Is Anopheles gambiae (sensu stricto), the principal malaria vector in Africa prone to resistance development against new insecticides? Outcomes from laboratory exposure of An. gambiae (s.s.) to sub-lethal concentrations of chlorfenapyr and clothianidin.

Indiscriminate use of pesticides in the public health and agriculture sectors has contributed to the development of resistance in malaria vectors following exposure to sub-lethal concentrations. To preserve the efficacy of vector control tools and prevent resistance from spreading, early resistance detection is urgently needed to inform management strategies. The introduction of new insecticides for controlling malaria vectors such as clothianidin and chlorfenapyr requires research to identify early markers of resistance which could be used in routine surveillance. This study investigated phenotypic resistance of Anopheles gambiae (sensu stricto) Muleba-Kis strain using both WHO bottle and tube assays following chlorfenapyr, clothianidin, and alpha-cypermethrin selection against larvae and adults under laboratory conditions. High mortality rates were recorded for both chlorfenapyr-selected mosquitoes that were consistently maintained for 10 generations (24-h mortality of 92-100% and 72-h mortality of 98-100% for selected larvae; and 24-h mortality of 95-100% and 72-h mortality of 98-100% for selected adults). Selection with clothianidin at larval and adult stages showed a wide range of mortality (18-91%) compared to unselected progeny where mortality was approximately 99%. On the contrary, mosquitoes selected with alpha-cypermethrin from the adult selection maintained low mortality (28% at Generation 2 and 23% at Generation 4) against discrimination concentration compared to unselected progeny where average mortality was 51%. The observed resistance in the clothianidin-selected mosquitoes needs further investigation to determine the underlying resistance mechanism against this insecticide class. Additionally, further investigation is recommended to develop molecular markers for observed clothianidin phenotypic resistance.

Monitoring of Fabric Integrity and Attrition Rate of Dual-Active Ingredient Long-Lasting Insecticidal Nets in Tanzania: A Prospective Cohort Study Nested in a Cluster Randomized Controlled Trial.

Pyrethroid-treated long-lasting insecticidal nets (LLINs) have been the main contributor to the reduction in malaria in the past two decades in sub-Saharan Africa. The development of pyrethroid insecticide resistance threatens the future of LLINs, especially when nets become holed and pyrethroid decays. In this study, three new classes of dual-active ingredient (AI) LLINs were evaluated for their physical durability: (1) Royal Guard, combining pyriproxyfen, which disrupts female fertility, and a pyrethroid, alpha-cypermethrin; (2) Interceptor G2, which combines the pyrrole chlorfenapyr and a pyrethroid (alpha-cypermethrin); (3) Olyset Plus, which incorporates the pyrethroid permethrin and the synergist piperonyl butoxide, to enhance the pyrethroid potency; and Interceptor, a reference net that contains alpha-cypermethrin as the sole active ingredient. About 40,000 nets of each type were distributed in February 2019 to different villages in Misungwi. A total of 3072 LLINs were followed up every 6-12 months up to 36 months to assess survivorship and fabric integrity. The median functional survival was less than three years with Interceptor, Interceptor G2, and Royal Guard showing 1.9 years each and Olyset Plus showing 0.9 years. After 36 months, 90% of Olyset Plus and Royal Guard and 87% of Interceptor G2 were no longer in use (discarded) due to wear and tear, compared to 79% for Interceptor. All dual-AI LLINs exhibited poor textile durability, with Olyset Plus being the worst.

Effectiveness of long-lasting insecticidal nets with pyriproxyfen-pyrethroid, chlorfenapyr-pyrethroid, or piperonyl butoxide-pyrethroid versus pyrethroid only against malaria in Tanzania: final-year results of a four-arm, single-blind, cluster-randomised trial.

