Northern Senegal is a zone of very low malaria transmission, with an annual incidence of < 5/1000 inhabitants. This area, where the Senegal National Malaria Control Programme has initiated elimination activities, hosts Fulani, nomadic, pastoralists that spend the dry season in the south where malaria incidence is higher (150–450/1000 inhabitants) and return to the north with the first rains. Previous research demonstrated parasite prevalence of < 1% in this Fulani population upon return from the south, similar to that documented in the north in cross-sectional surveys.19) antigen of the four primary Plasmodium species, as well as circumsporozoite protein (CSP) and liver stage antigen (LSA-1) of Plasmodium falciparum.A modified snowball sampling survey of nomadic pastoralists was conducted in five districts in northern Senegal during September and October 2014. Demographic information and dried blood spots were collected. Multiplex bead-based assays were used to assess antibody responses to merozoite surface protein  and the lowest observed in Podor (28%). Low seroprevalence was observed for non-falciparum species in all the study sites: 0.4, 0.7 and 1.8%, respectively, for Plasmodium ovale, Plasmodium vivax and Plasmodium malariae MSP-1. Antibody responses to P. vivax were observed in all study sites except Kanel.In the five study districts, 1472 individuals were enrolled, with a median age of 22 years (range 1 to 80 years). Thirty-two percent of subjects were under 14 years and 57% were male. The overall seroprevalence of P. falciparum MSP-119 antibodies and increases by study participant age provided data for low levels of exposure among this transient nomadic population. In addition, antibody responses to P. falciparum short half-life markers (CSP and LSA-1) and non-falciparum species were low. Further investigations are needed to understand the exposure of the Fulani population to P. vivax.Prevalence of  Malaria continues to cause significant morbidity and mortality, and according to the World Health Organization (WHO), an estimated 219 million malaria cases and 435,000 malaria deaths occurred worldwide in 2017  carbodiimide hydrochloride) and 5 mg/mL sulfo-NHS (sulfo N-hydroxysulfosuccinimide) and incubated with rotation for 20 min at room temperature (RT) protected from light. After a wash with coupling buffer (50 mM 2-(4-morpholino)-ethane sulfonic acid, 0.85% NaCl at pH 5.0), antigens were coupled to beads in presence of coupling buffer for 2 h at an antigen concentration of 20 μg/mL for all MSP-119 antigens, 60 μg/mL for LSA1, 30 μg/mL for CSP, and GST at 15 μg/mL. Beads were washed once with PBS, and suspended in PBS with 1% bovine serum albumin (BSA) with incubation for 30 min at RT by rotation. Beads were then resuspended in storage buffer  and stored at 4 °C.All antigens were coupled to magnetic beads  in the same manner as prior studies . BrieflyEscherichia coli extract to prevent non-specific binding, and stored at 4 °C until analysis. For the immunoassay, in 5 mL of reagent buffer , a bead mix was prepared with all regions included, and 50 μL bead mix was pipetted into each well of a BioPlex Pro plate (BioRad). Beads were washed 2 × with 100 μL wash buffer , and 50 μL reagent mix  was added to all wells, then 50 µL samples (or controls) were added to the appropriate wells in singlet at a sample dilution of 1:50. Plates were incubated overnight with gentle shaking at RT and protected from light. The next morning , plates were washed 3×, and beads were re-suspended with 100 µL PBS and read on a MAGPIX machine . Median fluorescence intensity (MFI) assay signal was generated for a minimum of 50 beads/region. To give the final signal used for analyses, MFI from wells incubated with Buffer B alone was subtracted from each samples’ MFI to give a final value of MFI minus background (MFI-bg).The bead-based multiplex technology was used. Briefly, a 6-mm circular punch was taken from the centre of each blood spot, corresponding to 10 μL of whole blood, for elution. Samples were diluted in 200 μL blocking Buffer B , 0.8% Polyvinylpyrrolidine (Sigma), 0.1% casein (ThermoFisher), 0.5% BSA (Millipore), 0.3% Tween-20, 0.1% sodium azide, and 0.01% 19 antibodies among the different malaria species, for generating estimates for seropositivity to the non-falciparum Plasmodium MSP-119 antigens, the MFI for the non-Pf MSP-119 was required to be both greater than the cut-off generated by the finite mixture model and greater than the PfMSP-119 MFI for that sample. Differences between groups were evaluated using the Chi-square test for dichotomous variables and the nonparametric Kruskal–Wallis test followed by post hoc test to analyse pairs of groups for continuous variables. A p-value < 0.05 was considered significant. A reversible catalytic model was fit to the seropositivity by age data for each antigen, and the estimates for the serological conversion rate (SCR) and serological reversion rate (SRR) per year were directly calculated from the likelihood model [Data were analysed using SPSS Statistics version 17 for Windows . After log-transforming MFI-bg values for the IgG data for each antigen, a two-compartment finite mixture model was applied to each antigen to determine means and standard deviation of the putative seronegative population for each antigen’s signal intensity data. The seropositivity threshold cut-off was defined as the lognormal mean of the lower distribution plus three standard deviations. Results of the finite mixture model for calculation of cut-offs are presented in Additional file od model .A total of 1472 individuals were enrolled from study sites in five districts: Podor, Dagana, Pete, Kanel, and Ranerou. The median age of the study cohort was 22 years (IQR: 12–37). Other characteristics of the study population are detailed in Table 19 was 45.0% (95% CI 42.5–47.7). The distribution of antibody response by district showed the highest seroprevalence to PfMSP-119 in Kanel  and Pete , followed by Ranerou , Dagana , and Podor . The highest estimated serological conversion rates (eSCR) for PfMSP-119 were observed in Kanel and Pete, and lowest in Dagana and Podor . Overall, positive antibody response was higher in males  compared to females .The overall seroprevalence of PfMSP-1 95% CI 4.5–47.7.  95% CI 4.5–47.7. ). Overall, CSP antibody responses increased significantly with age from 0.7% in 0–9 year olds to 29.6% among those 40 years and older (95% CI 24.8–34.9) , respectively. At 20.4%, seroprevalence was highest in Pete, as was the eSCR . No antibody response was observed in children less than 10 years old and seroprevalence among older children and adults increased significantly with age Table . The disP. malariae, MSP-119 positivity was 1.8% and was observed in every site. Plasmodium ovale MSP-119 positivity was 0.4%, and was observed only in the three northernmost sites on the Senegal River , which border Mauritania. Of the 10 individuals positive for P. vivax MSP-119 (0.7%), all but one were found in Dagana, Podor, or Pete , PfCSP and PfLSA-1 (pre-erythrocytic antigens) demonstrated low current levels of recent exposure to falciparum malaria in this population. In addition, very low seroprevalence (< 2%) to non-falciparum IgG antibodies also shows that exposure to these other human malarias is sparse in this population of nomadic persons. Both parasite prevalence and malaria serology suggest that this population in fact has low malaria burden and low exposure, despite spending dry season in a higher transmission zone.This work was part of an investigation to assess the degree of access to malaria control interventions and the burden of malaria among nomadic pastoralists in northern Senegal, as there was concern that this population could potentially contribute to ongoing transmission in very low transmission districts. Previously published analysis demonstrated that while access of nomadic pastoralists to malaria control interventions was low compared to non-nomadic populations in Senegal, malaria prevalence was also low, at 0.6% by nested PCR . Assessm19) allows an assessment of malaria over time, rather than a snapshot in time. Seroprevalence rates are thus higher than parasite prevalence and provide greater discrimination that can be used to provide a finer measure of malaria transmission than parasite prevalence.In low-transmission areas, parasite prevalence estimation using light microscopy is limited by detection sensitivity . Even usP. falciparum MSP-119 seroprevalence rates were found in Kanel (63.3%), Pete (55.8%) and Ranérou (39.9%), the three districts in which malaria infections were detected. These districts also have higher reported annual incidence of confirmed malaria than the other two districts, Dagana and Podor, two of the northernmost districts in Senegal [19 seropositivity is known to increase with age [P. falciparum in the context of recent declines in transmission.The highest  Senegal . Howeverwith age , and pos19 seroprevalence rates (obtained by ELISA) of 53.1 and 31.5%, were reported in Velingara (south) and Keur Socé , respectively, among children under 10 years in 2010 [At two sites in the south and central regions of Senegal, where nomadic pastoralists spend the dry season, PfMSP-119 antibody and protection from clinical malaria has been reported [Low susceptibility to malaria among Fulani compared to sympatric populations has been documented in Burkina Faso  and Malireported –32. In treported . RegardiP. falciparum are exposed to the immune system for only short periods after inoculation. Antibodies to PfCSP and PfLSA-1 were rare among children under 10 years, and somewhat more common among those 10 years and older, with relationship between seroprevalence of PfCSP and PfLSA-1 antibodies and age similar in all study sites. The PfCSP and PfLSA-1 seroprevalence by study site follows the same trends as PfMSP-1, with levels in Kanel and Pete higher than the other three sites.Seroprevalence to the pre-erythrocytic antigens PfCSP and PfLSA-1 was very low in this study, indicating the relative rarity of recent infections in the study population. These antibodies would most commonly be detected among individuals with frequent or recent exposure , 34. Unl19 antibodies was noted in all study sites, with carriage of anti-P. malariae IgG slightly more prevalent than P. ovale and P. vivax. While individuals seropositive for P. malariae MSP-119 were found in all study sites, all the individuals seropositive for P. ovale MSP-119, and 9 of the 10 persons seropositive for P. vivax MSP-119 were from the Podor, Dagana, and Pete sites along the Senegal River, bordering Mauritania, a country where P. vivax and P. ovale have both been reported [Very low seroprevalence to the non-falciparum MSP-1reported , 36.Plasmodium vivax has traditionally been considered virtually absent from Western and Central Africa, due to the absence of the Duffy blood group, a blood receptor hitherto considered indispensable for the invasiveness of P. vivax into red blood cells, as more than 90% of the population in this area are believed to be Duffy negative [P. vivax, albeit at low levels, consistent with other recent reports suggesting the circulation of P. vivax in sub-Saharan Africa [P. vivax was considered absent, recent studies have indicated its presence in the country [P. vivax in a small sample of asymptomatic children from Kédougou in the southeastern Senegal, with a seroprevalence of 58% by ELISA [P. vivax, especially in the northern areas where the NMCP is conducting pre-elimination strategies for malaria, would require diagnostic tools capable of detecting non-falciparum malaria and mixed infections, as well as updated treatment recommendations for non-falciparum malaria.negative . Howevern Africa , 39. In  country . A recenby ELISA . AdditioThis study was not designed to compare nomadic pastoralists to surrounding populations, thus there are no available comparators among non-nomadic peoples in space and time. However, available evidence suggests that antibody response among nomadic pastoralists is more similar to resident populations in the north than to southern populations. While the majority of the sample of nomadic pastoralists are at least 15 years old, adult resident populations have not been sampled to permit comparison. While efforts were made to recruit a representative sample of nomadic pastoralists, due to their mobility, generating a sampling frame was not possible and a modified snowball approach was adopted. As this is a transient population, the differences between the population demographics by district are difficult to interpret since malaria exposure for an individual could have occurred in that district, or when the person was travelling outside of the district into another part of Senegal.P. falciparum among Fulani nomadic pastoralists in northern Senegal was consistent with low exposure to malaria in the population. Due to the low seroprevalence of short-term antigens, and the previously reported low PCR prevalence in this population, it is likely the pastoralists would not pose a substantial risk of malaria re-introduction if local transmission is interrupted. Antibodies against to P. vivax, P. ovale, and P. malariae were very low in the entire population, but were found in persons enrolled at nearly all study sites. As Senegal moves towards pre-elimination in the north of the country, further investigations are needed to understand the circulation of non-falciparum Plasmodium.Antibody response to short- and long-term antigenic markers to Additional file 1. Fixed methods model distribution of two compartments for calculation of cut off values.


