A cluster-randomized trial of client and provider directed financial interventions to align incentives with appropriate case management in private medicine retailers: Results of the TESTsmART trial in Lagos, Nigeria.

Malaria remains a major health priority in Nigeria. Among children with fever who seek care, less than a quarter gets tested for malaria, leading to inappropriate use of the recommended treatment for malaria; Artemisinin-based Combination Therapy (ACT). Here we test an innovative strategy to target ACT subsidies to clients seeking care in Nigeria's private retail health sector who have a confirmed malaria diagnosis. We supported point-of-care malaria testing (mRDTs) in 48 Private Medicine Retailers (PMRs) in the city of Lagos, Nigeria and randomized them to two study arms; a control arm offering subsidized mRDT testing for USD $0.66, and an intervention arm where, in addition to access to subsidized testing as in the control arm, clients who received a positive mRDT at the PMR were eligible for a free (fully subsidized) first-line ACT and PMRs received USD $0.2 for every mRDT performed. Our primary outcome was the proportion of ACTs dispensed to individuals with a positive diagnostic test. Secondary outcomes included proportion of clients who were tested at the PMR and adherence to diagnostic test results. Overall, 23% of clients chose to test at the PMR. Test results seemed to inform treatment decisions and resulted in enhanced targeting of ACTs to confirmed malaria cases with only 26% of test-negative clients purchasing an ACT compared to 58% of untested clients. However, the intervention did not offer further improvements, compared to the control arm, in testing rates or dispensing of ACTs to test-positive clients. We found that ACT subsidies were not passed on to clients testing positive in the intervention arm. We conclude that mRDTs could reduce ACT overconsumption in Nigeria's private retail health sector, but PMR-oriented incentive structures are difficult to implement and may need to be complemented with interventions targeting clients of PMRs to increase test uptake and adherence. Trials registration: Clinical Trials Registration Number: NCT04428307. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816435/ Correction: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9476591/.

bistro: An R package for vector bloodmeal identification by short tandem repeat overlap.

Measuring vector-human contact in a natural setting can inform precise targeting of interventions to interrupt transmission of vector-borne diseases. One approach is to directly match human DNA in vector bloodmeals to the individuals who were bitten using genotype panels of discriminative short tandem repeats (STRs). Existing methods for matching STR profiles in bloodmeals to the people bitten preclude the ability to match most incomplete profiles and multi-source bloodmeals to bitten individuals.We developed bistro, an R package that implements 3 preexisting STR matching methods as well as the package's namesake, bistro, a new algorithm described here. bistro employs forensic analysis methods to calculate likelihood ratios and match human STR profiles in bloodmeals to people using a dynamic threshold. We evaluated the algorithm's accuracy and compared it to existing matching approaches using a publicly-available panel of 188 single-source and 100 multi-source samples containing DNA from 50 known human sources. Then we applied it to match 777 newly field-collected mosquito bloodmeals to a database of 645 people.The R package implements four STR matching algorithms in user-friendly functions with clear documentation. bistro correctly matched 99% (187/188) of profiles in single-source samples, and 62% (224/359) of profiles from multi-source samples, resulting in a sensitivity of 0.75 (vs < 0.51 for other algorithms). The specificity of bistro was 0.9998 (vs. 1 for other algorithms). Furthermore, bistro identified 79% (720/906) of all possible matches for field-derived mosquitoes, yielding 1.4x more matches than existing algorithms.bistro identifies more correct bloodmeal-human matches than existing approaches, enabling more accurate and robust analyses of vector-human contact in natural settings. The bistro R package and corresponding documentation allow for straightforward uptake of this algorithm by others.

À la carte: how mosquitoes choose their blood meals.