BACKGROUND: New classes of long-lasting insecticidal nets (LLINs) containing two active ingredients have been recently recommended by WHO in areas where malaria vectors are resistant to pyrethroids. This policy was based on evidence generated by the first 2 years of our recently published trial in Tanzania. In this Article, we report the final third-year trial findings, which are necessary for assessing the long-term effectiveness of new classes of LLIN in the community and the replacement intervals required. METHODS: A third year of follow-up of a four-arm, single-blind, cluster-randomised controlled trial of dual active ingredient LLINs was conducted between July 14, 2021, and Feb 10, 2022, in Misungwi, Tanzania. Restricted randomisation was used to assign 84 clusters to the four LLIN groups (1:1:1:1) to receive either standard pyrethroid (PY) LLINs (reference), chlorfenapyr-PY LLINs, pyriproxyfen-PY LLINs, or piperonyl butoxide (PBO)-PY LLINs. All households received one LLIN for every two people. Data collection was done in consenting households in the cluster core area with at least one child between 6 months and 15 years of age who permanently resided in the selected household. Exclusion criteria were householders absent during the visit, living in the cluster buffer area, no adult caregiver capable of giving informed consent, or eligible children who were severely ill. Field staff and study participants were masked to allocation, and those analysing data were not. The primary 24-month endpoint was reported previously; here, we present the secondary outcome, malaria infection prevalence in children at 36 months post LLIN distribution, reported in the intention-to-treat analysis. The trial was registered with ClinicalTrials.gov (NCT03554616) and is now complete. FINDINGS: Overall usage of study nets was 1023 (22·3%) of 4587 people at 36 months post distribution. In the standard PY LLIN group, malaria infection was prevalent in 407 (37·4%) of 1088 participants, compared with 261 (22·8%) of 1145 in the chlorfenapyr-PY LLIN group (odds ratio 0·57, 95% CI 0·38-0·86; p=0·0069), 338 (32·2%) of 1048 in the PBO-PY LLIN group (0·95, 0·64-1·42; p=0·80), and 302 (28·8%) of 1050 in the pyriproxyfen-PY LLIN group (0·82, 0·55-1·23; p=0·34). None of the participants or caregivers reported side-effects. INTERPRETATION: Despite low coverage, the protective efficacy against malaria offered by chlorfenapyr-PY LLINs was superior to that provided by standard PY LLINs over a 3-year LLIN lifespan. Appropriate LLIN replacement strategies to maintain adequate usage of nets will be necessary to maximise the full potential of these nets. FUNDING: Department for International Development, UK Medical Research Council, Wellcome Trust, Department of Health and Social Care, and Bill & Melinda Gates Foundation via the Innovative Vector Control Consortium.

Factors associated with malaria infection among children after distribution of PBO-pyrethroid synergist-treated nets and indoor residual spraying in north-western Tanzania.

BACKGROUND: After a decade of successful control, malaria is on the rise again. The prevalence of malaria in Tanzania has increased from 7% in 2017 to 8% in 2022 and reached 18% in Kagera region in the North West of Tanzania. Malaria vectors in Muleba district Kagera have high level of pyrethroid resistance. The aim of this paper is to explore factors associated with malaria infection prevalence in children aged 6 months to 14 years in Muleba, where Long Lasting Insecticidal Net (LLIN) combining a pyrethroid insecticide and synergist piperonyl butoxide (PBO) that counteract resistance in the mosquitoes, was first distributed under trial conditions in 2015. METHODS: The trial was a community randomized control in which there were two malaria prevalence cross-sectional household surveys each year (June and December) from 2015 to 2017 in Muleba. In this study we conducted a secondary data analysis of the December surveys only. Multilevel Poisson regression analysis was used to assess factors associated with malaria infection. RESULTS: A total of 10,941 children and 4,611 households were included in this study. Overall malaria prevalence was 35.8%, 53.3% and 54.4% in the year 2015, 2016 and 2017 respectively. Living in an area with standard LLIN as opposed to the novel PBO synergist LLIN, being a male child, above 5 years of age, living in a house with open eaves, living in house without IRS, having head of household with no formal education, lower socioeconomic status and survey year were associated with increased risk of malaria infection. CONCLUSIONS: Using PBO LLIN reduced the risk of malaria infection. However, additional measures could further reduce malaria infection in areas of insecticide resistance such as housing improvement.

Assessing risk factors for malaria and schistosomiasis among children in Misungwi, Tanzania, an area of co-endemicity: A mixed methods study.

Malaria and schistosomiasis are two major parasitic vector-borne diseases that are a particular threat to young children in Sub-Saharan Africa. In the present study, we investigated factors that are associated with malaria, schistosomiasis, and co-infection among school-aged children, using an explanatory sequential mixed-methods approach. A cross-sectional study was conducted in January 2022 in Misungwi, Tanzania, that sampled 1,122 children aged 5 to 14 years old for malaria and schistosomiasis infection. Mixed-effect logistic regression models were used to assess the association between infection prevalence or seroprevalence, and environmental determinants that create favorable conditions for vectors and parasites and social determinants that relate to disease exposure. Community mapping combined with direct field observations were conducted in August 2022 in three selected villages from the cross-sectional study to understand specific water use behaviors and to identify potential malaria mosquito larval breeding sites and freshwater snail habitat. The prevalence of malaria, seroprevalence of schistosomiasis, and co-infection in this study were 40.4%, 94.3%, and 38.1%, respectively. Individual-level factors emerged as the primary determinants driving the association with infection, with age (every one-year increase in age) and sex (boys vs girls) being statistically and positively associated with malaria, schistosomiasis, and co-infection (P<0.05 for all). Community maps identified many unimproved water sources in all three villages that were used by humans, cattle, or both. We found that children primarily fetched water, and that unprotected wells were dedicated for drinking water whereas ponds were dedicated for other domestic uses and cattle. Although not identified in the community maps, we found hand pumps in all three villages were not in use because of unpleasant taste and high cost. This study improves our understanding of individual, social and environmental factors that are associated with malaria, schistosomiasis, and co-infection, which can inform potential entry points for integrated disease prevention and control.

