Malaria remains endemic in Bangladesh, with the majority of cases occurring in forested, mountainous region in the Chittagong Hill Tracts (CHT). This area is home to Bengali and diverse groups of indigenous people (Pahari) residing largely in mono-ethnic villages.msp2 and deep sequencing of 5 amplicons  for Plasmodium falciparum (n = 20), and by microsatellite (MS) typing of ten loci and amplicon sequencing of msp1 for Plasmodium vivax (n = 21). Population structure was analysed using STRUCTURE software. Identity-by-state (IBS) was calculated as a measure of parasite relatedness and used to generate relatedness networks.1002 individuals of the 9 most prominent Pahari and the Bengali population were randomly selected and screened by RDT and qPCR. Parasites were genotyped by P. falciparum and P. vivax infection was 0.7% by RDT  and 4% by qPCR . Infections were highly clustered, with 64% (27/42) of infections occurring in only two Pahari groups, the Khumi and Mro. Diversity was high; expected heterozygosity was 0.93 for P. falciparum and 0.81 for P. vivax. 85.7% (18/21) of P. vivax and 25% (5/20) of P. falciparum infections were polyclonal. No population structure was evident for either species, suggesting high transmission and gene flow among Pahari groups.The prevalence of High subclinical infection prevalence and genetic diversity mirror ongoing transmission. Control activities should be specifically directed to Pahari groups at greatest risk.The online version contains supplementary material available at 10.1186/s12936-022-04236-0. Plasmodium falciparum account for 86% of all clinical cases, Plasmodium vivax for 12%, and Plasmodium malariae and Plasmodium ovale account for remaining cases [The Bangladesh National Malaria Control Programme has made significant progress towards controlling malaria within the country, reducing the incidence of malaria and mortality by 40% between 2015 and 2020 [P. falciparum infection vary among Pahari groups, likely due to differences in lifestyle and geographic location [The CHTs are home to at least 12 diverse indigenous groups, collectively known as Pahari, as well as increasing numbers of non-indigenous Bengali. Specific risk factors for malaria include jhum cultivation (the practice of forest-based shifting cultivation) and residing close to dense forest and at higher elevations –5. The rlocation , 5, 6.P. falciparum infection by light microscopy, which persisted year-round [P. falciparum and P. vivax is moderate or high; however, few studies have been conducted in low endemic, pre-elimination settings, and in regions where P. malariae and P. ovale are also present.To ensure ongoing progress towards malaria elimination, it is critical that malaria control efforts are targeted to those at the greatest risk of infection, including parasite reservoirs that sustain ongoing transmission , 8. Subcar-round . The truParasite genotyping is being applied increasingly to provide a better understanding of residual transmission –14. If iPlasmodium spp. infection using qPCR [To understand transmission patterns and differences in infection prevalence, residents of the CHT were enrolled into a cross sectional survey and blood samples were screened for any ing qPCR . GenotypThe study obtained ethical approval from the Ethics Review Committee of the icddr,b, Bangladesh (PR-15021), the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research, Australia (HREC 2015–2336), and the University of Notre Dame Institutional Review Board (18-09-4875). Written informed consent was collected from all participants or their legal guardians prior to enrollment and in addition, written assent was collected from all minors above the age of 11 years .As the aim of this study was to understand malaria epidemiology among different ethnic groups in the CHT, villages were purposively selected to represent different ethnicities . Most viP. falciparum using the varATS assay [P. vivax using the cox1 assay [varATS assay targets multicopy genes and amplifies approximately 20 copies per genome. Cox1 is a mitochondrial gene and present in approximately 10 copies per genome. Samples were screened for P. ovale and P. malariae using single gene assays [DNA was extracted from 200 µL blood using the Qiagen DNeasy kit according to manufacturer’s instructions. 4 µL of DNA, corresponding to 4 µL of blood, were screened by qPCR for x1 assay . The vare assays .Plasmodium falciparum-positive samples were genotyped by size-polymorphic msp2 and 5 highly diverse amplicons. msp2 typing was done by nested PCR followed by capillary electrophoresis [apical membrane antigen 1 , circumsporozoite surface protein , merozoite surface protein 7 , a conserved Plasmodium membrane protein , and a conserved Plasmodium protein  [phoresis . Amplico1475800) .Plasmodium vivax-positive samples were typed by 10 microsatellite markers  [Pvmsp1). Peaks higher than 300 units of relative fluorescent intensity and above background noise were considered true clones. Peaks less than 1/3 the height of the highest peak were not recorded. Alleles were grouped using TANDEM software [Pvmsp1, PVP01_0728900) as described [5, MS20)  and one software . Amplicoescribed  with thephiX control v3 (Illumina).PCR products were run on a 2% agarose gel, then 4uL of PCR product from each sample was combined to create pools of similar marker concentration as estimated from band intensities on the gel. These pools were purified using AMPure XP beads (Beckman Coulter) and DNA concentrations were measured using a Qubit fluorometer (Thermo Fisher Scientific). Pools were diluted to 0.1ug/uL and combined to form the sequencing library. The library was sequenced in paired-end mode in one run using the Illumina MiSeq reagent kit v3 600 cycle (2 × 300 bp) with 15% Enterobacteria phage Plasmodium falciparum positive samples were typed for pfhrp2 deletion using a protocol based on droplet digital PCR (ddPCR) [ (ddPCR) .Analyses were conducted using R software version 4.0.0  and a modified identity-by-state (IBS) metric were calculated using R software version 3.6.3  [P. falciparum, IBS was calculated using data from the 5 amplicons. For P. vivax, IBS was calculated using data from the 10 MSs and the one amplicon. Structure plots were created with the rmaverick package in R, using no admixture and considering only the dominant clone in each sample. Relatedness networks were generated using R package igraph, with an IBS threshold of 0.5 for each link. This value is the theoretical relatedness of meiotic siblings; however, it is a less restrictive threshold than the more rigorous IBS ≥ 0.6 used for identification of highly related infections in some studies [P. falciparum and at 1 amplicon and 10 MS markers for P. vivax.Statistical analyses were done in the R base package. Prevalence and parasitaemia were compared across age groups  by analysis of variance (ANOVA) and Tukey Post Hoc tests. Pearson’s Chi-squared tests were performed to test for differences in prevalence by sex, ethnicity, and village. Simple logistic regression was performed to separately compare participants’ village and ethnicity as predictors of infection. Multivariate regression was not performed due to high correlation between villages and ethnicities. Multiplicity of infection (MOI) for Country) . A modifCountry)  was used studies , 34. PriIn total, 1002 samples were collected from 28 villages, with 9–100 individuals sampled per village   individuals tested positive by RDT or microscopy, one of which was not confirmed by qPCR. RDT and microscopy detected 6/26 P. falciparum infections and 1/5 mixed infections  of individuals tested positive by qPCR for p = 0.045; Tukey Post Hoc: p = 0.043) (Table p = 0.021). Parasitaemia did not differ among age groups for either P. falciparum (p = 0.545) or P. vivax (p = 0.749).Prevalence by qPCR was highest in individuals aged between 11 and 20 years , and lowest in individuals older than 60 years . The only significant difference was between the 11–20 years and 21–30 years  age groups (ANOVA: P < 0.0001), with 0% (0/100) of Bengali participants infected and 64% (27/42) infections concentrated among two of the ten Pahari groups , with no infections detected in 50% (14/28) of villages. All Mro were sampled in one village (Ampu Para), where prevalence was 9% (9/100). Khumi were sampled in four villages, and prevalence was high in two of them: 12% (6/51) in Murungu Bazar, and 36% (12/33) Masim Hostel  and Mro (P = 0.001), while village was not a significant predictor (p = 0.006).Simple logistic regression identified two ethnicities as significant predictors of infection, Khumi  and 7 alleles for msp1. MOI ranged from 1 to 3 (median = 2.0) and 85% (18/21) were polyclonal.All 21 individuals with P. vivax , 0.31 for the Mro (n = 5), and 0.29 for Tripura (n = 2)  per amplicon marker and sample. Genetic diversity was high with 14 msp2 alleles (3 FC27 and 11 3D7) and 8–15 alleles per amplicon marker . The MOI ranged from 1 to 5 (median = 1) and 20% (4/20) of infections were polyclonal. None of the isolates had a pfhrp2 deletion.In total 76.9% (20/26) of individuals with P. vivax, STRUCTURE analysis revealed no population structure for P. falciparum  had identical haplotypes. The mean IBS within ethnicities was 0.12 for the Khumi (n = 8), 0.50 for Mro (n = 4), 0.0 for Tripura (n = 3), and 0.05 Tanchangya (n = 2)  of isolates linked in a small network including samples from the Khumi (n = 3), Mro (n = 4), and Tripura (n = 1)  and Mro . The 1% overall prevalence measured by RDT in this study was the same as that measured by RDT and microscopy on samples collected from the CHT in 2009 to 2012 . As obsePlasmodium spp. infections were far more prevalent among the Mro in Ampu Para and the Khumi in Murungu Bazar than in any of the nearby villages in northern Bandarban. Additionally, prevalence was elevated in a second Khumi village Masim Hostel, despite its location roughly 50 km to the south. In the dense forests of Indian subdistrict or Keshkal, a cross-sectional survey of three closely situated tribal groups demonstrated similar small-scale heterogeneity, with a significantly higher incidence of malaria in one group compared to the other two groups [There is significant spatial heterogeneity in the prevalence of infectious diseases, including that for malaria –38. Whero groups .Plasmodium species. Locally increased prevalence of infection in low transmission settings can arise from localized, small outbreaks, in which case, expansion of clonal or nearly related lineages would be expected [Despite the clustering of infections within ethnic groups, there was high genetic diversity and limited population structure for both expected –44; howeexpected , 45, 46,P. falciparum and from 11 to 34% for P. vivax [The causes for the pronounced differences in paras ite prevalence yet absence of any indication of small-scale transmission are unknown. Differences in behaviour, housing structure, and treatment-seeking are likely to play a role. A survey of health-seeking behaviour among five ethnic groups in the CHT in 2001 reported that the Mro had the lowest incidence of malaria (based on self-reported symptoms) and were the least likely to seek external healthcare when ill; this could reflect the greater distance to health facilities for the Mro . AlthougP. vivax . These vpfhrp2 deletions in P. falciparum samples from the CHT, a deletion has been detected in Sylet in northeastern Bangladesh near the Indian border [pfhrp2 and pfhrp3 genes have been detected in northern India [Although this study did not find any n border . Additiorn India . ContinuPlasmodium infections clustered among the Khumi and Mro Pahari groups in the CHT. Infections were highly diverse and showed no population structure, indicating sustained transmission of parasites rather than a single, clonal outbreak. To ensure sustained progress towards malaria elimination in Bangladesh, the potential reservoir for ongoing transmission should be identified and targeted for public health control interventions.This study detected a substantial burden of subclinical Additional file 1: Ethnicity, sex, age, and infection data for each participant; Haplotype data for P. vivax and P. falciparum.Additional file 2: R code used for haplotyping and statistical analysis.


Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) target night-time indoor biting mosquitoes and effectively reduce malaria transmission in rural settings across Africa, but additional vector control tools are needed to interrupt transmission. Attractive targeted sugar baits (ATSBs) attract and kill mosquitoes, including those biting outdoors. Deployment of ATSBs incorporating the insecticide dinotefuran was associated with major reductions in mosquito density and longevity in Mali. The impact of this promising intervention on malaria transmission and morbidity now needs to be determined in a range of transmission settings.We will conduct three similar stand-alone, open-label, two-arm, cluster-randomized, controlled trials (cRCTs) in Mali, Kenya, and Zambia to determine the impact of ATSB + universal vector control versus universal vector control alone on clinical malaria. The trials will use a “fried-egg” design, with primary outcomes measured in the core area of each cluster to reduce spill-over effects. All household structures in the ATSB clusters will receive two ATSBs, but the impact will be measured in the core of clusters. Restricted randomization will be used. The primary outcome is clinical malaria incidence among children aged 5–14 years in Mali and 1–14 years in Kenya and Zambia. A key secondary outcome is malaria parasite prevalence across all ages. The trials will include 76 clusters (38 per arm) in Mali and 70 (35 per arm) in each of Kenya and Zambia. The trials are powered to detect a 30% reduction in clinical malaria, requiring a total of 3850 person-years of follow-up in Mali, 1260 person-years in Kenya, and 1610 person-years in Zambia. These sample sizes will be ascertained using two seasonal 8-month cohorts in Mali and two 6-month seasonal cohorts in Zambia. In Kenya, which has year-round transmission, four 6-month cohorts will be used . The design allows for one interim analysis in Mali and Zambia and two in Kenya.Strengths of the design include the use of multiple study sites with different transmission patterns and a range of vectors to improve external validity, a large number of clusters within each trial site, restricted randomization, between-cluster separation to minimize contamination between study arms, and an adaptive trial design. Noted threats to internal validity include open-label design, risk of contamination between study arms, risk of imbalance of covariates across study arms, variation in durability of ATSB stations, and potential disruption resulting from the COVID-19 pandemic.NCT04800055. Registered on March 15, 2021Zambia: ClinicalTrials.gov NCT04149119. Registered on November 4, 2019Mali: ClinicalTrials.gov NCT05219565. Registered on February 2, 2022Kenya: ClinicalTrials.gov The online version contains supplementary material available at 10.1186/s13063-022-06555-8. Anopheles gambiae s.s. and Anopheles funestus to the more zoophagic outdoor-biting and outdoor-resting Anopheles arabiensis [Indoor residual spraying of insecticides [IRS] and long-lasting insecticidal nets [LLINs] target indoor, night-time-biting, and indoor-resting mosquitoes and have contributed to a 68% reduction in malaria parasite prevalence in Africa between 2000 and 2015 guanidine)  is a neonicotinoid insecticide effective at rapidly killing mosquitoes Fig. . ATSBs aDurability studies have recently been completed. These studies involved installing the bait station on the exterior walls of structures and assessing bait station integrity over a 6–8-month period.Anopheles with ≥3 gonotrophic cycles, and an overall younger age structure of the mosquito population, resulting in a 97.8% reduction in sporozoite rate and EIR [The ATSB is designed to provide population-wide protection by killing mosquitoes and thereby reducing their population size and longevity. Initial results from Mali show that high ATSB coverage is associated with a 70% reduction in the density of target vectors, a 97.1% reduction in female  and EIR , 21.Two ATSBs will be installed per eligible structure. Structures are eligible to receive an ATSB if they meet the following criteria: a complete roof, walls at least 1 m high and wide, at least three complete exterior walls, the stations cannot be easily accessed by peri-domestic animals or children, the structure is not under active construction, and the structure is not a working platform . Stations will be mounted on the outside of exterior walls, with the perforated membrane facing outwards, at least 1 m off the ground, ideally immediately under the eaves where possible.A cadre of trained monitoring assistants will provide information to households on the ATSB. ATSBs will be installed subject to informed consent by the householder. Bait stations will be marked with a unique barcode to allow tracking over time and association with specific household information. In Mali and Kenya, ATSBs will be deployed continuously for the 2-year trial, replaced every 6 months, or earlier if they are damaged or removed Table . In ZambPrior to and throughout ATSB deployment, community sensitization will be conducted through community meetings, posters, pamphlets, or other printed materials. Qualitative research has been conducted in the study sites, including stakeholder mapping, in-depth interviews, and focus group discussions, whose results will feed into the community sensitization and mobilization strategies.E malaria vaccine which is being routinely implemented by the Kenya National Vaccines and Immunizations Programme through a WHO-sponsored pilot as a four-dose schedule at 6, 7, 9, and 24 months of age . All sites use a combination of RDTs and microscopy for diagnosing malaria in the health system, and all sites use artemether-lumefantrine (AL) as the first-line treatment for uncomplicated malaria. All sites provide community case management for malaria for children aged < 5 years.The standard of care for malaria prevention in Mali includes universal coverage with LLINs as well as seasonal chemoprevention for malaria for children under five (SMC). The standard of care in Zambia is universal vector control coverage with either LLIN coverage or IRS. The standard of care in Kenya includes universal LLIN coverage. In approximately two-thirds of the study area in Kenya, children aged 6 to 12 months of age are eligible to receive the RTS,S/AS01Each of the three stand-alone trials will be an open-label, two-arm, cluster-randomized, controlled trial (cRCT) design comparing ATSB + universal vector control against universal vector control alone. A cluster trial design is indicated given the intended community-level effect of ATSBs on malaria transmission. Each trial is independently powered.The trial design incorporates adaptive elements. Planned adaptations for this trial include sample size refinement based on parameters estimated from baseline cohorts and cross-sectional surveys, including baseline incidence, prevalence, coefficient of variation (CV), loss to follow-up, and non-response. Secondly, the adaptive design will include an interim analysis of the primary endpoint of clinical malaria incidence , with a In Mali and Kenya, single villages, or groups of adjacent villages, form clusters consisting of on average 149 households in Mali and 100–400 households in Kenya. In Zambia, communities of approximately 250–350 households in close proximity to one another will form clusters; these clusters do not necessarily abide by administrative boundaries  and assignment of arms to intervention or control will be conducted separately for each site by an independent statistician.The primary outcome is the incidence of clinical malaria, defined as a history of fever within the previous 48 h or a measured temperature of ≥37.5°C, plus a positive malaria RDT in cohorts aged 1–14 years in Kenya and Zambia and 5–14 years in Mali. These outcomes will be ascertained during monthly follow-up visits or through cohort participant sick visits.P. falciparum infection among participants aged 6 months and older in Zambia and Mali (among participants aged 1 month and older in Kenya), detected by RDT in cross-sectional surveysPrevalence of Incidence rate of passively reported confirmed malaria among participants of all ages, defined as the number of confirmed malaria cases (by RDT or microscopy) detected at health facilities Entomological outcomes, including daily female vector survival, vector species abundance, composition, sex, sporozoite rate, EIR, and resistance to dinotefuran, among other insecticidesSocial and behavioral outcomes related to the acceptability and correct knowledge of ATSBsCost of ATSB deployment and incremental cost-effectiveness ratiosHuman safety and adverse eventsKey secondary outcomes include:For all data collection procedures involving human subjects, written informed consent and assent (where age-appropriate) will be obtained. An overview of the planned data collection activities can be found in Fig. Baseline estimates of clinical malaria incidence, malaria prevalence, within-cluster homogeneity, loss to follow-up, and refusal will be ascertained. The baseline measures will be used to revise sample sizes prior to the trial, develop and refine data collection instruments, organize data collection logistics, assess where additional quality control measures are required, and provide data for restricted randomization.Mali will have two 8-month seasonal cohorts, totaling 16 months of follow-up time  or individual-level  sampling frame. In Kenya, a mapping and census exercise will be repeated within 2 months before each cohort is enrolled to update the sampling frame. Cohort households will be selected via simple random sampling (SRS) from the household enumeration sampling frame within the core area of clusters in Kenya and Zambia. One individual meeting the inclusion criteria will be selected via SRS to participate in the cohort from each selected household at any given time. In Mali, eligible children will be directly sampled from census lists for each cluster.At the enrollment visit in Mali and Zambia, information about the households will be captured, including LLIN ownership and use, housing characteristics, and household demographic information. In Kenya, enrolment will occur at study clinics, and separate visits will be made to the households to collect data on housing characteristics. All cohort participants will be cleared of malaria parasite infection at enrollment with artemether-lumefantrine. In Zambia and Mali, confirmation of parasite clearance by blood smear will be ascertained 2 weeks after enrollment. In Kenya, RDTs will be conducted at the enrollment visits. Confirmation of parasite clearance will be ascertained by blood smear or RDT 2 weeks after enrollment . Those found positive at the clearance confirmation visit will be treated and removed from the cohort.Cohort participants will then be seen monthly. In Mali and Zambia, these visits will be conducted at the household. In Kenya, these will be conducted at study clinics. At each scheduled visit, a dried blood spot (DBS) will be collected on filter paper. Study participants with a history of fever in the last 48 h or current fever (axillary temperature of ≥ 37.5° C) will have an RDT performed . Those without fever or history of fever will not have an RDT performed. A child with fever plus a positive RDT test (or positive blood smear) will be considered to have clinical malaria and contribute to the numerator of the primary outcome measure. Episodes of uncomplicated clinical malaria will receive the standard of care ACT treatment. Children with severe malaria will be referred for treatment with parenteral artesunate. Participants who are treated for malaria will have 2 weeks of follow-up time censored from the subsequent follow-up period due to the prophylactic effect of the standard of care anti-malarial.Among all participants with a positive RDT in Mali, a repeat RDT will be conducted during the next monthly scheduled visits to confirm parasite clearance (no clearance confirmation will be conducted in Kenya and Zambia). If the RDT remains positive, the individual will be treated again. Due to the persistence of HRP-2 antigenemia following malaria infections, however, determination of whether there is an active malaria infection for the purpose of the primary outcome will be ascertained by microscopy in Mali and by PCR in Zambia.Inclusion criteria include currently living in the study household and aged 1–14 years (Zambia and Kenya) or 5–14 years  at the time of enrollment. Exclusion criteria comprise the following: household location/location of residence located in a buffer area or outside all cluster boundaries, pregnant at the time of enrollment, detection of patent malaria parasitemia 2 weeks after initial parasite clearance dose, ill and needing referral to the nearest health center at time of enrollment , those for which AL is contraindicated, known sickle cell disease and taking daily cotrimoxazole prophylaxis (Kenya), and current enrollment in another interventional study (Kenya). Pregnant women are excluded because they are eligible for monthly intermittent preventative therapy (IPT). Any participant found to become pregnant at any time during the cohort study will be omitted from further follow-up and referred to the nearest clinic for antenatal