A Cohort Study on the Duration of Plasmodium falciparum Infections During the Dry Season in The Gambia.

BACKGROUND: In areas where Plasmodium falciparum malaria is seasonal, a dry season reservoir of blood-stage infection is essential for initiating transmission during the following wet season. METHODS: In The Gambia, a cohort of 42 individuals with quantitative polymerase chain reaction-positive P falciparum infections at the end of the transmission season (December) were followed monthly until the end of the dry season (May) to evaluate infection persistence. The influence of human host and parasitological factors was investigated. RESULTS: A large proportion of individuals infected at the end of the wet season had detectable infections until the end of the dry season (40.0%; 16 of 40). At the start of the dry season, the majority of these persistent infections (82%) had parasite densities >10 p/µL compared to only 5.9% of short-lived infections. Persistent infections (59%) were also more likely to be multiclonal than short-lived infections (5.9%) and were associated with individuals having higher levels of P falciparum-specific antibodies (P = .02). CONCLUSIONS: Asymptomatic persistent infections were multiclonal with higher parasite densities at the beginning of the dry season. Screening and treating asymptomatic infections during the dry season may reduce the human reservoir of malaria responsible for initiating transmission in the wet season.

Getting to zero: micro-foci of malaria in the Solomon Islands requires stratified control.

BACKGROUND: The Solomon Islands has made significant progress in the control of malaria through vector control, access and use of improved diagnostics and therapeutic drugs. As transmission is reduced there is a need to understand variations in transmission risk at the provincial and village levels to stratify control methods. METHODS: A cross-sectional survey of malaria in humans was conducted in the Solomon Islands during April 2018. Nineteen villages across 4 provinces were included. The presence of Plasmodium species parasites in blood samples was detected using PCR. RESULTS: Blood samples were analysed from 1,914 participants. The prevalence of DNA of Plasmodium falciparum was 1.2 % (n = 23) and for Plasmodium vivax was 1.5 % (n = 28). 22 % (n = 5/23) of P. falciparum DNA positive participants were febrile and 17 % of P. vivax DNA positive participants (n = 5/28). The prevalence of both P. falciparum and P. vivax was extremely spatially heterogeneous. For P. falciparum, in particular, only 2 small foci of transmission were identified among 19 villages. Plasmodium falciparum infections were uniformly distributed across age groups. Insecticide-treated bed net use the night prior to the survey was reported by 63 % of participants and significantly differed by province. CONCLUSIONS: Malaria transmission across the Solomon Islands has become increasingly fragmented, affecting fewer villages and provinces. The majority of infections were afebrile suggesting the need for strong active case detection with radical cure with primaquine for P. vivax. Village-level stratification of targeted interventions based on passive and active case detection data could support the progress towards a more cost-effective and successful elimination programme.

Prevalence and diversity of enteric pathogens among cholera treatment centre patients with acute diarrhea in Uvira, Democratic Republic of Congo.

BACKGROUND: Cholera remains a major global health challenge. Uvira, in the Democratic Republic of the Congo (DRC), has had endemic cholera since the 1970's and has been implicated as a possible point of origin for national outbreaks. A previous study among this population, reported a case confirmation rate of 40% by rapid diagnostic test (RDT) among patients at the Uvira Cholera Treatment Centre (CTC). This study considers the prevalence and diversity of 15 enteric pathogens in suspected cholera cases seeking treatment at the Uvira CTC. METHODS: We used the Luminex xTAG® multiplex PCR to test for 15 enteric pathogens, including toxigenic strains of V. cholerae in rectal swabs preserved on Whatman FTA Elute cards. Results were interpreted on MAGPIX® and analyzed on the xTAG® Data Analysis Software. Prevalence of enteric pathogens were calculated and pathogen diversity was modelled with a Poisson regression. RESULTS: Among 269 enrolled CTC patients, PCR detected the presence of toxigenic Vibrio cholerae in 38% (103/269) of the patients, which were considered to be cholera cases. These strains were detected as the sole pathogen in 36% (37/103) of these cases. Almost half (45%) of all study participants carried multiple enteric pathogens (two or more). Enterotoxigenic Escherichia coli (36%) and Cryptosporidium (28%) were the other most common pathogens identified amongst all participants. No pathogen was detected in 16.4% of study participants. Mean number of pathogens was highest amongst boys and girls aged 1-15 years and lowest in women aged 16-81 years. Ninety-three percent of toxigenic V. cholerae strains detected by PCR were found in patients having tested positive for V. cholerae O1 by RDT. CONCLUSIONS: Our study supports previous results from DRC and other cholera endemic areas in sub-Sahara Africa with less than half of CTC admissions positive for cholera by PCR. More research is required to determine the causes of severe acute diarrhea in these low-resource, endemic areas to optimize treatment measures. TRIAL REGISTRATION: This study is part of the impact evaluation study entitled: "Impact Evaluation of Urban Water Supply Improvements on Cholera and Other Diarrheal Diseases in Uvira, Democratic Republic of Congo" registered on 10 October 2016 at clinicaltrials.gov Identification number: NCT02928341 .