Effectiveness of piperonyl butoxide and pyrethroid-treated long-lasting insecticidal nets (LLINs) versus pyrethroid-only LLINs with and without indoor residual spray against malaria infection: third year results of a cluster, randomised controlled, two-by-two factorial design trial in Tanzania.

BACKGROUND: After decades of success in reducing malaria through the scale-up of pyrethroid long-lasting insecticidal nets (LLINs), the decline in the malaria burden has stalled, coinciding with the rapid spread of pyrethroid resistance. In a previously reported study, nets treated with a pyrethroid and a synergist, piperonyl butoxide (PBO), demonstrated superior efficacy compared to standard pyrethroid LLINs (std-LLINs) against malaria. Evidence was used to support the public health recommendation of PBO-Pyrethroid-LLIN by the World Health Organization in 2018. This study looks at the third year of rollout of these nets in Muleba district, Tanzania to inform whether policy guidelines need to be updated. METHODS: A four-group cluster randomized trial (CRT) using a two-by-two factorial design was carried out between January 2014 and December 2017. A total of 48 clusters, were randomized in a 1:1:1:1 ratio to the following treatment groups, each intervention being provided once in 2015: 1/std-LLIN; 2/PBO-pyrethroid LLIN; 3/std-LLIN + Indoor Residual Spraying (IRS) and 4/PBO-Pyrethroid-LLIN + IRS. During the third year follow-up, malaria infection prevalence in 80 children per cluster, aged 6 months to 14 years, was measured at 28- and 33-months post-intervention and analysed as intention-to-treat (ITT) and per protocol (PP). Mosquito collections were performed monthly in all clusters, using CDC light traps in 7 randomly selected houses per cluster. RESULTS: At 28 and 33 months, study net usage among household participants was only 47% and 31%, respectively. In ITT analysis, after 28 months malaria infection prevalence among 7471 children was 80.9% in the two std-LLIN groups compared to 69.3% in the two PBO-Pyrethroid-LLIN (Odds Ratio: 0.45, 95% Confidence Interval: 0.21-0.95, p-value: 0.0364). After 33 months the effect was weaker in the ITT analysis (prevalence 59.6% versus 49.9%, OR: 0.60, 95%CI:0.32-1.13, p-value: 0.1131) but still evident in the PP analysis (57.2% versus 44.2%, OR: 0.34, 95%CI: 0.16-0.71, p-value: 0.0051). Mean number of Anopheles per night collected per house was similar between PBO-Pyrethroid-LLIN groups (5.48) and std-LLIN groups (5.24) during the third year. CONCLUSIONS: Despite low usage of PBO- Pyrethroid LLIN, a small impact of those nets on malaria infection prevalence was still observed in the 3rd year with the most protection offered to children still using them. To maximize impact, it is essential that net re-distribution cycles are aligned with this LLIN lifespan to maintain maximum coverage. TRIAL REGISTRATION: The trial was registered with ClinicalTrials.gov (registration number NCT02288637).

Evaluation of Durability as a Function of Fabric Strength and Residual Bio-Efficacy for the Olyset Plus and Interceptor G2 LLINs after 3 Years of Field Use in Tanzania.

Long-lasting insecticidal nets (LLINs) are prone to reduction in insecticide content and physical strength due to repeated washes and usage. The significant loss to these features jeopardizes their protection against bites from malaria vectors. Insecticide washout is attributed to routine use, friction, and washing, while fabric damage is associated with routine use in households. To maintain coverage and cost-effectiveness, nets should maintain optimal bio-efficacy and physical strength for at least 3 years after distribution. In this study, the bio-efficacy and fabric strength of Olyset plus (OP) LLINs and Interceptor G2 (IG2), that were used for 3 years, were assessed in comparison to untreated and new unwashed counterparts. Both IG2 and OP LLINs (unused, laboratory-washed, and 36 months used) were able to induce significant mortality and blood feeding inhibition (BFI) to mosquitoes compared to the untreated nets. Significantly higher mortality was induced by unused IG2 LLIN and OP LLIN compared to their 36-month-old counterparts against both pyrethroid resistant and susceptible Anopheles gambiae sensu strito. The physical strength of the IG2 LLIN was higher than that of the Olyset Plus LLIN with a decreasing trend from unwashed, laboratory-washed to community usage (36 months old). Malaria control programs should consider bio-efficacy and physical integrity prior to an LLINs' procurement and replacement plan.