care.Cross-sectional household surveys will be used to estimate parasite infection prevalence among participants aged 6 months and older in Zambia and Mali and among participants aged 1 month and older in Kenya. In Zambia and Mali, three cross-sectional household surveys will be conducted to estimate point parasite prevalence during peak malaria transmission season , at baseline and in years 1 and 2 of trial implementation  registers at facilities and by CHWs, which capture individual-level patient data that are then captured through a photo via a Smartphone App and are converted to a working database. Data will be transmitted at least once per month.Confirmed passive malaria case incidence will be calculated from data from public health facilities and community health workers operating under each facility. The population denominator for incidence calculations will be derived from the census enumeration listing conducted prior to the trial start in each cluster. In Mali, facility-based assistants will be provided with electronic tablets with a custom application for collecting case data. Training will be provided for dispensary personnel and community health workers (CHWs) to capture case data. Data will be transmitted weekly to the field data manager. In Kenya, health care data are entered into routine Ministry of Health registers that have been converted into ScanForm  with community members and ATSB monitoring assistants and in-depth interviews (IDI) with community members will be conducted in intervention areas to understand the potential factors influencing ATSB and LLIN acceptability and coverage. Early results will be used to guide community engagement before the first ATSB deployment and inform strategies to ensure high community engagement and coverage levels throughout the trial.ATSB product, delivery, and deployment costs will be collected to estimate the financial and economic costs of the intervention. Cost data collection will include a review of program records and reports, invoices, budgets, expenditure reports, and interviews with intervention implementers. Interviews with trial staff will be focused on resource use during the implementation of the study interventions.Entomological monitoring activities will take place in the three trial sites using indoor and outdoor CDC UV light traps and human landing catches. The mosquito collections will be conducted in 16 clusters in Kenya, 24 clusters in Mali, and 20 clusters in Zambia. The clusters will be equally assigned to each study arm. Entomological monitoring in Zambia will be aligned with the deployment of ATSBs and will be carried out seasonally, while in Mali and Kenya, entomological monitoring will be carried out year-round. Specimens will be used to estimate key indicators including daily female vector survival (parity as determined by ovarian dissection), vector abundance, sporozoite rate, and calculated EIR. Specimens will also be used to monitor species composition and sex as well as indoor and outdoor landing rates. Insecticide resistance for dinotefuran and insecticides used in LLINs and IRS will be monitored annually in a subset of clusters. Samples of bait stations will be routinely collected from the field (and replaced) for laboratory testing to confirm ongoing target vector attraction and susceptibility to the bait stations.Adverse events (AEs) and severe adverse events (SAEs) will be monitored through both passive and active data collection systems. AEs will be collected for those related to AL for treatment of uncomplicated malaria in the cohort, as well as exposure to the ATSBs in the general community (non-ingestion). An SAE will be considered any death of a cohort participant, or any ingestion of the bait from an ATSB in the general community.The trials will include 76 clusters 38 per arm) in Mali and 70 (35 per arm) in each of Kenya and Zambia. All sample size estimates for epidemiological measures are based on the formula for community randomized control trial designs as outlined in Hayes and Moulton . All par8 per armA sample size of 3850 person-years is required in Mali, 1260 person-years in Kenya, and 1610 person-years in Zambia, to detect a 30% reduction in the primary outcome allowing for an overall 5% type-1 error rate following Haybittle-Peto boundaries allowing for interim analyses, and after accounting for the coefficient of variation (CV) of 0.4  or 75% (n = 558) number of expected primary outcome events have occurred in the control arm. The number of events will be tracked by an independent statistician not involved in the trial. Following the Haybittle-Peto rule, the interim analysis will be considered significant if p < 0.001 and the final null-hypothesis significance testing will be conducted with alpha levels of 0.05 in Mali and Zambia  and at 0.049 in Kenya  [A single interim analysis of the primary endpoint is planned in Mali and Zambia after the first transmission season regardless of the total number of events. In Kenya, the interim analyses will be either event or time driven, depending on which comes first, occurring after approximately 50% and 75% of person-time has accrued or after 50%  , 26. TheThe primary outcome analysis of the incidence rate of clinical malaria in the cohort will use a multi-level random intercept (variance components) model constructed on a generalized linear model framework with a Poisson likelihood and a log link function. The study arm will be included as a fixed effect independent variable.P. falciparum infection prevalence among participants aged 6 months  or 1 month (Kenya) and older detected by RDT in the household surveys will be analyzed using a multi-level random intercept (variance components) model constructed on a generalized linear model framework with a Bernoulli likelihood and a logit link function. The study arm will be included as a fixed effect independent variable.Data quality monitoring will be conducted to ensure that the trial is continuously operated according to protocol. The analysis will consist of a blinded review of all datasets generated for quality and completeness. Continuous corrective actions will occur based on these data, as needed.The ATSB project is governed by a steering committee with members from the Bill & Melinda Gates Foundation, IVCC (Innovative Vector Control Consortium), Westham Co., PATH, the Liverpool School of Tropical Medicine, the University of Bamako, and representatives from IVCC’s external expert advisors. An independent Data Safety and Monitoring Board (DSMB) has been established for each study site. The primary responsibilities of the DSMB are to periodically review and evaluate the accumulated study data for participant safety, study conduct and progress, and, when appropriate, efficacy. The DSMBs are responsible for making recommendations to investigators and research ethics committees concerning the trials. The DSMBs are empowered to advise to stop the trial early if there is evidence of harm or mismanagement.The projects will take place over 3 years, with the trials occurring over 2 years and the baseline conducted during the first year  at the University Teaching Hospital, the PATH Research Ethics Committee, and the Institutional Review Board at Tulane University. For the Mali trial, ethics review was undertaken by the Comite D’Ethique of the University of Sciences, Techiques and Technologies of Bamako and by the Ethics Committee of the London School of Hygiene and Tropical Medicine. For the trial in Kenya, ethical approval has been obtained from the Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU), the Liverpool School of Tropical Medicine; the Centers for Disease Control and Prevention IRB is operating on a reliance agreement with the KEMRI SERU. Protocol amendments will be sought for each study site for all relevant ethics committees if a protocol change is made.Individuals greater than 18 years of age will provide individual consent. For individuals aged 6 months to less than 18 years of age, consent will be sought from the parent or guardian of the child. For children greater than 6 years and less than 18, oral assent will be sought from the child. All concent and assent will be ascertained by trained data collectors.Confidentiality will be ensured through removal of all uniquely identifiable data. All data stored and managed on password-protected electronic platformsThese three trials are designed to establish the efficacy of ATSBs for the prevention of clinical malaria in a range of transmission settings in malaria-endemic Africa. Results will be used to inform national and global health policy regarding the use of the ATSB for malaria vector control.The use of three stand-alone but standardized cRCTs in different geographic areas with differing vectorial systems, seasonal patterns, human-built environments, vegetation patterns, and cultures will ensure that the results of the three trials will have high levels of external validity for sub-Saharan African settings. The use of restricted randomization, ITT analyses, measurement of endpoints along the causal pathway for the intervention, and other rigorous quality control methods will ensure the study’s internal validity. Analyst and investigator blinding will also strengthen internal validity. Finally, the use of buffer zones or adequate separation between clusters will also enhance the study’s internal validity by reducing contamination that could bias results towards the null effect.There are several potential threats to the internal validity of the trial. First, the use of sham ATSBs deployed in control clusters was deemed infeasible, and thus, the trial will not be blinded to the study participants. Potential bias due to the absence of blinding will be partially offset through highly standardized protocols for entomological field procedures. Laboratory processing of mosquito and human blood specimens will be blinded. Second, the potential for changes in human behavior in intervention clusters (or control clusters) affecting seeking treatment for fevers and malaria control intervention coverage, namely LLIN use, will be minimized through comprehensive community engagement and communications in all clusters. Additionally, study teams will monitor LLIN use in intervention and control areas to document changes throughout the course of the trial. There is always the potential for error in the measurement of outcomes, exposure to the interventions, and potential confounding factors in large and complex trials. We have gone to considerable lengths to minimize such errors with standardized outcomes and data collection instruments, electronic data capture systems for all fieldwork, and robust diagnostic methods for ascertaining disease and infectious status in humans.The trial results will be submitted to the World Health Organization (WHO) Vector Control Advisory Group (VCAG) for review of the public health value of the ATSB. Should VCAG determine that the ATSB class has public health value, a vector control guidelines development group (GDG) would subsequently develop a recommendation around the use of products in this class. These recommendations may include guidelines for a pathway for second-in-class products to achieve WHO prequalification status, which may require entomological data demonstrating non-inferiority to the first-in-class product . A WHO pIn Mali, the most recent version of the protocol was approved on 20 April 2021 by the ethical institution of record the Comite D’Etique of the Universite des Sciences, des Techniques et des Technologies de Bamako (Ref # 2021/124/CE/USTTB). Baseline recruitment for the seasonal cohort took place in the month of May 2021; enrolled participants were followed up for 8 months (through January 2022). Recruitment for the first seasonal cohort post-ATSB deployment took place in the month of May 2022; enrolled participants are currently being followed up for 8 months (through January 2023). The second season cohort is planned to take place from May 2023 through January 2024, with enrollment of participant conducted in the first month of the cohort. In Kenya, the latest protocol approval is for an amendment on 25 February 2022 by the ethical institution of record the Scientific Ethics Review Unit at the Kenya Medical Research Institute (Ref # KEMRI/CGHR/368/4189). Recruitment for the baseline cohort took place during the month of August 2021 with those recruited followed up for 5 months. Recruitment for the main trial cohort study took place in the months of April–May 2022; enrolled participants will be followed up for 24 months with new participants enrolled each 6 months . In Zambia, the latest protocol approval is for an amendment on 27 November 2021 (Ref # 1197-2020) by the ethical institution of record the National Health Research Ethics Board (NHREB) at the University Teaching Hospital. Recruitment for the baseline season cohort took place in the month of December 2020; enrolled particiapnts were followed up through June 2021. Recruitment for the first follow-up seasonal cohort took place in the month of December 2021; enrolled participants were followed up for 6 months through June 2022. The second season cohort is planned to take place in January–June 2023, with recruitment taking place in December 2022.Additional file 1. SPIRIT checklist.