Natural Human Infections With Plasmodium cynomolgi and Other Malaria Species in an Elimination Setting in Sabah, Malaysia.

To determine the presence and species composition of malaria infections, we screened a subset of samples collected during a cross-sectional survey in Northern Sabah, Malaysia using highly sensitive molecular techniques. Results identified 54 asymptomatic submicroscopic malaria infections, including a large cluster of Plasmodium falciparum and 3 P. knowlesi infections. We additionally identified 2 monoinfections with the zoonotic malaria Plasmodium cynomolgi, both in individuals reporting no history of forest activities or contact with macaques. Results highlight the need for improved surveillance strategies to detect these infections and determine public health impacts.

Markers of sulfadoxine-pyrimethamine resistance in Eastern Democratic Republic of Congo; implications for malaria chemoprevention.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) is a cornerstone of malaria chemoprophylaxis and is considered for programmes in the Democratic Republic of Congo (DRC). However, SP efficacy is threatened by drug resistance, that is conferred by mutations in the dhfr and dhps genes. The World Health Organization has specified that intermittent preventive treatment for infants (IPTi) with SP should be implemented only if the prevalence of the dhps K540E mutation is under 50%. There are limited current data on the prevalence of resistance-conferring mutations available from Eastern DRC. The current study aimed to address this knowledge gap. METHODS: Dried blood-spot samples were collected from clinically suspected malaria patients [outpatient department (OPD)] and pregnant women attending antenatal care (ANC) in four sites in North and South Kivu, DRC. Quantitative PCR (qPCR) was performed on samples from individuals with positive and with negative rapid diagnostic test (RDT) results. Dhps K450E and A581G and dhfr I164L were assessed by nested PCR followed by allele-specific primer extension and detection by multiplex bead-based assays. RESULTS: Across populations, Plasmodium falciparum parasite prevalence was 47.9% (1160/2421) by RDT and 71.7 (1763/2421) by qPCR. Median parasite density measured by qPCR in RDT-negative qPCR-positive samples was very low with a median of 2.3 parasites/µL (IQR 0.5-25.2). Resistance genotyping was successfully performed in RDT-positive samples and RDT-negative/qPCR-positive samples with success rates of 86.2% (937/1086) and 55.5% (361/651), respectively. The presence of dhps K540E was high across sites (50.3-87.9%), with strong evidence for differences between sites (p < 0.001). Dhps A581G mutants were less prevalent (12.7-47.2%). The dhfr I164L mutation was found in one sample. CONCLUSIONS: The prevalence of the SP resistance marker dhps K540E exceeds 50% in all four study sites in North and South Kivu, DRC. K540E mutations regularly co-occurred with mutations in dhps A581G but not with the dhfr I164L mutation. The current results do not support implementation of IPTi with SP in the study area.

Bead-based assays to simultaneously detect multiple human inherited blood disorders associated with malaria.

