Plasmodium falciparum among some falciparum malaria-infected patients in Southeastern Iran.To overcome human malaria problem several solutions have been employed including extensive studies in the field of Plasmodia relevant antigens. The aim of this study was to determine allelic variation in the MSP1 gene of P. falciparum positive cases were enrolled from Sistan and Baluchistan Province, southeastern Iran in 2013–15. From each case, 1.5ml of peripheral blood was collected into EDTA contained tubes. Thick and thin blood smears were stained with standard Giemsa stain and were checked with conventional microscopical method. DNA was extracted from blood samples and amplification of block 2 MSP1 was performed using specific primers. Gel electrophoresis was done and results showed some amplification fragments corresponding to block 2 regions of Pf MSP1 gene. Finally, four samples from different allelic types were sent for sequencing process.Twenty Fragments were different in size, so classified into six allelic types as kinds of 1–6 based on happening frequencies. Digestion of PCR products revealed two sub allelic types (A and B) within allelic types 2 and 3, but not in allelic types 1, 4, 5 and 6. Twenty percent of samples were sent for sequencing. Sequence alignment showed 78.95% to 91.83% identity between samples.Identity between samples and phylogenetic tree revealed that there is an extensive diversity range among isolates. Fifty percent of the isolates were under the risk of complicated malaria. Two of these patients (10%) needed special care and recovery was obtained after getting hospital services. Plasmodium falciparum. Cerebral malaria, blackwater fever, severe anemia and some other disorders occur in individuals who infected with P. falciparum. Such problems can result in death among the infected people if is not being treated with suitable drug.Malaria is an ancient problem created many calamities against human for many centuries. Parasites of the infection cause illness to 216 million persons and almost 445000 deaths per year. Pregnant women and children under 5yr old are two greatest groups damaged from this infection . The feaP. falciparum isolates. However, diversities in MSP1 gene demonstrate different alleles which such diversities can reveal different aspects of phenomena in P. falciparum. One of the most important aspects of the phenomena is emergence a number of complications in patients who infected with P. falciparum  according to the manufacturer instructions.DNA of PCR amplification of the block2 MSP1 was performed in 25µl reaction that containing, 10 µl Distilled water, 10µl master mix, 3ng DNA, 1µl forward primer, 1µl revers primer. Designing primers and PCR condition were performed .The sets of following primers were used for amplification: Forward 5′CTAGAAGCTTTA GAAGATGCAGTATTG3′ Revers 5′GTACG TCTAATTCATTTGCACG AAT3′ PCR amplification was carried out using initial denaturation about 5min in 95 °C, 30 cycles of 1 min at 94 °C, 2min in 57 °C, 2min in 72 °C and final extension 5min in 72 °C in a thermal cycler. Four µl of PCR product was run on 1.5% agarose gel with simply safe . Finally the process was visualized with gel duct and resulted in some photographs.Product amplification of MSP1 gene block 2 zones was digested with Dra-1 endonuclease with active site of TTT AAA (Cat: FD0224). The digestion process was carried out in a 30 µl composition of 17µl distilled water, 2µl 10 X fast digest buffer, 10µl PCR product and 1 µl fast digest enzyme. The compound was incubated at 37 °C for 5min and the incubation was continued at 65 °C for 5min to inactive the enzyme. Overall 18µl of the digested PCR product was analyzed on a 3% agarose gel and then stained with simply safe stain. Visualizing process was conducted as mentioned previously.Four PCR products were selected for sequence processes and analyzed with ClustalW2 and Blast software.P. falciparum block2 MSP1 gene was considered from clinical isolates collected from Sistan and Baluchistan Province in southeastern Iran  within allelic types 2 and 3. While allelic types 1, 4, 5, 6 did not show any subtype , Fig. 2.analysis .PfMSP1 gene, a few of them have been focused on relationship between the gene and probable malignancy of P. falciparum (Investigations about genetic diversities in parasite population can prepare much information in the field of geographical distribution, genomic markers, clinical manifestation, drug resistance and gene polymorphism for relevant researchers –12. Genelciparum . There ilciparum , 7, 10.P. falciparum have shown an extended variation in size and pattern (P. falciparum. There was no significant relationship between genetic diversity of MSP1 and severity in malaria, however, Mad20 allelic type increased with the severity of malaria. On the other hand, there are a few alleles in Pf MSP1 gene which are more predominant in cerebral malaria when they used PCR-RFLP to investigate diversity in the gene. Five allelic types including eight genetic types were identified, they implied that sub allelic types 2A and 3A with 300–200 and 280–200bp were more prevalence in patients got involved with cerebral malaria these sub allelic types were recognized when they used PCR-RFLP with Dra-1 restriction enzyme. Moreover, sub allelic 2B with 250–25bp was seen in both cerebral and non-cerebral malaria cases. Presence of certain alleles may cause severe disease in patients.Obtaining different results about the mentioned subject are due to the different employed methods to found diversity in this gene. For example nested PCR was used to found frequency of K1, Mad20 and Ro33 alleles in the parasite population –20. Howe pattern . Any corfalciparum malaria in endemic areas.In the present study six allelic types were classified by PCR-RFLP, also two sub allelic types were identified when PCR products digested with Dra-1, results showed that there is a large diversity in the weight of the products fragments when compared with Farooq et al results . So, divP. falciparum 3D7. There is high diversity in the gene in the specific population of the parasite.Moreover, sequence alignment among the samples showed 78.95% to 91.83% identity. There is an extension of diversity among the samples. Phylogenetic tree revealed that there is an extensive diversity range between isolates so that some of the isolates achieved in the present study showed a close relationship with isolate HN2 from China and the others have this association P. falciparum in Iranian isolates showed an extensive diversity, moreover, potential characteristics to produce sever malaria particularity cerebral malaria is a burglar alarm for the health authorities to provide accessible facilities for residents in malaria endemic area.MSP1 gene of


E. histolytica CRT (EhCRT) in the processes of pathogenicity or in the modulation of the host immune response. The aim of this study was to evaluate the role of CRT in the proliferation and the cytokine profile in peripheral blood mononuclear cells (PBMCs) from patients with amebic liver abscess (ALA) during the acute phase (AP-ALA) of the disease compared to patients during the resolution phase (R-ALA). The PBMCs from each participant were cocultured with EhCRT and tested by the colorimetric method to evaluate their proliferation index (PI). The supernatants were subjected to an enzyme-linked immunosorbent assay (ELISA) to evaluate the concentration of cytokines. The mean values of all groups were compared using the independent t-test. When the PIs of individuals without diagnosis of liver abscess (NEG) were compared, there were no statistically significant differences in the proliferation of PBMCs between patients with AP-ALA and R-ALA when stimulated with EhCRT or concanavalin A (ConA). However, the levels of interleukins  were higher in patients with AP-ALA, whereas in patients with R-ALA, higher levels of interferon gamma (IFNγ) were detected. These results suggest that EhCRT acts as a mitogen very similar to the activity of ConA. In addition, EhCRT is an excellent immunogen for the specific activation of PBMCs, inducing the differential expression of ILs depending on the outcome of disease, determining the type of immune response: a Th2 cytokine profile during the acute phase and a Th1 profile during the resolution phase.Calreticulin (CRT) is a highly conserved protein in the endoplasmic reticulum that plays important roles in the regulation of key cellular functions. Little is known about the participation of  E. histolytica is one of the leading causes of death worldwide. The disease is a consequence of the parasite's abilities to invade the colon, causing amebic colitis. E. histolytica can disseminate to the liver via the portal venous system, resulting in amebic liver abscess (ALA). However, approximately 90% of infected people are asymptomatic cyst carriers  from E. histolytica, which promotes the accumulation of mononuclear cells, including neutrophils, inflammatory monocytes, and macrophages, at the site of infection , growth-regulated protein alpha (GRO-α), granulocyte macrophage colony-stimulating factor (GMCSF), and IL-1  results in the secretion of chemoattractant and proinflammatory cytokines  that breakdown IgA1 and IgA2 antibodies. These proteins cleave the Fc region that interacts with parasite surface receptors and mediates effector functions that can mask immunogenic surface molecules with inert Fab fragments, thus helping to prevent the parasite expulsion from the intestinal lumen  have been cloned and sequenced  in host–parasite interactions has recently become a major area of research. The CRT genes from many parasites  and that this protein induces an important immunogenic response in the human host. More than 90% of patients with ALA develop high levels of serum antibodies against EhCRT  cultures isolated from ALA patients during the acute phase of the disease and from individuals who resolved ALA in order to characterize the cytokines profiles produced in response to The present work was designed according to the guidelines for the management of human samples for experimental purposes as indicated in the Official Regulation NOM-12SSA3-2007 included in the General Health Law of Mexican Health Ministry. In addition, the project was approved by the Scientific and Ethics Committee of the Faculty of Medicine from the National Autonomous University of Mexico. Patients were informed about the purposes of the project, the sampling, and the potential risks during procedure, and the patients were invited to voluntarily participate by signing an informed consent letter.E. histolytica by enzyme-linked immunosorbent assay (ELISA), using an OD490nm ≥0.5 as an indicator of a positive result . The patients in the resolution phase of ALA (R-ALA) were recruited from a search of the archives of discharged patients from the General Hospital of Mexico, and they formed the ALA resolution group (R-ALA). Three fecal samples were collected from each patient from both groups at 1-week intervals after their hospitalization for microscopic examination for parasites. At the time of collection of the third sample, 10 ml of blood was drawn to evaluate serum antibodies against A third group, named the negative control (NEG), was formed with clinically healthy individuals from the Blood Bank of the General Hospital of Mexico, with ELISA serum levels of antiamebic antibodies below the OD threshold (OD < 0.5). Using the same protocol for ALA groups, three fecal samples and 10-ml blood samples were taken for microscopic examination and PBMC isolation, respectively. Ten individuals for the group were included.6 cells/ml) were incubated with rEhCRT (5 μg/ml) at 37°C with 5% CO2; concanavalin A (ConA)  was used as a stimulating factor at 10 μg/ml for different periods of time . Each experiment was performed in duplicate. At each time point, the cultures were centrifuged for 10 min at 1,000 × g, and the supernatant was reserved for cytokine analysis.The PBMCs were isolated from 