Mosquitoes are important vectors for human diseases, transmitting pathogens that cause a range of parasitic and viral infections. Mosquito blood-feeding is heterogeneous, meaning that some human hosts are at higher risk of receiving bites than others, and this heterogeneity is multifactorial. Mosquitoes integrate specific cues to locate their hosts, and mosquito attraction differs considerably between individual human hosts. Heterogeneous mosquito biting results from variations in both host attractiveness and availability and can impact transmission of vector-borne diseases. However, the extent and drivers of this heterogeneity and its importance for pathogen transmission remain incompletely understood. Here, we review methods and recent data describing human characteristics that affect host-seeking behavior and host preferences of mosquito disease vectors, and the implications for vector-borne disease transmission.

Analytic optimization of Plasmodium falciparum marker gene haplotype recovery from amplicon deep sequencing of complex mixtures.

Molecular epidemiologic studies of malaria parasites and other pathogens commonly employ amplicon deep sequencing (AmpSeq) of marker genes derived from dried blood spots (DBS) to answer public health questions related to topics such as transmission and drug resistance. As these methods are increasingly employed to inform direct public health action, it is important to rigorously evaluate the risk of false positive and false negative haplotypes derived from clinically-relevant sample types. We performed a control experiment evaluating haplotype recovery from AmpSeq of 5 marker genes (ama1, csp, msp7, sera2, and trap) from DBS containing mixtures of DNA from 1 to 10 known P. falciparum reference strains across 3 parasite densities in triplicate (n = 270 samples). While false positive haplotypes were present across all parasite densities and mixtures, we optimized censoring criteria to remove 83% (148/179) of false positives while removing only 8% (67/859) of true positives. Post-censoring, the median pairwise Jaccard distance between replicates was 0.83. We failed to recover 35% (477/1365) of haplotypes expected to be present in the sample. Haplotypes were more likely to be missed in low-density samples with <1.5 genomes/µL (OR: 3.88, CI: 1.82-8.27, vs. high-density samples with ≥75 genomes/µL) and in samples with lower read depth (OR per 10,000 reads: 0.61, CI: 0.54-0.69). Furthermore, minority haplotypes within a sample were more likely to be missed than dominant haplotypes (OR per 0.01 increase in proportion: 0.96, CI: 0.96-0.97). Finally, in clinical samples the percent concordance across markers for multiplicity of infection ranged from 40%-80%. Taken together, our observations indicate that, with sufficient read depth, the majority of haplotypes can be successfully recovered from DBS while limiting the false positive rate.

Plasmodium falciparum infection in humans and mosquitoes influence natural Anopheline biting behavior and transmission.

The human infectious reservoir of Plasmodium falciparum is governed by transmission efficiency during vector-human contact and mosquito biting preferences. Understanding biting bias in a natural setting can help target interventions to interrupt transmission. In a 15-month cohort in western Kenya, we detected P. falciparum in indoor-resting Anopheles and human blood samples by qPCR and matched mosquito bloodmeals to cohort participants using short-tandem repeat genotyping. Using risk factor analyses and discrete choice models, we assessed mosquito biting behavior with respect to parasite transmission. Biting was highly unequal; 20% of people received 86% of bites. Biting rates were higher on males (biting rate ratio (BRR): 1.68; CI: 1.28-2.19), children 5-15 years (BRR: 1.49; CI: 1.13-1.98), and P. falciparum-infected individuals (BRR: 1.25; CI: 1.01-1.55). In aggregate, P. falciparum-infected school-age (5-15 years) boys accounted for 50% of bites potentially leading to onward transmission and had an entomological inoculation rate 6.4x higher than any other group. Additionally, infectious mosquitoes were nearly 3x more likely than non-infectious mosquitoes to bite P. falciparum-infected individuals (relative risk ratio 2.76, 95% CI 1.65-4.61). Thus, persistent P. falciparum transmission was characterized by disproportionate onward transmission from school-age boys and by the preference of infected mosquitoes to feed upon infected people.

Relationship between malaria vector survival, infectivity and insecticide treated net use in western Kenya.

