Plasmodium falciparum merozoite antigens and antibody-mediated effector functions were associated with reduced odds of developing severe malaria at different time points during the first year of life. We conducted a case-control study of well-defined severe falciparum malaria nested within a longitudinal birth cohort of Kenyan children. We measured cord blood total IgG levels against five recombinant merozoite antigens and antibody function in the growth inhibition activity and neutrophil antibody-dependent respiratory burst assays. We also assessed the decay of maternal antibodies during the first 6 months of life. The mean antibody half-life range was 2.51 months : 2.19–2.92) to 4.91 months (95% CI: 4.47–6.07). The rate of decline of maternal antibodies was inversely proportional to the starting concentration. The functional assay of antibody-dependent respiratory burst activity predicted significantly reduced odds of developing severe malaria during the first 6 months of life (Odds ratio (OR) 0.07, 95% CI: 0.007–0.74, P = 0.007). Identification of the targets of antibodies mediating antibody-dependent respiratory burst activity could contribute to the development of malaria vaccines that protect against severe episodes of malaria in early infancy.Young infants are less susceptible to severe episodes of malaria but the targets and mechanisms of protection are not clear. Cord blood antibodies may play an important role in mediating protection but many studies have examined their association with the outcome of infection or non-severe malaria. Here, we investigated whether cord blood IgG to  We inve22.1n = 5,949) set up between 2001 and 2008 to study the risk factors of invasive pneumococcal disease in young children. As the study was primarily set up to study pneumococcal disease, malaria-specific indices such as intermittent preventive treatment during pregnancy (iPTp) and bed net usage were not recorded. The children were followed up quarterly at the Outpatient Department of KCH until 2 years of age. During the quarterly visits, a blood sample was collected and thick and thin blood smears prepared for detection of parasites by microscopy. In the event of an illness outside the scheduled 3-monthly visits, parents were advised to seek care at KCH and the children were treated according to national guidelines. Children who were admitted to hospital were identified using a unique number that linked their clinical, demographic and laboratory information.The study was conducted in Kilifi County, on the Kenyan coast. The area experiences two seasonal peaks in malaria transmission (May to August and October to November). The study setting and study population are described in detail elsewhere . Briefly2.1.1n = 130). Following informed consent, baseline information  and a cord blood sample were obtained  impaired consciousness (Blantyre Coma Score <5), (ii) chest indrawing or deep breathing or (iii) severe anemia (Hb <5 g/dL).2.1.3msp2) gene followed by capillary electrophoresis  was expressed as a Histidine (His)-tagged protein in Pichia pastoris  was expressed as a glutathione S-transferase (GST)-fusion protein in Escherichia coli (E. coli (19) (Wellcome) and a fragment of P. falciparum reticulocyte-binding homolog 2 (PfRh2 (3D7)) were expressed as GST- 1  were added to individual wells, followed by the dialyzed plasma at a ratio of 1:10. The plates were incubated in a humidified chamber containing 5% CO2, 5% 02 and 90% N2 for 80 h. Ten microliters of culture medium were added to each well after the first growth cycle. A positive and negative control well containing 10 mg/ml of purified Malaria Immune Globulin (MIG) and a pool of plasma from UK adults, respectively, were included. After two cycles, the parasites were stained with 10 μg/ml of ethidium bromide for 30 min, washed with 1 X PBS and acquired on an FC500  flow cytometer.Cord plasma were dialyzed in 1 X PBS using 20 kDa MWCO mini dialysis units and incubated at 56 °C for 30 min to inactivate complement proteins. Highly synchronous trophozoite stage parasites from the 3D7 2.4.2Plasmodium falciparum 3D7 parasite cultures were maintained at <10% parasitemia and 2% hematocrit. Highly synchronous mature trophozoite stages were enriched by magnetic separation and allowed to mature into early schizont stages. Thereafter, a protease inhibitor (trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64)) was added to allow development into late schizonts without rupture. The schizonts were pelleted, resuspended in 1 X PBS and stored at −20 °C.2.4.3g for 15 min at room temperature (RT). The pellet containing PMNs and red blood cells (RBCs) was resuspended in 3% dextran solution and incubated for 30 min at RT in the dark. Thereafter, the supernatant was carefully removed and centrifuged at 500g for 7 min. Residual RBCs were lysed using ice cold 0.2% NaCl followed by 1.6% NaCl. PMNs were washed in PMN buffer  supplemented with 0.1% BSA and 1% D-(+)-glucose) and resuspended in PMN buffer at a concentration of 1x107 PMNs/ml.Whole blood samples from healthy donors were collected into heparin tubes, layered onto Ficoll-Histopaque 1077  and centrifuged at 6002.4.45/ml. Following three washes with 1 X PBS, the plates were blocked and incubated with plasma (1:50) for 1 h at 37 °C. The plates were washed and 50 μl of PMNs (1 × 107 PMNs/ml) added, followed by 50 μl of isoluminol (0.04 mg/ml). Chemiluminescence was measured for 1 s every 2 min over an hour. Control wells containing a pool of UK adult plasma and a pool of plasma from Kilifi adults were included in each plate. Readings were expressed relative to those obtained from the pool of plasma from Kilifi adults.The ADRB assay was performed as previously described . Briefly2.510-transformed values of the antibody titres collected at ages 0, 3 and 6 months. To assess the natural decay of maternal IgG in the absence of boosting due to infection, we excluded children that had antibody titres boosted at 3 months of age compared with baseline titres as well as those with boosted titres at 6 months of age compared with titres at 3 months of age. Half-life estimates were calculated using the equation:T1/2 is the estimated half-life and m is the slope component of the mixed-effects model  and GraphPad prism Version 6 . A linear regression model that included an indicator of the case-control status was used to determine the influence of maternal age, parity, birth weight, year of birth, season of birth and gestation period on the levels of specific antibodies and antibody function. The Benjamini-Hochberg method was used to adjust for the false discovery rate. The rate of antibody decay was determined using a longitudinal mixed-effects model . The modts model . A condits model . P < 0.033.1Of the 222 children recruited to the matched case-control study of severe malaria described previously , 130/222P. falciparum infections as measured by PCR or microscopy in the quarterly samples collected up to 2 years of age. Of these, 11/32 (34%) were identified among the children who subsequently developed severe malaria and 16/98 (16%) among the controls.Twenty-seven out of 130 (20.0%) acquired asymptomatic 3.2n = 32) and controls (n = 98) for all antigens. Among the cases, the seroprevalence against schizont lysate, AMA1(3D7), MSP-2(Dd2), MSP-3(3D7), MSP-119 and PfRh2 was 98%, 93%, 87% 69%, 46% and 33%, respectively, compared with 100%, 93%, 90%, 68%, 43% and 37% among the controls , P = 0.004, respectively) (P = 0.02). Surprisingly, we observed an increase in GIA and ADRB levels over the duration of recruitment (2002–2006) (We used a linear regression model to determine which factors were positively or negatively correlated with increasing antibody levels and function in cord blood plasma . Maternactively)  and (Supctively)  but not ctively) . However02–2006) , a perio02–2006) . Other f02–2006) .3.410 transformed antibody titres for both cases and controls aged less than 6 months. All the index samples were collected prior to the severe malaria episode. To assess the decay of antibodies in the absence of boosting of P. falciparum infection, we excluded results from five cases and five controls who had detectable parasites at either month 3 or 6. In addition, all samples from individuals whose antibody titres at 3 months of age were higher than cord titres were excluded and for those whose titres at 6 months were higher than month 3, only the result for the 6-monthly sample was excluded. A total of 56 (43%) samples (12 cases and 44 controls) were excluded in the analysis. The mean half-life range was 2.51 months : 2.19–2.92) to 4.91 months (95% CI: 4.47–6.07)  and 4.91 months   . There wctively) . The ratctively) .10 AU reduction per month in the 1st, 2nd, 3rd and 4th cord quartiles, respectively , as it was highly unlikely that cord blood antibodies were responsible for protection. Thus, we analysed samples from a total of 16 severe cases admitted to hospital during the first 12 months of life. These were individually matched to four controls per case based on date of birth. Three cases were born a few days apart and were therefore matched to a similar set of four controls.We evaluated the relationship between cord antibody titres and their functional activity (GIA and ADRB) with the odds of developing severe malaria at different time points after birth  . AlthougSeropositivity for antibody titres and ADRB were defined as a cut-off above the mean + 3 S.D. of 20 European plasma samples whereas the GIA cut-off for positivity was defined as being above the median GIA level of the cord plasma samples . Of the PfRh2  and MSP-3 and PfRh2  among the controls but not cases  but not the cases  (P < 0.009) but not GIA activity  with increase in the breadth of merozoite-specific cord blood antibodies (seropositivity against the number of antigens tested) (P = 0.007) and this association remained strong and significant until 9 months of age  but was lost thereafter  . There w tested) . Antibod tested) A. Childrereafter B. In conereafter B. We havereafter . Here, aereafter B.4We designed a case control study of severe malaria nested within a longitudinally monitored birth cohort. This design provided a unique opportunity to determine whether antibodies present in cord blood were associated with a reduced risk of severe malaria during infancy. We found that cord blood IgG in the assay of ADRB activity was significantly associated with lower odds of developing severe malaria. This association was only observed for severe malaria cases occurring within 9 months of birth and fits expectations of the half-life of passively transferred maternal antibodies . Cord blThe ADRB assay has been assessed in a limited number of studies  and has Our study differs from previously published reports that investigated the protective role of maternal antibodies against the outcomes of infection and/or clinical or febrile malaria (reviewed in A key and interesting finding in our study was that antibody decay rates are inversely proportional to the initial titres present in cord blood, a finding that is in contrast to a study by We speculate that the rate of decay of maternal antibodies may have been influenced by the presence of sub-patent infections. Moderate to high maternal antibody titres may have a masking effect on infections in infants and this could contribute to the rapid decay of circulating antibodies. On the other hand, infections in the presence of low titre maternal antibodies could result in seroconversion and persistence of antibody titres . DetectiThe distribution of different IgG subtypes could also affect the rate of antibody decline. For instance, IgG3 is more rapidly degraded than other isotypes . In otheWe found that anti-merozoite antibodies in cord blood were not associated with parity, maternal age, birth weight or gestational age. In contrast, other studies demonstrated a positive relationship between parity and placental malaria infection  with incIn summary, we found that cord blood IgG activity in the ADRB assay was strongly associated with lower odds of developing severe malaria in the first 9 months of life. Although larger studies are needed, these data suggest that ADRB could be useful for the identification of targets of protective antibodies that could be translated to the clinic as candidate vaccines for infants and young children who are most susceptible to death due to severe malaria.