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PDd), haemoglobin C (HbC) and S (HbS) are inherited blood disorders (IBD) common in populations in malaria endemic areas. All are associated to some degree with protection against clinical malaria whilst additionally G6PDd is associated with haemolysis following treatment with 8-aminoquinolines. Measuring the prevalence of these inherited blood disorders in affected populations can improve understanding of disease epidemiology. Current methodologies in epidemiological studies commonly rely on individual target amplification and visualization; here a method is presented to simultaneously detect the polymorphisms and that can be expanded to include other single nucleotide polymorphisms (SNPs) of interest. METHODS: Human DNA from whole blood samples was amplified in a novel, multiplex PCR reaction and extended with SNP-specific probes in an allele specific primer extension (ASPE) to simultaneously detect four epidemiologically important human markers including G6PD SNPs (G202A and A376G) and common haemoglobin mutations (HbS and HbC). The products were hybridized to magnetic beads and the median fluorescence intensity (MFI) was read on MAGPIX® (Luminex corp.). Genotyping data was compared to phenotypical data generated by flow cytometry and to established genotyping methods. RESULTS: Seventy-five samples from Burkina Faso (n = 75/78, 96.2%) and 58 samples from The Gambia (n = 58/61, 95.1%) had a G6PD and a HBB genotype successfully assigned by the bead-based assay. Flow cytometry data available for n = 61 samples further supported the concordance between % G6PD normal/deficient cells and genotype. CONCLUSIONS: The bead based assay compares well to alternative measures of genotyping and phenotyping for G6PD. The screening is high throughput, adaptable to inclusion of multiple targets of interest and easily standardized.

Haemolysis and haem oxygenase-1 induction during persistent "asymptomatic" malaria infection in Burkinabé children.

BACKGROUND: The haemolysis associated with clinical episodes of malaria results in the liberation of haem, which activates the enzyme haem oxygenase-1 (HO-1). HO-1 has been shown to reduce neutrophil function and increase susceptibility to invasive bacterial disease. However, the majority of community-associated malaria infections are subclinical, often termed "asymptomatic" and the consequences of low-grade haemolysis during subclinical malaria infection are unknown. STUDY DESIGN AND RESULTS: As part of an ongoing study of subclinical malaria in Burkina Faso, 23 children with subclinical Plasmodium falciparum infections (determined by qPCR) were compared with 21 village-matched uninfected control children. Infected children showed evidence of persistent haemolysis over 35 days, with raised plasma haem and HO-1 concentrations. Concentrations of IL-10, which can also directly activate HO-1, were also higher in infected children compared to uninfected children. Regression analysis revealed that HO-1 was associated with haemolysis, but not with parasite density, anaemia or IL-10 concentration. CONCLUSIONS: This study reveals that subclinical P. falciparum malaria infection is associated with sustained haemolysis and the induction of HO-1. Given the association between HO-1, neutrophil dysfunction and increased risk of Salmonella bacteraemia, prolonged HO-1 induction may explain epidemiological associations and geographic overlap between malaria and invasive bacterial disease. Further studies are needed to understand the consequences of persistent subclinical malaria infection, low-grade haemolysis and raised HO-1 on immune cell function and risk of comorbidities.

Low and heterogeneous prevalence of glucose-6-phosphate dehydrogenase deficiency in different settings in Ethiopia using phenotyping and genotyping approaches.

BACKGROUND: 8-Aminoquinolines such as primaquine clear mature Plasmodium falciparum gametocytes that are responsible for transmission from human to mosquitoes and bring radical cure in Plasmodium vivax by clearing dormant liver stages. Deployment of primaquine is thus of relevance for malaria elimination efforts but challenged by the widespread prevalence of glucose-6-phosphate dehydrogenase deficiency (G6PDd) in endemic countries since primaquine in G6PDd individuals may lead to acute haemolysis. In this study, the prevalence of G6PDd was investigated in different settings in Ethiopia using phenotyping and genotyping approaches. METHODS: Community and school based cross-sectional surveys were conducted from October to December 2016 in four administrative regions (Gambela, Benishangul Gumuz, Oromia, and Amhara) in Ethiopia. Finger prick blood samples were collected for G6PD enzyme activity using the CareStart™ G6PD screening test and genotyping of 36 selected single nucleotide polymorphisms (SNPs) located in the G6PD gene and its flanking regions. RESULTS: Overall, the prevalence of phenotypic G6PDd was 1.4% (22/1609). For the first time in the Ethiopian population, the African variant (A-) was detected in 3.5% (7/199) of the limited set of genotyped samples, which were all phenotypically normal. Interestingly, all of these individuals had a variation at the rs2515904 locus. Strong geographical variation was observed for both phenotypic and genotypic G6PDd; three-quarters of the phenotypically G6PDd individuals were detected in Gambela. CONCLUSION: A very low prevalence of G6PDd was detected in the present study populations. The presence of the A- variant alongside other G6PD mutants and the patchy distribution of G6PDd indicate that larger studies specifically designed to unravel the distribution of G6PDd at small geographical scale may be needed to tailor malaria elimination efforts in Ethiopia to the local context.

