Northern Uganda hosts a large population of refugees from South Sudan, and malaria is one of the major health problems in the area. In 2015, intermittent preventive treatment for malaria (IPTc) was implemented in two refugee camps among children aged 6 months to 14 years. Three distributions of dihydroartemisinin–piperaquine (DP) were conducted at 8-week intervals. The first dose was directly administered at IPTc distribution sites and the second and third doses were given to caregivers to administer at home. A multi-faceted evaluation was implemented, including coverage surveys, malaria prevalence surveys, reinforced surveillance, and pharmacovigilance. Programme coverage exceeded 90% during all three distributions with a total of 40,611 participants. Compared to same period during the previous year (only available data), the incidence of malaria in the target populations was reduced . Among those not targeted for intervention, the incidence between the 2 years increased . Cross-sectional surveys showed a prevalence of parasitaemia (microscopy or PCR) of 12.9–16.4% (95% CI 12.6–19.3) during the intervention, with the highest prevalence among children aged 5–14 years, but with a large increase 8 weeks after the final distribution. A total of 57 adverse events were reported during the intervention period, including one severe adverse event (death from varicella). Adverse events were of mild to moderate severity, and were mainly dermatologic and gastrointestinal. This is the first documentation of an IPTc programme in a refugee camp. The positive impact of DP on the incidence of malaria, together with its favourable safety profile, should lead to further use of IPTc in similar settings. Expanding coverage groups and decreasing intervals between distributions might provide more benefit, but would need to be balanced with the operational implications of a broader, more frequent distribution schedule. Clear recommendation and guidelines exist for several preventive treatment strategies for malaria, including seasonal malaria chemoprevention (SMC) with sulfadoxine–pyrimethamine and intermittent preventive treatment of infants (IPTi) and pregnant women (IPTp) –3. Such Médecins Sans Frontières was present in several camps in the Adjumani District of northern Uganda between January 2014 and July 2015, providing both curative and preventive care to over 40,000 persons. Over 38,000 cases of malaria were notified in 2014 in two camps located in Ayilo (Ayilo 1 and Ayilo 2), Adjumani District, despite distribution of long-lasting insecticide treated bed nets (LLINs) upon arrival into camps. Like much of Uganda, the Adjumani District is situated in an area of high, stable transmission of malaria, and the predominant species is Plasmodium falciparum, responsible for 90–98% of malaria episodes. In the West Nile region of Uganda (which includes Adjumani District), the prevalence of malaria among children aged 0–59 months was 51.3% by rapid diagnostic test (RDT) and 27.5% by microscopy in the 2014–2015 malaria indicator survey . Because the camps were new settlements, prior historical data were not available to compare incident malaria cases in a time series. The overall number of incident malaria cases for the period March–August 2015 (corresponding to the likely protective period of DP) was compared to the same period of 2014, during which time the overall population of the camps was stable.A total of four malaria prevalence surveys were carried out: the first three were performed in the week prior to each of the three IPTc distributions, and the fourth was performed 8 weeks after the final IPTc distribution. The population was stratified into three groups . Systematic sampling was used to include 275 households; one person from each age group was randomly selected for participation. After capillary fingerstick, thick and thin smears were prepared and a set of dried blood spots was collected. Point-of-care haemoglobin testing was performed  among children aged 6–59 months. In a research laboratory in Uganda, microscopy was performed on all slides following WHO guidelines  in a WHOCommunity-based programme coverage surveys 2 days after each distribution used systematic sampling, and were stratified by age group (6–59 months and 5–14 years). A total of 250 households were targeted in each survey. Parents of non-participating children were asked why they did not participate in the distribution, and also to describe adherence to the second and third doses of DP at home.Because of the logistic difficulty of weighing children in a mass treatment context , and because of the desire to more easily distribute DP in similar settings, an attempt was made to create a height-based dosing scale. During the first distribution, weight and height data were recorded for all children attending at two distribution sites. A posteriori, a logarithmic curve was fit to these data, and the dose of DP actually administered based on weight was compared to the dose of DP that would have been administered if the dosing had been based on the child’s height.An existing community-based mortality reporting system was reinforced to ensure that all deaths and serious illnesses in the community were investigated. Programme participants were instructed to return to the health centre for any illness in the days after taking DP. Health centre staff received training about side-effects associated with DP and a dedicated register was put into place to capture adverse events (AE). The causal relationship between DP and the reported AE was judged by the treating physician as related or unrelated –13 basedComité de Protection des Personnes, Ile-de-France XI of Saint-Germain-en-Laye, France.Participation in the IPTc distributions was voluntary. Participants in the malaria prevalence surveys provided written informed consent prior to sample collection. The study protocol was reviewed and approved by the Research Ethics Committee of the Mbarara University of Science and Technology, the Uganda National Council for Science and Technology and the A total of 40,611 children received DP during the programme between 21 March and 13 July, 2015, an average of 13,537 per distribution.Among children <5 years, the 6-month incidence was 0.71 cases/person in 2014 and 0.52 cases/person in 2015 . Among children aged 5–14 years, the 6-month incidence was 0.96 cases/person in 2014 and 0.67 in 2015 . Among persons aged 15 years or older (who did not receive DP in 2015), the 6-month incidence was 0.37 cases/person in 2014 and 0.55 cases/person in 2015 .When considering both card-confirmed and verbally confirmed receipt of IPTc, programme coverage exceeded 90% during each of the three distributions Table . No signThe three principal reasons for non-participation in all three distributions were that the child was ill, that the child or household was absent, and that there was no parent/chaperone available to take the child to the distribution site. Some children were present at the site but refused to take the medication; this proportion rose from 4% of non-participating children during the first distribution to 14% during the third distribution. Correct adherence to the three-day course of DP was described in over 90% of recipients during each of the three distributions (Table 2 = 0.89). As Table Data were collected on 2268 participating children during the first IPTc distribution. Figure Among all age groups, the prevalence of parasitaemia increased by the final survey, approximately 2 months after the third and final IPTc distribution Table . ChildreIn any given age group in any given survey, the rate of parasitaemia detectable by PCR that was not seen on microscopy ranged between 5 and 10%. For the most part, these cases represented low-level parasitaemia: the 90th percentile value of quantitative PCR for microscopy-negative, PCR-positive samples was 101 parasites/μl, and only 5% of microscopy-negative PCR-positive samples had quantitative levels over 200 parasites/μl.10-transformed densities p = 0.04).As expected, overall levels of parasitaemia were lowest among adults over the age of 15 Fig. . On the Plasmodium species changed over the course of the programme . The sole SAE was a 12 year- old female who died unexpectedly at home 18 days after taking the third daily dose of DP during the first IPTc distribution. The death was noted in the community surveillance system. The information that follows was collected from her mother approximately 2 weeks after her death. She correctly took one dose of DP per day during 3 days of the first IPTc distribution. Approximately 10 days later, she developed fever and an itchy vesicular rash on her entire body, and after 2 days of these symptoms, she was seen in the health centre and diagnosed with varicella (an epidemic was ongoing in the camp at that time). She was prescribed zinc oxide cream, paracetamol and tepid sponging for symptomatic relief. She was seen in a follow-up visit 4 days later with no change in her rash; zinc oxide cream and paracetamol were continued. Two days later (18 days after receiving DP and 8 days after the beginning of her varicella), she developed swelling of her face and body and then experienced a sudden loss of consciousness at home. She went to the health centre and was immediately sent by ambulance to the hospital, where she was pronounced dead on arrival. Her mother reported that she had taken no other medications, including traditional medications. This SAE was judged as being possibly related to DP, but her concurrent illness with varicella provides an alternative cause to the SAE, and no definitive causal relationship could be established.Among the AEs, 18 occurred after the first distribution, 31 after the second distribution, and seven after the third. All AEs reported were notified within 10 days of the given distribution. All AEs were judged to have either mild or moderate-intensity symptoms. After the first distribution, eight out of 18 reported AEs were judged as probably or definitely related to DP Fig. . One casThis is the first description of the successful implementation of an IPTc programme in a refugee camp. The reduction in the incidence of malaria among children in the target population was marked, especially in comparison to those persons not targeted by the programme, who saw their incidence of malaria increase significantly compared to the previous year.The overall effect on parasite burden in the community was less impressive. Adults, who made up approximately 50% of the camp population, may have provided an ample parasite reservoir for continued transmission. Indeed, even with high coverage among the under-15 population as documented in the surveys, only 40–45% of the overall population of the camp was treated. Taken together, these data underscore the importance of expanding the target age groups for preventive interventions in similar contexts. Children aged 5–14 had considerable parasite burden in the prevalence surveys and a high incidence of clinical malaria. Mass Drug Administration (MDA) among all age groups would also have been a reasonable strategy in this context, and could be implemented in a future humanitarian emergency. Decreasing the interval between distributions (to 4 or 6 weeks) would also be a reasonable strategy, but would need to be weighed with the additional logistical and operational burdens in the setting of a humanitarian emergency.In this context, a large increase in the prevalence of parasitaemia was seen among all age groups after the final IPTc distribution. This late-season peak occurred in the setting of particularly intense transmission across northern Uganda in the autumn of 2015, when multiple nearby districts declared malaria epidemics. This provides an example of the difficulty of planning such an intervention around a seasonal peak: a delay of one or two weeks in implementing the IPTc programme might have made a considerable difference at the end of the programme.While specific details about the epidemiology of malaria in South Sudan are lacking, malaria remains a major problem there . It is tThe size of this intervention provides important programmatic and operational conclusions about similar programmes in terms of acceptability and safety. Programme coverage remained high throughout the distributions, and DP was well tolerated. The most important results of the intervention may well be about the safety profile of repeated doses of DP. Over 40,000 courses were administered , and only one SAE was reported during the study period, a sudden death which is difficult to link directly to DP, particularly since it occurred in a child with an underlying medical condition after only one course. The overall pharmacovigilance system in the camp was strong during the intervention, and these detailed results are quite reassuring about DP’s safety profile.Its long prophylactic period makes DP an obvious choice for such an intervention, but it does not come without difficulties. Largely because of concerns about under-dosing, recommendations have recently changed , and theThese results come with certain limitations. The lack of historical data makes it difficult to interpret the overall impact of the intervention on malaria incidence. Specimen collection in the prevalence surveys was done in participants’ homes. While the quality of specimens appeared good, it is possible that some samples were sub-standard. All surveys were carried out using systematic sampling, which was at times challenging in the two camps because of the layout and orientation of homes.IPTc was successfully implemented in a refugee camp for the first time at a large scale. The knowledge gained should inform operational responses to crises of displaced persons both in Uganda and other high malaria transmission settings.


