Applying the ESPT
The ESPT was piloted by the NVDCP in 2018 and 2019. The ESPT-based entomological surveillance plan was based on NVDCP’s priority program question: ‘What are the Anopheles species temporal compositions, bionomic characteristics, and susceptibility to insecticides in Namibia?’ The ESPT was used to select question-based minimal essential indicators and outline a sampling design grounded in available capacity, and served as a framework for data analysis and the interpretation of findings. Towards answering this question, several sampling methods were selected and evaluated in 2018 with the aim to capture key data elements, including species occurrence and density, human biting rate (HBR), biting time and location, indoor resting density and resistance frequency. Data-based evaluations of sampling methods in 2018 determined sampling methods utilized in 2019.
Study sites
Annual entomological surveillance was conducted in 2018 and 2019 in nine and four sentinel sites, respectively, located in the malaria endemic regions of northern Namibia. The specific sentinel sampling site in each region was a malaria hotspot, as determined by the NVDCP based on malaria risk maps generated from malaria surveillance data of the past three consecutive years. Each sentinel site constituted a village in a malaria endemic region and included a sampling area of about 10-km radius from a central location of the village. The nine regions with the sentinel sites (in brackets) that were included in the 2018 entomological surveillance were Kunene (Otjimuhaka), Omusati (Etaka), Oshana (Onamutai), Ohangwena (Okanghudi), Oshikoto (Onayena), Otjozondjupa (Otjituuo), Kavango West (Mukekete), Kavango East (Shadikongoro) and Zambezi (Makanga) (Fig. 1a). The four sentinel sites studied in 2019 were Ohangwena (Okanghudi), Kavango West (Mukekete), Kavango East (Shadikongoro) and Zambezi (Sibbinda) (Fig. 1b).
Sampling methods
Mosquito sampling was conducted between March and April (malaria transmission months) to determine the following entomological indicators: site-specific Anopheles species occurrence and density, indoor and outdoor HBRs, time of biting, indoor resting behavior and susceptibility to insecticides (deltamethrin, Actellic, bendiocarb, DDT) used in IRS. In 2018, a baseline survey was conducted in nine sentinel sites that represented a sampling village in each of the nine malaria endemic regions. In 2019, the same entomological parameters were studied between April and May but in only four out of the nine sentinel sites. The 2019 entomological surveillance was carried out in two rounds, with the first round conducted in April and the second round conducted in May.
Human landing catches
Human landing catches (HLCs) [23, 24] were conducted both inside and outside structures. A set of four sentinel households, with at least one type of each local house construction type represented (i.e. mud, zinc, traditional), was randomly selected from each sentinel village. Host-seeking adult mosquitoes were sampled for an entire night (from 1800 hours to 0600 hours) in each household, with variable numbers of nights based on the site and year ( as described below). Two community volunteers (CVs) sampled mosquitoes (one indoors and one outdoors) at each of the sampling households. The CVs collected landing mosquitoes for 45 min every hour, with resting breaks of 15 min in between collections, and swapped positions (inside and outside) every hour to minimize personal bias. A team of eight CVs worked a 6-h shift (1900 hours to 0100 hours) and were then replaced by another team of eight CVs for the remaining 6 h (a total of 16 volunteers were used per collection night). Mosquito samples from each household were placed in different holding cups and the cups labeled according to hour collected and location.
In 2018, there were a total of 16 trapping nights per site in Kavango East, Kavango West, Kunene, Ohangwena, Omusati, Oshana, Otjozondjupa and Zambezi and 12 trapping nights at the site in Oshikoto. There were two rounds of collections in 2019: in round 1, there were a total of 16 trapping nights per site each in Kavango East, Ohangwena and Zambezi in round 1 (March; summer) and only 12 trapping nights in Kavango West; in round 2, there were 16 trapping nights per site in all sites (May; autumn). Hence, there was a total of 32 trapping nights each in 2019 for Kavango East, Ohangwena and Zambezi and 28 trapping nights in Kavango West. In 2019 samples were collected in two sampling rounds to account for seasonal variation.
Pyrethrum spray catches
Houses where pyrethrum spray catches (PSCs) were conducted were not the same houses as those where HLCs were conducted; there was a buffer distance of at least 30 m between any house included in the entomology collections. Collections using PSCs were conducted in 2019 only. After first obtaining consent by the head of the household, field technicians removed all major obstacles in the rooms under study. All wall crevices and roof openings were covered with cloth to prevent mosquitoes from escaping. The floors of the rooms were then covered with white sheets and an insecticide (aerosol pyrethroid) was sprayed on the walls and roofs of the rooms for about 2 min. After 10 min, the white sheets were examined for knocked-down mosquitoes, which were collected and placed in holding cups. Different houses were sampled each night. There were two rounds of collections in 2019: in round 1, PSCs were conducted in a total of 16 houses each in Kavango East, Ohangwena and Zambezi, and only four PSCs were conducted in Kavango West; in round 2, there was a total of 16 PSCs in all sites. Hence, there was a total of 32 trapping nights each in 2019 for Kavango East, Ohangwena and Zambezi and 20 trapping nights in Kavango West.
