Considerations for first field trials of low-threshold gene drive for malaria vector control.

Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a 'causal pathway' would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

Will a lack of fabric durability be their downfall? Impact of textile durability on the efficacy of three types of dual-active-ingredient long-lasting insecticidal nets: a secondary analysis on malaria prevalence and incidence from a cluster-randomized trial in north-west Tanzania.

BACKGROUND: The Dual-Active Ingredient long-lasting insecticidal nets (Dual-AI LLIN) have been developed to counteract the reduced efficacy of pyrethroid (PY)-only nets due to widespread pyrethroid insecticide resistance in malaria vector mosquitoes. They constitute half of the nets distributed in sub-Saharan Africa between 2022 and 2024. However, their effectiveness once they develop holes is unclear, particularly in pyrethroid-resistant settings. This study evaluates the textile integrity of three dual- AI LLINs compared to standard PY LLN, over 3 years of use in a community in Tanzania and the associated impact on malaria prevalence and incidence. METHODS: A secondary analysis of data from a randomized controlled trial (RCT) in North-western Tanzania was conducted to evaluate the effectiveness of α-cypermethrin only; pyriproxyfen and α-cypermethrin (PPF-PY); chlorfenapyr and α-cypermethrin (chlorfenapyr-PY); and the synergist piperonyl butoxide and permethrin (PBO-PY) LLINs on malaria infection prevalence and case incidence. The association between the net textile condition and 1/malaria prevalence over 3 years of use between 2019 and 2022, and 2/malaria case incidence in a cohort of children over 2 years of follow-up was assessed between 2019 and 2021. RESULTS: There was no significant association between damaged (OR 0.98, 95% CI 0.71-1.37, p-value = 0.655) and too-torn (OR 1.07, 95% CI 0.77-1.47, p-value = 0.694) compared to intact nets on malaria prevalence for all net types. However, there were reduced rates of malaria case incidence in children sleeping under a net in good condition compared to too-torn nets (incidence rate ratio (IRR) 0.76 [95% CI 0.63-0.92], p = 0.005). Malaria incidence was also consistently lower in too-torn PBO-PY LLIN (IRR = 0.37 [95% CI 0.19-0.72], p = 0.003) and chlorfenapyr-PY LLIN (IRR = 0.45 [95% CI 0.33-0.97], p = 0.053) compared to an intact PY-only LLIN during the first year of follow up. In year 2, the incidence was only significantly lower in intact chlorfenapyr-PY LLIN (IRR = 0.49 [95% CI 0.29-0.81], p = 0.006) compared to intact PY LLIN. CONCLUSION: The study confirmed that sleeping under a chlorfenapyr-PY LLIN or PBO-PY LLIN offered superior protection to pyrethroid-only nets even when torn. Preventing the development of holes is essential as they impact the level of protection offered against malaria infection. TRIAL REGISTRATION: ClinicalTrials.gov, number (NCT03554616).

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.

Prevalence of G6PD deficiency and submicroscopic malaria parasites carriage in malaria hotspot area in Northwest, Tanzania.

