, the quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH) to determine the amount applied by each volunteer.
The MAÏA® a shea butter-based ointment containing 15% DEET (N, N-Diethyl-3-methylbenzamide) was received from Maïa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control) and a negative control of 70% ethanol. The DEET is known as the standard repellent reference.
Strains of mosquitoes
Four strains of mosquitoes were used in the laboratory tests. This including Kisumu F57 and Bora bora F58 susceptible strains of respectively Anopheles gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso were also used hereafter named as “An-Goden” (An. gambiae local strain, F418) and “Loc-Aedes” (local Aedes aegypti, F318). These species are being maintained under a 12:12 (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 25 - 28 °C and 60 - 80% respectively. Moreover, the individual mosquitoes used in these experiments were five to ten-day-old nulliparous females starved from sugar solution for twelve hours before the experiment.
Evaluation in the laboratory
The laboratory experiments were conducted following the WHO guidelines for the arm-in-cage test (21). Cages were 45 x 45 x 45 cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200, 5 to 10-day-old females of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out on at temperature ranging between 25 and 28 °C with the relative humidity being between 60 and 80%.
Overall, 2 mg of ointment were applied per square-centimetre (cm2) on left forearm of each volunteer. A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment for avoiding absorption of part of the ointments through applier’s skin during the application. Though, the positive control consisting of 1ml of the 20% DEET solution was applied to the right forearm of each volunteer.
Before exposure to the mosquito probing, the forearm was washed with odorless soap, dried, rinsed using 70% ethanol solution and then dried again. All volunteers wore latex gloves to protect their hands from mosquitoes biting. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30 seconds (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow too with the MAÏA® ointment. Thirty minutes after application of the repellent, the participant exposed the treated forearm in the test cage for 3 minutes. The procedure was then repeated every 30 minutes until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Aedes Aegypti strains were carried out during the day between 09:00 am to 18:00 pm, whereas that of Anopheles gambiae were conducted between 5 pm and 5 am (21).
Here, the lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs treated volunteers were also asked to avoid rubbing, touching or wetting the repellent-treated area. Then, 2 mg of the MAÏA® per cm2 (2.4 ± 0.2 g per 1189 ± 79.2 cm2) and 2 ml per cm2 of the 20% DEET (2ml ± 0.1 ml per 1189 ± 79.2 cm2, as a positive control) were applied to volunteers lower legs, from the knee to the ankle. A total of twenty volunteers were recruited from the Goden village and trained for the nocturnal mosquito’s collection using Human Landing Catches (HLC). Each volunteer was later randomly allocated to one of the five groups (2 for the MAIA®, 2 for positive control and 1 negative control) of four volunteers according to the treatment received. The experiments took place at five different households at each night of collection that were at least 20 meters apart as per WHO guidelines (21). This, in order to avoid biases due to competition in attractiveness to the mosquitoes
Mosquito collection started thirty minutes following treatments. Here, volunteers acting as bait, sat on a chair by pair and actively collected mosquito that landed on their treated lower leg using mouth aspirator and flash torch(22) for 45 minutes followed by a fifteen-minute break. During this collection, volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head. However, the treated lower leg was exposed to mosquito probing by rolling out the trousers up to the knee. During these experiments, mosquitoes were collected simultaneously at both indoor and outdoor environments between 7 pm to 6 am. Here to avoid biases introduced by individual attractiveness and skills(23,24) volunteers at the same household rotated between indoor and outdoor hourly. At the each of the household two groups of two people were constituted. A group collecting from 6 to 12 pm and the second from 12 to 6 am. The treatment rotated between households following a Williams balanced Latin Square design.
Collected mosquitoes were transferred into plastic caps covered, using a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of the CNRFP and morphologically identified using a stereo microscope and the identification keys (25).
No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.
Written informed consent was obtained from all volunteers recruited and household owners in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.
All data were collected on standard forms and were entered twice in a database by different people. Databases have been compared using Epi Info ™ 3.5.3, and inconsistencies were verified using the printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time.
A Generalized Linear Mixed Model was used to further analyze the effect of the location (indoor versus outdoor) on the performance of the treatments. We also assessed whether there was any variation in the average number of bites received between treatments.
The median complete protection time (CPT) is defined as the interval of time between the beginning of the collection/test and the first mosquito landing. To estimate the median complete protection time of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the lab experiments and on field data through ‘survival function’ from R software - version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed only on Anophelesgambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C-T) / C) * 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of stinging mosquitoes on the volunteers legs treated with the test product (21).