Malaria transmission is highly seasonal in Niger. Despite the introduction of seasonal malaria chemoprevention (SMC) in the Magaria District, malaria incidence remains high, and the epidemiology of malaria in the community is not well-understood.Four cross-sectional, household-based malaria prevalence surveys were performed in the Magaria District of Niger between October 2016 and February 2018. Two occurred during the peak malaria season and two during the low malaria season. Individuals in each of three age strata  were sampled in randomly-selected households. Capillary blood was collected by fingerprick, thick and thin blood films were examined. Microscopy was performed at Epicentre, Maradi, Niger, with external quality control. The target sample size was 396 households during the high-season surveys and 266 households during the low-season surveys.Prevalence of parasitaemia was highest in children aged 5–9 years during all four surveys, ranging between 53.6% (95%CI 48.8–63.6) in February 2018 and 73.2% (66.2–79.2) in September 2017. Prevalence of parasitaemia among children aged 3–59 months ranged between 39.6% (33.2–46.4) in February 2018 and 51.9% (45.1–58.6) in October 2016. Parasite density was highest in children aged 3–59 months during all four surveys, and was higher in high season surveys than in low season surveys among all participants. The prevalence of gametocytaemia in children aged 3–59 months ranged between 9.9% (6.5–14.8) in February 2018 and 19.3% (14.6–25.2) in October 2016. The prevalence of gametocytaemia in children aged 5–9 years ranged between 6.3% (3.5–11.1) in February 2018 and 18.5% (12.7–26.1) in October 2016.Asymptomatic malaria infection is highly prevalent in this area, even during the season with low incidence of clinical malaria. The high prevalence of parasitaemia in children aged 5–9 years warrants considering their inclusion in SMC programmes in this context. Niger is a vast landlocked country in West Africa, with an estimated population of over 21 million . The MagBecause of the highly seasonal nature of malaria transmission, seasonal malaria chemoprevention was introduced in Niger in 2013, and at the time of this study in 2016–2018 targeted the entire Magaria District . This strategy is recommended in areas with strong seasonal malaria, and consists of up to four monthly courses of single-dose sulfadoxine-pyrimethamine (SP) and 3 daily doses of amodiaquine (AQ) during the period of highest malaria risk .Evidence shows that SMC is well-tolerated and acceptable to the general population in programme settings . NonetheGiven the substantial residual burden of malaria in Magaria despite the benefits of SMC, we performed a series of community-based surveys to better understand malaria epidemiology in the Magaria District, and to potentially inform future policy decisions about preventive strategies.Four cross-sectional, household-based malaria prevalence surveys were performed in the Magaria District of Niger between October 2016 and February 2018  at a fixed-point distribution site, and the child’s caregiver was given the remaining doses of the blisterpack with instructions for administration at home the following 2 days. This strategy is referred to as the directly observed therapy strategy (DOT). In villages in the second contiguous zone, no doses of SMC were directly observed by health workers at the fixed-point distribution sites, and all doses were administered by caregivers at home. This strategy is referred to as the non-directly observed strategy (non-DOT). During the 2017 season, the DOT strategy was used throughout the study zone. An independent SMC coverage survey performed in 2016 estimated programme coverage of 88% for the first distribution of the season, 90% for the second, 72% for the third and 70% for the fourth. The 2016 high-season prevalence survey described here was performed at the end of the third distribution cycle. No independent SMC coverage surveys were performed in 2017 . Those points which fell within 5 m of a building were retained, and numbered sequentially in the village, until the target number of households to include in each village was attained. These points were then loaded into handheld GPS devices and used during the surveys. If the selected building did not contain a household (either because of vacancy or day-long absence), the nearest building to the selected household was visited to replace the missing household. If this second building was also empty, the household was not replaced.The survey sample consisted of three age-based strata: children aged 3–59 months, children aged 5–9 years, and persons aged 10 years and older. In each household, one person from each stratum was offered enrollment. In a given household, if there were multiple individuals in a given stratum, one person was randomly selected using a random numbers table. All household members (including those who were temporarily absent) were eligible for inclusion. If a temporarily-absent household member was selected, study staff revisited the household later in the day or the following day.After obtaining informed consent (and assent from minor participants aged ≥ 10 years), the study nurse collected basic demographic information  about the participant on the study register. The participant was also asked whether they slept under a bed net the previous night, and whether they had been febrile in the preceding 24 h.The study nurse then collected capillary blood by a fingerstick, preparing thick and thin smears on two different slides following study standard operating procedures (SOP). Slides were allowed to dry in a carrier protected from dust and flies and were then transported to the study lab in Magaria at the end of each day.If a participant or other household member was febrile or reported a history of fever in the preceding 24 h, the study nurse also performed a rapid diagnostic test for malaria (CareStart pLDH(pan), AccessBio, Somerset, NJ, USA). If the RDT was positive and there were no signs of severity, the participant was treated with artemether-lumefantrine according to national guidelines. If a participant or other household member had signs of severe malaria, they were immediately transported to the nearest health centre.Plasmodium species identified, or parasite count differing by > 50% between the first and second reads), the laboratory supervisor performed a third examination of the slide. The final reported parasite density was the average of the value reported by the supervisor and the microscopist whose value was closest to that of the supervisor.At the end of each day, study staff returned to the central laboratory with the samples collected in the field. Thick and thin smears were stained following study SOPs and were then independently examined by two microscopists following study SOPs and WHO guidance . A minimExternal quality control on a random sample of slides read as positive and negative from each survey was performed at the Centre de Recherche Médicale et Sanitaire (CERMES) in Niamey.The target sample size for each survey was calculated to provide an estimate of the prevalence of microscopy-confirmed parasitaemia in each of the three different age strata, under the assumption that the prevalence would differ among the age groups and between the two surveys.While it would have theoretically been possible to enroll different numbers of individuals per stratum, this would have been difficult to implement practically. Therefore, for simplicity’s sake, and to reduce the risk of error, to the target was to enroll 360 individuals in each stratum during the high-season surveys , and 242 in each stratum during the low-season surveys . To account for non-response and households missing members of a given stratum, these figures were increased by 10%. The final target sample sizes were therefore set at 396 households (with one member of each age stratum) in the high transmission season surveys and 266 households in the low transmission season surveys.Plasmodium species were considered to be parasitaemic.For each survey, the prevalence of parasitaemia was independently estimated in each stratum taking into account the survey design, and 95% confidence intervals were calculated. The same procedures were used to describe the prevalence of gametocytaemia in each age group. Participants infected with any Comparison between groups took into account survey design. Multiple comparisons between groups in a single survey were tested using chi-square, Wald tests and the Kruskall-Wallis tests. Comparisons of the same groups between the four surveys were made using the t-test and the Mann–Whitney