U.S. Centers for Disease Control and Prevention miniature light traps
Sampling of host-seeking mosquitoes using U.S. Centers for Disease Control and Prevention miniature light traps (CDC-LTs) [25, 26] was conducted in two houses (houses in which other entomological sampling methods were not performed). For indoor sampling, a CDC-LT was hung 1 m above the floor and next to a bed in which a household member slept under a bed net. For outdoor sampling, a CDC-LT was hung about 10 m from a house where indoor sampling was conducted. CDC-LT catches were conducted from 1900 hour to 0700 hour. On the following morning, all mosquitoes trapped were removed from the trap and placed in holding cups. In 2018, there were a total of 16 trapping nights indoors and outdoors each in Kavango East, Kavango West, Kunene, Ohangwena, Omusati, Oshana, Oshikoto, Otjizondjupa and Zambezi. CDC-LT catches were not conducted in 2019.
Resting Box Collections
Two sentinel houses were used for catches with resting box collections (RBCs) [27, 28]. One resting container (a bucket lined with black cloth) was placed indoors and another was placed about 10 m away from the house. The RBs remained in their positions for 12 h (1900 hour to 0700 hour) after which mosquitoes found to rest inside the buckets were aspirated into holding cups. The total number of RB collection nights were site specific, with 16 indoor and 16 outdoor RB collections each in Kavango East, Kavango West, Kunene, Ohangwena, Omusati and Otjizondjupa, 12 indoor and 12 outdoor RB collections each in Zambezi and Oshikoto and only four indoor and four outdoor RB collections in Oshana. RB collections were not conducted in 2019.
Larval sampling and rearing
Larval sampling was conducted after the rainy season, during the months of March and April, using larval dippers [29,30,31]. Sampling of larvae was conducted in the same sentinel villages where adult mosquitoes were sampled. Anopheles mosquito larvae and pupae were identified morphologically and reared to adulthood at the NDVCP insectary in Oshakati and Zambezi. The larvae were fed on dog biscuits and yeast mixture, and emerging adults were fed on a 10% sugar solution ad libitum. Temperature and relative humidity within the insectary were maintained between 28 °C and 29 °C and 70–80%, respectively.
Insecticide susceptibility assays
Larvae sampling for the insecticide resistance (IR) tests was conducted after the rainy season, during the months of March and April, using larval dippers [29,30,31]. Sampling of larvae was conducted in the same sentinel villages where adult mosquitoes were sampled. Anopheles mosquito larvae were identified morphologically and reared to adulthood at the NDVCP insectary in Oshakati and Zambezi under the same conditions as described in section Larval sampling and rearing. The WHO bioassays were conducted following WHO protocols [31]. Female Anopheles mosquitoes aged 3–5 days which had been raised from larvae and had never had a blood meal were used for the WHO IR bioassays. Susceptible An. arabiensis mosquitoes (KGB strain) were used for the control WHO bioassays. Live and dead mosquitoes at the end of the assay were separated and placed in tubes with labels specifying outcome. IR tests were conducted in 2018 across the nine sentinel sites for the following IRS insecticides deltamethrin 0.05%, DDT 4%, bendiocarb 1.25% and pirimiphos-methyl (Actellic) 0.25%. Resistance tests were done only in Zambezi in 2019 using deltamethrin 0.05%, DDT 4%, bendiocarb 1.25% and pirimiphos-methyl (Actellic) 0.25%.
Species identification
All mosquitoes sampled were morphologically identified to species [32] where possible and placed individually in Eppendorf tubes containing silica gel separated from the mosquito by plain paper. PCR-based species identification was also conducted for morphologically identified members of the An. gambiae s.l. and An. funestus s.l. complexes [33, 34], while sequencing-based species identification using internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit 1 (CO1) sequences [35] was conducted on a subsample of all specimens. Successful PCR identifications were obtained for only a portion of the collected mosquitoes (n = 1873 out of n = 3188). Therefore, we multiplied the number of An. gambiae s.l. without a confirmed molecular identification (disaggregated by village, biting location and hour) by the proportions of An. arabiensis and An. gambiae s.s. that were successfully identified at that location/time. Subsequent analyses used this estimated dataset.
Analysis
Insecticide susceptibility data were analyzed according to WHO protocols. Variability in the number of anophelines caught by HLC was investigated using generalized linear mixed models (GLMM) with a negative binomial distribution and log link function. All analyses were performed in R v4.3.2 using the lme4 package (R Foundation for Statistical Computing, Vienna, Austria). Separate models were fit for each of the following response variables: total anophelines, An. gambiae s.s., An. arabiensis and An. funestus s.s. Random effects included village, date and their interaction. For the total anopheline, An. arabiensis, and An. funestus s.s. models, fixed effects included biting location (indoors/outdoors), biting time, year and year × location interaction. For the An. gambiae s.s. model, fixed effects including the year variable were removed because An. gambiae s.s. was only caught in 2018. For the remaining descriptive statistics, mosquito catches were standardized to bites per night (bpn).
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