Rapid diagnostic tests (RDTs) are alternative methods for microscopy in the diagnosis of malaria in resource limited settings. Among commercially available RDTs, CareStart™ Malaria test was found to show reliable results. This study evaluated the performance of CareStart™ Malaria Combo test kit in Northwestern Tigray in Ethiopia.Blood samples were collected from 320 malaria-suspected patients at Mayani Hospital in Northwestern Tigray from December 2015 to March 2016. All blood samples were examined using both light microscopy and CareStart™ Malaria HRP2/pLDH Combo Test kit. Statistical analyses were performed using SPSS version 20.Plasmodium falciparum or mixed infection and non-falciparum malaria parasites was 94.4 and 85.0%, respectively while the specificity was found to be 98.9 and 99.7%, respectively. The agreement between the two test methods was “excellent” with a kappa value of 0.92.The overall parasite positivity using light microscopy and CareStart™ RDT was 41 (12.8%) and 43 (13.4%), respectively. The sensitivity and specificity of CareStart™ RDT, regardless of species, were found to be 95.4 and 99.3%, respectively. Furthermore, the sensitivity of CareStart™ RDT for CareStart™ RDT has very good sensitivity and specificity for malaria diagnosis. The test kit also has an excellent agreement with light microscopy. It is therefore useful in resource-limited areas where microscopy is not available. Plasmodium falciparum and Plasmodium vivax are the dominant Plasmodium species with 60–70% and 30–40% reported cases, respectively [With an estimated 214 million cases worldwide, and 438, 000 deaths annually, malaria still causes high morbidity and mortality . It is rectively , 10. Unaectively –11. HoweP. falciparum-specific protein and pan-Plasmodium protein. It fulfills the performance criteria set for the rapid diagnosis of malaria by attaining sensitivity of greater than 95% for samples with parasitemia ≥ 100 parasites/μl of blood [RDTs are based on the detection of histidine-rich protein 2 (HRP-2), plasmodium specific lactate dehydrogenase (pLDH) and aldolase antigens , 13. AmoIn Ethiopia, CareStart™ Malaria Combo test was evaluated in Afar, Kola Debi Health Center and Southern Ethiopia. In these studies the overall sensitivity and specificity of CareStart™ malaria combo test was 94.2–98.5% , 14. CarThis cross-sectional study was carried out in Mayani Hospital Northwestern Tigray from December 2015 to March 2016. Mayani Hospital is located in Sheraro town; Sheraro is a town and separate district in Northern Ethiopia , d = Margin of error (5%);2*0.238(1–0.238)/ (0.05)2N = (1.96)N = 3.8146*0.18/0.0025N = 276.7 = 277 However, to enhance the reliability of the test, an additional 43 more malaria-suspected patients were recruited.Two (2) ml of venous blood sample was collected using EDTA-anticoagulated tube for the detection of malaria parasite using both microscopy and CareStart™HRP2/pLDH Combo rapid malaria test methods. Two experienced laboratory technologists were assigned in the study hospital to collect blood sample, prepare blood film and perform RDT. Staining and microscopic examination of blood films were performed in Aksum University Referral Hospital diagnostic laboratory by another two experienced microscopists who had training and are certified in malaria microscopy. They were unaware about CareStart™HRP2/pLDH Combo rapid malaria test results. The discordant between the two microscopists was examined by a third highly experienced expert microscopist and the result was taken as final. Microscopy and RDT performers had training before the start of the study. In addition, the investigators were in close contact with data collectors to assess the quality of data throughout the data collection period. To assess the consistency of microscopic examination, 30 blood smears were examined blindly by a laboratory technologist who was not part of the study. He detected malaria parasites in three of the blood smears previously reported as negative. However, only one of the three blood smears was positive by the experienced expert microscopist.Plasmodium was observed in 200 high-power fields. On the other hand, thick blood films were taken as positive if one or more malaria parasites have been observed; and, negative if no parasites were seen after examining 1000 white blood cells [Thin and thick blood smears were prepared from each clinically-suspected malaria patients. Blood films were sent to Aksum University Referral Hospital in a standard slide box for staining with 3% Giemsa and microscopic examination. Thin smears were considered positive for malaria if one or more malarial parasites were seen; and, negative if no asexual form of od cells .Plasmodium falciparum-specific protein and pan-Plasmodium protein. The storage temperatures of the kits were between 4 and 30 °c.CareStart™HRP2/pLDH Combo rapid malaria test  was based on lateral flow immunochromatography in cassette format which can detect doubtful and repeated. If the control line did not appear, the test was considered invalid and was repeated [Five (5) μl blood samples and diluents were added on the kit and reading was performed at 20 min according to the manufacturer’s directions. Results were recorded as negative (no test line visible) or positive (at least one test line visible). If the test line was barely visible, the result was scored as repeated . All RDTMicroscopically-confirmed and malaria-free blood samples were used as a positive and negative control respectively to check the validity of CareStart™ RDTs.p-value of less than 0.05 was considered as significant in all comparisons.The data were entered in Microsoft Excel spreadsheet, exported and analyzed by SPSS version 20. Two-way contingency table analysis and cross tabulations were used to show the association and odds ratio (OR) of study population characteristics and parasite positivity by light microscope. Sensitivity, specificity and predictive values for the detection of different Plasmodium species were calculated and compared against each other. Kappa value was determined to evaluate the agreement between CareStart™ RDT and light microscopy. A Consent form was used to ask patients’ or guardians’ (in case of children) willingness. Ethical clearance was obtained from the institutional review board (IRB) of Aksum University, College of Health Sciences. Microscopically-confirmed malaria positive study participants were treated with appropriate anti-malaria drug in the study hospital.A total of 320 suspected malaria patients were tested using light microscopy and the CarStart™ malaria RDT. The male-to-female ratio was 1.3:1. The age range of study participants was from 6 to 53 years and majority of them (166 (51.9%)) were between 15 and 45 years age group. Most of the study participants 227 (70.9%) were from the rural areas of the district. The age groups 15–45  and above 45 years old  were more likely to be malaria positive using microscopy  for P. vivax and 5 (1.6%) for mixed infections. While using the CareStart™ RDT, the overall parasite positivity was 43 (13.1%): 19 (5.6%) for P. falciparum, 19 (5.9%) for P. vivax and 5 (1.6%) for mixed infections : 16 (5.0%) for .8%: 16  and negative predictive value (NPV) were 99.6 and 98.92% respectively. The agreement between the CareStart™ RDT and light microscopy tests was excellent with a kappa value of 0.92  or the light microscope (12.8%), the overall prevalence of malaria in this study was very low compared to Moges et al. report (40.9%) . The lowCareStart™ RDT has very good sensitivity and specificity for malaria diagnosis. It has also an excellent agreement with light microscopy. Therefore, continued use of this RDT is highly recommended in resource-limited areas where microscopy is not available.