test. These comparisons should be regarded as exploratory, as the study was not designed to make these comparisons. Data were analysed using Stata v15.0 .393 random points were selected for the first high-season survey. At 129 of the points, both the household located at the random point and its replacement were absent. After inclusion, one household withdrew its consent. A total of 263 households and 591 individuals were therefore included and microscopy results were available for 588 individuals .Because of the high absentee rate during the first high-season survey, 300 random points were selected for the first low-season survey. At 32 of the points, both the household located at the random point its replacement were absent. A total of 268 households and 634 individuals were therefore included.410 random points were selected for the second high-season survey. At 4 points, both the household and its replacement were absent. After inclusion, 3 households withdrew consent. At 30 random points, households refused to participate. A total of 373 households and 818 individuals were therefore included.266 random points were selected for the second low-season survey. At 6 random points, the household refused to participate. A total of 260 households and 643 individuals were therefore included.Participants were evenly split by gender, and during each of the surveys, the stratum containing school-aged children had the most missing members from the households Table .Table 1DPlasmodium falciparum. During the second high-season survey, of 379 participants with positive blood smears, 361 participants had mono-infection with P. falciparum, 10 participants had mixed infection with P. falciparum and Plasmodium ovale, 2 participants had mixed infection with P. falciparum and Plasmodium malariae, 5 participants had mono-infection with P. ovale, and 1 participant had mono-infection with P. malariae. During the second low-season survey, 213 participants had mono-infection with P. falciparum, 1 participant had mixed infection with P. falciparum and P. ovale, 2 participants had mixed infection with P. falciparum and P. malariae, 2 participants had mono-infection with P. ovale, and 2 participants had mono-infection with P. malariae.The prevalence of parasitaemia is summarized in Table Within individual surveys, there were no differences in the prevalence of parasitaemia between males and females. Similarly, there was no difference in parasitaemia between areas receiving different SMC delivery strategies in 2016. While the overall prevalence of parasitaemia did not differ between the first high-season and first low-season surveys conducted in 2016, the prevalence was higher in the second high season survey compared to the second low-season survey, which held across gender and age groups.In all four surveys, children aged 5–9 years had the highest prevalence of parasitaemia, ranging from a low of 56.3% (95% CI 48.8–63.6) during the second low-season survey to a high of 73.2% (95% CI 66.2–79.2) during the second high-season survey. They were followed by children aged 3–59 months, and then participants ≥ 10 years of age  in 99% of samples sent for testing.This series of community-based malaria prevalence surveys provides valuable information that adds to our understanding of malaria in an area receiving SMC, and sheds light on new potential strategies for malaria prevention.Two main findings were expected: that children are more likely to be parasitaemic than adults and that younger children who are infected are more likely to have higher parasite burdens. On the other hand, the fact that children aged 5–9 had a higher prevalence of parasitaemia than children aged 3–59 months in each survey was mildly surprising. Simple explanations would be that older children are less likely to sleep under mosquito nets and have some degree of acquired immunity, but the most obvious explanation for this trend in this area is that the younger children are in the target age group for SMC, while the older group are not. Another explanation would be that children under 5 years old have better overall access to health care because of national policy dictating that their care is free of charge.Approximately half of children in the community were carrying parasites during the high-season surveys, which occurred just under 4 weeks after the previous SMC distribution. The following distribution might have decreased the overall parasite burden among this group, but the results from the low-season surveys clearly show that even with SMC there is a significant residual parasite burden in this population at the end of the high-transmission season. These findings should help counter fears that SMC may lead to increased rates of severe malaria in older children , because children in this area are clearly exposed to malaria parasites, even with a functional SMC programme.Malaria transmission is driven by the presence of gametocytes in peripheral blood. The high prevalence of gametocytaemia among children aged 3–59 months during the high season surveys is not particularly surprising, but the fact that there was no difference in the prevalence of gametocytaemia between children aged 3–59 months and children aged 5–9 years shows that this older population is significantly contributing to malaria transmission in the community. These school-aged children have likely developed significant natural immunity to malaria after repeated infections in early childhood, hence their lower parasite densities.These surveys were undertaken to better understand the epidemiology of malaria in this area with extremely strong and seasonal transmission. A case–control study performed in the same study zone suggested that SMC was generally effective at preventing clinical malaria, despite poor adherence to AQ at home . DespitePlasmodium carriage high in school-aged children [Plasmodium infection over time and are, therefore, likely not at high risk of developing severe malaria, but given the high prevalence of parasitaemia, expanding the target age group of SMC to children aged 5–9 years might prevent significant morbidity in this population.The coverage of SMC in the study area was reasonably high, though it could certainly have been improved. Efforts should be made to consolidate coverage in the current target group of children. Nonetheless, given the results of these surveys, it is reasonable to question whether SMC is targeted at the optimal group of children in rural Niger. A survey in another part of rural Niger showed similar results, with asymptomatic children . These cThe largest potential benefit of an SMC expansion would likely be indirect. With a high prevalence of gametocytaemia, children aged 5–9 are helping to drive transmission in their communities, in turn causing malaria infections (more likely to be severe) among their younger siblings and neighbours. Evidence supporting this hypothesis has already been shown in a stepped-wedge trial in Senegal, where the expansion of SMC to children under 10 was associated with a 27% reduction in malaria in persons who had not received SMC, an effect that was not seen when SMC was limited to children 3–59 months [SMC was never intended to be a magic bullet, there are many logistical challenges inherent with implementing SMC year in and year out in the context of the rural Sahel. DoublinThere are limits to these surveys. The most important is that the target sample sizes were not met for all strata in the surveys. In the first survey, the absentee rate was much higher than expected. This was overcome with more intensive preparation in subsequent surveys, but many households with adult respondents did not have children in one of the two age groups. In some cases, this was because of older or younger heads of household without children present, and in others it was due to the absence of school-aged children. This has led to larger confidence intervals than expected, particularly for the children aged 5–9 years, but it is reassuring that the overall trends are consistent between the surveys. Information about receipt of (or adherence to) SMC in the children in the youngest age group was not collected, though it has been previously shown that adherence in the study area was poor in 2016 . The timThese surveys showed a high prevalence of asymptomatic parasitaemia among children under 10 years old in an area of rural Niger where children under 5 were receiving SMC. The high prevalence of parasitaemia in school-aged children could justify expansion of SMC to this group, but this decision would need to consider clinical disease as well as financial and logistic concerns.


