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.

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.

Risk of Malaria Following Untreated Subpatent Plasmodium falciparum Infections: Results Over 4 Years From a Cohort in a High-Transmission Area in Western Kenya.

BACKGROUND: People with suspected malaria may harbor Plasmodium falciparum undetected by rapid diagnostic test (RDT). The impact of these subpatent infections on the risk of developing clinical malaria is not fully understood. METHODS: We analyzed subpatent P. falciparum infections using a longitudinal cohort in a high-transmission site in Kenya. Weighted Kaplan-Meier models estimated the risk difference (RD) for clinical malaria during the 60 days following a symptomatic subpatent infection. Stratum-specific estimates by age and transmission season assessed modification. RESULTS: Over 54 months, we observed 1128 symptomatic RDT-negative suspected malaria episodes, of which 400 (35.5%) harbored subpatent P. falciparum. Overall, the 60-day risk of developing clinical malaria was low following all episodes (8.6% [95% confidence interval, 6.7%-10.4%]). In the low-transmission season, the risk of clinical malaria was slightly higher in those with subpatent infection, whereas the opposite was true in the high-transmission season (low-transmission season RD, 2.3% [95% confidence interval, .4%-4.2%]; high-transmission season RD, -4.8% [-9.5% to -.05%]). CONCLUSIONS: The risk of developing clinical malaria among people with undetected subpatent infections is low. A slightly elevated risk in the low-transmission season may merit alternate management, but RDTs identify clinically relevant infections in the high-transmission season.

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.

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.

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.

Plasmodium falciparum Genetic Diversity in Coincident Human and Mosquito Hosts.

Population genetic diversity of Plasmodium falciparum antigenic loci is high despite large bottlenecks in population size during the parasite life cycle. The prevalence of genetically distinct haplotypes at these loci, while well characterized in humans, has not been thoroughly compared between human and mosquito hosts. We assessed parasite haplotype prevalence, diversity, and evenness using human and mosquito P. falciparum infections collected from the same households during a 14-month longitudinal cohort study using amplicon deep sequencing of two antigenic gene fragments (ama1 and csp). To a prior set of infected humans (n = 1,175/2,813; 86.2% sequencing success) and mosquito abdomens (n = 199/1,448; 95.5% sequencing success), we added sequences from infected mosquito heads (n = 134/1,448; 98.5% sequencing success). The overall and sample-level parasite populations were more diverse in mosquitoes than in humans. Additionally, haplotype prevalences were more even in the P. falciparum human population than in the mosquito population, consistent with balancing selection occurring at these loci in humans. In contrast, we observed that infections in humans were more likely to harbor a dominant haplotype than infections in mosquitoes, potentially due to removal of unfit strains by the human immune system. Finally, within a given mosquito, there was little overlap in genetic composition of abdomen and head infections, suggesting that infections may be cleared from the abdomen during a mosquito's lifespan. Taken together, our observations provide evidence for the mosquito vector acting as a reservoir of sequence diversity in malaria parasite populations. IMPORTANCE Plasmodium falciparum is the deadliest human malaria parasite, and infections consisting of concurrent, multiple strains are common in regions of high endemicity. During transitions within and between the parasite's mosquito and human hosts, these strains are subject to population bottlenecks, and distinct parasite strains may have differential fitness in the various environments encountered. These bottlenecks and fitness differences may lead to differences in strain prevalence and diversity between hosts. We investigated differences in genetic diversity and evenness between P. falciparum parasites in human and mosquito hosts collected from the same households during a 14-month longitudinal study in Kenya. Compared to human parasite populations and infections, P. falciparum parasites observed in mosquito populations and infections were more diverse by multiple population genetic metrics. This suggests that the mosquito vector acts as a reservoir of sequence diversity in malaria parasite populations.

Experience and confidence in health technologies: evidence from malaria testing and treatment in Western Kenya.

BACKGROUND: Low adoption of effective health technologies increases illness morbidity and mortality worldwide. In the case of malaria, effective tools such as malaria rapid diagnostic tests (RDTs) and artemisinin-combination therapies (ACTs) are both under-used and used inappropriately. Individuals' confidence in RDTs and ACTs likely affects the uptake of these tools. METHODS: In a cohort of 36 households (280 individuals) in Western Kenya observed for 30 months starting in June 2017, we examined if experience with RDTs and ACTs changes people's beliefs about these technologies and how those beliefs affect treatment behavior. Household members requested a free RDT from the study team any time they suspected a malaria illness, and positive RDT results were treated with a free ACT. We conducted annual, monthly, and sick visit surveys to elicit beliefs about the accuracy of malaria RDT results and the effectiveness of ACTs. Beliefs were elicited on a 5-point Likert scale from "very unlikely" to "very likely." RESULTS: Over the study period, the percentage of survey respondents that said a hypothetical negative RDT result was "very likely" to be correct increased from approximately 55% to 75%. Controlling for initial beliefs, people who had been tested at least once with an RDT in the past year had 3.6 times higher odds (95% CI [1 1.718 7.679], P = 0.001) of saying a negative RDT was "very likely" to be correct. Confidence in testing was associated with treatment behavior: those who believed a negative RDT was "very likely" to be correct had 1.78 times higher odds (95% CI [1.079 2.934], P = 0.024) of adhering to a negative RDT result (by not taking ACTs) than those who were less certain about the accuracy of negative RDTs. Adherence to a negative test also affected subsequent beliefs: controlling for prior beliefs, those who had adhered to their previous test result had approximately twice the odds (OR = 2.19, 95% CI [1.661 2.904], P < 0.001) of saying that a hypothetical negative RDT was "very likely" to be correct compared to those who had not adhered. CONCLUSIONS: Our results suggest that greater experience with RDTs can not only increase people's confidence in their accuracy but also improve adherence to the test result.