BACKGROUND: The use of primaquine for mass drug administration (MDA) is being considered as a key strategy for malaria elimination. In addition to being the only drug active against the dormant and relapsing forms of Plasmodium vivax, primaquine is the sole potent drug against mature/infectious Plasmodium falciparum gametocytes. It may prevent onward transmission and help contain the spread of artemisinin resistance. However, higher dose of primaquine is associated with the risk of acute haemolytic anaemia in individuals with a deficiency in glucose-6-phosphate dehydrogenase. In many P. falciparum endemic areas there is paucity of information about the distribution of individuals at risk of primaquine-induced haemolysis at higher dose 45 mg of primaquine. METHODS: A retrospective cross-sectional study was carried out using archived samples to establish the prevalence of G6PD deficiency in a malaria hotspot area in Misungwi district, located in Mwanza region, Tanzania. Blood samples collected from individuals recruited between August and November 2010 were genotyped for G6PD deficiency and submicroscopic parasites carriage using polymerase chain reaction. RESULTS: A total of 263 individuals aged between 0 and 87 were recruited. The overall prevalence of the X-linked G6PD A- mutation was 83.7% (220/263) wild type, 8% (21/263) heterozygous and 8.4% (22/263) homozygous or hemizygous. Although, assessment of the enzymatic activity to assign the phenotypes according to severity and clinical manifestation as per WHO was not carried out, the overall genotype and allele frequency for the G6PD deficiency was 16.4% and 13. 2%, respectively. There was no statistically significant difference in among the different G6PD genotypes (p > 0.05). Out of 248 samples analysed for submicroscopic parasites carriage, 58.1% (144/248) were P. falciparum positive by PCR. G6PD heterozygous deficiency were associated with carriage of submicroscopic P. falciparum (p = 0.029). CONCLUSIONS: This study showed that 16.4% of the population in this part of North-western Tanzania carry the G6PD A- mutation, within the range of 15-32% seen in other parts of Africa. G6PD gene mutation is widespread and heterogeneous across the study area where primaquine would be valuable for malaria control and elimination. The maps and population estimates presented here reflect potential risk of higher dose of primaquine being associated with the risk of acute haemolytic anaemia (AHA) in individuals with a deficiency in glucose-6-phosphate dehydrogenase and call further research on mapping of G6PD deficiency in Tanzania. Therefore, screening and education programmes for G6PD deficiency is warranted in a programme of malaria elimination using a higher primaquine dose.

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).

Durability of three types of dual active ingredient long-lasting insecticidal net compared to a pyrethroid-only LLIN in Tanzania: methodology for a prospective cohort study nested in a cluster randomized controlled trial.

BACKGROUND: Progress achieved by long-lasting insecticidal nets (LLINs) against malaria is threatened by widespread selection of pyrethroid resistance among vector populations. LLINs with non-pyrethroid insecticides are urgently needed. This study aims to assess the insecticide and textile durability of three classes of dual-active ingredient (A.I.) LLINs using techniques derived from established WHO LLIN testing methods to set new standards of evaluation. METHODS: A WHO Phase 3 active ingredients and textile durability study will be carried out within a cluster randomized controlled trial in 40 clusters in Misungwi district, Tanzania. The following treatments will be evaluated: (1) Interceptor®G2 combining chlorfenapyr and the pyrethroid alpha-cypermethrin, (2) Royal Guard® treated with pyriproxyfen and alpha-cypermethrin, (3) Olyset™ Plus which incorporates a synergist piperonyl butoxide and the pyrethroid permethrin, and (4) a reference standard alpha-cypermethrin only LLIN (Interceptor®). 750 nets will be followed in 5 clusters per intervention arm at 6, 12, 24 and 36 months post distribution for survivorship and hole index assessment. A second cohort of 1950 nets per net type will be identified in 10 clusters, of which 30 LLINs will be withdrawn for bio-efficacy and chemical analysis every 6 months up to 36 months and another 30 collected for experimental hut trials every year. Bio-efficacy will be assessed using cone bioassays and tunnel tests against susceptible and resistant laboratory strains of Anopheles gambiae sensu stricto. Efficacy of field-collected nets will be compared in six experimental huts. The main outcomes will be Anopheles mortality up to 72 h post exposure, blood feeding and egg maturation using ovary dissection to assess impact on fecundity. CONCLUSIONS: Study findings will help develop bio-efficacy and physical durability criteria for partner A.I., in relation to the cRCT epidemiological and entomological outcomes, and refine preferred product characteristics of each class of LLIN. If suitable, the bioassay and hut outcomes will be fitted to transmission models to estimate correlation with cRCT outcomes. TRIAL REGISTRATION NUMBER: NCT03554616.

Performance evaluation of the highly sensitive histidine-rich protein 2 rapid test for plasmodium falciparum malaria in North-West Tanzania.