The Indian state of Odisha has a longstanding battle with forest malaria. Many remote and rural villages have poor access to health care, a problem that is exacerbated during the rainy season when malaria transmission is at its peak. Approximately 62% of the rural population consists of tribal groups who are among the communities most negatively impacted by malaria. To address the persistently high rates of malaria in these remote regions, the Odisha State Malaria Control Program introduced ‘malaria camps’ in 2017 where teams of health workers visit villages to educate the population, enhance vector control methods, and perform village-wide screening and treatment. Malaria rates declined statewide, particularly in forested areas, following the introduction of the malaria camps, but the impact of the intervention is yet to be externally evaluated. This study protocol describes a cluster-assigned quasi-experimental stepped-wedge study with a pretest-posttest control group design that evaluates if malaria camps reduce the prevalence of malaria, compared to control villages which receive the usual malaria control interventions , as detected by PCR. Plasmodium vivax cases globally [Despite declines over the past decade, malaria remains an important global public health problem in India. India is one of twenty countries accounting for 85% of all malaria cases and deaths in 2018 and 47% of all globally  and 3.5%’ to refer to these communities [Odisha is known for its hilly, forested, and inaccessible regions, and for the indigenous ‘tribal’ people who live there.  Tribalmunities ,8. Odishmunities , 314 blomunities . The climunities ,12.Plasmodium falciparum is the major parasite species (>90% of cases), and the major malaria vectors are Anopheles fluviatilis found in inaccessible, hilly, and forested regions [Anopheles culicifacies sp. C and E in plains villages where they breed in rice fields [Malaria transmission occurs predominantly in forested and forest fringe regions in Odisha, with many communities experiencing poor access to health care, especially during the rainy season.  regions ,14, and e fields ,16. Evene fields . DistribP. falciparum infections at various sites in Odisha, primarily through molecular studies that sequence the parasite genes known to be involved. For example, mutations in P. falciparum genes associated with chloroquine resistance [P. falciparum kelch13 gene, implicated in resistance to artemisinin, have arisen in multiple parts of Odisha [Antimalarial drug resistance has been reported in sistance  and sulpsistance ,20 have sistance . More ref Odisha ,22, but The persistently high malaria burden in remote forested areas of Odisha has led to the introduction of ‘malaria camps’ (MCs) by the Odisha State Malaria Control Program (MCP), where teams of health workers visit villages to educate the population, enhance vector control with long-lasting insecticide-treated nets (LLINs) and IRS, and perform village-wide screening with rapid diagnostic tests (RDTs) and treatment for malaria. The MCs are part of a program, Durgama Anchalare Malaria Nirakaran (DAMaN) ‘malaria control in inaccessible areas,’ and have two main components, mass screening and treatment of all malaria positive cases and simultaneous intensive vector control using LLIN and IRS, allowing for both the parasite and mosquito to be targeted in parallel. The camps appear to be effective, but this has been hard to assess in the context of ongoing changes such as LLIN introduction. The goal of the study protocol described here is to evaluate the effectiveness of MCs on malaria prevalence.The goal of this protocol is to evaluate the effectiveness of malaria camps (MCs) by determining if they reduce malaria and to characterize malaria transmission in intervention villages over time. Specifically, this pilot study is designed to demonstrate proof-of-concept and feasibility, and aims to assess if MCs in intervention villages reduce the prevalence of malaria compared to standard malaria control efforts in control villages. In addition, the study aims to investigate the out-of-pocket costs and impact of malaria from the patient’s perspective in these hard-to-reach tribal communities.Plasmodium parasites in each study arm using molecular and genomic methods; characterizing the social factors affecting the implementation of MCs using interviews; surveying mosquito vectors in and around the houses of subjects enrolled in each arm; evaluating the impact of the interventions using epidemiological compartmental modeling of host-vector interactions; and building research capacity in Odisha and India to help train the next generation of malaria and mosquito vector biologists.Secondary objectives of this study include: determining if MCs reduce the prevalence of subpatent, symptomatic, and asymptomatic malaria as detected in the study surveys as well as in local clinics ; assessing malaria exposure, immunity, and risk among participants in each arm of the study through high-throughput detection of antibodies; assessing the genetic epidemiology  of Plasmodium infection as detected by PCR. At baseline, villages are assigned to one of three study arms. In year 1, Arm A villages receive MCs for the first time; Arm B villages serve as control villages where no MCs but routine malaria control activities are implemented; and Arm C are villages that have already had at least one round of MCs prior to initiation of this study that continue to receive the MCs, providing an opportunity to study the longer term effects of the MCs. In the second year, both Arm A and Arm B villages receive the intervention (i.e. a non-randomized stepped-wedge design). MC effectiveness is evaluated from epidemiologic surveys and PCR detection of malaria prevalence in villages with MCs as compared to those without MCs.The effectiveness of the MC intervention is being evaluated with a quasi-experimental cluster-assigned stepped-wedge study with a pretest-posttest control group design; clusters (i.e. villages) are not randomized to the intervention. The main aim of the study is to determine if MCs reduce the prevalence of This proof-of-concept pilot study was designed in part to build and test the relationship with the Odisha Malaria Control Program (MCP) in the Ministry of Health. Because of the relatively short timeline from funding to field implementation, the districts needed to make decisions on where the malaria camps were going to be implemented rather quickly. While randomization was not possible at that time, the MCP did balance the assignment of villages with respect to ecology. The stepped wedge design was chosen for the same logistical restraints that resulted in the decision to not randomize the villages for this pilot study.P. falciparum as per Indian national guidelines is artesunate 4 mg/kg body weight per day (3 days); sulfadoxine-pyrimethamine (SP) 25 and 1.25 mg/kg body weight, respectively (one dose) on day 1; and primaquine 0.75 mg/kg body weight or 45 mg (one dose) on day two. Similarly, the first-line treatment for P. vivax is chloroquine (CQ) 10 mg/kg body weight (day 1 and day 2) and 5 mg/kg body weight (day 3), along with primaquine 0.25 mg/kg body weight (one dose) for 14 days.The MCs implemented by the Odisha State MCP in remote forested areas of the state involve teams of health workers visiting villages to educate the population, enhance vector control with LLINs and indoor residual spraying (IRS), and perform village-wide testing with rapid diagnostic tests and treatment for malaria. The MCP has dramatically scaled up efforts to prevent, diagnose, and treat malaria under the DAMaN program, with apparently rapid and impressive results . The firThe program consists of two and up to three MC rounds administered per annum. The first round (round 1) during the pre-monsoon season includes testing for the whole village population with treatment of RDT+ individuals along with LLIN distribution, IRS, and intensified campaigning with ASHAs. The second round  screens only febrile adults but all children (<5 yr age) and lactating/pregnant mothers regardless of fever status. This is because round 1 is expected to reduce malaria by >90%, and subsequent rounds need only target the remaining febrile cases or potentially asymptomatic cases that have become symptomatic in the interim. The necessity of round 3 MCs is determined based on the coverage and success of rounds 1 and 2 and is, therefore, optional.For each MC round, the DAMaN team is in each village for one to three days, depending on the size of the village. The MCs also offer a range of malaria awareness activities such as plays containing malaria prevention messages performed by traveling troupes, bednet use ‘performances’ in government high schools with hostels, and vans with loudspeakers that play popular local songs that incorporate malaria prevention guidance in the lyrics and have mosquito control messages displayed on their sides. In addition, the MCs include nutritional screening of children and pregnant women.The eastern state of Odisha is a predominantly (83%) rural state of approximately 42 million people  where thA recent study in the adjacent Sundargarh district found that behavioral risk factors for malaria include being outside in the evening (34.5% among adult men and 7.9% among adult women aged 15+ years and 0.7% among children) and sleeping outside . Although women were more likely to get up before 6 a.m. , men were more likely to be outside in the early morning . More children used insecticide treated nets the previous night (73.4%) than men (45.6%) or women (39.6%), and repellents were used by 29.5% of 234 households  [Tribal areas such as the Jharsuguda and Keonjhar districts, where this study is situated, are the most seriously afflicted with malaria, displaying meso- to hyperendemic transmission. IRS coverage in malarious regions is approximately 90% and the last ITN distribution was in July/August 2017 . These cVillage residents are eligible for the study if they are (1) aged 12 months to 69 years and (2) have an apparent complete understanding of the study procedures as outlined in the consent form. There are no exclusion criteria based on residency length, pregnancy, social class, or health conditions.steppedwedge command [Statistical significance is not the central goal of this two-year pilot study. The sample size was estimated based on the stated requirement of a minimum of 2,000 participants in the National Institutes of Health request for proposals in concert with study logistics . We used command  which isPlasmodium infection prevalence by PCR in the forest villages around Rourkela was 16% during the rainy season [Plasmodium infection prevalence by PCR after two rounds of MCs between control and intervention clusters measured during the rainy season in the second year, to be a meaningful reduction. For our sample size calculations, we compared between clusters in arm A (intervention) and arm B (control), and used an intracluster correlation coefficient (ICC) of 0.02  households and 1,200  people, and we aim to enroll 163 people per cluster. In our previous studies, y season , and we evalence ).Plasmodium infection by PCR with 80% power and 5% significance level; we use a 15% higher sample size  to allow for loss- to - follow - up.We calculated power for the stepped wedge design using Arms A and B; Arm C was not included in the power calculations because we do not have pre-intervention data for that arm. Two study arms of 5 clusters each and an average cluster size of 163  allows us to detect a reduction from 16.0 to 10.0% prevalence of Plasmodium infection by PCR with 80% power and 5% significance level; we use a 18% higher sample size  to allow for loss-to-follow-up. The increase in sample size allows us to maintain study power, and the rationale for the increase is twofold. First, two of the villages were inhabited by the Juang People, one of the most geographically and culturally isolated tribal groups specific to the Keonjhar district [Revised sample size: Based on our experiences recruiting in the field in August and September 2019, we increased the sample size from 2244 to 2700 and add two new clusters (villages). To achieve the sample size of 2700, we are enrolling one additional village in each arm for a total of six villages each in Arms A and B and three in Arm C . Six clusters each in Arms A and B and an average cluster size of 163  allows us to detect a reduction from 16.0 to 10.5% prevalence of district . WillingWe selected one or more district blocks with a high API (>2) from the study districts. Villages/hamlets are the units of the MCs and are therefore the clusters in this study. We assign the villages/hamlets into two study arms: Arm A ‘new-MC’ (n = 6 villages that start MCs in year 1) and Arm B ‘delayed-MC’ . A third study arm (Arm C ‘old-MC’) is comprised of n = 3 villages where MCs have already been implemented, providing an opportunity to assess the effects of the MCs over a longer period of time. After assignment to study arms, a cohort is enrolled during a baseline visit in all the clusters. In the old-MC clusters (where MCs have occurred) and in the clusters not receiving MCs, malaria activities continue as planned by the MCP. In the new-MC clusters, the MC intervention is introduced . The exact villages and districts were chosen in consultation with the Odisha MCP which balanced the assignments in terms of ecotype . Because this is a community-based intervention, participants are not blinded.To recruit participants, a field team travels to the villages at the same time when the DAMaN team delivers the intervention. Within each village, the DAMaN team sets up their screening and treatment camps in a central location. Our team sets up their operations adjacent to the DAMaN team. As each village resident completes their DAMaN visit, they are informed of the nature and purpose of the study and invited to enroll.For those who express interest in the study, field staff read aloud a consent form in one of the local languages (i.e. Oriya or Hindi). Eligible participants are offered a copy of the consent form in the local language to read along with the field staff member. Persons aged 18 years or older who are mentally capable of consenting are provided a consent form to review and sign. For children under seven years of age, a consent form signed by a parent or guardian is required. For children between seven and 17 years of age, parental consent and an additional child assent is required for participation. If a person cannot write, they are asked to place a thumbprint on the consent form in the presence of a literate witness (i.e. another adult in the household or a neighbor), who also signs the form. Ethical approval for the study was obtained from the institutional review board at New York University  and the institutional ethics committee at Community Welfare Society Hospital . The study was also approved by the Indian Council of Medical Research Health Ministry’s Screening Committee.A brief survey is administered to consenting adults and older children (aged 17 and older) via Samsung Galaxy tablets  loaded with a REDCap  mobile application instrument. A parent or guardian provides proxy responses for children aged under 17 years. The survey includes measures of demographics, socioeconomic status (SES), malaria knowledge, malaria prevention, and previous malaria and treatment. With respect to demographics, participants are asked to provide their age, sex, and number of people living in the household; proxy interviewees for children are asked to describe their relationship to the child. SES measures include educational attainment and occupation.Malaria knowledge focuses on signs and symptoms; respondents are asked open-ended questions and field staff record responses. For malaria prevention, participants are asked if they slept outside and whether they slept under a bednet when they did so. For previous malaria and treatment, participants are asked if they have ever had malaria, when was the last time they had it, whether they sought advice or treatment last time, the time to treatment, whether they were tested for malaria last time, and the result of the last malaria test.At the time of enrollment, each study participant receives a blue study card with their study participant number. The study card is a means to provide anthropometric and blood testing results to the study participant at each visit and also allows for participant tracking in subsequent visits.Field staff measure each participant’s body temperature, height, weight, and mid upper arm circumference (MUAC). Body temperature is measured temporally in degrees Fahrenheit with a non-contact infrared thermometer  to the nearest tenth of a degree. Height is measured with a height measuring tape  in centimeters. Weight is measured in kilograms with a scale . MUAC is the circumference of the left upper arm, measured at the mid-point between the tip of the shoulder and the tip of the elbow (i.e. olecranon process and the acromion). Participants are asked to remove any clothes that may hinder the measurement . MUAC is measured in centimeters to one decimal using a Myotape/tape measure .Participants who self-report having malaria in the past eight weeks are administered a malaria cost survey to assess the total out-of-pocket cost of a malaria illness episode in our study setting alongside the trial. The malaria cost survey has questions eliciting information on treatment-seeking behavior and associated out-of-pocket spending, work and school absenteeism, and income loss incurred by participants or their caregivers during the illness episode.Out-of-pocket costs include direct and indirect costs borne by households during a malaria illness episode. Direct costs include all out-of-pocket medical  and non-medical  expenditures for seeking and receiving treatment for malaria while indirect costs consist of lost paid work for participants, or, where applicable, their caregivers during malaria illness. We will value lost paid work due to malaria illness as the higher of the self-reported or estimated income loss; the latter is estimated conservatively as the product of the number of reported work-days lost by the patient or their caregivers and the minimum daily wage for Odisha state set by India’s Labour Bureau . We willThe total out-of-pocket cost per malaria episode is obtained by summing direct and indirect costs. If there are missing cost data, depending on the amount and the reasons for the data being