Plasmodium falciparum infection during pregnancy is a major cause of poor maternal health, adverse foetal outcome and infant mortality in sub-Saharan Africa. Genetic disposition is involved in susceptibility to malaria in pregnancy and its manifestation. MicroRNAs (miRNAs) influence gene regulation including that of innate immune responses. A miRNA-146a rs2910164 G > C single nucleotide polymorphism (SNP) has been associated with increased risks of several diseases, but no data as to malaria are available.P. falciparum infection among 509 Ghanaian women attending antenatal care (ANC) and 296 delivering Ghanaian primiparae was investigated. Malaria parasites were diagnosed by microscopy and PCR. Leukocyte-associated hemozoin in placental samples was recorded as well. Proportions were compared between groups by Fisher’s exact test, and logistic regression models were used to adjust for possible confounders.The association between miRNA-146a rs2910164 and P. falciparum infection was detected in 63% and 67% of ANC attendees and delivering primiparae, respectively. In both groups, two in three women were either heterozygous or homozygous for miRNA-146a rs2910164. Among ANC attendees, homozygosity conferred increased odds of infection (adjusted odds ratio (aOR), 2.3; 95% CI, 1.3–4.0), which was pronounced among primigravidae  but only marginal in multigravidae. Likewise, homozygosity for miRNA-146a rs2910164 in primiparae increased the odds of past or present placental P. falciparum infection almost six-fold .By PCR, P. falciparum infection in first-time pregnant women who are considered to lack sufficient acquired immune responses against pregnancy-specific strains of P. falciparum. These findings suggest that miRNA-146a is involved in protective malarial immunity, and specifically in the innate component.These results indicate that SNP rs2910164 G > C is associated with increased odds for  Plasmodium falciparum infection during pregnancy is a major cause of poor maternal health, miscarriage, stillbirth, low birth weight (LBW), preterm delivery and infant mortality in sub-Saharan Africa. Primiparous woman exhibit an increased susceptibility to P. falciparum infection and consequently bear a higher risk for placental malaria  discernible in placental tissue or blood), malarial anaemia and malaria-related morbidity and mortality as compared to multigravidae. The increased risk of malaria and complications is largely due to parasites exhibiting specific variants of the P. falciparum erythrocyte membrane protein-1, which facilitate adhesion to the syncytiotrophoblast , followed by the accumulation of infected erythrocytes and inflammatory cells in the placental intervillous space [P. falciparum strains increases with every consecutive pregnancy, resulting in better parasite recognition and reduced susceptibility and manifestation in multigravidae [P. falciparum infection in pregnancy [gravidae . Moreoveregnancy .Host genetic variation plays an important role in susceptibility to and manifestation of malaria. The association of single nucleotide polymorphisms (SNPs) in genes encoding toll-like receptors (TLRs) and other members of the innate immune system with susceptibility to (severe) malaria in Sub-Saharan African populations , 5 suggeP. falciparum infection. Plasmodium falciparum infection was assessed and the miRNA-146a SNP was genotyped in 805 Ghanaian pregnant women, a group at high risk of malaria.In this cross-sectional study, the presence of miRNA-146a SNP rs2910164 G > C was hypothesized to affect susceptibility to P. falciparum [In November and December 1998 and between January 2000 and January 2001, respectively, 530 pregnant women attending antenatal care (ANC) and 893 delivering woman were recruited at the Presbyterian Mission Hospital in Agogo, Ashanti Region, Ghana, a region holoendemic for lciparum . Informelciparum , 16. BriPlasmodium parasite density in venous and intervillous blood samples were microscopically counted on Giemsa-stained think films per 500 white blood cells (WBC) and per 100 high-power fields (HPF), respectively. The presence of leukocyte-associated hemozoin in the intervillous samples was also recorded. For ANC attendees, WBCs were counted using a Cell Counter  and the peripheral blood parasite density was calculated as parasites per microliter, deducing the multiplicator from the individual WBC count. Plasma and blood cells were separated by centrifugation. Genomic DNA was extracted from blood  and plasmodial infections and species were diagnosed by nested PCR assays [P. falciparum infection by PCR, microscopy, and/or hemozoin detection. MiRNA-146a SNP rs2910164 genotyping was carried out by melting-curve analysis applying commercially available primers and probes .R assays . “Past oP. falciparum infection among women with and without the miRNA-146a SNP were compared by a two-tailed Fisher’s exact test, and odds ratio (OR) and 95% confidence intervals (95% CIs) were computed. Additionally, miRNA-146a genotypes were compared with respect to the outcomes of malaria, i.e., fever, anaemia, LBW and preterm delivery. Trends, e.g., increasing infection prevalence in women with wild type alleles over heterozygosity to homozygosity for the miRNA-146a SNP, were tested by the Cochran Armitage test. Logistic regression models were used to adjust ORs of infection for known predictors, i.e., age (years), presence of PYR, and rainy or dry season (only in delivering women). All analysis was done in R version 3.4.3. A P-value < 0.05 was considered statistically significant.Haemoglobin (Hb) was measured by a HemoCue photometer , and anaemia was defined as Hb level < 11 g/dL . Gestati2 = 0.24; P = 0.62, c2 = 0.18; P = 0.67).Typing of the miRNA-146a SNP was successful in 96.0% 509/530) of ANC attendees and 97.4% 296/304) of delivering women. The miRNA-146a polymorphism was present in 67.7%  of ANC attendees and 69.2%  of delivering women  was higher in primigravidae  than in multigravidae . The geometric mean parasite density (GMPD) of microscopically positive samples was 304/μL  for all ANC attendees, 165/μL  for primigravidae and 719/μL  for multigravidae. 14.7% (75/509) of the ANC attendees were febrile, and 53.6% (273/509) had anaemia .In peripheral blood samples of ANC attendees , malaria parasites were detected by microscopy in 32.8% (167/509), and 63.3% (322/509) were found to harbour P. falciparum PCR, these figures were 59.1% (175/296) and 64.9% (192/296). Past or present placental malaria, i.e. considering also deposited hemozoin, was identified in 67.9% (201/296). The GMPD of microscopically positive peripheral and placental blood samples was 709/μL  and 1.15/HPF , respectively. 4.1% (12/293) of the delivering women were febrile and 38.8% (115/296) had anaemia . 25.7% (76/296) of the neonates had LBW and 26.4% (78/296) were preterm.Among delivering primiparae, malaria parasites were detected microscopically in 26.4% (78/296) and 45.6% (135/296) of peripheral and placental blood films, respectively. By P. falciparum infection in both ANC attendees . Of note, increased P. falciparum prevalence was also observed among multigravid miRNA-146a SNP carriers, however, only weakly and statistically not significant.Carriage of the miRNA-146a SNP was associated with increased odds of es Table , 3. ThisP = 0.3) of SNP carriers, and anaemia was present in 49.4% (81/164) and 55.7% , respectively. Likewise, among primiparae, proportions did not differ between wildtype women and SNP carriers for fever , anaemia , and preterm delivery . Stratification by infection status did not change this observation.In terms of clinical manifestation of infection, the miRNA-146a SNP did not show any significant association. In ANC attendees, fever occurred in 17.1% (28/164) of wildtype individuals and 13.6% , affects the processing of pre-miRNA-146a into mature miRNA-146a. Homozygosity for this polymorphism is associated with reduced expression of the downstream mediators, and heterozygosity with the expression of additional miRNA-146a: one from the leading strand and two from the passenger strand (miRNA-146a*G and miRNA-146a*C), which all three give rise to a mature miRNA [Consequently, altered TLR and cytokine signalling might influence the innate immune response to re miRNA , 27. There miRNA . Whereasre miRNA  and leprre miRNA , in addiTLR-4 variants or mannose-binding lectin deficiency [Expanding on Haldane’s malaria hypothesis, a polymorphism increasing malaria risk should be expected to be rare in endemic regions. However, in sub-Saharan Africa, miRNA-146a rs2910164 occurs in 67%  , similarficiency .TLR-4 SNP in Ghana, increased susceptibility to severe malaria but a trend towards reduced mortality was found in a previous study [Potential explanations include alleles or genotypes, which may have become deleterious after the out-of-Africa-migration of humans, possibly because of increased susceptibility to severe bacterial infections and sepsis . Alternaus study . Consideus study , more reP. falciparum infection in first-time pregnant Ghanaian women. This suggests that miRNA-146a plays an important role in the respective innate immune response but further studies are required to detail the actual pathophysiology involved. Understanding protective immunity towards malaria in pregnancy is essential to improve maternal health and for decreasing the huge share of malaria in infant mortality in sub-Saharan Africa. MiRNA-based biopharmaceuticals are an active field of research. Enhanced antimicrobial immune responses have been observed after silencing or administration of miRNAs [Homozygosity for the miRNA-146a rs2910164 SNP predisposes to f miRNAs , 35. The