BACKGROUND: Precise detection of Plasmodium infections in community surveys is essential for effective malaria control. Microscopy and rapid diagnostic tests (RDTs) are the major techniques used to identify malaria infections in the field-based surveys. Although microscopy is still considered as the gold standard, RDTs are increasingly becoming versatile due to their rapid and adequate performance characteristics. METHODS: A malaria prevalence cross-sectional survey was carried out in north-western Tanzania in 2016, aimed at appraising the performance of high sensitivity Plasmodium falciparum (HSPf) tests compared to SD Bioline Pf and microscopy in detecting P. falciparum infections. A total of 397 individuals aged five years and above were tested for P. falciparum infections. The sensitivity, specificity, positive, and negative predictive values (PPV and NPV) of microscopy, Pf RDT and HSPf RDT was determined using PCR as the gold standard method. RESULTS: The prevalence of P. falciparum infections determined by microscopy, SD Bioline Pf, HSPf and PCR was 21.9, 27.7, 33.3 and 43.2%, respectively. The new HSPf RDT had significantly higher sensitivity (98.2%) and specificity (91.6%) compared to the routinely used SD Bioline Pf RDT(P < 0.001). The positive predictive value (PPV) was 81.8% and the negative predictive value (NPV) was 99.2% for the routinely used SD Bioline Pf RDT. Moreover, HSPf RDT had sensitivity of 69% and specificity of 76.8% compared to microscopy. The PPV was 45.5% and the NPV was 89.8% for microscopy. Furthermore, the analytical sensitivity test indicated that the newly developed HSPf RDT had lower detection limits compared to routinely used SD Bioline RDT. CONCLUSIONS: HSPf RDT had better performance when compared to both microscopy and the currently used malaria RDTs. The false negativity could be associated with the low parasite density of the samples. False positivity may be related to the limitations of the expertise of microscopists or persistent antigenicity from previous infections in the case of RDTs. Nevertheless, HS PfRDT performed better compared to routinely used Pf RDT, and microscopy in detecting malaria infections. Therefore, HS Pf RDT presents the best alternative to the existing commercial/regularly available RDTs due to its sensitivity and specificity, and reliability in diagnosing malaria infections.

Novel genetic polymorphisms associated with severe malaria and under selective pressure in North-eastern Tanzania.

Significant selection pressure has been exerted on the genomes of human populations exposed to Plasmodium falciparum infection, resulting in the acquisition of mechanisms of resistance against severe malarial disease. Many host genetic factors, including sickle cell trait, have been associated with reduced risk of developing severe malaria, but do not account for all of the observed phenotypic variation. Identification of novel inherited risk factors relies upon high-resolution genome-wide association studies (GWAS). We present findings of a GWAS of severe malaria performed in a Tanzanian population (n = 914, 15.2 million SNPs). Beyond the expected association with the sickle cell HbS variant, we identify protective associations within two interleukin receptors (IL-23R and IL-12RBR2) and the kelch-like protein KLHL3 (all P<10-6), as well as near significant effects for Major Histocompatibility Complex (MHC) haplotypes. Complementary analyses, based on detecting extended haplotype homozygosity, identified SYNJ2BP, GCLC and MHC as potential loci under recent positive selection. Through whole genome sequencing of an independent Tanzanian cohort (parent-child trios n = 247), we confirm the allele frequencies of common polymorphisms underlying associations and selection, as well as the presence of multiple structural variants that could be in linkage with these SNPs. Imputation of structural variants in a region encompassing the glycophorin genes on chromosome 4, led to the characterisation of more than 50 rare variants, and individually no strong evidence of associations with severe malaria in our primary dataset (P>0.3). Our approach demonstrates the potential of a joint genotyping-sequencing strategy to identify as-yet unknown susceptibility loci in an African population with well-characterised malaria phenotypes. The regions encompassing these loci are potential targets for the design of much needed interventions for preventing or treating malarial disease.

In Vivo Imaging of the Buccal Mucosa Shows Loss of the Endothelial Glycocalyx and Perivascular Hemorrhages in Pediatric Plasmodium falciparum Malaria.