missing, we will apply naive or multiple imputation methods to the dataset to derive unbiased and efficient estimates . We willPlasmodium infection by RDT and species-specific PCR. Vacutainers  of venous blood are requested from individuals found to be Plasmodium positive by RDT. All vacutainers and small volume microvettes are immediately refrigerated on ice and shipped overnight in insulated coolers to the main laboratory where they are processed. Blood components (e.g. red blood cells (RBC) pellet, plasma) are isolated and stored at −80°C until assayed.In addition to completion of a study questionnaire and anthropometric measurements, study enrollment procedures include a finger prick by sterile lancet for small blood volume sampling , thin and thick blood smears, blood spots (Whatman FTA Blood Stain Cards), and point of care (POC) testing . All individuals are screened for P. falciparum and P. vivax using the point-of-care (POC) test FalciVax . FalciVax is a lateral flow RDT that detects species-specific Plasmodium antigens in the peripheral blood. Using the sample loop included in each RDT kit, five microliters of blood and two drops of buffer are added to the cartridge. The results for each RDT is read after 20 minutes at room temperature and recorded.Finger prick blood samples from all study subjects are tested for Thin smears are fixed in methanol and dried prior to Giemsa staining alongside thick smears. Stained smears are evaluated quantitatively and qualitatively for asexual  and sexual (i.e. gametocytes) stage parasites at the species level by two independent microscopists as previously described .P. falciparum and/or P. vivax is determined by PCR amplifcation of multicopy target loci  as described [The RBC pellet isolated from the small blood volume obtained from each study participant is stored at −80°C until assayed. Upon thawing, DNA is extracted using QIAGEN’s QIAamp DNA mini Kit. A final elution volume of 50 ul is used. Molecular detection of P. falciparum and P. vivax proteins and/or peptides  and converted to Net MFI (Net MFIAg = raw MFIAg – background MFIAg) where background MFIAg is the mean MFI of a given antigen in the blank wells. For each antigen, seropositivity is defined as mean Net MFInegative pool plus three standard deviations (SD).Plasma pellets isolated from the small blood volume obtained from each study subject are prepared at 1/200 in a buffer of PBS, 0.05% Tween, 0.5% BSA, 0.02% sodium azide, 0.1% casein, 0.5% PVA, 0.5% PVP, and ocedures . Plates Plasmodium species composition and complexity of infection for each arm are determined. We use deep sequencing methods [P. falciparum drug resistance alleles to infer the resistance of circulating parasites to antimalarial drugs, and (2) P. falciparum and P. vivax highly polymorphic loci for genetic diversity measurements. The latter assay determines if, as transmission decreases, there is a decrease in parasite genetic diversity, as has been shown to occur with P. falciparum [P. falciparum and P. vivax isolates to determine their genetic diversity and phylogenetics in comparison to our previously sequenced Indian isolates [The  methods  to run mlciparum . We alsoisolates ,45.Plasmodium positive by RDT.Follow-up visits are conducted immediately prior to the MC camps in each intervention village and during roughly the same time period in the comparison villages. Participant IDs are confirmed via presentation of the individual’s blue study card. Follow-up visit procedures parallel those employed during the baseline enrollment visit, including completion of the study questionnaire, anthropometric measurements, malaria RDT and hemoglobin POC testing, and collection of a small volume of blood by finger prick. Vacutainers  of venous blood are requested from individuals found to be In each study village, eight randomly selected households and eight cattle sheds are surveyed for mosquitoes using light trap, hand catch, and pyrethrum space spray collection methods. UV light traps are set for a 12 hour window (6 pm to 6 am) both inside and outside of the selected households. Mosquitoes caught in the traps are identified morphologically to the species level, and mosquito density per trap per night is calculated. Hand catch collections of resting mosquitoes are performed inside each household and cattle shed for 15 minutes via sucking tubes. Hand caught mosquitoes are also identified morphologically to the species level, and per hour species mosquito density is calculated. Pyrethrum spray collection of the resting mosquitoes in one bedroom of each village is performed. The collected mosquitoes are identified morphologically to the species level, and per room density is calculated. Blood meals of the vectors are collected on filter paper to identify their feeding behavior. All mosquito collection data are tabulated manually. The selected households are evaluated using a questionnaire to document features and characteristics of the dwelling and surrounding region relevant for parasite transmission and increased risk of infection. The household data are collected via the REDCap Mobile App on Samsung Galaxy tablets.Plasmodium presence and species. Parasite presence is defined as PCR-positive for any malaria parasite species, and parasite species are defined as PCR-positive for P. falciparum and/or P. vivax.Primary outcome measures are Plasmodium-specific serology, and Plasmodium parasite genetic epidemiology. Malaria by RDT is defined as RDT-negative vs. RDT-positive. We further identify asymptomatic and subpatent infections. Asymptomatic infections are defined as PCR and/or RDT positive for any malaria species with absence of documented fever or self-reported fever in the last 48 hours. Subpatent malaria is defined as RDT-negative and PCR-positive. Gametocyte density is measured by quantifying the number of gametocyte infected erythrocytes and dividing by the number of leukocytes. To account for a mixed-infection (either by species or by multiple strains), we take the gametocyte to trophozoite ratio to reduce any apparent bias. Plasmodium-specific serology is analyzed as a continuous antibody titer variable as well as a categorical (seropositive vs. seronegative) variable. In terms of Plasmodium parasite genomic epidemiology, variables include clonality, genetic diversity, and molecular force of infection.Secondary malaria outcome measures include malaria as detected by RDT, gametocyte density, 2 and analyzed as a continuous and categorical variable. BMI categories  are based on adult and child standards for Asian Indians [Secondary anthropometric outcome measures include hemoglobin, body mass index (BMI), body temperature, and MUAC. Hemoglobin is measured by POC testing which provides both continuous and categorical data  regarding anemia status. BMI is calculated as kilograms/meters Indians . Body tePlasmodium infection prevalence by PCR at the third follow-up visit is the main outcome and will be compared across Arms A and B, taking clustering into account with generalized estimating equations in Stata version 16.1 or higher . The unit of analysis is the individual. Potential confounders will be examined including age, gender, antimalarial use, type of housing, and indicators of socio-economic status at the individual level; malaria intervention use at the household and individual levels, and elevation, village size, and mosquito-habitats  at the village level. We will conduct intent-to-treat analyses. If it is decided that an interim analysis needs to be conducted, the study design will be adjusted for the p-value spent.Mathematical modeling and simulation analyses have the potential to assist with analysis and interpretation of randomized trials of interventions, particularly if trial results are unexpected , as wellBy definition of the intervention program criterion, the villages selected by the Odisha state MCP for MCs are located in remote and ‘hard to reach’ parts of the state. Most of the selected villages lack telephone and internet networks and cannot be visited via standard automobile, requiring the study teams to travel long distances on foot. The posed challenges are exacerbated in the months of peak malaria transmission (e.g. monsoon seasons) when rains are frequent and heavy and many of the roadways are inaccessible or washed out entirely. Close coordination of study visits and continued collaboration with the DAMaN teams, the local health centers and personnel including the village ASHAs, and the Odisha state MCP have so far aided greatly in tackling these logistic challenges.These remote and rural regions of Odisha are largely populated by tribal groups. Some of the study villages in the Keonjhar district are home to the Juang and Munda tribes, two populations that operate largely independent of surrounding communities and are not accustomed to outside contact. Villagers of the stated tribes demonstrated a greater reluctance to participate in the study than observed in other study villages, and many of the individuals who gave initial written consent to participate were not willing to give a blood sample.On 25 March 2020, the study was temporarily suspended following Prime Minister Narendra Modi’s national lockdown in response to the COVID-19 global pandemic. The national lockdown