\ud Detection and Monitoring of Insecticide Resistance in Malaria Vectors in Tanzania Mainland\ud

\ud Vector control is a major component of the global strategy for malaria control which aims to prevent parasite transmission mainly through interventions targeting adult Anopheline vectors. Insecticide treated nets (ITNs) and indoor residual spraying (IRS) are the cornerstone of malaria vector control programmes. These major interventions in most cases use pyrethroid insecticides which are also used for agricultural purposes. With widespread development of resistance to pyrethroid insecticides in malaria vectors raises concern over the sustainability of insecticide-based interventions for malaria control. Therefore, close monitoring of performance of the insecticides against malaria vectors is essential for early detection and\ud management of resistance. To measure pyrethroid susceptibility in populations of malaria vectors in Tanzania and to test the efficacy of LLINs/ITNs and insecticide residues on sprayed wall substrates in the IRS operation areas. In 2011 the National Institute for Medical Research (NIMR) in collaboration with National Malaria Control Programme (NMCP) conducted large scale surveillance to determine the countrywide susceptibility levels of malaria vectors to insecticides used for both public health and agricultural purposes. Anopheles gambiae Giles s.l. were collected during national surveys and samples of LLINs/ITNs in the 14 sentinel sites and houses from the IRS areas were randomly selected for bioassays to test the efficacy and insecticide residual effects on sprayed wall substrates respectively. Wild adult mosquitoes for susceptibility testing were collected by resting catches indoors. Net traps (outdoors and indoors) were set up to enhance catches. WHO Susceptibility kits were used to test for resistance status using test papers: Lambdacyhalothrin 0.05%, Deltamethrin 0.05%, Permethrin 0.75%, DDT 4%, Propoxur 0.1% and Fenitrothion 1%. The quality of the test paper was checked against a laboratory susceptible An. gambiae Kisumu strain. Knockdown effect and mortality were measured in standard WHO susceptibility tests and cone bio-efficacy tests. Whereas, con bioassays on treated walls and ITNs were conducted using the laboratory susceptible An. gambiae Kisumu strain. The results from the surveillance recorded continued susceptibility of malaria vectors to commonly used insecticides. However, there were some isolated cases of resistance and/or reduced susceptibility to pyrethroid insecticides which may not compromise the current vector control interventions in the country. Anopheles gambiae s.l. showed resistance (15-28%) to each of the pyrethroids and to DDT but not to Organophosphates (Propoxur 0.1%), and Carbamates (Fenitrothion 1%). The information obtained from this surveillance is expected to be used to guide the National Malaria Control Programme on the rational selection of insecticides for malaria vector control and for the national mitigation plans for management and containment of malaria vector resistance in the country. The current observation warrants more vigilant monitoring of the susceptibility of malaria mosquitoes to commonly used insecticides in areas found with resistance and/or reduced levels of susceptibility of malaria vectors to insecticides, particularly in areas with heavy agricultural and/or public health use of insecticides where resistance is likely to develop. The current survey covered malaria vectors only and not the non malaria vectors (nuisance) mosquitoes such as Culex. Similar monitoring of insecticide susceptibility of this non malaria vectors may be needed to ensure public motivation for sustained use of ITNs/LLINs in the country. The surveillance leading to these results received funding from PMI/USAID through RTI International with Sub Agreement Number 33300212555.\u

Genetic Sequence Variation in the Plasmodium falciparum Histidine-Rich Protein 2 Gene from Field Isolates in Tanzania: Impact on Malaria Rapid Diagnosis

Malaria rapid diagnosis test (RDT) is crucial for managing the disease, and the effectiveness of detection depends on parameters such as sensitivity and specificity of the RDT. Several factors can affect the performance of RDT. In this study, we focused on the pfhrp2 sequence variation and its impact on RDTs targeted by antigens encoded by Plasmodium falciparum histidine-rich protein 2 (pfhrp2). Field samples collected during cross-sectional surveys in Tanzania were sequenced to investigate the pfhrp2 sequence diversity and evaluate the impact on HRP2-based RDT performance. We observed significant mean differences in amino acid repeats between current and previous studies. Several new amino acid repeats were found to occur at different frequencies, including types AAY, AHHAHHAAN, and AHHAA. Based on the abundance of types 2 and 7 amino acid repeats, the binary predictive model was able to predict RDT insensitivity by about 69% in the study area. About 85% of the major epitopes targeted by monoclonal antibodies (MAbs) in RDT were identified. Our study suggested that the extensive sequence variation in pfhrp2 can contribute to reduced RDT sensitivity. The correlation between the different combinations of amino acid repeats and the performance of RDT in different malaria transmission settings should be investigated further

Dramatic decreases of malaria transmission intensities in Ifakara, south-eastern Tanzania since early 2000s.