Severe malaria is mostly caused by Plasmodium falciparum, resulting in considerable, systemic inflammation and pronounced endothelial activation. The endothelium forms an interface between blood and tissue, and vasculopathy has previously been linked with malaria severity. We studied the extent to which the endothelial glycocalyx that normally maintains endothelial function is involved in falciparum malaria pathogenesis by using incident dark-field imaging in the buccal mucosa. This enabled calculation of the perfused boundary region, which indicates to what extent erythrocytes can permeate the endothelial glycocalyx. The perfused boundary region was significantly increased in severe malaria patients and mirrored by an increase of soluble glycocalyx components in plasma. This is suggestive of a substantial endothelial glycocalyx loss. Patients with severe malaria had significantly higher plasma levels of sulfated glycosaminoglycans than patients with uncomplicated malaria, whereas other measured glycocalyx markers were raised to a comparable extent in both groups. In severe malaria, the plasma level of the glycosaminoglycan hyaluronic acid was positively correlated with the perfused boundary region in the buccal cavity. Plasma hyaluronic acid and heparan sulfate were particularly high in severe malaria patients with a low Blantyre coma score, suggesting involvement in its pathogenesis. In vivo imaging also detected perivascular hemorrhages and sequestering late-stage parasites. In line with this, plasma angiopoietin-1 was decreased while angiopoietin-2 was increased, suggesting vascular instability. The density of hemorrhages correlated negatively with plasma levels of angiopoietin-1. Our findings indicate that as with experimental malaria, the loss of endothelial glycocalyx is associated with vascular dysfunction in human malaria and is related to severity.

Risk factors for malaria infection prevalence and household vector density between mass distribution campaigns of long-lasting insecticidal nets in North-western Tanzania.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the most widely deployed vector control intervention in sub-Saharan Africa to prevent malaria. Recent reports indicate selection of pyrethroid insecticide resistance is widespread in mosquito vectors. This paper explores risk factors associated with malaria infection prevalence and vector density between mass distribution campaigns, changes in net coverage, and loss of protection in an area of high pyrethroid resistance in Northwest Tanzania. METHODS: A cross sectional malaria survey of 3456 children was undertaken in 2014 in Muleba district, Kagera region west of Lake Victoria. Vector density was assessed using indoor light traps and outdoor tent traps. Anophelines were identified to species using PCR and tested for Plasmodium falciparum circumsporozoite protein. Logistic regression was used to identify household and environmental factors associated with malaria infection and regression binomial negative for vector density. RESULTS: LLIN use was 27.7%. Only 16.9% of households had sufficient nets to cover all sleeping places. Malaria infection was independently associated with access to LLINs (OR: 0.57; 95% CI 0.34-0.98). LLINs less than 2 years old were slightly more protective than older LLINs (53 vs 65% prevalence of infection); however, there was no evidence that LLINs in good condition (hole index < 65) were more protective than LLINs, which were more holed. Other risk factors for malaria infection were age, group, altitude and house construction quality. Independent risk factors for vector density were consistent with malaria outcomes and included altitude, wind, livestock, house quality, open eaves and LLIN usage. Indoor collections comprised 4.6% Anopheles funestus and 95.4% Anopheles gambiae of which 4.5% were Anopheles arabiensis and 93.5% were Anopheles gambiae sensu stricto. CONCLUSION: Three years after the mass distribution campaign and despite top-ups, LLIN usage had declined considerably. While children living in households with access to LLINs were at lower risk of malaria, infection prevalence remained high even among users of LLINs in good condition. While effort should be made to maintain high coverage between campaigns, distribution of standard pyrethroid-only LLINs appears insufficient to prevent malaria transmission in this area of intense pyrethroid resistance.

Malaria vector species composition and entomological indices following indoor residual spraying in regions bordering Lake Victoria, Tanzania.