Background: Much effort and resources have been invested to control malaria transmission in Sub-Saharan Africa, but it remains a major public health problem. For the disease to be transmitted from one person to another, the female Anopheles vector must survive 10-14 days following an infective bite for the Plasmodiumgametocytes to develop into infectious sporozoites which can be transmitted to the next person during a bloodmeal. The goal of this investigation was to assess factors associated with wild-caught Anopheles survival and infection following host-seeking and indoor resting. Methods: The study was conducted in a longitudinal cohort of 75 households in 5 villages including a total of 755 household members in Bungoma County, Kenya. Monthly adult mosquito collection was conducted by attenuated aspiration in all the enrolled households, and the mosquitoes were reared in the insectary for 7 days. The daily mortality rate was determined through day 7, and all the mosquitoes were morphologically identified. Female Anopheline mosquitoes were dissected, and species-level members of the Anopheles gambiae complex were resolved by molecular methods. The abdomen for all samples were processed for P. falciparum detection by PCR. Results: Within a period of 25 months, the total number of culex and Anopheles mosquitoes collected indoors were 12,843 and 712 respectively. Anopheles gambiaeand Anopheles funestus were the major vectors though their population varied between different villages. 61.2% (n=436/712) of the Anopheles species survived up to day 7 with the lowest mortality rate recorded on day 5 of captivity. The survival rate also varied between the different Anophelesspecies. 683 of 712 mosquito abdomens were tested for P. falciparumdetection and 7.8% (53/683) tested positive for P. falciparum with An. funestus having a higher (10%) prevalence than An. gambaie s.s.(6.0%, p=0.095, Pearson Chi square test). The proportion of household members sleeping under a bednet the night before mosquito collection varied across time and village. An. funestus survival times were refractory to household ITN coverage and An. gambaie s.s. survival was reduced only under very high (>95%) ITN coverage. Conclusion: Despite ITN coverage, mosquitoes still acquired bloodmeals and P. falciparum infections. Survival differed across species and was inversely correlated with high ITN exposure in the household, but not oocyst development.

Characterizing mobility patterns and malaria risk factors in semi-nomadic populations of Northern Kenya.

While many studies have characterized mobility patterns and disease dynamics of settled populations, few have focused on more mobile populations. Highly mobile groups are often at higher disease risk due to their regular movement that may increase the variability of their environments, reduce their access to health care, and limit the number of intervention strategies suitable for their lifestyles. Quantifying the movements and their associated disease risks will be key to developing interventions more suitable for mobile populations. Turkana, Kenya is an ideal setting to characterize these relationships. While the vast, semi-arid county has a large mobile population (>60%) and was recently shown to have endemic malaria, the relationship between mobility and malaria risk in this region has not yet been defined. Here, we worked with 250 semi-nomadic households from four communities in Central Turkana to 1) characterize mobility patterns of travelers and 2) test the hypothesis that semi-nomadic individuals are at greater risk of malaria exposure when migrating with their herds than when staying at their semi-permanent settlements. Participants provided medical and travel histories, demographics, and a dried blood spot for malaria testing before and after the travel period. Further, a subset of travelers was given GPS loggers to document their routes. Four travel patterns emerged from the logger data, Long Term, Transient, Day trip, and Static, with only Long Term and Transient trips being associated with malaria cases detected in individuals who carried GPS devices. After completing their trips, travelers had a higher prevalence of malaria than those who remained at the household (9.2% vs 4.4%), regardless of gender and age. These findings highlight the need to develop intervention strategies amenable to mobile lifestyles that can ultimately help prevent the transmission of malaria.

A cluster-randomized trial of client and provider-directed financial interventions to align incentives with appropriate case management in retail medicine outlets: Results of the TESTsmART Trial in western Kenya.

