Entomological survey of sibling species in the Anopheles funestus group in Tanzania confirms the role of Anopheles parensis as a secondary malaria vector.

BACKGROUND: Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an anthropophilic vector, which is now strongly resistant to public health insecticides, and several sibling species, which remain largely understudied despite their potential as secondary vectors. This paper provides the initial results of a cross-country study of the species composition, distribution and malaria transmission potential of members of the Anopheles funestus group in Tanzania. METHODS: Mosquitoes were collected inside homes in 12 regions across Tanzania between 2018 and 2022 using Centres for Disease Control and Prevention (CDC) light traps and Prokopack aspirators. Polymerase chain reaction (PCR) assays targeting the noncoding internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA (18S rDNA) were used to identify sibling species in the An. funestus group and presence of Plasmodium infections, respectively. Where DNA fragments failed to amplify during PCR, we sequenced the ITS2 region to identify any polymorphisms. RESULTS: The following sibling species of the An. funestus group were found across Tanzania: An. funestus s.s. (50.3%), An. parensis (11.4%), An. rivulorum (1.1%), An. leesoni (0.3%). Sequencing of the ITS2 region in the nonamplified samples showed that polymorphisms at the priming sites of standard species-specific primers obstructed PCR amplification, although the ITS2 sequences closely matched those of An. funestus s.s., barring these polymorphisms. Of the 914 samples tested for Plasmodium infections, 11 An. funestus s.s. (1.2%), and 2 An. parensis (0.2%) individuals were confirmed positive for P. falciparum. The highest malaria transmission intensities [entomological inoculation rate (EIR)] contributed by the Funestus group were in the north-western region [108.3 infectious bites/person/year (ib/p/y)] and the south-eastern region (72.2 ib/p/y). CONCLUSIONS: Whereas An. funestus s.s. is the dominant malaria vector in the Funestus group in Tanzania, this survey confirms the occurrence of Plasmodium-infected An. parensis, an observation previously made in at least two other occasions in the country. The findings indicate the need to better understand the ecology and vectorial capacity of this and other secondary malaria vectors in the region to improve malaria control.

Distribution and dynamics of Anopheles gambiae s.l. larval habitats in three Senegalese cities with high urban malaria incidence.

Urban malaria has become a challenge for most African countries due to urbanization, with increasing population sizes, overcrowding, and movement into cities from rural localities. The rapid expansion of cities with inappropriate water drainage systems, abundance of water storage habitats, coupled with recurrent flooding represents a concern for water-associated vector borne diseases, including malaria. This situation could threaten progress made towards malaria elimination in sub-Saharan countries, including Senegal, where urban malaria has presented as a threat to national elimination gains. To assess drivers of urban malaria in Senegal, a 5-month study was carried out from August to December 2019 in three major urban areas and hotspots for malaria incidence (Diourbel, Touba, and Kaolack) including the rainy season (August-October) and partly dry season (November-December). The aim was to characterize malaria vector larval habitats, vector dynamics across both seasons, and to identify the primary eco- environmental entomological factors contributing to observed urban malaria transmission. A total of 145 Anopheles larval habitats were found, mapped, and monitored monthly. This included 32 in Diourbel, 83 in Touba, and 30 in Kaolack. The number of larval habitats fluctuated seasonally, with a decrease during the dry season. In Diourbel, 22 of the 32 monitored larval habitats (68.75%) were dried out by December and considered temporary, while the remaining 10 (31.25%) were classified as permanent. In the city of Touba 28 (33.73%) were temporary habitats, and of those 57%, 71% and 100% dried up respectively by October, November, and December. However, 55 (66.27%) habitats were permanent water storage basins which persisted throughout the study. In Kaolack, 12 (40%) permanent and 18 (60%) temporary Anopheles larval habitats were found and monitored during the study. Three malaria vectors (An. arabiensis, An. pharoensis and An. funestus s.l.) were found across the surveyed larval habitats, and An. arabiensis was found in all three cities and was the only species found in the city of Diourbel, while An. arabiensis, An. pharoensis, and An. funestus s.l. were detected in the cities of Touba and Kaolack. The spatiotemporal observations of immature malaria vectors in Senegal provide evidence of permanent productive malaria vector larval habitats year-round in three major urban centers in Senegal, which may be driving high urban malaria incidence. This study aimed to assess the presence and type of anopheline larvae habitats in urban areas. The preliminary data will better inform subsequent detailed additional studies and seasonally appropriate, cost-effective, and sustainable larval source management (LSM) strategies by the National Malaria Control Programme (NMCP).