BACKGROUND: Vector control through long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) is a major component of the Tanzania national malaria control strategy. In mainland Tanzania, IRS has been conducted annually around Lake Victoria basin since 2007. Due to pyrethroid resistance in malaria vectors, use of pyrethroids for IRS was phased out and from 2014 to 2017 pirimiphos-methyl (Actellic® 300CS) was sprayed in regions of Kagera, Geita, Mwanza, and Mara. Entomological surveillance was conducted in 10 sprayed and 4 unsprayed sites to determine the impact of IRS on entomological indices related to malaria transmission risk. METHODS: WHO cone bioassays were conducted monthly on interior house walls to determine residual efficacy of pirimiphos-methyl CS. Indoor CDC light traps with or without bottle rotator were hung next to protected sleepers indoors and also set outdoors (unbaited) as a proxy measure for indoor and outdoor biting rate and time of biting. Prokopack aspirators were used indoors to capture resting malaria vectors. A sub-sample of Anopheles was tested by PCR to determine species identity and ELISA for sporozoite rate. RESULTS: Annual IRS with Actellic® 300CS from 2015 to 2017 was effective on sprayed walls for a mean of 7 months in cone bioassay. PCR of 2016 and 2017 samples showed vector populations were predominantly Anopheles arabiensis (58.1%, n = 4,403 IRS sites, 58%, n = 2,441 unsprayed sites). There was a greater proportion of Anopheles funestus sensu stricto in unsprayed sites (20.4%, n = 858) than in sprayed sites (7.9%, n = 595) and fewer Anopheles parensis (2%, n = 85 unsprayed, 7.8%, n = 591 sprayed). Biting peaks of Anopheles gambiae sensu lato (s.l.) followed periods of rainfall occurring between October and April, but were generally lower in sprayed sites than unsprayed. In most sprayed sites, An. gambiae s.l. indoor densities increased between January and February, i.e., 10-12 months after IRS. The predominant species An. arabiensis had a sporozoite rate in 2017 of 2.0% (95% CI 1.4-2.9) in unsprayed sites compared to 0.8% (95% CI 0.5-1.3) in sprayed sites (p = 0.003). Sporozoite rates were also lower for An. funestus collected in sprayed sites. CONCLUSION: This study contributes to the understanding of malaria vector species composition, behaviour and transmission risk following IRS around Lake Victoria and can be used to guide malaria vector control strategies in Tanzania.

Effectiveness of a long-lasting piperonyl butoxide-treated insecticidal net and indoor residual spray interventions, separately and together, against malaria transmitted by pyrethroid-resistant mosquitoes: a cluster, randomised controlled, two-by-two factorial design trial.

BACKGROUND: Progress in malaria control is under threat by wide-scale insecticide resistance in malaria vectors. Two recent vector control products have been developed: a long-lasting insecticidal net that incorporates a synergist piperonyl butoxide (PBO) and a long-lasting indoor residual spraying formulation of the insecticide pirimiphos-methyl. We evaluated the effectiveness of PBO long-lasting insecticidal nets versus standard long-lasting insecticidal nets as single interventions and in combination with the indoor residual spraying of pirimiphos-methyl. METHODS: We did a four-group cluster randomised controlled trial using a two-by-two factorial design of 48 clusters derived from 40 villages in Muleba (Kagera, Tanzania). We randomly assigned these clusters using restricted randomisation to four groups: standard long-lasting insecticidal nets, PBO long-lasting insecticidal nets, standard long-lasting insecticidal nets plus indoor residual spraying, or PBO long-lasting insecticidal nets plus indoor residual spraying. Both standard and PBO nets were distributed in 2015. Indoor residual spraying was applied only once in 2015. We masked the inhabitants of each cluster to the type of nets received, as well as field staff who took blood samples. Neither the investigators nor the participants were masked to indoor residual spraying. The primary outcome was the prevalence of malaria infection in children aged 6 months to 14 years assessed by cross-sectional surveys at 4, 9, 16, and 21 months after intervention. The endpoint for assessment of indoor residual spraying was 9 months and PBO long-lasting insecticidal nets was 21 months. This trial is registered with ClinicalTrials.gov, number NCT02288637. FINDINGS: 7184 (68·0%) of 10 560 households were selected for post-intervention survey, and 15 469 (89·0%) of 17 377 eligible children from the four surveys were included in the intention-to-treat analysis. Of the 878 households visited in the two indoor residual spraying groups, 827 (94%) had been sprayed. Reported use of long-lasting insecticidal nets, across all groups, was 15 341 (77·3%) of 19 852 residents after 1 year, decreasing to 12 503 (59·2%) of 21 105 in the second year. Malaria infection prevalence after 9 months was lower in the two groups that received PBO long-lasting insecticidal nets than in the two groups that received standard long-lasting insecticidal nets (531 [29%] of 1852 children vs 767 [42%] of 1809; odds ratio [OR] 0·37, 95% CI 0·21-0·65; p=0·0011). At the same timepoint, malaria prevalence in the two groups that received indoor residual spraying was lower than in groups that did not receive indoor residual spraying (508 [28%] of 1846 children vs 790 [44%] of 1815; OR 0·33, 95% CI 0·19-0·55; p<0·0001) and there was evidence of an interaction between PBO long-lasting insecticidal nets and indoor residual spraying (OR 2·43, 95% CI 1·19-4·97; p=0·0158), indicating redundancy when combined. The PBO long-lasting insecticidal net effect was sustained after 21 months with a lower malaria prevalence than the standard long-lasting insecticidal net (865 [45%] of 1930 children vs 1255 [62%] of 2034; OR 0·40, 0·20-0·81; p=0·0122). INTERPRETATION: The PBO long-lasting insecticidal net and non-pyrethroid indoor residual spraying interventions showed improved control of malaria transmission compared with standard long-lasting insecticidal nets where pyrethroid resistance is prevalent and either intervention could be deployed to good effect. As a result, WHO has since recommended to increase coverage of PBO long-lasting insecticidal nets. Combining indoor residual spraying with pirimiphos-methyl and PBO long-lasting insecticidal nets provided no additional benefit compared with PBO long-lasting insecticidal nets alone or standard long-lasting insecticidal nets plus indoor residual spraying. FUNDING: UK Department for International Development, Medical Research Council, and Wellcome Trust.