In addition, toxic metabolites, e.g., ammonium, accumulate due to liver and kidney deficiency.Prostration: Lack of perfusion of skeletal muscles due flow restriction in their capillaries. Flooding the patient with cytokines like IL1β.Multiple convulsions: NET-driven disseminated intravascular coagulation (DIC) and microthrombi cause multiple focal brain lesions and convulsions.Acidosis: NET formation and acidosis are mutually influencing each other. Reduced blood flow causes hypoxia and drop in the plasma pH. However, acidic conditions interfere with NET formation : Infected as well as ruptured erythrocytes are trapped by intravascular NETs, escaping sequestration in liver and spleen.Severe malarial anemia, jaundice, pulmonary edema, and shock: There is no obvious specific contribution of NETs, besides the fact that these symptoms are the consequence of decreased end organ perfusion.Each year, 240 million people develop symptomatic malaria infections, placing malaria as one of the most serious diseases of mankind. Treatment options in affected areas are challenging and casualties remain high. Here, we propose NET formation as key event determining the severity of disease progression. Parasites within erythrocytes reportedly produce uric acid, which is released as MSU crystals together with crystalline hemozoin synchronously upon rupture of the infected cells. MSU crystals may induce the formation of intravascular NETs, and hemozoin pigments activate neutrophils, immobilizing parasites and crystals at the endothelium. We discussed three possible outcomes Figure . (i) ThePlasmodium infections, especially during gestation. This includes a significant reduction in peripheral neutrophil count  in 50% of patients with malaria supports the role of NETs in the etiopathogenesis of this, often fatal, disease , 24. TheThe involvement of NETs in severe malaria has not been studied in detail. Utilizing NET formation, MSU, and the coagulation cascade as targets of new therapies harbors the potential to reduce the cases with mortality outcomes and alleviate severe forms of malaria infection.SB wrote the manuscript. LM, MH, and TF supervised the project and wrote the manuscript. All the authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HIV protease inhibitors kill Thirty-one children from Malawi aged 4–62 months were followed every 3 months and at intercurrent illness visits for ≤47 months (September 2009-December 2011). We compared malaria parasite carriage by blood smear microscopy (BS) and confirmed clinical malaria incidence  in children initiated on HIV antiretroviral therapy (ART) with zidovudine, lamivudine, and either nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, or lopinavir-ritonavir (LPV-rtv), a protease inhibitor.We found an association between increased time to recurrent positive BS, but not CCM, when anti-malarial treatment and LPV-rtv based ART were used concurrently and when accounting for a LPV-rtv and antimalarial treatment interaction ; p = 0.03).LPV-rtv in combination with malaria treatment was associated with lower risk of recurrent positive BS, but not CCM, in HIV-infected children. Larger, randomized studies are needed to confirm these findings which may permit ART optimization for malaria-endemic settings.NCT00719602ClinicalTrials.gov  Malaria is highly prevalent in many areas of the world where HIV-infected children live, especially sub-Saharan Africa. Studies have shown that when HIV and malaria are present as co-infections, each disease can enhance the pathogenicity of the other. Moreovein vitro and in vivo data with Plasmodium suggest that HIV PIs kill various life cycle stages of malaria parasites [The World Health Organization (WHO) recommends HIV management with combination antiretroviral therapy (ART), with first line therapy including a non-nucleoside reverse transcriptase inhibitor (NNRTI) and 2 nucleoside reverse transcriptase inhibitors (NRTIs), with few exceptions, and second line therapy including an HIV protease inhibitor (HIV PI) and 2 NRTIs [arasites –7, indicarasites .Because transmission intensity influences the dynamics of malaria infection and intervention efficacy , we sougP1068s was substudy to the International Maternal Pediatric Adolescent AIDS Clinical Trials Network (IMPAACT) study P1060, details of which have been reported elsewhere . BrieflyThe malaria substudy described herein, P1068s, was conducted at three sites with endemic malaria transmission according to published data at the time, which included Kampala, Uganda; Lusaka, Zambia; and Lilongwe, Malawi –14. AnalAll participants from the parent HIV treatment study, P1060 were eligible for inclusion in this malaria substudy. Although not randomized for the primary objectives of the P1068s substudy described herein, it was originally intended the randomization of P1060 would be used for P1068s; however, due to later initiation of this substudy, a number of subjects had switched from the regimen to which they had been originally randomized. Thus, most data collected under P1068s that links treatment to outcome is observational and most analysis was conducted using an “as-treated” approach.Study visits were conducted 2 and 4 weeks after the initiation of treatment, every 4 weeks until week 16, at week 24, and every 12 weeks thereafter, and patients were encouraged to come in for intercurrent illness. Follow up for P1068s paralleled P1060 .History and physical exams were completed at all scheduled and intercurrent illness visits, and children were seen by study physicians at all visits. Heel or finger stick or venous blood were used to prepare thick blood smear using 2% Giemsa for 30 minutes, in addition to dried blood spots  collected on Whatman 903 paper  and handled as previously described  for confIn Malawi, trimethoprim-sulfamethoxazole prophylaxis was administered as per WHO guidelines , 20, andP. falciparum rate (PAPfr2-10) in 2010 of 32%; this encompasses part of the period in which this study was conducted [Malaria transmission in Malawi is perennial and holoendemic, with seasonal increases after the rains from November to April. Malaria mapping analysis in Malawi showed lack of significant changes in transmission between 2000–2010, with a population-adjusted onducted .This substudy was approved by site-specific institutional review boards (IRBs), including the New York University School of Medicine (NYU) IRB  and the Malawian Ministry of Health and Population National Health Sciences Research Committee  and the NIH/NIAID IRB through a reliance agreement with NYU IRB . Each child’s parent or legal guardian provided written informed consent. The study was first opened to accrual on August 19, 2009 with the first patient enrolled September 25, 2009.P. malariae sample from Lilongwe, were reported as P. falciparum. The diagnosis and management of malaria was based readings of blood smears on site, but reported results herein are from reads performed at NYU, with a primary and secondary reads by two microscopists, with discrepant results resolved and a random subset of smears reviewed by a third microscopist.A blood smear was deemed negative if no parasites were seen in > = 200 high-powered fields. All blood smears in the study, with the exception of one http://hydasworldhealth.org/), whose stock smears are read by WHO Level 1-Certified microscopists [Plasmodium falciparum, vivax, ovale or malariae. For the total 60 test slides, the readers’ overall sensitivity was 100%, specificity 92%, and density determination was within +/-25% of the accepted value in 96% of the slides. During the final 2 years of the study, the sensitivity and specificity were both tested at 100%. Separately, DNA was isolated from DBS and parasite genomic DNA was detected using real time from DBS collected for confirmation in parallel to smears.Quality control of blood smear reading was maintained by 30 hours of training which covered both slide preparation, cover slipping, microscope care and maintenance and identification and quantification of malaria parasites, both at NYU and at study sites. Over the time period of the study the NYU microscopists were proficiency tested four times using archived smears provided by Hydas World Health  or confirmed clinical malaria (CCM), defined as a positive BS with diagnosed malaria symptoms, as per WHO guidelines . Only epThirty-one of 288 subjects from the P1060 parent study enrolled in P1068s, and 8 of these 31 children changed regimens based on the primary study before or after enrollment. For this reason we undertook an as-treated analysis, attempting to link observed treatment and observed outcomes. Per-protocol analysis, censoring subjects when they switched regimens, and as-randomized analysis (as randomized for the P1060 parent study) analysis are also provided in Supporting Information  Tables.During this study, another clinical trial suggested that a drug interaction between LPV-rtv and lumefantrine  reduced clinical malaria burden in children on LPV-rtv ART in an area of high transmission intensity . For thiIn addition to evaluation of recurrent malaria events, comparisons of the rate of incident positive BS and CCM per time at risk by HIV treatment type was analyzed using negative binomial models including an offset for time-on-trial and adjusted for potential confounders. HIV treatment was accounted for using the following three time categories of LPV-rtv ART use: LPV-rtv ART assigned at P1060 baseline; LPV-rtv ART duration in months; or majority of time on LPV-rtv ARTReported p-values are not corrected for the number of analyses conducted. Statistical analysis was performed with R software, version 3.1.3.st positive BS and CCM are presented in Supporting Information, From Kamuzu Central Hospital, Lilongwe, Malawi, 31 children were enrolled between September 2009 and December 2011, and demographic information for these patients is summarized in In the as-treated recurrent event analysis, time on LPV-rtv ART prior to positive BS or CCM was not significantly different from NVP ART ; p-value 0.05 . The othIn our P1068s substudy, which was the first of its kind conducted in an area of low to moderate malaria transmission intensity, LPV-rtv in combination with malaria treatment is associated with lower risk of recurrent events of BS, but not CCM, in HIV-infected children.Plasmodium species in the laboratory because of inhibition of these enzymes, of which there are 10 in Plasmodium falciparum, although further investigation is required to clarify the target [HIV protease inhibitors are aspartyl protease inhibitors, and have been hypothesized to exhibit the killing activity for e target , 27, 28.e target , the latA previous study that was conducted in an area of high-intensity transmission has shown similar trends in children: HIV PIs were associated with an overall reduction in malaria for children on LPV-rtv ART . This fiOf note, the as treated recurrent events model also showed that malaria