G6PD deficiency alleles in a malaria-endemic region in the Western Brazilian Amazon.

BACKGROUND: Plasmodium vivax parasites are the predominant cause of malaria infections in the Brazilian Amazon. Infected individuals are treated with primaquine, which can induce haemolytic anaemia in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals and may lead to severe and fatal complications. This X-linked disorder is distributed globally and is caused by allelic variants with a geographical distribution that closely reflects populations exposed historically to endemic malaria. In Brazil, few studies have reported the frequency of G6PD deficiency (G6PDd) present in malaria-endemic areas. This is particularly important, as G6PDd screening is not currently performed before primaquine treatment. The aim of this study was to determine the prevalence of G6PDd in the region of Alto do Juruá, in the Western Brazilian Amazon, an area characterized by a high prevalence of P. vivax infection. METHODS: Five-hundred and sixteen male volunteers were screened for G6PDd using the fluorescence spot test (Beutler test) and CareStart™ G6PD Biosensor system. Demographic and clinical-epidemiological data were acquired through an individual interview. To assess the genetic basis of G6PDd, 24 SNPs were genotyped using the Kompetitive Allele Specific PCR assay. RESULTS: Twenty-three (4.5%) individuals were G6PDd. No association was found between G6PDd and the number of malaria cases. An increased risk of reported haemolysis symptoms and blood transfusions was evident among the G6PDd individuals. Twenty-two individuals had the G6PDd A(-) variant and one the G6PD A(+) variant. The Mediterranean variant was not present. Apart from one polymorphism, almost all SNPs were monomorphic or with low frequencies (0-0.04%). No differences were detected among ethnic groups. CONCLUSIONS: The data indicates that ~1/23 males from the Alto do Juruá could be G6PD deficient and at risk of haemolytic anaemia if treated with primaquine. G6PD A(-) is the most frequent deficiency allele in this population. These results concur with reported G6PDd in other regions in Brazil. Routine G6PDd screening to personalize primaquine administration should be considered, particularly as complete treatment of patients with vivax malaria using chloroquine and primaquine, is crucial for malaria elimination.

High heterogeneity of malaria transmission and a large sub-patent and diverse reservoir of infection in Wusab As Safil district, Republic of Yemen.

BACKGROUND: Yemen remains the country with the highest malaria transmission within the Arabian Peninsula and a source of imported cases to neighbouring countries. METHODS: This study collected samples from individuals resident in a valley in Western Yemen as a baseline to examine infection prevalence for a future trial. As well as rapid diagnostic test (RDT) and microscopy, a filter paper blood spot was collected for molecular and serological analyses. RESULTS: Samples were collected from 2261 individuals from 12 clusters across a study area of approximately 100 km(2). Plasmodium falciparum infection prevalence was 12.4, 11.1 and 19.6% by RDT, microscopy and polymerase chain reaction (PCR), respectively. RDT and microscopy did not detect 45% of infections present, suggesting many infections were low-density. Infection prevalence and seroprevalence were highly heterogeneous between clusters, with evidence of higher exposure in clusters close to the wadi. The mean multiplicity of infection (MOI) was 2.3 and high heterozygosity and allelic richness were detected. CONCLUSIONS: This highly diverse parasite population suggests a high degree of transmissibility and coupled with the substantial proportion of low-density infections, may pose challenges for malaria control and elimination efforts.

Focal Screening to Identify the Subpatent Parasite Reservoir in an Area of Low and Heterogeneous Transmission in the Kenya Highlands.