Susceptibility testing of Anopheles malaria vectors with the neonicotinoid insecticide clothianidin; results from 16 African countries, in preparation for indoor residual spraying with new insecticide formulations.

BACKGROUND: In 2017, more than 5 million house structures were sprayed through the U.S. President's Malaria Initiative, protecting more than 21 million people in sub-Saharan Africa. New IRS formulations, SumiShield™ 50WG and Fludora Fusion™ WP-SB, became World Health Organization (WHO) prequalified vector control products in 2017 and 2018, respectively. Both formulations contain the neonicotinoid active ingredient, clothianidin. The target site of neonicotinoids represents a novel mode of action for vector control, meaning that cross-resistance through existing mechanisms is less likely. In preparation for rollout of clothianidin formulations as part of national IRS rotation strategies, baseline susceptibility testing was conducted in 16 countries in sub-Saharan Africa. METHODS: While work coordinated by the WHO is ongoing to develop a suitable bottle bioassay procedure, there was no published guidance regarding clothianidin susceptibility procedures or diagnostic concentrations. Therefore, a protocol was developed for impregnating filter papers with 2% w/v SumiShield™ 50WG dissolved in distilled water. Susceptibility tests were conducted using insectary-reared reference Anopheles and wild collected malaria vector species. All tests were conducted within 24 h of treating papers, with mortality recorded daily for 7 days, due to the slow-acting nature of clothianidin against mosquitoes. Anopheles gambiae sensu lato (s.l.) adults from wild collected larvae were tested in 14 countries, with wild collected F0 Anopheles funestus s.l. tested in Mozambique and Zambia. RESULTS: One-hundred percent mortality was reached with all susceptible insectary strains and with wild An. gambiae s.l. from all sites in 11 countries. However, tests in at least one location from 5 countries produced mortality below 98%. While this could potentially be a sign of clothianidin resistance, it is more likely that the diagnostic dose or protocol requires further optimization. Repeat testing in 3 sites in Ghana and Zambia, where possible resistance was detected, subsequently produced 100% mortality. Results showed susceptibility to clothianidin in 38 of the 43 sites in sub-Saharan Africa, including malaria vectors with multiple resistance mechanisms to pyrethroids, carbamates and organophosphates. CONCLUSIONS: This study provides an interim diagnostic dose of 2% w/v clothianidin on filter papers which can be utilized by National Malaria Control Programmes and research organizations until the WHO concludes multi-centre studies and provides further guidance.

High birth prevalence of sickle cell disease in Northwestern Tanzania.