Plasmodium falciparum importation does not sustain malaria transmission in a semi-arid region of Kenya.

Human movement impacts the spread and transmission of infectious diseases. Recently, a large reservoir of Plasmodium falciparum malaria was identified in a semi-arid region of northwestern Kenya historically considered unsuitable for malaria transmission. Understanding the sources and patterns of transmission attributable to human movement would aid in designing and targeting interventions to decrease the unexpectedly high malaria burden in the region. Toward this goal, polymorphic parasite genes (ama1, csp) in residents and passengers traveling to Central Turkana were genotyped by amplicon deep sequencing. Genotyping and epidemiological data were combined to assess parasite importation. The contribution of travel to malaria transmission was estimated by modelling case reproductive numbers inclusive and exclusive of travelers. P. falciparum was detected in 6.7% (127/1891) of inbound passengers, including new haplotypes which were later detected in locally-transmitted infections. Case reproductive numbers approximated 1 and did not change when travelers were removed from transmission networks, suggesting that transmission is not fueled by travel to the region but locally endemic. Thus, malaria is not only prevalent in Central Turkana but also sustained by local transmission. As such, interrupting importation is unlikely to be an effective malaria control strategy on its own, but targeting interventions locally has the potential to drive down transmission.

The prevalence and density of asymptomatic Plasmodium falciparum infections among children and adults in three communities of western Kenya.

BACKGROUND: Further reductions in malaria incidence as more countries approach malaria elimination require the identification and treatment of asymptomatic individuals who carry mosquito-infective Plasmodium gametocytes that are responsible for furthering malaria transmission. Assessing the relationship between total parasitaemia and gametocytaemia in field surveys can provide insight as to whether detection of low-density, asymptomatic Plasmodium falciparum infections with sensitive molecular methods can adequately detect the majority of infected individuals who are potentially capable of onward transmission. METHODS: In a cross-sectional survey of 1354 healthy children and adults in three communities in western Kenya across a gradient of malaria transmission (Ajigo, Webuye, and Kapsisywa-Kipsamoite), asymptomatic P. falciparum infections were screened by rapid diagnostic tests, blood smear, and quantitative PCR of dried blood spots targeting the varATS gene in genomic DNA. A multiplex quantitative reverse-transcriptase PCR assay targeting female and male gametocyte genes (pfs25, pfs230p), a gene with a transcriptional pattern restricted to asexual blood stages (piesp2), and human GAPDH was also developed to determine total parasite and gametocyte densities among parasitaemic individuals. RESULTS: The prevalence of varATS-detectable asymptomatic infections was greatest in Ajigo (42%), followed by Webuye (10%). Only two infections were detected in Kapsisywa. No infections were detected in Kipsamoite. Across all communities, children aged 11-15 years account for the greatest proportion total and sub-microscopic asymptomatic infections. In younger age groups, the majority of infections were detectable by microscopy, while 68% of asymptomatically infected adults (> 21 years old) had sub-microscopic parasitaemia. Piesp2-derived parasite densities correlated poorly with microscopy-determined parasite densities in patent infections relative to varATS-based detection. In general, both male and female gametocytaemia increased with increasing varATS-derived total parasitaemia. A substantial proportion (41.7%) of individuals with potential for onward transmission had qPCR-estimated parasite densities below the limit of microscopic detection, but above the detectable limit of varATS qPCR. CONCLUSIONS: This assessment of parasitaemia and gametocytaemia in three communities with different transmission intensities revealed evidence of a substantial sub-patent infectious reservoir among asymptomatic carriers of P. falciparum. Experimental studies are needed to definitively determine whether the low-density infections in communities such as Ajigo and Webuye contribute significantly to malaria transmission.

Epidemiology of Plasmodium falciparum Infections in a Semi-Arid Rural African Setting: Evidence from Reactive Case Detection in Northwestern Kenya.

In northwestern Kenya, Turkana County has been historically considered unsuitable for stable malaria transmission because of its unfavorable climate and predominantly semi-nomadic population; consequently, it is overlooked during malaria control planning. However, the area is changing, with substantial development, an upsurge in travel associated with resource extraction, and more populated settlements forming. Recently, numerous malaria outbreaks have highlighted the need to characterize malaria transmission and its associated risk factors in the region to inform control strategies. Reactive case detection of confirmed malaria cases at six health facilities across central Turkana was conducted from 2018 to 2019. Infections in household members of index cases were detected by malaria rapid diagnostic tests (RDTs) and PCR tests, and they were grouped according household and individual characteristics. The relationships between putative risk factors and infection were quantified by multilevel logistic regression models. Of the 3,189 household members analyzed, 33.6% had positive RDT results and/or PCR test results. RDT-detected infections were more prevalent in children; however, PCR-detected infections were similarly prevalent across age groups. Recent travel was rarely reported and not significantly associated with infection. Bed net coverage was low and net crowding was associated with increased risks of household infections. Infections were present year-round, and fluctuations in prevalence were not associated with rainfall. These findings indicate year-round, endemic transmission with moderate population immunity. This is in stark contrast to recent estimates in this area. Therefore, further investigations to design effective intervention approaches to address malaria in this rapidly changing region and other similar settings across the Horn of Africa are warranted.