treatment was significantly associated with increased hazard of repeated positive BS and CCM in the presence of NVP ART. This finding was likely due to the strong correlation between receiving a malaria treatment in the current period and having a malaria event in the previous period. We investigated this by including malaria treatment in the model only if there was not a malaria event in the previous period for a subject. This modified version of the model suggested that malaria treatment was associated with reduced hazard of malaria outcomes under all ART regimens, albeit non-significantly due to the small number episodes observed (data not shown). Therefore, one might describe our malaria treatment measure as a mixture of previous illness and malaria treatment.A limitation of our study is our small sample size and the number of children who switched ART regimens. However, because we detected significant findings in a small sample size, and over a long period of time, we believe the effect of LPV-rtv ART may be considerable and as such, a larger study is warranted to investigate and validate this finding.WHO Guidelines now support the use of LPV-rtv ART in children less than 3 years of age because of longer life-expectancy and cost-effectiveness compared to NNRTI ART . WhetherS1 FigA and B: Kaplan Meier Curves depicting time to first (1st) positive blood smear (BS) and time to 1stconfirmed clinical malaria (CCM) case. Overall, we observe a BS positive rate of 0.55 and 0.87 for the 1st year and the 1st two years of follow-up, respectively; for CCM rates are, 0.52 and 0.77 for the 1st year and the 1st two years, respectively. We observe 123 positive BS events, and 109 CCM events over the 97.89 observed person-years. In 1A, shown is the survival proportion and times to 1st positive BS. The median survival times are 408 and 135 days in the randomized to-LPV-rtv ART and randomized to-NVP ART groups, respectively. However, there was an 0.88 infection rate in the LPV-rtv ART group, and an 0.87 rate in the NVP-ART group over follow-up. In 1B, shown is the survival proportion and times to 1st CCM. The median time to 1st CCM, is 417 and 135 days in the randomized to-LPV-rtv ART and randomized to-NVP ART groups, respectively. However, there was an 0.81 CCM rate in the LPV-rtv group, and a 0.73 rate in the NVP-based group over follow-up. Note that for BS, the longest period while on NVP-ART prior to 1st positive BS is shorter compared with LPV-ART, and the figure incorporates subject censoring.(TIFF)Click here for additional data file.S1 File(DOC)Click here for additional data file.S2 File(DOC)Click here for additional data file.S1 TableAs-randomized in P1060 analyses .(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Plasmodium falciparum exposure on the acquisition of high-avidity antibodies to P. falciparum antigens, which may be associated with protection.People living in malaria endemic areas acquire protection from severe malaria quickly, but protection from clinical disease and control of parasitaemia is acquired only after many years of repeated infections. Antibodies play a central role in protection from clinical disease; however, protective antibodies are slow to develop. This study sought to investigate the influence of P. falciparum transmission intensity . Sandwich ELISA was used to measure antibody responses to two P. falciparum merozoite surface antigens: merozoite surface protein 1-19 (MSP1-19) and apical membrane antigen 1 (AMA1). In individuals with detectable antibody levels, guanidine hydrochloride (GuHCl) was added to measure the relative avidity of antibody responses by ELISA.Cross-sectional surveys were performed in children and adults at three sites in Uganda with varied Within a site, there were no significant differences in median antibody levels between the three age groups. Between sites, median antibody levels were generally higher in the higher transmission sites, with differences more apparent for AMA-1 and in ≥5 year group. Similarly, median avidity index (proportion of high avidity antibodies) showed no significant increase with increasing age but was significantly lower at sites of higher transmission amongst participants ≥5 years of age. Using 5 M GuHCl, the median avidity indices in the ≥5 year group at the highest and lowest transmission sites were 19.9 and 26.8, respectively (p = 0.0002) for MSP1-19 and 12.2 and 17.2 (p = 0.0007) for AMA1.P. falciparum antigens was lower in areas of high transmission intensity compared to areas with lower transmission. Appreciation of the mechanisms behind these findings as well as their clinical consequences will require additional investigation, ideally utilizing longitudinal data and investigation of a broader array of responses.Avidity to two different  Plasmodium falciparum is a major global public health challenge, accounting for an estimated 214 million clinical cases and 438,000 deaths in 2015 [P. falciparum is rarely if ever achieved. Passive transfer of immunoglobulin from clinically immune donors to non-immune individuals with P. falciparum infection alleviated clinical symptoms and reduced the levels of blood stage parasites, indicating that antibodies play a central role in clinical immunity to malaria [P. falciparum antigens with conflicting results regarding correlates of protection [Malaria caused by otection . The quaP. falciparum infection, avidity to whole schizont extract, as well as to a number of specific antigens, has been shown to correlate with protection [P. falciparum antigens with increasing age and varied P. falciparum exposure intensity is poorly understood.Antibody properties including breadth of response, isotype composition, and avidity appear to play an important role in protective immunity –13. Antiotection –21. HoweP. falciparum infection directly interferes with B cell function [P. falciparum antigens with increasing exposure [P. falciparum leads to increased avidity of antibodies directed against plasmodium antigens.In general, repeated exposure to an antigen results in germinal centre reactions which lead to affinity maturation and increases in antibody avidity –24. Howefunction , 25 and function , 27, potfunction , 27. Onefunction . In contexposure , 29. OveP. falciparum exposure on the natural acquisition of high-antibody avidity, avidity indices to two P. falciparum merozoite surface antigens were measured in individuals across a wide range of ages from cross-sectional surveys performed in three sites in Uganda with varying transmission intensity. Antibody avidity indices were then compared between ages and sites to determine whether there were differences in antibody avidity associated with age and P. falciparum exposure intensity.To determine the influence of P. falciparum transmission intensity. Walukuba, a peri-urban area near Lake Victoria, had relatively low transmission intensity, with an entomological inoculation rate (EIR) estimated at 3.8 infectious bites per person per year (IBPPY) [This study took place in three sub-counties in Uganda with varied  (IBPPY) . Kihihi,Cross-sectional surveys were conducted between January and June 2012 at all three sites . Survey Ethical approval was obtained from the Makerere University School of Medicine Research and Ethics Committee (REC REF 2011–203), Uganda National Council for Science and Technology (HS 1074), London School of Hygiene & Tropical Medicine Ethics Committee (Reference 6012), and the University of California, San Francisco Committee on Human Research (Reference 027911). Written informed consent was obtained from the parents/guardians on behalf of the children enrolled in the study and from all participants above 18 years of age. Assent was obtained from all children 8–17 years.DBS were collected on Whatman 3MM filter paper and stored at −20 °C. For this study, a 3 mm diameter punch of DBS was hydrated in 200 µl of phosphate buffered saline (PBS) containing 0.005% Tween 20 and 0.01% sodium azide. The samples were left on a plate shaker overnight at room temperature before storage at 4 °C. The excised spot was estimated to contain approximately 2 µl plasma, resulting in a serum dilution factor of 1:200 .o-phenylenediamine (OPD)–H2O2. The reaction was stopped after 15 min with 25 μl 2 M sulphuric acid. ODs were measured at 492 nm using a VERSAmax plate reader, with Softmax software .Antibodies to MSP1-19 (Wellcome strain)  and AMA-To evaluate antibody avidity, the sandwich ELISA assay described above was modified to include an antibody disassociation step prior to addition of HRP-conjugated secondary antibody , 35, 36.In order to overcome the limitations of avidity measurement by ELISA (poor resolution at the lower OD), and to select for participants with a relatively recent antibody boost across the three sites, only samples with titers above a normalized OD (adjusted OD based on a positive control to correct for inter-plate variations) of 0.5 were included in the assessment of the avidity index. In each plate, hyper-immune sera was included at a dilution 1:3200 as a positive control.Participants were stratified into age groups 1–4, 5–15 and >15 years. Non-parametric comparison of medians across groups within a site and across sites for the different age groups was performed using the Mann–Whitney test. Correlations were performed using the Pearson test. Graphs were plotted using Graphpad PRISM version 5 .Antibody levels and avidity index (AI) were measured in samples from cross sectional surveys conducted at three sites in Uganda. Consistent with published data on antibody prevalence , the proMedian avidity index to MSP1-19 at 2 and 5 M GuHCl was not significantly different between age groups at all three sites. Similarly, median avidity index to AMA-1 at 2 and 5 M GuHCl showed no difference between age groups at all three sites, with the exception of 1–4 versus 5–15 year olds in Walukuba (73.6 versus 85.1 p = 0.04) . Overall, these findings suggest an inverse relationship between avidity index and both transmission intensity and antibody response to MSP1-19 and AMA-1 across sites for those above 5 years.In contrast to the trends observed in antibody levels, the avidity index to MSP1-19 was significantly lower in the highest malaria transmission site of Nagongera than Kihihi and Walukuba in those over five, at both GuHCl concentrations 2 and 5 M)  near constant exposure to antigen may impair maturation/persistence of avid antibodies via a number of potential mechanisms; and (2) recent infection may result in addition of low avid antibodies to the circulation, reducing the proportion which have high avidity.P. falciparum transmission sites, where exposure to antigen is most frequent. Animal studies suggest that germinal centre development is temporally ordered, with short lived plasma cells (SLPC) emerging from germinal centres initially, followed by memory B cells (MBC) and then, weeks to months later, long lived plasma cells (LLPC) that have undergone extensive affinity maturation [P. falciparum infection is associated with dysfunction of the B cell compartment, including accumulation of atypical memory B cells [Affinity maturation is acquired through somatic hypermutation during germinal centre reactions, with B cell affinity to antigen driving selection that results in the production of higher affinity antibodies , 38. Claturation , 39. Disturation , 40–42.  