Febrile symptoms in children are a leading cause of health-care seeking behaviour worldwide. The majority of febrile illnesses are uncomplicated and self-limited, without the need for referral or hospital admission. However, current diagnostic tools are unable to identify which febrile children have self-limited infection and which children are at risk of progressing to life-threatening infections, such as severe malaria. This paper describes the need for a simple community-based tool that can improve the early recognition and triage of febrile children, with either malarial or non-malarial illness, at risk of critical illness. The integration of a disease severity marker into existing malaria rapid diagnostic tests (RDT) could enable detection of children at risk of severe infection in the hospital and community, irrespective of aetiology. Incorporation of a disease severity marker could inform individualized management and early triage of children at risk of life-threatening infection. A child positive for both malaria and a disease severity marker could be prioritized for urgent referral/admission and parenteral therapy. A child positive for malaria and negative for a disease severity marker could be managed conservatively, as an out-patient, with oral anti-malarial therapy. An RDT with a disease severity marker could facilitate an integrated community-based approach to fever syndromes and improve early recognition, risk stratification, and prompt treatment of severe malaria and other life-threatening infections. Febrile syndromes account for over 1 billion episodes annually and are one of the most common reasons to seek medical care worldwide . A childThis paper advocates for the development of rapid and simple tools to enhance the early recognition and triage of severe malaria and other life-threatening infections in the community setting. This approach would permit integrated community-based management of “all cause” fever syndromes. By avoiding unnecessary referral of self-limited and uncomplicated infections, it would decompress health care facilities, while focusing health resources on those at risk of severe infection that are most likely to benefit from referral, admission and supportive care. This strategy could save lives and health dollars.Malaria remains a major contributor to childhood death and disability . In 2016As a result of current malaria control and elimination efforts, malaria prevalence is changing and the proportion of fevers due to malaria is decreasing in many regions of Africa ) predict clinical outcome in patients with non-malarial febrile illness, and could be used to risk-stratify patients and inform clinical management irrespective of aetiology , 39, 47.Many deaths in children under five in low resource settings occur in remote regions. Rural place of residence is associated with an increased risk of delayed access to medical treatment and death before the age of five . More thThe majority of febrile illness in children under the age of five is self-limited and, once malaria and critical illness are excluded, can be managed conservatively. The current inability to rapidly identify the small proportion of children who are at risk of progressing to life-threatening infection is a major obstacle to management of fever syndromes, rational antimicrobial use and effective health resource allocation. This paper proposes the incorporation of disease severity markers into existing RDTs as an approach to enable early recognition, risk stratification, and prompt treatment of severe malaria and other life-threatening infections. While additional studies are needed, this strategy could enhance triage, improve case management, resource allocation, and ultimately health outcomes for children presenting with both malarial and non-malarial febrile illness in hospital and community-based settings.


Plasmodium life stage infectious to mosquitoes. Here, we summarize evidence for naturally acquired anti‐gametocyte immunity and the current state of transmission blocking vaccines (TBV). Although gametocytes are intra‐erythrocytic when present in infected humans, developing Plasmodium falciparum gametocytes may express proteins on the surface of red blood cells that elicit immune responses in naturally exposed individuals. This immune response may reduce the burden of circulating gametocytes. For both P. falciparum and Plasmodium vivax, there is a solid evidence that antibodies against antigens present on the gametocyte surface, when co‐ingested with gametocytes, can influence transmission to mosquitoes. Transmission reducing immunity, reducing the burden of infection in mosquitoes, is a well‐acknowledged but poorly quantified phenomenon that forms the basis for the development of TBV. Transmission enhancing immunity, increasing the likelihood or intensity of transmission to mosquitoes, is more speculative in nature but is convincingly demonstrated for P. vivax. With the increased interest in malaria elimination, TBV and monoclonal antibodies have moved to the center stage of malaria vaccine development. Methodologies to prioritize and evaluate products are urgently needed.The efficient spread of malaria from infected humans to mosquitoes is a major challenge for malaria elimination initiatives. Gametocytes are the only  While P. falciparum is the dominant Plasmodium species in most of Africa and is associated with the most severe morbidity and mortality, P. vivax is more widely distributed and is increasingly recognized as an important source of morbidity and restrained economic productivity.Malaria is one of the few infectious diseases earmarked for worldwide eradication by the World Health Organization (WHO).Plasmodium life stages that are infectious to mosquitoes, so the uptake of these specialized forms by blood‐feeding female Anopheles mosquitoes is essential for human‐to‐mosquito transmission. Plasmodium falciparum gametocytes form when asexual schizonts become committed to produce sexual progeny by the activation and expression of the Apatella2‐g gene (AP2‐G).P. falciparum heterochromatin protein 1 (PfHP1).Plasmodium species; all P. falciparum ApiAP2 proteins have syntenic homologues in P. vivax and are expressed at a similar stage of development.P. falciparum, gametocyte formation is a 10‐12 day process during which the parasite passes through five morphologically distinct forms (stages I‐V) Gametocytes are the only Plasmodium gametocytes rapidly egress from the host erythrocyte and develop into gametes. Gametogenesis is induced by a reduction in temperature, increase in pH and exposure to xanthurenic acid.Plasmodium life cycle.In the mosquito midgut, P. falciparum than for P. vivax, although it is likely that many factors are shared between Plasmodium species. General parasite characteristics that have been associated with differences in transmission potential and infectivity include gametocyte densityMany factors influence the likelihood of gametocytes being transmitted to mosquitoes and establishing a successful mosquito stage infection.P. falciparum genomeP. falciparum gametocytesP. vivax revealed that the expression of 1613 genes was correlated with the expression of known gametocyte genes.The first empirical evidence that human immune responses to gametocytes could affect their infectiousness to mosquitoes came from immunization studies in birds,P. falciparum and P. vivax, discuss the effect of these responses on transmission, and propose strategies for transmission blocking interventions. Immature and mature gametocytes have markedly different biology, morphology, and preferential localization in human tissues. Immune responses to early and late gametocytes therefore have the potential to affect transmission differently; early gametocyte immunity could reduce the number of gametocytes achieving maturity in the peripheral blood, while late gametocyte immunity may affect gametocyte number and their likelihood of undergoing successful sporogonic development in the mosquito. While this review will focus on P. falciparum, we will also summarize the current state of knowledge for the less‐studied P. vivax and indicate major knowledge gaps with regard to anti‐gametocyte immunity, implications for transmission dynamics and potential vaccine strategies.This review will discuss the evidence for the existence of naturally acquired human immune responses against the sexual parasite stages of P. falciparum gametocytes sequester outside the peripheral circulation, primarily in the bone marrow and spleen. Their maturation (10‐12 days) involves five distinct developmental stages (I‐V); mature stage V are released in the peripheral circulation. Plasmodium vivax gametocytogenesis may also involve a blood marrow phase, but in contrast to P. falciparum only takes 48 hours. After ingestion by a blood‐feeding Anopheles mosquito, gametocytes rapidly egress from the host RBC and develop into gametes. Male gametocytes exflagellate to form eight microgametes that subsequently fertilize a “round‐up” microgamete to form a zygote leading to the formation of a motile ookinete. The ookinete penetrates the midgut wall and forms an oocyst that produces hundreds to thousands of sporozoites. Upon oocyst rupture, sporozoites are released and migrate to the salivary glands from where they can be transmitted to a new human host. Boxes represent surface antigens that are under consideration for vaccine development.Merozoites that are released from infected liver cells invade RBCs to initiate the erythrocytic cycle. The sexual development is initiated by a subset of parasites that are committed to produce gametocytes. Immature 22.1P. falciparum sequester to avoid prolonged circulation in the blood. Asexual parasites sequester in the tissue microvasculature through well‐defined ligand‐receptor interactions; Knob‐associated histidine rich protein (KAHRP) is critical to the formation of “knobs” on the infected erythrocyte surface,P. falciparum erythrocyte membrane protein 1 (PfEMP1) family accumulate on these knobs and mediate cytoadherence.P. falciparum gametocyte development are marked by changes in gene expression. Plasmodium falciparum gametocyte exported protein 5 (PfGEXP5) is detectable 14 hours after RBC invasion by a sexually committed merozoiteP. falciparum gametocytes sequester primarily in the bone marrow and spleen.Both the asexual and sexual intra‐erythrocytic forms of P. berghei parasites in mice demonstrated direct evidence of homing by merozoites to the extravascular niche of the bone marrow and spleen. Using specific inhibitors, de Niz et al demonstrated that this extravasation (movement from the bone marrow/spleen vasculature to the organ's extravascular space) is mediated via a receptor‐ligand interactionP. berghei study supports the hypothesis that a subset of sexual merozoites bearing specific surface ligands home to the bone marrow and spleen, bind the epithelium, move into the extravascular space, and invade erythrocyte precursors (abundant in this niche) to become young gametocytes.P. falciparum. These data provide evidence for the presence of surface molecules on sexually committed merozoites (or schizonts containing merozoites) that are involved in cytoadhesion during the process of homing and retention in the bone marrow. During the acute phase of infection, both asexual and sexual infected erythrocytes accumulate in the bone marrow, suggesting that early gametocytes can form in the peripheral blood and may specifically home to the bone marrow/spleen. The targeting of any parasite ligands that mediate gametocyte sequestration by immune factors could possibly inhibit transmission potential.A study using intravital imaging of P. berghei gametocytes pass freely into and out of the vascular spaces of the bone marrow, and that a switch in host cell deformability  underlies this freedom of movement.Numerous studies have shown that immature giRBCs are rigid and that a change in host cell deformability occurs in the transition to maturity.Naturally induced antibodies may affect gametocyte morphology and fitness. In one study, serum antibodies from Thai malaria patients were incubated with stage I gametocytes, and were observed to reduce their numbers, interfere with maturation, distort their morphology and reduce the number of oocysts developing in subsequent mosquito feeding assays.P. falciparum 3D7 gametocytes, and observed reactivity of immune sera from Gambian children with the surface of mature giRBCs, but not with