Bio-efficacy of Olyset® Plus, PermaNet® 3.0 and Interceptor® G2 on pyrethroid-resistant populations of Anopheles gambiae s.l. prior to the June 2023 net distribution campaign in Benin, West Africa | Tropical Medicine and Health

Study area

The study was carried out in the departments of Ouémé (communes of Akpro-Missérété and Porto-Novo), Atlantique (commune of Allada), Zou (commune of Bohicon), Mono (commune of Lokossa) (Fig. 1). These five communes have a subequatorial climate with two rainy seasons (April to June, and September to November) and two dry seasons (July to August, and December to March). The annual rainfall ranges between 1200 and 1300 mm and the humidity is about 75%. All the communes feature rivers, marshes, swamps and lowlands which fostering activities such as market gardening and fish farming.

Additionally, the commune of Djougou, situated in the Donga department, was included in the survey. Djougou exhibits a Sudano-Guinean climate marked by a single rainy season from April to October and a sole dry season from October to April. The annual rainfall in Djougou varies between 900 and 1100 mm, and agriculture stands as the predominant activity in this region.

The selection of these study communes was deliberate, driven by their elevated malaria prevalence and the prevalent vector resistance to pyrethroids [24,25,26]. Moreover, the extensive use of insecticides in these areas is remarkable, primarily for safeguarding crops against pests.

Larvae collection, rearing and identification

Larvae and pupae of Anopheles mosquitoes were meticulously gathered from identified positive breeding sites in both central and peripheral areas of each commune, employing a combination of dippers, ladles, pipettes, and larval containers. Subsequently, these specimens were transported to the insectary at the Centre de Recherche Entomologique de Cotonou (CREC) for further examination. Within the controlled environment of the insectary, the larvae and pupae were reared at a temperature of 26 °C ± 1 °C and a relative humidity of 80% until reaching adulthood. Morphological identification exclusively utilized the Coetzee taxonomic key [27], focusing solely on members of the An. gambiae s.l. species for subsequent tests.

WHO susceptibility tube tests

The susceptibility status of An. gambiae s.l. populations to pyrethroid insecticides was evaluated through WHO tube tests. Batches of 20–25 unfed female mosquitoes, aged 2–5 days, were exposed to papers treated with deltamethrin 0.05%, permethrin 0.75%, and alpha-cypermethrin 0.05% for a duration of 60 min. Concurrently, control batches of 20–25 mosquitoes were exposed to untreated papers. Throughout the exposure period, the number of mosquitoes knocked down by the insecticide was recorded at 15-min intervals. Following exposure, the mosquitoes were transferred to observation tubes, where they were provided with a 10% sugar solution and maintained at a temperature of 27 °C ± 2 °C with a humidity level of 75% ± 10% for 24 h. The mortality rate was determined 24 h post-exposure [28].

CDC bottle bioassays

The susceptibility of wild populations of An. gambiae s.l. to chlorfenapyr was determined utilizing the CDC bottle bioassay. To conduct this assay, 250 ml glass Wheaton bottles were coated with 1 ml of chlorfenapyr (100 μg/ml), while a bottle coated with 1 ml of acetone served as a control. Batches of 20–25 mosquitoes were introduced into the coated bottles for a 60-min exposure period, during which the number of knocked-down mosquitoes was recorded every 15 min. Subsequent to the exposure, the mosquitoes were gently transferred to observation cups and provided with a 10% sugar solution. Immediate mortality was recorded after 1 h of exposure, and delayed mortalities were subsequently documented at 24, 48, and 72 h post-exposure. Mosquitoes that expired immediately were preserved in RNA later at − 80 °C, whereas those succumbing after 24-, 48- and 72-h post-exposure were preserved in silica gel [29].

Molecular and biochemical assays

Dead and live mosquitoes from the WHO susceptibility tube tests underwent PCR analysis to ascertain the molecular species within the An. gambiae complex [30] and to detect the presence of the Kdr L1014F mutation [31].

For biochemical analyses, thirty unexposed female An. gambiae s.l., 2 to 5 days old, from each commune underwent biochemical analyses. These analyses aimed to compare the expression levels of detoxification enzymes, including mixed function oxidases, non-specific esterases, and glutathione S-transferases, across diverse field populations of An. gambiae s.l. and the reference susceptible strain (An. gambiae Kisumu), in accordance with the protocol described by Hemingway et al. [32].

Description of tested mosquito nets

The three types of new-generation nets include:

• Olyset^® Plus: Manufactured by Sumitomo Chemicals, Japan, this polyethylene LLIN incorporates 2% permethrin (800 mg permethrin ai/m²), and 1% PBO (400 mg PBO ai/m²).

• PermaNet^® 3.0: Produced by Vestergaard Frandsen SA, Denmark this LLIN features a polyethylene roof coated with 2.8 g/kg ± 25% deltamethrin and 4.0 g/kg ± 25% PBO. Its polyester sides are coated with 2.8 g/kg ± 25% deltamethrin.

• Interceptor^® G2: Manufactured by BASF SE, Ludwigshafen, Germany, this polyester LLIN is coated with a mixture of 200 mg/m² chlorfenapyr and 100 mg/m² alpha-cypermethrin.

In comparison, the standard pyrethroid-only net used as a control is Interceptor^®, a polyester netting manufactured by BASF SE, Ludwigshafen, Germany, incorporating 200 mg/m² of alpha-cypermethrin.

WHO cone bioassay

A susceptible laboratory strain (An. gambiae Kisumu), and field populations of An. gambiae s.l. were utilized to assess the bio-efficacy of new-generation nets according to the WHO cone test protocol [33].

