MAÏA® Topical Repellent Ointment Provides Long-Lasting Protection Against Anopheles Gambiae s.s., Anopheles Arabiensis and Aedes Aegypti Under Semi-Field Conditions in Tanzania

Background: N,N-Diethyl-3-methylbenzamide (DEET) topical mosquito repellents are effective personal protection tools. However, DEET repellents tend to have low consumer acceptability because they are cosmetically unappealing. More attractive formulations are needed to encourage regular compliance. This study evaluated the protective ecacy and duration of protection of a new topical repellent ointment, MAÏA ® compared to 20% DEET in ethanol using malaria and dengue mosquito vectors in Tanzania. Methods: Fully balanced 3x3 Latin square design studies were conducted in large semi-eld chambers using laboratory strains of Anopheles gambiae s.s, Anopheles arabiensis and Aedes aegypti. Human volunteers applied either MAÏA ® ointment, 20% DEET or ethanol to their lower limbs six hours before the start of tests. Approximately 100 mosquitoes per strain per replicate were released inside each chamber, with 25 mosquitoes released at regular intervals during the collection period to maintain adequate biting pressure throughout the test. Volunteers recaptured mosquitoes landing on their lower limbs for six hours over a period of six to 12-hours post-application of repellents. Data analysis was conducted using mixed-effects logistic regression. Results: The protective ecacy of MAÏA ® and 20% DEET was not different for each of the mosquito strains: 95.9% vs 97.4% against An. gambiae (OR=1.53 [95% CI: 0.93–2.51] p=0.091); 96.8% vs 97.2% against An. arabiensis (OR =1.08 [95% CI: 0.66 –1.77] P=0.757); 93.1% vs 94.6% against Ae. aegypti (OR=0.76 [95% CI: 0.20-2.80] p=0.675). Average complete protection time (CPT) of MAÏA ® and that of DEET was similar for each of the mosquito strains: 571.6 minutes (95% CI: 558.3-584.8) vs 575.0 minutes (95% CI: 562.1-587.9) against An. gambiae; 585.6 minutes (95% CI: 571.4-599.8) vs 580.9 minutes (95% CI: 571.1-590.7) against An. arabiensis; 444.1 minutes (95% CI: 401.8-486.5) vs 436.9 minutes (95% CI: 405.2-468.5) against Ae. aegypti. Conclusions: MAÏA ® repellent ointment provides complete protection for 9 hours against both An. gambiae and An. arabiensis, and 7 hours against Ae. aegypti similar to 20% DEET (in ethanol). MAÏA ® repellent ointment can be recommended as a tool for prevention of outdoor biting mosquitoes in tropical locations with a suggested reapplication time of 6 hours. The study results demonstrated that MAÏA® repellent ointment is comparable to unformulated 20% DEET in terms of mean repellency over 12 hours as well as the duration of complete protection time (CPT) against both malaria and dengue vectors. MAÏA® repellent ointment and 20% DEET had CPT of more than 9-hours against An. gambiae (Kisumu) and An. arabiensis (Kingani) and more than 7-hours for Ae. aegypti post-application of the repellents. These results are similar to another study which evaluated the effectiveness of MAÏA® repellent ointment under eld conditions in Burkina Faso in which authors concluded that MAÏA® and 20% DEET are comparable in terms of duration of complete protection time against Anopheles strain and Aedes aegypti (Guelbeogo et al., Manuscript under preparation). The study results indicated that MAÏA® repellent ointment is an effective mosquito repellent suitable for the use even under high mosquito biting pressure. According to Goodyer et al, an ideal mosquito repellent should provide CPT above 6-hours under the highest mosquito biting pressure The present study fullled this by demonstrating the mean CPT above 9-hours against An. gambiae (Kisumu) and An. arabiensis (Kingani) and above 7-hours against Ae. aegypti for both MAÏA® and 20% DEET with an average of 10 mosquito landings per strain per hour in the control, 30 landings per hour in total.

Conclusions: MAÏA ® repellent ointment provides complete protection for 9 hours against both An. gambiae and An. arabiensis, and 7 hours against Ae. aegypti similar to 20% DEET (in ethanol). MAÏA ® repellent ointment can be recommended as a tool for prevention of outdoor biting mosquitoes in tropical locations with a suggested reapplication time of 6 hours.