BACKGROUND: Worldwide, hemoglobinopathies affect millions of children. Identification of hemoglobin disorders in most sub-Saharan African countries is delayed until clinical signs of the disease are present. Limited studies have been conducted to understand their prevalence and clinical presentation among newborns in resource-limited settings. METHODOLOGY: This was a prospective cohort study. Newborns (aged 0-7 days) at two hospitals in Northwestern Tanzania were enrolled and followed prospectively for 6 months. Clinical and laboratory information were collected at baseline. Participants were screened for hemoglobinopathies using high-performance liquid chromatography. Clinical and laboratory follow-up was performed at 3 and 6 months for those with hemoglobinopathies as well as a comparison group of participants without hemoglobinopathies. RESULTS: A total of 919 newborns were enrolled. Among these, 1.4% (13/919) had sickle cell anemia or Hb S/ß0 -thalassemia (Hb FS), and 19.7% (181/919) had sickle cell trait or Hb S/ß+ thalassemia (Hb FAS). Furthermore, 0.2% (two of 919) had ß+ -thalassemia. Red cell indices compared between Hb FS, Hb FAS, and Hb FA were similar at baseline, but hemoglobin was lower and red cell distribution width was higher in children with Hb FS at 3- and 6-month follow-up. Febrile episodes were more common for children with Hb FS at 3- and 6-month follow-up. CONCLUSION: The prevalence of sickle cell disease among neonates born in Northwestern Tanzania is one of the highest in the world. Newborn screening is needed early in life to identify neonates with hemoglobinopathies so that clinical management may commence and morbidity and mortality related to hemoglobinopathies be reduced.

African glucose-6-phosphate dehydrogenase alleles associated with protection from severe malaria in heterozygous females in Tanzania.

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers.

Malaria Host Candidate Genes Validated by Association With Current, Recent, and Historical Measures of Transmission Intensity.

Background: Human malaria susceptibility is determined by multiple genetic factors. It is unclear, however, which genetic variants remain important over time. Methods: Genetic associations of 175 high-quality polymorphisms within several malaria candidate genes were examined in a sample of 8096 individuals from northeast Tanzania using altitude, seroconversion rates, and parasite rates as proxies of historical, recent, and current malaria transmission intensity. A principal component analysis was used to derive 2 alternative measures of overall malaria propensity of a location across different time scales. Results: Common red blood cell polymorphisms (ie, hemoglobin S, glucose-6-phosphate dehydrogenase, and α-thalassemia) were the only ones to be associated with all 3 measures of transmission intensity and the first principal component. Moderate associations were found between some immune response genes (ie, IL3 and IL13) and parasite rates, but these could not be reproduced using the alternative measures of malaria propensity. Conclusions: We have demonstrated the potential of using altitude and seroconversion rate as measures of malaria transmission capturing medium- to long-term time scales to detect genetic associations that are likely to persist over time. These measures also have the advantage of minimizing the deleterious effects of random factors affecting parasite rates on the respective association signals.

The Severity of Plasmodium falciparum Infection Is Associated with Transcript Levels of var Genes Encoding Endothelial Protein C Receptor-Binding P. falciparum Erythrocyte Membrane Protein 1.

By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites leave blood circulation and avoid splenic clearance. This sequestration is central to pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct P. falciparum erythrocyte membrane protein 1 (PfEMP1) subset mediating binding to endothelial receptors. Previous studies indicate that PfEMP1 adhesins with so-called CIDRα1 domains capable of binding endothelial protein C receptor (EPCR) constitute the PfEMP1 subset associated with severe pediatric malaria. To analyze the relative importance of different subtypes of CIDRα1 domains, we compared Pfemp1 transcript levels in children with severe malaria (including 9 fatal and 114 surviving cases), children hospitalized with uncomplicated malaria (n = 42), children with mild malaria not requiring hospitalization (n = 10), and children with parasitemia and no ongoing fever (n = 12). High levels of transcripts encoding EPCR-binding PfEMP1 were found in patients with symptomatic infections, and the abundance of these transcripts increased with disease severity. The compositions of CIDRα1 subtype transcripts varied markedly between patients, and none of the subtypes were dominant. Transcript-level analyses targeting other domain types indicated that subtypes of DBLß or DBLζ domains might mediate binding phenomena that, in conjunction with EPCR binding, could contribute to pathogenesis. These observations strengthen the rationale for targeting the PfEMP1-EPCR interaction by vaccines and adjunctive therapies. Interventions should target EPCR binding of all CIDRα1 subtypes.