BACKGROUND: Ongoing epidemiological transitions across Africa are particularly evident in fast-growing towns, such as Ifakara in the Kilombero valley, south-eastern Tanzania. This town and its environs (population ~ 70,000) historically experienced moderate to high malaria transmission, mediated mostly by Anopheles gambiae and Anopheles funestus. In early 2000s, malaria transmission [Plasmodium falciparum entomological inoculation rate (PfEIR)] was estimated at ~ 30 infectious bites/person/year (ib/p/yr). This study assessed the PfEIR after 15 years, during which there had been rapid urbanization and expanded use of insecticide-treated nets (ITNs). METHODS: Randomly-selected 110 households were sampled across Ifakara town and four adjacent wards. Mosquitoes were trapped nightly or monthly (June.2015-May.2016) using CDC-light-traps indoors, Suna® traps outdoors and human landing catches (HLC) indoors and outdoors. All Anopheles mosquitoes were morphologically identified and analysed by ELISA for Plasmodium circumsporozoite proteins. Mosquito blood meals were identified using ELISA, and sub-samples of An. gambiae and An. funestus examined by PCR to distinguish morphologically-similar siblings. Insecticide resistance was assessed using WHO-susceptibility assays, and some Anopheles were dissected to examine ovariole tracheoles for parity. RESULTS: After 3572 trap-nights, one Plasmodium-infected Anopheles was found (an An. funestus caught outdoors in Katindiuka-ward by HLC), resulting in overall PfEIR of 0.102 ib/p/yr. Nearly 80% of malaria vectors were from Katindiuka and Mlabani wards. Anopheles gambiae densities were higher outdoors (64%) than indoors (36%), but no such difference was observed for An. funestus. All An. funestus and 75% of An. gambiae dissected were parous. Anopheles gambiae complex consisted entirely of Anopheles arabiensis, while An. funestus included 84.2% An. funestus s.s., 4.5% Anopheles rivulorum, 1.4% Anopheles leesoni and 9.9% with unamplified-DNA. Anopheles gambiae were susceptible to bendiocarb and malathion, but resistant to pyrethroids, DDT and pirimiphos-methyl. Most houses had brick walls and/or iron roofs (> 90%), and 52% had screened windows. CONCLUSION: Malaria transmission in Ifakara has decreased by > 99% since early-2000s, reaching levels nearly undetectable with current entomological methods. These declines are likely associated with ITNs use, urbanization and improved housing. Remaining risk is now mostly in peri-urban wards, but concerted efforts could further decrease local transmission. Parasitological surveys are required to assess actual prevalence, incidence and importation rates

Deletions of the Plasmodium falciparum histidine-rich protein 2/3 genes are common in field isolates from north-eastern Tanzania.

Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3 (pfhrp2/3) genes have been reported in several parts of the world. These deletions are known to compromise the effectiveness of HRP2-based malaria rapid diagnostic tests (HRP2-RDT). The National Malaria Control Programme (NMCP) in Tanzania adopted HRP2-RDTs as a routine tool for malaria diagnosis in 2009 replacing microscopy in many Health facilities. We investigated pfhrp2/3 deletions in 122 samples from two areas with diverse malaria transmission intensities in Northeastern Tanzania. Pfhrp2 deletion was confirmed in 1.6% of samples while pfhrp3 deletion was confirmed in 50% of samples. We did not find parasites with both pfhrp2 and pfhrp3 deletions among our samples. Results from this study highlight the need for systematic surveillance of pfhrp2/3 deletions in Tanzania to understand their prevalence and determine their impact on the performance of mRDT

Using a New Odour-Baited Device to Explore Options for Luring and Killing Outdoor-Biting Malaria Vectors: A Report on Design and Field Evaluation of the Mosquito Landing Box.