Background
The use of insecticide-treated bed nets (ITNs) and Indoor Residual Sprays (IRS) has almost halved malaria burden throughout sub-Saharan Africa [1,2]. However, a considerable proportion of malaria transmission occurs outside of sleeping hours when ITNs are not in use [3]. Topical mosquito repellents are one of the oldest interventions used to prevent contact between humans and mosquito vectors [4,5], and may be useful for people who spend their time outdoors in the evening or overnight for occupational or social activities [6][7][8][9]. Some studies have shown that people who use topical repellents consistently in addition to ITNs are protected from malaria [10,11]. However, their utility as a disease prevention tool is limited by poor user compliance [12]. Therefore, deployment of topical mosquito repellents for malaria prevention is not recommended by the World Health Organisation (WHO) as an intervention with public health value but maybe bene cial as an intervention to provide personal protection against malaria [13,14].
Topical repellents must be applied daily or even several times a day, and poor user compliance is a major limitation to the effectiveness of topical repellents [15][16][17]. Common reasons for poor compliance include forgetting to apply the repellent [15,18], poor acceptability including unpleasant smell or greasy "feel" on the skin [18] and the perception that repellents are poisonous [15]. DEET is a common active ingredient of most of the topical mosquito repellents present in the market today. Over 200 million persons use DEET-containing repellents every year with the market growing annually due to increases in vector borne diseases such as dengue and zika [19]. DEET has an excellent safety pro le and is safe for use among children and pregnant women [20,21].
According to Carroll et al, "a good repellent ointment should be effective against target vector strain, easy to apply and has a nice odour and the residual feeling after application" [22]. To obtain better compliance, consideration of human customs and behaviour that may encourage consistent use of repellents is essential. In a study conducted in Burkina Faso, it was observed that about 91% of children under 5-years are washed in the evening and 80% of them receive ointment on their skin after bathing and before bedtime (Traoré et al; manuscript under preparation). During this time, mosquitoes are actively host-seeking and interacting with humans outdoors [23][24][25]. Therefore, a well-formulated topical mosquito repellent with skin softening properties for daily use after bathing may improve user compliance as well as protect against vector-borne diseases such as dengue and malaria when people are outdoors in the early evening.
The Maïa Africa SAS [26], a company based in Burkina Faso worked in collaboration with local mothers to develop MAÏA® repellent ointment that is formulated with petroleum jelly, shea butter, cotton oil, beeswax, fragrance, and 15% DEET. Shea butter-based ointments are widely used for skin softening purposes [27,28] and MAÏA® repellent is designed to repel mosquitoes in addition to softening the human skin. This study evaluated the protective e cacy and duration of repellency of MAÏA® repellent ointment comparing with the gold standard, unformulated 20% DEET against Anopheles and Aedes mosquitoes under semi-eld system conditions in Tanzania. The semi-eld evaluation of topical repellents generates data that are comparable to a full-eld evaluation [29].

Study area
This study was conducted under ambient conditions in a semi-eld system measuring 29 x 21 meters built from a fabricated greenhouse frame modi ed to make two compartments with a central corridor and an opaque polyethylene roof for rain protection (Fig. 1) [29] . The semi-eld system is located at 6° 8' S, 30° 37' E at the Ifakara Health Institute in Bagamoyo district in Tanzania. Bagamoyo district experiences annual rainfall between 800 mm and 1000 mm, temperatures between 22ºC and 33ºC, and mean relative humidity of 73%. This evaluation followed the WHO Guidelines for e cacy testing of topical repellents [30].

Study design
This study was divided into two parts to accommodate the circadian rhythm of mosquitoes investigated. Anopheles gambiae s.s. (Kisumu) and Anopheles arabiensis (Kingani) were tested at night and Aedes aegypti in the early morning. A pilot study for the rst six hours after repellent application was conducted and found that a longer testing period was required. The nal study tested the repellent from six to and twelve hours post-application.