10 ml peripheral blood samples collected from each participant in tubes with K2 ethylenediaminetetraacetic acid (EDTA) as anticoagulant . Cells were separated on a Ficoll-Hypaque gradient , and the PBMC pellet was separated and washed three times with phosphate-buffered saline PBS. After separation, PBMCs were centrifuged and resuspended in Roswell Park Memorial Institute medium RPMI culture medium supplemented with 10% fetal bovine serum. The cells   was used to purify the recombinant protein, and, briefly, the cells were harvested by centrifugation at 3,000 × g for 12 min, and the bacterial pellet was resuspended in 5 ml lysis buffer . The lysate was added to a 50% suspension of Ni-NTA agarose . The mixture was passed through a filtration column, and the recombinant protein was eluted with 8 M-urea buffer pH 4.5. The selected fractions were dialyzed against 19 mM PBS. Protein concentration was determined by the Bradford method, and the quality was evaluated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The reactivity grade of the recombinant protein was tested against sera from patients with ALA and for anti-E. coli lipopolysaccharide antibody (LPS) antibody  by Western blot.Full-length rEhCRT or ConA and incubated at 37°C for 1 h in a humidified incubator with 5% CO2. After incubation, the plates were centrifuged at 1,000 × g for 10 min and the supernatant was discarded. The cells were resuspended in 300 μl dimethyl sulfoxide (Sigma) and the optical density (OD) was measured at 570 nm in an ELISA plate reader (EL × 800 BioTek). The proliferation index was calculated by [OD of test sample—OD of negative control/OD of negative control]. -2,5-diphenyltetrazolium bromide] (MTT) (Sigma) was added to each well containing PBMCs incubated with rEhCRT, ConA, or without stimulus (RPMI-10% SFB), were tested for detection of interleukins IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17A, interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), granulocyte colony stimulating factor (GCSF), and transforming growth factor beta1 (TGFβ1) using a multianalytic ELISA array kit , according to manufacturer's instructions. The concentrations of the cytokines are given in pg/ml and were calculated using the standard curve provided in the kit.The supernatants of the PBMC cultures, treated with rt test for unpaired results was used for the evaluation of differences between cytokine concentrations in each group. Differences were statistically significant when P ≤ 0.05.All values are expressed as the means ± S.D. The student's 490nm values, considering negative results when values were under the cut value of 0.520. The mean value for the negative group was 0.15, the AP-ALA group was 0.87, and the R-ALA group was 1.0.The demographic features of each group are shown in Table EhCRT from E. coli appeared as a single band at 60 kDa on 12% SDS-PAGE after bromophenol blue staining   assays, comparing the stimulatory capacity of rEhCRT vs. the mitogen activity of ConA. The differences in the PI values were not statistically significant for any time point when comparing between the NEG and ALA groups   Figure .The concentrations of cytokines in PBMC samples obtained for sera from the ALA and NEG groups and treated with EhCRT were measured through ELISA only at 72 h Figure . The patThe trend of the data agrees with an overexpression of all interleukins analyzed in all groups.p = 0.0035, 0.0023, 0.019, 0.006, 0.0076, 0.0049, 0.0039, 0.0035, 0.0059, respectively).However, in the ALA groups, the overexpression was, in general, larger than in the negative group, and significant differences were measured in the expression of the following interleukins; IL-2, IL-5, IL-6, IL-10, IL-17, IFNγ, TNFα, GCSF, and TGFβ1 , which were higher in the AP-ALA group. In the R-ALA group, we observed higher overexpression of the interleukins IL-17, INFγ, and TNFα; however, statistically significant differences were detected only for INFγ .The comparisons of differential expressions of the interleukins between groups of AP-ALA and R-ALA are shown in Figure in vitro with rEhCRT or ConA and determine the cytokine profiles induced in the different groups. The responses of the PBMC show that EhCRT is one of the many immunogenic proteins in E. histolytica that can induce activation and proliferation of PBMCs similarly to ConA. These results reinforce our previous observations that the EhCRT is highly immunogenic in humans, mice, and rabbits  and anti-inflammatory cytokines such as IL-10, as well as the low expression of chemokines CCL1, CCL4, and the receptor CCR1 in human monocytes  is produced by In the group of patients with AP-ALA, the cytokines IL2, IL5, Il6, IL17A, IFNγ, and TNFα displayed an increase in their concentration and demonstrated that the immune response had a pro-inflammatory profile. This response has been observed in stimulation assays using Gal/GalNAc-lectin in intestinal cell cultures  secreted TNF-α, IL-6, IL-8, IL-12, and IL-10 via TLR2 (Maldonaldo-Bernal et al., E. histolytica is not clear. One of these mechanisms could be associated with the ability of parasites to modulate cytokine expression in the inflammatory process, which is initiated by expression of proinflammatory cytokines. E. histolytica infections induce a state of transient suppression of cell-mediated immunity in early stages of inflammation in amebic hepatic abscess, and a complex signaling system of cytokines is triggered by pathogen invasion (Eckmann et al., Host protective immunity involves participation of both humoral and cellular responses; however, the mechanism involved in the immune evasion of EhCRT behaves like an amebic immunogenic protein for humans and suggest that the EhCRT participates in the specific stimulation of immune cells.The data obtained in this study confirmed that the EhCRT can stimulate human PBMC proliferation independently of the presence of E. histolytica trophozoites, acting as a specific costimulator of the immune response like that induced by ConA. In addition, these results underline EhCRT as a parasitic factor that can modulate the immune response, from the stimulation of proinflammatory cytokines to the immunosuppressive effects, depending on the progression of the ALA, thus inducing the development of a Th2 cytokine profile in the acute phase of disease and a Th1 profile once the individuals had resolved the ALA.Our results suggest that the rEhCRT and its role in the pathogenesis of ALA need further research, particularly on the interaction with the cells of immune system and the induction of chemokines and cytokines regulators that hopefully will allow a better understanding of the pathogenesis of ALA.The functions of CX conceptualized the manuscript. EG, MN-R, UM. PM, OP-R, EH, LR, and AS curated the data. CX, EG, MN-R, PM, OP-R, EH, LR, and AS performed the formal analysis. CX acquired the funding. EG, MN-R, and PM performed the investigation. MN-R, PM, AS, and UM provided the resources. EG, OP-R, and EH performed the software analysis. CX, EG, MN-R, and PM provided the supervision. CX, EG, MN-R, and PM executed the validation. CX, EG, MN-R, and PM wrote the original draft of the manuscript. CX, EG, MN-R, PM, OP-R, EH, UM, and AS wrote, reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum malaria syndrome requiring urgent blood transfusion, exhibits inflammatory and hemolytic pathology. Differentiating between hypo-haptoglobinemia due to hemolysis or that of genetic origin is key to understand SMA pathogenesis. We hypothesized that while malaria-induced hypo-haptoglobinemia should reverse at recovery, that of genetic etiology should not. We carried-out a case-control study of children living under hyper-endemic holoendemic malaria burden in the sub-Saharan metropolis of Ibadan, Nigeria. We show that hypo-haptoglobinemia is a risk factor for childhood SMA and not solely due to intravascular hemolysis from underlying schizogony. In children presenting with SMA, hypo-haptoglobinemia remains through convalescence to recovery suggesting a genetic cause. We identified a haptoglobin gene variant, rs12162087 , to be associated with plasma haptoglobin levels (p = 8.5 × 10−6). The Homo-Var:(AA) is associated with high plasma haptoglobin while the reference Homo-Ref:(GG) is associated with hypo-haptoglobinemia (p = 2.3 × 10−6). The variant is associated with SMA, with the most support for a risk effect for Homo-Ref genotype. Our insights on regulatory haptoglobin genotypes and hypo-haptoglobinemia suggest that haptoglobin screening could be part of risk-assessment algorithms to prevent rapid disease progression towards SMA in regions with no-access to urgent blood transfusion where SMA accounts for high childhood mortality rates.Severe Malarial Anemia (SMA), a life-threatening childhood  The mortality rate of malaria was estimated to be 90% in Africa, and 80% of these deaths occur in sub-Saharan Africa and mostly in the under five years–of-age group due to severe, and often-overlapping syndromes such as Severe Malarial Anemia (SMA) and Cerebral Malaria (CM)2. A quarter of the global cases and a third of malaria-attributable childhood deaths occur in Nigeria, the most populous country of Africa5.The annual global incidence of clinical malaria is estimated at about 216 million cases leading to approximately 445,000 deaths in 20168. Heme, the molecule bearing the iron moiety of fHb, can be oxidized by nitric oxide (NO) leading to oxidative stress-mediated damage through mediators such as lipid and hydrogen peroxides10. To avoid this, fHb is scavenged by plasma Haptoglobin (HP)11 forming a complex, thereby preventing fHb to be oxidized and potentially preventing further heme-mediated nitric oxide damage, renal excretion of iron and further oxidative damage12.Malaria is characterized by increased inflammation and intravascular hemolysis due to schizogony leading to the release of free Hemoglobin (fHb) into the plasmaHP gene is comprised of two co-dominant alleles, HP1 and HP2, located on the long arm of chromosome 1615. The HP1 allele has five exons and the HP2 allele has seven exons  during an acute inflammatory response16. The HP-fHb complex, formed by the binding of HP to fHb, is removed from circulation by CD163 receptors expressed on the surface of macrophages followed by internalization, where fHb is degraded and heme catabolized by heme oxygenase-117.The 19 and therefore the circulatory level of HP has been used as a surrogate marker of intravascular hemolysis23. Similarly, Lactate Dehydrogenase (LDH) is known to be abundant in erythrocytes and its plasma level is elevated in the event of erythrocyte lysis. An elevated plasma level of LDH is also a surrogate marker of hemolysis in vascular pathophysiology27.A high level of fHb during severe hemolysis has been reported to be responsible for low HP plasma levels28. Circulatory HP level shows ethnic and geographic variation with a high prevalence of hypo-haptoglobinemia in Africans compared with Europeans. Recent studies report that the average frequency of hypo-haptoglobinemia in Africa might be as high as 40%29 although with different and unknown etiologies32.In some individuals, a complete lack of HP (ahaptoglobinemia) or low circulating HP (hypo-haptoglobinemia) can be observed35, while on the other hand the low level has been attributed to the malaria infection causing intravascular hemolysis36 and other immune pathological processes37. HP levels have been reported to recover in 75% in a study population following anti-malarial treatment38. While malaria infection induced hypo-haptoglobinemia should be reversed with anti-malarial treatment32, that of genetic etiology should not. Differentiating between these two etiological scenarios should provide insights into the role of HP in the establishment of mild or severe malaria syndromes.Studies on hypo-haptoglobinemia have provided conflicting views regarding its etiology in childhood malaria. On one hand, the low level of plasma HP found in malaria