In this study, the merozoite surface proteins  had the highest prevalence of 41.5 and 38.5% in rural and urban settings, respectively. Overall, 82.1 and 70.0% of samples had multiclonal infection with msp-1 gene resulting in a mean multiplicity of infection (MOI) of 2.8 and 2.6 for rural and urban samples, respectively. Msp-1 and msp-2 genes displayed higher levels of diversity and higher MOI rates than the glurp gene.Three-hundred and nine samples were successfully genotyped for msp-1 allele, compared to previous reports, thereby confirming the changing face of malaria transmission in these communities. This study provides important baseline information required for monitoring the impact of malaria elimination efforts in this region and data points useful in revising current protocols.Significant genetic diversity was observed between rural and urban parasite populations in Ibadan, southwestern Nigeria. The results of this study show that malaria transmission intensity in these regions is still high. No significant difference was observed between rural and urban settings, except for a completely different  Plasmodium falciparum remains a major public health problem in most sub-Saharan African regions. Nigeria, one of the biggest nations by population in sub-Saharan Africa, accounts for the highest burden of the disease worldwide with about 30% under-5 years death, 25% infant death and 11% maternal mortality [P. falciparum in sub-Saharan Africa, the development of insecticide-resistant mosquitoes and failure to develop effective malarial vaccines has all added to the disease burden and eradication challenge [P. falciparum populations remain unclear. Dramatic variation in diversity and geographical differentiation have been reported in areas of different transmission intensities [P. vivax that the relationship between diversity and transmission intensity is not simply linear because many patients could be infected by either related parasites resulting from inbreeding or distinct new parasites [P. falciparum, a feature that has helped the parasite in overcoming anti-malarial drugs, vaccines and vector control strategies, but the dynamics and genetics of P. falciparum populations remain unclear. Moreover, geographic genetic differentiation exists which appear to be inconsistent, warranting the need to constantly assess the structure and diversity of P. falciparum in Africa.Malaria caused by hallenge . The hugensities . Regionsensities . Despitearasites . Also, tarasites . Many stP. falciparum merozoite surface proteins 1 and 2 (msp-1 and msp-2) and glutamate-rich protein (glurp) genes are significant molecular markers, highly effective in differentiating monoclonal from multiclonal infections within infecting parasite strains, including their utilization in discriminating recrudescence from re-infecting alleles [msp-1 and 2 are erythrocyte membrane-bound proteins abundantly found on the blood stages of P. falciparum infected erythrocytes, and reported to play major roles in erythrocyte invasion [msp-1-block 2 regions which has three allelic groups [msp-2 is dimorphic, existing in two main allelic forms of 3D7-like and FC27-like family, sharing N- and C-terminal regions, with strain-specific variable regions. Merozoite surface protein 2 is suggested to be essential during parasite invasion and remains on the surface throughout invasion only to be degraded upon invasion completion [glurp gene is composed of a rather conserved region (R0 region), a central repeat region (R1 fragment) and a C-terminal immunodominant repeat region (R2 fragment) [msp-1, the central repetitive domain of the msp-2 (block 3), and R2 region of the glurp gene have been well characterized in most malaria-endemic regions globally to study the parasite genetic structure and the allelic frequency distribution [The  alleles , 10. The alleles –14. The invasion . It is hc groups . On the mpletion . The gluragment) . The polragment) . The bloribution , 19.P. falciparum infection from rural and urban areas. To this end, this study report the results of the genetic analysis of msp-1, msp-2 and glurp genes among children under 5 years of age, presenting with acute uncomplicated P. falciparum infection, recruited from rural and urban areas of Nigeria.Since malaria is endemic in Nigeria, it is important to characterize these markers from time to time in order to elucidate the genotypic pattern of the circulating parasite populations, understand the mechanism underlying disease pathogenesis, transmission and acquisition of drug resistance in parasite strains with the intent of establishing an effective control strategy. The assemblage of baseline data for these polymorphic biomarkers in the parasite population from different geographical regions and different levels of malaria transmission in urban and rural areas are essential tools for the current efforts on malaria control strategies in sub-Saharan Africa , 21. Sigvs urban 146), presenting with fever (axillary temperature ≥ 37.5 °C) or a history of fever in the past 48 h, and other inclusion criteria, were enrolled into the study. Using standard aseptic technique, capillary blood obtained from a fingerprick on the middle finger after cleaning with 70% alcohol was used to prepare thick blood smears and spotted on filter paper (Whartman 3MM™) for future molecular analysis. Thick blood smears were air dried and stained with 10% fresh Giemsa, following standard procedures, and parasite density calculated by counting the number of asexual parasites against 200 leukocytes in the same high power fields, assuming a leukocyte count of 8000 white cells/µl [This was a cross-sectional study conducted in a primary healthcare facility at Idi-Ayunre  and St Mary Catholic Hospital, Eleta (urban), Ibadan from November 2013 to November 2014. Idi-Ayunre is the headquarters of Oluyole Local Government Area, with an estimated area of 629 sq km and a population of approximately 202,725 based on 2006 population census . St Marycells/µl .Finger-prick blood spotted on Whatman™ 3MM filter paper, were dried in dust free area and subsequently placed in a zip-lock bag with silica gel to prevent DNA degradation. Dried blood spots were utilized for DNA isolation and PCR amplification. DNA extraction was performed with the QIAamp DNA Mini kit (QIAGEN Germany), according to manufacturer’s instructions with slight modification. Genomic DNA eluates were stored at − 20 °C until further PCR analysis.P. falciparum was amplified in a nested PCR using primary genus-specific primers and a secondary P. falciparum species-specific primers following the previously described standard protocol [P. falciparum were further characterized for msp-1, msp-2 and glurp genes, with specific oligonucleotides primer pairs.The small sub-unit ribosomal RNA (ssrRNA) gene of protocol  (Table 1msp-1 and 2 (block 2 and 3), as described previously [msp-1  and the block 3 region of msp-2 (FC27 and 3D7) genes were amplified with a nested PCR amplification in a final reaction volume of 20 µl containing 10X PCR buffer , 0.125 mM of dNTPs, 0.25 mM of each primer, and 1 U Taq DNA polymerase  and 5 µl of DNA in the first reaction and 3 µl of the first-round product in the second (nested) reaction was added as template. The PCR cycling conditions for both primary and nested reactions are as previously described [Primers specific for the polymorphic region of eviously , were usescribed .glurp gene, a semi-nested PCR reaction in a final volume of 25 µl for the polymorphic R2 region was performed with specific primers containing PCR reagent concentrations as in msp-1 and msp-2 genotyping above [® Green 1 nucleic acid gel stain  and resolved by gel electrophoresis in 1.5% agarose gel. DNA sizes were determined using 100 bp DNA ladder , and photographed under UV trans-illuminator .For the ng above . The cycP. falciparum infection. Specifically, 68.5% (250/365) of the PCR confirmed P. falciparum cases was from the rural community while 40.4% (59/146) was from the urban community. The difference was statistically significant (p < 0.001)  were confirmed positive by nested PCR for msp-1 alleles were observed in this study, with RO33 being the most common allele in the rural community with a monoclonal infection frequency of 7.3%. The frequency of multiclonal infection (RO33 + MAD20 + K1) was 82.1%. The multiplicity of infection (MOI) for merozoite surface proteins-1 in the rural community was 2.8. For the urban community, both RO33 and MAD20 had a monoclonal infection frequency of 10.0% each, frequency of multiclonal infection (RO33 + MAD20 + K1) at 70.0% and an overall MOI of 2.6  were identified  and coded as genotype I–XII with genotype XI the most predominant (41.5%). In the urban setting however, fewer genotypes were recorded with only six genotypes  detected. Genotype XI (1000–1050 bp) was also the most common. Genotype VI (750–800 bp) did not appear in both rural and urban communities and the MOI in both communities was 1.0  while 3D7 recorded 81.8% (36/44), respectively.When parasite density was compared with prevalence of the different allelic families at parasite density ≥ 2000 parasites/µl, the K1, MAD20 and RO33 allelic families were amplified in 87.6% (85/97), 90.7% (88/97) and 94.8% (92/97) of positive samples, respectively. Similarly, FC27 was positive for 71.4% (65/91) while 3D7 accounted for 86.8% (79/91). At parasite density ≤ 2000 parasites/µl however, there was a reduction in the allelic frequencies, but not statistically significant, as K1, MAD20 and RO33 had 84.3% (70/83), 83.1% (69/83) and 91.6% (76/83), respectively. For glurp gene was 100% (64/64) positive at parasite density ≥ 2000 parasites/µl, and 97.1% (66/68) at parasite density ≤ 2000 parasites/µl blood, an MOI of 1.1 (Table p > 0.05).The allelic frequency for .1 Table . There wP. falciparum polymorphic markers  in children from urban and rural communities of Oyo State, southwest Nigeria. Understanding the genetic diversity of P. falciparum in different geographical settings is of critical importance in resolving the population structure of the parasites in endemic areas and in order to develop effective control strategies [P. falciparum is also considered a major obstacle in the acquisition of natural immunity and vaccine development efforts. The findings in this study are consistent with previous reports that showed that these genes are highly polymorphic among the parasite population in southwestern Nigeria [P. falciparum populations in Idi Ayunre  and Eleta (urban) exhibited an overall similar level of high genetic diversity, with MOI of 2.8 and 2.6, respectively.This study evaluated the genetic diversity of rategies , 30. On  Nigeria , 31, 32.msp-1 gene, RO33 was the most predominant allelic family in both rural (7.3%) and urban (10.0%) settings, contrary to previous reports by others from the same region where K1 allelic family was reported as the commonest [P. falciparum parasite in this study may be due to high transmission intensity and the possible impact of different control measures, mainly chemotherapy and insecticide-treated nets (ITNs). Change in genetic make-up of P. falciparum has been reported in endemic communities following change in anti-malarial drugs and introduction of ITNs [msp-2 locus, the 3D7 allelic family was the most abundant single allele in both communities. This is similar to that reported from the Thai-Myanmar border [msp alleles has been associated with clinical status yielding conflicting reports. Some studies have reported that RO33 and FC27 alleles are typically frequently observed in asymptomatic malarial