Mosquitoes that bite people outdoors can sustain malaria transmission even where effective indoor interventions such as bednets or indoor residual spraying are already widely used. Outdoor tools may therefore complement current indoor measures and improve control. We developed and evaluated a prototype mosquito control device, the 'Mosquito Landing Box' (MLB), which is baited with human odours and treated with mosquitocidal agents. The findings are used to explore technical options and challenges relevant to luring and killing outdoor-biting malaria vectors in endemic settings. Field experiments were conducted in Tanzania to assess if wild host-seeking mosquitoes 1) visited the MLBs, 2) stayed long or left shortly after arrival at the device, 3) visited the devices at times when humans were also outdoors, and 4) could be killed by contaminants applied on the devices. Odours suctioned from volunteer-occupied tents were also evaluated as a potential low-cost bait, by comparing baited and unbaited MLBs. There were significantly more Anopheles arabiensis, An. funestus, Culex and Mansonia mosquitoes visiting baited MLB than unbaited controls (P<=0.028). Increasing sampling frequency from every 120 min to 60 and 30 min led to an increase in vector catches of up to 3.6 fold (P<=0.002), indicating that many mosquitoes visited the device but left shortly afterwards. Outdoor host-seeking activity of malaria vectors peaked between 7:30 and 10:30pm, and between 4:30 and 6:00am, matching durations when locals were also outdoors. Maximum mortality of mosquitoes visiting MLBs sprayed or painted with formulations of candidate mosquitocidal agent (pirimiphos-methyl) was 51%. Odours from volunteer occupied tents attracted significantly more mosquitoes to MLBs than controls (P<0.001). While odour-baited devices such as the MLBs clearly have potential against outdoor-biting mosquitoes in communities where LLINs are used, candidate contaminants must be those that are effective at ultra-low doses even after short contact periods, since important vector species such as An. arabiensis make only brief visits to such devices. Natural human odours suctioned from occupied dwellings could constitute affordable sources of attractants to supplement odour baits for the devices. The killing agents used should be environmentally safe, long lasting, and have different modes of action (other than pyrethroids as used on LLINs), to curb the risk of physiological insecticide resistance

Indoor application of attractive toxic sugar bait (ATSB) in combination with mosquito nets for control of pyrethroid-resistant mosquitoes.

BACKGROUND: Attractive toxic sugar bait (ATSB) sprayed onto vegetation has been successful in controlling Anopheles mosquitoes outdoors. Indoor application of ATSB has yet to be explored. The purpose of this study was to determine whether ATSB stations positioned indoors have the potential to kill host-seeking mosquitoes and constitute a new approach to control of mosquito-borne diseases. METHODS: Insecticides were mixed with dyed sugar solution and tested as toxic baits against Anopheles arabiensis, An. Gambiae s.s. and Culex quinquefasciatus in feeding bioassay tests to identify suitable attractant-insecticide combinations. The most promising ATSB candidates were then trialed in experimental huts in Moshi, Tanzania. ATSB stations were hung in huts next to untreated mosquito nets occupied by human volunteers. The proportions of mosquitoes killed in huts with ATSB treatments relative to huts with non-insecticide control treatments huts were recorded, noting evidence of dye in mosquito abdomens. RESULTS: In feeding bioassays, chlorfenapyr 0.5% v/v, boric acid 2% w/v, and tolfenpyrad 1% v/v, mixed in a guava juice-based bait, each killed more than 90% of pyrethroid-susceptible An. Gambiae s.s. and pyrethroid-resistant An. arabiensis and Cx. quinquefasciatus. In the hut trial, mortality rates of the three ATSB treatments ranged from 41-48% against An. arabiensis and 36-43% against Cx. quinquefasciatus and all were significantly greater than the control mortalities: 18% for An. arabiensis, 7% for Cx. quinquefasciatus (p<0.05). Mortality rates with ATSB were comparable to those with long lasting insecticidal nets previously tested against the same species in this area. CONCLUSIONS: Indoor ATSB shows promise as a supplement to mosquito nets for controlling mosquitoes. Indoor ATSB constitute a novel application method for insecticide classes that act as stomach poisons and have not hitherto been exploited for mosquito control. Combined with LLIN, indoor use of ATSB has the potential to serve as a strategy for managing insecticide resistance