was imposed in all Indian states with regulations including the closure of all non-essential services and businesses including places of worship, suspension of all education and research operations, suspension of non-essential transportation, prohibition of all social, political, sports and entertainment, and cultural activities, and bans on leaving one’s household. The nationwide lockdown was first extended through 3 May 2020 and subsequently until 8 June 2020. All districts were classified into risk zones  and re-evaluated on a weekly basis. The lockdown was extended through May 31 for all ‘orange’ and ‘red’ zones. The study site districts were ‘orange zones’ at the time of the extension, so study operations remained suspended. The study region began re-opening on 8 June 2020, and data collection for the study resumed with the second follow-up visit during the week of 22 June 2020 . Due to During the writing of this manuscript, the study protocol of a complementary project ‘DAMaN Assessment’ was published by colleagues at the Indian Council of Medical Research/Regional Medical Research Centre, Bhubaneswar, India .There are several important limitations to this study. Villages were not randomized to study arms, which may result in potential selection bias. However, our assignment of villages aimed to ensure that the distribution of villages across the arms was comparable in terms of ecology and terrain type, presence/absence of mining activity, population size, and API. Given the current COVID-19 pandemic, loss-to-follow-up could lead to selection bias as well. With respect to information bias, all self-reported survey measures, including malaria symptoms and diagnoses, may be subject to recall bias. We note that all participants are surveyed prior to receiving the MC screen and treat protocol, but other activities may be occurring including the educational components of the MC intervention package. This could result in possible information bias, resulting in an overestimation of malaria symptoms knowledge in the study sample.Implementation research is intended to combine research approaches and real-life practices to expedite both the general development and delivery of effective public health policies and programs . Here, w


Plasmodium falciparum and Plasmodium vivax infections in the multidrug-resistant areas along the Thai-Myanmar border. The association between gene polymorphisms and parasite density was also investigated. Genomic DNA (gDNA) of P. falciparum and P. vivax were extracted from whole blood and dried blood spot (DBS). Gene amplification and genotyping were performed by PCR and PCR-RFLP analysis, respectively. Of these samples, 178 and 209 samples were, respectively, mono-infection with P. falciparum and P. vivax. The ratio of P. falciparum: P. vivax was 46%:54%. Results showed marked variation in the frequency distribution and patterns of the genotypes and gene alleles of the nine immune response genes or human host genes. The SNPs of TGFβ1, IL10 and ICAM1, were significantly associated with P. falciparum, but not P. vivax parasite density. TGFβ1, IL10 and ICAM1, may play more significant roles in modulating P. falciparum than P. vivax parasitemia. The prevalence of the genotypes and gene alleles of these genes, including their association with parasite density, may vary depending on patient ethnicity and endemic areas. Information obtained from each endemic area is essential for treatment strategies and the development of vaccines for malaria prophylaxis in specific areas.Polymorphisms of the genes encoding proteins involved in immune functions and the binding of malaria parasites to human host cells have been the focus of research in recent years, aiming to understand malaria pathogenesis and case severity and to exploit this knowledge to assert control over malaria. This study investigated the genetic diversity of the human host genes encoding proteins that are involved in immune functions and malaria parasite binding, i.e., MCP1 (−2518), TGFβ1 (−509), TNFα (−308), IL4 (VNTR), IL6 (−174), IL10 (−3575), TLR4 (299), CD36 (−188), and ICAM1 (469) in patients with mono-infection of  Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi. P. falciparum is the most virulent and widespread infectious species in tropical and subtropical countries due to the parasite’s resistance to the most available antimalarial drugs. The species P. vivax is the next most significant malaria species, which usually causes benign uncomplicated malaria with relapse from dormant hypnozoites in the liver. Its clinical features differ from those of P. falciparum malaria. Both P. falciparum and P. vivax often co-exist in several parts of the world. The signs and symptoms of malaria vary from uncomplicated to complicated or severe malaria. Severe malaria manifests life-threatening clinical signs and symptoms, which are defined according to criteria of the World Health Organization (WHO) as impaired consciousness, respiratory distress, multiple convulsions, circulatory collapse, abnormal bleeding, jaundice, hemoglobinuria, severe anemia, hypoglycemia, acidemia, hyperlactatemia, renal impairment, and hyperparasitemia  . Conversely, the parasite density in the isolates with the heterozygous or homozygous mutant genotype for IL10 was significantly lower than the wild type genotype . The parasite density in patients carrying the heterozygous genotype of ICAM1 was significantly lower than the wild type genotype [median (range): 620  vs. 3780  /μL)]. No association between genotypes and P. vivax parasite density was found.The association between the genotypes of the nine genes in patients with P. falciparum infection. The parasite density in the isolates carrying the mutant allele of TGFβ1 was significantly higher than those with the wild type allele [median (range): 4568  vs. 1109  /µL)]. Conversely, the parasite density in patients carrying heterozygous alleles of IL10 and ICAM1 was significantly lower than those with the wild type allele . No association between genotypes and P. vivax parasite density was found.For the gene alleles, similarly to those observed with genotypes, significant associations were observed between the gene alleles of TGFβ1, IL10 and ICAM1, and parasite density in patients with P. falciparum compared with P. vivax infection for malaria parasite density, one of the criteria for malaria severity. The HWE deviation observed with TGFβ1, TNFα, IL10, CD36 and ICAM1 could be due to gene mutation, population migration, and/or selection of genotypes/alleles of the study populations. In the present study, the SNPs of the two host genes involved in immune functions, i.e., TGFβ1 and IL10, and the one involved in malaria parasite binding (ICAM1) were significantly associated with P. falciparum parasitemia. The SNPs of TGFβ1 (CT genotype) was significantly associated with high parasite density, whereas that of IL10  and ICAM1  were significantly associated with low parasite density compared with their corresponding wild type alleles/genotypes. However, this association was not observed in patients with P. vivax infection. This may suggest the significant contribution of these genes in host susceptibility to infection and disease severity in P. falciparum cases but not P. vivax.The frequencies and distribution patterns of genotypes and gene alleles of most genes under investigation were markedly different, depending on the species of the malaria parasite. It is noteworthy that results may suggest a more significant contribution of these genes in patients with P. falciparum, while the CT genotype is linked with high parasite density. It is possible that the homozygous genotype (CC) results in the decrease in TGFβ cytokine production for malaria eradication [P. falciparum and P. vivax infections [The CC genotype of TGFβ has been reported to link with low parasite density in dication , whereasdication . The T adication ,23,24 thdication . Neverthfections ,27,28.P. falciparum infection compared with the wild type genotype/allele. It is hypothesized that the T allele and TT genotype may increase the production of IL10 and severe malaria [P. vivax infection, in spite of the high plasma levels of IL10 and TGFβ during the infection phase [Plasmodium vivax has a hypnozoite stage, mono-invasion and long incubation period of the asexual stage. These factors result in low parasitemia and pathogenicity [P. vivax infection produces non-severe symptoms, patients usually do not seek treatment with antimalarial drugs. This may lead to chronic morbidity and high plasma cytokine levels. The association between human genetics and plasma levels of these cytokines remains conflicting. The result of the current study in the Thai-Myanmar area, however, showed no association between the genetic polymorphisms of the human immune cytokines and P. vivax parasite density.For IL10 gene, the A allele and TA and AA genotypes were associated with low parasite density in patients with  malaria ,30. This malaria ,32, whic malaria . In addi malaria . It is l malaria ,32, ther malaria . For P. on phase , minimumon phase . Plasmodgenicity . Since PPfEMP1 , leading to an enhanced risk of severe clinical outcomes [P. falciparum density. The mutation of the ICAM1 gene at this position may result in an