infections [In the ommonest , 33. Inc of ITNs . The fin of ITNs , Sudan [ of ITNs , Uganda  of ITNs , and Tha of ITNs , which wr border , Nigeriar border , Congo Bnd urban .0% settir border , 40, butr border  and nortr border . Differefections , others fections , 43, 44.msp-1 and msp-2 genotypes in both community settings. This persistent high level of genetic diversity is an indication that the parasite population size remained large enough to allow effective mixing of genotypes. There is also the possibility of human migration, which may also bring about increase and changes in genetic diversity by introducing additional parasite genotypes [msp-1 variants (up to 350 bp) and similar msp-2 sizes viz a viz previous report [High prevalence of multiclonal infection was observed with enotypes . Signifis report , contribglurp gene also demonstrated a high degree of polymorphism in the parasite population, with 12 distinct allelic fragment sizes, out of which 11 were observed in the rural setting and six observed in the urban community. The high number of glurp alleles in the study area is not unexpected, considering the high malaria endemicity, which is also in agreement with previous reports [glurp alleles observed in the urban area in comparison to the rural area could also be an indication of higher transmission intensity in the rural area, including significant changes in the parasite genome happening at a faster pace in the rural than urban areas.The RII region of  reports , 46. TheP. falciparum in the study areas. The association of P. falciparum genetic diversity on clinical malaria (indicated as parasitaemia in this study) has been yielding conflicting report. Previous work failed to establish a significant association between different allelic families of msp-2 and parasite density [msp-1 and msp-2 genotypes. Additionally, previous association between the msp-2 locus of P. falciparum and clinical severity of malaria in children, reported from Ibadan [Interestingly, when parasite density was categorized into two groups (> 2000 and < 2000 parasites/µl), the genetic diversity was not influenced by parasite density. This is indicative of the fact that high malaria transmission and parasitaemia may not have strong association with genetic diversity of  density , with th density  and Cong density , showingm Ibadan , was notm Ibadan –50. ContP. falciparum parasite population remains highly diverse at the three antigenic loci  analysed in this study. The high MOI is suggestive of effective genetic recombination of the parasite population, which may be related to the high transmission of P. falciparum in the study area, leading to continued maintenance of genetic diversity. The data could serve as an important baseline information for understanding the population structure of P. falciparum and can be harnessed into malaria control strategies in an effort aimed at eliminating malaria in Nigeria.Despite the ongoing extensive malaria control efforts in Nigeria, the prevalence of malaria remain high, especially in the rural communities and the


Malaria infection during pregnancy can result in placental malaria and is associated with adverse pregnancy outcomes particularly among primigravidae. The aim of this study was to assess the prevalence and risk factors for placental malaria and its effect on pregnancy outcomes in Blue Nile state, Sudan.A cross-sectional hospital-based study was conducted consecutively during January 2012–December 2015 in three main hospitals in Blue Nile State, Sudan. At delivery, peripheral and placental blood samples were collected from consenting women. Finger prick blood was used for preparation of peripheral smears and for haemoglobin measurement. Smears were stained with Giemsa and examined microscopically for malaria parasites. Pregnancy outcomes in association to placental malaria were investigated.Plasmodium falciparum as the only species detected. In multivariate analysis, younger age ≤23.2 years old , primiparae , secundiparae  visits  and not using bed nets  were risk factors for placental malaria. Education and residence were not associated with placental malaria infection. Placental malaria was significantly associated with maternal anaemia  and low birth weight (LBW) .A total of 1149 mothers and their newborns were recruited. The mean (SD) of the age was 23.3 (5.2) years. Detection of malaria parasites was confirmed in 37.8% of the peripheral blood films and 59.3% of the placental films with During the study, there was a high prevalence of placental malaria in Blue Nile State-Sudan, as the enhanced control activities were not practiced, leading to adverse pregnancy outcomes, such as maternal anaemia and LBW. Plasmodium falciparum, which is found in tropical and subtropical regions [Malaria during pregnancy is a serious public health problem in sub-Saharan Africa and about 10,000 women and 200,000 babies die annually because of malaria in pregnancy. Most of these deaths are caused by igravidae) are highly susceptible to malaria compared with multigravidae [In malaria endemic areas, at least one in four pregnant women has evidence of peripheral or placental malaria at delivery . It has gravidae –7. Malargravidae , 8. PM igravidae , 10, lowgravidae . Congenigravidae . Moreovegravidae , 12.In Sudan, malaria is one of the deadliest endemic diseases, and the increased susceptibility of pregnant women to malaria is a long-standing public health problem , 13.Maternal mortality rate in Sudan in 2013 was 311/100,000 live birth . The infPrimary health care in Blue Nile State is the worst in central Sudan in terms of health indicators accompanied by the role of migration due to political unrest. Infant and child mortality are higher than in other neighbouring states. Malaria is one of the major problems that increase mortality in the state with more than 30,000 cases reported in 2010 . Moreover, malaria is the main cause of common morbidity and mortality in the state . In SudaThe current study was conducted to assess the prevalence and risk factors of placental malaria and its effect on pregnancy outcomes in Sudanese women from Blue Nile State, Sudan.2 and an estimated population of 832,000; 75% of them reside in rural areas, and 25% in the four urban centres. Women represent 47% of the people in the state with maternal mortality rate of 258/100,000 live births. The relevant environmental, anthropological, political unrest and geographic characteristics of Blue Nile State, which shares an international border with Ethiopia and South Sudan, impact uniquely on the epidemiology and control of malaria [A cross-sectional hospital-based study was conducted in the Blue Nile State between January 2012 and December 2015. Blue Nile State lies in the tropical climate zone between latitude 9°30 and 12°30′ and longitude 33°5′ and 35°3 East. Average annual rainfall is around 700 mm with the southern part of the state being the wettest. The State has an area of 45,844 km malaria . The malThe study was conducted at the same time in three main maternity units of the hospitals: Ed-Damazin Hospital, El-Roseires Hospital and the Surgical Complex.The study sites conducted around 6700 deliveries during the study period with the highest number (3968) of deliveries were at the Surgical Complex and the least (789) were at El Roseires Hospital.Women were recruited after explaining to them the purpose and nature of the study and signing informed consent forms. Women were included irrespective of age, educational background, and socio-economic, cultural or religious condition. Women were approached to participate consented to join the study and to provide the suggested 3 samples .The study protocol was discussed and agreed upon initially with health authorities of the Ministry of Health, Blue Nile State prior to being submitted to and reviewed by the Ethical and Scientific Committees of the Tropical Medicine Research Institute and the Directorate of Research, Federal Ministry of Health, Sudan. A signed informed consent was obtained from each woman prior to their enrolment in the study. Women positive for malaria were treated with artesunate-SP according to the National Malaria Control Programme protocol.Consenting parturient women with singleton pregnancies and normal vaginal were recruited. Pregnancy with twins and mothers with serious delivery complications including hypertension, diabetes mellitus, antepartum haemorrhage or post-partum bleeding, eclampsia and other complicated conditions were excluded. Women were clinically examined and their socio-demographic data, relevant medical and obstetric history were collected. These data were all summarized on a predesigned questionnaire which included information on maternal age, gravidity and gestation, history of previous malaria episodes, use of anti-malarial drugs, IPTp usage and ITNs ownership and use during pregnancy and ANC visits. Birth weight was measured by the attending midwife for all births within 1 h of delivery, using an electronic scale accurate to ±10 grams and calibrated weekly. Newborns were classified as normal birth weight (≥2.5 kg) or LBW (<2.5 kg) according to WHO guidelines .At delivery, peripheral and placental blood samples were collected. A finger prick was used to test for malaria microscopy and to measure haemoglobin (Hb) level. Placental blood smears were obtained from the maternal side intervillous space of the placenta using a sterile disposable lancet.Blood smears were prepared on grease-free, clean glass slides and allowed to dry before being stained with 10% Giemsa at pH 7.2 for 10 min and examined under the ×100 oil immersion objective lens of a light microscope. Microscopic examination of thick films, using high power magnification was used to detect the presence of parasites, while the thin films were used to determine species when thick films were positive. The staining techniques and blood film examination were conducted employing WHO guidelines .Parasite density was determined using the plus system , by counMaternal haemoglobin concentrations were estimated with a portable HemoCu device . Anaemia was defined as Hb < 11 g/dL and Hb < 7 g/dL: severe anaemia, Hb 7–9.9 g/dL: moderate anaemia, and Hb 10–10.9 g/dL: mild anaemia .All of the selected research team were centrally trained on the important aspects of the research work and had continuous supervision by the site supervisor. Quality of slide reading was ensured at each of the three study sites then rechecked in Khartoum by an experienced microscopist, blinded to original diagnosis.2 test were used to compare the mean and proportions between two groups, respectively. Binary logistic regressions were built where placental malaria was the dependent variable, the site of samples collection and socio-demographic characteristics were the independent variables. In order to investigate the effect of placental malaria on maternal anaemia and LBW binary logistic regression models were built where these were the independent variables and the site, socio-demographic  and placental malaria were the independent variables. P < 0.05 was considered significant. Parity was categorized as primiparae, secundiparae and multiparae (≥3). For the research purposes, women were diagnosed as malaria positive if parasites were detected by light microscopy in either the peripheral blood or samples collected from the placenta.The collected data were analysed using SPSS V.20.0 . Student’s t test and XA total of 1149 mothers and their newborns fulfilled the inclusion criteria and were had complete data. The mean (SD) of the age was 23.2 (5.2) years. Five