infections has been proposed to have a genetic basisHP genes to identify genotypes associated with both circulatory HP levels and the malarial disease syndromes studied.We hypothesized that if low HP levels are exclusively a consequence of malaria infection they would return to normal on patients’ recovery. To test this hypothesis, we carried out a prospective case-control study of children  living in the sub-Saharan 3-million inhabitants densely-populated urban metropolis of Ibadan, Nigeria, that is under hyper-endemic and holoendemic malaria burden. We assayed for levels of plasma HP and the markers of intravascular hemolysis fHb and LDH from admission through convalescence to recovery. Furthermore, we performed high throughput amplicon-based sequencing of the HP gene rs12162087 to be associated with circulatory HP level. We provide evidence that the variant is also associated with SMA.Here we show, by longitudinal follow-up of malaria-positive pediatric cohorts, that hypo-haptoglobinemia is a risk factor for acute onset childhood SMA, rather than the unique consequence of this syndrome, and it is likely of genetic etiology. We report for the first time a variant in the distal upstream region of the The internationally recognized ethical committee at the Institute for Advanced Medical Research and Training (IMRAT) of the College of Medicine, University of Ibadan (COMUI) approved this research on the platform of the Childhood Malaria Research Group (CMRG) within the academic Department of Paediatrics, University College Hospital (UCH), as well as at school and Primary Care centers throughout the city of Ibadan with permit number: UI/EC/10/0130.Parents and/or guardians of study participants gave informed written consent in accordance with the World Medical Association ethical principles for research involving human subjects.All study participants were recruited under the auspices of the CMRG at the 800-bed tertiary hospital, UCH in the city of Ibadan, Nigeria, in west sub-Saharan Africa. UCH Ibadan academic prestige, quality of care, costs and excellent accessibility from any point in the large metropolis makes it the preferred choice for all socio-economic sectors of the population. UCH Ibadan provides tertiary, secondary and it is associated with primary care centers across the city.39.Ibadan is the second most densely populated urban metropolis in Nigeria with nearly 3 Million inhabitants. The city has a lengthy 8-month rainy season, with the average of 10 rainy days per month between May and October. Malaria transmission and severe disease occur throughout the year. Although severe malaria syndromes are predominant in children under 5 years-of-age, there is a significant large burden of severe disease in children up to 16 years-of-age40, we recruited children with median age of 26–48 months-of-age between 2010 and 2013. The defined clinical cases for the malaria-positive groups include Uncomplicated Malaria (UM), Severe Malaria Anemia (SMA) and Cerebral Malaria (CM) according to WHO criteria for diagnosis of P. falciparum complications42. UM cases were defined as patients with fever and P. falciparum parasitemia who did not require hospital admission. The SMA group was defined as conscious children with packed cell volume (PCV) <16%, or hemoglobin concentration <5 g/dL, with asexual forms of P. falciparum in circulation and no other overt etiology. All SMA patients were transfused within two hours of admission and confirmation of malaria diagnosis. However, samples were collected prior to the blood transfusion. CM cases were defined as children with unarousable coma persisting for more than an hour with generalized convulsion in the presence of asexual forms of P. falciparum. A Blantyre coma score <2 was used to define coma status. Children presenting with both CM and SMA, belonging to the CM-SMA clinical group, did not form part of this study.As part of a larger case-control study of severe malariaThe UM group was recruited as part of daily routine malaria parasite screening at our children outpatient clinics at UCH Ibadan. All severe CM and SMA cases were recruited on admission from our emergency department at UCH Ibadan, the Otunba Tunwase Children’s Emergency Ward (OTCHEW). The Community Control (CC) group was comprised of malaria-negative healthy children.43. Uncomplicated malaria is treated with a 3-day oral course of Artemisinin-based Combination Therapy (ACT). On the other hand, severe malaria is treated with intravenous artesunate for at least 24 hours and until they can tolerate oral therapy after which treatment is then completed with three days of oral ACT. Overall, successful antimalarial treatment is completed in less than one week. At our clinics at UCH Ibadan, an urban academic tertiary-care center, guidelines are strictly followed and monitored weekly.All children diagnosed with malaria were treated and followed through convalescence to recovery following Nigerian antimalarial guidelines which are based on the WHO guidelines3.7 deletion, which is of little or no clinical significance, is observed in Nigerians44. Beta-thalassemia trait is known to have a very low gene frequency of 0.008 in this population45 and recent authors report inconclusive higher prevalence within healthy individuals46.All cohorts excluded children presenting with: moderate to severe malnutrition and/or iron deficiency; hypochromic microcytic anemia; spherocytosis; sickle cell anemia (SS); haemoglobin (SC) disease; known beta-thalassemia status. Only the alpha-thalassemia –αThe study was prospectively performed on subjects recruited from 2010 to 2012 (Discovery Cohort) and 2013 . The entire cohort is predominantly of the same Yoruba ethnicity and within the same population geopolitical zone.50. As shown by these studies and our data, the PD28 recovery time-point is sufficient for the HP level and other clinical parameters to normalize as it happens in the CM and UM clinical groups.All clinical malaria-positive groups received anti-malaria treatment. A child was considered recovered if, and only if, the PCV and reticulocyte counts returned to normal ranges; there were no clinical nor laboratory signs of hemolysis with fHb and LDH within normal range and; with a negative malaria Giemsa blood-film at day 28–35, day 28 (PD28) on the average. All these are well-defined clinical and physiological signs that a child has recovered from the acute malaria episode and are consistent with previous studies showing that Hb, reticulocyte counts and haptoglobin levels return to normal 2–3 weeks after commencement of antimalarial treatment40 at University College Hospital Ibadan. The experiments were conducted using samples from 3 well-defined cohorts namely the discovery, the validation and the High-throughput Sequencing (HTS) cohorts.Clinical data were collected using a malaria-tailored questionnaire designed by the CMRGFor the discovery cohort, plasma samples were collected at acute onset of the disease termed as PD0 (Plasma at Day zero) as well as from parasite-negative healthy community controls; CC.For the validation cohort plasma samples were collected at acute onset of the disease at day zero (PD0) during convalescence at day 7 (PD7) and day 14 (PD14); and at recovery at day 28–32 (PD28).HP genes were sequenced.The HTS cohort is drawn from both the discovery and validation cohorts, including malaria-negative healthy controls, whose All plasma samples from malaria-positive clinical groups within all these cohorts were collected prior to onset of anti-malarial drug treatment or blood transfusion in the case of SMA.g, 10 minutes), aliquoted and stored at −80 °C within 2 hours of collection.A 2.5 ml blood sample was obtained from each participant. A very short needle was used to collect the blood into a tube, which was firmly screwed to the syringe. This was to avoid dispensing blood into EDTA tubes through the syringe, which might have caused hemolysis of some red blood cells. Blood samples in an EDTA collection tube were kept on ice and plasma for each subject was harvested following centrifugation. Plasma for this study was harvested by centrifugation  were detected and counted by microscopy following Giemsa staining of thick and thin blood films. The criterion for declaring a participant to be malaria parasite-free was no detectable parasites in 100 high-power (1000x) fields in both thick and thin films. We validated the diagnosis outcome by randomly selecting one in ten thick blood films for independent review by local experienced senior microscopists who were not part of the study research team2O2 was added. A 2M tetraoxosulfate-VI-acid solution was used as the stop solution. Plates were read within 5 minutes at 490 nm using a FLUorStar-Omega (BMG-Labtech) ELISA plate reader. HP concentrations were computed using the five to four parameters logistic (5/4-PL) curve-fit .HP levels in plasma were quantified using an HP sandwich enzyme-linked immunosorbent assay (sELISA) kit (Genway). Plasma samples diluted 1 in 20 in phosphate buffered saline (PBS) as stock were further diluted to 1/50, 1/100, 1/250, 1/5,000, 1/10,000, 1/20,000 and 1/40,000. The low to high dilutions were to ensure detection of HP that could be present at low to high concentration, respectively. Standards were prepared according to the manufacturer’s instructions but diluted in PBS. Anti-human HP antibody (Genway) was diluted in coating buffer  to 5 µg/mL. Plates were incubated with this antibody for 60 minutes at room temperature. Thereafter, a blocking step was performed with 400 μL of blocking buffer  followed by a washing step in washing buffer . Diluted plasma samples were added to the plates and incubated for 60 minutes, followed by another washing step. Bound HP was detected with a secondary antibody coupled with horseradish peroxidase, which was diluted 1 in 2,000 in 50 mM Tris-HCl containing 140 mM NaCl, 1% BSA (wt/vol), and 0.05% Tween 20 (vol/vol), pH 8.0. The substrate, OPD was prepared at 1 mg/mL by dissolving one OPD tablet (Acros Organics) in 20 mL citrate-phosphate buffer (50 mM) to which 20 µL of 30% HWe performed genotyping and phenotyping of HP in individual subjects and ascertained whether the circulatory level of HP varies with the HP1-1, HP2-1 and HP2-2 phenotypes.HP gene isoform genotyping, a forward primer, Sa5 (5′ GAGGGGAGCTTGCCTTTCCATTG 3′) and a reverse primer, Sa6 (5′ GAGATTTTTGAGCCCTGGCTGGTG 3′) were designed to amplify both HP1 and HP2 alleles  DNA polymerase (0.5 μL); 10 mM Kappa dNTP mix (0.75 μL); Kapa HiFi fidelity buffer (0.5 μL); forward and reverse primers (10 μM each); template DNA (50 ng) and distilled water to a total reaction volume of 25 μL. The PCR for LS1 and LS2 was performed at 95 °C for 3 minutes followed by 35 cycles of 98 °C for 20 seconds, 61.5 °C for 15 seconds and 72 °C for 2 minutes with a final extension step at 72 °C for 2 minutes. The PCR conditions for the Sa5 and Sa6 primers were similar to that for LS1 and LS2 but with an additional 2 mM of MgCl2 and at an annealing temperature of 69.2 °C or 71.5 °C. Amplified products were resolved by agarose gel electrophoresis and detected by fluorescence following staining with ethidium bromide. Subjects with only the 1.7 kb band produced with primers Sa5/Sa6 are designated as having the HP1-1 genotype, and subjects with a 1.7 kb band from both the Sa5/Sa6 and LS1/LS2 primers sets are designated as having the HP2-1 genotype while those with a 3.4 kb band from the Sa5/Sa6 and a 1.7 kb band from the LS1/LS2 primer pairs are designated as having the HP2-2 genotype  and at a constant voltage of 30 V for approximately 15–16 hours. Polyclonal rabbit anti-human HP (Dako-Cytomation) diluted 1 in 10,000 in blocking solution (PBS containing 0.1% Tween 20 and 2–3% dried milk) was used as the primary antibody at room temperature for 2 hours or at 4 °C overnight. Alexa Fluor 488-labelled goat anti-rabbit diluted 1 in 5000 in blocking solution or HRP-conjugated goat anti-rabbit IgG diluted 1 in 10,000 in blocking