Study sites
The first part of the study was conducted in Kinshasa Province in 2016 and 2017. Four sites were selected for mosquito larval collection to monitor changes in Anopheles gambiae sensu lato (s.l.) pyrethroid resistance intensity following mass LLIN (DawaPlus 2.0 coated with deltamethrin at a target dose of 80 mg/m2) distribution in December 2016 (Fig. 1). A fifth site, Kasangulu, in neighbouring Kongo Central province was selected to provide a “control” site, which would not be included in the Kinshasa Province LLIN distribution campaign in 2016. However, a limitation is that PermaNet 2.0 LLINs (formulation of deltamethrin at a target dose of 55 mg/m2)) were distributed in a mass campaign in Kongo Central Province in 2014. More details of the study sites and previous mass LLIN distributions in each region are presented in Supplemental Table 1.
The second part of the study involved nationwide testing of pyrethroid resistance intensity. Deltamethrin and permethrin resistance intensity tests were conducted in seven sites in 2017 using Centers for Disease Control and Prevention (CDC) bottle bioassays. In 2018, testing was expanded to eleven sites, with resistance intensity to permethrin, alpha-cypermethrin and deltamethrin monitored using WHO tube tests for intensity (Fig. 2). More site details are included in Supplemental Table 1.
Insecticide susceptibility tests
Mass LLIN distribution took place in December 2016 in Kinshasa Province. CDC intensity assays were conducted once before the mass distribution of LLINs (June 2016) and two, six, and ten months after the distribution (February 2017, June 2017, and October 2017). For each round of bioassays, An. gambiae s.l. larvae were collected from the five sites (Fig. 1) and returned to the laboratory at the Institut National de Recherche Biomédicale (INRB) in Kinshasa city, where they were reared to adults. Adult mosquitoes were kept in cages and provided with 10% sugar solution ad libitum until the time of testing at the age of 2-5 days.
The intensity assays conducted nationwide followed the same protocol, but tests were conducted once per year (all tests between January and August in 2017 and 2018) and mosquitoes were reared and tested in field insectaries.
CDC bottle bioassays
CDC bottle bioassays were conducted to determine the intensity of pyrethroid resistance, following standard guidelines [29, 30]. Pre-measured vials of technical grade active ingredient were supplied by CDC and made into stock solutions for each insecticide dose by diluting with acetone. Stock solutions were stored in the refrigerator (4°C) in light-proof bottles for future use. Glass Wheaton bottles (250ml) were washed with warm soapy water and rinsed thoroughly with water at least three times and left to dry overnight. A disposable pipette was used to transfer 1ml of acetone into the negative control bottle and 1ml of each stock solution into the respective treatment bottle. Bottles were swirled so that the glass bottom and inside cap were coated before being placed on their side and rotated while rocking so that the sides were evenly coated with insecticide. The bottles were protected from sunlight, and caps removed before being left to dry overnight.
An aspirator was used to gently add twenty-five mosquitoes into each bottle per replicate. Four replicates of each dose were done to reach approximately 100 mosquitoes tested. Mosquitoes were exposed for the diagnostic time of 30 minutes, with knock-down being recorded at the end of exposure. A knocked-down mosquito was defined as not being able to stand. Deltamethrin was tested at 1× (12.5μg/bottle), 2× (25μg/bottle), 5× (62.5µg/bottle), and 10x (125µg/bottle) the diagnostic dose for Anopheles. Permethrin was also tested at 1× (21.5μg/bottle), 2× (43μg/bottle), 5× (107.5µg/bottle), and 10 times (215µg/bottle) the diagnostic dose for Anopheles.
WHO susceptibility tests
In 2018, insecticide susceptibility and resistance intensity testing were conducted in 11 sentinel sites (Fig. 1) using the WHO tube test. The insecticides tested in 2018 were: deltamethrin ×1, ×5, ×10 (0.05%, 0.25%, 0.5%); permethrin ×1, ×5, ×10 (0.75%, 3.75%, 7.5%) and alpha-cypermethrin ×1, ×5, ×10 (0.05%, 0.25%, 0.5%). In all sites, susceptibility testing was conducted with adult An. gambiae s.l., following WHO protocols [22]. INRB entomologists traveled to each site to collect larvae and pupae, which were reared to female adult mosquitoes aged 2-5 days and exposed for one hour to insecticide-treated filter papers provided by the WHO (prepared by Universiti Sains Malaysia). All tests were accompanied by negative control tests where mosquitoes were exposed to filter papers impregnated with oil or solvent. Testing was done according to WHO protocols, with mortality read 24 hours after exposure. Four replicates of 25 An. gambiae s.l. were exposed to each concentration.
Identification of species and target site mutations
A subset of An. gambiae s.l. that were collected from the four sites in Kinshasa (Bu, Kimpoko, Kingasani and Kinkole) and 1 ‘control’ site in Bas-Congo (Kasungulu) in October 2017 and tested in CDC control bottles, were sent to CDC, Atlanta, USA for molecular analysis. In addition, 100 mosquitoesused for WHO bioassays in each of the eleven nationwide sites in 2018 were used for molecular analysis at INRB, Kinshasa, DRC. PCR was used to determine the species of mosquitoes from the An. gambiae complex and to determine the frequency of the voltage-gated sodium channel mutation (VGSC) 1014S (formerly known as kdr-east) and VGSC1014F (formerly known as kdr-west).
Genomic DNA was extracted from whole mosquitoes at CDC using ExtractaTM DNA Prep for PCR-Tissue kits (QuantaBio, USA) and at INRB using the CTAB method [31]. Species identification was performed according to the protocol of Wilkins et al. [32] at CDC and using the protocol of Santolamazza et al. [33] at INRB. The VGSC-1014S and 1014F alleles were detected using adapted protocols for allele-specific PCR (AS-PCR)[34-36]. Anopheles coluzzii AKDR and An. gambiae sensu stricto (s.s.) RSP-ST strains from the Malaria Research and Reference Reagent Resource Center (MR4), were used as positive controls, alongside negative (no template) controls.
Analysis
Scoring of bottle bioassays using the diagnostic dose followed WHO and CDC criteria, with mortality of 98-100% indicating susceptibility, 90-97% indicating possible resistance that should be confirmed, and less than 90% indicating resistance [22, 29]. Mortality of 98–100% at the 5× concentration (but <90% at 1×) indicates low resistance intensity. Mortality <98% at the 5× concentration but 98–100% at the 10× concentration indicates moderate resistance intensity. Mortality <98% at the 10× concentration indicates high resistance intensity [22].
The comparison of bioassay results prior to the LLIN mass distribution and after distribution in Kinshasa were made using a logistic regression model, taking into account the dose, site, time period, and an interaction between dose and site as fixed effects and bottle as a random effect. Analysis was done using the glmm function in R (version 3.2.3). Pearson’s Chi squared test was used to determine deviations from Hardy-Weinberg equilibrium for L1014F allele frequencies.