Plasmodium falciparum infection and malaria disease, the Malawi International Center of Excellence for Malaria Research (ICEMR) has developed and implemented diverse and robust surveillance and research projects. Descriptive studies in ICEMR Phase 1 increased our understanding of the declining effectiveness of long-lasting insecticidal nets (LLINs), the role of school-age children in malaria parasite transmission, and the complexity of host–parasite interactions leading to disease. These findings informed the design of ICEMR Phase 2 to test hypotheses about LLIN use and effectiveness, vector resistance to insecticides, demographic targets of malaria control, patterns and causes of asymptomatic to life-threatening disease, and the impacts of RTS,S vaccination plus piperonyl butoxide-treated LLINs on infection and disease in young children. These investigations are helping us to understand mosquito-to-human and human-to-mosquito transmission in the context of Malawi's intransigent malaria problem.Despite the scale-up of interventions against malaria over the past decade, this disease remains a leading threat to health in Malawi. To evaluate the epidemiology of both  Plasmodium falciparum infection existing throughout the country. Although interventions have been scaled-up over the past decade, malaria infection risk and morbidity remain high, with some reports showing increasing malaria burden.,Malaria continues to be one of the greatest threats to health in Malawi, with the risk of Anopheles vector abundance and behavior, Plasmodium infections of vectors and humans, and behavioral risk factors for transmission and disease. Using several longitudinal cohorts, we aim to understand population-level factors that affect malaria prevention and control interventions as well as individual-level host and parasite contributions that influence the spectrum of outcomes, ranging from asymptomatic infection to uncomplicated malaria illness and severe malaria disease , we conducted 2 years of surveillance for malaria illness in health facilities in southern Malawi. Across all sites and in both the rainy and dry seasons, use of mRDTs was widespread with over 80% of patients with any symptoms of malaria having a mRDT test performed. One-quarter of tests were positive, implying a high burden of disease.This finding highlights the need for a clinical algorithm that includes evaluation for other causes of fever even in the presence of a positive mRDT as well as diagnostics for other common causes of fever.Plasmodium genotype at the time of clinical presentation. Genotypes at time of clinical presentation were often mixed (both a persistent genotype and a new genotype) suggesting that although the presence of a persistent asymptomatic infection does not proceed to clinical disease, it also does not protect against the acquisition of an infection that leads to clinical disease.The majority of infections detected in community-based surveillance are submicroscopic, and these low-density infections are not associated with fever.,After a change in Malawi’s first-line therapy from chloroquine to SP in 1993, parasites regained chloroquine sensitivity within 8 years.,Throughout both phases of our ICEMR grants, we have evaluated the burden of malaria in school-age children and described their disproportionate contribution to perpetuating transmission.,,,School-age children acquire new infections and maintain higher density infections like younger children. However, school-age children have acquired some immunity to clinical malarial disease and hence experience fewer symptoms and thus do not receive treatment to clear infections.Anopheles vectors based on LLIN use and body surface area suggest that school-age children are responsible for more than 60% of mosquito infections perpetuating transmission  are core vector control interventions recommended by WHO in all malaria-endemic settings. LLINs constitute the most widely used vector control intervention and remain the cornerstone of malaria control in Malawi. Since 2011, three LLIN mass distribution campaigns have been conducted. These are supplemented by continuous distribution through antenatal and immunization clinics. Despite this massive distribution of LLINs, the decline in the burden of malaria has plateaued, and the risk of disease remains high.In Malawi, mass LLIN distribution campaigns have historically been conducted every three years. Following each net distribution campaign, overall net ownership peaks near 90% and reported use approaches 80% (ICEMR unpublished).P. falciparum infection compared with those using a newer LLIN. Another study in Malawi demonstrated that among individuals 1 to 2 years old, there was an 18% reduction in malaria risk in users of LLINs with no holes (of any size) compared with users of LLINs with at least one hole.The deterioration in net condition  over time can affect the LLIN effectiveness. After the 2012 distribution campaign, we assessed how physical integrity and age impact net effectiveness.–Anopheles vector populations are resistant to pyrethroids (permethrin and deltamethrin) and only susceptible to pirimiphos-methyl, chlorfenapyr, and clothianidin. These findings are consistent with reports from across multiple sites in the countryMalaria vector control in Malawi relies primarily on use of LLINs, and because LLINs may be compromised in the face of pyrethroid resistance, there are concerns that the high burden of disease in Malawi could be due to LLIN failure from high insecticide resistance. Insecticide resistance, largely to pyrethroid insecticides, is intense and widespread in Malawi.Anopheles funestus s.s. and An. arabiensis in terms of disease transmission, population abundance, and geographic distribution.,An. gambiae s.s. was the most important malaria vector species in the country.An. gambiae s.l. largely exhibits outdoor biting behavior in both districts before and after the IRS campaign. However, An. funestus s.l., a vector that was largely indoor biting before the start of 2020 IRS campaign, exhibited increased outdoor biting behavior after IRS; this behavior gradually reversed over time. Results also show that the absolute abundance of both Anopheles species dropped sharply right after the IRS and gradually increased over the rainy season. Changes were also observed in the relative abundance of both vectors in Balaka district where An. funestus s.l. were progressively replaced by An. gambiae s.l., from the dry to the rainy season. The hourly biting rates showed that both species, in the two districts , had a peak of activities between 2 and 4 am, a time of night when most people are sleeping. These results suggest that An. funestus s.l., known as a highly anthropophilic and indoor biting vector species, reduced in abundance due to the insecticide and concomitantly developed a tendency to bite outside. When the effect of the insecticide decreased, this species switched back to its natural indoor biting behavior. As for An. gambiae s.l., this species was not heavily affected by IRS because it is able to bite both indoor and outdoor and feeds on humans and other animals. Ongoing studies and further analyses of available data will determine which Anopheles species of the gambiae complex and funestus group are involved and clarify whether the change in the relative abundance of An. funestus s.l. over time might also be due to the change in climatic conditions.Our work has shown that the malaria vector landscape in Malawi is currently dominated by P. falciparum infection and transmission reservoirs is forthcoming.To address concerns about the impact of increasing insecticide resistance on LLIN effectiveness, the National Malaria Control Program distributed PBO nets to some districts during the 2018 campaign. One of our ICEMR sites (Machinga District) received PBO nets, whereas our other site  received pyrethroid-only LLINs. Our ongoing studies in ICEMR Phase 2 are evaluating the impact of this net distribution campaign on malaria disease and infection as well as entomologic indicators of transmission. Before the campaign, the health centers serving these sites had similar numbers of monthly malaria cases. After the mass distribution, there was a dramatic decrease in malaria cases in sites that received PBO nets Figure . HoweverAn. arabiensis, An. gambiae, and An. funestus) reveal high rates of feeding on humans regardless of deployment of LLINs containing the synergist PBO or LLINs without this synergist.Anopheles, a lower proportion blood-fed in Machinga district compared with Balaka, suggesting that PBO nets reduced successful blood-feeding . Infection rates in these vector populations for P. falciparum, as assessed using molecular detection, was a combined 15.6% resulting in an estimated entomological inoculation rate of ca. 0.09 infectious bites per person per night. Together with the health center data, these data suggest that PBO nets are more effective than pyrethroid-only LLINs, but, similar to pyrethroid-only LLINs, their effectiveness wanes over time. Our ongoing data analysis should reveal insights into why this waning effectiveness occurs.Entomologic evaluations also demonstrate some impact of PBO nets, but transmission is sustained. Analysis of blood host selection patterns of the major vectors of human malaria in Malawi  to clinical disease remain a major research interest. Is the introduction of any new parasite clone sufficient to disturb this relationship? Is the immune response somehow “primed” to move to clinical disease if another parasite clone is already present? Is there a certain subset of parasites that will always lead to clinical disease? Our previous work focused heavily on differentiating infections causing uncomplicated clinical malaria from those causing cerebral malaria as well as differentiating disease severity within cerebral malaria. In our ongoing ICEMR, we broadened our focus to include elucidating the host and parasite factors that lead to a broader spectrum: from asymptomatic infection to uncomplicated clinical malaria and severe disease. Elucidating the host and parasite factors that determine the clinical consequences of infection should help appropriately target malaria control efforts, as well as generate immunologic and pathogenesis insights relevant to the vaccine and adjunctive treatment options.Although the majority of PfEMP1 is the major family of parasite-produced proteins responsible for the binding of infected erythrocytes to vascular endothelial cells, a phenomenon that leads to microvascular obstruction and subsequent organ damage. In research completed at the ICEMR-funded laboratory, we have shown that the expression of the EPCR-binding subtype of the protein PfEMP1 is associated with the deadliest form of cerebral malaria, a disease that includes brain swelling.P. falciparum infections. This clinical immunity is thought to require a repertoire of immune responses to multiple alleles of diverse parasite antigens. However, not all these responses are protective. Using longitudinal samples from symptomatic participants to differentiate protective and nonprotective responses, we evaluated changes in parasite allele frequencies in individuals over time. This approach allowed identification of subdominant, but protective, epitopes that might otherwise