B cells , which c B cells . It is aP. falciparum infection is associated with non-specific, polyclonal B cell activation that may result in the expansion of low avidity, short lived plasma cells [In the presence of low antigen levels, affinity matured plasma cells that secrete high affinity antibodies should be favored over lower affinity plasma cells and circulating antibodies for survival. These affinity matured plasma cells would therefore maintain the blood antibody levels. However, in the presence of frequent re-infection, a relatively large antigen load may be more likely to promote survival of poorly affinity-matured plasma cells, including T cell-independent or extra-follicular B cell reactions. Furthermore, acute ma cells . These fP. falciparum antigens were less likely to experience malaria during follow-up [P. falciparum transmission, based on reductions in the incidence of disease with age [The impact of the differences in avidity index observed in this study on acquisition of clinical or anti-parasite immunity is not known. While antibody avidity is theoretically important in the function of an antibody response , there aollow-up , 46. In ollow-up , 48. Thewith age . Howeverwith age .P. falciparum proteins, and it is not yet well established which combinations of responses are most relevant for acquisition of immunity. In addition, while providing an overall estimate of the proportion of the polyclonal antibody pool which is highly avid, the ELISA-based methods used in this study were not able to measure avidity in participants with OD <0.5 or further dissect binding characteristics of the antibodies.The cross-sectional nature of this study limited the ability to investigate the mechanisms behind the acquisition of antibody avidity. Furthermore, this study only investigated responses to two of many P. falciparum antigens was lower in areas of high versus low transmission intensity. The mechanisms behind these findings as well as their clinical consequences, if any, are not yet clear. A more detailed investigation, ideally linked to longitudinal investigation of P. falciparum exposure as well as a more comprehensive analysis of the B cell and antibody response, will be valuable in helping to further illuminate these findings.In conclusion, this study showed that avidity to two different


Plasmodium falciparum antigens is a major hurdle in developing an effective malaria vaccine. Protective efficacy of the vaccine is dependent on the polymorphic alleles of the vaccine candidate antigens. Therefore, we investigated the genetic diversity of the potential vaccine candidate antigens i.e. msp-1, msp-2, glurp, csp and pfs25 from field isolates of P.falciparum and determined the natural immune response against the synthetic peptide of these antigens. Genotyping was performed using Sanger method and size of alleles, multiplicity of infection, heterogeneity and recombination rate were analyzed. Asexual stage antigens were highly polymorphic with 55 and 50 unique alleles in msp-1 and msp-2 genes, respectively. The MOI for msp-1 and msp-2 were 1.67 and 1.28 respectively. A total 59 genotype was found in glurp gene with 8 types of amino acid repeats in the conserved part of RII repeat region. The number of NANP repeats from 40 to 44 was found among 55% samples in csp gene while pfs25 was found almost conserved with only two amino acid substitution site. The level of genetic diversity in the present study population was very similar to that from Asian countries. A higher IgG response was found in the B-cell epitopes of msp-1 and csp antigens and higher level of antibodies against csp B-cell epitope and glurp antigen were recorded with increasing age groups. Significantly, higher positive responses were observed in the csp antigen among the samples with ≥42 NANP repeats. The present finding showed extensive diversity in the asexual stage antigens.The genetic diversity in  Plasmodium falciparum contributes 67% of the total malaria cases in India with a greatly varied proportion from 0% to 93% in different states [Globally about 3.2 billion people are at risk of malaria and 214 million new malaria cases and 438,000 deaths was reported in 2015[P. falciparum are under various stages of the development [P. falciparum antigens currently under consideration for vaccine development, exhibit polymorphism in the field isolates from various part of the world [P. falciparum [P.falciparum polymorphic proteins. Given the importance of antigenic diversity in influencing the outcome of any vaccine, we have conducted genetic polymorphism of vaccine candidate antigens  and antibody responses in P. falciparum infected individual from Chhattisgarh state, Central India which is second malarious state contributing 12% of malaria cases in India.Therefore, highly effective malaria vaccine is definitely needed to achieve the target of malaria elimination. Although there is no effective vaccine as on date, however a number of potential stage specific vaccine candidate antigens of elopment . Understelopment . Almost he world –14. Furtlciparum –16. The lciparum . In endelciparum . In addilciparum . TherefoP. falciparum is the predominant species followed by P. vivax and both Anopheles culicifacies and An. fluviatilis are responsible for transmission the disease in this area. A malaria clinic of National Institute for Research in Tribal Health (NIRTH) of Indian Council of Medical Research (ICMR) was established at Janakpur CHC , Baikunthpur district, during August 2013 to March 2015. Symptomatic patients were screened for malaria parasites by microscopy using thick and thin blood smears stained with JSB stain [P. falciparum malaria after taking written informed consent. Plasma and red blood cells were separated and stored at -80°C for further use in immunological and molecular assays, respectively.This study was carried out at Janakpur Community Health Care (CHC), district Baikunthpur, Chhattisgarh, Central India . This isSB stain . The patThe study protocol for patient participation and collection of blood samples for laboratory testing was reviewed and approved by the institutional ethics committee of NIRTH, Jabalpur. All study participants’ provided written informed consent prior to their participation, accroding to ICMR guidelines. A copy of the consent form in the local language was also provided and explained to the patients or parents/Guardian of children. The participation of other institutes was approved by the ICMR, New Delhi, India.P. falciparum, P. vivax, P. ovale, and P. malariae), as described previously [Genomic DNA was isolated by QIAamp DNA blood mini kit as per the manufacturer's instructions  and stored at -20°C for further use. Genus and species-specific nested PCR was performed using 18S rRNA gene to detect malaria parasite species (eviously .pfmsp1 (block -2 region), pfmsp2 , pfglurp (polymorphic RII region), pfcsp  and pfs25 was performed in GeneAmp® PCR System 2700 (Applied Biosystems). The PCR reaction mixture and amplification conditions were summarized in 2 free buffer, 1.5 mM of MgCl2, 200 μM of dNTPs, 0.2 mM of each primer and 2.5U of Taq polymerase enzyme . The appropriate bands excised from the gel and purified using AccuPrep gel extraction kit, according to manufacturer’s protocol (Bioneer Corporation). The purified products were sequenced using both forward and reverse primers, using ABI Big Dye Terminator Ready Reaction Kit Version 3.1  on an ABI-3130xL genetic analyzer. The BioEdit Sequence Alignment Editor and Gene Doc Version 2.6.002 were used to analyze the sequencing electropherograms and generate sequence alignment, respectively. The sequence data generated in this study have been submitted into the NCBI GenBank database. Amino acid sequences of P. falciparum msp1 (block-2), msp2 , csp  from present study and partial/complete amino acid sequences from NCBI database were retrieved using BATCH Entrez and considered for further analysis. Blast clust tool was used to identify the variants from globally available sequence. The details about these isolates have been provided in pfmsp1, pfmsp2 and pfcsp, Minimal Spanning Tree (MST) was constructed using MLST Clustering algorithm implemented in BioNumerics version 7.6.1 . The haplotype with the highest numbers of single locus variants (SLVs) was considered as a root haplotype and all other haplotypes as relatives.The amplification of highly polymorphic antigens; P. falciparum genotypes per infected individual. The MOI was calculated as the proportion of the total number of P. falciparum genotypes for the same gene and the number of PCR positive isolates.The multiplicity of infection (MOI) was defined as the mean number of P. falciparum gene specific allelic types were determined by different methods implemented MEGA 7 Software [w = 100), and the statistical significance of Φw values were assessed using a permutation test with 1,000 iterations {H0  rejected at α = 0.05 in favor of H1 (recombination)} [Genetic parameters of Software . The aveSoftware ,25. IntrSoftware ,27. TajiSoftware . Window nation)} .2SO4. The OD was measured at 450 nm using an ELISA plate reader.The total IgG response against the synthetic peptides of pfmsp1 B & T cell epitopes, pfmsp2 B cell epitope, pfcsp B & T cell epitopes and pfglurp were quantified by direct ELISA. Details of peptide sequence were given in the supplementary table . BrieflyTo determine the specificity of the assay, sera samples of 16 healthy individuals from non-endemic area  were used as negative control. Prevalence of total IgG immune response for the different antigens were considered positive if their OD values were higher than the mean plus two standard deviations of negative control after subtraction. The percentage prevalence was calculated as follows:  x 100. Odd ratio (OR) was calculated for association between the different age groups.P.falciparam (n = 271), P.vivax (n = 79) and two mixed infection of P.falciparam and P.vivax}. Out of 271, polymerase chain reaction (PCR) positive 180 mono-infected P. falciparam patients who fulfilled the enrolment criteria were included in the study. The age range across the sample was 01 to 75 years with a mean age of 20.34±15.19 year. Fifty two percent (n = 93) of the patients were female and the rest were male, only 7% (n = 13) of the patients had history of previous malaria infection. Additionally, majority of the participants were from the tribal community . Majority (94%) of the patients had fever at the time of enrollment followed by headache (90%), vomiting (75%) and