Durability associated efficacy of long-lasting insecticidal nets after five years of household use

<p>Abstract</p> <p>Background</p> <p>Long-lasting insecticidal nets (LLINs) have been strongly advocated for use to prevent malaria in sub-Saharan Africa and have significantly reduced human-vector contact. PermaNet^®2.0 is among the five LLINs brands which have been given full approval by the WHO Pesticide Evaluation Scheme (WHOPES). The LLINs are expected to protect the malaria endemic communities, but a number of factors within the community can affect their durability and efficacy. This study evaluated the durability, efficacy and retention of PermaNet^®2.0 after five years of use in a Tanzanian community.</p> <p>Method</p> <p>Two to three day- old non blood-fed female mosquitoes from an insectary susceptible colony (<it>An. gambiae </it>s.s, this colony was established at TPRI from Kisumu, Kenya in 1992) and wild mosquito populations (<it>An. arabiensis </it>and <it>Culex quinquefasciatus</it>) were used in cone bioassay tests to assess the efficacy of mosquito nets.</p> <p>Findings</p> <p>The knockdown effect was recorded after three minutes of exposure, and mortality was recorded after 24 hours post-exposure. Mortality of <it>An. gambiae </it>s.s from insectary colony was 100% while <it>An. arabiensis </it>and <it>Cx.quinquefasciatus </it>wild populations had reduced mortality. Insecticide content of the new (the bed net of the same brand but never used before) and used PermaNet^®2.0 was determined using High Performance Liquid Chromatography (HPLC).</p> <p>Conclusion</p> <p>The results of this study suggest that, in order to achieve maximum protection against malaria, public health education focusing on bed net use and maintenance should be incorporated into the mass distribution of nets in communities.</p

Long-Lasting Control of Anopheles arabiensis by a Single Spray Application of Micro-encapsulated Pirimiphos-methyl (Actellic(R) 300 CS).

Pyrethroid-resistant mosquitoes are an increasing threat to malaria vector control. The Global Plan for Insecticide Resistance Management (GPIRM) recommends rotation of non-pyrethroid insecticides for indoor residual spraying (IRS). The options from other classes are limited. The carbamate bendiocarb and the organophosphate pirimiphos-methyl (p-methyl) emulsifiable concentrate (EC) have a short residual duration of action, resulting in increased costs due to multiple spray cycles, and user fatigue. Encapsulation (CS) technology was used to extend the residual performance of p-methyl. Two novel p-methyl CS formulations were evaluated alongside the existing EC in laboratory bioassays and experimental hut trials in Tanzania between 2008-2010. Bioassays were carried out monthly on sprayed substrates of mud, concrete, plywood, and palm thatch to assess residual activity. Experimental huts were used to assess efficacy against wild free-flying Anopheles arabiensis, in terms of insecticide-induced mortality and blood-feeding inhibition. In laboratory bioassays of An. arabiensis and Culex quinquefasciatus both CS formulations produced high rates of mortality for significantly longer than the EC formulation on all substrates. On mud, the best performing CS killed >80% of An. arabiensis for five months and >50% for eight months, compared with one and two months, respectively, for the EC. In monthly bioassays of experimental hut walls the EC was ineffective shortly after spraying, while the best CS formulation killed more than 80% of An. arabiensis for five months on mud, and seven months on concrete. In experimental huts both CS and EC formulations killed high proportions of free-flying wild An. arabiensis for up to 12 months after spraying. There was no significant difference between treatments. All treatments provided considerable personal protection, with blood-feeding inhibition ranging from 9-49% over time. The long residual performance of p-methyl CS was consistent in bioassays and experimental huts. The CS outperformed the EC in laboratory and hut bioassays but the EC longevity in huts was unexpected. Long-lasting p-methyl CS formulations should be more effective than both p-methyl EC and bendiocarb considering a single spray could be sufficient for annual malaria control. IRS with p-methyl 300 CS is a timely addition to the limited portfolio of long-lasting residual insecticides