be difficult to detect in immunological studies because they are masked by responses to immunodominant, but not protective, loci. Using whole genome sequence data generated from 140 P. falciparum isolates, we identified 25 genes that encode possible targets of allele-specific acquired immunity to clinical malaria.Beyond PfEMP1, our group has used genomic approaches to identify possible targets of allele-specific malaria immunity. In high malaria transmission areas, individuals acquire immunity to clinical disease after repeated exposure to In addition to heterogeneity in protein expression on the parasite, heterogeneity in the host response to parasites could explain the development of varying clinical states. Previous work has shown that a wide range of different genes are transcribed when the same host has an episode of uncomplicated or cerebral malaria.Plasmodium transmission and infection patterns and pathways that operate at various functional scales  in an effort to understand and improve malaria control in Malawi. The first phase of our ICEMR generated key insights into the role of school-age children in Plasmodium transmission, the declining effectiveness of LLINs, and the complex host–parasite interactions leading to the severity of disease. These insights generated hypotheses for the recent and ongoing studies reported here, including evaluating the impact of PBO-treated LLINs and IRS. In addition, we are deepening understanding of Plasmodium transmission dynamics by assessing infectiousness of humans, the role of transmission-reducing immunity, and patterns of human–vector interactions. Results of these studies will inform development of interventions to decrease transmission and improve targeting of existing approaches. Examples of innovative strategies we are pursuing include school-based chemoprevention studies and implementation research to develop and evaluate an electronic vaccination registry to improve the cost-effectiveness of RTS,S vaccination of young children. Our ongoing cohort and case–control studies will define specific host and parasite factors that determine whether infections are asymptomatic, lead to clinical illness, or result in severe disease. These results should help develop prevention and therapeutic targets to decrease the most life-threatening cases of malaria infection. The diversity of basic and applied investigations at multiple levels of biological organization, involving questions and methods from epidemiology, ecology, parasitology, and immunology, will be refined and focused on future investigations as we continue to develop evidence to control disease and eliminate infection in Malawi.The Malawi ICEMR has been testing hypotheses about


Placental parasitic infections continue to be a public health problem despite numerous interventions put in place. Placental parasitic infections reported are Toxoplasma, Trypanosome, Borrelia, Schistosoma, Hookworm and Plasmodia. The infections persist to cause poor pregnancy outcomes such as maternal anaemia, low birth weight and stillbirth. This study aimed to determine the prevalence and pregnancy outcomes associated with placental parasitic infections at a tertiary hospital in northern Tanzania.A cross sectional study was conducted at Kilimanjaro Christian Medical Centre between June and July 2016. Pregnant women were interviewed before delivery and additional information obtained from their medical files. Blood samples as well as placental material were collected from each mother. Malaria was tested using a malaria rapid diagnostic test (mRDT). A total of 80 placental slide sections were made following histological protocols. After staining, slide sections were examined for the presence of parasites microscopically. Pearson's Chi-square and Fisher's exact tests were used to test for differences between groups.p=0.035). Stillbirth, maternal anaemia and pre-eclampsia were significantly associated with placenta malaria (p<0.05).Placental malaria parasites were found on histological examination of 8(10%) mothers' placental sections, none of whom had a positive mRDT. Education status was significantly associated with placental malaria (Placental malaria was found to be prevalent in the studied population and was associated with stillbirth, maternal anaemia and pre-eclampsia. Efforts for developing malaria tests that will detect subclinical infections are needed in order to identify infections early and offer prompt treatment to prevent poor pregnant outcomes. In Latin America 1, 1250,000 women are infected annually with Trypanosomacruzi.2 Each year, 25 million pregnant women in sub-Saharan countries are at risk of placental infections, the majority reported are from Plasmodium falciparum.3 In Tanzania, the prevalence of placental parasitemia has been reported to range from 8%4 to 63.5%.5 In Kilimanjaro, the Malaria Survey conducted in 2017 reported the maximum monthly prevalence of placental infection in antenatal care to be ≥2% to <5%.6 The reported placental infections include; Toxoplasma, Trypanosome, Borrelia, Schistosoma, Hookworm and Plasmodia.5Placental parasites infect millions of pregnant women in the world each year, and either directly or indirectly lead to fetal complications like intrauterine growth retardation, congenital malformations and fetal loss.9 as well as morbidity.11 It is estimated that up to 200,000 deaths per year in sub-Saharan Africa result directly from placental parasitic infections.7Maternal anemia,10 stillbirth,11 premature delivery, intrauterine growth retardation and low birth weight11are among the outcomes of placental parasites.Placental infections have been associated with maternal and neonatal mortality12 Successful control of placental parasites especially malaria in pregnant women is a major step towards reducing the disease burden in Africa. Control of these parasites in pregnancy involves preventing infection as well as clearing parasitemia when it occurs.13 Preventive measures put in place by the World Health Organization (WHO) include keeping a clean environment, use of Insecticide Treated Nets (ITN), intermittent preventive treatment in pregnancy and effective case management.14 The use of preventive treatment like Sulfadoxine Pyrimethamine (SP) has been shown to be effective in pregnancy.14Malaria incidence fell by 37% from 2000 to 2015 and one of the targets of the Sustainable Development Goal (SDG) number 3.3 is to end the epidemics of Acquired Immune Deficiency Syndrome (AIDS), tuberculosis, malaria and neglected tropical diseases by 2030.15 However, there are limited studies in Tanzania to quantify these infections especially after the introduction of various interventions. The majority of researchers would use daily routine (eosin and hematoxylin) or Giemsa stain to investigate placental parasite infections.16 The use of both stains may provide more accurate findings, whereby Giemsa stain would reveal parasites like Leishmania,17 while eosin and hematoxylin would reveal malaria parasites.18 This study was carried out to determine the prevalence, risk factors and peripartum maternal outcomes associated with placental parasitic infections among women who delivered at Kilimanjaro Christian Medical Centre (KCMC). This information can be used to plan effective measures for reducing maternal and fetal risk factors and outcomes resulting from parasitic infections.The Ministry of Health (MOH) of the Government of United Republic of Tanzania adopted SP as the preferred chemo preventive method in pregnancy with set guidelines for its use. Despite these interventions, placental parasitic infections, especially malaria, may still occur. The presence of placental receptors for parasites may enable their sequestration, which may then cause re-infections or ultimately lead to maternal and neonatal complications.A hospital based cross sectional study recruited delivering mothers from June to July 2016 at the KCMC referral and consultant hospital serving the northern zone in Tanzania. Pregnant women aged 18 to 40 years who delivered and were admitted to the labour ward were invited to participate in this study after providing written, informed consent. Pregnant women who did not consent, had planned abortion and those that experienced miscarriage were excluded from this study. A total of 80 pregnant women met the inclusion criteria and consented to participate in this study. A non-probability convenient sampling technique was used to select study participants. A hemoglobin test using Haemoglobinometer HemoCue 201+ machine and the rapid malaria detection test (mRDT)using SD BIOLINE Malaria Antigen P.F HRP2/PLDH, followed by microscopic examination of blood slides on positive samples using Olympus CX31 Binocular Microscope, was done for each participant at enrollment. Hemoglobin test, rapid malaria detection test and microscopic examination of blood slides were performed the clinical laboratory. After delivery, the maternal surface of the placenta was washed with normal saline and then incised with a scalpel and specimens fixed in 10% neutral buffered formalin. One full placental block with 4-5μm thickness was prepared by dehydration using acetone for two hours using Semi-Automated Rotary Microtome M-240, clearing by using Xylene for two hours, and paraffin infiltration with paraffin wax. Two slides' sections were made and stained differently; one slide stained with Hematoxylin and Eosin (H&E) as the routine stain and other slide stained with Giemsa stain as the special stain. The slides were then examined by light microscopy. Placental parasites were recorded during examination and all diagnoses confirmed by an experienced pathologist at Bugando Medical Centre (BMC) pathology laboratory for External Quality Control (EQC). Pre-tested questionnaires were used to collect socio demographic information where examination record files and clinic card were used to collect clinical characteristics.19 Pre-eclampsia was defined as a blood pressure of greater than 140/90 and protein in urine. Stillbirth, the death of an infant before delivery in a term pregnancy, was based on the first day after the mother's last menstrual period. The exposures for placental parasitic infections included maternal age, maternal occupation, marital status, area of residence; types of diet often used (nutrition), consumption of soil, education status and gravidity. Analysis was conducted using SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc. Pearson's Chi-square test was used to determine the association between the exposures and outcomes of interest. A p value of less than 0.05 was considered statistically significant.Maternal anemia, stillbirth, low birth weight and preeclampsia were the main outcomes assessed. Low birth weight was defined as an infant born with a weight of less than 2.5kg while maternal anemia as a pregnant mother with hemoglobin level less than 11g/dl. This was further categorized into mild anemia 10.0-10.9g/dl, moderate anemia 7.0-9.9 g/dl, and severe anemia <7.0 g/dl as adapted from a study conducted in Jordan.Approval to conduct this study was obtained from the Kilimanjaro Christian Medical College Research and Ethical Review Committee (CRERC) with ethical clearance certificate code number 2103, an independent review board for the medical college. Pregnant women aged 