immature stages.Several studies have aimed to identify the erythrocyte surface antigens of immature gametocytes that could be involved in sequestration. The first study used a flow cytometry‐based method with purified Chan et al quantified antibody reactivity to erythrocytes infected with gametocytes and asexual stages using microscopy in order to better understand the difference in humoral response against these two life stages.Interestingly, a recent study demonstrated that surface recognition by naturally acquired antibodies was only present on erythrocytes infected with immature forms (I to III) of NF54 and a genetic strain with a Pf2004 background. In this study, no measurable reactivity with the surface of giRBC infected with mature stage V gametocytes was observed.P. berghei model it seems likely that a subset of merozoites directly translocate into the bone marrow, spleen, and possibly other areas of low vascular flow, but it is also possible that some degree of homing occurs by early gametocytes.P. falciparum mature gametocytes P. vivax  taken at the point of infection “crisis” or paroxysm can kill gametocytes, and this appears to be mediated by inflammatory cytokines including tumor necrosis factor‐α (TNF‐α) which stimulates leukocytes to produce toxic nitric oxides.P. vivax‐infected humans, cytokine concentrations were insufficient to induce killing factors during paroxysm,P. vivax schizont extract was able to kill P. falciparum gametocytes, and vice versa.The role of cellular immune mechanisms in the clearance of circulating gametocytes is contentious. As erythrocytes lack major histocompatibility complex molecules, direct targeting of giRBCs by T lymphocytes is not possible. However, CD4 + T cells clearly respond to gametocyte antigens3In contrast to the sparse and partially conflicting evidence for immune responses against intact giRBCs that may reduce circulating gametocyte density, there is a large and cohesive body of data demonstrating that humoral responses to intra‐erythrocytic gametocyte proteins can inhibit parasite development inside mosquitoes.P. vivax infections produced gamete specific antibodies, and that antibodies from these patients inhibited transmission in direct membrane feeding assays (DMFA). Shortly after, Graves et al showed that similar mechanisms prevented P. falciparum gamete viability using the standard membrane feeding assay (SMFA), in which the effect of sera from patients in Papua New Guinea was tested on cultured gametocytes.The proof of principle for the existence of transmission reducing (TR) antibody responses comes from experiments in which birds were immunized with whole inactivated gametocytes or gametes in the 1950’s and 1970’s.Parasite antigens that can be targeted by antibodies to inhibit transmission can be divided into two broad classes; prefertilization and the postfertilization antigens. Prefertilization antigens are expressed during gametocyte development and contribute to the viability of mosquito stage gametes, zygotes and/or ookinetes. Antibodies to prefertilization antigens are naturally acquired in the human host only because the majority of gametocytes die in circulation, releasing their intra‐erythrocytic proteins. Postfertilization antigens are expressed solely in the mosquito vector; though transcription may occur in circulating gametocytes, the resulting mRNA is held in translational repression until the gametocytes activate in the mosquito midgut.Pb48/45 by its vivax orthologue seemed to reduce oocyst development, but did not abolish it, indicating that there is probably functional conservation of P48/45 in these two parasite strains since fertilization was not affected.P. falciparum gametocytes, Pfs230 is present on the gametocyte surface, and a 50 kDa fragment is proteolytically cleaved from the surface‐bound protein after the parasite’s emergence from the RBC in the mosquito gut.P. falciparum Pfs48/45 knock‐outs (KO) produce Pfs230, but it is not retained on the gametocyte surface, indicating that Pfs48/45 mediates Pfs230 retention.The most studied prefertilization antigens are P48/45 and P230, which belong to the 6‐cysteine protein familyIn alignment with their exposure on the surface of gametes and importance in gamete fertility, studies have shown that the presence and titer of naturally acquired antibody responses to Pfs48/45 and Pfs230 are statistically associated with serum transmission reducing activity (TRA).P. berghei did not result in any defects throughout the life cycle, indicating that the protein is dispensable.p230p locus in P. bergheiP. knowlesi P. falciparum mutants lacking Pf230p have a strongly reduced ability to bind erythrocytes to form exflagellation centers, similar to the observed phenotype in Pfs230 KO parasites.P. falciparum or P. vivax have neither been tested, nor have proteins been produced for immunization studies with a view to TBV testing. Naturally acquired Pfs230p antibody responses have been assessed in one study, which linked the TRA of serum antibodies from individuals living in malaria endemic areas with the same individual's antibody responses to 315 gametocyte enriched proteins.https://doi.org/10.5061/dryad.8bp05). The magnitude of α‐Pfs230p responses was not significantly different between individuals with evidence of blocking transmission in the SMFA and individuals whose antibodies had no notable transmission reduction activity. However, the proportion of individuals deemed “sero‐reactive” in a mixture model was borderline significantly higher in blockers (13.6%) than in non‐blockers (3.9%) for one of the two Pfs230p peptides tested . It should be noted that this analysis was not adjusted for false discovery from multiple comparisons, and the overall prevalence of responses was low (13/276). Further serological studies with Pfs230p in its native conformation will be valuable.Besides P230, rodent and human parasites also encode a paralog P230p. This protein is only expressed by mature male gametocytes.A. gambiae.A. stephensi.P. berghei the opposite has been demonstrated, with gene disruption resulting in a significant reduction of oocyst numbers.A. gambiae and A. stephensi.Pfs47 is another member of the 6‐cysteine protein familyP. berghei and P. falciparum after disruption of P230p and P47 suggest that there are functional differences in these proteins between these two Plasmodium spp. There are no data available with respect to the function of these proteins in P. vivax, and it remains unknown whether they have an essential role in mosquito stage development.The observed differences in phenotype between HAP2) was first identified in Arabidopsis thaliana,P. berghei.P. berghei HAP2 is expressed in gametocytes and is present on the surface of intra‐erythrocytic gametocytes and microgametes. Disruption of the gene results in reduced transmission by blocking gamete fertilization.P. falciparum gametocytes sourced from naturally infected donors.P. vivax, though it seems likely that the function is conserved in P. vivax based on the observed functional conservation of HAP2 throughout species.The male‐specific sterility gene  are potentially vulnerable to direct phagocytosis. It has been shown in vitro that extracellular gametes can be phagocytosed and that the addition of immune serum leads to an increase in phagocytosis.44.1Immune modulation of malaria transmission can be assessed directly using mosquito feeding assays,The read‐out of the SMFA and serum‐replacement DMFA is generally reported as percent TRA. This is the percent inhibition of oocyst density  in test mosquitoes relative to the experimental control. For example, if mosquitoes that have fed on a blood meal containing test plasma results in a mean oocyst load of ten, and control mosquitoes have a oocyst load of 100, the percentage TRA would 90%. Transmission inhibition by immune factors is referred to as either transmission reducing (TR) immunity or transmission blocking (TB) immunity. Specifically, “blocking” should refer only to the total annulment of mosquito infection. Transmission enhancing (TE) immunity is less commonly reported than TR immunity, and though random variation of mosquito infection rate around the baseline (control) is likely to be the cause of many observations of low level TE, there is substantial evidence for immune mediated enhancement of immunity.4.2P. falciparum develops rapidly after malaria exposureP. falciparum TR immunity,P. falciparum gametocyte carriers. SMFA showed that TR immunity was present in 48% of sera, while TE immunity was present in 7%. The reproducibility of these results was variable, but significant numbers retained their TE and TR activity in repeat feeds. For P. falciparum, studies with serum‐replacement DMFA generally show increases in mosquito infection rates between 14% and 66% in the absence of host immune factors,In experiments with sera from Papua New Guineans performed by Graves et al, serum TRA in SMFA experiments varied from −124% TRA (TE immunity) to 99.4% (TR immunity). TE immunity was apparent in 10/41 sera (5/33 tested in duplicate).P. falciparum gametocyte antigens.P < .0001]).P = .001). TR immunity correlated with antibodies against these en others antigens (including α‐Pf11‐1 and α‐PfGEST) were also associated with reduced infectivity in field based DMFA. P. falciparum gametocyte immune responses may therefore contribute to the substantial variation in mosquito infectivity from natural gametocyte donors  were able to block P. vivax gametocyte transmission in the DMFA.P. vivax infection, Carter and Mendis reported complete suppression of mosquito infection by 22% of sera from Sri Lankans (n = 196), and enhancement of infectivity in 12% of sera.P. vivax infections either completely blocked transmission or showed varying levels of reduction (n = 41). Individuals with primary infections produced a similar proportion of enhancing and reducing effects in the serum‐replacement DMFA (n = 63).In 1987, Mendis et al showed that naturally acquired antibody‐mediated immune responses to the sexual stages of P. vivax sexual stage antibodies,P. falciparum. Various studies however have assessed P. vivax gamete recognition using immunofluorescence assays; MendisP. vivax‐infected (n = 105) and uninfected individuals (n = 44) from Colombia also examined antibody titer and TRA using serum‐replacement DMFAs. Among infected individuals, 44.6% had 50%‐89% TRA and 35.2% had ≥90% TRA. The correlation between anti‐gamete antibody titre and TRA was clear in exposed, currently uninfected, individuals; individuals with low titres tended to enhance transmission, and individuals with higher titres either had no effect or blocked transmission.Few studies assessed the presence of antigen‐specific 4.4Plasmodium transmission.P. cynomolgi infection in macaques, anti‐gamete antibody titer was shown to increase steadily from baseline, the peak level coinciding with a period of serum TR immunity.P. vivax anti‐gamete antibodies in P. vivax‐infected human blood led to enhanced oocyst infection in mosquitoes compared to controls; highly dilute immune