BACKGROUND: Mass screening and treatment currently fails to identify a considerable fraction of low parasite density infections, while mass treatment exposes many uninfected individuals to antimalarial drugs. Here we test a hybrid approach to screen a sentinel population to identify clusters of subpatent infections in the Kenya highlands with low, heterogeneous malaria transmission. METHODS: Two thousand eighty-two inhabitants were screened for parasitemia by nested polymerase chain reaction (nPCR). Children aged ≤ 15 years and febrile adults were also tested for malaria by rapid diagnostic test (RDT) and served as sentinel members to identify subpatent infections within the household. All parasitemic individuals were assessed for multiplicity of infections by nPCR and gametocyte carriage by nucleic acid sequence-based amplification. RESULTS: Households with RDT-positive individuals in the sentinel population were more likely to have nPCR-positive individuals (odds ratio: 1.71, 95% confidence interval, 1.60-1.84). The sentinel population identified 64.5% (locality range: 31.6%-81.2%) of nPCR-positive households and 77.3% (locality range: 24.2%-91.0%) of nPCR-positive individuals. The sensitivity of the sentinel screening approach was positively associated with transmission intensity (P = .037). CONCLUSIONS: In this low endemic area, a focal screening approach with RDTs prior to the high transmission season was able to identify the majority of the subpatent parasite reservoirs.

Submicroscopic carriage of Plasmodium falciparum and Plasmodium vivax in a low endemic area in Ethiopia where no parasitaemia was detected by microscopy or rapid diagnostic test.

BACKGROUND: Motivated by the success in malaria control that was documented over the last decade Ethiopia is aiming at malaria elimination by 2020 in selected districts. It is currently unknown if asymptomatic, submicroscopic malaria parasite carriage may form a hurdle to achieve elimination. The elimination effort may further be complicated by possible glucose-6 phosphate dehydrogenase (G6PD) deficiency which would hinder the use of 8-aminoquinolines in the elimination efforts. METHOD: In February 2014 a community-based cross-sectional survey was conducted in Malo, southwest Ethiopia. Finger-prick blood samples (n = 555) were tested for presence of Plasmodium falciparum and Plasmodium vivax with microscopy, rapid diagnostic test (RDT), and nested polymerase chain reaction (nPCR). Multiplicity of P. falciparum infections was determined based on genotyping the polymorphic merozoite surface protein-2 (MSP-2) gene. Individuals were also genotyped for mutations in the gene that produces G6PD. RESULTS: All study participants were malaria infection negative by microscopy and RDT. Nested PCR revealed P. falciparum mono-infection in 5.2% (29/555), P. vivax mono-infection in 4.3% (24/555) and mixed infection in 0.2% (1/555) of individuals. All parasitemic individuals were afebrile (axillary temperature <37.5°C). None of the study participants carried mutations for the G6PD African A-(202GA) and Mediterranean (563CT) variants. All infections, except one, were single-clone infection by MSP-2 genotyping. CONCLUSION: The detection of a substantial number of subpatent malaria infections in an apparently asymptomatic population without evidence for malaria transmission by conventional diagnostics raises questions about the path to malaria elimination. It is currently unknown how important these infections are for sustaining malaria transmission in the study sites. The absence of G6PD deficiency indicates that 8-aminoquinolines may be safely deployed to accelerate elimination initiatives.

Glucose-6-phosphate dehydrogenase status and risk of hemolysis in Plasmodium falciparum-infected African children receiving single-dose primaquine.

Glucose-6-phosphate dehydrogenase (G6PD) enzyme function and genotype were determined in Ugandan children with uncomplicated falciparum malaria enrolled in a primaquine trial after exclusion of severe G6PD deficiency by fluorescent spot test. G6PD A- heterozygotes and hemizygotes/homozygotes experienced dose-dependent lower hemoglobin concentrations after treatment. No severe anemia was observed.

Malaria seroepidemiology in very low transmission settings in the Peruvian Amazon.