ACTs are responsible for a substantial proportion of the global reduction in malaria mortality over the last ten years, made possible by publicly-funded subsidies making these drugs accessible and affordable in the private sector. However, inexpensive ACTs available in retail outlets have contributed substantially to overconsumption. We test an innovative, scalable strategy to target ACT-subsidies to clients with a confirmatory diagnosis. We supported malaria testing(mRDTs) in 39 medicine outlets in western Kenya, randomized to three study arms; control arm offering subsidized mRDT testing (0.4USD), client-directed intervention where all clients who received a positive RDT at the outlet were eligible for a free (fully-subsidized) ACT, and a combined client and provider directed intervention where clients with a positive RDT were eligible for free ACT and outlets received 0.1USD for every RDT performed. Our primary outcome was the proportion of ACT dispensed to individuals with a positive diagnostic test. Secondary outcomes included proportion of clients tested at the outlet and adherence to diagnostic test results. 43% of clients chose to test at the outlet. Test results informed treatment decisions, resulting in targeting of ACTs to confirmed malaria cases- 25.3% of test-negative clients purchased an ACT compared to 75% of untested clients. Client-directed and client+provider-directed interventions did not offer further improvements, compared to the control arm, in testing rates(RD = 0.09, 95%CI:-0.08,0.26) or dispensing of ACTs to test-positive clients(RD = 0.01,95% CI:-0.14, 0.16). Clients were often unaware of the price they paid for the ACT leading to uncertainty in whether the ACT subsidy was passed on to the client. This uncertainty undermines our ability to definitively conclude that client-directed subsidies are not effective for improving testing and appropriate treatment. We conclude that mRDTs could reduce ACT overconsumption in the private retail sector, but incentive structures are difficult to scale and their value to private providers is uncertain. Trial registration: ClinicalTrials.gov NCT04428307.

Characterizing mobility patterns and malaria risk factors in semi-nomadic populations of Northern Kenya.

While many studies have characterized mobility patterns and disease dynamics of individuals from settled populations, few have focused on more mobile populations. Highly mobile groups are often at higher disease risk due to their regular movement that may increase the variability of their environments, reduce their access to health care, and limit the number of intervention strategies suitable for their lifestyles. Quantifying the movements and their associated disease risks will be key to developing intervention strategies more suitable for mobile populations. Here, we worked with four semi-nomadic communities in Central Turkana, Kenya to 1) characterize mobility patterns of travelers from semi-nomadic communities and 2) test the hypothesis that semi-nomadic individuals are at greater risk of exposure to malaria during seasonal migrations than when staying at their semi-permanent settlements. From March-October, 2021, we conducted a study in semi-nomadic households (n=250) where some members traveled with their herd while others remained at the semi-permanent settlement. Participants provided medical and travel histories, demographics, and a dried blood spot for malaria testing before and after the travel period. Further, a subset of travelers was given GPS loggers to document their routes. Four travel patterns emerged from the logger data, Long Term, Transient, Day trip, and Static, with only Long Term and Transient trips being associated with malaria cases detected in individuals who carried GPS devices. After completing their trips, travelers had a higher prevalence of malaria than those who remained at the household (9.2% vs 4.4%), regardless of gender, age group, and catchment area. These findings highlight the need to develop intervention strategies amenable to mobile lifestyles that can ultimately help prevent the transmission of malaria.

Detection of Anopheles stephensi Mosquitoes by Molecular Surveillance, Kenya.

The Anopheles stephensi mosquito is an invasive malaria vector recently reported in Djibouti, Ethiopia, Sudan, Somalia, Nigeria, and Ghana. The World Health Organization has called on countries in Africa to increase surveillance efforts to detect and report this vector and institute appropriate and effective control mechanisms. In Kenya, the Division of National Malaria Program conducted entomological surveillance in counties at risk for An. stephensi mosquito invasion. In addition, the Kenya Medical Research Institute conducted molecular surveillance of all sampled Anopheles mosquitoes from other studies to identify An. stephensi mosquitoes. We report the detection and confirmation of An. stephensi mosquitoes in Marsabit and Turkana Counties by using endpoint PCR and morphological and sequence identification. We demonstrate the urgent need for intensified entomological surveillance in all areas at risk for An. stephensi mosquito invasion, to clarify its occurrence and distribution and develop tailored approaches to prevent further spread.

Plasmodium vivax Prevalence in Semiarid Region of Northern Kenya, 2019.