solution were used as secondary antibody. The blots were then placed in blocking solution for one hour and washed in PBS-Tween. The protein bands were visualized using the Pharos FX Plus (Bio-Rad) molecular imager or chemi-luminescent detection  as follows. Plasma samples used for the assay were diluted 1:500 in PBS and the absorbance measured at 450 nm. LDH concentrations were calculated from the standard curve using the best-fit parameter in the MasterPlex ReaderFit v2.0 software .The level of fHb was quantified by sELISA using a human fHb-specific antibody (Mybiosource) assay MBS700075 and the manufacturer’s instructions. Plasma samples were diluted 1:800 in PBS, the absorbance measured at 450 nm and fHb concentrations calculated from the standard curve using the 5-PL function in the MasterPlex ReaderFit v2.0 software .HP gene high throughput sequencing was prepared from packed blood cells with a QIamp blood kit as described previously51. Primers specific to different locations on the HP gene were designed to produce overlapping amplicons for the entire HP gene  to a concentration of 100 μM, and then further diluted 1:10 to a working concentration of 10 μM. The final concentration of each primer in the PCR mix was about 0.3 μM in a 25 μL reaction volume.The lyophilized primers purchased from Sigma-Aldrich were reconstituted in distilled water  using MatLab to visualize a projection that best represents the structure of the multivariate data from all of the clinical groups of the Discovery Cohort (PD0). The multivariate data has five dimensions including the following parameters at admission: age; PCV; reticulocyte count; plasma haptoglobin and; plasma fHb). Clinical group labels and parasite-density values were excluded from the PCA analysis as it would strongly bias the results towards a malaria-positive vs. malaria-negative projection. The two orthogonal components that captured most of the variance were then plotted on two-dimensional space and clinical groups where imposed thereafter by labelling as follows: UM (blue); SMA (red); CM (grey) and; CC (yellow) Fig. .Figure 1Unpaired non-parametric Mann-Whitney tests were used to compare clinical demographic data between two disease groups Table . Non-parFor the genomic data and the identification of variants, FASTQ output files were aligned to the reference genome using Burrows-Wheeler Aligner (BWA). Samtools was used on the BAM file to produce the output VCF file, containing the variants. This was used for downstream computation such as the measure of association , 95% CI and genotype frequency. Minimum Allele Frequency greater or equals 10% (MAF ≥ 10%) were used to select variants  in a previous cohort from Ibadan50, we did not observe this in our present cohort (data not shown).As expected, PCV values were lowest in the SMA clinical group. In SMA cases, low PCV at recruitment (PD0) was attributed to the anemia as the consequence of the malaria infection. In all clinical malaria-positive groups receiving anti-malaria treatment the PCV returned to normal at recovery (PD28) Table The mean parasite densities were not significantly different among the malaria-positive groups; the CM, SMA and UM. Females and males were reasonably represented in this study. The clinical demographics of the subjects within the Discovery Cohort Table , ValidatTo explore the structure of the multivariate data and to obtain the most visually useful projection of its variance in two-dimensional space, we created a scatter-plot with the two most informative principal components coefficients of all study groups in the discovery cohort Fig.  followed4 ng/ml, median of 4 × 103 ng/ml and mean of 6 × 103 ng/ml ; SMA < CM (p < 0.001); SMA < UM (p < 0.0001) Fig. . All chi/ml Fig. .The low plasma HP level in the SMA group does not appear to be dependent on the fHb level since the levels of fHb did not differ among the malaria groups Fig. . HP plas3 ng/ml) compared to the other clinical groups CM, UM and CC (p < 0.001)  than those of the UM, CM and CC group (mean 105 ng/ml) (p < 0.001)  Fig. . Overall01) Fig. . Althoug01) Fig. . The val01) Fig.  despite 01) Fig.  and decr01) Fig. . Unlike 01) Fig. .The levels of both fHb and LDH through convalescence to recovery in the SMA group in the validation cohort do not appear to be higher than those of the UM and CM groups Fig. . LDH levThe plasma HP levels significantly inversely correlated with parasite density in the UM group  with an estimated OR of 2.1 (1.04–4.4); there is a higher frequency of HP1-1 in the CM group (0.46) compared to the CC (0.29), UM (0.35) and SMA (0.29) groups Fig. . The obsHP gene, all located 3.4 kb upstream of the translation start site, were analyzed (Table −6)  Fig. . The GG −6) Fig. .Table 2HTo evaluate whether rs12162087 is associated with malaria syndromes, we performed a genetic association study. The analysis revealed a significant association between SMA and rs12162087 (p = 0.03). The (Homo-Ref: GG) genotype frequency is higher in SMA (19.8%) compared to CC (8.2%) with an OR of 2.74 (95% CI = 1.11–6.78). All together our findings in the Nigerian cohort show that the genotype HP rs12162087 GG is more common in SMA and is associated with lower HP levels  and also lower than normal in the CM group, an observation that appears consistent with the hemolysis-induced hypo-haptoglobinemia hypothesis52. Similarly, other studies have reported that as parasite disappears from the blood, either by antimalarial therapy or host mechanism, both hemoglobin level and bone marrow morphology returns to normal53 2–3 weeks which is within the convalescence phase54. Furthermore, in acquired hypo-haptoglobinemia, induced by malaria or hemolytic disease, HP levels return to normal 2 weeks after the commencement of antimalarial treatment47. In line with these previous findings and coupled with the clinical and laboratory evidence of recovery provided in our study, the PD28 recovery time-point is sufficient for the HP level and other clinical parameters to normalize as it happens in the UM and CM clinical groups. By measuring HP levels at these time-points we have been able to study the role of congenital hypo-haptoglobinemia over that of other etiologic causes. Although hypo-haptoglobinemia can be induced by hemolysis, a genetic contribution to this phenomenon can also be equally important as our data suggest. Our study provides evidence that the etiology of hypo-haptoglobinemia observed in children with acute-onset SMA could be due to genetic factors as HP levels fail to rise after the underlying cause of pathology has been successfully treated and the children recovered four weeks after diagnosis.It is intractable to carry-out a large clinical study where HP levels are measured at the initial day of infection, especially in holoendemic high-transmission malaria settings such as ours. To tackle this challenge, we focused on the disease trajectory of the malaria-positive clinical groups from day of diagnosis (PD0) through convalescence  to recovery (PD28) in order to establish a possible genetic etiology of the observed hypo-haptoglobinemia. In our study, children recovery at four weeks (PD28) post-diagnosis is strictly defined by clinical and laboratory parameters such as return to normal values of PCV, fHb, LDH and reticulocyte counts. Our definition of recovery at PD28 is also consistent with previous studies that show that hemoglobin levels as low as 5.0 g/dL in SMA returns to normal 2–3 weeks after commencement of treatmentThe evidence that the rs12162087 (g.-1203G > A) genetic variant, with an observed frequency of 0.67 in this predominantly Yoruba Ibadan’s cohort, is associated with plasma HP levels in the malaria-negative CC group is fairly compelling. First, the association is determined within a single cohort, the malaria-negative well-children cohort, reducing the possibility that confounders play a role. Second, this variant is the only common variant that has such strong evidence suggesting little confounding acting across SNPs. Third, the variant is not out of Hardy-Weinberg equilibrium, suggesting that it is well genotyped by our HTS protocol and there is not stratification bias in the CC group. We show that children with rs12162087 GG genotype showed a risk of 2.74 to develop SMA compared to those with the rs12162087 GA and AA genotypes.HP gene is reported to be associated with both circulatory HP level and the disease, SMA55. The rs12162087 (g.-1203G > A) variant has not been associated with another trait before. The variant is upstream of the transcriptional start site of the HP gene, raising the likelihood that it might be a regulatory variant affecting gene expression. Furthermore, in HaploReg this variant is highlighted as enhancer histone marks and it is associated with eQTL. We suggest that there is a possible interaction between the distal upstream region and the proximal upstream (promoter) region of the HP gene through DNA looping that could affect the expression of the protein.This is the first time a variant in the distal upstream region of the 56, we found that the Hp1-1 isoform is significantly more prevalent in children presenting with CM than in the CC group in our Nigerian cohort. However, our larger sample size than the previous study56 did not detect any significant differences between Hp1-1 isoform in the SMA when compared to CC. Consistent to a study carried out in Kenya57, we found that the Hp2-1 isoform does not confer protection against SMA. In the Kenyan cohort57, the Hp2-2 isoform is associated with the greatest risk of CM and Hp2-1 with greatest protection against CM. In our west-Africa CM cohorts we found that Hp1-1 was more common in the CM patients, implying a potential for greater risk factor, whilst Hp2-1 and Hp2-2 seem to be associated with protection. Further multi-site studies are needed to dissect the differences between these west- and east- African cohorts and to investigate if there is a correlation between HP isoform and the reported rs12162087 variant.Aside the hypo-haptoglobinemia observed in SMA patients, we also showed that plasma HP level is independent of HP isoform phenotype. Replicating the findings of a previous study in GhanaHP gene known to be associated with hypo-haptoglobinemia36 are rare in this population. This deletion was found in only 1 out of 401 subjects whose HP genes were sequenced. These deletions appear not to be the major cause of hypo-haptoglobinemia in this predominantly Yoruba population.The reported long heterozygous and homozygous deletions of the 58 is predominantly driven by increased clearance of uninfected erythrocytes by the reticulo-endothelial system49 as intravascular hemolysis of infected erythrocytes due to schizogony cannot explain by itself this dramatic change. In line with our previous SMA study49, we suggest that, in the cohorts with baseline low plasma HP and Hb, particularly the SMA group, clearance of non-infected erythrocytes is driven by the reticulo-endothelial system in the spleen through an as yet unknown CD8(+) T-cell-dependent mechanism49. This splenic clearance of erythrocytes could be responsible for the low Hb level in the SMA group. This further indicates that extravascular hemolysis is implicated more strongly in the pathology of SMA rather than intravascular hemolysis.Our data shows that the degree of intravascular hemolysis in the SMA group is not different to that of other malaria positive groups. In acute-onset life-threatening childhood SMA rapid PCV decreaseIt is unknown why some children develop severe forms of malaria such as SMA whilst in others the infection is restricted to the mild form of the disease. Our study suggests that a low circulatory HP level is a risk factor for the occurrence of SMA in contrast to the other malaria syndromes studied. Children bearing the genotype at risk rs12162087 g.-1203G/G had one order of magnitude lower HP levels which is consistent also with plasma HP levels observed to be low in SMA children at disease onset and at recovery.HP genotypes with hypo-haptoglobinemia, our findings provide evidence that the hypo-haptoglobinemia phenotype and the presence of the rs12162087 g.-1203G/G genotype could be used as predictors for acute onset SMA. We expect that our findings will prompt these multi-site studies, not only to replicate association, but to dissect what other characteristics of other clinical settings can reinforce the significance of genetic control of HP plasma levels in determining acute onset severe anemia phenotypes including that induced by malaria.Although multi-site studies within Africa and outside Africa, with populations under different malaria burden, are needed to further dissect the relationship between of Finally, our study contributes to the development of risk assessment algorithms to identify children with higher risk to develop SMA, which is lethal without urgent access to safe blood transfusion, this being the case in all  areas in high-transmission malaria holoendemic regions in sub-Saharan Africa. Improved risk assessment algorithms will also have a substantial impact on the use and management of health-care resources such as blood banks across low- and middle-income regions of the World under malaria burden.Supplementary Information