impaired binding property of the ICAM1 molecule on the infected erythrocytes, which was supported by the results of an in vivo study [P. vivax, the parasite exports vir protein encoded by vir gene (variant interspersed repeats gene), which binds to ICAM1 receptor molecule. Unlike PfEMP1, vir protein is not a potent cytoadherence and sequestering ligand [P. vivax parasitemia in the Thai-Myanmar border area.The adhesion of infected red blood cells to the microvasculature of the brain is a key feature of cerebral malaria ,40. The vo study . This levo study ,43. Howevo study . For P. g ligand ,46; therP. falciparum or P. vivax parasite density and SNPs of the MCP1, TNFα, IL4, IL6, IL10, TLR4, or CD36 genes. Current reports on the association between malaria parasite density and severity and the SNPs of these genes are controversial. High MCP1 levels during P. falciparum and P. vivax infection were associated with severe malaria and mortality [There was no significant association between ortality ,50,51,52ortality  and reduortality . The SNPortality ,54. The ortality ,56. A heortality ,58.It was noted that hyperparasitemia is only one criterion for malaria severity. Malaria symptoms and disease severity can be influenced by many factors such as parasite mixed strain infection, endemic malaria area, host genetic background, time of infection (the duration from infection and sample collection), medication history, nutrition, prior infections, and co-infection with other pathogens (viruses and bacteria). These factors could be confounding factors for conflicting results reported in this as well as other studies in other malaria-endemic areas. This should be considered when analyzing and interpreting the results. An additional limitation of the current study is that the number of samples used in each association analysis is small, particularly for the TNFα and MCP1 genes. Information obtained from each endemic area, therefore, is essential for treatment strategies and the development of vaccines for malaria prophylaxis in those specific areas. Furthermore, other potential genes were identified in the large-scale genome-wide association studies, e.g., gene expression profiles related to neutrophil and erythroid activity, sickle cell hemoglobin S variant, interleukin receptors and kelch-like protein KLHL3 ,60,61, sP. falciparum than in P. vivax infection. The SNPs of these genes were associated with P. falciparum parasite density. The prevalence of the genotypes and alleles of these genes reported in various studies, including their association with malaria parasite density and disease severity, is conflicting due to confounding factors. Interpretation and conclusion on the association of the polymorphisms of genes involved in host immune response and binding of the malaria parasite and P. falciparum and P. vivax parasitemia should be based on studies conducted in each endemic area with adequate sample size and minimized confounding factors.The genes involved in host immune response and binding of the malaria parasite, particularly TGFβ1, IL10 and ICAM1 may play a more significant role in malaria parasitemia in


Plasmodium spp. Consequently, this study aimed to evaluate the genetic diversity of P. falciparum and the decrease in allelic richness in this population. The polymorphic regions of the pfmsp-1 and pfmsp-2 genes of patients with falciparum malaria from Honduras and Nicaragua were analyzed using nested PCR and sequencing. Most of the samples were classified into the K1 allelic subfamily of the pfmsp-1 gene and into the 3D7 subfamily of the pfmsp-2 gene. Despite the low genetic diversity found, more than half of the samples presented a polyclonal K1/RO33 haplotype. No sequence polymorphisms were found within each allelic subfamily. This study describes a notable decrease in the genetic diversity of P. falciparum in the Moskitia region after a bottleneck phenomenon. These results will be useful for future epidemiological investigations and the monitoring of malaria transmission in Central America.The countries of Central America and the island of Hispaniola have set the goal of eliminating malaria in less than a decade. Although efforts to reduce the malaria burden in the region have been successful, there has been an alarming increase in cases in the Nicaraguan Moskitia since 2014. The continuous decrease in cases between 2000 and 2014, followed by a rapid expansion from 2015 to the present, has generated a potential bottleneck effect in the populations of  The incidence of malaria in the Americas has decreased by 53% in the last decade, reaching 0.9 million cases in 2019 . T. Tpfmsp-Honduras . These dBased on extensive global data collection by Lê et al. , subfamipfmsp-2 gene, our results reveal a clear predominance of the 3D7 subfamily (92.7%) over FC27 (7.3%). This result coincides with what was reported 10 years earlier in Honduras by López et al. [pfmsp-2 gene in the Americas are scarce. Two studies conducted in Colombia and Brazil reported that both FC27 and 3D7 allelic subfamilies were found in almost the same proportions [Regarding the z et al.  with 91.portions ,39. On tportions  carried The most surprising result of the present study is the low genetic diversity found in the parasite population. All 124 samples that amplified the K1 subfamily showed a single allele of approximately 235 bp that was confirmed by sequencing. The same happened with the RO33 subfamily, with a single allele of approximately 335 bp. Only the MAD20 subfamily revealed the presence of three alleles with size polymorphism. No sequence polymorphisms were found within each allele. The only similar study conducted with 35 samples from Honduras and collected a decade ago described a total of 23 alleles  . This inpfmsp-1, especially K1/RO33. This proportion is higher than previously reported in Honduras, where 23% of mixed samples are described [pfmsp-2 gene. If we consider the recent bottleneck, the homogeneity of the mixed genotypes and the low transmission that continues to prevail in the region, it is very likely that these mixed genotypes are propagated by a single multiple inoculation event rather than by superinfection [Despite the homogeneity in the population demonstrated in this study, 96/147 (65.3%) samples showed a polyclonal infection with at least two alleles of escribed . Mixed infection . This hynfection  when anaFinally, the allele frequencies of the parasite populations of Honduras were compared with those of Nicaragua. Statistical analysis showed that there were no significant differences between the two populations. Our results are consistent with the results of a previous study conducted with 110 samples collected in the Moskitia (Honduras and Nicaragua) between 2009 and 2012 that evaluated seven neutral microsatellite markers . Those aP. falciparum. The samples were collected between 2018 and 2021.This study included 160 blood samples from febrile patients diagnosed with falciparum malaria and who sought medical assistance in national health establishments in two municipalities of Honduras and five municipalities of Nicaragua belonging to the ecological and cultural region called the Moskitia . All samBlood samples were collected by fingerstick on Whatman FTA filter paper at the time of patient recruitment. The ethics committee (CEI-MEIZ) of the National Autonomous University of Honduras (UNAH) reviewed and approved the study under protocol number 03-2020. Consent to participate was waived for the following reasons: (a) No personal information was included; (b) the study was beneficial to public health and, (c) did not harm the participants. Blood samples were collected for parasite species identification and analysis of genes associated with drug resistance, in accordance with national regulations and for routine malaria surveillance purposes.DNA was extracted from blood on filter paper cards using a Chelex-100 based method  . Microscpfmsp-1 gene was amplified by nested PCR as described previously [® 2X Master Mix , 2 μL of each primer .® 9.1.7 software  and queried against international databases contained in NCBI to confirm the identity of the sequences. Subsequently, the sequences were analyzed for sequence and/or size polymorphisms. The sequences obtained were deposited in the NCBI database. The nucleotide sequences were translated in silico in the correct open reading frame to obtain the amino acid sequences of K1, MAD20, RO33, 3D7 and FC27. The peptide sequences were analyzed with the NCBI Protein Blast tool.Sequences were trimmed at both 5′ and 3′ ends with the GeneiousPlasmodium falciparum populations in the Moskitia region of Central America. The drastic decrease in the number of malaria cases reported in the region in recent years could have been responsible for a notable decrease in the genetic diversity of the parasite, generating highly homogeneous populations as revealed by the analysis of the pfmsp-1 and pfmsp-2 genes. These results will be useful for future epidemiological investigations and the monitoring of malaria transmission in Central America.This study describes a bottleneck effect suffered by