In urban and rural areas of Turkana County, Kenya, we found that 2% of household members of patients with Plasmodium falciparum infections were infected with P. vivax. Enhanced surveillance of P. vivax and increased clinical resources are needed to inform control measures and identify and manage P. vivax infections.

Adherence to national malaria treatment guidelines in private drug outlets: a cross-sectional survey in the malaria-endemic Kisumu County, Kenya.

BACKGROUND: Malaria prevalence in Kenya is 6%, with a three-fold higher prevalence in western Kenya. Adherence to malaria treatment guidelines improves care for suspected malaria cases and can reduce unnecessary anti-malarial use. Data on adherence to guidelines in retail drug outlets (DOs) is limited, yet approximately 50% of people with fever access treatment first in these outlets. This study assessed adherence to the national malaria treatment guidelines among DOs in a high transmission area of Western Kenya. METHODS: In a cross-sectional survey of DOs in Kisumu Central and Seme sub-counties in 2021, DO staff were interviewed using structured questionnaires to assess outlet characteristics (location, testing services), staff demographics (age, sex, training), and health system context (supervision, inspection). Mystery shoppers (research assistants disguised as clients) observed malaria management practices and recorded observations on a standardized tool. Adherence was defined as dispensing artemether-lumefantrine (AL) to patients with a confirmed positive test, accompanied by appropriate medication counseling. Logistic regression was used to test for association between adherence to guidelines and DO-related factors. RESULTS: None of the 70 DOs assessed had a copy of the guidelines, and 60 (85.7%) were in an urban setting. Staff adhered to the guidelines in 14 (20%) outlets. The odds of adherence were higher among staff who had a bachelor's degree {odds ratio (OR) 6.0, 95% confidence interval (95% CI) 1.66-21.74}, those trained on malaria rapid diagnostic test (RDT) {OR 4.4, 95% CI 1.29-15.04}, and those who asked about patient's symptoms {OR 3.6, 95% CI 1.08-12.25}. DOs that had higher odds of adherence included those with functional thermometers {OR 5.3, 95% CI 1.46-19.14}, those recently inspected (within three months) by Pharmacy and Poisons Board (PPB) {OR 9.4, 95% CI 2.55-34.67}, and those with all basic infrastructure {OR 3.9, 95% CI 1.01-15.00}. On logistic regression analysis, recent PPB inspection {adjusted OR (AOR) 4.6, 95% CI 1.03-20.77} and malaria RDT-trained staff (aOR 4.5, 95% CI 1.02-19.84) were independently associated with adherence. CONCLUSION: Most outlets didn't adhere to malaria guidelines. Regular interaction with regulatory bodies could improve adherence. Ministry of Health should enhance private sector engagement and train DOs on RDT use.

bistro: An R package for vector bloodmeal identification by short tandem repeat overlap.

1. Measuring vector-human contact in a natural setting can inform precise targeting of interventions to interrupt transmission of vector-borne diseases. One approach is to directly match human DNA in vector bloodmeals to the individuals who were bitten using genotype panels of discriminative short tandem repeats (STRs). Existing methods for matching STR profiles in bloodmeals to the people bitten preclude the ability to match most incomplete profiles and multi-source bloodmeals to bitten individuals. 2. We developed bistro, an R package that implements 3 preexisting STR matching methods as well as the package's namesake, bistro, a new algorithm described here. bistro employs forensic analysis methods to calculate likelihood ratios and match human STR profiles in bloodmeals to people using a dynamic threshold. We evaluated the algorithm's accuracy and compared it to existing matching approaches using a publicly-available panel of 188 single-source and 100 multi-source samples containing DNA from 50 known human sources. Then we applied it to match 777 newly field-collected mosquito bloodmeals to a database of 645 people. 3. The R package implements four STR matching algorithms in user-friendly functions with clear documentation. bistro correctly matched 99% (184/185) of profiles in single-source samples, and 63% (225/359) of profiles from multi-source samples, resulting in a sensitivity of 0.75 (vs < 0.51 for other algorithms). The specificity of bistro was 0.9998 (vs. 1 for other algorithms). Furthermore, bistro identified 80% (729/909) of all possible matches for field-derived mosquitoes, yielding 1.4x more matches than existing algorithms. 4. bistro identifies more correct bloodmeal-human matches than existing approaches, enabling more accurate and robust analyses of vector-human contact in natural settings. The bistro R package and corresponding documentation allow for straightforward uptake of this algorithm by others.