Urban malaria vector bionomics and human sleeping behavior in three cities in Senegal.

BACKGROUND: Malaria is endemic in Senegal, with seasonal transmission, and the entire population is at risk. In recent years, high malaria incidence has been reported in urban and peri-urban areas of Senegal. An urban landscape analysis was conducted in three cities to identify the malaria transmission indicators and human behavior that may be driving the increasing malaria incidence occurring in urban environments. Specifically, mosquito vector bionomics and human sleeping behaviors including outdoor sleeping habits were assessed to guide the optimal deployment of targeted vector control interventions. METHODS: Longitudinal entomological monitoring using human landing catches and pyrethrum spray catches was conducted from May to December 2019 in Diourbel, Kaolack, and Touba, the most populous cities in Senegal after the capital Dakar. Additionally, a household survey was conducted in randomly selected houses and residential Koranic schools in the same cities to assess house structures, sleeping spaces, sleeping behavior, and population knowledge about malaria and vector control measures. RESULTS: Of the 8240 Anopheles mosquitoes collected from all the surveyed sites, 99.4% (8,191) were An. gambiae s.l., and predominantly An. arabiensis (99%). A higher number of An. gambiae s.l. were collected in Kaolack (77.7%, n = 6496) than in Diourbel and Touba. The overall mean human biting rate was 14.2 bites per person per night (b/p/n) and was higher outdoors (15.9 b/p/n) than indoors (12.5 b/p/n). The overall mean entomological inoculation rates ranged from 3.7 infectious bites per person per year (ib/p/y) in Diourbel to 40.2 ib/p/y in Kaolack. Low anthropophilic rates were recorded at all sites (average 35.7%). Of the 1202 households surveyed, about 24.3% of household members slept outdoors, except during the short rainy season between July and October, despite understanding how malaria is transmitted and the vector control measures used to prevent it. CONCLUSION: Anopheles arabiensis was the primary malaria vector in the three surveyed cities. The species showed an outdoor biting tendency, which represents a risk for the large proportion of the population sleeping outdoors. As all current vector control measures implemented in the country target endophilic vectors, these data highlight potential gaps in population protection and call for complementary tools and approaches targeting outdoor biting malaria vectors.

Evaluation of the Efficacy of Fludora® Fusion WP-SB 56.25 (Mixture of Clothianidin and Deltamethrin) against Anopheles coluzzii Laboratory and An. arabiensis Wild Colonies.

For malaria control, the application of long-lasting insecticidal nets and indoor residual spraying has led to a significant reduction in morbidity and mortality. However, the sustainability of these gains is hampered by the increase in insecticide resistance. It is therefore judicious to evaluate new insecticide formulations. In comparison to clothianidin and deltamethrin, the efficacy and residual effect of Fludora® Fusion was evaluated using an Anopheles coluzzii laboratory and An. arabiensis wild colonies in huts from August 2016 to June 2017 on cement and mud walls. Mortality was recorded at 24, 48, 72, and 96 h post exposure. Like deltamethrin and clothianidin, Fludora® Fusion showed delayed mortality rates above the WHO's 80% threshold over a period of 11 months with the laboratory strain. With the wild strain, while residual efficacy was observed at 2 months for the three insecticides, no residual efficacy was observed at 8 months at 24 h in both substrates. However, the increased efficacy was observed with increased holding periods (72 h and 96 h). These findings suggest that Fludora® Fusion could be an alternative candidate since this duration covers the transmission period in most areas in Senegal.

Evolution of the Ace-1 and Gste2 Mutations and Their Potential Impact on the Use of Carbamate and Organophosphates in IRS for Controlling Anopheles gambiae s.l., the Major Malaria Mosquito in Senegal.