Effects of next-generation, dual-active-ingredient, long-lasting insecticidal net deployment on insecticide resistance in malaria vectors in Tanzania: an analysis of a 3-year, cluster-randomised controlled trial.

BACKGROUND: Insecticide resistance among malaria-vector species is a pervasive problem that might jeopardise global disease-control efforts. Novel vector-control tools with different modes of action, including long-lasting insecticidal nets (LLINs) incorporating new active ingredients, are urgently needed to delay the evolution and spread of insecticide resistance. We aimed to measure phenotypic and genotypic insecticide-resistance profiles among wild Anopheles collected over 3 years to assess the longitudinal effects of dual-active-ingredient LLINs on insecticide resistance. METHODS: For this analysis, data nested in a 3-year, four parallel-arm, superiority cluster-randomised controlled trial (cRCT) in Tanzania, collected from 84 clusters (39 307 households) formed of 72 villages in the Misungwi district, were used to measure insecticide-resistance profiles among female Anopheles mosquitoes via insecticide-resistance bioassays and quantitative RT-PCR of metabolic-resistance genes. Wild, blood-fed, indoor-resting mosquitoes were collected annually during the rainy seasons from house walls in clusters from all four trial groups. Mosquitoes were morphologically identified as An gambiae sensu lato (SL) or An funestus SL before separate bioassay testing. The primary outcomes were lethal-dose values for α-cypermethrin, permethrin, and piperonyl butoxide pre-exposure plus permethrin-resistance intensity bioassays, mortality 72 h after insecticidal exposure for chlorfenapyr bioassays, fertility reduction 72 h after insecticidal exposure for pyriproxyfen bioassays, and fold change in metabolic-enzyme expression relative to an insecticide-susceptible laboratory strain. All primary outcomes were measured in An funestus SL 1 year, 2 years, and 3 years after LLIN distribution. Primary outcomes were also assessed in An gambiae SL if enough mosquitoes were collected. The cRCT is registered with ClinicalTrials.gov (NCT03554616). FINDINGS: Between May 24, 2019, and Oct 25, 2021, 47 224 female Anopheles were collected for resistance monitoring. In the pyrethroid (PY)-LLIN group, there were significant increases in α-cypermethrin-resistance intensity (year 1 LD50=9·52 vs year 2 76·20, p<0·0001) and permethrin-resistance intensity (year 1 13·27 vs year 2 35·83, p=0·0019) in An funestus SL. In the pyriproxyfen PY-LLIN group, there was similar increase in α-cypermethrin-resistance intensity (year 1 0·71 vs year 2 81·56, p<0·0001) and permethrin-resistance intensity (year 1 5·68 vs year 2 50·14, p<0·0001). In the piperonyl butoxide PY-LLIN group, α-cypermethrin-resistance intensity (year 1 33·26 vs year 3 70·22, p=0·0071) and permethrin-resistance intensity (year 1 47·09 vs year 3 2635·29, p<0·0001) also increased over time. In the chlorfenapyr PY-LLIN group, there were no effects on α-cypermethrin-resistance intensity (year 1 0·42 vs year 3 0·99, p=0·54) or permethrin-resistance intensity (data were not estimable due to nearly 100% mortality). There were also minimal reductions in chlorfenapyr susceptibility. However, in the chlorfenapyr PY-LLIN group, a significant decline in piperonyl-butoxide synergy was seen by year 3 (year 1 0·02 vs year 3 0·26, p=0·020). Highly over-expressed detoxification enzymes showed dynamic patterns of selection throughout the trial. INTERPRETATION: Our phenotypic data supports trial epidemiological findings; chlorfenapyr PY-LLINs provided superior protection from malaria across multiple transmission seasons, with few effects on insecticide-resistance selection. Rapid pyrethroid-resistance intensification in the piperonyl butoxide PY-LLIN group and pre-existing tolerance of pyriproxyfen in vector populations might explain the poorer performance of these two interventions regarding malaria outcomes. Further work is required to elucidate the potential mechanisms driving cross-resistance between pyrethroids and novel active ingredients to better inform the design of pre-emptive resistance-management strategies. FUNDING: UK Department for International Development; UK Medical Research Council; Wellcome Trust; UK Department of Health and Social Care; UK Foreign, Commonwealth and Development Office; and The Bill and Melinda Gates Foundation via the Innovative Vector Control Consortium.