USP38, FREM3, SDC1, DDC, and LOC727982 Gene Polymorphisms and Differential Susceptibility to Severe Malaria in Tanzania.

Populations exposed to Plasmodium falciparum infection develop genetic mechanisms of protection against severe malarial disease. Despite decades of genetic epidemiological research, the sickle cell trait (HbAS) sickle cell polymorphism, ABO blood group, and other hemoglobinopathies remain the few major determinants in severe malaria to be replicated across different African populations and study designs. Within a case-control study in a region of high transmission in Tanzania (n = 983), we investigated the role of 40 new loci identified in recent genome-wide studies. In 32 loci passing quality control procedures, we found polymorphisms in USP38, FREM3, SDC1, DDC, and LOC727982 genes to be putatively associated with differential susceptibility to severe malaria. Established candidates explained 7.4% of variation in severe malaria risk (HbAS polymorphism, 6.3%; α-thalassemia, 0.3%; ABO group, 0.3%; and glucose-6-phosphate dehydrogenase deficiency, 0.5%) and the new polymorphisms, another 4.3%. The regions encompassing the loci identified are promising targets for the design of future treatment and control interventions.

Genetic determinants of anti-malarial acquired immunity in a large multi-centre study.

BACKGROUND: Many studies report associations between human genetic factors and immunity to malaria but few have been reliably replicated. These studies are usually country-specific, use small sample sizes and are not directly comparable due to differences in methodologies. This study brings together samples and data collected from multiple sites across Africa and Asia to use standardized methods to look for consistent genetic effects on anti-malarial antibody levels. METHODS: Sera, DNA samples and clinical data were collected from 13,299 individuals from ten sites in Senegal, Mali, Burkina Faso, Sudan, Kenya, Tanzania, and Sri Lanka using standardized methods. DNA was extracted and typed for 202 Single Nucleotide Polymorphisms with known associations to malaria or antibody production, and antibody levels to four clinical grade malarial antigens [AMA1, MSP1, MSP2, and (NANP)4] plus total IgE were measured by ELISA techniques. Regression models were used to investigate the associations of clinical and genetic factors with antibody levels. RESULTS: Malaria infection increased levels of antibodies to malaria antigens and, as expected, stable predictors of anti-malarial antibody levels included age, seasonality, location, and ethnicity. Correlations between antibodies to blood-stage antigens AMA1, MSP1 and MSP2 were higher between themselves than with antibodies to the (NANP)4 epitope of the pre-erythrocytic circumsporozoite protein, while there was little or no correlation with total IgE levels. Individuals with sickle cell trait had significantly lower antibody levels to all blood-stage antigens, and recessive homozygotes for CD36 (rs321198) had significantly lower anti-malarial antibody levels to MSP2. CONCLUSION: Although the most significant finding with a consistent effect across sites was for sickle cell trait, its effect is likely to be via reducing a microscopically positive parasitaemia rather than directly on antibody levels. However, this study does demonstrate a framework for the feasibility of combining data from sites with heterogeneous malaria transmission levels across Africa and Asia with which to explore genetic effects on anti-malarial immunity.

On the performance of multiple imputation based on chained equations in tackling missing data of the African α3.7 -globin deletion in a malaria association study.

Multiple imputation based on chained equations (MICE) is an alternative missing genotype method that can use genetic and nongenetic auxiliary data to inform the imputation process. Previously, MICE was successfully tested on strongly linked genetic data. We have now tested it on data of the HBA2 gene which, by the experimental design used in a malaria association study in Tanzania, shows a high missing data percentage and is weakly linked with the remaining genetic markers in the data set. We constructed different imputation models and studied their performance under different missing data conditions. Overall, MICE failed to accurately predict the true genotypes. However, using the best imputation model for the data, we obtained unbiased estimates for the genetic effects, and association signals of the HBA2 gene on malaria positivity. When the whole data set was analyzed with the same imputation model, the association signal increased from 0.80 to 2.70 before and after imputation, respectively. Conversely, postimputation estimates for the genetic effects remained the same in relation to the complete case analysis but showed increased precision. We argue that these postimputation estimates are reasonably unbiased, as a result of a good study design based on matching key socio-environmental factors.