Plasmodium falciparum density was  × 103/µL red blood cell (RBC). P. falciparum prevalence was not significantly dependent on gravidity, parity, trimester, age, and BMI status of the women (p > 0.05). Difference in P. falciparum density per µL RBC in primigravidae (268.13 ± 58.23) × 103 vs. secundi- (92.14 ± 4.72) × 103 vs. multigravidae (65.22 ± 20.17) × 103; and in nulliparous (225.00 ± 48.25) × 103 vs. primiparous (26.25 ± 8.26) × 103 vs. multiparous (67.50 ± 20.97) × 103 was significant (p < 0.05). Majority of attendees were at 3rd trimester at time of first antenatal visit. Prevalence of malaria parasitaemia in the first-time (48.6%), and multiple-time (52.6%) antenatal attendees was not significantly different . The higher prevalence of malaria among bed net owners ) and users ) at multiple antenatal visits vs. first timers was not significant. None of the pregnant women examined used malaria preventive chemotherapy. Antenatal services at the hospital were not effective at preventing PAM. Holistic reviews reflecting recommendations made here can be adopted for effective service delivery.Pregnancy-associated malaria (PAM) is a major public health concern constituting a serious risk to the pregnant woman, her foetus, and newborn. Management of cases and prevention rely partly on effective and efficient antenatal services. This study examined the effectiveness of antenatal service provision in a major district hospital in sub-Saharan Africa at preventing PAM. A cross-sectional hospital based study design aided by questionnaire was used. Malaria diagnosis was by microscopy. Overall prevalence of PAM was 50.7% (38/75). Mean  Plasmodium falciparum, further subjects the woman to physiological and pathological stress, in addition to that arising from pregnancy. This combination of stresses which, on one hand, is directed at ensuring the survival of the foetus and, on the other, at combating the parasite, may overwhelm the pregnant woman leading to her death, abortion of the foetus [Pregnancy-associated malaria (PAM) is a major public health concern constituting a serious risk in sub-Saharan Africa to pregnant women, her foetus, and the newborn . Infectie foetus , or stile foetus .P. falciparum-infected red blood cells (RBC). This effect on the foetus results from interference with placento-foetal exchange, with shortage of glucose and oxygen supplies to foetus [P. falciparum-infected RBC have been suggested [Low birth weight is another effect attributable to PAM ,5,6 as io foetus . Disturbuggested . Low biruggested .Appropriate and prompt diagnosis and chemotherapy requiring stringent consideration for the duration of pregnancy remains a standard management procedure . IntermiP. falciparum prevalence and density in women attending antenatal clinics in a hospital in sub-Saharan Africa . Treated mosquito bed net ownership and usage, and the practice of intermittent preventive treatment for malaria were assessed also. The aim of the assessment was to ascertain the effectiveness of antenatal services in the hospital at combating PAM. It was assumed that where the antenatal service is effective, (i) PAM prevalence and P. falciparum density will be low; (ii) ITN ownership will be greater and its effective usage higher in multiple antenatal attendees than first-timers; (iii) where malaria preventive chemotherapy is in use, it should be effective at reducing PAM prevalence; (iv) prevalence and density of malaria parasitaemia by obstetric characteristics, such as gravidity, parity, trimester, age, and BMI will highlight groups that need significant attention concerning PAM management. Information from this study is important for policy-makers who have continued to highlight the need to reduce the high maternal and neonatal mortality rate in Nigeria. Among other objectives, this study assessed The study was conducted at the district hospital, Enugu-Ezike in Igbo-Eze North Local Government Area (LGA). Igbo-Eze North LGA is located between latitude 7°00′ and 7°12′ N and longitude 7°12′ and 7°36′ E . Enugu sEnugu-Ezike where the district hospital is situated is a semi-urban area. According to the 2006 National Census figures, Igbo-Eze North has a population size of 258,829 which is about 8.0% of Enugu State population, placed at 3,267,837 . Number Antenatal procedure at the District Hospital Ogrute commences with registration. Antenatal services are provided Wednesday and Friday, weekly. It involved a sensitization session on food consumption and vaccination processes, blood pressure measurement, general pregnancy-related clinical examinations, compulsory HIV/AIDS screening for first timers, and voluntary rapid diagnostic tests (RDTs) for malaria. The voluntary malaria RDTs involved payment of 50 naira (<$0.17) as of the time of this study. ITN distribution was not a regular part of antenatal services, but distribution was made whenever there was a donation or special provision. All antenatal attendees who participated in this study complained that their ITNs were very old due to prolonged use.A cross-sectional hospital-based study design was used. Sample collection took place between August and early December 2015, encompassing the rainy season and early dry season. Sample size estimation projected 48–84 as possible number of attendees for the duration of the study. Therefore 60–80 participants were taken as acceptable sample size. A total of 75 pregnant women out of a total of 78 that reported for antenatal at the hospital for the duration of the study consented to the study. All of the women were examined for malaria parasitaemia. Only two (2.78%) of the women were in the first trimester, 32 (44.44%) were in the second, while 38 (52.78%) were in the third. The two first trimester participants were excluded for comparisons between trimesters.The study protocol was approved by the Department of Zoology and Environmental Biology, University of Nigeria, Nsukka, Enugu State, Nigeria. Ethical clearance for the conduct of the study on human subjects was obtained from the University of Nigeria Teaching Hospital (UNTH) Enugu State, Nigeria. It was assigned ethical clearance number NHREC/05/01/200B-FWA00002458-1RB00002323. Approval was also obtained from the Department of Health, Enugu-Ezike LGA secretariat. Informed approval was obtained from the Chief Medical Officer at the district hospital, Enugu-Ezike for the conduct of the study in the hospital. Data were collected with the consent of subjects.A questionnaire which was pretested for this study was designed to reduce the time expended and stress caused to the participant. This was done by making the questions concise and administered when the subject was waiting for blood sample collection. The questions asked covered maternal age, gravidity, parity, duration of pregnancy, community of residence, ownership and usage of treated mosquito bed nets, and use of intermittent preventive therapy for malaria. Questions were closed and open-ended and administered in English. The participants understood English, but where cases of difficulty in reading or comprehension of English arose, questions were read out to the participants and interpreted. The response to the duration of ITNs ownership and usage was by interview.P. falciparum in blood samples were specifically identified using a guide from the CDC [For the study purpose, blood samples were collected by a qualified nurse well-trained in venepuncture. Four drops were immediately used for malaria diagnosis. Thin and thick blood smears were prepared and stained with 3% Giemsa at pH 7.2. Blood films were examined using a light microscope and recorded as negative only after 100× oil immersions was used to count >1900 RBCs in thin smears.  the CDC . The ass the CDC :(1) Mala® version 20.0  and Microsoft Office Excel® . Data was carefully entered and cross-examined twice before analysis. Parasite density was not normally distributed, hence, a Kruskal-Wallis H test was used for its comparison. Chi-square analysis was used for calculation and comparison of malaria prevalence, ITNs ownership and usage by obstetric characteristics. Spearman’s correlation was used to assess relationship between P. falciparum density, maternal age, and BMI. Logistic regression was used to evaluate the risk of malaria among ITNs owners and users. All tests were two-sided and p < 0.05 considered significant.Data was analysed using SPSS2, respectively. Thirty-eight (50.7%) of the women were positive for P. falciparum malaria parasitaemia. Overall, P. falciparum density per microliter of red blood cells (RBC) was (112.89 ± 22.90) × 103. Prevalence of P. falciparum malaria was not significantly dependent on the gravidity, parity, trimester, age, and BMI status of the women (p > 0.05) . Difference in P. falciparum density per µL RBC in primigravidae (268.13 ± 58.23) × 103 vs. secundi- (92.14 ± 4.72) × 103 vs. multigravidae (65.22 ± 20.17) × 103; and in nulliparous (225.00 ± 48.25) × 103 vs. primiparous (26.25 ± 8.26) × 103 vs. multiparous (67.50 ± 20.97) × 103 was significant (p < 0.05). The statistical significant difference in P. falciparum density between primi-, secundi-, and multigravidae from Kruskal-Wallis H test  was due mainly to the significant difference between primi- (ranked 28.94) and multigravidae (ranked 15.91); similarly, the parasite density in nulliparous (ranked 27.21) was significantly higher than multiparous (ranked 16.16) women . Younger participants (<20–29 years) vs. older (30–39 years) had higher P. falciparum density. P. falciparum density had a strong negative relationship with the age of the women from Spearman’s correlation .For the duration of the study, 75 (96.2%) out of a total of 78 antenatal attendees at the district hospital accepted to participate in the study. The mean age and BMI of the participants were 27.60 ± 0.71 (18–38) years and 27.65 ± 0.52 (20.1–41.9) kg/m > 0.05) . Prevale2 = 0.119, p = 0.730). P. falciparum load was higher in those that had attended antenatal service more than once for same pregnancy.Antenatal attendees at the district hospital were mainly those at second and third trimesters of pregnancy. The majority of attendees were at the third trimester at time of first antenatal visit. Thirty-seven (49.3%) out of the 75 participants were at first antenatal visit. Out of these first timers, 28 (76%) were already at the third trimester, seven (18%) at the second trimester, and only two (6%) at the first trimester . Prevale2 = 6.199, p = 0.013), and in the age brackets 20–29 and 30–39 years . Usage of bed nets was, however, neither dependent on the trimester of pregnancy nor maternal age (p > 0.05).Thirty-seven (49.3%) of the women examined owned a mosquito bed net, while 24 (32.0%) used