hundred and fifty-three (48.1%), 297 (25.8%) and 299 (26.0%) women were primiparae, secundiparae and multiparae, respectively. Less than one-third of the women (30.2%) were rural residents and 922 (80.2%) had education less than secondary level (<9 years). Out of these 1149 women, 452 (39.3%) had antenatal attendance, 102 (8.9%) used bed nets in the index pregnancy and 94 (8.2%) used IPTp.Five hundred and fifty (47.9%), 227 19.8%) and 372 (32.4%) women were recruited from the Surgical Complex (549), El Roseires Hospital and Ed-Damazin Hospital, respectively  of the smears diagnosed by peripheral blood microscopy, while 677 (58.9%) had placental infection diagnosed by placental blood smear examination. Four hundred and sixteen subjects (36.2%) were both peripheral and placental malaria positive, while 261 of the mothers with negative peripheral blood had placental malaria positive. Mild (+) parasitaemia was recorded in 271 (62.4%) and 236 (34.9%) of the positive peripheral blood and placental blood films, respectively, while moderate (++) parasitaemia was found in 142 (32.6%) and 260 (38.4%) of the positive peripheral and placental blood films, respectively. Hyperparasitaemia > +++ was found in 21 (5%) of the maternal peripheral blood and 181 (26.7%) of the placental blood films.Plasmodium falciparum parasitaemia by microscopy in their peripheral smears at delivery were placental malaria positive.Only 18 women were peripheral smear positive while placental smears negative showing mild (+) parasitaemia and 14 of them (67%) were primiparae. The vast majority of the women (95.9%) who had P < 0.001), primiparae , secundiparae , no ANC visits , not using bed nets  and peripheral blood positivity was significantly associated with placental malaria at delivery  were risk factors for placental malaria. Education, residence and site of samples collection were not associated with infection  of the haemoglobin among these women was 10.6 (3.3) g/dL. A high rate of anaemia where 743 (64.7%) women had anaemia, and 22 of them (3%) had severe anaemia (Hb < 7 g/dL). Placental malaria was the highest risk factor for maternal anaemia  (Table P < 0.001), placental malaria , and no ANC visits  were significant risk factors for low birth weight  of the birth weight of the neonates was 2.5 0.38) kg and the overall frequency of LBW was 56.4% (n = 684). Malaria infection was significantly associated with LBW with the mean (SD) birth weight in malaria negative women being 2.8 (0.33) kg while that in peripheral malaria positive women was 2.3 (0.20) kg and in placental malaria positive women was 2.2 kg (0.20 kg). Maternal anaemia , and young age , with placental malaria. This finding is consistent with those of Brabin [P = 0.049). Poor women from rural areas in the study area usually deliver at home and tend to be less aware of the need to avoid infection because most of the education programmes are concentrated in the urban areas. This factor must be an important driver towards the design and implementation of programmes at community level in rural areas. Ezebialu et al. [The findings of this study were in accordance with those of Uneke , who fouf Brabin  and Tacof Brabin , 34. A cu et al.  reportedIn consistent with previous studies, the present study showed that neonates of mothers with placental malaria were LBW , 25, 35,In this study, there was an association between placental malaria and maternal anaemia similar to previously published studies from several countries in sub-Saharan Africa which indicated that malaria infected women had higher prevalence of anaemia than uninfected women , 38. TheThe study extended from 2012 to 2015 and samples were collected in order to investigate the prevalence of placental malaria at the study sites. This extended duration was due to availability of field expenses and resources. Moreover, recruitment and samples collection was not carried on a daily basis due to the limited number of midwives participated in the study. Parasite density was the existing routine practice in the laboratories of the study sites as the technicians participated the study were not researchers.There was a high prevalence of placental malaria in this part of Sudan especially younger age and primiparae. Placental malaria was associated with maternal anaemia and LBW.Community based rigorous education programme to increase awareness of the importance of ANC services and the safety of anti-malarial drugs during pregnancy should be encouraged among all pregnant women, especially the primigravidae. An assessment of the control measures available in the area particularly providing free ITNs should be urgently implemented. Further community based studies should follow.


The role of these cells in mediating clinical immunity (i.e. protection against symptoms) to malaria remains unclear. We measured Vδ2+ T cell absolute counts at acute and convalescent malaria timepoints (n = 43), and Vδ2+ counts, cellular phenotype, and cytokine production following in vitro stimulation at asymptomatic visits (n = 377), among children aged 6 months to 10 years living in Uganda. Increasing age was associated with diminished in vivo expansion following malaria, and lower Vδ2 absolute counts overall, among children living in a high transmission setting. Microscopic parasitemia and expression of the immunoregulatory markers Tim-3 and CD57 were associated with diminished Vδ2+ T cell pro-inflammatory cytokine production. Higher Vδ2 pro-inflammatory cytokine production was associated with protection from subsequent Pf infection, but also with an increased odds of symptoms once infected. Vδ2+ T cells may play a role in preventing malaria infection in children living in endemic settings; progressive loss and dysfunction of these cells may represent a disease tolerance mechanism that contributes to the development of clinical immunity to malaria.Vδ2 Children residing in endemic areas eventually acquire ‘clinical’ immunity to malaria (i.e. they are protected against symptoms)5, but they commonly harbor parasites as asymptomatic and transmitting carriers7. Although individuals generally do not appear to develop sterilizing immunity that prevents any infection, blood-stage parasite density declines with age and repeated exposure8, suggesting the development of immune responses that are able to limit blood stage replication. Importantly, pro-inflammatory responses that limit parasitemia may also lead to clinical symptoms; thus, ‘clinical’ immunity could depend upon the ability to down-modulate such responses, as suggested by recent data from our group and others11.Despite declines in malaria morbidity in parts of sub-Saharan AfricaPf antigen stimulation and to markedly expand following malaria infection in naïve hosts17. These cells (hereafter termed Vδ2 T cells) rapidly react to phosphoantigens produced by the plasmodial apicoplast, and have been shown to inhibit parasite growth in vitro via the release of cytotoxic granules containing granulysin19. Given these attributes, Vδ2 T cells may function as ready-made effector cells, and may be most important early in response to malaria infection, potentially before the adaptive immune response to Pf has developed. Supporting this hypothesis, cytokine production from these cells has been associated with protection from high density P falciparum infection20, and higher baseline percentages of these cells have recently been associated with protection from subsequent Pf infection among individuals receiving an experimental attenuated sporozoite vaccine21.The Vγ9 Vδ2 subset of γδ T cells, which constitute 0.5 to 5% of peripheral T cells in humans, have been shown to robustly proliferate and produce pro-inflammatory cytokines in response to 22. Thus, curtailing excessive Vδ2 T cell activation may be required for the development of clinical immunity to malaria. We have previously shown that repeated malaria was associated with a loss of Vδ2+ T cells in peripheral blood, decreased proliferation and cytokine production of these cells in response to malaria antigen stimulation, and upregulation of numerous genes associated with dampening of the immune response23. Furthermore, loss and dysfunction of Vδ2+ T cells was associated with a lower likelihood of symptoms upon subsequent infection9. Notably, we did not find a significant association between Vδ2+ T cell parameters and protection from subsequent infection, although our prior studies were limited to small cohorts of children <5 years of age and were unable to fully account for heterogeneous exposure to mosquitoes.While Vδ2 T cells may play role in limiting parasite replication, their production of pro-inflammatory cytokine has been implicated in the pathogenesis of severe symptoms from malaria+ T cells in mediating clinical immunity to malaria, leveraging large and comprehensively characterized cohorts of children age 6 months to 10 years from two regions of Eastern Uganda with differing transmission intensities [17]. We first evaluated Vδ2+ T cell absolute counts following symptomatic malaria episodes, hypothesizing that older children – who have sustained more cumulative malaria exposure in a high transmission setting – would exhibit diminished in vivo Vδ2+ T cell proliferation. We then evaluated Vδ2+ T cell absolute counts, cellular phenotype and Pf stimulation-induced IFNγ and TNFα-production from asymptomatic children living in both high and low transmission settings, assessing relationships between these parameters with age, parasitemia, and malaria infection. Finally, we analyzed the relationship between Vδ2+ T cell parameters and prospective protection from both Pf infection and the likelihood of symptoms once infected. We adjusted our analyses for heterogeneity in exposure to mosquitos using household-level mosquito capture data . We hypothesized that higher Vδ2+ T cell numbers and cytokine production would be associated with protection from infection, but that greater cytokine production from these cells would also be associated with symptoms among children who are infected.In the present study, we extend our prior observations regarding the potential role of Vδ2+ T cells expand following a symptomatic malaria infection in naïve and malaria-susceptible adults24. Thus it is somewhat paradoxical that we recently found Vδ2+ T cell frequencies to be markedly lower among two cohorts of Ugandan children following chronic and repeated malaria exposure23. To address these seemingly contradictory observations, we measured absolute Vδ2+Vγ9+ CD3+ cell counts at the time of an acute malaria episode and 3, 6 and 9 weeks post-infection among children aged 6 months to 10 years living in a highly endemic region of Uganda. In this setting, parasite prevalence (including both microscopic and sub-microscopic parasitemia) is very high, but the probability of symptoms if blood smear positive decreases dramatically with increasing age , with stable counts thereafter, although there was significant heterogeneity among individuals . Together, these data indicate that there are significant age-associated differences in expansion of Vδ2+ T cells following acute malaria in endemic settings, with younger children having significantly greater expansion than older children.It has previously been shown that both the absolute count and percentage of Vδ2age Fig. , consistals Fig. . When st+ T cells among children ≤4 years of age23, but it is not clear whether this is due to an absolute vs. relative loss, as total lymphocyte counts decline gradually during childhood25. We assessed the absolute count of Vδ2+ T cells/μl among children aged 1–11 