diarrhoea (5%). The mean parasite density was 5827.05 ± 17295.A total of 6718 patients were screened, of which 5.2% (n = 352) were found positive for malaria parasite {pfmsp1 gene. The overall allelic prevalence was higher in MAD20 (42%) followed by K1 (32%) and RO33 (26%)  samples were successfully sequenced and analyzed for block-2 region of 33 (26%) . Fifty f33 (26%) . The ave33 (26%) . The dif33 (26%) . Thirty pfmsp2 gene was successfully analyzed in 112 (62%) samples , Pfmsp2 (882 sequences) and Pfcsp (985 sequence) from global isolates  were included and used to construct global population structures  analysis of ructures . The genP. falciparum infected patients by Enzyme linked Immunosorbent assay. The overall prevalence of total IgG antibody varied greatly between 37% to 97% to different antigens /epitopes (pfcsp and pfglurp antigens in the ≥15 years age group than ≤5 year age group (pfcsp response to NANP repeat showed significantly high positive response where the average NANP repeats was ≥42 repeats (p<0.05).The levels of total IgG was assessed against different antigens/epitope in 180 plasma samples of epitopes . The higge group . Furtherge group . AlthougP. falciparum  in India. Although, P. falciparum has been one of the most extensively investigated parasites but there is limited information on genetic diversity of each stage along with antibody response. Present study showed that encoding protein expressed on the surface of the merozoite and sporozoite are more polymorphic than they are expressed during the sexual stage. These results are in agreement with the observation that stage specific surface proteins exhibit high polymorphism when compared with internal antigens [P. falciparum msp1, msp2 and glurp were highly diverse from central India region in respect of length as well as sequence motifs with prevalence of all the allelic families of msp1 and msp2, which corroborates with earlier reports on Indian isolates [pfmsp1 exhibit repetitive tri-nucleotides and appears to be subjected to a rapid intra-genic recombination process. Besides these variability, Cavanaugh et.al. (2004) observed that the antibody against block-2 region of pfmsp1 are also associated with protection from clinical malaria [msp1 also show many variants in this study as initially it was considered as limited diversified family [pfmsp1, it is considered as one of the good malaria vaccine candidate genes. Although, the block-2 of pfmsp1 is considered highly polymorphic but Cavanagh et al 2004 demonstrated that various construct of block 2 region of pfmsp1 showed good antibody response compared to block 1 region of pfmsp1. It was further confirmed that block 2 region showed protective potential of the immunity [Almost all vaccine candidate antigens present a great challenge for successful development of malaria vaccine due to antigenic diversity in the parasite resulting immune selection in most of the parasite. In this study, we determined the antigenic diversity of sporozoite, merozoite surface proteins, sexual stage antigen and antibody response against these antigens in Indian samples. To the best of our knowledge, this is the first report to analysed genetic polymorphism and antibody responses of five potential vaccine candidates of antigens . In presisolates ,14,31,32 malaria . The hig malaria ,10. One  malaria  but in td family ,36. The d family . The litimmunity .P. falciparum msp2 allelic family are also associated with malaria severity [msp2 prevent re-infection with the parasite carrying alleles of the same family [pfmsp1, pfmsp2 and the ring –infected erythrocyte surface antigen (RESA) are combination vaccine known as combination B, which is immunogenic [pfmsp2. In addition, both FC27 and 3D7 family were associated with the extensive polymorphism and immune response. Furthermore, Fluck C et al 2007 demonstrated that family specific dimorphic domain is more likely to account for the pfmsp2 family specific selective effect [P. falciparum glurp has also been considered an important antigen that playing an important role in the induction of protective immunity [severity  and coulseverity . It has severity . In triae family . The aseunogenic ,42 and de effect . In addiimmunity . Our resimmunity ,44. R2 rP. falciparum csp antigen is a part of RTS’S vaccine, which showed only partial protection (50%) in the phase- III trial [pfcsp. A large number of haplotypes observed in the present study is consistent with this region of the circumsporozoite gene being under diversifying selection. The selection of non-vaccine variant of CSP following immunogenicity with RTS, S/AS01 vaccine, which is CSP based was not allele specific [pfs25 was found to be conserved and only two site mutated one is Alanine with Glycine and other Valine with Alanine, which is in agreement with previous study [II trial , and no II trial ,46. In aspecific . Therefous study .P. falciparum diversity. Therefore, the understanding the population structure and diversity of P. falciparum has implication for vaccine, drug and epidemiological studies including monitoring of malaria [In the present study, agreement with large parasite diversity in these genes may be due to higher malaria transmission intensity as the extent of polymorphism may vary with transmission intensity by applying inter-clonal parasite mating and intragenic recombination . Other f malaria ,50.P. falciparum in Central India, where malaria is endemic [P. falciparum infected samples has been demonstrated, however their role in protection was not established [P. falciparum populations is complex and distribution of different parasite strains differ from region to region and evolves over time possibly because of immune selection. Therefore, a study from a range of different settings are required to show how the vaccine candidates dependent to the different alleles of the vaccine candidate antigens.The blood stage  and liver stage (csp) vaccine candidates have been reported to have B cell and T cell recognition domain. In the present investigation, we determined the IgG antibody response against synthetic peptides representing the conserved region of B cell & T cell of msp1, csp and B cell of msp2, glurp among patients infected with  endemic . In this endemic . However endemic . The exiablished . Furtherablished . Althougablished . In the P. falciparum vaccine candidate antigens. In the present study, we aimed for such analysis to be relevant for epidemiologic investigations, malaria transmission dynamics and vaccine design.The strengths of this study is the comprehensive use of sensitive and standardized assays, rendering our findings amenable to a detailed analysis of genetic polymorphisms as well as antibody response in P. falciparum csp T-cell (Th2 and Th3) epitopes. However, the present study has robustly genotypes the P. falciparum merozoite, sporozoite surface and sexual stage protein to obtain a conclusive genetic diversity data set for this region. It has been proposed that diversity in the repeat region might have evolved as an immune evasion mechanism by the parasite in which the host mount the protective immune response against this region while the parasite escapes into the liver [The limitation of present study is that we could not assess the genetic polymorphism of he liver .P. falciparum vaccine candidate antigens from Chhattisgarh, Central India, where malaria is endemic. There is probably some association between malaria hyper endemicity and the parasite mixture as well as the number of alleles present in the area. This data would be helpful in the future malaria vaccine trial in India and to monitor changes in the parasite population. Furthermore, the characterization of the pattern of antibody response to malarial antigens/epitopes may also be useful in monitoring the effect of malaria vaccine. The simultaneous presence of antibody response, in our population, to more than one antigen is indicative of a lower frequency of malarial infection and suggests that a successful malaria vaccine should contain multiple antigens. Therefore, similar study should be carried out on a large scale to cover the entire country to map the different endemic regions. Finally, the global analysis of allelic variants reported in this study would be helpful in the identifying the predominant allele in the region and may aid in designing region specific vaccine and would pave the way for an effective malaria intervention measures.This is a first extensive study that contributes in understanding the type and distribution of naturally evolved genetic polymorphism in the S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Plasmodium ovale. The LAMP kit (LoopAMP®) can be used in the field without special equipment and could have an important role in malaria control programmes in endemic areas and for malaria diagnosis in returned travellers. The performance of the Pan primer of the kit in detecting malaria by P. ovale was compared with the results of standard nPCR in samples of patients returning from P. ovale endemic areas.Microscopy and rapid diagnosis tests have a limited sensitivity in diagnosis of malaria by Plasmodium ovale positive samples  and malaria negative specimens  were collected in different hospitals of Europe from June 2014 to March 2016 and frozen at −20 °C. Boil and spin method was used to extract DNA from all samples and amplification was performed with LoopAMP® MALARIA kit  in an automated turbidimeter (Eiken 500). The results of LAMP read by turbidimetry and with the naked eye were compared.P. ovale was between 0.8 and 2 parasites/µl.The kit showed a sensitivity of 100% and a specificity of 97.24% with positive and negative predictive values of 72.5 and 100%, respectively. Naked eyed readings were in accordance with turbidimetry readings . The limit of detection of LAMP assay for P. ovale at very low-levels and showed a predictive negative value of 100%. This tool can be useful in malaria control and elimination programmes and in returned travellers from P. ovale endemic areas. Naked eye readings are equivalent to automated turbidimeter readings in specimens obtained with EDTA.The Pan primer of the Malaria kit LoopAMP® can detect The online version of this article (doi:10.1186/s12936-016-1669-8) contains supplementary material, which is available to authorized users. Plasmodium ovale infection may represent up to 8% of imported malaria cases, as shown in some published series of mainly West African patients  .P. ovale positive and 398 malaria negative) were used to evaluate the performance of the LoopAMP malaria kit for the detection of P. ovale parasites in clinical samples. Of the 30 patients with P. ovale malaria previously confirmed by nPCR, one sample was excluded due to lack of amplification by LAMP as well as by nPCR when retested. This is possibly due to degradation of DNA over time or an error in initial diagnosis. The other 29 samples were positive with the Pan probe of LAMP, obtaining