theirs . Ownersh2 = 3.861, p = 0.049, OR (odd ratio) = 2.519 (95% CI 0.994–6.383)) and use ) mosquito bed nets than those at their first antenatal attendance ) and users ) at multiple antenatal visits vs. first timers was not significant.Relationships between numbers of antenatal visits to ownership and usage of mosquito bed nets, and the effect of such ownership and usage on infection by malaria parasites are indicated in tendance a. The prtendance b. The hiNone of the pregnant women examined used intermittent preventive treatment with sulphadoxine-pyrimethamine (IPTp-SP) or malaria prevention chemotherapy of any kind. The nurses also confirmed that no medication for malaria was given without diagnostic confirmation of malaria.P. falciparum endemicity and associated morbidity is a major public health challenge. The overall prevalence of malaria parasitaemia of 50.7% is high. A similar, but slightly higher, prevalence of 52% was recently reported from Yemetu-Adeoyo, a semi-urban community in Ibadan, Nigeria , 72% in The relatively high overall prevalence of PAM reported in the present study may be attributed to several factors that encompass human hosts, mosquito vectors, malaria parasites, and the environment. Of importance to this study is the human factor. The non-usage of intermittent preventive treatment and poor usage of mosquito bed nets probably contributed to the high PAM prevalence. Though 49.3% of the pregnant women examined had an insecticide-treated bed net, only 32.0% used it. Also, all of the women complained that their bed nets were already too old. This may have contributed significantly to the ineffectiveness of the mosquito bed nets in the prevention of malaria among users. While these malaria-preventive procedures were not strongly adhered to, there was a routine rapid diagnostic test (RDT) for malaria at the district hospital. The RDTs were, however, subject to the consent of the pregnant women. This routine diagnosis, and ownership and usage of old mosquito bed nets, were apparently not effective at malaria prevention. Where antenatal service is effective, prevalence of PAM should be low; multiple antenatal attendees should not only own and use ITNs, but should equally have much lower PAM prevalence compared to first-time attendees. This was not the case at the district hospital. High prevalence of PAM, despite antenatal attendance and ITN usage highlights the need for enlightenment on ITN usage and incorporation of PAM preventive chemotherapy into antenatal services at the hospital. The season of sampling may be another contributing factor to the high prevalence of malaria. Sample collection encompassed the rainy season  and the early dry season  when mosquito population and malaria transmission is usually high ,26,27. HP. falciparum parasitaemia may be attributed to delayed diagnosis and treatment [P. falciparum density was higher in those that had two or more antenatal visits than those visiting for the first time. This suggests that non-commitment to regular malaria diagnosis may be the more important contributor  to the high parasite load. In addition, pregnant women with multiple antenatal attendances did not even have a lower prevalence of malaria. Therefore, the voluntary consent allowed the pregnant women before they were diagnosed of malaria at the hospital is not effective at combating PAM. The mean parasite density of 112,894.74 ± 22,901.73/µL RBC is high . The higreatment ,30, and P. falciparum malaria is acquired in a gravidity-dependent manner; multigravidae usually develop protective immunity, but it is entirely absent in primigravidae, or poorly developed in secundigravidae [P. falciparum density in primigravidae and secundigravidae. Therefore, P. falciparum PAM causes more serious morbidity in primigravidae and secundigravidae [P. falciparum load was significantly higher in the primigravidae (p < 0.05) and nulliparous (p < 0.05) compared to their multigravid and multiparous counterparts, respectively. This suggests that limited attention was paid to PAM in the antenatal service provided at the hospital. Studies on P. falciparum PAM have consistently highlighted the need for more stringent attention to be given to primigravidae and secundigravidae. If the outcomes of those studies were taken seriously at the District Hospital Ogrute, primigravidae and secundigravidae would have had statistically significantly lower cases of PAM, and possibly would not have had significantly higher parasite loads. This highlights the need for a robust review of antenatal care at the district hospital with a view to effectively incorporate PAM prevention and management procedures.Maternal immunologic state also contributes to high parasite density . Chandragravidae ,33. Thisgravidae ,35. As oP. falciparum load observed in the third trimester compared to the second may have arisen from progressive immunomodulation in pregnancy. During pregnancy, maternal systemic immune response is skewed toward the humoral arm [P. falciparum clearance [The higher oral arm ,37, a colearance . It may Sensitization on malaria prevention during the morning antenatal enlightenment class should be introduced;Malaria diagnosis during pregnancy should be made routine and carried out at least once every month; and efforts should be made at making it completely free;Effort should be made at providing treated mosquito bed nets;Finally, antenatal attendees should be enlightened and encouraged to take malaria preventive chemotherapy in accordance with WHO recommendations.Antenatal practices at the District Hospital Ogrute at the time of this study were not effective at preventing pregnancy-associated malaria. The following actions may help enhance PAM prevention and/or reduction of cases among the antenatal attendees at the hospital:


The current PCR-based assays present limitations as most protocols still rely on amplification of few-copies target gene. Here, the hypothesis was that amplification of different plasmodial targets—ribosomal (NR-qPCR) was established and compared with three additional PCR protocols, two of them based on 18S rRNA gene amplification (Nested-PCR and R-qPCR) and one based on Pvr47/Pfr364 targets (NR-cPCR). The limit of detection of each PCR protocol, at single and artificial mixed P. vivax/P. falciparum infections, was determined by end-point titration curves. Field samples from clinical (n = 110) and subclinical (n = 324) malaria infections were used to evaluate the impact of using multiple molecular targets to detect malaria infections.A non-ribosomal real-time PCR assay targeting Pvr47/Pfr364  of infections among asymptomatic Amazonian residents, with a high proportion of infections (~ 20%) identified only by the NR-qPCR.The results demonstrated that an association of ribosomal and non-ribosomal targets did not increase sensitivity to detect submicroscopic malaria infections. Despite of that, artificial mixed-malaria infections demonstrated that the NR-qPCR was the most sensitive protocol to detect low-levels of P. vivax and P. falciparum infections. As the majority of subclinical infections was caused by P. vivax, the commonest form of malaria in the Amazon area, future studies should investigate the potential of Pvr47/Pfr364 to detect mixed-malaria infections in the field.This study presents a new species-specific non-ribosomal PCR assay with potential to identify low-density The online version of this article (10.1186/s12936-019-2781-3) contains supplementary material, which is available to authorized users. Plasmodium vivax is the most widely distributed parasite causing malaria, Plasmodium falciparum accounts for the most severe forms of the disease [Malaria is a worldwide public health concern that is present in roughly 90 countries, mainly in tropical and subtropical regions . While P disease . Althoug disease , 3.Plasmodium infections in endemic areas have confirmed previous finding of high frequencies of malaria infections at densities below the limit of conventional microscopic diagnostics [Plasmodium parasites, acting as reservoirs for malaria [In order to progress towards malaria control and elimination, it is critical to understand the sources of transmission (the infectious reservoir) and those at risk of infection at the population level . In thisgnostics –9. It isgnostics , 11. Accgnostics . Many ar malaria , 14. Bey malaria .18S rRNA) has been the most common target used for molecular diagnosis of malaria [Plasmodium parasites [18S rRNA gene amplification have been described [Plasmodium parasites has allowed the discovery of new species-specific multi-copy targets which show potential for molecular diagnosis of P. vivax and P. falciparum malaria [P. vivax and P. falciparum, respectively [18S rRNA gene by the conventional nested-PCR assay [Historically, the small subunit 18S of the ribosomal RNA gene  could increase the chances of detecting submicroscopic malaria infections. For that, field samples  were amplified by four different PCR assays, two of them targeting Pvr47/Pfr364 sequences [18S rRNA gene [Since most malaria PCR-based protocols still relies on amplification of phoresis , here a equences  and two RNA gene , 17.Ethical and methodological aspects of this study were approved by the Ethical Committee of Research on Human Beings from the René Rachou Institute/Oswaldo Cruz Foundation , according to the Brazilian National Council of Health (Resolutions 196/96 and 466/12). All participants were informed about the objectives and procedures of the study, with voluntary participation through written informed consent.This group included individuals with clinical suspicion of malaria who sought care at Brazilian malaria reference healthcare facilities located in both endemic (States of Rondônia and Mato Grosso) and non-endemic areas (Minas Gerais). After short-trips to malaria transmission areas, these individuals presented symptoms suggestive of uncomplicated malaria infection, such as fever, myalgia, chills, and headaches. Non-inclusion criteria included: (i) refusal or inability to sign the informed consent; (ii) age below 5 years-old; (iii) pregnant women; and (iv) any other co-morbidity that could be traced. One-hundred-and-ten patients were enrolled in the study, which gives 90% statistical power at 5% significance level assuming 30% of prevalence by light microscopy and an estimative of 50% increase by molecular assays; the majority of study population were adults, with a median age of 40 years (IQR 30.75–48.25), and a proportion female:male of 1:5. For each individual, blood sample was collected at a single time point by venipuncture or finger prick. The period of blood collection varied from 2008 to 2017, and since there, all samples from participants have been maintained in the biorepository of malaria research group at FIOCRUZ-MINAS , Brazilian Ministry of Health, according to the local legislation.P. vivax infection molecular assays is expected to increase malaria prevalence by a factor of 2 [This group included malaria-exposed individuals who had participated of cross-sectional surveys carried-out between 2008 and 2015 in a rural community of the Brazilian Amazon rainforest, Rio Pardo , Presidente Figueiredo municipality, State of Amazonas. The study site and malaria transmission patterns have been described in details elsewhere –29. In ttor of 2 , sample tor of 2 , samplesAt the time of blood collection, all individuals  were submitted to a finger-prick for malaria diagnosis by light microscopy. The Giemsa-stained thick blood smears were prepared and examined by experienced local microscopists, according to the malaria diagnosis guidelines of the Brazilian Ministry of Health . ParasitThe experimental approach to the molecular detection of clinical and subclinical malaria infections was included in Fig. P. falciparum (3D7 strain) from in vitro continuous blood-stage cultures maintained in the routine of the laboratory, according to protocol previously described [P. vivax, P. falciparum