Fetal growth and birth weight are independently reduced by malaria infection and curable sexually transmitted and reproductive tract infections in Kenya, Tanzania, and Malawi: A pregnancy cohort study.

OBJECTIVES: Malaria and sexually transmitted and reproductive tract infections (STIs/RTIs) are highly prevalent in sub-Saharan Africa and associated with poor pregnancy outcomes. We investigated the individual and combined effects of malaria and curable STIs/RTIs on fetal growth in Kenya, Tanzania, and Malawi. METHODS: This study was nested within a randomized trial comparing monthly intermittent preventive treatment for malaria in pregnancy with sulfadoxine-pyrimethamine vs dihydroartemisinin-piperaquine, alone or combined with azithromycin. Fetal weight gain was assessed by serial prenatal ultrasound. Malaria was assessed monthly, and Treponema pallidum, Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia trachomatis, and bacterial vaginosis at enrollment and in the third trimester. The effect of malaria and STIs/RTIs on fetal weight/birthweight Z-scores was evaluated using mixed-effects linear regression. RESULTS: In total, 1435 pregnant women had fetal/birth weight assessed 3950 times. Compared to women without malaria or STIs/RTIs (n = 399), malaria-only (n = 267), STIs/RTIs only (n = 410) or both (n = 353) were associated with reduced fetal growth (adjusted mean difference in fetal/birth weight Z-score [95% confidence interval]: malaria = -0.18 [-0.31,-0.04], P = 0.01; STIs/RTIs = -0.14 [-0.26,-0.03], P = 0.01; both = -0.20 [-0.33,-0.07], P = 0.003). Paucigravidae experienced the greatest impact. CONCLUSION: Malaria and STIs/RTIs are associated with poor fetal growth especially among paucigravidae women with dual infections. Integrated antenatal interventions are needed to reduce the burden of both malaria and STIs/RTIs.

Ovary Dissection Is a Sensitive Measure of Sterility in Anopheles gambiae Exposed to the Insect Growth Regulator Pyriproxyfen.

Pyriproxyfen (PPF) is an insect growth regulator used in the co-treatment of long-lasting insecticidal nets for its ability to sterilize female mosquitoes. To evaluate the efficacy of PPF-treated nets on mosquito reproductivity, most studies observe oviposition (egg-laying) rates in the laboratory. This technique has several technical disadvantages. Our study assessed if ovarial dissection could serve as an effective proxy for evaluating sterility in Anopheles gambiae mosquitoes. Blood-fed females were exposed to untreated or PPF-treated nets in cylinder assays and followed over several days to observe oviposition rates or egg development by dissection. For identifying PPF-exposed mosquitoes, both techniques demonstrated high sensitivity (oviposition: 99.1%; dissection: 100.0%), but for identifying non-exposed mosquitoes, specificity was significantly higher in the dissection group (52.5% vs. 18.9%). To assess whether dissection could be applied to nets treated with a pyrethroid or co-treated with a pyrethroid and PPF in tunnel tests, a blinded investigator performed dissections to predict the PPF exposure status across different treatment groups. The exposure status of dissected females was predicted with >90% accuracy. We report that dissection is a sensitive technique to assess sterility in female Anopheles gambiae mosquitoes and can be used as a predictor of PPF exposure.

Differential impact of dual-active ingredient long-lasting insecticidal nets on primary malaria vectors: a secondary analysis of a 3-year, single-blind, cluster-randomised controlled trial in rural Tanzania.