Analytic optimization of Plasmodium falciparum marker gene haplotype recovery from amplicon deep sequencing of complex mixtures.

Molecular epidemiologic studies of malaria parasites commonly employ amplicon deep sequencing (AmpSeq) of marker genes derived from dried blood spots (DBS) to answer public health questions related to topics such as transmission and drug resistance. As these methods are increasingly employed to inform direct public health action, it is important to rigorously evaluate the risk of false positive and false negative haplotypes derived from clinically-relevant sample types. We performed a control experiment evaluating haplotype recovery from AmpSeq of 5 marker genes (ama1, csp, msp7, sera2, and trap) from DBS containing mixtures of DNA from 1 to 10 known P. falciparum reference strains across 3 parasite densities in triplicate (n=270 samples). While false positive haplotypes were present across all parasite densities and mixtures, we optimized censoring criteria to remove 83% (148/179) of false positives while removing only 8% (67/859) of true positives. Post-censoring, the median pairwise Jaccard distance between replicates was 0.83. We failed to recover 35% (477/1365) of haplotypes expected to be present in the sample. Haplotypes were more likely to be missed in low-density samples with <1.5 genomes/µL (OR: 3.88, CI: 1.82-8.27, vs. high-density samples with ≥75 genomes/µL) and in samples with lower read depth (OR per 10,000 reads: 0.61, CI: 0.54-0.69). Furthermore, minority haplotypes within a sample were more likely to be missed than dominant haplotypes (OR per 0.01 increase in proportion: 0.96, CI: 0.96-0.97). Finally, in clinical samples the percent concordance across markers for multiplicity of infection ranged from 40%-80%. Taken together, our observations indicate that, with sufficient read depth, haplotypes can be successfully recovered from DBS while limiting the false positive rate.

Symptomatic malaria enhances protection from reinfection with homologous Plasmodium falciparum parasites.

A signature remains elusive of naturally-acquired immunity against Plasmodium falciparum. We identified P. falciparum in a 14-month cohort of 239 people in Kenya, genotyped at immunogenic parasite targets expressed in the pre-erythrocytic (circumsporozoite protein, CSP) and blood (apical membrane antigen 1, AMA-1) stages, and classified into epitope type based on variants in the DV10, Th2R, and Th3R epitopes in CSP and the c1L region of AMA-1. Compared to asymptomatic index infections, symptomatic malaria was associated with reduced reinfection by parasites bearing homologous CSP-Th2R (adjusted hazard ratio [aHR]:0.63; 95% CI:0.45-0.89; p = 0.008) CSP-Th3R (aHR:0.71; 95% CI:0.52-0.97; p = 0.033), and AMA-1 c1L (aHR:0.63; 95% CI:0.43-0.94; p = 0.022) epitope types. The association of symptomatic malaria with reduced hazard of homologous reinfection was strongest for rare epitope types. Symptomatic malaria provides more durable protection against reinfection with parasites bearing homologous epitope types. The phenotype represents a legible molecular epidemiologic signature of naturally-acquired immunity by which to identify new antigen targets.

Supply-side and demand-side factors influencing uptake of malaria testing services in the community: lessons for scale-up from a post-hoc analysis of a cluster randomised, community-based trial in western Kenya.