serum and mAbs (which caused significant TRA at higher concentrations) promoted infection in experiments where gametocyte density was insufficient to cause infection without additional factors.P. vivax exposed individuals from Colombia, sera with varying levels of TRA were titrated to study the effect of dilution on transmission modulation.We recently reviewed the evidence for immune enhancement of P. vivax, enhancing antibodies have the same specificity as reducing antibodies. Carter and Mendis suggested that antibody‐mediated TE is the result of both low antibody titers and low inherent gametocyte infectivity (ie low gametocyte densities).P. cynomolgi,P. falciparum, the evidence for enhancement is less clear. Although multiple studies show low level immune‐mediated enhancement for P. falciparum,P. falciparum; mAbs against the central peptides of the D2 region of Pfs47  blocked transmission to mosquitoes, while mAbs against proteins at the regions N‐terminus gave rise to twice the number of midgut oocysts.These studies collectively promote the hypothesis that ingestion of low sexual stage antibody titers may lead to enhanced mosquito stage infection. The work of Peiris et al55.1P. falciparum RBC surface antigens results from the constant re‐organization and variable expression of var genes.var genes family is the most intensively studied, the genetic variation in non‐var genes is increasingly appreciated. This is largely due to recent sequencing efforts and the availability of sequence data in open access databases  has been tested in malaria naive US adults, and is the only Pfs25 construct tested in malaria‐exposed individuals. Four doses were required to induce higher functional transmission reducing antibody responses in a subset of vaccinated Malian adults compared to the control group and antibody levels waned rapidly.The post‐fertilisation antigen P25 is the most extensively studied TBV candidate to date with a variety of vaccine constructs evaluated in both pre‐clinical studies and clinical trials P. berghei parasites.The development of vaccines against P48/45 and P230 has been challenging due to their complex native conformation. Pfs48/45 is a 51.6 kDa protein and contains three domains with up to six conserved cysteines in each domainP. falciparum macrogametes in mice.The expression of Pfs230 has been even more challenging and has long lagged behind Pfs48/45. Pfs230 is over 300 kDa in size and contains 14 six cysteine motifs, which has hampered the expression of the full‐length protein  that induces functional antibody response.E. coli,Nicotiana benthamiana,Similar to Pfs48/45, many different expression systems have been used to overcome difficulties of expressing native epitopes of Pfs230. These include P. falciparum NF54 and two Thai patient isolates in the SMFA. Safety studies have been performed in US and Malian adults with this construct formulated in Alhydrogel®  and AS01 . The results of these trials have to date not been reported. Recruitment is ongoing for a phase 2 trial using the Pfs230D1M‐EPA/AS01 vaccine. In this trial, groups of healthy Malian children of decreasing age will be recruited .Currently, the most advanced Pfs230 construct in the clinical pipeline is the Pfs230D1M construct expressed in yeast. This construct comprises amino acids 542 to 736 and has been conjugated to EPA.While clinical trials are ongoing, more potent Pfs230‐based formulations are being developed, including the conjugation of Pfs230D1M to the outer membrane protein complex which induces higher TRA responses in mice than Pfs230D1M‐EPA.Plasmodium vivax TBV research has moved along the same path as that of P. falciparum, although the inability to culture P. vivax‐infected RBC in vitro (and thus gametocytes) has limited vaccine discovery and evaluation.P. vivax TBV candidate to date is Pvs25.Saccharomyces cerevisiae (Pvs25H)The best characterized E. coli with intact native epitopes as demonstrated by the reactivity of antibodies in sera from Colombian individuals.Pvs48/45 and the C‐terminal cysteine rich domain equivalent to Pfs48/45 6C. Antibodies against full‐length Pvs48/45 showed superior reactivity against protein lysates and reduced oocyst numbers. Whether antibodies against Pvs48/45 6C were able to reduce oocyst numbers has not been tested. Pvs47, orthologue of Pvs45/45, has also been tested in this study and antibodies induced after DNA immunization were also able to reduce oocyst number. Using a similar approach, rodent immunizations with a fragment of Pvs230 based on the Pfs230C region (excluding the pro‐domain) also demonstrated the induction of functional antibody responses.Full‐length Pvs48/45 has been recombinantly expressed in Several TBV candidates eg Pfs25,, 251 hav6The importance of naturally acquired immunity in malaria was demonstrated in the 1960s by the passive transfer of polyclonal antibodies from adults to infected children, reducing parasitemia and alleviating (severe) clinical disease.The first human mAbs against a sexual stage antigen were derived from transgenic mice, ie mice expressing human immunoglobulins, that were immunized with Pfs25. Characterization of these mAbs revealed sites associated with transmission blocking activity but also demonstrated that these mAbs have low potency.P. falciparum strains, a finding that can be confirmed using DMFA with genetically diverse strains in field settings. Interestingly, TB31F appears more stable than 85RF45.1, with higher aggregation and melting temperatures,P. vivax have been described. Crystallization of one of these in complex with Pvs25 revealed its binding site; however, the potency of this mAb has not been established.P. vivax target unknown antigens and/or have very limited information on potency.P. vivax transmission.Monoclonal antibody 63F2A2.2a is the most potent Pfs230 mAb to date. The most potent Pfs48/45 mAbs described to date target epitope I on the C‐terminal 6‐Cys domain.Isolation and characterization of human mAbs can also guide vaccine design, an approach that has been pioneered in the quest for a broadly neutralizing HIV vaccine.It is anticipated that many human mAbs against sexual stage targets will become available in the near future, either from individuals who are naturally exposed to gametocytes and exhibit high levels of TRA77.1TBVs and mAbs are recognized as tools with great promise for malaria elimination initiatives. As a consequence of the increased interest in elimination, the pipeline of candidate TBVs and mAbs with transmission blocking properties has expanded considerably in recent years,P. falciparum and not for P. vivax for which parasite culture is currently not possible. In addition, SMFA typically relies on a single gametocyte‐producing P. falciparum parasite line at unnaturally high gametocyte densities in combination with a single mosquito strain.P. falciparum TBV with published results from a field study to date (Pfs25‐EPA as described above) showed excellent results in in vitro assays but failed to induce substantial transmission blocking activity when serum of vaccinated volunteers from the United States was offered to mosquitoes with cultured gametocytes.Importantly, these in vitro assays are only available for P. falciparum TBVs Pfs48/45, Pfs230 and Pfs25P. vivax TBVs Pvs48/45, Pvs230 and Pvs47 as described above.One approach that allows preclinical samples, eg serum samples from rodents, in a real‐life context is to add these antibody samples to giRBCs of naturally infected individuals in the DMFA. These natural giRBC gametocyte donors can be infected with multiple gametocyte‐producing clones,8An early evaluation model has recently been developed that may allow a direct assessment of vaccine efficacy against gametocytes that are induced in the vaccinated donor.P. falciparum 3D7 parasites. Treatment of asexual parasites with a subcurative dose of piperaquine monotherapy allows the production of viable mature gametocytes whose infectivity can be assessed by DMFA or direct skin feeding experiments. Both mosquito biteP. falciparum in small groups of 10‐20 malaria naive volunteers per study arm, all infected with a single well‐characterized parasite line . A CHMI‐transmission model with similar efficiency in terms of induction of transmissible gametocytes has been developed for P. vivax . At present, CHMI‐transmission studies have only been conducted in malaria naive volunteers, with the advance of CHMI studies in naturally exposed populations,Controlled human malaria infection (CHMI) models provide powerful tools for early evaluation of malaria vaccine candidates and have been used predominantly for pre‐erythrocyticConventional phase II trials in adult volunteers in endemic settings may complement the clinical development pipeline for TBVs. In addition to immunogenicity and activity of serum samples of vacinnees in the SMFA, such studies can examine the transmissibility of possible naturally acquired infections by DMFA or direct skin feeding.8.1Plasmodium sporozoite rates in field‐caught mosquitoes are among the obvious secondary endpoints, and should provide insight into the mechanism of actions of TBV. As surrogate endpoints, it has been debated whether these outcome measures would allow for accelerated approval,Definitive evidence on the public health endpoints of TBVs will require larger studies that are complex due to the intended outcome of vaccination that aims to reduce incident infections by reducing the reproductive number (R0) of malaria and overall exposure to infective mosquitoes.9P. falciparum has been the primary focus of investigations to date, and our understanding of P. vivax gametocyte biology is limited. Several independent studies reported the presence of gametocyte antigens on the P. falciparum giRBC membrane surface. While data are in part conflictingP. vivax giRBC immunity exists remains unknown.Early malaria research was dominated by investigations of the asexual blood stage that is the cause of clinical disease and mortality. Following the release of the first global malaria elimination framework in 2007,P. vivax where very few studies have examined the occurrence of gametocyte immunity and TRA in endemic populations or whether the kinetics of TRA differs with respect to mosquito‐derived infection versus relapsing infection.While the existence of TRA is beyond dispute, and several key antigens are firmly established, the importance of TRA in determining natural transmission dynamics remains to be quantified.Compared to TRA, the potential importance of TE for the transmissibility of natural infections is very poorly understood. Understanding the dynamics of TRA and TE following natural exposure and following vaccination may be particularly important for malaria elimination initiatives. Some evidence suggests that malaria transmission efficiency may increase as the burden of malaria decreasesTBVs may play important roles in the malaria endgame, preventing secondary infections from the remaining pockets of transmission, preventing malaria outbreaks and protecting other interventions from escape mutants.