BACKGROUND: Gains in malaria control are threatened by widespread pyrethroid resistance in malaria vectors across sub-Saharan Africa. New long-lasting insecticidal nets (LLINs) containing two active ingredients (dual active-ingredient LLINs) have been developed to interrupt transmission in areas of pyrethroid resistance. We aimed to evaluate the effectiveness of three dual active-ingredient LLINs compared with standard pyrethroid LLINs against pyrethroid-resistant malaria vectors in rural Tanzania. METHODS: In this study, we did a secondary analysis of entomological data from a four-group, 3 year, single-blind, cluster-randomised controlled trial carried out between Feb 18, 2019, and Dec 6, 2021. We conducted quarterly indoor mosquito collections using the Centers for Disease Control and Prevention light trap, in eight houses in each of the 84 study clusters in the Misungwi district, northwestern Tanzania. Anopheles vectors were then tested for malaria parasites and identified at species level, to distinguish between sibling species of the Anopheles gambiae and Anopheles funestus groups, using molecular laboratory techniques. The primary outcomes were density of different malaria vector species measured as the number of female Anopheles collected per household per night, the entomological inoculation rate (EIR), an indicator of malaria transmission, and sporozoite rate. Entomological outcomes were assessed on the basis of intention to treat, and the effect of the three dual active-ingredient LLINs was compared with the standard pyrethroid LLINs at household level. FINDINGS: Dual active-ingredient LLINs had the greatest effect on Anopheles funestus sl, the most efficient vector in the study area, with comparatively weak effect on An arabiensis. An funestus density was 3∙1 per house per night in the pyrethroid LLIN group, 1∙2 in the chlorfenapyr pyrethroid LLIN group (adjusted density ratio [aDR]=0∙26, 95% CI 0∙17-0∙14, p<0∙0001), 1∙4 in the piperonyl-butoxide pyrethroid LLIN group (aDR=0∙49, 0∙32-0∙76, p=0∙0012), and 3∙0 in the pyriproxyfen pyrethroid LLIN group (aDR=0∙72, 0∙47-1∙11, p=0∙15). Malaria transmission intensity was also significantly lower in the chlorfenapyr pyrethroid group, with 0∙01 versus 0∙06 infective bites per household per night in the pyrethroid LLIN group (aDR=0∙21, 0∙14-0∙33, p<0∙0001). Ecological niche models indicated that vector-species distribution was stable following LLIN intervention despite the reductions observed in An funestus sl density. INTERPRETATION: Chlorfenapyr pyrethroid LLINs were the most effective intervention against the main malaria vector An funestus sl over 3 years of community use, whereas the effect of piperonyl-butoxide pyrethroid LLIN was sustained for 2 years. The other vector, An arabiensis, was not controlled by any of the dual active-ingredient LLINs. Additional vector control tools and strategies targeted to locally prevalent vector species evading dual active-ingredient LLINs should be deployed to further reduce malaria transmission and achieve elimination. FUNDING: The Department for International Development, UK Medical Research Council, Wellcome Trust, the Department of Health and Social Care, and The Bill & Melinda Gates Foundation via the Innovative Vector Control Consortium.

Laboratory and semi-field efficacy evaluation of permethrin-piperonyl butoxide treated blankets against pyrethroid-resistant malaria vectors.

To control pyrethroid-resistant malaria vectors, Indoor Residual Spraying (IRS) and Long-Lasting Insecticidal Nets (LLINs) that include additional ingredients to pyrethroid are being developed. Same progress needs to be made to the pyrethroid-treated blankets, which are more compatible with shelter structures found in emergency settings such as displaced populations. In the current study, efficacy of blankets treated with permethrin and piperonyl butoxide (PBO) was evaluated against pyrethroid-resistant Anopheles gambiae sensu stricto. Efficacy was compared with that of Olyset LLIN, Olyset Plus LLIN and untreated blanket in terms of mortality and blood-feeding inhibition against pyrethroid-resistant Anopheles gambiae mosquitoes. The current study indicates that, in emergency shelters such as migrant and refugee camps where LLINs cannot be used, PBO-permethrin blankets may provide protection against resistant mosquitoes if widely used. No side effects related to the use of the treated blankets were reported from the participants. These results need validation in a large-scale field trial to assess the epidemiological impact of the intervention, durability and acceptability of this new vector control strategy for malaria vector control.

Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations.

Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N'Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126-54.00 µg/ml or LC95 × 3 = 0.7437-17.82 µg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

The Impact of Submicroscopic Parasitemia on Malaria Rapid Diagnosis in Northeastern Tanzania, an Area with Diverse Transmission Patterns.

Global malaria epidemiology has changed in the last decade with a substantial increase in cases and deaths being recorded. Tanzania accounts for about 4% of all cases and deaths reported in recent years. Several factors contribute to the resurgence of malaria, parasite resistance to antimalarials and mosquito resistance to insecticides being at the top of the list. The presence of sub-microscopic infections poses a significant challenge to malaria rapid diagnostic tests (mRDT). Our cross-sectional surveys in Handeni and Moshi, Tanzania assessed the effect of low parasite density on mRDT. Handeni had higher malaria prevalence by mRDT (39.6%), light microscopy (LM) (16.9%) and polymerase chain reaction (PCR) (18.5%), compared to Moshi with prevalence of 0.2%, 1.3% and 2.3%, respectively. A significant difference (p ˂ 0.001) in malaria prevalence by mRDT, LM and nested PCR was found among age groups. In comparison to all other groups, school-age children (5-15 years) had the highest prevalence of malaria. Our results show that mRDT may miss up to 6% of cases of malaria mainly due to low-density parasitemia when compared to LM and PCR. Routinely used mRDT will likely miss the sub-microscopic parasitemia which will ultimately contribute to the spread of malaria and hinder efforts of elimination.