Despite progress towards malaria reduction in Peru, measuring exposure in low transmission areas is crucial for achieving elimination. This study focuses on two very low transmission areas in Loreto (Peruvian Amazon) and aims to determine the relationship between malaria exposure and proximity to health facilities. Individual data was collected from 38 villages in Indiana and Belen, including geo-referenced households and blood samples for microscopy, PCR and serological analysis. A segmented linear regression model identified significant changes in seropositivity trends among different age groups. Local Getis-Ord Gi* statistic revealed clusters of households with high (hotspots) or low (coldspots) seropositivity rates. Findings from 4000 individuals showed a seropositivity level of 2.5% (95%CI: 2.0%-3.0%) for P. falciparum and 7.8% (95%CI: 7.0%-8.7%) for P. vivax, indicating recent or historical exposure. The segmented regression showed exposure reductions in the 40-50 age group (ß1 = 0.043, p = 0.003) for P. vivax and the 50-60 age group (ß1 = 0.005, p = 0.010) for P. falciparum. Long and extreme distance villages from Regional Hospital of Loreto exhibited higher malaria exposure compared to proximate and medium distance villages (p < 0.001). This study showed the seropositivity of malaria in two very low transmission areas and confirmed the spatial pattern of hotspots as villages become more distant.

Understanding and maximising the community impact of seasonal malaria chemoprevention in Burkina Faso (INDIE-SMC): study protocol for a cluster randomised evaluation trial.

INTRODUCTION: Seasonal malaria chemoprevention (SMC) involves repeated administrations of sulfadoxine-pyrimethamine plus amodiaquine to children below the age of 5 years during the peak transmission season in areas of seasonal malaria transmission. While highly impactful in reducing Plasmodium falciparum malaria burden in controlled research settings, the impact of SMC on infection prevalence is moderate in real-life settings. It remains unclear what drives this efficacy decay. Recently, the WHO widened the scope for SMC to target all vulnerable populations. The Ministry of Health (MoH) in Burkina Faso is considering extending SMC to children below 10 years old. We aim to assess the impact of SMC on clinical incidence and parasite prevalence and quantify the human infectious reservoir for malaria in this population. METHODS AND ANALYSIS: We will perform a cluster randomised trial in Saponé Health District, Burkina Faso, with three study arms comprising 62 clusters of three compounds: arm 1 (control): SMC in under 5-year-old children, implemented by the MoH without directly observed treatment (DOT) for the full course of SMC; arm 2 (intervention): SMC in under 5-year-old children, with DOT for the full course of SMC; arm 3 (intervention): SMC in under 10-year-old children, with DOT for the full course of SMC. The primary endpoint is parasite prevalence at the end of the malaria transmission season. Secondary endpoints include the impact of SMC on clinical incidence. Factors affecting SMC uptake, treatment adherence, drug concentrations, parasite resistance markers and transmission of parasites will be determined. ETHICS AND DISSEMINATION: The London School of Hygiene & Tropical Medicine's Ethics Committee (29193) and the Burkina Faso National Medical Ethics Committee (Deliberation No 2023-05-104) approved this study. The findings will be presented to the community; disease occurrence data and study outcomes will also be shared with the Burkina Faso MoH. Findings will be published irrespective of their results. TRIAL REGISTRATION NUMBER: NCT05878366.

Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies.

BACKGROUND: Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution. METHODS: Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays. RESULTS: Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not homogenously distributed throughout blood spots. CONCLUSION: Combined DNA extraction and antibody elution is an operationally attractive approach for high throughput assessment of cumulative malaria exposure and current infection prevalence in endemic settings. Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure. The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity.

Serological evaluation of risk factors for exposure to malaria in a pre-elimination setting in Malaysian Borneo.

Malaysia has reported no indigenous cases of P. falciparum and P. vivax for over 3 years. When transmission reaches such low levels, it is important to understand the individuals and locations where exposure risks are high, as they may be at greater risk in the case of a resurgence of transmission. Serology is a useful tool in low transmission settings, providing insight into exposure over longer durations than PCR or RDT. We ran blood samples from a 2015 population-based survey in northern Sabah, Malaysian Borneo on a multiplex bead assay. Using supervised machine learning methods, we characterised recent and historic exposure to Plasmodium falciparum and P. vivax and found recent exposure to P. falciparum to be very low, with exposure to both species increasing with age. We performed a risk-factor assessment on environmental, behavioural, demographic and household factors, and identified forest activity and longer travel times to healthcare as common risk-factors for exposure to P. falciparum and P. vivax. In addition, we used remote-sensing derived data and geostatistical models to assess environmental and spatial associations with exposure. We created predictive maps of exposure to recent P. falciparum in the study area and showed 3 clear foci of exposure. This study provides useful insight into the environmental, spatial and demographic risk factors for P. falciparum and P. vivax at a period of low transmission in Malaysian Borneo. The findings would be valuable in the case of resurgence of human malarias in the region.