Mass screening and treatment (MST) aims to reduce malaria risk in communities by identifying and treating infected persons without regard to illness.A cluster-randomized trial evaluated malaria incidence with and without MST. Clusters were randomized to 3, 2, or no MST interventions: MST3, 6 clusters ; MST2, 5 clusters ; and MST0, 5 clusters . All clusters completed the study with 14 residents withdrawing. In a cohort of 324 schoolchildren  negative by microscopy at enrollment, we evaluated the incidence density of malaria during 3 months of MST and 3 months following. The MST intervention involved community-wide expert malaria microscopic screening and standard therapy with dihydroartemisinin-piperaquine and primaquine for glucose-6 phosphate dehydrogenase–normal subjects. All blood examinations included polymerase chain reaction assays, which did not guide on-site treatment.P. falciparum and P. vivax) infections. Microscopically subpatent, untreated infections accounted for 72% of those infected.The risk ratios for incidence density of microscopically patent malaria in MST3 or MST2 relative to that in MST0 clusters were 1.00  and 1.22 , respectively. Similar results were obtained with molecular analysis and species-specific (Two or 3 rounds of MST within 3 months did not impact the force of anopheline mosquito-borne infection in these communities. The high rate of untreated microscopically subpatent infections likely explains the observed poor impact.NCT01878357. This cluster-randomized trial demonstrated a negligible impact of mass microscopic screening in guiding treatment as a means to reduce malaria transmission at a mesoendemic site in Indonesia. Retrospective polymerase chain reaction analysis revealed that the majority of infections were microscopically subpatent. Plasmodium falciparum ) or mixed infections   were positive, either by microscopy (21 [3.9%]), PCR (391 [72.4%]), or both (128 [23.7%]). Only 27.6% (149/540) of malaria infections were treated in connection with MST activities. Subpatent microscopic infections (n = 391) consisted of 48  [0.8%]) .Drug administration during this study did not prompt withdrawal of any subject, and no serious AEs occurred. The most common AEs during treatment were fever (0.023/person-day), headache (0.008/person-day), vomiting (0.006/person-day), cough (0.004/person-day), shivering (0.003/person-day), and nasal congestion (0.002/person-day).P. vivax, appear to have sustained transmission during the 3 months of intervention and the following 3 months. Thus, despite far lower transmission intensity, expert microscopy, and the use of primaquine as a transmission-blocking gametocytocide and hypnozoitocide, this study demonstrated similar results in the African setting of intense P. falciparum transmission diagnosed by RDT and treated with artemisinin-based combination therapy alone [This study demonstrated that MST may have little or no impact on malaria transmission in endemic communities where the majority of malaria infections cannot be detected by standard point-of-care diagnostics. Unrestricted malaria infection occurred with 2 or 3 rounds of MST, despite screening coverage >80%—higher than that typically attained in practice. Dominant subpatent and asymptomatic reservoirs, along with the latent hypnozoite reservoir of py alone . Our attP. falciparum prevalence was observed at the last round of MST by both microscopy and PCR diagnosis. This may be attributed to the greater efficiency of diagnosis of P. falciparum relative to P. vivax . In the cohort of schoolchildren, 10.5% (9/86) had recurrences within 5 months. All subjects received 0.25 mg/kg per national treatment guidelines; this relatively low dose may be inadequate [P. vivax, likely due to the inability to diagnose and treat the majority of subpatent infections. The latent reservoir of vivax malaria was not significantly impacted by MST that included a hypnozoitocide.In adequate . The sigThere are several important limitations to this study. Presumptive radical cure of the schoolchildren cohorts—to eliminate subpatent malaria infections—did not occur. These preinfections, rather than biting infectious mosquitoes, may account for some of the new infections detected and counted. Nonetheless, these were assumed to be balanced among the randomized arms, and therefore minimally impacting the MST effectiveness endpoints of RR. Another weakness may be the relatively small number of clusters per MST intervention (5 or 6), thus limiting the statistical power (<80%) to detect relatively small differences. Subtle but real differences are unimportant in the context of practice, where impacts must be relatively large for justifying costly and labor-intensive MST operations.Additionally, the enrollment targets based on detecting a 50% reduction were not met. The RR near unity pointed to little difference of impact between arms, and the proportionate CI supported this finding . Thus, tIn summary, 2 or 3 rounds of MST had no impact on the force of malaria infections in these communities. Effective MST requires improved sensitivity in point-of-care diagnostics . Both hiClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary MaterialsClick here for additional data file.


Plasmodium falciparum infection of the red blood cells (RBCs), the parasite replicates and consumes haemoglobin resulting in the release of free heme which is rapidly converted to hemozoin crystallites. The bulk magnetic susceptibility of infected RBCs (iRBCs) is changed due to ferric (Fe3+) paramagnetic state in hemozoin crystallites which induce a measurable change in spin-spin relaxation (transverse relaxation) rate in proton nuclear magnetic resonance (NMR) of iRBCs. Earlier, our group reported that this transverse relaxation rate (R2) can be measured by an inexpensive, portable 0.5 Tesla bench top magnetic resonance relaxometry (MRR) system with minimum sample preparation and is able to detect very low levels of parasitemia in both blood cultures as well as animal models. However, it was challenging to diagnose malaria in human blood using MRR, mainly due to the inherent variation of R2 values of clinical blood samples, caused by many physiological and genotypic differences not related to the parasite infection. To resolve the problem of baseline R2 rates, we have developed an improved lysis protocol for removing confounding molecular and cellular background for MRR detection. With this new protocol and by processing larger volume of blood (>1 ml), we are able to reliably detect very low level of parasitemia  with a stable baseline and improved sensitivity using the current MRR system.Upon  Malaria is caused by Plasmodium species parasite like Plasmodium falciparum, P. vivax, P. malaria, P. ovale3 and P. knowlesi4. Particularly in sub-Saharan Africa, P. falciparum causes high mortality rates in children under the age of 56. Accurate diagnosis of a malaria parasite infection along with rapid treatment significantly reduces the risk of developing severe disease7. In addition, ultrasensitive detection of asymptomatic carriers of the parasite is a critical tool for the control and elimination of the parasite from a particular country or region9.In spite of the significant progress in the elimination of malaria, it still remains as a major mosquito-borne infectious disease burden in the world, especially in Africa10 with Giemsa stained blood smear microscopy being the gold standard for malaria diagnosis11. Using thick smear, this approach is able to detect levels as low as 0.001% of parasitemia (50–100 parasites/µl of blood)12. The reliability of this approach depends on the quality of the microscopist and can be prone to human error13. Alternative diagnostic approaches based on protein biomarkers (dipstick), which are easier to use and able to detect 100–1000 parasites per micro liter14, often suffer from relatively high rate of false positive results16. Polymerase chain reaction (PCR) based detection is the most sensitive malaria diagnosis method, but requires sophisticated laboratory equipment/reagents/skilled labor, making it less suitable for rapid detection in resource poor settings18.Currently, diagnosis of malaria exclusively focuses on the parasite blood stage19. This is based on the increased hemozoin crystallites being formed in the erythrocyte as the parasites develops from ring, trophozoite to schizont stage  relative to the R2 rates of healthy RBCs . So far, such highly sensitive detection was made possible by comparing the R2 rate of iRBCs with that of healthy RBCs from the same source of blood samples1, limiting its application to clinical diagnostic settings. The main reason for this is that the R2 value of healthy RBCs varies significantly depending on the metabolic conditions of each person, resulting in the possibility of false positive diagnosis due to the presence of different paramagnetic Hb states (e.g. presence of met-Hb). To overcome this challenge and thereby improve the quality of MRR based diagnosis of malaria in clinical settings, it is necessary to overcome the variations in the baseline R2 value that are not directly attributable to parasite infection and growth.Recently, label free and rapid diagnosis of malaria was possible by using portable benchtop micro magnetic resonance relaxometry (MRR) Fig. 1,19. Thiage Fig. . During BCs Fig. 1,19. TheP. falciparum parasites from the iRBCs using saponin lysis, followed by MRR analysis of this enriched parasites for more reliable and sensitive detection. The MRR measurements of saponin lysed healthy RBCs show well-defined and stable baseline R2 value, regardless of sample’s metabolic or storage conditions. We have applied two types of sample preparation methods for the measurements of R2 using micro MRR that allow rapid and reliable micro MRR detection of iRBCs in as little as 30 minutes. With this new approach, we are able to detect ring stage P. falciparum iRBCs at 0.001% parasitemia from RBCs equivalent of 100 µl of untreated blood, or 0.0001% in 1 ml of RBCs equivalent of untreated blood. Reliable detection of parasitemia can also be achieved in cryopreserved samples as well as in whole blood containing all the blood cells, with well-defined, universal baseline R2 levels. This data shows that micro MRR can be used as an efficient detection and surveillance method for malaria infection in human, in low resource clinical settings.In this work, we report a simple and practical solution to this baseline challenge by the enrichment of Plasmodium falciparum