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.

Efficacy of bednets with dual insecticide-treated netting (Interceptor® G2) on side and roof panels against Anopheles arabiensis in north-eastern Tanzania.

BACKGROUND: Optimising insecticide use and managing insecticide resistance are important to sustain gains against malaria using long-lasting insecticidal nets (LLINs). Restricting insecticides to where mosquitoes are most likely to make multiple contacts could reduce the quantity of insecticide needed to treat the nets. Previous studies have shown that nets partially treated with a pyrethroid insecticide had equivalent mortality compared to a fully treated net. This study compared the efficacy of: (i) whole Interceptor® G2 nets (IG2; a dual-active LLIN containing alpha-cypermethrin and chlorfenapyr), (ii) nets with roof panels made of IG2 netting, (iii) nets with side panels made of IG2 netting and (iv) whole untreated nets as test nets. METHODS: The study was conducted in cow-baited experimental huts, Moshi Tanzania, using a four-arm Latin square design. Test nets had 30 holes cut in panels to simulate a typical net after 2-3 year use. The trial data were analysed using generalized linear models with mortality, blood-feeding, exophily and deterrence against wild free-flying Anopheles arabiensis as outcomes and test nets as predictors. RESULTS: Mortality was significantly higher in the nets with roof IG2 [27%, P = 0.001, odds ratio (OR) = 51.0, 95% CI = 4.8-546.2), side IG2 (44%, P < 0.001, OR = 137.6, 95% CI = 12.2-1553.2] and whole IG2 (53%, P < 0.001, OR = 223.0, 95% CI = 19.07-2606.0) nettings than the untreated (1%) nets. Mortality was also significantly higher in the whole IG2 net compared to the net with roof IG2 netting (P = 0.009, OR = 4.4, 95% CI = 1.4-13.3). Blood feeding was 22% in untreated, 10% in roof IG2, 14% in side IG2 and 19% in whole IG2 nets. Exiting was 92% in untreated, 89% in roof IG2, 97% in side IG2 and 94% whole IG2 nets. CONCLUSION: The results show that although the roof-treated IG2 net induced greater mortality compared to untreated nets, its efficacy was reduced compared to whole IG2 nets. Therefore, there was no benefit to be gained from restricting dual-active ingredient IG2 netting to the roof of nets.

Enhancing the Quality of Spray Application in IRS: Evaluation of the Micron Track Sprayer.

Indoor residual spraying (IRS) has changed little since its introduction in the 1940s. Manual spraying is still prone to variation in insecticide dose. To improve the application of IRS in experimental hut trials, an automated track sprayer was developed, which regulates the speed of application and the distance of the nozzle from the wall, two key sources of variation. The automated track sprayer was compared to manual spraying, firstly using fluorescein solution in controlled indoor settings, and secondly in experimental huts in Tanzania using several IRS products. Manual spraying produced greater variation with both fluorescein and insecticide applications. Both manual and automated spray methods under-dosed the actual dose sprayed compared to the target dose. Overall, the track sprayer treats surfaces more consistently, offering a potential improvement over manual spraying for experimental hut evaluation of new IRS formulations.

Implementing OECD GLP principles for the evaluation of novel vector control tools: a case study with two novel LLINs, SafeNet® and SafeNet NF®.

BACKGROUND: To sustain high universal Long-Lasting Insecticidal Nets (LLINs) coverage, affordable nets that provide equivalent or better protection than standard LLINs, are required. Test facilities evaluating new LLINs require compliance to Good Laboratory Practice (GLP) standards to ensure the quality and integrity of test data. Following GLP principles allows for the reconstruction of activities during the conduct of a study and minimizes duplication of efficacy testing. This case study evaluated the efficacy of two LLINs: SafeNet NF® and SafeNet® LLIN. METHODS: The study was conducted according to GLP principles and followed World Health Organization guidelines for evaluating LLINs. The LLINs were assessed in experimental huts against wild, pyrethroid-resistant Anopheles arabiensis mosquitoes. Nets were either unwashed or washed 20 times and artificially holed to simulate a used torn net. Blood-feeding inhibition and mortality were compared with a positive control (Interceptor® LLIN) and an untreated net. RESULTS: Mosquito entry in the huts was reduced compared to negative control for the unwashed SafeNet NF, washed Safenet LLIN and the positive control arms. Similar exiting rates were found for all the treatment arms. Significant blood-feeding inhibition was only found for the positive control, both when washed and unwashed. All insecticide treatments induced significantly higher mortality compared to an untreated net. Compared to the positive control, the washed and unwashed SafeNet NF® resulted in similar mortality. For the SafeNet® LLINs the unwashed net had an equivalent performance, but the mortality for the washed net was significantly lower than the positive control. Internal audits of the study confirmed that all critical phases complied with Standard Operating Procedures (SOPs) and the study plan. The external audit confirmed that the study complied with GLP standards. CONCLUSIONS: SafeNet NF® and SafeNet® LLIN offered equivalent protection to the positive control (Interceptor® LLIN). However, further research is needed to investigate the durability, acceptability, and residual efficacy of these nets in the community. This study demonstrated that GLP-compliant evaluation of LLINs can be successfully conducted by African research institutions.