Widespread of insecticide resistance amongst the species of the Anopheles gambiae complex continues to threaten vector control in Senegal. In this study, we investigated the presence and evolution of the Ace-1 and Gste2 resistance genes in natural populations of Anopheles gambiae s.l., the main malaria vector in Senegal. Using historical samples collected from ten sentinel health districts, this study focused on three different years (2013, 2017, and 2018) marking the periods of shift between the main public health insecticides families (pyrethroids, carbamates, organophosphates) used in IRS to track back the evolutionary history of the resistance mutations on the Ace-1 and Gste2 loci. The results revealed the presence of four members of the Anopheles gambiae complex, with the predominance of An. arabiensis followed by An. gambiae, An. coluzzii, and An. gambiae-coluzzii hybrids. The Ace-1 mutation was only detected in An. gambiae and An. gambiae-coluzzii hybrids at low frequencies varying between 0.006 and 0.02, while the Gste2 mutation was found in all the species with a frequency ranging between 0.02 and 0.25. The Ace-1 and Gste2 genes were highly diversified with twenty-two and thirty-one different haplotypes, respectively. The neutrality tests on each gene indicated a negative Tajima's D, suggesting the abundance of rare alleles. The presence and spread of the Ace-1 and Gste2 resistance mutations represent a serious threat to of the effectiveness and the sustainability of IRS-based interventions using carbamates or organophosphates to manage the widespread pyrethroids resistance in Senegal. These data are of the highest importance to support the NMCP for evidence-based vector control interventions selection and targeting.

Multiple insecticide resistance target sites in adult field strains of An. gambiae (s.l.) from southeastern Senegal.

BACKGROUND: High coverage of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the cornerstones of vector control strategy in Senegal where insecticide resistance by the target vectors species is a great of concern. This study explores insecticide susceptibility profile and target-site mutations mechanisms within the Anopheles gambiae complex in southeastern Senegal. METHODS: Larvae of Anopheles spp. were collected in two sites from southeastern Senegal Kedougou and Wassadou/Badi in October and November 2014, and reared until adult emergence. Wild F0 adult mosquitoes were morphologically identified to species. Susceptibility of 3-5-day-old An. gambiae (s.l.) samples to 11 insecticides belonging to the four insecticide classes was assessed using the WHO insecticide susceptibility bioassays. Tested samples were identified using molecular techniques and insecticide resistance target-site mutations (kdr, ace-1 and rdl) were determined. RESULTS: A total of 3742 An. gambiae (s.l.) were exposed to insecticides (2439 from Kedougou and 1303 from Wassadou-Badi). Tests with pyrethroid insecticides and DDT showed high level of resistance in both Kedougou and Wassadou/Badi. Resistance to pirimiphos-methyl and malathion was not detected while resistance to bendoicarb and fenitrothion was confirmed in Kedougou. Of the 745 specimens of An. gambiae (s.l.) genotyped, An. gambiae (s.s.) (71.6%) was the predominant species, followed by An. arabiensis (21.7%), An. coluzzii (6.3%) and hybrids (An. gambiae (s.s.)/An. coluzzii; 0.4%). All target site mutations investigated (Vgsc-1014F, Vgsc-1014S, Ace-1 and Rdl) were found at different frequencies in the species of the Anopheles gambiae complex. Vgsc-1014F mutation was more frequent in An. gambiae (s.s.) and An. coluzzii than An. arabiensis. Vgsc-1014S was present in An. gambiae (s.l.) populations in Wassadou but not in Kedougou. Ace-1 and rdl mutations were more frequent in An. gambiae (s.s.) in comparison to An. arabiensis and An. coluzzii. CONCLUSIONS: Resistance to all the four insecticide classes tested was detected in southeastern Senegal as well as all target site mutations investigated were found. Data will be used by the national Malaria Control Programme.

Insecticide resistance in Anopheles arabiensis populations from Dakar and its suburbs: role of target site and metabolic resistance mechanisms.

BACKGROUND: Urban malaria is an increasing concern in most of the sub-Saharan Africa countries. In Dakar, the capital city of Senegal, the malaria epidemiology has been complicated by recurrent flooding since 2005. The main vector control measure for malaria prevention in Dakar is the community use of long-lasting insecticide-treated nets. However, the increase of insecticide resistance reported in this area needs to be better understood for suitable resistance management. This study reports the situation of insecticide resistance and underlying mechanisms in Anopheles arabiensis populations from Dakar and its suburbs. RESULTS: All the populations tested showed resistance to almost all insecticides except organophosphates families, which remain the only lethal molecules. Piperonil butoxide (PBO) and ethacrinic acid (EA) the two synergists used, have respectively and significantly restored the susceptibility to DDT and permethrin of Anopheles population. Molecular identification of specimens revealed the presence of An. arabiensis only. Kdr genotyping showed the presence of the L1014F mutation (kdr-West) as well as L1014S (kdr-East). This L1014S mutation was found at very high frequencies (89.53%) in almost all districts surveyed, and in association with the L1014F (10.24%). CONCLUSION: Results showed the contribution of both target-site and metabolic mechanisms in conferring pyrethroid resistance to An. arabiensis from the flooded areas of Dakar suburbs. These data, although preliminary, stress the need for close monitoring of the urban An. arabiensis populations for a suitable insecticide resistance management system to preserve core insecticide-based vector control tools in this flooded area.