Nets that were brand new, and never used in the community (Olyset^® Plus, Interceptor^® G2 and PermaNet^® 3.0) were tested in this study. Five pieces of net, measuring 30 cm × 30 cm, including a piece from the roof and a piece from each of the four lateral sides were sampled on Olyset^® Plus (mixture PBO and Permethrin) and Interceptor^® G2 (mixture Chlorfenapyr and alphacypermethrin). For PermaNet^® 3.0, four pieces of netting were sampled, two from the roof and two from the sides (one from the length and one from the width), due to the difference in insecticide types between the roof (PBO + Deltamethrin) and the lateral sides (Deltamethrin). All net pieces were individually labelled, securely wrapped in foil, and stored in a refrigerator before testing. During the tests, each net piece had two standard cones affixed using a plastic plate. We introduced five unfed female An. gambiae s.l., aged 2 to 5 days, into each cone for a 3-min exposure. Post-exposure, mosquitoes were gently transferred into cups, provided with a 10% sweetened juice, and observed for 24 h at room temperature (27 °C ± 2 °C) with a relative humidity of 75% ± 10%. The number of knocked-down mosquitoes was recorded every 5 min during and one hour after exposure. Mortality rates were determined 24 h post-exposure.

For Olyset^® Plus and Interceptor^® G2 nets, a total of 50 mosquitoes were tested per net, while 40 were tested for PermaNet^® 3.0. This comprehensive testing approach aimed to provide a thorough assessment of the nets’ effectiveness against An. gambiae s.l. populations.

WHO tunnel test

The bio-efficacy assessment of Interceptor^® G2 and Interceptor^® LLINs also included tunnel tests, with an untreated mosquito net serving as a negative control. In this experiment, unfed female An. gambiae s.l., aged 7 days, were released into a tunnel with two square sides (25 cm × 25 cm) and a length of 60 cm. The tunnel was partitioned into two sections: section A, constituting 2/3 of the tunnel where mosquito releases occurred, and section B, encompassing the remaining portion of the tunnel where an immobilized bait (a guinea pig) was placed. At the end of section B, a 25 cm side square cage covered with polyester netting was installed. The netting to be tested was positioned just after section A. The area of the net accessible to mosquitoes measured 400 cm2 (20 cm × 20 cm), featuring nine holes of 1 cm in diameter. In the evening, one hundred unfed female mosquitoes, held without food for a minimum of 6 h before the test, were introduced into the cage through the end of section A. Three separate tunnels were employed for the Interceptor^® G2, Interceptor^® (positive control), and the untreated net (negative control). Following 12 h of exposure, mosquitoes were carefully removed from each section of the tunnel in the early morning using a mechanical aspirator and placed in veiled and labeled cups. Mosquitoes in these cups were provided with a 10% sugar solution and observed for 72 h to determine the delayed mortality. The number of live, dead, unfed or blood-fed mosquitoes in each section of the tunnel was recorded to determine the entry, mortality, and blood-feeding inhibition rates. The tests were conducted in total darkness overnight, maintaining a constant temperature of 27 ± 2 °C and a relative humidity of 75% ± 10% [34].

Data analysis

The mortality rates observed 24 h after exposure to various insecticides were interpreted in accordance with WHO criteria [28]:

• Mortality rate between 98 and 100%: Susceptible mosquito population

• Mortality rate ≥ 90% and < 98%: Possible resistance in the mosquito population

• Mortality rate < 90%: Insecticide-resistant mosquito population.

The allelic frequencies of the kdr L1014F mutation were determined by the following formula: F = (2nRR + nRS) / (2(nRR + nRS + nSS)).

n = number of genotypes, RR: homozygous resistant, RS: Heterozygous, SS: homozygous susceptible.

The exact binomial test was used to calculate confidence intervals for mortality rates and allelic frequencies of kdr L1014F mutations. To assess the resistance activity of metabolic enzymes, their expression level was compared between field populations of An. gambiae s.l. and the laboratory susceptible strain, An. gambiae (Kisumu strain). GraphPad Prism8 software was used to draw the graphs and calculate the p-values.

The Mann–Whitney U test enabled comparison of the activity of enzymes, between the field mosquito populations and the laboratory susceptible strain (An. gambiae Kisumu). Statistical analyses were conducted using R 3.3.2 software [35].

Data on the bioefficacy of LLINs tested with the laboratory susceptible strain An. gambiae (Kisumu strain) was analyzed according to WHO criteria:

Furthermore, mortality rates displayed by LLINs using wild populations of An. gambiae s.l. was also presented.

The indicators evaluated through the tunnel tests are as follows:

• Blood-feeding rate (%) = (A/B) × 100, where A is the number of blood-fed mosquitoes collected in the tunnel, and B is the total number of mosquitoes exposed to the insecticide-incorporated net.

• Blood-feeding inhibition rate (%) = ((C − D))/C × 100, where C and D are the blood feeding rates obtained with the untreated, and insecticide-treated nets, respectively.

• Immediate mortality (%) = (E/F) × 100, where E is the number of dead mosquitoes collected in the tunnel just after the 12-h-exposure time, and F is the total number of mosquitoes exposed to the insecticide-incorporated net.

• 24-h mortality (%) = (G/H) × 100, where G is the number of dead mosquitoes 24 h post-exposure, and H, the total number of mosquitoes exposed to the insecticide-treated net

• 72-h mortality (%) = (I/J) × 100, where I is the number of dead mosquitoes within 72 h, and J, the total number of mosquitoes exposed to the insecticide-treated net.