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.

Effectiveness and cost-effectiveness against malaria of three types of dual-active-ingredient long-lasting insecticidal nets (LLINs) compared with pyrethroid-only LLINs in Tanzania: a four-arm, cluster-randomised trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) have successfully reduced malaria in sub-Saharan Africa, but their effectiveness is now partly compromised by widespread resistance to insecticides among vectors. We evaluated new classes of LLINs with two active ingredients with differing modes of action against resistant malaria vectors. METHODS: We did a four-arm, cluster-randomised trial in Misungwi, Tanzania. Clusters were villages, or groups of hamlets, with at least 119 households containing children aged 6 months to 14 years living in the cluster's core area. Constrained randomisation was used to allocate clusters (1:1:1:1) to receive one of four types of LLIN treated with the following: α-cypermethrin only (pyrethroid-only [reference] group); pyriproxyfen and α-cypermethrin (pyriproxyfen group); chlorfenapyr and α-cypermethrin (chlorfenapyr group); or the synergist piperonyl butoxide and permethrin (piperonyl butoxide group). At least one LLIN was distributed for every two people. Community members and the field team were masked to group allocation. Malaria prevalence data were collected through cross-sectional surveys of randomly selected households from each cluster, in which children aged 6 months to 14 years were assessed for Plasmodium falciparum malaria infection by rapid diagnostic tests. The primary outcome was malaria infection prevalence at 24 months after LLIN distribution, comparing each of the dual-active-ingredient LLINs to the standard pyrethroid-only LLINs in the intention-to-treat population. The primary economic outcome was cost-effectiveness of dual-active-ingredient LLINs, based on incremental cost per disability-adjusted life-year (DALY) averted compared with pyrethroid-only LLINs, modelled over a 2-year period; we included costs of net procurement and malaria diagnosis and treatment, and estimated DALYs in all age groups. This study is registered with ClinicalTrials.gov (NCT03554616), and is ongoing but no longer recruiting. FINDINGS: 84 clusters comprising 39 307 households were included in the study between May 11 and July 2, 2018. 147 230 LLINs were distributed among households between Jan 26 and Jan 28, 2019. Use of study LLINs was reported in 3155 (72·1%) of 4378 participants surveyed at 3 months post-distribution and decreased to 8694 (40·9%) of 21 246 at 24 months, with varying rates of decline between groups. Malaria infection prevalence at 24 months was 549 (45·8%) of 1199 children in the pyrethroid-only reference group, 472 (37·5%) of 1258 in the pyriproxyfen group (adjusted odds ratio 0·79 [95% CI 0·54-1·17], p=0·2354), 512 (40·7%) of 1259 in the piperonyl butoxide group (0·99 [0·67-1·45], p=0·9607), and 326 [25·6%] of 1272 in the chlorfenapyr group (0·45 [0·30-0·67], p=0·0001). Skin irritation or paraesthesia was the most commonly reported side-effect in all groups. Chlorfenapyr LLINs were the most cost-effective LLINs, costing only US$19 (95% uncertainty interval 1-105) more to public providers or $28 (11-120) more to donors per DALY averted over a 2-year period compared with pyrethroid-only LLINs, and saving costs from societal and household perspectives. INTERPRETATION: After 2 years, chlorfenapyr LLINs provided significantly better protection than pyrethroid-only LLINs against malaria in an area with pyrethroid-resistant mosquitoes, and the additional cost of these nets would be considerably below plausible cost-effectiveness thresholds ($292-393 per DALY averted). Before scale-up of chlorfenapyr LLINs, resistance management strategies are needed to preserve their effectiveness. Poor textile and active ingredient durability in the piperonyl butoxide and pyriproxyfen LLINs might have contributed to their relative lack of effectiveness compared with standard LLINs. FUNDING: Joint Global Health Trials scheme (UK Foreign, Commonwealth and Development Office; UK Medical Research Council; Wellcome; UK Department of Health and Social Care), US Agency for International Development, President's Malaria Initiative.