Copy number variants underlie the major selective sweeps in insecticide resistance genes in Anopheles arabiensis from Tanzania.

To keep ahead of the evolution of resistance to insecticides in mosquitoes, national malaria control programmes must make use of a range of insecticides, both old and new, while monitoring resistance mechanisms. Knowledge of the mechanisms of resistance remains limited in Anopheles arabiensis, which in many parts of Africa is of increasing importance because it is apparently less susceptible to many indoor control interventions. Furthermore, comparatively little is known in general about resistance to non-pyrethroid insecticides such as pirimiphos-methyl (PM), which are crucial for effective control in the context of resistance to pyrethroids. We performed a genome-wide association study to determine the molecular mechanisms of resistance to deltamethrin (commonly used in bednets) and PM, in An. arabiensis from two regions in Tanzania. Genomic regions of positive selection in these populations were largely driven by copy number variants (CNVs) in gene families involved in resistance to these two insecticides. We found evidence of a new gene cluster involved in resistance to PM, identifying a strong selective sweep tied to a CNV in the Coeae2g-Coeae6g cluster of carboxylesterase genes. Using complementary data from An. coluzzii in Ghana, we show that copy number at this locus is significantly associated with PM resistance. Similarly, for deltamethrin, resistance was strongly associated with a novel CNV allele in the Cyp6aa / Cyp6p cluster. Against this background of metabolic resistance, target site resistance was very rare or absent for both insecticides. Mutations in the pyrethroid target site Vgsc were at very low frequency in Tanzania, yet combining these samples with three An. arabiensis individuals from West Africa revealed a startling diversity of evolutionary origins of target site resistance, with up to 5 independent origins of Vgsc-995 mutations found within just 8 haplotypes. Thus, despite having been first recorded over 10 years ago, Vgsc resistance mutations in Tanzanian An. arabiensis have remained at stable low frequencies. Overall, our results provide a new copy number marker for monitoring resistance to PM in malaria mosquitoes, and reveal the complex picture of resistance patterns in An. arabiensis.

Dynamics of malaria vector composition and Plasmodium falciparum infection in mainland Tanzania: 2017-2021 data from the national malaria vector entomological surveillance.

BACKGROUND: In 2015, Tanzania National Malaria Control Programme (NMCP) established a longitudinal malaria vector entomological surveillance (MVES). The MVES is aimed at a periodical assessment of malaria vector composition and abundance, feeding and resting behaviours, and Plasmodium falciparum infection in different malaria epidemiological strata to guide the NMCP on the deployment of appropriate malaria vector interventions. This work details the dynamics of malaria vector composition and transmission in different malaria epidemiological strata. METHODS: The MVES was conducted from 32 sentinel district councils across the country. Mosquitoes were collected by the trained community members and supervised by the NMCP and research institutions. Three consecutive night catches (indoor collection with CDC light trap and indoor/outdoor collection using bucket traps) were conducted monthly in three different households selected randomly from two to three wards within each district council. Collected mosquitoes were sorted and morphologically identified in the field. Thereafter, the samples were sent to the laboratory for molecular characterization using qPCR for species identification and detection of P. falciparum infections (sporozoites). ELISA technique was deployed for blood meal analysis from samples of blood-fed mosquitoes to determine the blood meal indices (BMI). RESULTS: A total of 63,226 mosquitoes were collected in 32 district councils from January 2017 to December 2021. Out of which, 39,279 (62%), 20,983 (33%) and 2964 (5%) were morphologically identified as Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l., and as other Anopheles species, respectively. Out of 28,795 laboratory amplified mosquitoes, 13,645 (47%) were confirmed to be Anopheles arabiensis, 9904 (34%) as An. funestus sensu stricto (s.s.), and 5193 (19%) as An. gambiae s.s. The combined average entomological inoculation rates (EIR) were 0.46 (95% CI 0.028-0.928) for An. gambiae s.s., 0.836 (95% CI 0.138-1.559) for An. arabiensis, and 0.58 (95% CI 0.165-0.971) for An. funestus s.s. with variations across different malaria transmission strata. Anopheles funestus s.s. and An. arabiensis were predominant in the Lake and South-Eastern zones, respectively, mostly in high malaria transmission areas. Monthly mosquito densities displayed seasonal patterns, with two peaks following the rainy seasons, varying slightly across species and district councils. CONCLUSION: Anopheles arabiensis remains the predominant vector species followed by An. funestus s.s. in the country. Therefore, strengthening integrated vector management including larval source management is recommended to address outdoor transmission by An. arabiensis to interrupt transmission particularly where EIR is greater than the required elimination threshold of less than one (< 1) to substantially reduce the prevalence of malaria infection.