OBJECTIVES: Maximising the impact of community-based programmes requires understanding how supply of, and demand for, the intervention interact at the point of delivery. DESIGN: Post-hoc analysis from a large-scale community health worker (CHW) study designed to increase the uptake of malaria diagnostic testing. SETTING: Respondents were identified during a household survey in western Kenya between July 2016 and April 2017. PARTICIPANTS: Household members with fever in the last 4 weeks were interviewed at 12 and 18 months post-implementation. We collected monthly testing data from 244 participating CHWs and conducted semistructured interviews with a random sample of 70 CHWs. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome measure was diagnostic testing before treatment for a recent fever. The secondary outcomes were receiving a test from a CHW and tests done per month by each CHW. RESULTS: 55% (n=948 of 1738) reported having a malaria diagnostic test for their recent illness, of which 38.4% were tested by a CHW. Being aware of a local CHW (adjusted OR=1.50, 95% CI: 1.10 to 2.04) and belonging to the wealthiest households (vs least wealthy) were associated with higher testing (adjusted OR=1.53, 95% CI: 1.14 to 2.06). Wealthier households were less likely to receive their test from a CHW compared with poorer households (adjusted OR=0.32, 95% CI: 0.17 to 0.62). Confidence in artemether-lumefantrine to cure malaria (adjusted OR=2.75, 95% CI: 1.54 to 4.92) and perceived accuracy of a malaria rapid diagnostic test (adjusted OR=2.43, 95% CI: 1.12 to 5.27) were positively associated with testing by a CHW. Specific CHW attributes were associated with performing a higher monthly number of tests including formal employment, serving more than 50 households (vs <50) and serving areas with a higher test positivity. On demand side, confidence of the respondent in a test performed by a CHW was strongly associated with seeking a test from a CHW. CONCLUSION: Scale-up of community-based malaria testing is feasible and effective in increasing uptake among the poorest households. To maximise impact, it is important to recognise factors that may restrict delivery and demand for such services. TRIAL REGISTRATION NUMBER: NCT02461628; Post-results.

Symptomatic malaria enhances protection from reinfection with homologous Plasmodium falciparum parasites.

A signature remains elusive of naturally-acquired immunity against Plasmodium falciparum . We identified P. falciparum in a 14-month cohort of 239 people in Kenya, genotyped at immunogenic parasite targets expressed in the pre-erythrocytic (circumsporozoite protein, CSP) and blood (apical membrane antigen 1, AMA-1) stages, and classified into epitope type based on variants in the DV10, Th2R, and Th3R epitopes in CSP and the c1L region of AMA-1. Compared to asymptomatic index infections, symptomatic malaria was associated with a reduced reinfection by parasites bearing homologous CSP-Th2R (adjusted hazard ratio [aHR]:0.63; 95% CI:0.45-0.89; p=0.008) CSP-Th3R (aHR:0.71; 95% CI:0.52-0.97; p=0.033), and AMA-1 c1L (aHR:0.63; 95% CI:0.43-0.94; p=0.022) epitope types. The association of symptomatic malaria with reduced risk of homologous reinfection was strongest for rare epitope types. Symptomatic malaria more effectively promotes functional immune responses. The phenotype represents a legible molecular epidemiologic signature of naturally-acquired immunity by which to identify new antigen targets.

Exploring how space, time, and sampling impact our ability to measure genetic structure across Plasmodium falciparum populations.

A primary use of malaria parasite genomics is identifying highly related infections to quantify epidemiological, spatial, or temporal factors associated with patterns of transmission. For example, spatial clustering of highly related parasites can indicate foci of transmission and temporal differences in relatedness can serve as evidence for changes in transmission over time. However, for infections in settings of moderate to high endemicity, understanding patterns of relatedness is compromised by complex infections, overall high forces of infection, and a highly diverse parasite population. It is not clear how much these factors limit the utility of using genomic data to better understand transmission in these settings. In particular, further investigation is required to determine which patterns of relatedness we expect to see with high quality, densely sampled genomic data in a high transmission setting and how these observations change under different study designs, missingness, and biases in sample collection. Here we investigate two identity-by-state measures of relatedness and apply them to amplicon deep sequencing data collected as part of a longitudinal cohort in Western Kenya that has previously been analysed to identify individual-factors associated with sharing parasites with infected mosquitoes. With these data we use permutation tests, to evaluate several hypotheses about spatiotemporal patterns of relatedness compared to a null distribution. We observe evidence of temporal structure, but not of fine-scale spatial structure in the cohort data. To explore factors associated with the lack of spatial structure in these data, we construct a series of simplified simulation scenarios using an agent based model calibrated to entomological, epidemiological and genomic data from this cohort study to investigate whether the lack of spatial structure observed in the cohort could be due to inherent power limitations of this analytical method. We further investigate how our hypothesis testing behaves under different sampling schemes, levels of completely random and systematic missingness, and different transmission intensities.