Randomized Trials Comparing Inactivated Vaccine After Medium- or High-titer Measles Vaccine With Standard Titer Measles Vaccine After Inactivated Vaccine: A Meta-analysis.

BACKGROUND: Observational studies have suggested that girls have higher mortality if their most recent immunization is an inactivated vaccine rather than a live vaccine. We therefore reanalyzed 5 randomized trials of early measles vaccine (MV) in which it was possible to compare an inactivated vaccines [after medium-titer MV (MTMV) or high-titer MV (HTMV)] and a live standard titer MV (after an initial inactivated vaccine). METHODS: The trials were conducted in Sudan, Senegal, The Gambia and Guinea-Bissau. The intervention group received live MTMV or HTMV from 4 to 5 months and then an inactivated vaccine from 9 to 10 months of age; the control children received inactivated vaccine/placebo from 4 to 5 months and standard titer MV from 9 to 10 months of age. We compared mortality from 9 months until end of study at 3 to 5 years of age for children who received inactivated vaccine (after MTMV or HTMV) and standard titer MV (after inactivated vaccine), respectively. The original datasets were analyzed using a Cox proportional hazards model stratified by trial. RESULTS: The mortality rate ratio (MRR) was 1.38 (95% confidence interval: 1.05-1.83) after an inactivated vaccine (after MTMV or HTMV) compared with a standard titer MV (after inactivated vaccine). Girls had a MRR of 1.89 (1.27-2.80), whereas there was no effect for boys, the sex-differential effect being significant (P = 0.02). Excluding measles cases did not alter these conclusions, the MRR after inactivated vaccines (after MTMV or HTMV) being 1.40 (1.06-1.86) higher overall and 1.92 (1.29-2.86) for girls. Control for variations in national immunization schedules for other vaccines did not modify these results. CONCLUSIONS: After 9 months of age, all children had been immunized against measles, and mortality in girls was higher when they had received inactivated vaccines (after MTMV or HTMV) rather than live standard titer MV (after an inactivated vaccine).

Investigating molecular basis of lambda-cyhalothrin resistance in an Anopheles funestus population from Senegal.

BACKGROUND: Anopheles funestus is one of the major malaria vectors in tropical Africa, notably in Senegal. The highly anthropophilic and endophilic behaviours of this mosquito make it a good target for vector control operations through the use of insecticide treated nets, long-lasting insecticide nets and indoor residual spraying. However, little is known about patterns of resistance to insecticides and the underlying resistance mechanisms in field populations of this vector in Senegal. METHODS: Here, we assessed the susceptibility status of An. funestus populations from Gankette Balla, located in northern Senegal and investigated the potential resistance mechanisms. RESULTS: WHO bioassays indicated that An. funestus is resistant to lambda-cyhalothrin 0.05 % (74.64 % mortality), DDT 4 % (83.36 % mortality) and deltamethrin 0.05 % (88.53 % mortality). Suspected resistance was observed to permethrin 0.75 % (91.19 % mortality), bendiocarb 0.1 % (94.13 % mortality) and dieldrin 4 % (96.41 % mortality). However, this population is fully susceptible to malathion 5 % (100 % mortality) and fenitrothion 1 % (100 % mortality). The microarray and qRT-PCR analysis indicated that the lambda-cyhalothrin resistance in Gankette Balla is conferred by metabolic resistance mechanisms under the probable control of cytochrome P450 genes among which CYP6M7 is the most overexpressed. The absence of overexpression of the P450 gene, CYP6P9a, indicates that the resistance mechanism in Senegal is different to that observed in southern Africa. CONCLUSIONS: This study represents the first report of pyrethroid and DDT resistance in An. funestus from Senegal and shows that resistance to insecticides is not only confined to An. gambiae as previously thought. Therefore, urgent action should be taken to manage the resistance in this species to ensure the continued effectiveness of malaria control.