Community knowledge, attitude, practices and beliefs associated with persistence of malaria transmission in North-western and Southern regions of Tanzania.

BACKGROUND: Despite significant decline in the past two decades, malaria is still a major public health concern in Tanzania; with over 93% of the population still at risk. Community knowledge, attitudes and practices (KAP), and beliefs are key in enhancing uptake and utilization of malaria control interventions, but there is a lack of information on their contribution to effective control of the disease. This study was undertaken to determine KAP and beliefs of community members and service providers on malaria, and how they might be associated with increased risk and persistence of the disease burden in North-western and Southern regions of Tanzania. METHODS: This was an exploratory study that used qualitative methods including 16 in-depth interviews (IDI) and 32 focus group discussions (FGDs) to collect data from health service providers and community members, respectively. The study was conducted from September to October 2017 and covered 16 villages within eight districts from four regions of mainland Tanzania (Geita, Kigoma, Mtwara and Ruvuma) with persistently high malaria transmission for more than two decades. RESULTS: Most of the participants had good knowledge of malaria and how it is transmitted but some FGD participants did not know the actual cause of malaria, and thought that it is caused by bathing and drinking un-boiled water, or consuming contaminated food that has malaria parasites without warming it. Reported barriers to malaria prevention and control (by FGD and IDI participants) included shortage of qualified health workers, inefficient health financing, low care-seeking behaviour, consulting traditional healers, use of local herbs to treat malaria, poverty, increased breeding sites by socio-economic activities and misconceptions related to the use of bed nets and indoor residual spraying (IRS). Among the misconceptions, some participants believed that bed nets provided for free by the government came with bedbugs while others reported that free bed nets caused impotence among men. CONCLUSION: Despite good knowledge of malaria, several risk factors, such as socio-economic and behavioural issues, and misconceptions related to the use of bed nets and IRS were reported. Other key factors included unavailability or limited access to health services, poor health financing and economic activities that potentially contributed to persistence of malaria burden in these regions. Relevant policies and targeted malaria interventions, focusing on understanding socio-cultural factors, should be implemented to reduce and finally eliminate the disease in the study regions and others with persistent transmission.

Trends of insecticide resistance monitoring in mainland Tanzania, 2004-2020.

BACKGROUND: Insecticide resistance is a serious threat to the continued effectiveness of insecticide-based malaria vector control measures, such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). This paper describes trends and dynamics of insecticide resistance and its underlying mechanisms from annual resistance monitoring surveys on Anopheles gambiae sensu lato (s.l.) populations conducted across mainland Tanzania from 2004 to 2020. METHODS: The World Health Organization (WHO) standard protocols were used to assess susceptibility of the wild female An. gambiae s.l. mosquitoes to insecticides, with mosquitoes exposed to diagnostic concentrations of permethrin, deltamethrin, lambdacyhalothrin, bendiocarb, and pirimiphos-methyl. WHO test papers at 5× and 10× the diagnostic concentrations were used to assess the intensity of resistance to pyrethroids; synergist tests using piperonyl butoxide (PBO) were carried out in sites where mosquitoes were found to be resistant to pyrethroids. To estimate insecticide resistance trends from 2004 to 2020, percentage mortalities from each site and time point were aggregated and regression analysis of mortality versus the Julian dates of bioassays was performed. RESULTS: Percentage of sites with pyrethroid resistance increased from 0% in 2004 to more than 80% in the 2020, suggesting resistance has been spreading geographically. Results indicate a strong negative association (p = 0.0001) between pyrethroids susceptibility status and survey year. The regression model shows that by 2020 over 40% of An. gambiae mosquitoes survived exposure to pyrethroids at their respective diagnostic doses. A decreasing trend of An. gambiae susceptibility to bendiocarb was observed over time, but this was not statistically significant (p = 0.8413). Anopheles gambiae exhibited high level of susceptibility to the pirimiphos-methyl in sampled sites. CONCLUSIONS: Anopheles gambiae Tanzania's major malaria vector, is now resistant to pyrethroids across the country with resistance increasing in prevalence and intensity and has been spreading geographically. This calls for urgent action for efficient malaria vector control tools to sustain the gains obtained in malaria control. Strengthening insecticide resistance monitoring is important for its management through evidence generation for effective malaria vector control decision.