Monthly sulfadoxine/pyrimethamine-amodiaquine or dihydroartemisinin-piperaquine as malaria chemoprevention in young Kenyan children with sickle cell anemia: A randomized controlled trial.

BACKGROUND: Children with sickle cell anemia (SCA) in areas of Africa with endemic malaria transmission are commonly prescribed malaria chemoprevention. Chemoprevention regimens vary between countries, and the comparative efficacy of prevention regimens is largely unknown. METHODS AND FINDINGS: We enrolled Kenyan children aged 1 to 10 years with homozygous hemoglobin S (HbSS) in a randomized, open-label trial conducted between January 23, 2018, and December 15, 2020, in Homa Bay, Kenya. Children were assigned 1:1:1 to daily Proguanil (the standard of care), monthly sulfadoxine/pyrimethamine-amodiaquine (SP-AQ), or monthly dihydroartemisinin-piperaquine (DP) and followed monthly for 12 months. The primary outcome was the cumulative incidence of clinical malaria at 12 months, and the main secondary outcome was the cumulative incidence of painful events by self-report. Secondary outcomes included other parasitologic, hematologic, and general events. Negative binomial models were used to estimate incidence rate ratios (IRRs) per patient-year (PPY) at risk relative to Proguanil. The primary analytic population was the As-Treated population. A total of 246 children were randomized to daily Proguanil (n = 81), monthly SP-AQ (n = 83), or monthly DP (n = 82). Overall, 53.3% (n = 131) were boys and the mean age was 4.6 ± 2.5 years. The clinical malaria incidence was 0.04 episodes/PPY; relative to the daily Proguanil group, incidence rates were not significantly different in the monthly SP-AQ (IRR: 3.05, 95% confidence interval [CI]: 0.36 to 26.14; p = 0.39) and DP (IRR: 1.36, 95% CI: 0.21 to 8.85; p = 0.90) groups. Among secondary outcomes, relative to the daily Proguanil group, the incidence of painful events was not significantly different in the monthly SP-AQ and DP groups, while monthly DP was associated with a reduced rate of dactylitis (IRR: 0.47; 95% CI: 0.23 to 0.96; p = 0.038). The incidence of Plasmodium falciparum infection relative to daily Proguanil was similar in the monthly SP-AQ group (IRR 0.46; 95% CI: 0.17 to 1.20; p = 0.13) but reduced with monthly DP (IRR 0.21; 95% CI: 0.08 to 0.56; p = 0.002). Serious adverse events were common and distributed between groups, although compared to daily Proguanil (n = 2), more children died receiving monthly SP-AQ (n = 7; hazard ratio [HR] 5.44; 95% CI: 0.92 to 32.11; p = 0.064) but not DP (n = 1; HR 0.61; 95% CI 0.04 to 9.22; p = 0.89), although differences did not reach statistical significance for either SP-AQ or DP. Study limitations include the unexpectedly limited transmission of P. falciparum in the study setting, the high use of hydroxyurea, and the enhanced supportive care for trial participants, which may limit generalizability to higher-transmission settings where routine sickle cell care is more limited. CONCLUSIONS: In this study with limited malaria transmission, malaria chemoprevention in Kenyan children with SCA with monthly SP-AQ or DP did not reduce clinical malaria, but DP was associated with reduced dactylitis and P. falciparum parasitization. Pragmatic studies of chemoprevention in higher malaria transmission settings are warranted. TRIAL REGISTRATION: clinicaltrials.gov (NCT03178643). Pan-African Clinical Trials Registry: PACTR201707002371165.