Drosophila melanogaster. Adult Drosophila fruit flies were fed cornmeal food containing various concentrations of 5‐ALA/SFC. Locomotor functions, life span, muscle architecture, and age‐associated changes in mitochondrial function were analyzed. We found that feeding 5‐ALA/SFC mitigated age‐associated declines in locomotor functions and extended organismal life span. Moreover, 5‐ALA/SFC preserved muscle architecture and maintained the mitochondrial membrane potential in aged animals. Since 5‐ALA phosphate/SFC is used as a human dietary supplement, our results suggest that it could be used to slow the age‐related declines in muscle functions, prevent age‐associated clinical conditions such as frailty, and extend healthspan and life span.Declines in mitochondrial functions are associated with aging. The combination of 5‐aminolevulinic acid (5‐ALA) and sodium ferrous citrate (SFC) improves mitochondrial functions in cultured cells. In this study, we investigated the effects of dietary supplementation with 5‐ALA and SFC (5‐ALA/SFC) on the healthspan and life span of  Drosophila melanogaster. 5‐ALA/SFC might be used to prevent age‐associated clinical conditions.Age‐related declines in physiological systems cause problematic features such as frailty. In this study, we found that 5‐aminolevulinic acid hydrochloride and sodium ferrous citrate (5‐ALA/SFC) mitigated age‐associated declines in locomotor functions and the muscle architecture, preserved the mitochondrial membrane potential, and extended life span in  CumulatThe age‐associated physiological decline is linked with mitochondrial dysfunctions , 5, 6. IMitochondrial respiratory chain complexes II, III, and IV, and cytochrome c contain heme; heme deficiency may be an underlying cause of age‐associated mitochondrial dysfunction . The bioDrosophila melanogaster. Drosophila is a genetically tractable model system with the relatively short life span and is used to study human diseases and aging. Age‐associated dysfunctions are prominently observed in the muscle . The primary antibodies used were rabbit anti‐citrate synthase. The secondary antibodies used were anti‐rabbit IgG HRP‐Linked Whole Ab Donkey .Thoraxes from the 20 flies were homogenized, and citrate synthase activity was measured by using Citrate Synthase Activity Colorimetric Assay Kit  according to the manufacturer's manual. Protein levels were measured with the BCA Protein Assay Kit (Thermo Fisher Scientific).Twenty thoraxes were homogenized, and bilirubin levels were measured by using Bilirubin Assay Kit  according to the manufacturer's instruction.rp49. Primers were designed using DRSC FlyPrimerBank . Primer sequences are shown in Table Total RNA of the 20 thoraxes was purified according to the protocol provided with the RNeasy Plus Universal Mini Kit . The amount and purity of the total RNA were measured using the NanoDrop One Spectrophotometer (Thermo Scientific). cDNA was synthesized from 0.5 μg of total RNA using the High Capacity RNA‐to‐cDNA Kit (Life Technologies). The expression level of each gene was measured using the PowerUp SYBR Green Master Mix (Life Technologies) and the StepOnePlus Real‐Time PCR System (Life Technologies). Expression of genes of interest was standardized relative to Male flies were anesthetized, and the indirect flight muscle was dissected in cold SDM. The muscles were then fixed for 30 min with 4% paraformaldehyde at room temperature. After fixation, samples were washed three times with 0.1% PBST (0.1% triton in 1× PBS) for 5 min each time. The samples were blocked with 1% normal goat serum (NGS)/0.1% PBST for 30 min at room temperature. The samples were then incubated with anti‐ATP5A antibody  in 1% NGS/0.1% PBST overnight at 4 °C. The next day, the samples were washed three times for 5 min in 0.1% PBST and then incubated in 1% NGS with 0.1% PBST containing Alexa Fluor 488 anti‐mouse IgG (Thermo Fisher) at room temperature for 3 h. Subsequent to this, the samples were washed three times in 0.1% PBST and mounted in VectaShield mounting medium . Images were captured using a laser confocal microscope (Zeiss LSM 710) and analyzed with ImageJ .m MitoTracker Deep Red FM (Thermo Fisher Scientific) and MT‐1 Dye  for 30 min at room temperature. Samples were washed three times for 5 min each time with SDM, and fixed with 4% paraformaldehyde for 40 min at room temperature. After fixation, the samples were washed three times for 5 min each time with SDM. The samples were imaged under a laser confocal microscope (Zeiss LSM 710) and analyzed with ImageJ [Male flies were anesthetized, and the indirect flight muscles were dissected in cold SDM. The muscles were then incubated in SDM containing 100 nh ImageJ .imagej [Male flies were anesthetized, and the indirect flight muscles were dissected in cold SDM. The muscles were then fixed for 40 min with 4% paraformaldehyde at room temperature. After fixation, samples were washed three times with 0.1% PBST (0.1% triton in 1× PBS) for 5 min each time. The samples were blocked with 1% NGS/0.1% PBST for 5 h at room temperature. The samples were then incubated with anti‐4‐HNE antibody  in 1% NGS/0.1% PBST overnight at 4 °C. The next day, the samples were washed three times for 15 min in 0.1% PBST. Alexa Fluor 488 anti‐rabbit IgG (Thermo Fisher) and phalloidin were then added to the samples, and kept at room temperature for 3 h. Subsequent to this, the samples were washed three times in 0.1% PBST and mounted in VectaShield mounting medium (Vector Laboratories). Images were captured using a laser confocal microscope (Zeiss LSM 710) and analyzed with imagej .sas version 9.4 , graphpad prism9 , and r  [P values < 0.05 were considered statistically significant.Statistics were done with Microsoft Excel , Statcel‐the Useful Addin Forms on Excel‐3rd ed. , Proc Lifetest of m or higher) and SFC to the culture medium of cells significantly increases the cellular oxygen consumption rate [m 5‐ALA hydrochloride combined with SFC at a ratio of 20 : 1.To examine the effect of 5‐ALA on aging and life span, we maintained flies on a diet of cornmeal food containing 5‐ALA and SFC. The addition of 5‐ALA . The maximum life span of control flies was 57 days, while the maximum life span of flies fed food containing 5 mm 5‐ALA and 0.25 mm SFC was 60 days, and the maximum life span of flies fed food containing 50 mm 5‐ALA and 2.5 mm SFC was 65 days . In flies fed food containing 5‐ALA/SFC, sarcomeres were more clearly observed .Muscle integrity is essential for locomotor function and declines during aging . We analm 5‐ALA/0 mm SFC and 35‐day‐old flies 0 mm 5‐ALA/0 mm SFC, P < 0.006). By contrast, there was no significant difference in myonuclear density between young and aged flies supplemented with 5‐ALA/SFC .The number, size, and arrangement of myonuclei are associated with muscle function . MyonuclThese results suggest that the muscle of aged flies treated with 5‐ALA/SFC maintains a key feature, namely myonuclear density, which is usually associated with young muscle.Mitochondrial dysfunctions occur in aged muscle tissues . We sougm 5‐ALA/0 mm SFC and 0.5 mm 5‐ALA/0.025 mm SFC or 50 mm 5‐ALA/2.5 mm SFC). We also used MT‐1, another cationic dye sequestered to mitochondria by their membrane potential [m 5‐ALA/0 mm SFC and 50 mm 5‐ALA/2.5 mm SFC).Finally, we analyzed the mitochondrial membrane potential by staining dissected tissues with mitochondrial membrane potential‐sensitive dyes. We used MitoTracker, whose accumulation in mitochondria is dependent upon their membrane potential and preserved after fixation. We found that the MitoTracker signal intensity was significantly increased in the muscles in aged flies and was reduced by 5‐ALA/SFC . By contrast, no or less significant differences in 4‐HNE signal were found between young flies and aged flies on the diet containing 5‐ALA/SFC . These results suggest that the supplementation of 5‐ALA/SFC counteracts the age‐associated decline in the mechanisms regulating mitochondrial activities.The rate of ROS production depends on mitochondrial membrane potential, and hyperpolarization of mitochondria could lead to an increase in ROS production . 5‐ALA/SAdministration of 5‐ALA/SFC ameliorates mitochondrial dysfunction in fibroblasts from individuals with mitochondrial diseases  and in wReactive oxygen species induce oxidative damage to cellular macromolecules due to their high chemical reactivity and are believed to be a significant cause of aging . The res5‐ALA is a natural delta amino acid widely present in nature . In addiDrosophila. Our results also suggest that 5‐ALA/SFC supports the maintenance of the mitochondrial membrane potential and suppresses ROS generation. Together, our findings suggest that 5‐ALA/SFC may be a possible therapeutic intervention for age‐related declines in muscle functions and for preventing important symptoms of aging such as frailty.Oral administration of 5‐ALA/SFC mitigates age‐dependent declines in locomotor function, extends life span, and maintains muscle architecture and mitochondrial membrane potential in aged Naoko Nozawa, Takuya Ishii, and Masahiro Ishizuka are employees of SBI Pharmaceuticals Co., Ltd. No other conflict of interest.NN, KMI, and KA designed the experiments. NN, MN, KS, MT, AA, TS, SA, and KA performed the experiments. NN and KA analyzed the data. NN, MN, TI, MI, KMI, and KA wrote the manuscript.Table S1. Primer sequences (5′–3′).Click here for additional data file.