Laboratory and semi-field evaluation of the efficacy of Bacillus thuringiensis var. israelensis (Bactivec®) and Bacillus sphaericus (Griselesf®) for control of mosquito vectors in northeastern Tanzania.

Bacterial larvicides Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) have been used extensively for mosquito control. However, their efficacy varies greatly mainly due to factors related to target mosquitoes, larval habitat conditions, and inherent larvicide properties. We evaluated the efficacy of Bti (Bactivec®) and Bs (Griselesf®) for control of Anopheles gambiae complex, Culex quinquefasciatus and Aedes aegypti larvae under laboratory and semi-field conditions in northeastern Tanzania. Laboratory bioassays were conducted with five to six different concentrations of Bti and Bs, replicated four times and the experiment repeated on three different days. Larvae mortality was recorded at 24 or 48 h after the application of larvicide and subjected to Probit analysis. Laboratory bioassays were followed by semi-field trials to establish initial and residual activity of Bti and Bs. Semi-field trials were conducted in artificial larval habitats in the open sunlit ground and in "mosquito spheres". These artificial larval habitats were colonized with mosquito larvae, treated with Bti and Bs, and the impact of treatments on mosquito larvae was monitored daily. Lethal concentration values that caused 50% and 95% mortalities of test larvae (LC50 and LC95) showed that An. gambiae complex and Cx. quinquefasciatus tested were highly susceptible to Bti and Bs under laboratory conditions. Likewise, larvae of Ae. aegypti were highly susceptible to Bti, with LC95 value as low as 0.052 mg/l. However, Ae. aegypti larvae were not susceptible to Bs under practical doses of laboratory settings. In semi-field trials, all treatment dosages for Bti provided 91.0-100% larval mortality within 24 h whereas Bs resulted in 96.8-100% larval mortality within the same time-frame. Bs had a more prolonged residual activity, with pupal reductions range of 55.7-100% for 9 days at all application rates while the corresponding pupal reduction with Bti was 15.4-100% for 5 days. Due to the low residual activity of Bti and Bs tested, weekly application at a maximum label rate would be appropriate to reduce mosquito larvae in natural larval habitats. Based on laboratory findings, Bs product tested would not be recommended for use in the control of Ae. aegypti.

Lymphatic filariasis transmission on Mafia Islands, Tanzania: Evidence from xenomonitoring in mosquito vectors.

INTRODUCTION: Lymphatic filariasis (LF) is a chronic nematode infection transmitted by mosquitoes and in sub-Saharan Africa it is caused by Wuchereria bancrofti. The disease was targeted for global elimination by 2020 using repeated community-wide mass drug administration (MDA) distributed in endemic areas. However, recently, there has been a growing recognition of the potential role of including vector control as a supplement to MDA to achieve elimination goal. This study was carried out to determine mosquito abundance and transmission of bancroftian filariasis on Mafia Islands in Tanzania as a prerequisite for a search for appropriate vector control methods to complement the ongoing MDA campaign. METHODS: Mosquitoes were collected indoor and outdoor using Centre for Disease Control (CDC) light and gravid traps, respectively. Collected mosquitoes were identified based on their differential morphological features and Anopheles gambiae complex and An. funestus group were further identified to their respective sibling species by polymerase chain reaction (PCR). Filarial mosquito vectors were then examined for infection with Wuchereria bancrofti by microscopy and PCR technique. RESULTS: Overall, a total of 35,534 filarial mosquito vectors were collected, of which Anopheles gambiae complex, An. funestus group and Culex quinquefasciatus Say accounted for 1.3, 0.5 and 98.2%, respectively. Based on PCR identification, An. gambiae sensu stricto (s.s) and An. funestus s.s sibling species accounted for 88.3% and 99.1% of the identified members of the An. gambiae complex and An. funestus group, respectively. A total of 7,936 mosquitoes were examined for infection with W. bancrofti by microscopy. The infection and infectivity rates were 0.25% and 0.08%, respectively. Using pool screen PCR technique, analysis of 324 mosquito pools (each with 25 mosquitoes) resulted to an estimated infection rate of 1.7%. CONCLUSION: The study has shown that Cx. quinquefasciatus is the dominant mosquito on Mafia Islands. By using mosquito infectivity as proxy to human infection, the study indicates that W. bancrofti transmission is still ongoing on Mafia Islands after more than a decade of control activities based on MDA.