a sensitivity of 100% . Of the 398 negatives controls remaining after excluding those samples with degraded DNA and abnormal curves  readings was possible in 384 LAMP negative specimens (of 387 negative controls only 3 were indeterminate by NE reading) and 40 turbidimetry positive specimens . All in all, turbidimetry and NE were in good agreement, with only 4 false positive and 3 false negative results by NE: Sensitivity, 92.5% ; Specificity, 98.96% [380/384 CI 95%: 98.81–100%]; positive and negative predictive values, respectively, 90.24% [CI 95%: 79.94–100%] and 99.22% [CI 95%: 98.20–100%] . No apparent breaking of the protocols nor incidents during pipetting procedures or leaking could be detected. To solve the problem, before the next amplification session, all working surfaces, equipments and reagents were decontaminated with sodium hypochlorite, a new working space for the amplification reaction was established, and the DNA of all false positive samples was re-extracted from archived specimens. The LAMP test was repeated with all these second DNA samples and the number of false positive results was reduced to 5.P. falciparum infections have lower parasite densities than P. falciparum infections [P. ovale and P. malariae infections can be very low [P. ovale endemic countries, an affordable molecular rapid test which no requires special equipment with a very high negative predictive value for all common species of malaria could be very useful.Malaria control and eradication programmes worldwide require reliable tools for detecting very low parasite densities in asymptomatic patients, where microscopy and RDTs can produce false negative results, as it is the case in areas of low transmission where submicroscopic malaria can be a key factor in the control programmes , 13. Nonfections  and the very low . On the P. ovale parasites using Plasmodium genus-specific LAMP assay. This is the hitherto largest reported study about LAMP in P. ovale malaria.In this study, it is demonstrated the accurate and sensitive detection of P. ovale infected patients with the PAN primer when comparing to nested PCR, as other studies showed previously with P. falciparum (99 and 93%) in Thailand [P. ovale. Of the 427 clinical samples tested, only 11 resulted in discordant results between LAMP and multiplex nested-PCR. All these discordant results were false positives compared to nPCR results. However, another explanation for these results classified as “false-positive” could be that they are actually true positive of very-low parasitaemia that were undetected by the nPCR reference standard, as has been previously reported [The obtained results confirm the high sensitivity and specificity of LAMP  for detecting Thailand , all fivThailand , and in reported .Naked-eye readings showed a very good correspondence with the results obtained by turbidimetry. This result confirm that LAMP can be used in field settings when there is no turbidimeter available, the blood is extracted with EDTA anticoagulant and the UV fluorescence reading method cannot be used .P. ovale malaria was determined between 2 and 0.8 parasites/µl of blood, even more sensitive than the results obtained in previous studies about LoDs for P. ovale, which were performed with malachite green-LAMP or with ultraviolet (UV) light as reading methods  [The limit of detection of ctively) , 18.The occurrence of a contamination episode could have had an impact in the specificity results, if it had remained undetected. Contamination could be due to the semi-automated nature of the test and the high sensitivity of the LAMP technique as has been also documented in previous studies , 15. In P. ovale is endemic, although the kit does not include a set of P. ovale specific primers and further testing as nested specific PCR would be necessary for final species identification. However, the specific identification of P. ovale in the field would not be crucial, as patients could be treated with chloroquine for a non-falciparum malaria and the final identification could be made later in a reference center.No false-negative results were detected and this suggests that the Pan malaria LoopAmp® can be used with confidence in malaria eradication or control programmes where P. ovale at very low-levels and it showed a predictive negative value of 100%. This tool can be useful in malaria control and elimination programmes and in returned travellers from P. ovale endemic areas. Naked eye readings are equivalent to automated turbidimeter readings in specimens obtained with EDTA. The relative simplicity of the LAMP procedure and the low infrastructure costs open a range of opportunities by bringing molecular-level parasite detection and capacity of using malaria LAMP in field settings [The Pan primer of the Malaria kit LoopAMP® can detect settings .


Plasmodium falciparum induced antibodies are key components of anti-malarial immunity in malaria endemic areas, but their antigen targets can be polymorphic. Induction of a high proportion of strain-specific antibodies will limit the recognition of a broad diversity of parasite strains by these responses. There are indications that circulating parasite diversity varies with malaria transmission intensity, and this may affect the specificity of elicited anti-malarial antibodies. This study therefore assessed the effect of varying malaria transmission patterns on the specificity of elicited antibody responses and to identify possible antibody correlates of naturally acquired immunity to malaria in children in an area of Ghana with seasonal malaria transmission.P. falciparum AMA 1 variants  and the 3D7 variant of the EBA 175 region II antigen and the levels compared between symptomatic and asymptomatic children. The relative proportions of cross-reactive and strain-specific antibodies against the four AMA 1 variants per sampling time point were assessed by Bland-Altman plots. The levels of antibodies against allelic AMA1 variants, measured by singleplex and multiplex luminex assays, were also compared.This retrospective study utilized plasma samples collected longitudinally at six time points from children aged one to five years. Multiplex assays were used to measure antibody levels against four The data show that increased transmission intensity is associated with higher levels of cross-reactive antibody responses, most likely a result of a greater proportion of multiple parasite clone infections during the high transmission period. Anti-AMA1 antibodies were however associated with a history of infection rather than protection in this age group.The data contribute to understanding the underlying mechanism of the acquisition of strain-transcending antibody immunity following repeated exposure to diverse parasite strains. The World Health Organization  reports P. falciparum has been shown in both experimental animal models and human challenge studies to be parasite strain-specific since functional responses elicited against a particular parasite strain do not yield comparable levels of inhibition against heterologous strains [Antibodies have been demonstrated to play an important role in the partial protection against clinical malaria –5. High  strains –17. This strains ,19. On t strains ,21.in vitro using antibodies from animals that were immunized with combinations of known polymorphic antigens [Studies in naturally infected humans however show that repeated exposure to malaria infection could potentially lead to a broadening of antibody specificity ,22–24. Iantigens –28.An assessment of the relevance of strain-specific and cross-reactive responses to clinical malaria immunity in naturally exposed individuals is however complex since the parasite exposure history in such individuals is usually unknown and the cross-reactive or strain-specific description is dependent on this history. We have however attempted an analysis of the relative proportions of cross-reactive and strain-specific antibodies to a set of polymorphic antigens in cross-sectional plasma samples from naturally exposed individuals . In the This study made use of archived human plasma samples which were obtained from a longitudinal cohort study conducted between 2004 and 2005 in the Kassena-Nankana District of the Upper East region of Ghana. The original study was approved by the Ghana Ministry of Health and ethical clearance was sought from the Ethics Committees of the Navrongo Health Research Centre (FWA00000250) and Noguchi Memorial Institute for Medical Research, Legon (FWAA00001824). Informed consent was obtained from parents of the participating children for the original study, and the current analyses of archived samples were done under renewed approvals from the two ethical bodies mentioned above.The study site (Kassena-Nankana District) is a savanna region with subsistence farming being the main occupation of the inhabitants. The region has had two main seasons; at the time the study was conducted, there was a dry season that spanned between November and April the following year, and a rainy season which was between May and October ,30. MalaFor the original study that collected plasma samples, a cohort of 325 children aged 1 to 5 years were enrolled at the end of the low malaria transmission season (May) in 2004 and were followed up over a one year period (till May 2005) that spans the high and low transmission seasons. At the beginning of the study, and at two-month intervals, 0.5–1.0 ml of finger-prick blood samples were collected from the participants whose parents had consented to enroll their children in the study. A total of seven samples per child were taken over the one year period and these were processed and stored for laboratory analysis. At each sampling time point, body temperature measurements were taken and parasite infection status was determined by microscopic examination of blood smears and by malaria RDTs, while haemoglobin levels were measured by Hemocue. Demographic information and data relating to the subjects’ malaria exposure during the transmission season was captured by a study questionnaire. Children who were febrile (axillary temperature ≥ 37.5°C) and tested positive for malaria parasites were referred to the nearest designated health facility for appropriate treatment.Symp) group, included children who had one or more clinical malaria episodes. Clinical malaria was defined as having blood film parasitaemia, a fever (temperature ≥ 37.5°C) and at least one other common malaria symptom at any time during the one year study period and no other obvious cause for the fever. The second category was the asymptomatic (Asymp) group, which included children who had blood film parasitaemia but with no fever and no other clinical symptom of malaria. In total, samples from 126 children  were selected for analysis. Sixty-four (64) of the 126 children were in the symptomatic study group and 62 were in the asymptomatic group.For the current study, archived plasma samples from