parasites were purified using a Percoll gradient centrifugation to remove uninfected RBCs and obtain a relatively pure schizont stage fraction (approximately 90% schizont stage). The schizont fraction was then diluted with uninfected RBCs (uRBCs) to generate a final parasitemia of 10%. This 10% parasitemia stock solution was subsequently further diluted with uRBCs to create final parasitemia concentrations between 0.0001–10%. Three different approaches to detect a parasite specific signal using the micro MRR  were lysed using 0.15% of saponin (Methods) and parasite pellets were collected after repeated washing23. Saponin permeablizes the RBC membranes while leaving the malaria parasite membrane intact. By repeated washing after saponin lysis, we can collect a pure parasite pellet. This purified parasite pellet was then processed using two different procedures. For the first procedure  most likely can be attributed to different metabolic conditions and different uncontrolled Hb states present in the different donors. Saponin lysis and subsequent washing of the samples to obtain purified parasites is a possibility to reduce or remove donor derived fluctuations. As can be seen both the spinning as well as the suspension method  in the R2 value between uRBCs and iRBCs even at the lowest parasitemia levels of 0.0001% used here indicating that the MRR assay coupled to either lysis protocols has the sensitivity needed for very early diagnosis of even asymptomatic parasite carriers.To compare the differences in signal obtained from 1 ml of synchronized rings or schizonts we prepared stock solutions with a parasitemia of 0.1%, 0.01%, 0.001%, and 0.0001%. A side by side comparison of both the spinning and the suspension Fig.  methods 2 value observed we prepared washed ring stage parasites with parasitemia of 0.1%, 0.01%. 0.001% and 0.0001% as well as uRBCs as above. The pellets were collected after repeated washings and stored in −140 °C at liquid nitrogen for two weeks. These cryogenically stored lysed samples were analysed by MRR and compared to freshly prepared ring stage parasite samples of similar parasitemias  were lysed by saponin and the pellet was collected after washing. The R2 values of the different samples were determined by MRR indicating that there is no significant difference in limit of detection of malaria for the samples diluted with purified uRBCs (100 µl) or whole fresh blood (200 µl)  Fig. . As stat2, it is also important to monitor the efficacy of drug treatment as a function of time, by measuring the active parasite burden directly, repeatedly, rapidly, and preferably at a low cost. Current malaria diagnostic options, including Giemsa microscopy, protein marker based malaria rapid diagnostic tests (RDTs), as well as PCR based detection, fall short of meeting all of the above diagnostic requirements for the future global malaria eradication efforts10. MRR based detection of malaria parasite described in this paper provides a potential viable tool to support the malaria eradication effort as it is rapid, sensitive, and cost effective while having the additional benefit of being suitable for automation. In addition, the R2 value obtained by MRR can be regarded as an alternative quantitative metric replacing parasitemia, which can be used for time-dependent monitoring of the live, active parasite burden for managing drug-resistant malaria19. Yet, in order to realize the full potential of MRR, it is necessary to overcome host specific variations in blood chemistry, variations in blood collection and storage in field clinics and other factors that could directly impact on the resulting R2 measurements independent of the parasite burden. Here we report a simple strategy that allows us to eliminate most of the non-parasite derived factors that would contribute to variations of the R2 measurements. This approach allowed the reliable detection of live parasites independently of host factors and is suitable to evaluate even frozen samples.The reliable diagnosis of malaria at the earliest stage of infection is an essential tool for the reliable treatment of infected individuals. The detection of asymptomatic carriers with very low levels of parasitemia is critical for the global effort to eliminate malaria. In addition, with the recent rise in drug-resistant parasites in certain parts of the world23. Combining this with a rigorous washing step allowed the removal of most of the host cell contents thereby removing potential confounding factors like cell-free Hb, RBC ghosts, white blood cells, etc., leaving only the parasite pellet behind  making it appropriate for malaria endemic countries. However, the current MRR system is still expensive and its electronics and polarizing magnets are the major cost drivers of this MRR instrument. However, the recent developments of on-chip NMR systems26 using inexpensive magnets and electronics will significantly reduce the cost of the detection equipment needed to below $500.The relatively large volume (100 µl/1 ml) of blood needed for the MRR diagnostics of malaria is a potential drawback as both Giemsa staining and RDT require a much smaller volume of blood. However, this is offset by the significantly higher sensitivity and range of detection, with Giemsa staining becoming unreliable when the parasitemia number is below 0.1%In conclusion, the results obtained in this study significantly strengthen the applicability of MRR as an efficient diagnostic method for malaria in clinical settings. Using the current enrichment method, sample preparation and use of MRR for diagnosis, more effective surveillance of this disease in malaria endemic regions will be possible.P. falciparum 3D7 parasites  were grown using human RBCs in RPMI 1640 (Life technologies) supplemented with 0.25% AlbuMAX II , 2.3 g/L sodium bicarbonate , and 0.1 mM of hypoxanthine  10 mg/L of gentamicin . The culture was maintained at 2.5% haematocrit at 37 °C, 5% CO2, 3% O2 and 92% N2.Blood stage 27, while schizont stage parasites were purified using 68% Percoll centrifugation as described29. Parasitemia was determined by standard Giemsa stain microscopy. For preparation of different parasitemia samples, iRBCs were spiked into uRBCs to obtain different final parasitemia. Fridge stored blood and freshly drawn blood were used based on different experiment design.Synchronized early ring-stage parasites were obtained using 5% D-sorbitol23. The sample was kept on ice for 15 minutes and vortexed every few minutes. The sample was then washed with PBS thrice and centrifuged at 5000 g for 5 min. After washing, sample pellet was collected and filled into micro capillary tube for MRR experiments. For experiments with cryogenically stored samples, RBCs were lysed with 0.15% saponin and washed as described above. The processed sample was then stored in −140 °C at liquid nitrogen for up to two weeks. Before MRR experiments, all the samples were thawed and brought back to room temperature.For enrichment of parasites from blood, 0.15% saponin was added to the RBCs in the volume ratio of 3 to 1For preparation of unlysed samples, the spiked iRBCs/uRBCs mixture was collected and adjusted to 10% haematocrit. The cell suspension was then filled into the micro capillary tube  for 4 cm length and sealed with an inert seal . The micro capillary tube was centrifuged at 3000 g, 3 min in a micro centrifuge  to make a 4 mm band of compact iRBCs/uRBCs which is in the detection range of the micro coil Fig. .2 value was calculated.For the preparation of saponin lysed samples, lysed pellet was diluted in 20 µl of PBS and filled in to micro capillary tube. For spinning method sample preparation, the micro capillary tube was sealed and centrifuged at 5000 g for 5 min to concentrate the saponin lysed pellet in to the 4 mm detection range of the micro coil Fig. . For susB0 = 0.5 T and a bench-top type NMR console . 1H MRR measurements were performed at the resonance frequency of 21.65 MHz inside the magnet1. A single resonance proton MRR probe with detection micro coil of 900-μm inner diameter was used for accommodating the MRR samples in to the micro capillary tubes  . In MRR probe, the electronic parts and coil were mounted on the single printed circuit board .MRR consists of a portable permanent magnet  with ard Fig. 1. All th2 were measured by standard Carr-Purcell-Meiboom-Gill (CPMG) pulse programme . Blood component collection service was provided by Blood Transfusion Service and Blood Donation Centre of National University Hospital. All individuals gave informed consent.Plasmodium falciparum in human bloodEnhancing the sensitivity of micro magnetic resonance relaxometry detection of low parasitemia


African populations are characterized by high degree of genetic diversity. This high genetic diversity could result from the natural selection pressure. Several studies have described an association between some genetic diversities and difference of susceptibility to infectious diseases like malaria. It seems therefore important to consider genetic diversity impact when interpreting results of clinical trials in malaria endemic areas. This study aimed to determine the genetic polymorphism with erythrocyte traits in different populations of malaria endemic area in Mali. The cross-sectional surveys were carried out in different ethnic groups living in malaria endemic areas in Mali. Six milliliters of whole blood were collected in EDTA vials from each participant after informed consent has been obtained. The ABO, RH, Kell, MNSs, Kidd and Duffy systems phenotypes were assessed by the technique of gel filtration. A total of 231 subjects were included from six villages. The blood groups phenotypes O (40.7%) and A (31.2%) were more frequent with respective allele frequencies of 0.65 and 0.21. In the RH system the haplotypes R0 (0.55), r (0.20) and R1 (0.13) were the most frequent. Seven percent (7%) of Duffy positive and 4% of Glycophorin B deficiency (S-s-) were observed among participants. All participants were Kell negative. ABO and RH systems were polymorphic in these ethnic groups in Mali. Their implication in susceptibility to malaria should be taken into account in clinical trials interpretation, and for prevention of blood transfusion risks during anemia frequently caused by malaria in children. The ABO and the other blood group systems remain of prime importance in transfusion medicine. These blood group antigens are mainly expressed on erythrocytes. They are the most immunogenic and the most common cause of life threatening