Experimental hut evaluation of a novel long-lasting non-pyrethroid durable wall lining for control of pyrethroid-resistant Anopheles gambiae and Anopheles funestus in Tanzania.

BACKGROUND: A novel, insecticide-treated, durable wall lining (ITWL), which mimics indoor residual spraying (IRS), has been developed to provide prolonged vector control when fixed to the inner walls of houses. PermaNet® ITWL is a polypropylene material containing non-pyrethroids (abamectin and fenpyroximate) which migrate gradually to the surface. METHODS: An experimental hut trial was conducted in an area of pyrethroid-resistant Anopheles gambiae s.l. and Anopheles funestus s.s. to compare the efficacy of non-pyrethroid ITWL, long-lasting insecticidal nets (LLIN) (Interceptor®), pyrethroid ITWL (ZeroVector®), and non-pyrethroid ITWL + LLIN. RESULTS: The non-pyrethroid ITWL produced relatively low levels of mortality, between 40-50% for An. funestus and An. gambiae, across all treatments. Against An. funestus, the non-pyrethroid ITWL when used without LLIN produced 47% mortality but this level of mortality was not significantly different to that of the LLIN alone (29%, P = 0.306) or ITWL + LLIN (35%, P = 0.385). Mortality levels for An. gambiae were similar to An. funestus with non-pyrethroid ITWL, producing 43% mortality compared with 26% for the LLIN. Exiting rates from ITWL huts were similar to the control and highest when the LLIN was present. An attempt to restrict mosquito access by covering the eave gap with ITWL (one eave open vs four open) had no effect on numbers entering. The LLIN provided personal protection when added to the ITWL with only 30% blood-fed compared with 69 and 56% (P = 0.001) for ITWL alone. Cone bioassays on ITWL with 30 min exposure after the trial produced mortality of >90% using field An. gambiae. CONCLUSIONS: Despite high mortality in bioassays, the hut trial produced only limited mortality which was attributed to pyrethroid resistance against the pyrethroid ITWL and low efficacy in the non-pyrethroid ITWL. Hut ceilings were left uncovered and may have served as a potential untreated refuge. By analogy to IRS campaigns, which also do not routinely treat ceilings, high community coverage with ITWL may still reduce malaria transmission. Restriction of eave gaps by 75% proved an inadequate barrier to mosquito entry. The findings represent the first 2 months after installation and do not necessarily predict long-term efficacy.

Evaluation of collection methods for Culex quinquefasciatus, Aedes aegypti, and Aedes simpsoni in northeastern Tanzania.

In East Africa, significant morbidity and mortality are caused by infections spread by Culex quinquefasciatus and Aedes aegypti. Sticky traps have been shown to be effective tools for sampling populations of Aedes mosquitoes and have been found to catch Cx. quinquefasciatus. Thus, they could potentially be used to sample populations of this species. This study compared Sticky ovitraps (SO) and MosquiTraps (MQT) with the CDC Gravid trap (CDC-GT) for collection of Culex and Aedes mosquito populations in Tanzania. A follow-up experiment was carried out using traps set for a 24-h period to accommodate the oviposition habits of Aedes aegypti and Ae. simpsoni s.l. mosquitoes. The results showed that the CDC-GT caught significantly more Cx. quinquefasciatus and Ae. aegypti than the SO or MQT, but there was no significant difference in the number of mosquitoes caught between the two sticky traps or of Ae. simpsoni s.l. caught among the three trap types. The results suggest that CDC-GTs are the most appropriate in sampling of Cx. quinquefasciatus. Although CDC-GTs collected more Ae. aegypti than the sticky traps, the simplicity and cost benefit of sticky traps facilitates large scale studies. All three trap types should be considered for monitoring Aedes mosquitoes.

The effectiveness of non-pyrethroid insecticide-treated durable wall lining to control malaria in rural Tanzania: study protocol for a two-armed cluster randomized trial.