18-40 years who were admitted to the labor ward and delivered were invited to participate in this study after providing written, informed consent. Individual level medical information obtained from those mothers before and after delivery was kept strictly confidential.The socio-demographic characteristics of the pregnant mothers are summarized in Histopathological examination revealed that 8(10%) of placenta were infected with malaria parasites, despite the fact all mothers had negative mRDTs test results. Placental malaria infection was significantly associated with level of education of the mother .p<0.01), pre-eclampsia , and stillbirth   .Malaria was the only placental parasitic infection observed among delivering mothers in this study population. Malaria positivity among women who delivered at KCMC referral hospital was significantly associated with level of education, stillbirth, anemia and pre-eclampsia.20 and lower than 16.4% reported in another study conducted in Tanzania.5 Variations in community acquired immunity, sociodemographic characteristics of the study population, as well as endemicity of parasitemia which can be attributed to behavioral and environmental exposure to malaria may explain the observed differences.21The prevalence observed in this study is higher than the 8% prevalence reported in the previous study22 A study in Dar es Salaam used immunosorbent agglutination assay, in Mwanza ELISA23 was used and in this study, manual histological staining was used.Additionally, geographical location, stage of pregnancy and the methods used to determine presence of parasites in the placenta may have also contribute to the observed difference between the current and previous studies. Dar es Salaam and Mwanza (high endemic area) are hotter than Kilimanjaro (low endemic area). Hotter weather has been found to favor the sporulation of Oocyst.Furthermore, the presence of placental malaria infections among participants was different based on their level of education while this difference was not seen with other socio-demographic characteristics. This revealed that pregnant women who are in the primary level of education were more likely than those with secondary and higher education to have placental malaria infection.P. falciparum malaria infection. A study conducted in Tanzania found that malaria infection during pregnancy contributed 15% of maternal anemia.24 The result of this outcome also corresponds with previous studies conducted in Sub-Saharan Africa.25 The pathogenesis of anemia by malaria parasites  includes the hemolysis of the infected red blood cells. This is thought to be due to the reduced production of red blood cells, rupture of infected red blood cells and the destruction of uninfected cells due to antibody sensitization and with the resulting pathological effects. Marrow hypoplasia occurs in acute infections of malaria which may reduce the production of red blood cells.26 The mentioned processes may also explain how placenta malaria is significantly associated with anemia in this study.In this study, placental malaria was associated with anemia. Anemia is the most common consequence of P. falciparum in pregnancy.27 Most stillbirths, however, occur where malaria transmission is low3 and the effect of malaria on stillbirth is likely to be greater in areas of low transmission where there is little or no maternal immunity.28 In Moshi, falciparum malaria detected at delivery, even at sub-microscopic levels may increase the risk of stillbirth. These findings suggest that even low-level, asymptomatic and/or sub-microscopic infections that might easily be missed during routine antenatal care could be detrimental to the developing fetus.28The study also showed an association between placental malaria and stillbirth. Eight (8) percent of stillbirths worldwide  were estimated to be contributed by malaria parasites especially 32Placental malaria also was significantly associated with pre-eclampsia. Seasonal changes in the incidence of pre-eclampsia have been described in tropics, which are consistent with malaria transmission periods. Placental malaria is likely to impair placental development and cause maternal hypertension and placental vascular dysfunction.This study used a standard histological examination, where this method can diagnose parasites which could not be detected by the rapid test (mRDT). Information was collected with the help of midwives who interviewed the women using a standardized questionnaire. To ensure data completeness and accuracy, information obtained from clinic cards was triangulated with responses from the questionnaire.This study also had some limitations which are important to be taken into account while interpreting theresults. Firstly, the sample size was small and therefore limits the power of the inference being made. Secondly, the method used, although highly specific, can miss some other parasite as compared with more sensitive techniques like immunosorbent assay e.g. ELISA.These findings add to the evidence of adverse the health outcomes of placental parasitic infections among delivering mothers. Malaria in pregnancy was found to be significant associated with anemia, stillbirth as well as contributing to increased risk of pre-eclampsia. There is a need for more sensitive tests forearly diagnosis and adequate treatment during pregnancy to prevent adverse pregnancy outcomes caused by submicroscopic malaria infection.


Plasmodium that do not carry pfhrp2 or pfhrp 3 genes. Plasmodium lactate dehydrogenase (pLDH)-based tests are promising alternatives, but less available. This study assessed the performances of HRP2 and pLDH(pan) tests under field conditions.Rapid diagnostic tests (RDT) for malaria are the primary tool for malaria diagnosis in sub-Saharan Africa but the utility of the most commonly used histidine-rich protein 2 (HRP2) antigen-based tests is limited in high transmission settings due to the long duration of positivity after successful malaria treatment. HRP2 tests are also threatened by the emergence of The study performed a prospective facility-based diagnostic evaluation of two malaria RDTs in Aweil, South Sudan, during the high transmission season. Capillary blood by fingerprick was collected from 800 children under 15 years of age with fever and no signs of severity. SD Bioline HRP2 and CareStart pLDH(pan) RDTs were performed in parallel, thick and thin smears for microscopy were examined, and dried blood was used for PCR testing.pfhrp2/3 deletions in the study population was 0.6%.Using microscopy as the gold standard, the sensitivity of both tests was estimated at  > 99%, but the specificity of each was lower: 55.0% for the pLDH test and 61.7% for the HRP2 test. When using PCR as the gold standard, the sensitivity of both tests was lower than the values assessed using microscopy (97.0% for pLDH and 96.5% for HRP2), but the specificity increased (65.1% for pLDH and 72.9% for HRP2). Performance was similar across different production lots, sex, and age. Specificity of both the pLDH and HRP2 tests was significantly lower in children who reported taking a therapeutic course of anti-malarials in the 2 months prior to enrollment. The prevalence of pfhrp2/3 deletions in the study area warrants continued monitoring and underscores the relevance of assessing deletion prevalence nationally. Improved malaria RDTs for high-transmission environments are needed.The low specificity of the pLDH RDT in this setting confirms previous results and suggests a problem with this specific test. The prevalence of The online version contains supplementary material available at 10.1186/s12936-022-04280-w. Plasmodium falciparum, and Plasmodium lactate dehydrogenase (pLDH), which is present in all Plasmodium species; currently available tests detect one or both antigens. HRP2-based tests have been preferred in areas where P. falciparum is predominant, due to a higher reported sensitivity [Because of their ease of use and relatively low price, rapid diagnostic tests (RDT) have become the first-line malaria diagnostic tests in most malaria-affected countries in sub-Saharan Africa. Current malaria RDTs are immunochromatographic tests that detect the presence of circulating parasite antigens, and their performance has been monitored by the World Health Organization (WHO), which runs a rigorous testing and quality assurance process . The twositivity . In vivositivity , 5.™ Malaria PAN (pLDH) Ag RDT (Reference RMNM-02571) in several high-transmission environments. In most of these settings, it replaced the previously used SD BIOLINE Malaria Antigen (Reference 05FK50). Both of these tests easily met overall WHO performance thresholds in their most recent evaluations, with good performance even at the lowest level parasitaemia [Because of the lower specificity of HRP2-based tests in high-transmission environments, which can lead to false positive results and overtreatment, pLDH-based malaria RDTs have been introduced in some settings, though supplies of pLDH-based tests are considerably lower than those of HRP2-based tests. Between 2016 and 2020, the international medical humanitarian organization Médecins Sans Frontières introduced the CareStartsitaemia .Plasmodium missing the pfhrp2 gene in multiple sub-Saharan African countries [Plasmodium that have deletions of both the pfhrp2 and pfhrp3 genes, the parasite is often undetectable by HRP2-based RDTs [Concerns about the accuracy of HRP2-based RDTs have arisen in recent years because of the increased prevalence of ountries , 10. In sed RDTs . These dsed RDTs –14, but pfhrp2/3 deletions was investigated in all samples.To accompany its introduction in South Sudan, the study performed a formal clinical diagnostic evaluation of the newly introduced pLDH-based RDT, in parallel with the previously used HRP2-based RDT, in field conditions in Aweil, South Sudan. Off-site microscopy by at least two blinded microscopists was the gold standard comparator, and RDT performance was also compared to quantitative PCR. The presence of Malaria transmission in northwest South Sudan has a marked seasonality, with a relatively long peak season that occurs between June and December, following the rainy season. In 2020, there were an estimated 3,211,331 cases and 7431 malaria deaths in South Sudan, including 457,888 cases in Northern Bahr el-Ghazal (NBeG) State, of which Aweil is the capital city .The study planned to perform a prospective health centre-based, clinical diagnostic evaluation in two phases: first during the peak malaria transmission season (October–November 2019) and then during the low malaria transmission season (February–April 2020), but the low season phase was cancelled after the issuance of the Notice of Concern by WHO. The study was conducted in Aweil State Hospital, which is the referral hospital for NBeG  or a history of fever in the preceding 48 h. Children from whom blood samples could not be safely obtained (i.e. low weight or severe anaemia) were excluded as were children with any sign of severity (including but not limited to decreased consciousness and seizure), who were referred immediately to the hospital emergency room for treatment. Because of logistic constraints in laboratory processing, enrollments were limited to the first 20 eligible children who presented each day.After