years of age living in high prevalence Nagongera and compared these with age-matched children living in the lower malaria transmission setting of Walukuba, Jinja. Children living in Nagongera had significantly fewer Vδ2+ T cells/μl  were similar in the two settings . Furthermore, we observed a subtle, but statistically significant, inverse association between age and Vδ2+ T cell production of the inflammatory cytokines IFNγ and TNF following in vitro stimulation with P. falciparum–infected RBCs among children living in Nagongera , but not in the lower transmission setting . These data suggest that increasing age – a surrogate for cumulative exposure to malaria in high transmission settings - is associated with loss, in both percentage and absolute count, and dysfunction of circulating Vδ2+ T cells among children living in a high transmission setting.Some studies performed in non-malaria endemic settings have observed an age-associated increase in Vδ2era Fig. , and no + T cell percentages or cytokine production from these cells following in vitro stimulation with P. falciparum–infected RBCs9. However, our earlier cohort had relatively few patients with microscopic parasitemia and a lack of data on sub-microscopic infection. In the present study, we found that children with microscopic parasitemia had significantly fewer circulating Vδ2+ T cells/μl than uninfected children or those with submicroscopic infection at the time of assay  following in vitro malaria antigen stimulation. Asymptomatic children with microscopic parasitemia had significantly lower overall Vδ2+ T cells percentages . Together, these data reveal that concurrent microscopic parasitemia, and higher parasite densities, are associated with both lower counts and diminished pro-inflammatory cytokine production of Vδ2+ T cells.Similar results were obtained when evaluating the overall percentage of Vδ2ges Fig. , and lowlls Fig. , than un9. These include the gene encoding CD57, a protein which has been associated with replicative senescence of CD8+ T cells in the setting of chronic antigen exposure28; and HAVCR2, which encodes the receptor Tim-3, a cell surface molecule that has been implicated in tolerance and exhaustion of Th130 and innate cells31. We assessed cell surface expression of CD57 and Tim-3 on Vδ2+ T cells and found expression of both markers to be significantly higher among children living in Nagongera in comparison to children living in Walukuba .We have previously shown that repeated malaria exposure is associated with increased Vδ2 expression of several genes associated with immunomodulationuba Fig. . Importaion Fig. . We also+ T cells increased with age among children living in Nagongera . We also evaluated associations with protection from higher density P. falciparum infection . After adjusting for age, children in the middle/highest strata of Vδ2+ T cell IFNγ/TNF co-production had a significantly higher odds of submicroscopic infection if infected than children in the lowest strata . These results are consistent with the hypothesis that Vδ2+ T cells contribute to protection against infection, possibly by limiting parasite replication.We found that higher frequencies of Vδ2ar Table . Childrear Table . These r+ T cell parameters and the prospective odds of having symptoms given microscopic infection. Higher percentages of Vδ2+ T cells producing inflammatory cytokines IFNγ and TNF following in vitro stimulation was associated with an increased odds of having symptoms given P. falciparum infection. Children in the middle and highest strata of Vδ2+ T cell cytokine production had a >3 times higher odds of symptoms given patent blood smear infection in the subsequent year compared to children in the lowest strata  and did not assess for submicroscopic infection.In the present study, we found that higher Vδ2+ T cells may possess effector functions that that can limit parasite replication, we found that higher frequencies of cytokine-producing Vδ2+ T cells were associated with a higher probability of symptoms given infection. This observation is consistent with published data suggesting that cytokine production from these and other cells may be responsible, in part, for mediating clinical symptoms22. Taken together, our observations are consistent with the hypothesis that repeated malaria may drive a disease tolerance pathway among Vδ2+ T cells that attempts to reduce the negative impact of infection on host fitness37. Mechanisms of malaria-induced Vδ2+ T cell dysfunction remain to be elucidated, but may include signaling through Tim-332 and/or other immunoregulatory pathways, and/or changes to the epigenetic landscape of these cells38, similar to what has been described in several innate cellular populations in response to infection40.Though Vδ2P. falciparum and their direct anti-merozoite effects in vitro, we speculate that these cells may play a beneficial role as ready-made effectors during primary acute malaria infection of infants and young children, before the development of an effective adaptive immune response41. Although the progressive loss and dysfunction of these cells with repeated exposure is associated with reduced symptoms, it may conceivably interfere with effective clearance of the infection in childhood, contributing to rising parasite prevalence with increasing age in high transmission settings42. Although individuals rarely if ever develop sterilizing immunity that prevents any infection, control of blood-stage parasite density improves with increasing age, which we speculate may be due to the eventual development of adaptive immune responses, including antibodies, that develop with repeated exposure. Furthermore, it is possible that as Vδ2 T cells downregulate production of pro-inflammatory cytokines, they acquire additional effector functions, perhaps mediated by CD16 (FcgRIII), which is upregulated following repeated exposure to malaria23. In other contexts, CD16+ Vδ2 T cells have been reported to mediate phagocytosis43; hence it is possible that in older children with chronic malaria exposure, Vδ2 T cells may work in concert with antimalarial antibodies to restrict parasite burden.Given the intrinsic reactivity of Vδ2 T cells to + T cells that parallel the development of clinical immunity to malaria, we cannot determine whether these changes are causally responsible for clinical immunity. Many immune effector populations are regulated in concert, any of which may contribute to this process. Furthermore, the age-associated loss and dysfunction of Vδ2+ T cells observed in peripheral blood in heavily malaria-exposed children could potentially reflect redistribution to the spleen, liver, or other tissue sites in response to malaria infections. Finally, given the cross-sectional nature of the phenotypic and functional assessments described, associations with protection from infection and symptoms given infection should be interpreted cautiously, and require validation in other cohorts and exploration in model systems.There were several limitations to this study. Although we observed age-related differences in Vδ2+ T cells may play an important role in protection from Pf infection36. However, clinical immunity to malaria may be mediated by disease tolerance mechanisms that down-modulate pro-inflammatory responses from Vδ2+ and/or other cellular subsets37 and may interfere with the ability of hosts to clear and/or prevent re-infection. Although clinical immunity may protect children from malaria-associated morbidity and mortality, asymptomatic infection is an important driver of Pf transmission7. Understanding the mechanisms and determinants of clinical immunity thus remains critically important for the success of malaria elimination strategies and for the development of novel vaccination strategies.In conclusion, our data support a growing body of studies which suggest that Vδ2Written informed consent was obtained from the parent or guardian of all study participants. The study protocols were approved by the Uganda National Council of Science and Technology (HS 1019), the Makerere University School of Medicine Research and Ethics Committee (Rec No. 2011–167), and the University of California, San Francisco Committee on Human Research (11–05995). All research was performed in accordance with the Declaration of Helsinki and Good Clinical Practice.44. In all households, all eligible children aged 6 months to 10 years were enrolled into the study beginning in August of 2011. The cohorts are dynamic, with children exiting the study at age 11, and new children born into study households enrolled at 6 months of age.Samples were obtained from children enrolled in the East African International Centers of Excellence in Malaria Research cohorts. These participants live in 100 randomly selected households from each of two study sites with different transmission intensities: 1) Nagongera sub-county in Tororo district, a setting with holoendemic malaria transmission and an estimated annual entomological inoculation rate (aEIR) of 310 bites per person year, and 2) Walukuba sub-county in Jinja district, a lower transmission setting with an aEIR of 2.8 infectious bites per yearPlasmodium parasites, the patient was diagnosed with malaria regardless of parasite density, and treated with artemether-lumefantrine. Routine assessments were performed in the study clinic every three months, including blood smears and dry blood spots to detect for parasite infection. Negative blood smears obtained at routine assessments were tested for the presence of submicroscopic malaria parasites using loop-mediated isothermal amplification (LAMP)45.Upon enrollment all study participants were given an insecticide treated bed net and followed for all medical care at a dedicated study clinic. All participants were reimbursed for travel to and from clinic, and participants agreed to avoid all antimalarial medications administered outside the study. Children who presented with a fever (tympanic temperature >38.0 °C) or history of fever in the previous 24 hours had blood obtained by finger prick for a thick smear. If the thick smear was positive for 44. Each month, miniature Centers for Disease Control and Prevention light traps  were positioned with the light 1 meter above the floor at the foot end of the bed where a cohort study participant slept. Traps were set at 7.00 PM and collected at 07.00 AM the following morning by field workers. A mosquito exposure rate was calculated for each individual based on the number of female Anopheles mosquitoes captured during the 12 months following the blood draw/number of house nights of collection44. Mosquito exposure rate was used in analysis as a categorical variable .Entomological surveys were conducted monthly beginning in October 2011P. falciparum infection with a parasite density >5,000/uL; 3) absence of complicated malaria; 4) no clinical suspicion of concurrent non-malarial illness; 5) no malaria in the prior month. Convenience sampling was used to select study participants to ensure that similar numbers of children in each of 3 age groups  were included. At the time of each blood draw, 3 to 5 mls of blood were obtained in acid citrate dextrose tubes. 