from a blood transfusion in which an incompatible type of ABO blood is transfused. They are polymorphic and are associated to susceptibility/protection against certain infectious disease such as malaria.Africa is a continent where higher population genetic diversity and the role of evolutionary processes that contributed to this diversity has been described ,2. It isThe Sahel-Saharan regions have a strong cultural diversity, with contrasting climatic areas in terms of rainfall, defining different environments and lifestyles. The populations living in these environments are constantly affected by numerous infectious diseases, leading to a strong pressure of natural selection ,2. This Mali is a country with different eco-climatic areas and an important ethnic diversity. Some genetic diversities associated with differences in susceptibility to malaria have already been described in sympatric Malian populations ,5,6,7,8Many clinical trials including therapeutic and vaccine studies are being conducted in such areas and blood transfusion is common due to severe malaria and sickle cell disease (reference). Studying the genetic structure of these populations (including erythrocyte systems) is crucial in interpreting the finding of clinical trials. In addition, the knowledge of genetic polymorphism map across the countries could have advantages in the prevention of risks related to blood transfusion in countries with limited resources where the technical platform cannot efficiently determine some erythrocyte phenotypes.This study aimed to assess the genetic polymorphism with erythrocyte traits in populations living in different environmental and sociocultural conditions in Mali. We found that the ABO and RH systems were polymorphic in different ethnic groups in Mali. Therefore, their implication in susceptibility to malaria should be considered in clinical trials interpretation, and for prevention of blood transfusion risks during anemia frequently caused by malaria in children.The study was carried out in six villages with different eco-climatic and sociocultural conditions in the northeast of Mali. These six villages are located in three districts  in the region of Mopti .Bandiagara: a city located at 700 km from the capital city in the northeast of Mali has 15,000 inhabitants. The population consists mostly of Dogon (64%) who live with Fulani and Tukulor (12%), and other ethnic groups. They practice ethnic exogamy and the caste endogamy. The languages are Niger-Congo-Atlantic, Niger-Congo-Mande and Afro-Asiatic-Berber.Manteourou: a village with 1156 inhabitants (according to the census conducted in 2011) is divided into two districts  separated by approximately one kilometer.Madougou: with 3128 inhabitants, is located at 12 kilometers from Manteourou and these two villages have the same configuration and characteristics (Madougou Dogon district and Fulani district).Binedama: is a Fulani’s village with only 510 inhabitants (2011 census).In these villages, the two ethnic groups (Dogon and Fulani) live in sympatry and practice ethnic endogamy. The language is Niger-Congo-Atlantic.Petaka and N'Gono are located at around 150 km in the northeast from previous villages, in the district of Douentzan. Dogon constitutes the predominant ethnic group of the population. Family is mainly based on ethnic endogamy, patriarchal society, and the language is Niger-Congo-Atlantic.The structure of these communities is patrilineal and patriarchal societies.Data were collected in cross-sectional surveys from July to September 2011. A total of 231 participants from permanent resident populations were included in our study. Participants were included after they signed consent forms. As main inclusion criteria participant should be at least 3 years old, with no clinically chronic disease, no immune deficiency or history of blood transfusion in the last three months. A blood sample (6 ml in EDTA tube) was collected from each participant by venipuncture. Interviewing participants, we also collected the socio demographic data of each participant.Determination of blood group phenotypes: the manual technique of gel filtration  was used for phenotyping of the blood groups systems according to the standardized operating procedure. Briefly, after opening filtration support, we added successively in each well one drop of saline solution, one drop of each reagent  and one drop of diluted blood with saline. A centrifugation was done for 5 minutes (Ortho BioVue System centrifuge), followed by 20 minutes incubation at 37°C, and then centrifugation for 5 minutes. For ABO system, diluted blood was directly added into the well of filtration support, which already contained the immunoglobulin anti-A and anti-B (+ anti-AB). The result was positive if there was agglutination or negative if there was no agglutination.All data were entered and analyzed in SPSS software version 16. The phenotype frequencies were calculated for all study participants while allele frequencies were determined only in unrelated participants.For ABO, Kidd, Duffy, MNSs and Kell systems, the Bernstein formula was used to estimate the frequencies of the alleles, with the assumption that the study population was in Hardy-Weinberg equilibrium.The algorithm EM (Expectation-Maximization) based on the maximum likelihood principle, and GENE [RATE] program were used to determine the genotypes of RH system.The study protocol was approved by the ethics committee ‘‘Comité d’étique de la faculté de médecine et d’ondontostomatologie” of Mali by the letter No. 2011-59-/FMPOS prior the study. Each participant signed two copies of informed consent form. The illiterate participants unable to sign used their fingerprint and were assisted by literate witness. All the study procedures were discussed in the participant’s desirable language in the presence of a witness. Parents or legal guardians signed the assent forms for the minors after having their consent. Each participant was given a signed copy of informed consent. To warrant the confidentiality, each participant was assigned a code, which has been used instead of the name on samples.Overall 231 participants, composed in majority of Dogon  and Fulani  were included. Other ethnic groups (Tamashek and Bambara) constituted 3.9% of the sample. The sex ratio was 1.22  and the median age was 38 years with respectively 3 and 88 years as minimal and maximal age. Binedama and Ngono villages had the lowest participant’s number with respectively 5.2 and 8.6% of participants . PopulatR0 (0.55) was the most frequent, followed by r (0.20) and R1 (0.13) , followed by A (31.2%), B (21.6%) and AB (6.5%) . The all1 (0.13) . Most of1 (0.13) . The alland FY*B . In the and FY*B , with reand JK*B . Phenotyand JK*B . All parThe study involved populations from six villages with different eco-climatic conditions and lifestyles. The lower numbers of participants from Binedama and N’gono are due to small total population size of these villages. Most of the study sites were Dogon and/or Fulani villages, leading to a high representation of these two ethnic groups in our sample. Except children under 3 years old, all the age groups (3–88 years) were represented in our sample, allowing the determination of erythrocytes polymorphisms distribution at all ages. Children less than 3 years old were excluded from the study to avoid confounder factors related to blood group systems immaturity.High frequencies of blood group O phenotypes in Sub-Saharan Africa are already reported in literature ,10. In MThe highest frequencies of O phenotype were observed in Fulani ethnic group with 48.6% . ReducedR0 had the higher frequencies in our study population. Previous studies have described the high frequencies of R0 in Sub-Saharan populations [R0. Carriers of this allele HbS have enhanced expression of the antigens D, c et e, leading to the high frequencies of haplotype R0 in high sickle cell prevalence area [The haplotype ulations –16. Thisulations ,16. The ulations ,18, may nce area ,15. Dogonce area . All theR1 was described in this study, compared to previous data published [Higher frequency of haplotype ublished . This haublished –22.FY*O allele (0.99) in our sample. However, 6.5% of our participants were Duffy positive. Menard et al. described 8.8% of asymptomatic Plasmodium vivax infection in Duffy negative people of Madagascar where 28% of the population are Duffy positive [P. vivax infection have been described [P. vivax. P. vivax may also develop alternative mechanism to bypass the obstacle due to Duffy binding protein deficit to infect erythrocyte. Investigating this new mechanism used by Plasmodium vivax to infect Duffy blood negative erythrocytes is crucial for malaria control and elimination. Malaria control programs should incorporate more sensitive tools for the diagnosis and surveillance of P. vivax in malaria endemic areas.As already described in black populations ,9,16,18,positive . Recentlescribed . The lowThe phenotype S-s+ (74%) was the most frequent in our study participants. Higher frequency of Glycophorin B deficiency (S-s-) was observed in Fulani (7.4%), compared to their neighbor Dogon (2.6%) . The GlyPolymorphisms among the different populations were mainly observed in the ABO and RH systems. The ethnicity and the environment of residence were the factors involved in polymorphism distribution; highlighting the role of environmental natural selection in the genetic diversity. The study of erythrocytes polymorphisms is a significant contribution in understanding the differences in susceptibility to malaria. We have described in Fulani ethnic group higher frequencies of O blood phenotype and glycophorines B deficiency known as genetic factors conferring natural protection against malaria. The natural selection pressure can involve several genetic markers . The natural resistance to disease could be the sum of polymorphisms of several genetic markers. Since some polymorphisms in erythrocyte systems have been described to be associated with susceptibility or resistance to diseases, their distribution and their impact should be considered in epidemiological studies of malaria. For an accurate interpretation of clinical trials data for efficacy of drugs or vaccine in malaria endemic areas, any ethnic group difference in the distribution of erythrocytes phenotypes should be considered. The knowledge of erythrocytes polymorphism countrywide could also contribute to reduce risks related to blood transfusion.S1 File(SAV)Click here for additional data file.