BACKGROUND: Despite considerable reductions in malaria achieved by scaling-up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), maintaining sustained community protection remains operationally challenging. Increasing insecticide resistance also threatens to jeopardize the future of both strategies. Non-pyrethroid insecticide-treated wall lining (ITWL) may represent an alternate or complementary control method and a potential tool to manage insecticide resistance. To date no study has demonstrated whether ITWL can reduce malaria transmission nor provide additional protection beyond the current best practice of universal coverage (UC) of LLINs and prompt case management. METHODS/DESIGN: A two-arm cluster randomized controlled trial will be conducted in rural Tanzania to assess whether non-pyrethroid ITWL and UC of LLINs provide added protection against malaria infection in children, compared to UC of LLINs alone. Stratified randomization based on malaria prevalence will be used to select 22 village clusters per arm. All 44 clusters will receive LLINs and half will also have ITWL installed on interior house walls. Study children, aged 6 months to 11 years old, will be enrolled from each cluster and followed monthly to estimate cumulative incidence of malaria parasitaemia (primary endpoint), time to first malaria episode and prevalence of anaemia before and after intervention. Entomological inoculation rate will be estimated using indoor CDC light traps and outdoor tent traps followed by detection of Anopheles gambiae species, sporozoite infection, insecticide resistance and blood meal source. ITWL bioefficacy and durability will be monitored using WHO cone bioassays and household surveys, respectively. Social and cultural factors influencing community and household ITWL acceptability will be explored through focus-group discussions and in-depth interviews. Cost-effectiveness, compared between study arms, will be estimated per malaria case averted. DISCUSSION: This protocol describes the large-scale evaluation of a novel vector control product, designed to overcome some of the known limitations of existing methods. If ITWL is proven to be effective and durable under field conditions, it may warrant consideration for programmatic implementation, particularly in areas with long transmission seasons and where pyrethroid-resistant vectors predominate. Trial findings will provide crucial information for policy makers in Tanzania and other malaria-endemic countries to guide resource allocations for future control efforts. TRIAL REGISTRATION: NCT02533336 registered on 13 July 2014.

Insecticide resistance mechanisms associated with different environments in the malaria vector Anopheles gambiae: a case study in Tanzania.

BACKGROUND: Resistance of mosquitoes to insecticides is a growing concern in Africa. Since only a few insecticides are used for public health and limited development of new molecules is expected in the next decade, maintaining the efficacy of control programmes mostly relies on resistance management strategies. Developing such strategies requires a deep understanding of factors influencing resistance together with characterizing the mechanisms involved. Among factors likely to influence insecticide resistance in mosquitoes, agriculture and urbanization have been implicated but rarely studied in detail. The present study aimed at comparing insecticide resistance levels and associated mechanisms across multiple Anopheles gambiae sensu lato populations from different environments. METHODS: Nine populations were sampled in three areas of Tanzania showing contrasting agriculture activity, urbanization and usage of insecticides for vector control. Insecticide resistance levels were measured in larvae and adults through bioassays with deltamethrin, DDT and bendiocarb. The distribution of An. gambiae sub-species and pyrethroid target-site mutations (kdr) were investigated using molecular assays. A microarray approach was used for identifying transcription level variations associated to different environments and insecticide resistance. RESULTS: Elevated resistance levels to deltamethrin and DDT were identified in agriculture and urban areas as compared to the susceptible strain Kisumu. A significant correlation was found between adult deltamethrin resistance and agriculture activity. The subspecies Anopheles arabiensis was predominant with only few An. gambiae sensu stricto identified in the urban area of Dar es Salaam. The L1014S kdr mutation was detected at elevated frequency in An gambiae s.s. in the urban area but remains sporadic in An. arabiensis specimens. Microarrays identified 416 transcripts differentially expressed in any area versus the susceptible reference strain and supported the impact of agriculture on resistance mechanisms with multiple genes encoding pesticide targets, detoxification enzymes and proteins linked to neurotransmitter activity affected. In contrast, resistance mechanisms found in the urban area appeared more specific and more related to the use of insecticides for vector control. CONCLUSIONS: Overall, this study confirmed the role of the environment in shaping insecticide resistance in mosquitoes with a major impact of agriculture activities. Results are discussed in relation to resistance mechanisms and the optimization of resistance management strategies.