obtaining written informed consent from the participant’s caregiver and documenting oral assent for participants aged 7 years and older, a study nurse collected demographic and clinical information about each participant. This included age, sex, village of origin, whether the child slept under a bed net the previous night, receipt of intermittent preventive treatment for malaria in infants (IPTi) or curative anti-malarial treatment in the preceding 2 months, the date and time of the performance of the test, and the production lot number of the RDT.The study nurse collected capillary blood by a fingerstick, preparing two thick and thin smears, and collecting 2–3 drops of blood directly onto filter paper. The study nurse wrote the patient ID number and time of testing on the RDT cassettes. The two tests (SD Bioline Ag P.f. (HRP2), Standard Diagnostics, Giheung-ku, Republic of Korea, catalog number 05FK50; and CareStart pLDH(pan), AccessBio, Somerset, NJ, USA, catalog number RMNM-02571) were performed following the manufacturer’s instructions, and the results were recorded in the study register. Following manufacturer’s instructions, the RDTs were stored at temperatures  < 30 °C between shipment and their arrival to the study site (confirmed by Logtag), where they were stored at temperatures  < 30 °C with a dedicated temperature monitor. Two different production lots of the HRP2-based test were used, but only one production lot of the pLDH-based RDT was available during the study period.Any child with a positive result on either test (pLDH or HRP2) was treated, free of charge, following national protocols, with antipyretics, artemether-lumefantrine, and any other treatment deemed necessary by the treating clinician. Febrile children with a negative RDT were treated according to the clinician’s best judgment.Plasmodium species and mixed infections were used for this assessment. Microscopists were considered competent and allowed to read study smears if they scored  ≥ 90% in the proficiency assessment. In brief, the microscopists performed blind double-reading of all slides, with 200 high-power fields for negativity, and a third reading by a blinded microscopist in case of discrepant results between the first two for detection, species identification or parasitaemia discrepancy  > 50%. For positive slides, results included parasitaemia  and species identification. The presence or absence of gametocytes was noted for all samples. Approximately 10% of negative and 10% of positive slides were sent for external quality control at the Shoklo Malaria Research Unit in Thailand.At the end of each day, both slides were stained (10% Giemsa solution for 15 min), and the slide judged to be of lesser quality was discarded. Slides were stored and transported to the Epicentre laboratory in Mbarara, Uganda at the end of the study period, where microscopy was carried out according to WHO recommendations . MicroscPlasmodium spp DNA was determined using real time PCR targeting the cytochrome b gene using previously-described methods [Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale) was determined using specific real-time PCR [P. falciparum below a threshold of 30 cycles in the PCR, the presence or absence of deletion of pfhrp2 and pfhrp3 genes was determined by RT-PCR detecting parasite DNA. Reference strain DNA from Dd2, HB3 and 3D7 were used as controls of pfhrp2 deletion, pfhrp3 deletion, and absence of deletion, respectively. Plasmodium falciparum tubulin gene was used as a control for amplification. Primer sequences and the PCR conditions are available in Additional file Dried blood spots (DBS) were dried, wrapped and sealed in plastic bags according to standard operating procedures, stored at the study site and then sent to Institute Pasteur Cambodia (IPC) for further PCR analysis at the end of data collection period. Genomic DNA was extracted from the DBS using a DNA blood kit  according to manufacturer’s instructions. Presence of  methods . Speciattime PCR , 18. ForThe target sample size was calculated based on the number of true-positive and true-negative cases needed to estimate a sensitivity of 95% with a total width of the 95%CI of 8%, and a specificity ranging between 80% and 95% with a 95%CI width of 10.5% (for a point estimate of 80%) and 6% (for a point estimate of 95%). Under this scenario, it would be necessary to enroll a minimum of 140 true positives  and 240 true negatives . Based on historical data from Aweil Hospital, a 70% positivity rate among febrile children during the high season was assumed, and the study therefore aimed to enroll 800 children in Phase I (expecting 560 true-positives and 240 true-negatives).P. falciparum (mono-infection or co-infection) asexual forms were considered positive for analytic purposes. The gold standard for the primary objectives was microscopy; pre-specified secondary analysis used qPCR as a gold standard. Pre-specified subgroup analysis included children  < 5, patients without recent history of anti-malarial treatment, and parasitaemia levels:  < 200, 200–1999, 2000–199999 and  ≥ 200000 parasites/µl by microscopy. Comparisons in these pre-specified subgroups should nonetheless be regarded as exploratory, as the target sample size was not set with the goal of making these comparisons. Data were analysed using Stata version 16 .The performance characteristics of each RDT  were calculated independently, and 95%CI were calculated using the exact binomial method. Only infections with P. falciparum parasitaemia by microscopy. A total of 792 participants (99%) reported sleeping under a bed net the night prior to enrollment, and 259 (32%) reported having taken an anti-malarial drug in the preceding 2 months, including 168 (21%) who had taken it in the 30 days prior to enrollment and 74 (9%) in the 7 days prior to enrollment. No participant reported having received IPTi. Full results of microscopy and PCR are presented in Table P. falciparum parasitaemia by microscopy, 459 were mono-infections, 11 had P. falciparum/P. malariae co-infection, and 1 had mixed P. falciparum/P. ovale co-infection. Parasite density did not differ by age group . A total of 542 samples (68%) were positive for P. falciparum by PCR, including 30 which had co-infection with at least one other Plasmodium species  with For the pLDH test, there were 616 positive results and 184 negative results. For the HRP2 test, there were 593 positive results and 207 negative results , as judged by microscopy. The sensitivity of the HRP2 test was above 99% at parasite densities  > 2000 parasites/µl, and 98% at parasite densities between 200 and 1999 parasites/µl Table . NonetheP. falciparum parasitaemia by PCR, 469 (86%) had a cycle threshold (CT)  < 30 and could, therefore, be reliably analysed for the presence of pfhrp2/3 deletions. One sample had a single pfhrp2 deletion, one sample had a single pfhrp3 deletion, and three samples (0.6%) had double pfhrp2/3 deletions. The pfhrp2 RDT was positive for 2 of the 3 samples with double pfhrp2/3 deletions, as well as for both samples with a single deletion  in 7 of the samples. Plasmodium falciparum was detected in 3 of those samples , with only one sample having a CT  < 30. That sample did indeed have pfhrp2/3 deletions and is shown in the second line of Table It is important to note that of the 26 samples positive by pLDH RDT but negative by HRP2 RDT  among febrile children presenting for care was in line with expectations and was higher than in other evaluations performed in areas receiving chemoprevention programs like SMC . The decreased specificity of the pLDH and HRP2 test among children who had received anti-malarials underscores the known limitations of HRP2 tests in high transmission environments, where persistent antigenaemia after successful treatment leads to diagnostic and therapeutic dilemmas for treating clinicians. pLDH-based RDTs are expected to mitigate these problems, but that effect was not seen in this setting, nor in the Niger study.This study used expert microscopy as the primary gold standard for evaluating RDT performance. As a secondary objective, RDT performance was evaluated against PCR, which should have a lower limit of detection. The main results suggest that this was indeed the case. The decreased sensitivity and NPV of both RDTs when PCR was used as the gold standard likely suggests that low or very low-level parasitaemia was responsible for most false negative results. The clinical implications of this are unclear, particularly given the high prevalence of malaria among febrile children seeking care in the study area, as well as their overall high parasite densities, though it is plausible that among children with very low densities their fevers were not malarial in origin , 20.pfhrp2/3 deletions in South Sudan is scarce. To consider a national switch to RDTs based on another antigen , the WHO recommends a threshold of ≥5% prevalence of pfhrp2/3 deletions among pfHRP RDT suspected false negatives confirmed by either pLDH RDT or microscopy among clinical cases in one location (including 370 samples from at least 10 facilities in site) [pfhrp2/3 deletion and given the high prevalence of deletions seen in the nearby Horn of Africa, the authors consider that the 0.6% prevalence seen in this study population supports increased vigilance in South Sudan. Further, it is curious that 2 of the 3 samples with the double pfhrp2/3 deletion were detected by the pfhrp2 RDT. The authors do not have a simple explanation for this unexpected result, though one possibility is multiple P. falciparum strains present in one sample, with persistent antigenaemia from a recent malaria episode from a strain without the pfhrp2/3 deletions leading to both a positive HRP2 RDT as well as a finding of double pfhrp2/3 deletion in that same sample.Data on the prevalence of in site) . This cuThis study has certain limitations. One is that the study did not obtain data about the specific timing of the use of curative anti-malarials in the time before presentation. Second, this study reports only on the RDT performance during the high transmission season, when parasite prevalence is at its highest; overall performance of both RDTs may be different in the low season, when parasite densities and overall prevalence tend to be lower.pfhrp2/3 deletions cannot be interpreted in the context of a national recommendation, but this area of concern should be followed closely both in NBeG and in other areas of South Sudan. The study findings support the continued use of the HRP2 RDT for routine malaria diagnosis in South Sudan until such point as a pan-pLDH RDT is developed that both performs well and can be feasibly incorporated into the national malaria strategy guidelines.This evaluation was undertaken to support the introduction of the pLDH RDTs in South Sudan, but the results showed specificity that was essentially the same as that seen with HRP2-based RDTs, similarly to the results seen in the Niger study. The ensuing overtreatments were not ideal, particularly given that the higher specificity of this more costly pLDH test was supposed to be its advantage over traditional HRP2 tests in high transmission environments. The prevalence of Additional file 1. Primer sequences and PCR conditions for PCR testing of study samples at Institute Pasteur Cambodia.