and Plasmodium malariae from peripheral blood of malaria infected individuals whose species-specific diagnosis was confirmed by PCR protocols ; (iii) Plasmodium brasilianum/P. malariae  kindly provided by the Malaria Research and Reference Reagent Resource Center, MR4 .The following parasites were used as reference in molecular assays: (i) escribed ; (ii) P.P. vivax  and P. falciparum  infections were used to determine the end-point detection for ribosomal and non-ribosomal PCR assays. For each plasmodial DNA, three-fold dilutions were prepared range from 2000 to 0.3 parasites/µL. Similarly, artificial mixed malaria infections were produced by mixed DNA samples from P. vivax and P. falciparum in different proportions . Each sample was assayed in triplicate and submitted to all PCR protocols  and 41 copies of Pfr364 . The set of oligonucleotides obtained for Pvr47 was 5′TCCGCAGCTCACAAATGTTC3′ (forward), 5′ACATGGGGATTCTAAGCCAATTTA3′ (reverse), and 5′HEX-TCCGCGAGGGCTGCAA3′ (probe), which binds respectively to positions 142–161, 245–222 and 189–204 of P. vivax Sal-I (GenBank accession number AAKM01000578), resulting in a 104 bp amplicon. For Pfr364, the set used was 5′ACTCGCAATAACGCTGCAT3′ (forward), 5′TTCCCTGCCCAAAAACGG3′ (reverse), and 5′FAM-GGTGCCGGGGGTTTCTACGC3′ (probe), which binds respectively to positions 313–331, 400–383 and 335–354 of P. falciparum 3D7  were used to define positivity to P. vivax and P. falciparum, respectively.Primers and MGB (minor groove binder) probes were designed for Pvr47 and Pfr364 targets using Primer-Blast (NCBI) and OLIGO  softwares, considering conserved regions in each species alignments. The alignments were based on the sequences previously described , composeP. vivax) and Pfr364  were conducted by using primers previously described [The amplification of the targets Pvr47  were included as Additional file Nested-PCR assay for amplification of escribed , using gescribed . The reaP. vivax . Primers2) test or Fisher’s exact test, as appropriate. The Probit Regression analysis was used to define the limit of detection (LOD) of NR-qPCR assay, with different input concentrations of plasmid DNA used to calculate the predicted proportion of positive results . Heat map of malaria prevalence was constructed using the heatmap.2 function available in the R package gplots. The assessment of sensitivity and specificity of PCR assays was determined as described [GraphPad InStat, version 3.0 . The significance at the 5% level was considered to all analysis.Differences in proportions were evaluated using the Chi square  through serial dilution ranging from 20,000 to 0.05 copies/μL  to detect P. vivax and P. falciparum monoinfections at low parasite densities was investigated through serial dilutions of field samples containing known amounts of either P. vivax or P. falciparum gDNA. In case of P. vivax monoinfection, no significant difference was observed between parasite targets amplified by PCR . While the amplification of Pfr364 by NR-qPCR was reproducible until 3 parasites/μL, the amplification of 18S rRNA was inconsistent, alternating between positive and negative results at similar levels of parasite density (both nested-PCR and R-qPCR).The ability of different targets  and varying the amount of P. falciparum (1489 to 0.7 parasites/μL), it was possible to demonstrate that the protocols based on non-ribosomal targets were much more precise to identify both parasite species, even when P. falciparum was present at very low densities  and varying P. vivax densities (1433 to 0.7 parasites/μL), a good performance of both targets was observed until 6 parasites/μL   identified a similar proportion of malaria infections  . In addition, the use of different PCR targets  did not increase malaria positivity . To confirm that an association of ribosomal and non-ribosomal targets did not increase sensitivity to detect submicroscopic malaria infections, the values of sensitivity and specificity were determined for molecular PCR-assays. In accordance, the performance of each PCR assay was similar with almost no detection of false positives or negatives  identified by LM  confirmed 35 32%) malaria infections  or non-ribosomal targets (NR-cPCR/NR-qPCR) detected a similar proportion of subclinical infections, being 22% (n = 72) and 27% (n = 89), respectively. As the predicted sensitivity of individual PCR assays varied significantly in this group of low-parasite densities, especially with high false negative rate for nested-PCR assay . Overall, while PCR protocols identified 92 (28%) subclinical infections, only 21 (7%) were identified by LM .Screening for subclinical malaria infections among individuals from cross-sectional surveys identified a positivity of 7% (21 out of 324) by LM  qPCR targeting the multi-copy Pvr47/Pfr364 sequences was developed and this new protocol was compared with the original non-ribosomal gel-stained PCR-based protocol [18S rRNA gene amplification.Although major advances have been reached for the molecular detection of malaria parasites , 36, 37,protocol  as well P. vivax and P. falciparum infections—in single and artificial mixed infections—producing reproducible results at the lowest parasite densities (1–3 parasites/µL). Although there was considerable variation between PCR protocols assayed, the non-ribosomal protocols (NR-cPCR and NR-qPCR) were more accurate than ribosomal (nested-PCR and R-qPCR) to detect mixed-species infections. Of interest, only NR-qPCR assay developed here were able to detect P. falciparum when this species was present in a proportion of 240-fold lower than P. vivax. As the sensitivity of any PCR protocol depends largely on the molecular target used [P. falciparum in co-infections as compared to 18S rRNA (around 5–8 copies). Although different multi-copy targets have been described as sensitive for molecular diagnosis of malaria [18S rRNA qPCR to detect low P. falciparum densities in situation where P. vivax was present in much higher densities was observed. The use of a single pair of primers to detect both species may have been a determinant factor in causing failure of R-qPCR to identify mixed infections. A similar phenomenon of primer competition was described in the original protocol [The end-point titration assays of field samples revealed that the NR-qPCR protocol was able to accurately detect both  malaria , 25, 36, malaria . More woprotocol , straighP. vivax and P. falciparum detected by amplification of non-ribosomal Pvr47/Pfr364 targets was not significantly different than that of either conventional microscopy or 18S rRNA gene amplification. The predicted sensitivity and specificity of each PCR protocol assayed here were also similar, and it was independent of the parasite target. Although the clinical sample size limited the statistical power to detect small differences between protocols, these results were not completely unexpected as symptomatic patients usually present high parasite densities in the peripheral blood; consequently, it may facilitate the confirmation of malaria infection by less sensitive protocols such as microscopy and rapid diagnostic tests (RDTs) [Plasmodium species is circulating [In clinical malaria suspects, the overall prevalence for s (RDTs) . These fs (RDTs) , 42. Nevculating , 44, a rP. vivax, the commonest malaria parasite in the Amazon basin, and frequently associated with low-density infections [P. vivax infections across different endemic settings, particularly areas with relatively low transmission intensity [P. vivax infections is unknown, it is probably associated with the unique biology of P. vivax that includes a fast acquisition of clinical immunity as compared with P. falciparum [18S rRNA gene has been described [18S rRNA gene was dependent on (i) large blood volume (2 mL); (ii) careful removal of plasma and buffy coat as prerequisite to avoid interference during PCR processing; (iii) concentration of purified DNA dehydrated in a centrifugal vacuum concentrator; additionally, these “high-volume” 18S rRNA PCR strategy did not allow the detection by species (only Plasmodium spp.) [While low frequencies of submicroscopic infections were observed in the group of clinical malaria cases (3–5%), screening for malaria in cross-sectional surveys demonstrated a large proportion (> 70%) of malaria cases in the study area that was not detected by conventional microscopy. The majority of subclinical infections were caused by fections , 45–47. ntensity –12, 26. lciparum . In the escribed , 49, theum spp.) .The apparent ability of Pvr47/Pfr364 NR-qPCR to increase sensitivity to investigate the true prevalence of malaria infection is relevant as an unexpectedly large reservoir of infections may hinder control and elimination of malaria in the Americas . These fPlasmodium species. Furthermore, considering the rules for quality assessment of diagnostic accuracy studies (QUADAS-2) [Assuming that the NR-qPCR developed here seems to be the most sensitive method—as it was positive in a number of samples not detected by other PCR protocols—the results suggested that NR-qPCR has a lower detection threshold. Despite of that, it is important to clarify the technical limitations that apply for the definition of “reference standard” for PCR-detection of submicroscopic malaria infections. In general, the estimative of test accuracy are based on the assumptions that the reference standard is 100% sensitive and that specific disagreements between the reference standard and the diagnostic test being evaluated (index test) result from incorrect classification by the index test . HoweverUADAS-2) , the risP. vivax being the predominant malaria parasite [P. falciparum cases in recent years [P. vivax/P. falciparum co-infection, as the accurate detection of malaria mixed-infections seems to be critical for control and management of malaria [P. falciparum reemergence from Amazonian neighboring countries with high transmission rates, a cross-border malaria study to evaluate the relevance of NR-qPCR in mixed-malaria infections are on progress.Finally, relatively low frequencies of mixed-malaria infections were detected here, which precluded a more detailed evaluation of the potential of Pvr47/Pfr364 to detect mixed-malaria infections in the field. In the Amazon area, besides parasite , 29, thent years . Notwith malaria , 44. In  malaria . As mala malaria . Due to P. vivax/P. falciparum infections, even though one of the species was present in a ratio of hundred-times lower than the other species.Although the simultaneous use of ribosomal and non-ribosomal PCR-targets did not impact the molecular diagnosis of malaria, the amplification of Pfr364 and Pvr47 multi-copy targets by the NR-qPCR seems to be a valuable tool in detection of subclinical and mixed Additional file 1. Analytical evaluation of NR-qPCR assay.Additional file 2. Amplification curves of NR-qPCR performed on field samples infected with P. vivax , P. falciparum , P. malariae , and P. brasilianum DNA . The amplification plots are shown for (A) Pvr47 and (B) Pfr364 assays.Additional file 3. Five-fold dilution of P. vivax-Pvr47 and P. falciparum-Pfr364 plasmids amplified by NR-qPCR.Additional file 4. Conditions of the Nested-PCR, R-qPCR, and NR-cPCR assays. The primers/probes used for P. vivax and P. falciparum targets were the original described. The products of nested-PCR and NR-cPCR were visualized by 2% agarose gel stained with ethidium bromide.Additional file 5. Limit of detection (LOD) for (A) Pvr47 and (B) Pfr364 targets amplified by NR-qPCR. Probit regression analysis was used on logarithmic scale using nine points of NR-qPCR standard curves . The calculated regression curves (blue lines) indicate the probability (y-axis) of obtaining positive results at any template concentration, and dashed brown lines shows 95% confidence intervals. Dashed black lines correspond to the lower DNA concentration in which 95% of positive samples were detected (0.66 copies/µL of Pvr47 and 3.27 copies/µL of Pfr364).