A study for Anopheles
In the pilot study of Anopheles strains, repellents were applied at 17:45 hours and mosquito collection was conducted between 18:00 hours to 00:00 hours ( Fig. 2A). In the nal study, repellents were applied at 17:45 hours and mosquito collection was conducted between 00:00 hours to 06:00 hours (i.e. 6-12 hours after the application of repellents) (Fig.   2B).
Both the pilot and nal study consisted of two fully balanced (3x3) Latin squares (LS) design conducted in two chambers of the SFS simultaneously over nine nights using six volunteers. In each LS, three volunteers rotated sequentially between the three mosquito collection positions each day inside the chamber and swapped repellents after every 3-days. After nine days of the study period, each volunteer had tested each of the repellents at each of the three mosquito collection positions inside the SFS chamber three times. The study ow plan for Anopheles gambiae s.s. (Kisumu) and Anopheles arabiensis (Kingani) is shown in (Fig. 2).

A study for Aedes
For Aedes aegypti repellents were applied at 05:50 hours in the pilot study and at 23:45 hours in the nal study. Mosquito collection was conducted from 06:00 hours to 10:00 hours in both the pilot and the nal study. The pilot study consisted of two fully balanced (3x3) LS conducted in two chambers of the semi-eld system simultaneously over nine nights using six volunteers. In the nal study, one fully balanced (3x3) LS was conducted in one chamber over nine nights using three additional volunteers. In both studies, volunteers rotated sequentially between three collection positions each day inside the chamber of the SFS and swapped repellents after every 3-days. After the nine-days study, each volunteer had tested each of the repellents at each of the three mosquito collection positions inside the SFS chamber three times. The study ow plan for Aedes aegypti is shown in (Fig. 3).

Mosquito strains
This study used nulliparous female laboratory-reared mosquitoes, aged 5-7 days old, sugar starved and reactive to human odour. Mosquito strains used were pyrethroid-resistant (20% mortality with pyrethroid) Anopheles arabiensis (Kingani, colonized Tanzania 2006), fully pyrethroid susceptible Anopheles gambiae (Kisumu, colonized Kenya 1975) and fully pyrethroid susceptible Aedes aegypti (Bagamoyo, colonized Tanzania 2018). Mosquitoes were reared following the MR4 guideline [31]. Before the experiment, An. arabiensis were lightly marked by placing them in a cup coated with a uorescent dye to make them distinguishable from the morphologically identical An. gambiae. By very lightly marking the mosquitoes there is no signi cant effect on their tness nor host preference [32]. Mosquitoes were then sugar-starved for 8-hours. About 30 minutes before the experiment, 100 female mosquitoes that were responsive to human odour were selected and transported in boxes to the SFS chambers to acclimatize with the ambient environmental conditions. Repellents tested MAÏA ® ointment and 20% DEET were shipped to Ifakara Health Institute (IHI) Vector Control Product Testing Unit (VCPTU) in plastic jars by MAÏA Africa SAS [26]. The amount of MAÏA® repellent ointment and DEET 97% (reference number 26028, lot number 2436308) received at IHI was 600 ml and 200 ml respectively. After receiving, they were stored the same day at room temperature between 25ºC and 29ºC until they were used in the experiment. The 20% DEET in ethanol (V/V) was prepared in-house before the experiment.

Volunteers
Nine male volunteers aged between 24 to 30 years were recruited after signing informed consent forms written in Swahili. All volunteers were tested for malaria parasite infection using SD BIOLINE Malaria Ag P.f [33] rapid malaria diagnostic kits before participating in the study and once per week during the study period as part of IHI health and safety procedures.

Allocation of volunteers
At the beginning of the study (pilot study), six male volunteers were assigned into two groups of 3 volunteers. One group was assigned to chamber '1' and the other in chamber '2' of the SFS (Fig. 1). Inside each chamber, three mosquito recapture positions were marked each 9 meters from the mosquito releasing point (Fig. 4). Each volunteer was randomly assigned to one of the three mosquito recapture positions inside one chamber and rotated between positions nightly.
After nine days of experiments it was discovered that the complete protection time of both repellents is above six hours. Therefore, the nal study of twelve hours recapture period was set up with an additional three volunteers recruited to run the Aedes aegypti which was conducted immediately after the Anopheles experiment.