In spite of the global reduction of 21% in malaria incidence between 2010 and 2015, the disease still threatens many lives of children and pregnant mothers in African countries. A correct assessment and evaluation of the impact of malaria control strategies still remains quintessential in order to eliminate the disease and its burden. Malaria follow-up studies typically involve routine visits at pre-scheduled time points and/or clinical visits whenever individuals experience malaria-like symptoms. In the latter case, infection triggers outcome assessment, thereby leading to outcome-dependent sampling (ODS). Commonly used methods to analyze such longitudinal data ignore ODS and potentially lead to biased estimates of malaria-specific transmission parameters, hence, inducing an incorrect assessment and evaluation of malaria control strategies.In this paper, a new method is proposed to handle ODS by use of a joint model for the longitudinal binary outcome measured at routine visits and the clinical event times. The methodology is applied to malaria parasitaemia data from a cohort of The results indicate that malaria parasite prevalence and force of infection (FOI) increase with age in the region of high malaria intensity with highest FOI in age group 5–10 years. For the region of medium intensity, the prevalence slightly increases with age and the FOI for the routine process is highest in age group 5–10 years, yet for the clinical infections, the FOI gradually decreases with increasing age. For the region with low intensity, both the prevalence and FOI peak at the age of 1 year after which the former remains constant with age yet the latter suddenly decreases with age for the clinically observed infections.Malaria parasite prevalence and FOI increase with age in the region of high malaria intensity. In all study sites, both the prevalence and FOI are highest among previously asymptomatic children and lowest among their symptomatic counterparts. Using a simulation study inspired by the malaria data at hand, the proposed methodology shows to have the smallest bias, especially when consecutive positive malaria parasitaemia presence results within a time period of 35 days were considered to be due to the same infection.The online version contains supplementary material available at 10.1186/s12936-022-04386-1. Plasmodium parasites that are transmitted through bites of infected female mosquitoes and is potentially life-threatening. In spite of the fact that malaria is a preventable and mostly curable disease for which increased efforts worldwide dramatically reduced malaria incidence  disease dynamics, study participants are asked to come to the clinic and get examined for malaria infection during scheduled (routine) visits. On top of that, unscheduled  visits can occur when participants develop symptoms for the disease under consideration, or when they experience symptoms similar to those typically observed for the infection at hand. If infection triggers outcome assessment in between pre-scheduled follow-up visits, the outcome and observation-time processes are said to be dependent, which in literature is often referred to as biased sampling or outcome-dependent sampling (ODS) . ConventDifferent models have been proposed to address ODS  in both prospective experimental settings as well as retrospective observational studies. One commonly used method to deal with ODS is to conduct a weighted analysis in which weights are inversely proportional to the probability of being sampled . AlternAlthough several authors developed methods to accommodate ODS in various settings, in this paper, a new method is proposed to cope with both the routine and clinical data on malaria infections from a cohort study in Uganda. To be more specific, a joint model for longitudinal binary outcomes (routine process) and time-to-event data  is considered, as proposed earlier by Rizopoulos et al. , howeverThe paper is organized as follows. A motivating example is introduced and briefly discussed in the following section. The general methodology to estimate malaria FOI from parasitaemia data is presented next. The impact of ignoring ODS is highlighted through a simulation study after which the proposed joint model is fitted to the available routine and clinical data on parasite presence in Ugandan children. Finally, the results are discussed in the Discussion section together with strengths and limitations of the proposed methodology.Plasmodium parasites using microscopy every three months from August 2011 to August 2014 (3 years). Furthermore, tests were also conducted at unscheduled clinical visits whenever individuals experience malaria-like symptoms. In the left panel of Fig. n-th infection is only known to lie between the point the child is tested positive and the point he/she first tested negative after recovering from the previous infection. More detailed information regarding the study design can be found in Kamya et al. [In this paper, longitudinal cohort data from children aged 0.5 to 10 years in three regions in Uganda  are considered. The data were collected as part of the Program for Resistance, Immunology, Surveillance and Modelling of malaria (PRISM) study . More spThroughout this paper, the outcome process refers to the occurrence of the longitudinal binary outcome (parasite presence), and the observation-time process relates to the timing of scheduled, i.e., routine, and unscheduled, i.e., clinical, visits over the entire follow-up period of the study.S(a) denote the proportion of susceptible individuals in the population and I(a) the proportion of infected individuals of age a, i.e., the (point) parasite prevalence, then the following set of ordinary differential equations (ODEs) describes transitions in the compartmental SIS model [a  can easily be derived in terms of the point prevalence I(a) [I(a) yields the FOI For the purpose of this paper, a simplified version of a realistic transmission model to describe malaria infection dynamics is considered. More specifically, following Mugenyi et al. , a so-caIS model :1\documence I(a) :2\documen individuals, denoted by individual i has a total of i, respectively, implying that First of all, the terminology and notation used to specify the proposed model to encompass ODS when modelling the data is introduced. Consider a sample of i at (routine) visit j. Consequently, for scheduled routine visits, i at visit j, ith individual, implying the prevalence, and equivalently the FOI, to depend on both age and calendar time [Consider the binary random variable dar time . In Tablmentclass2pt{minimi at routine visit In the absence of unscheduled clinical visits i for which the jth visit is clinical, and As mentioned before, clinical visits due to symptomatic malaria infections, or malaria-like events giving rise to symptoms similar to those observed for malaria, can not be treated in the same way as described previously. Let jth clinical visit and During a clinical visit, either a symptomatic malaria infection or a malaria-like event will cause the individual to have symptoms and to visit the health center. In the analysis, only symptomatic malaria infections are considered. Let Although some symptomatic infection times Different distributional assumptions can be made regarding the time at risk distribution, such as, e.g., exponential, Weibull, Gompertz, among others, which also relates to the selected functional form for mentclass2pt{minimjth clinical event, respectively, andFor outcomes Finally, the likelihood for the joint model including both information on routine and clinical visits is obtained by combining conditional likelihood contributions as described before:mentclass2pt{minimFollowing the development in the previous subsections, parameter estimation for the proposed combined model was performed in a Bayesian framework using MCMC sampling. The joint posterior distribution for the proposed models is then given byVague normal priors with mean zero and variance equal to 1000 are considered for all model parameters with support accuracy 23, 24. Maccuracy  implemenTo investigate the impact of ignoring ODS, a simulation study that is inspired by the PRISM data under consideration is conducted. More specifically, as a Bayesian approach is time-consuming, only entclass1pt{minimaAn important point is that if a single infection is contributing to both the routine and clinical process , i at visit j. Different parametric distributional assumptions regarding the infection times are explored  is applied to the observed Ugandan malaria parasitaemia data as previously presented. In particular, different models are fitted in order to select important covariates and the most appropriate functional form for  on WAIC  as point on WAIC  for deta on WAIC ), site-smentclass2pt{minimOne MCMC chain of total length 250,000 iterations is initiated with thinning factor = 15 and the first 40,000 iterations are removed as burn-in.Convergence of the chain is assessed using graphical diagnostic tools such as trace plots, histograms of posterior samples (representing the posterior density), and auto-correlation plots  process based on expression  is shownnsidered . On top In this paper, a new method has been proposed to account for ODS when estimating malaria transmission parameters such as, for example, the parasite prevalence and the FOI in case of longitudinal cohort data with routine (scheduled) and clinical (unscheduled) visits. A simulation study, inspired by parasitaemia data from a cohort of Ugandan children who were tested for malaria parasites (parasitaemia) during such visits, was conducted in which different parametric functions were considered to model the age-specific malaria prevalence and FOI while accounting for both observed and unobserved heterogeneity. The simulation results indicate that ignoring ODS tends to overestimate the marginal malaria infection risk and variance of the random effects. This is expected since children with a high disease activity are more likely to visit the clinic. Overestimation of such parameters may lead to incorrect inference and wrong conclusions. More specifically, from a public health point of view, ignoring ODS could potentially lead to overestimating the impact of efforts towards improved community-wide coverage of long-lasting insecticidal nets, indoor residual spraying of insecticides and additional malaria control interventions. Especially in settings where malaria elimination is achievable, reliable estimation of current malaria transmission is of crucial importance for the detection of malaria hotspots to be targeted. It has been demonstrated that the bias can be reduced by using a joint model in which both outcome (routine) and observation-time  components are present. In order to reduce the bias, the analysis performed here suggests that malaria events occurring within 35 days after a first malaria infection might be treated as being part of the same infection. This is supported by the results presented by Maiga et al.  and NdiaThe results regarding the data application show that both the malaria parasite prevalence and the FOI increase with increasing age in an areas of medium and high transmission intensities, Kihihi and Nagongera, respectively. In Walukuba which is an area of low transmission intensity, however, the prevalence peak at the age of about 2 years, after which remains constant. The FOI is highest in children aged 5–10 years and it becomes higher as children grow older or are at risk for a longer time. Unlike the areas of low  and medium (Kihihi) transmission intensities which were of high transmission intensity, the FOI in Nagongera is close to zero and constant with time at risk for children aged 1 year when becoming at risk. Further, both the prevalence and FOI are highest among the children with asymptomatic infections, and lower among the symptomatic ones or the previously treated children. These results are in line with those reported previously by Mugenyi et al. . The autOne way to avoid bias in estimating the epidemiological parameters of interest is the use of routine data only. This approach has been demonstrated in the past . HoweverThe proposed joint model can be extended to have a (shared) parameter ee, e.g, ), where To conclude, the combined model was applied to routine and clinical data on malaria infections from a cohort study in Uganda to estimate the parasite prevalence and FOI accounting for observed and unobserved heterogeneity, while dealing with ODS. Malaria parasite prevalence and FOI increase with age in the region of high malaria intensity with the highest FOI observed in age group 5–10 years. In all study sites, both the prevalence and FOI are highest among previously asymptomatic children and lowest among their symptomatic counterparts. Furthermore, inclusion of unscheduled clinical visits, which occur when individuals develop malaria-like symptoms, creates dependence between the study outcome and the observational time processes. Using a simulation study, the impact of ignoring this dependence and its potential to introduce bias in model parameter estimates was clearly demonstrated. This is confirmed by the fact that the combined model showed to have the smallest bias, especially when consecutive positive malaria parasitaemia presence results within a time period of 35 days were considered to be due to the same infection. Moreover, the proposed joint modelling approach can be used to model any longitudinal binary outcome data collected during scheduled routine observation times and linked to time to event data from the same subjects, obtained during unscheduled observation times.Additional file 1.Supplementary material to the manuscript including additional details concerning the simulation approach, fit andconvergence statistics for the PRISM data application and R code to fit the models.