Evaluation of Anopheline Diversity and Abundance across Outdoor Collection Schemes Utilizing CDC Light Traps in Nchelenge District, Zambia.

In the global fight against malaria, standard vector control methods such as indoor residual spraying (IRS) and insecticide-treated nets (ITNs) are intended to protect inside residential structures and sleeping spaces. However, these methods can still leave individuals vulnerable to residual transmission from vectors that they may be exposed to outdoors. Nchelenge District in northern Zambia experiences persistently high malaria transmission even with ITNs and IRS in place. However, very few studies have examined outdoor vector activity. To assess the diversity and abundance of outdoor foraging female anopheline mosquitoes, CDC light traps were used as proxy measures for mosquito host-seeking, set in three outdoor trapping schemes randomly assigned on different nights: (1) locations where people congregate at night outside of the house within the peri-domestic space, (2) animal pens or shelters, and (3) high-human-traffic areas, such as paths to latrines, where traps were baited with BG-Lure®. A total of 1087 total female anophelines were collected over a total of 74 trap nights. Anopheles funestus s.s. comprised the majority of the collection (86%), with An. gambiae s.s. (2%) and a highly diverse sampling of other anophelines (12%) making up the remainder. Plasmodium falciparum parasites were only detected in An. funestus (1%). No significant difference in species diversity or female anopheline abundance was detected between trapping schemes. Outdoor foraging anopheline mosquitoes, including a number of infectious An. funestus, may partially explain the difficulty of controlling malaria transmission in Nchelenge District, where vector control is only targeted indoors. BG-Lure® shows some promise as an alternative to human-baited landing catch collections in this resource-poor setting.

The first complete mitochondrional genome of Anopheles gibbinsi using a skimming sequencing approach.

Mosquitoes belonging to the genus Anopheles are the only vectors of human malaria. Anopheles gibbinsi has been linked to malaria transmission in Kenya, with recent collections in Zambia reporting the mosquito species exhibiting zoophilic and exophilic behavioral patterns with occasional contact with humans. Given the paucity of genetic data, and challenges to identification and molecular taxonomy of the mosquitoes belonging to the Anopheles genus; we report the first complete mitochondrial genome of An. gibbinsi using a genome skimming approach. An Illumina Novaseq 6000 platform was used for sequencing, the length of the mitochondrial genome was 15401 bp, with 78.5% AT content comprised of 37 genes. Phylogenetic analysis by maximum likelihood using concatenation of the 13 protein coding genes demonstrated that An. marshallii was the closest relative based on existing sequence data. This study demonstrates that the skimming approach is an inexpensive and efficient approach for mosquito species identification and concurrent taxonomic rectification, which may be a useful alternative for generating reference sequence data for evolutionary studies among the Culicidae.

The Impact of Household and Community Indoor Residual Spray Coverage with Fludora Fusion in a High Malaria Transmission Setting in Northern Zambia.

Zambia's National Malaria Elimination Program transitioned to Fludora Fusion in 2019 for annual indoor residual spraying (IRS) in Nchelenge District, an area with holoendemic malaria transmission. Previously, IRS was associated with reductions in parasite prevalence during the rainy season only, presumably because of insufficient residual insecticide longevity. This study assessed the impact of transitioning from Actellic 300CS to long-acting Fludora Fusion using active surveillance data from 2014 through 2021. A difference-in-differences analysis estimated changes in rainy season parasite prevalence associated with living in a sprayed house, comparing insecticides. The change in the 2020 to 2021 dry season parasite prevalence associated with living in a house sprayed with Fludora Fusion was also estimated. Indoor residual spraying with Fludora Fusion was not associated with decreased rainy season parasite prevalence compared with IRS with Actellic 300CS (ratio of prevalence ratios [PRs], 1.09; 95% CI, 0.89-1.33). Moreover, living in a house sprayed with either insecticide was not associated with decreased malaria risk (Actellic 300CS: PR, 0.97; 95% CI, 0.86-1.10; Fludora Fusion: rainy season PR, 1.06; 95% CI, 0.89-1.25; dry season PR, 1.21; 95% CI, 0.99-1.48). In contrast, each 10% increase in community IRS coverage was associated with a 4% to 5% reduction in parasite prevalence (rainy season: PR, 0.95; 95% CI, 0.92-0.97; dry season: PR, 0.96; 95% CI, 0.94-0.99), suggesting a community-level protective effect, and corroborating the importance of high-intervention coverage.

Expanded geographic distribution and host preference of Anopheles gibbinsi (Anopheles species 6) in northern Zambia.

BACKGROUND: Nchelenge District in northern Zambia suffers from holoendemic malaria transmission despite a decade of yearly indoor residual spraying (IRS) and insecticide-treated net (ITN) distributions. One hypothesis for this lack of impact is that some vectors in the area may forage in the early evening or outdoors. Anopheles gibbinsi specimens were identified in early evening mosquito collections performed in this study area, and further insight was gleaned into this taxon, including characterizing its genetic identity, feeding preferences, and potential role as a malaria vector. METHODS: Mosquitoes were collected in July and August 2019 by CDC light traps in Nchelenge District in indoor sitting rooms, outdoor gathering spaces, and animal pens from 16:00-22:00. Host detection by PCR, COI and ITS2 PCR, and circumsporozoite (CSP) ELISA were performed on all samples morphologically identified as An. gibbinsi, and a subset of specimens were selected for COI and ITS2 sequencing. To determine risk factors for increased abundance of An. gibbinsi, a negative binomial generalized linear mixed-effects model was performed with household-level variables of interest. RESULTS: Comparison of COI and ITS2 An. gibbinsi reference sequences to the NCBI database revealed > 99% identity to "Anopheles sp. 6" from Kenya. More than 97% of specimens were morphologically and molecularly consistent with An. gibbinsi. Specimens were primarily collected in animal pen traps (59.2%), followed by traps outdoors near where humans gather (24.3%), and traps set indoors (16.5%). Host DNA detection revealed a high propensity for goats, but 5% of specimens with detected host DNA had fed on humans. No specimens were positive for Plasmodium falciparum sporozoites. Animal pens and inland households > 3 km from Lake Mweru were both associated with increased An. gibbinsi abundance. CONCLUSIONS: This is the first report of An. gibbinsi in Nchelenge District, Zambia. This study provided a species identity for unknown "An. sp. 6" in the NCBI database, which has been implicated in malaria transmission in Kenya. Composite data suggest that this species is largely zoophilic and exophilic, but comes into contact with humans and the malaria parasites they carry. This species should continue to be monitored in Zambia and neighbouring countries as a potential malaria vector.