The malaria burden of Myanmar still remains high within the Greater Mekong Subregion of Southeast Asia. An important indicator of progress towards malaria elimination is the prevalence of parasite infections in endemic populations. Information about malaria epidemiology is mostly derived from reports of confirmed acute malaria cases through passive case detection, whereas the prevalence of baseline subclinical malaria infections is much less known.Plasmodium subclinical infections were estimated by light microscopy (LM), rapid diagnostic tests (RDTs) and nested PCR. The nested PCR analysis was performed with a modified pooling strategy that was optimized based on an initial estimate the infection prevalence.In this study, cross-sectional surveys were conducted during the rainy season of 2017 in four townships  of Myanmar with divergent annual malaria incidences. A total of 1991 volunteers were recruited from local villages and Plasmodium vivax was the most prevalent in all locations, while Plasmodium falciparum also accounted for 32% of infections in the western township Paletwa. Two RDTs based on the detection of the hrp2 antigen detected a total of 103 P. falciparum infections, and the ultrasensitive RDT detected 20% more P. falciparum infections than the conventional RDT. In contrast, LM missed the majority of the subclinical infections and only identified 14 Plasmodium infections.The overall malaria infection prevalence based on all methods was 13.9% (277/1991) and it differed drastically among the townships, with Paletwa in the western border having the highest infection rate (22.9%) and Thabeikkyin in central Myanmar having the lowest (3.9%). As expected, nested PCR was the most sensitive and identified 226 (11.4%) individuals with parasite infections. Among the parasite species, Plasmodium infections in endemic populations of Myanmar with P. vivax becoming the predominant parasite species. Geographical heterogeneity of subclinical infections calls for active surveillance of parasite infections in endemic areas. The pooling scheme designed for nested PCR analysis offers a more practical strategy for large-scale epidemiological studies of parasite prevalence. Such information is important for decision-makers to put forward a more realistic action plan for malaria elimination.Cross-sectional surveys identified considerable levels of asymptomatic The online version of this article (10.1186/s13071-019-3330-1) contains supplementary material, which is available to authorized users. Globally, malaria remains a substantial public health problem and significant economic burden with an estimated 219 million cases and 435,000 deaths in 2017 . TypicalConventionally, malaria diagnosis is done by light microscopy (LM), which normally has a detection limit of 10–20 parasites/μl blood in thick film and 100 parasites/μl blood in thin film , 9. In aThis study aimed to assess malaria prevalence at sentinel sites in Myanmar and provide baseline information for the evaluation of progress toward malaria elimination. Cross sectional surveys were conducted in four townships with different levels of endemicity , and parasite infection rates were estimated by using LM, two types of RDTs, and nested PCR incorporating a sample pooling design.P. falciparum and 10–50‰ P. vivax), followed by Banmauk , Bilin  and Thabeikkyin  [Four Myanmar townships Paletwa (western), Banmauk (northern), Thabeikkyin  and Bilin (southeastern) with different malaria transmission intensities were selected to conduct cross-sectional surveys to assess malaria prevalence during the rainy season of 2017 , a conventional RDT (cRDT); and Alere™ malaria Ag Pf RDT (Alere), an ultrasensitive RDT (uRDT); were employed to detect P. falciparum infections. Both RDTs were based on the detection of P. falciparum HRP-2 antigen.For LM diagnosis of malaria, Giemsa-stained blood smears were read by microscopists at the field sites following an established standard operating procedure . A slideTo detect parasite DNA, genomic DNA (gDNA) was isolated from the dried blood spots on filter paper using a QIAmp DNA Mini Kit . Isolated genomic DNA was eluted into 35 μl of elution buffer and used immediately or stored at -20 °C until further use. For parasite detection, genus-specific nested PCR and species-specific nested PCR assays were performed using 2 μl of gDNA and primers  and rPLU3/rPLU4 (Nest2). To design a pooling strategy for the remaining negative samples, a contingency table /(2+P×n1), Tindividual = S×2+P×S×n1=S×(2+P×n1), Tpooling = (S/N)×2+P×S×N×2+P×S×n2=S×(2/N+2P×N+P×n2), Tsave = Tindividual – Tpooling= S×(2+P×n1) –S×(2/N+2PN+P×n2) =S×(2+P×n1–2/N–2PN–P×n2), where Tindividual refers to the number of tests performed if the samples are tested individually, Tpooling the number of tests performed if the samples are pooled, and Tsave the number of tests saved if the samples are pooled versus tested individually. In the formula, P is the estimated infection rate, N number of samples in each pool, n1 number of species (5 in this study), and n2 the number of groups of the species are divided into for nested PCR (3 in this study). Rsave was estimated assuming that each pool contains no more than one positive sample. For each positive pool, samples were first tested individually by using genus-specific nested PCR  was calculated for binary variables and Chi-square or Fisher’s exact tests were used where appropriate. n = 315), Banmauk (n = 631), Thabeikkyin (n = 414) and Paletwa (n = 633). The overall parasite prevalence was 13.9% (277/1991) when all positive samples identified by at least one method were considered  and 5.0% (100/1991), respectively. For parasite species, P. vivax accounted for the majority of the infections 69.9% (158/226), while P. falciparum and P. ovale were responsible for 25.2% (57/226) and 0.4% (1/226), respectively. Mixed infections by P. vivax/P. falciparum and P. vivax/P. ovale accounted for 3.5% (8/226) and 0.9% (2/226), respectively  of P. falciparum infections  of asymptomatic malaria infections, followed by Bilin and Banmauk (~12%), and Thabeikkyin had the lowest prevalence 3.9%). LM only identified parasite infections in the western and northern townships, whereas it did not detect any in the eastern and central townships. In the southeastern and northern townships, .9%. LM o2.9% of aP. falciparum infections by at least one method, of which 3.3% (n = 65), 0.7% (n = 13), 3.7% (n = 73) and 5.0% (n = 100) were detected by nested PCR, LM, cRDT and uRDT, respectively. A Venn diagram shows the overlap in the number (percent) of individuals with P. falciparum infections detected by the four methods . uRDT detected all samples that were positive by cRDT. Among 65 P. falciparum positives from nested PCR, only 10.8% (n = 7), 55.4% (n = 36) and 43.1% (n = 28) were also positive by LM, uRDT and cRDT, respectively, whereas 44.6% (n = 29) was not detected by the rest of detection methods .Of the 1991 participants, 131 were diagnosed with ods Fig. . Of all P. falciparum infections in the cross-sectional surveys were evaluated using LM or nested PCR as the gold standard .Because the visits to the surveyed villages were done in the daytime, when adults and men were probably away from home engaging in agricultural activities, the study population were slightly female-biased (56.91%) and relatively young  . Excluding 103 positive samples identified by LM and/or RDTs, 1788 samples were divided into 596 pools and genus-specific nested PCR identified 126 pools as positive. All samples from the 126 positive pools were amplified individually by genus-specific nested PCR (756 reactions), of which 158 were positive. To confirm parasite species, each of the 158 samples was re-tested in three reactions of species-specific PCR . With this pooling design, a total of 2170 PCR reactions were performed as compared to 4050 if all samples had been analyzed individually (1788 × 2 + 158 × 3). In another word, this pooling strategy reduced the workload by 46.4%.To obtain highly accurate infection prevalence by molecular methods in low-endemic areas, sample pooling is necessary. Table P. falciparum or P. vivax infections, most of which were afebrile and asymptomatic [Plasmodium infections. Compared with the confirmed acute malaria infections reported in 2017, the different prevalence of subclinical infections in these endemic areas appeared to correspond to the levels of acute malaria and very well reflected the local malaria transmission intensities. The majority of the asymptomatic infections were present at parasite densities below the detection limit of LM. Such submicroscopic infections could develop into much higher-density infections at a later time and cause acute diseases [It is believed that low-density asymptomatic infections are expected to be common in high-endemic areas with high herd immunity and premunition in the population . Contrarptomatic . Similardiseases , 30. In diseases –33. Therdiseases . AsymptoPlasmodium infections in the four townships surveyed ranged from 3.9 to 22.9%. Paletwa, located in the south-west site of Chin State had the highest parasite prevalence. Its mountainous terrain and poor accessibility thwart malaria control efforts. Bordering malarious areas of Bangladesh and India further promotes travel-associated malaria and malaria introduction [This study identified significant heterogeneity in the prevalence of subclinical malaria infections in different geographical regions of Myanmar, a country with the heaviest malaria burden in the GMS. The prevalence of oduction . InteresPlasmodium infections detected, accounting for 5% (14/277) of total infections identified by other methods. This is likely due to the low parasite densities associated with the asymptomatic infections, especially by P. vivax. Therefore, the use of microscopy is likely to result in a gross underestimation of the malaria burden [Plasmodium antigens are commonly used for malaria diagnosis due to its simplicity. Conventional RDTs have similar detection limits as microscopy, but uRDTs may be ten times more sensitive than cRDTs. This study showed that uRDTs identified 20% more P. falciparum infections. However, one limitation for the P. falciparum RDTs is the high prevalence of P. vivax infections in the GMS, and uRDTs to detect P. vivax are not available. Moreover, other disadvantages of HRP-2 based RDTs are the presence of P. falciparum parasites with hrp2 gene deletion in the study region  [This study assessed several available malaria diagnostic tools for screening asymptomatic malaria infections. LM examination, the traditional gold standard for diagnosis of clinical malaria, performed poorly in this study with only 14 a burden . RDTs fogatives) , and congatives) , 18. A pgatives) –39. In tP. vivax was the predominant species. In the western border township P. falciparum was also substantially prevalent. Whereas LM was deemed not suitable for detecting subclinical infections in these low-endemicity areas, the nested PCR method incorporating a pooling strategy would enable the implementation of molecular epidemiology studies on a large scale. Such information is important to decision-makers for future plans in the national malaria elimination program.This study shows that large proportions of individuals in malaria endemic areas of Myanmar carried subclinical malaria infections with pronounced geographical heterogeneity. In all four townships, Additional file 1:Table S1. Primers for pooling strategy of nested PCR (Plasmodium spp. detection). (DOCX 15 kb)