25 μl of fresh whole blood was stained with CD3 PerCP, CD4 FITC, Vδ2 PE, and Vγ9 APC  for 20 minutes in a Trucount Tubes (Becton Dickinson) and then lysed and permeabilized with BD FACS Lyse buffer prior to acquisition on an Accuri C6 Cytometer (BD Biosciences).Repeated assessments of T cell absolute counts were performed at the time of symptomatic malaria and 3,6, and 9 weeks following the malaria episode, in a subset of children in the Nagongera cohort (n = 43). Children were enrolled in this “symptomatic malaria cohort” if they met the following criteria: 1) duration of fever <48hrs; 2) in vitro stimulation were made using cryopreserved specimens collected between February and August of 2013 at a scheduled “immunology visit.” For this “immunology visit”, children were bled if they were currently afebrile and did not have a recorded episode of malaria in the last 7 days. For this “immunology” visit, negative blood smears were tested for the presence of submicroscopic malaria parasites using PCR.Vδ2 phenotypic, absolute count, and functional assessments were obtained using convenience sampling at the time of routine assessments in age-matched children enrolled in both the high  and lower transmission  cohorts. Not all assessments were performed in all individuals due to sample availability. Measurements of cell surface phenotype and absolute counts were made utilizing fresh samples obtained at routine visits occurring between February of 2014 and July of 2015. Measurements of cytokine production following —PerCP (B6), anti-Vγ9 APC (B3), and anti-CD57 FITC (HCD57) and from R&D, anti-Tim-3-PE (344823), fixed with 1% paraformaldehyde, and acquired on a FACS Canto flow cytometer (BD Biosciences) with FACSDiva software.At the time of routine visit, 3 to 5 mls of blood were obtained in acid citrate dextrose tubes. Absolute count measurements were performed on 25 μl of fresh whole blood as above. Peripheral blood mononuclear cells (PBMC) were then isolated by density gradient centrifugation  and counted. 1 × 10^6 freshly isolated PBMC were stained for 30 minutes with the following cell-surface antibodies: from BD Pharmingen, anti-CD8-APC-H7 (SK1); from Biolegend, anti-CD3-Pacific Blue (SK7), anti-Vδ2falciparum infected red blood cell (RBC) stimulation via intracellular cytokine staining was performed as previously described9. Briefly, thawed PBMCs were rested overnight in 10% fetal bovine serum (Gibco) and counted before stimulation with P. falciparum infected RBCs or uninfected RBCs. A total of 106 PBMCs were stimulated with intact purified trophozoite/schizont-stage P. falciparum (clone 3D7)–infected RBCs or uninfected RBCs at an effector to target ratio of 1:2. Parasite cultures were routinely confirmed to be mycoplasma negative. Following 6 hours of stimulation, Brefeldin A and monensin (BD Pharmingen) were added (10 μg/mL). At 24 hours, cells were washed, and surface and intracellular staining was performed with the following antibodies: from BD Pharmingen, anti-CD3-PerCP (SK7), anti-CD8-APC-H7 (SK1), anti-IFNγ- PE-Cy7 (B27), and anti-TNFα- FITC (6401.1111); from Biolegend, anti-CD4-BV650 (OKT4), anti-CD14-Alexa700 (M5E2), anti-CD19-Alexa700 (HIB19); from Miltenyi anti- Vδ2—APC (123R3); and from Invitrogen, Live/Dead aqua amine. Additional experiments utilized anti-Tim-3 PE  and anti-CD57 BV 421 . Samples were acquired on a BD LSR2 flow cytometer with FACSDiva software.At the time of the “immunology visit,” 6 to10 mls of blood were obtained in acid citrate dextrose tubes. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation , counted, and cryopreserved in liquid nitrogen prior to use. Analysis of Vδ2 T cell responses to P. −CD19−Aqua−/CD3+/Vδ2+. For cell surface phenotypes, gates were set using fluourescence-minus one controls. Cytokine production was gated as the percentage of Vδ2+ T cells producing IFNγ and TNFα. To calculate frequencies of P. falciparum–specific T cells, background responses to uninfected RBCs were subtracted from each subset. Thresholds for cell surface phenotype were set based on fluorescence-minus-one controls.Flow cytometry data were analyzed using FlowJo software . Color compensation was performed using single-color cell controls or beads stained for each fluorochrome. Absolute cell counts were calculated using the manufacturer recommendations (BD Biosciences). For surface phenotype and functional assays, cells were gated as lymphocytes/singlets/CD14All statistical analyses were performed using Prism 6.0 (GraphPad) and/or STATA version 14 (College Station).Comparisons of absolute counts between timepoints were made using the Wilcoxon matched pairs signed-rank test. Associations between age strata and Vδ2 T cell absolute counts at 0, 3, 6, and 9 weeks following acute malaria were measured using generalized estimating equations regression analysis with robust standard errors to account for repeated measures in the same child, after adjustment for day 0 parasite density. These models were assessed for significant interaction between age strata and weeks following the acute malaria episode.Comparisons of immune parameters between groups were made using the Wilcoxon rank sum test. Continuous variables were compared using Spearman correlation. Associations between Vδ2 T cell responses and the subsequent odds of infection or symptoms when infected were assessed in the year following the assessment. For these analyses, only the infection status at the time of routine quarterly surveillance was considered. At each routine assessment, children were divided into one of four mutually exclusive categories: 1) Symptomatic malaria, with a window of 21 days prior to and 7 days following the routine visit to ensure capture of malaria episodes that were recently treated or infections that soon became symptomatic; 2) Asymptomatic, microscopic (blood smear+) infection; 3) Asymptomatic, submicroscopic (LAMP+) infection; or 4) No evidence of parasite infection. Associations between Vδ2 T cell responses and the prospective odds of any infection (including submicroscopic and microscopic infections), sub-microscopic infection (given infection), or symptoms when infected (detected by microscopy) were calculated using multilevel mixed-effects logistic regression. This analysis accounted for repeated measures within individuals and was clustered on household to account for multiple children living in the same household. In multivariate analysis, odds ratios for infection risk were adjusted for age  and mosquito exposure ; odds ratios for symptoms when infected were adjusted for age. In all models, non-normal variables were log-transformed where appropriate. In all analyses, a 2-tailed P value < 0.05 was considered to be statistically significant.Supplementary Figures


Control of malaria increasingly involves administration of 8-aminoquinolines, with accompanying risk of haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Few data on the prevalence and genotypic basis of G6PD deficiency are available from Bangladesh, where malaria remains a major problem in the South (Chittagong Division). The aim of this study was to determine the prevalence of G6PD deficiency, and associated G6PD genotypes, in adults with falciparum malaria in southern Bangladesh.G6PD status was assessed via a combination of fluorescent spot testing (FST) and genotyping in 141 Bengali patients admitted with falciparum malaria to two centres in Chittagong Division from 2012 to 2014. In addition, an analysis of genomic data from 1000 Genomes Project was carried out among five healthy Indian subcontinent populations.One male patient with uncomplicated malaria was found to have G6PD deficiency on FST and a genotype associated with deficiency (hemizygous Orissa variant). In addition, there were two female patients heterozygous for deficiency variants . These three patients had a relatively long duration of symptoms prior to admission compared to G6PD normal cases, possibly suggesting an interaction with parasite multiplication rate. In addition, one of 27 healthy local controls was deficient on FST and hemizygous for the Mahidol variant of G6PD deficiency. Examination of 1000 Genomes Project sequencing data across the Indian subcontinent showed that 19/723 chromosomes (2.63%) carried a variant associated with deficiency. In the Bengali from Bangladesh 1000 Genomes population, three of 130 chromosomes (2.31%) carried deficient alleles; this included single chromosomes carrying the Kerala-Kalyan and Orissa variants.In line with other recent work, G6PD deficiency is uncommon in Bengalis in Bangladesh. Further studies of particular ethnic groups are needed to evaluate the potential risk of wide deployment of primaquine in malaria control efforts in Bangladesh. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzyme deficiency present in more than 400 million people worldwide . The simThe coincident distribution of G6PD deficiency and malaria leads to a practical problem since the 8-aminoquinoline primaquine, the only drug active against hypnozoites used as radical treatment of vivax malaria, can cause severe haemolysis in G6PD deficient individuals . Thus, dIn Bangladesh, approximately 14 million people are at risk for malaria, with over 85% of cases occurring in Chittagong Division (80% in the Chittagong Hill Tracts) . At the The study was undertaken at two sites in southern Bangladesh (Chittagong Division) as part of a prospective observational study assessing the pathophysiology of falciparum malaria and a randomized controlled trial of paracetamol in severe and moderately severe malaria from 2012 to 2014. Ramu Upazila Health Complex is a rural centre in the District of Cox’s Bazar, ten miles east of Cox’s Bazar town, close to the Chittagong Hill Tracts and Myanmar border. Chittagong Medical College Hospital is an urban tertiary care centre in the city of Chittagong. Written, informed consent was obtained from all patients or attending relatives. Ethical approval was obtained from the Oxford Tropical Research Ethics Committee and Chittagong Medical College Hospital. ClinicalTrials.gov registration: NCT01641289.P. falciparum slide confirmed malaria were enrolled consecutively upon diagnosis. Criteria for severe malaria were: coma (Glasgow Coma Score <11), shock [systolic blood pressure (SBP) <80 mmHg with cool extremities], anaemia, , jaundice , hyperparasitaemia (>10%), acidosis (bicarbonate <15 mmol/l), hyperlactataemia (lactate >4 mmol/l), hypoglycaemia (glucose <2.22 mmol/l), convulsions (≥2/24 h), pulmonary oedema, and/or AKI (creatinine >265 μmol/l). Uncomplicated malaria was defined as slide positive malaria without severity criteria. Patients were managed according to WHO treatment guidelines . None of the G6PD deficient patients had visible haemoglobinuria on enrolment or during admission. Of the G6PD normal patients that had haemoglobinuria (n = 18), the median cell-free haemoglobin concentration on admission was 14.7 μM (10.7–40.8 μM).Clinical data for the three malaria cases with evidence of G6PD deficiency mutations were examined Table . ParasitPfHRP2 concentration. The cell-free haemoglobin was lower on enrolment compared to G6PD normal uncomplicated malaria patients and exhibited a bimodal pattern with a peak of 6.0 μM 42 h after G6PD normal patients. The creatinine was normal on admission and he did not develop kidney dysfunction. His recovery was unremarkable although he presented again 6 weeks later with Plasmodium vivax infection.Case 1 was a 27-year old male with G6PD deficiency Orissa variant and uncomplicated malaria. Haemoglobinuria was absent on admission and did not develop during admission. In the absence of prior anti-malarial treatment, his parasitaemia was above the 75% percentile of G6PD normal uncomplicated malaria patients, which was accompanied by an elevated Case 2 was a 32-year old female heterozygous for the Orissa mutation (FST not done) with severe malaria, acute kidney injury (creatinine >265 μmol/l), and acidosis (bicarbonate <15 mmol/l) presenting after 