Plasmodium falciparum gametocyte carriage in longitudinally monitored incident infections is associated with duration of infection and human host factors.

Malaria transmission depends on the presence of Plasmodium gametocytes that are the only parasite life stage that can infect mosquitoes. Gametocyte production varies between infections and over the course of infections. Infection duration is highly important for gametocyte production but poorly quantified. Between 2017 and 2019 an all-age cohort of individuals from Tororo, eastern Uganda was followed by continuous passive and routine assessments. We longitudinally monitored 104 incident infections from 98 individuals who were sampled once every 28 days and on any day of symptoms. Among infections that lasted ≥ 3 months, gametocyte appearance was near-universal with 96% of infections having detectable gametocytes prior to clearance. However, most infections were of much shorter duration; 55.7% of asymptomatic infections were detected only once. When considering all asymptomatic infections, regardless of their duration, only 36.3% had detectable gametocytes on at least one time-point prior to parasite clearance. Infections in individuals with sickle-cell trait (HbAS) were more likely to have gametocytes detected (Hazard Rate (HR) = 2.68, 95% CI 1.12, 6.38; p = 0.0231) and had gametocytes detected at higher densities (Density Ratio (DR) = 9.19, 95% CI 2.79, 30.23; p = 0.0002) compared to infections in wildtype (HbAA) individuals. Our findings suggest that a large proportion of incident infections is too short in duration and of too low density to contribute to onward transmission.

Prevalence and temporal changes of mutations linked to antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Palawan, Philippines.

OBJECTIVE: This study provides 2016 data on the prevalence of key single nucleotide polymorphisms (SNPs) associated with antimalarial drug resistance in Palawan, Philippines. Findings were combined with historical data to model temporal changes in the prevalence of these SNPs in Plasmodium isolates. METHODS: Plasmodium isolates were genotyped using drug resistance markers pfmdr1, pfcrt, pfdhfr, pfdhps, kelch-13, pvmdr1, pvdhfr, and pvdhps. Temporal trends in the probability of mutations were estimated as a function of time using a binomial generalised linear model. RESULTS: All samples sequenced for Plasmodium falciparum chloroquine markers pfmdr1 and pfcrt had wild-type alleles. Varying mutation patterns were observed for the sulphadoxine/pyrimethamine markers pfdhps and pfdhfr; complete quintuplet mutations were not found. No SNPs were observed for the artemisinin marker kelch-13. For Plasmodium vivax, differing patterns were detected for pvmdr1, pvdhfr, and pvdhps. CONCLUSIONS: The study findings suggest that the current drugs remain effective and that there is limited importation and establishment of resistant parasites in the area. Clear temporal trends were recognised, with prominent decreases in the proportions of pfcrt and pfmdr mutations detected within the past 15 years, consistent with a change in antimalarial drug policy. Continuous surveillance of antimalarial drug resistance is important to support malaria elimination efforts.

Improved Detectability of Plasmodium falciparum Clones with Repeated Sampling in Incident and Chronic Infections in Burkina Faso.

We evaluated the detectability of Plasmodium falciparum clones when assessed on 3 consecutive days in incident and chronic infections in naturally exposed children living in an area of intense malaria transmission in Burkina Faso. The median number of clones by merozoite surface protein 2 (MSP2) genotyping was 3 (interquartile range [IQR] 2-5) in incident infections compared with 6 (IQR 4-8) in chronic infections (P < 0.0001). When all clones detected on days 1-3 were considered as true complexity of infection, sampling on day 1 detected only 69.4% (109/157) or 68.3% (228/334) of all clones in incident and chronic infections, respectively. Our findings demonstrate that a large proportion of clones are missed by single time-point sampling. In addition, because of the high complexity of infection early in incident infections, our data suggest many infections may be caused by genetically complex inocula.