six of the seven contact time points (excluding the baseline samples) were retrieved from storage for analysis. Samples were grouped into two categories based on the clinical and parasitological data from the original study. The first group, described as the symptomatic (Pichia pastoris under good manufacturing practice (GMP) conditions and purified by a methodology that has been previously described [in silico designed AMA1 Diversity Covering (DiCo) proteins [P. pastoris as a non-glycosylated antigen under GMP conditions.The full length ectodomain (amino acids 25–545) of the FVO (Genbank accession number AJ2642667) variant of AMA1 was expressed as a recombinant protein in escribed . The thrproteins . The hydproteins  and wereThe four AMA1 variant antigens and the EBA-175 RII antigen were coupled to microspheres or beads  with unique spectral addresses  for the measurement of antigen-specific antibody levels by the Luminex xMAP technology. Bovine serum albumin (BSA) was coupled to a sixth bead set (106) for the assessment of non-specific binding in all assays.The coupling reaction was performed as previously described . Brieflyhttps://www.protocols.io/private/587a8b11e41cf045b36ffa515bb5a632).Protocol for antigen coupling and for performance of the multiplex assay has been deposited in protocols.io . Plasma samples from the 126 children as well as pools of positive and negative control plasma samples were assayed for antibodies specific to the four AMA1 allelic variants and the EBA 175RII antigen. Plasma samples were diluted 1:400 for test and negative control plasma samples, and 1:10,000 for positive control pool. Antigen-specific coupled beads were pooled, mixed and 50 μl aliquots added to wells in a washed Multiscreen filter base plate . Diluted plasma samples were subsequently added to duplicate wells and the plate vortexed and incubated at 4°C in the dark for one hour. After this period, the plate was washed three times and incubated with 25 μl/well of 5 μg/ml of biotin-labelled goat anti-human detection antibody in the dark at 4°C for 1 hour. This step was followed by incubation with 1:50 dilution of streptavidin-conjugated phycoerythrin (25 μl/well) for 30 minutes at 4°C in the dark. The plate was subsequently developed by addition of 25 μl/well of 0.5% formaldehyde in PBS and read on a programmed Luminex 200 system that runs on the xPonent 3.1 software (Luminex Corporation). Results were expressed as mean fluorescent intensity (MFI) and this is directly proportional to the antigen-specific antibody concentration. Bovine serum antigen (BSA) was included in all assays to account for nonspecific antibody binding by subtracting the average BSA antibody level for each plate from the antibody level of test wells before statistical analysis was performed.The feasibility of measuring the levels of antibodies against allelic variants of the same antigen (AMA1) using a multiplex assay was assessed by comparing AMA1-specific antibodies levels (expressed as MFI) from such multiplex assays with levels against the same alleles using singleplex assays. A subset of 42 plasma samples from one of the sampling time points was selected for this comparative analysis. Fifty microlitres (50 μl) of the antigen-coupled bead mixture were distributed into microtitre wells and 50 μl of 1:400 diluted plasma samples added to duplicate wells in the plate. For the multiplexed assay, equal numbers of the four AMA 1 variant coupled beads were mixed together and used to determine antigen-specific antibody levels in the selected plasma samples. Each AMA1 variant coupled bead was concurrently run as a singleplex using the same diluted plasma samples for comparison, and beads coupled with BSA were included in both multiplex and singleplex assays as a negative control. The median MFI of data from singleplex and multiplex assays were subsequently compared.Non-parametric analyses were performed since the antibody data were not normally distributed. Differences in median antibody MFI between singleplex and multiplex assays for each AMA1 allelic variant were assessed by Mann-Whitney U tests. For each antigen, the multiplex and singleplex MFI data were also subjected to Spearman rank correlation analysis.At each of the six sampling time points, the median antigen-specific antibody levels were compared between the two study groups. Antigen-specific antibody levels were also compared across the six sampling time points by Kruskal-Wallis test followed by the Bonferroni pair-wise post-hoc test.ggplot2  and sampling time point did not significantly contribute to the models. P-values for the intercept and fixed effect were obtained by likelihood ratio tests (with Laplace Approximation). All analyses and graphics were done using either the R statistical software (Version 3.4.0) or the GraphPad Prism software  and differences were considered to be statistically significant when p values were less than 0.05.For the assessment of potential antibody correlates of protection against clinical malaria, a generalized mixed effects regression model for repeated measurements was fitted to antigen-specific antibody data using the R package lme4 . InitialPlasma samples collected from 126 children at six different time points  were analyzed in this study. This number included 64 children who had clinical malaria symptoms at least once over the six sampling time points and 62 children who had high asymptomatic parasitaemia at least once over the six time points. There were 108 clinical malaria episodes over the entire study period; 25 clinical episodes were recorded in July 2004 and another 25 in September 2004, 18 episodes were recorded in November 2004, 13 episodes in January 2005, 18 episodes in March 2005 and 9 episodes in May 2005 . OverallAn initial assessment of the capacity of multiplex assays to accurately estimate the levels of specific antibodies to allelic variants of the same antigen was performed by measuring and comparing antibody levels in selected samples by singleplex and multiplex assays. Median MFI levels were not significantly different between singleplex and multiplex assays for any of the four allelic AMA1 variants . For all antigens, there were no significant differences in median antigen-specific antibody levels (MFI) between the two study groups at any of the six time points . CompariAlthough parasite density was not significantly different between symptomatic and asymptomatic children at any of the six sampling time points , parasitPlasma antibody levels against the four variants of AMA1 were log transformed (base 2) and the levels compared in a pair-wise manner using Bland-Altman plots . For eacOverall, the narrowest 95% limits were observed for plots between the HB3/CAMP paired variants of AMA1 in November 2004 and March 2005 , suggestFor pair-wise comparisons that included antibodies against the 3D7 AMA1 variant, the 95% limits observed in July 2004, March 2005 and May 2005 were at least two-fold wider compared to the corresponding 95% limits in September 2004, November 2004 and January 2005. These collectively suggest the predominance of a more cross-reactive anti-AMA1 antibody profile during periods of moderate to high disease transmission  and an increase in the levels of strain-specific anti-AMA1 antibodies during the dry season and before the next major transmission season begin.The extent of antibody cross-reactivity between any two AMA1 variants may be directly related to the degree of sequence similarity between the antigen vaiants. Alignment of the amino acid sequences of the four AMA1 variants used in this study  shows thOn the whole, there was no clear trend of differences between symptomatic and asymptomatic children with respect to the relative proportions of cross-reactive and strain-specific antibodies across all six sampling time points.To identify potential antibody correlates of protection against malaria, a mixed effect linear regression model was fitted for each antigen with clinical malaria status as the binary outcome variable, antigen-specific antibody data over the six time points entered as fixed effects and study subject as a random effect. Data from the asymptomatic study group were used as the reference for comparison with data from individuals who suffered at least one clinical malaria episode over the six time points. For all antigens (EBA 175RII and the four AMA1 variants), there was no association between measured antibody levels and clinical malaria status . HoweverThe acquisition of malaria-specific antibodies is very important for the establishment of partial clinical immunity to malaria since antibodies play a crucial role in parasite clearance and a reversal of clinical disease symptoms ,5. ThougThe measured antigen-specific antibody levels for all study subjects were compared across the six sampling time points. Antibody levels against variant AMA1 allele types were generally higher during the high transmission season compared to the low transmission season, while anti-EBA 175RII levels did not change considerably between seasons . The meaParasite density was high in July 2004 and peaked in September 2004, beyond which there was a decline to intermediate density in November 2004 and January 2005 . ParasitThe lack of statistically significant differences in antibody levels between symptomatic and asymptomatic children at all time points corroborate the outcome of the mixed effects logistic models, that none of the measured antibodies showed an association with clinical malaria outcome . BaselinFor assessment of the relative proportions of strain-specific and cross reactive antibodies, Bland-Altman plots revealed a relatively higher preponderance of cross-reactive antibodies from September 2004 to January 2005, which cover the peak rainfall season and into the early part of the dry season, while relatively higher levels of strain-specific antibodies were elicited during the low transmission season and into the early part of the rainy season . This obThough the parasite exposure history of the study population is not known, the pattern of antibody responses to the four AMA1 variants suggests possible exposure to a diversity of parasite variants over the one year follow up period. The observation of narrow limits of agreement during the high transmission period and broad limits of agreement during the low transmission period is consistent for antigen pairs that include 3D7 AMA1 but not paired comparisons involving any of the other three variants (XLSX)Click here for additional data file.S2 TableResults of pair-wise post-hoc tests for anti-AMA1 antibodies across the six sampling time points. Bonferroni post-hoc test was used to assess pair-wise differences in antibody levels across the different sampling time points.(XLSX)Click here for additional data file.S1 Appendix(TXT)Click here for additional data file.S2 Appendix(CSV)Click here for additional data file.S3 Appendix(TXT)Click here for additional data file.