2 days of oral artemether/lumefantrine. She did not present with or develop blackwater fever but gave a history of red urine prior to admission and had haemoglobin in her urine by dipstick on enrolment and at 76 h. Anaemia was present on enrolment, requiring three blood transfusions during admission. The fractional excretion of sodium was >2% suggesting acute tubular injury, while the urine pH (<5.3) and urine anion gap suggested a type 2 renal tubular acidosis. Cell-free haemoglobin on enrolment was below the 75% percentile compared to G6PD normal severe malaria patients but increased to 6.7 μM at 72 h. Dialysis was initiated 6 h after enrolment and an additional 5 cycles were required. She was discharged 3 weeks after enrolment and kidney function returned to baseline at 6 weeks.Case 3 was a 55-year-old female heterozygous for the Kerala-Kalyan mutation with uncomplicated malaria enrolled after three doses of intravenous quinine. Anaemia was present at enrolment but no blood transfusion was required. Haemoglobin was absent from the urine throughout. Cell-free haemoglobin was low on admission but increased to 16.2 μM at 24 h. Creatinine was normal on enrolment and acute kidney injury did not develop during admission. Although the FST was intermediate at admission, at 4-week follow-up a normal result was obtained.P. falciparum malaria in two centres in southern Bangladesh. One out of 141  P. falciparum patients was G6PD deficient on the basis of fluorescent spot testing followed by genotyping, while two patients were heterozygous at known deficiency alleles. A combination of phenotyping and genotyping was used for several reasons. Firstly, the FST accurately diagnoses severe G6PD deficiency [This study found that G6PD deficiency was uncommon in Bengalis admitted to hospital with ficiency  at activficiency , 29, 30.One particular factor that might have led to intermediate FST results (but no genotypic evidence of G6PD deficiency) could have been the observed heterogeneity in FST appearances according to haemoglobin level. It was noted that the haemoglobin concentration was significantly higher among the intermediate group compared to the G6PD normal group; this is consistent with the observation that G6PD-normal anemic subjects  tend to show a brighter fluorescence compared with G6PD-normal subjects without anaemia . This deTwo studies of G6PD deficiency in Bangladesh were published during the conduct of our study. A phenotypic survey of G6PD deficiency in malaria patients undertaken in Bandarban Division, Chittagong Hill Tracts, Bangladesh , showed P. falciparum. However, 1000 Genomes Project data  also show a low prevalence of deficient genotypes in Bengalis from Bangladesh as well as several other populations from the Indian subcontinent. An alternative explanation is that the prevalence of deficiency may be significantly higher within relatively isolated ethnic groups owing to the forces of positive selection as well as genetic drift. Methodological differences in terms of blood storage and measurement might also explain the difference in prevalence. Further studies of the genotypic basis of G6PD deficiency in various ethnic groups in the Chittagong Hill Tracts are needed to shed further light on this area, and are indicated given the potential risk in these populations of haemolysis with pro-oxidant drugs such as primaquine .In contrast, a study of randomly sampled individuals without malaria from the Marma and Khyang ethnic groups  also living in the Chittagong Hill Tracts (CHT) showed that 59% had normal G6PD activity, 35% had mild deficiency and 6.5% had severe deficiency ; similarThe three variants identified in this study were Orissa, Kerala-Kalyan, and Mahidol. While the study did not quantitatively assess G6PD deficiency, other studies have found these mutations to be associated with ~10% , ~20% 3 and <10%P. vivax administered daily 0.5 mg/kg primaquine, among those with a creatinine measurement 5/16 (31%) developed acute kidney injury [Due to the low number of G6PD deficient malaria patients detected in this study, it was not possible to examine in any detail the potential interaction between the forms of deficiency found and the severity of falciparum malaria or its form of illness. Evidence largely gathered from Africa indicates that G6PD deficiency protects against cerebral malaria –7 and agy injury .G6PD deficiency is not common in Bengalis with falciparum malaria assessed in two centres near Chittagong Hill Tracts, Bangladesh. One of 141 patients was deficient on FST screening and hemizygous for the Orissa G6PD variant, and there were also two heterozygote females , while a single case of hemizygous Mahidol deficiency was found among healthy controls. The overall prevalence of deficient alleles was similar to that in a healthy population of Bengalis from Bangladesh studied in the 1000 Genomes Project, and other populations across the wider Indian subcontinent. Further studies are required to determine whether the studied variants have a protective effect against malaria infection, to determine the haemolytic risk in response to malaria infection and 8-aminoquinolines, and to explore further the prevalence and genetic basis of G6PD deficiency in other ethnic groups in Bangladesh.


Plasmodium falciparum malaria have been observed across much of Africa in the past decade. However, regions of high relative prevalence and transmission that remain unaddressed or unrecognized provide a threat to this progress. Difficulties in identifying such localized hotspots include inadequate surveillance, especially in remote regions, and the cost and labor needed to produce direct estimates of transmission. Genetic data can provide a much-needed alternative to such empirical estimates, as the pattern of genetic variation within malaria parasite populations is indicative of the level of local transmission. Here, genetic data were used to provide the first empirical estimates of P. falciparum malaria prevalence and transmission dynamics for the rural, remote Makira region of northeastern Madagascar.Encouraging advances in the control of P. falciparum prevalence at three seasonal time points separated by 4-month intervals. Genomic loci in a panel of polymorphic, putatively neutral markers were genotyped for 94 P. falciparum infections and used to characterize genetic parameters known to correlate with transmission levels.Longitudinal surveys of a cohort of 698 total individuals  were performed in two communities bordering the Makira Natural Park protected area. Rapid diagnostic tests, with confirmation by molecular methods, were used to estimate P. falciparum over the 10-month course of the study, a rate approximately sevenfold higher than the countrywide average for Madagascar. Among those P. falciparum infections, a high level of genotypic diversity and a high frequency of polygenomic infections (68.1%) were observed, providing a pattern consistent with high and stable transmission.Overall, 27.8% of individuals tested positive for P. falciparum transmission in Madagascar. This suggests that the area should be highlighted for future interventions and that additional areas of high transmission may be present in ecologically similar regions nearby.Prevalence and genetic diversity data indicate that the Makira region is a hotspot of The online version of this article (doi:10.1186/s12936-016-1644-4) contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria are recorded annually in Madagascar, an incidence rate that, at the reported level, places Madagascar and its population of 24.9 million among the lower tier of African countries in terms of malaria burden . Thdagascar .Apart from the two study sites, approximately 127,000 people live in the Makira region, all in rural settings with comparable ecological conditions. Additionally, an estimated 86.8% of the 1 million people living in the broader Analanjirofo administrative region (containing the Makira region) also live within similar rural districts . FurtherP. falciparum transmission such as Panama [An alternative possibility is that the prevalence and incidence seen at the two Makira region sites studied here merely reflects a transient spike. This possibility seems unlikely, because if transmission were sporadic and we simply happened to observe a temporary, unstable peak in transmission in this study, then a pattern of low diversity, frequently repeated MLGs, and fewer polygenomic infections would be expected in the genetic data. In settings of low, unstable transmission, sometimes termed clonal transmission, few MLGs predominate and the reshuffling of those MLGs by recombination or re-assortment is rare. This pattern of clonal transmission has been observed in locations at the periphery of s Panama  and in as Panama , 19.The pattern observed here is the opposite, as we see very limited evidence of repeated MLGs and a high frequency of polygenomic infections for all subgroups of the sample set , then it would be expected that the average similarity among polygenomic infections would be similar to the average between MLGs randomly chosen from the population. Genetic similarity among polygenomic infections was significantly higher than the average between monogenomic infections, consistent with the presence of co-transmission.If these two confounders were present in this study, then it would be expected to see (1) a higher average level of genetic similarity among polygenomic infections than between monogenomic infections and (2) evidence that P. falciparum populations and therefore their effect is accounted for when comparing populations. Further study of co-transmission and spatial heterogeneity, especially in Madagascar, would serve to better characterize this relationship.As evidence against unusually extreme spatial heterogeneity in our sample, a high percentage (63.0%) of households contained an individual infected at one time point or more. Additionally, there was no evidence that genetic similarity was partitioned between communities. It seems more likely that co-transmission and/or spatial heterogeneity are widespread phenomena in P. falciparum genetic diversity in Madagascar have been performed, the identification of variable markers, the estimates of minor allele frequencies, and the calculation of the number of loci needed for a given level of resolution will be useful in future studies. Such studies are needed to better define the relative contributions of co-transmission and small-scale spatial heterogeneity to patterns of parasite genetic diversity. Remaining hotspots of P. falciparum transmission pose a threat to the recent gains in malaria control in Madagascar, and across Africa, and a better understanding of the genetic correlates of transmission can provide a better method for their identification and monitoring.Despite the abundance of estimates for low to moderate levels of malaria transmission on average regionally and countrywide, we show evidence that the Makira region in northeastern Madagascar is a hotspot of malaria transmission. First, RDT surveys, with RT-PCR confirmation generally validating the results, were performed and an overall level of infection approximately sevenfold higher than the national average and 2 to fivefold higher than existing regional estimates was observed. Next, a panel of SNP markers was validated as variable and informative in Madagascar, and these SNPs were used to estimate the allele and MLG frequencies as well as the frequency of polygenomic infections. A pattern consistent with high, stable transmission was observed. Because few previous studies of