Application of repellents
Volunteers wore shorts and washed their lower limbs using water without soap before starting the experiment and they also wore closed shoes and a mesh bug jacket to ensure mosquitoes have access to the lower limbs only. Volunteers were non-smokers, and were requested to not drink alcohol or use perfumed soaps or ointment during the entire study period. We calculated a lower limb-skin surface area for each volunteer using the following formula at the beginning of the study; Where "L" represents the leg length between the knee and the ankle and "K" represents the circumference at the knee and "A" represents the circumference of the ankle area. The average lower limb skin-surface area of volunteers was 1259.2 cm 2. The amount of repellents used on the day of experiment were measured using Ohaus CS200 weighing scale (Ohaus Corporation, USA) The average amount of MAÏA ® repellent ointment applied per limb was 2.52 grams corresponding to a target dose of 2 mg/cm2. All volunteers applied repellents using latex-gloved hands to minimize absorption onto the hands. Empty plastic cups that contained MAÏA ® ointment were weighed to determine the amount left in the cups after application. Repellents for the Anopheles study were applied at 17:45 hours and for the Aedes study at 05:50 hours during the pilot study, while during the nal study repellents were applied at 17:45 hours for the Anopheles study and at 23:45 hours for the Aedes study. In the nal study, all participants rested with their trousers rolled up to prevent abrasion of the repellents after repellent application Duration of the study Six-hour pilot test During the rst 9 days, 50 An. arabiensis (Kingani) and 50 An. gambiae (Kisumu) were released in each of the SFS chambers and testing proceeded for 6 hours. During the 6 hours recapture period, no mosquitoes were recaptured by volunteers who applied MAÏA ® and 20% DEET. Therefore, we extended the recapture period to 12-hours in order to con dently determine the duration of complete protection of MAÏA ® repellent ointment and 20% DEET.

Twelve-hour test
During the LS with 12-hours of recapture period, the same volunteers, chambers and rotation schedule was used for Anopheles experiment: 100 An. arabiensis (Kingani) and 100 An. gambiae (Kisumu) were released (25 per release) starting at 00:00 hours. Three additional volunteers were recruited following study procedure and were assigned to the Ae. aegypti experiments in which 100 Ae. aegypti were released with 25 released every hour between 06:00 and 10:00hours.

Mosquito recapture
Volunteers recorded the time of a rst mosquito recapture of each experiment (Anopheles or Aedes) and placed in a separate cup labelled with the time of recapture, volunteer's code, position and repellent (treatment) code. Volunteers collected subsequent mosquitoes that landed for a 6 hour collection period up to 12-hours post-application of repellent with cups labelled with repellent code, position and hour of collection. Cups were changed after every hour. At the end of recapture time, mosquitoes were killed by refrigeration at -4ºC for about 40 minutes and then sorted to species level. If fewer than 50% of mosquitoes were recaptured by a negative control volunteer, the data were discarded and the replicate was repeated. Temperature, relative humidity and wind speed was recorded on the day at the beginning of the experiment.

Data management and statistical analysis
Data were recorded in paper forms and then double entered and cleaned in Microsoft Excel 2016. Data analyses were performed in Stata 15.1 (Stata Corp, USA). Descriptive analyses of mosquitoes recaptured by repellents was performed.
The mean complete protection time (CPT) of each repellent for each mosquito strain was estimated using the Kaplan-Meier survival analysis. The protective e cacy (PE) was established for the data collected up to 12-hours and calculated using this formula; P = ((C -T)/C)*100; where 'P' represents the percentage protection, 'C' represents the number of mosquitoes recaptured on the negative control (ethanol) and 'T' represents the number of mosquitoes recaptured on volunteer's lower limbs treated with either MAÏA ® or 20% DEET.
Statistical analysis was performed using a mixed-effects binary logistic regression to compare the protective e cacy between MAÏA ® and 20% DEET (as the reference in the statistical model). Several models were tested using the proportion of recaptured mosquitoes as the outcome variable and repellent type (treatments), volunteer, position of the volunteer and time of recapture as xed effects and day of test as random effects. The best-t model was determined using the Aikaike's Information Criterion (AIC) and the model with the smallest AIC value was selected. In the Anopheles data, repellent and time after application were xed effects and the day of test was a random effect. In the Aedes data, the type of repellent, time of recapture and position of the volunteer were xed effects and the day of test was a random effect.