Plasmodium falciparum (Pf) infection and multiclonality on subsequent risk of clinical malaria have been reported, but the relationship between these 2 parameters and their relative impacts on the clinical outcome of infection are not understood. A longitudinal cohort study was conducted in a seasonal and high-transmission area of Mali, in which 500 subjects aged 1–65 years were followed for 1 year. Blood samples were collected every 2 weeks, and incident malaria cases were diagnosed and treated. Pf infection in each individual at each time point was assessed by species-specific nested-PCR, and Pf longitudinal prevalence per person  was calculated. Multiclonality of Pf infection was measured using a 24-SNP DNA barcoding assay at 4 time-points  over one year. PfLP was positively correlated with multiclonality at each time point . When host factors , PfLP, and multiclonality (at the beginning of the transmission season) were analyzed together, only increasing age and high PfLP were associated with reduced clinical malaria occurrence or reduced number of malaria episodes . When age, PfLP and baseline Pf positivity were analyzed together, the effect of high PfLP remained significant even after adjusting for the other two factors . In addition to host age and baseline Pf positivity, both of which have been reported as important modifiers of clinical malaria risk, our results demonstrate that persistent parasite carriage, but not baseline multiclonality, is associated with reduced risk of clinical disease in this population. Our study emphasizes the importance of considering repeated parasite exposure in future studies that evaluate clinical malaria risk.The effects of persistent  Plasmodium falciparum (Pf) transmission and infection burden in endemic areas will be facilitated by a better understanding of the true distribution of infections, and the host, parasite and vectorial factors that influence transmission and infection outcome. The application of updated, sensitive diagnostic tools to understanding how these factors interact and influence infection dynamics in different transmission settings will be essential for maximizing the impact of malaria control and elimination programs.While major gains have been made in the control and elimination of malaria, this disease remains a major threat to global health with an estimated 438,000 global malaria-related deaths in 2015 [Studies investigating the dynamics of Pf prevalence have shown varying degrees of association between host factors, such as age, gender and red blood cell (RBC) polymorphisms, and Pf prevalence and clinical malaria in many epidemiological settings –9. HowevP. falciparum infections in Papua New Guinea [One important feature of Pf infections, especially in areas of high transmission intensity, is that an individual could be concurrently infected with multiple, genetically-distinct clones of Pf. Multiclonality has been reported to influence the duration and outcome of infections –13, but w Guinea . Furtherw Guinea , 15, 16.w Guinea , 18.In the present study, we followed a cohort of 500 individuals aged 1–65 years living in Kenieroba, Mali, where malaria transmission is highly seasonal , 20. UsiThis study was conducted in Kenieroba, a village of about 2,000 people located approximately 75 kilometers southwest of Bamako. In this high-transmission area of Mali, the annual rainy season occurs from June to December. The study cohort of 500 individuals aged 1–65 years was selected in proportion to the age distribution of the entire village population based on a village-wide census conducted in May 2012. Study participants were recruited based on their age and residency in Kenieroba for the full study year.This study was approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases and the Ethics Committee of the Faculty of Medicine, Pharmacy, and Odontostomatology, University of Bamako, and is registered with Clinicaltrials.gov, number NCT01829737. Written informed consent was obtained from study participants, or the parents or guardians of children aged <18 years.® (Qiagen) and stored at –80°C. If participants had malaria symptoms  at the time of a scheduled visit or self-referral, parasitological malaria diagnosis was performed by experienced study microscopists, and if confirmed, patients were referred to Kenieroba’s health center for standard-of-care antimalarial treatment. It should be noted that microscopy was not performed for the entire study. Parasite detection by Giemsa staining and subsequent microscopic analysis was only performed for the purpose of diagnosing clinical malaria in participants who presented with malaria symptoms as listed above, during the study period. The Kenieroba health center is the only health facility in the entire village and all study participants were encouraged to visit there when they developed a fever or other malaria symptoms. For the purpose of our analysis, clinical malaria was defined as axillary temperature ≥37.5°C and asexual parasite density >5000/μL by microscopy [Finger-prick blood samples, collected every 2 weeks from June 2013 to May 2014, were spotted on Whatman 3MM filter paper, and then dried and stored at room temperature until used. At the same time, 100 μL of whole blood were collected in 500 μL RNAprotectcroscopy . At the croscopy .P. falciparum, P. malariae, P. vivax and P. ovale infections was performed using species-specific nested-PCR [Plasmodium spp., [Molecular diagnostic detection of sted-PCR , with drsted-PCR . Brieflyum spp., . The nes®, parasite genomic DNA (gDNA) was extracted as follows: gDNA samples were eluted from a gDNA eliminator column using the QIAmp 96 Blood Kit (Qiagen) protocol, as modified by Wampfler et al [Pf-positive samples (by nested-PCR) from June 2013 (Jun-V1), November 2013 (Nov-V1), February 2014 (Feb-V1), and April 2014 (Apr-V1) were selected for DNA barcoding . For eacer et al . Sampleser et al . Each prer et al . PolymorComplexity of infection (COI) was determined for each DNA sample using the COIL program as described previously , with a P. falciparum longitudinal prevalence (PfLP) was calculated for each individual as the proportion (%) of the number of Pf-positive samples among the total number of samples collected over 1 year. Only data from individuals with available samples from at least two-thirds of the 22 time-points  were analyzed. At each of the 4 selected time-points, Polymorphic Proportion (PmP) was calculated for each Pf-positive individual (sample), as the proportion (%) of polymorphic reactions among the total number of successful reactions  from the 24 reactions that make up the 24-SNP barcoded assay. Only data from samples with ≥13 successful assays  were analyzed.At each time-point, population-level Pf prevalence was calculated as the proportion (%) of individuals who were Pf-positive by nested-PCR among the total number of individuals who provided blood samples, regardless of whether they received antimalarial treatment before sample collection. Age- and gender-stratified PfLP were analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparison test and Mann-Whitney test, respectively. COI and PmP distributions were analyzed between time-points using Chi-squared test and Kruskal-Wallis test, respectively. Associations between PfLP and PmP were analyzed using Spearman rank correlation test. A multiple linear regression analysis was performed to evaluate the association between response and multiple explanatory variables . For clinical data, a logistic regression model was used to analyze the risk of experiencing clinical malaria over 1 year, and Poisson and Cox's proportional hazard regression models were used to analyze the number of malaria episodes and time to first clinical malaria episode, respectively.Plasmodium spp. in this area were also assessed at 2 wet season (Jun-V1 and Nov-V1) and 2 dry season (Feb-V1 and Apr-V1) time-points using species-specific nested-PCR. No P. ovale or P. vivax infections were detected while P. malariae infections were observed at all 4 time-points but at low levels . Therefore, further analysis was performed exclusively on P. falciparum infections.The 500-subject cohort, representing approximately a quarter of the population in Kenieroba, was drawn to reflect the age distribution of the entire village population. The demographic characteristics of the cohort are summarized in P = 0.002), even after adjusting for age (P = 0.010). Thus, host age and gender were significantly associated with PfLP in this population.We calculated the PfLP for each individual, and determined how it was affected by age, gender, and RBC polymorphisms including ABO/Rh types, Hb phenotype , and G6PD deficiency and α-thalassemia genotypes. When host factors were evaluated individually, or together using multiple linear regression analysis (data not shown), only age and gender were significantly associated with PfLP. PfLP increased with age until 9–16 years, after which it declined with increasing age . IndividPlasmodium spp. were measured . For this purpose, we used the 24-SNP DNA barcoding assay developed by Daniels et al [P = 0.192). Consistent with this analysis, there was no significant difference in the PmP distributions among the 4 time-points (P = 0.557). In addition, we examined the geographical distributions of Pf positivity, COI, and PmP within the study area at all 4 time-points and found no clustering of infections representing significant hotspots (data not shown).We measured the multiclonality of Pf infections at the same wet and dry season time-points at which the prevalence of other ls et al , and PmPP = <0.011). Since there was a significant correlation between PmP and PfLP, similar age and gender effects on PmP were also observed, as expected (data not shown). Furthermore, the associations between PmP and PfLP remained significant after adjusting for age and gender . Thus, these results indicate a positive relationship between multiclonality and the frequency of Pf positivity in this population.The relationship between PfLP and multiclonality was analyzed using PmP instead of COI because PmP yielded a higher resolution of multiclonality than the 3-level COI, which is the limit of COIL analysis . PmP andth percentile, and low = below 75th percentile). Since ABO/Rh types, Hb phenotype, G6PD deficiency, and α-thalassemia showed no significant impact on the clinical malaria readouts, those factors were excluded in the final analysis (data not shown). Logistic regression analysis showed that increasing age (P<0.001) and high PfLP (P = 0.005) were associated with reduced risk of malaria occurrence. Similarly, Poisson regression analysis showed that increasing age (P<0.001) and high PfLP (P = 0.005) were associated with decreasing number of malaria episodes. PmP and gender were not significantly associated with malaria occurrence or number of malaria episodes, and none of the factors were associated with time to first malaria episode. To further evaluate the impact of PfLP on clinical malaria risk, we included baseline Pf positivity as a covariate in the analysis (P = 0.001) and number of malaria episodes (P<0.001). Thus, in addition to host age, high PfLP was associated with reduced risk of clinical malaria.Using the Jun-V1 time-point as a baseline, we analyzed the impact of host factors, PfLP, and PmP on 3 measures of malaria risk over 1 year: 1) occurrence of malaria, 2) number of malaria episodes, and 3) time to first malaria episode . For thianalysis . This anP. falciparum longitudinal prevalence was positively correlated with multiclonality . Furthermore, >70% of Pf infections in our cohort at all 4 time-points were found to be polygenomic, which is about 25% higher than that measured by MSP2 genotyping in Kambila, Mali, where transmission dynamics are similar to those in Kenieroba [in vivo. Therefore, we cannot address whether there were any differences in the duration of infection by individual clones between wet and dry seasons.Genotyping of polymorphic genes, such as thus far –39, thisenieroba . In addiOur analysis revealed a significant correlation between PmP and PfLP, highlighting the importance of multiclonality as an indicator of parasite carriage, particularly in high-transmission areas where the prevalence of polygenomic infections may exceed 70%. However, the correlation coefficients were ≤0.47, meaning that one measure can explain ≤47% of variance in another measure. As a result, PfLP was significantly associated with the occurrence of clinical malaria and the number of episodes over 1 year, but PmP at baseline was not. These findings are consistent with previous reports and support a model of protection against clinical malaria resulting from persisting or repeated parasite carriage, but not necessarily the number of clones at any specific time-point , 16, 40.P. malariae which was the only other Plasmodium spp. detected in our cohort occurred only rarely, and it has been reported that other less commonly detected species such as P. ovale wallikeri are missed by the detection method used in this study [Plasmodium spp. co-infections on persistent carriage and malaria risk should be evaluated in a different setting with more targeted methodologies.Our study had a number of limitations. We did not measure parasite density as an endpoint and therefore we are unable to assess correlations between parasite density and host factors as well as parasite factors such as COI in this population. Moreover, future studies will need to investigate the impacts of other extrinsic determinants of transmission and parasite carriage such as rainfall, temperature, and usage of transmission-control tools  on PfLP. Our study was limited to 1 year and will need to be extended to determine the stability of the age and gender effects on PfLP with changing transmission dynamics. More participants may also be needed to properly evaluate the effects of several underrepresented host factors  on PfLP and clinical malaria risk. In addition, is study . TherefoIn conclusion, our study demonstrates that in the context of high seasonal malaria transmission, multiclonality (PmP) was positively associated with persistent Pf positivity (PfLP), but only persistent Pf carriage was significantly associated with clinical malaria risk when age and baseline Pf positivity were adjusted for in this population. Our results suggest that individuals with persistent Pf carriage are likely to have acquired a more effective protective immunity against clinical malaria. Our study emphasizes the importance of considering repeated parasite exposure, in addition to Pf positivity at baseline, in future studies that evaluate clinical malaria risk.S1 FigA-D), individuals with high COI also had high PmP .At all 4 time-points ((PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.