Discussion
DEET based topical mosquito repellents provide personal protection against numerous arthropods of medical importance including malaria vectors such as An. gambiae and An. arabiensis and the dengue vector Aedes aegypti [34]. The evaluation of the protective e cacy and duration of protection of MAÏA® repellent ointment compared to a standard unformulated 20% DEET in ethanol under semi-eld conditions was done in the SFS. Sangoro et al, demonstrated that a semi-eld evaluation of topical mosquito repellent gives similar results to eld studies but is far safer as only disease-free laboratory-reared mosquitoes are used [29]. This is especially important in the case of Ae. aegypti evaluations in areas of active dengue transmission such as Tanzania [35]. Moreover, semi-eld environments allow volunteers to be accessible to a known number of mosquitoes of known age, physiological status, and avidity and it minimizes heterogeneity in the data allowing a more precise estimation of true repellent e cacy. The study is also conservative as it used young, never blood-fed mosquitoes raised under optimal conditions to maximize body size and then sugar starved, which are less repelled by DEET than older or smaller mosquitoes [36,37].
The study results demonstrated that MAÏA® repellent ointment is comparable to unformulated 20% DEET in terms of mean repellency over 12 hours as well as the duration of complete protection time (CPT) against both malaria and dengue vectors. MAÏA® repellent ointment and 20% DEET had CPT of more than 9-hours against An. gambiae (Kisumu) and An. arabiensis (Kingani) and more than 7-hours for Ae. aegypti post-application of the repellents. These results are similar to another study which evaluated the effectiveness of MAÏA® repellent ointment under eld conditions in Burkina Faso in which authors concluded that MAÏA® and 20% DEET are comparable in terms of duration of complete protection time against Anopheles strain and Aedes aegypti (Guelbeogo et al., Manuscript under preparation).
The study results indicated that MAÏA® repellent ointment is an effective mosquito repellent suitable for the use even under high mosquito biting pressure. According to Goodyer et al, an ideal mosquito repellent should provide CPT above 6-hours under the highest mosquito biting pressure [38]. The present study ful lled this characteristic by demonstrating the mean CPT above 9-hours against An. gambiae (Kisumu) and An. arabiensis (Kingani) and above 7-hours against Ae. aegypti for both MAÏA® and 20% DEET with an average of 10 mosquito landings per strain per hour in the control, 30 landings per hour in total.
User compliance is a major limitation of most topical insect repellents [15][16][17] and since topical repellents are applied to the skin, most users prefer insect repellents which are cosmetically pleasant in terms of odour and feel on the skin; in addition to providing protection from biting insects [39,40]. In this study, all study volunteers preferred to use MAÏA® repellent ointment compared to 20% DEET because those who applied MAÏA® repellent reported that the ointment felt better on their skin. This observation con rms that some repellent users may be in uenced by product characteristics such as texture, skin feel, and odour [40,41]. DEET tends to damage some plastics [42] which is a disadvantage in an area where plastic footware is commonly worn. An additional bene t observed by the users was that MAÏA® repellent ointment did not damage plastics when intentionally applied to a plastic watch strap. Therefore, the use of MAÏA® repellent ointment may be a suitable alternative to a less cosmetically appealing DEET-based formulations. However, more studies are required to speci cally assess user acceptability comparing between MAÏA® repellent ointment and other formulated products available on the market.

Study Limitations
After the initial nine days of the experiment, the study was extended for another nine days in order to con dently determine the CPT of MAÏA® repellent ointment and 20% DEET. This led to the addition of volunteers to work exclusively on the Aedes experiment conducted in one chamber of the SFS and only one Latin square design conducted. Therefore, the mean CPT of MAÏA® repellent ointment and 20% DEET was achieved using different and fewer (3) volunteers in the Aedes experiment compared to six volunteers and two SFS chambers used simultaneously in the Anopheles experiments. Ideally, the experiment would use more volunteers to capture the repellent e cacy against a wide range of people [43]. The study did not asses the e cacy of the repellent against nuisance mosquitoes such as Culex quinquefasciatus, which may also be important when assessing the consumer acceptance of repellents.

Conclusion
In conclusion, MAÏA® repellent ointment is comparable to unformulated 20% DEET under high biting pressure. Therefore, it may be recommended for use in disease-endemic areas. We recommend a reapplication interval of six hours based on the shortest complete protection time of 7 hours observed for Aedes aegypti in this study. It is a cosmetically appealing mosquito bite protection tool that also nourishes and moisturizes the skin, which may improve consumer acceptability and t into daily life if used every evening after bathing. A semi-eld system (SFS) A semi-eld system (SFS) with 29 x 21 meters built from a fabricated greenhouse frame modi ed to make two chambers with a central corridor and an opaque polyethylene roof for rain protection.