Approximately one million malaria cases were reported in India in 2015, based on microscopy. This study aims to assess the malaria prevalence among hospitalised fever patients in India identified by PCR, and to evaluate the performance of routine diagnostic methods.During June 2011-December 2012, patients admitted with acute undifferentiated fever to seven secondary level community hospitals in Assam (Tezpur), Bihar (Raxaul), Chhattisgarh (Mungeli), Maharashtra (Ratnagiri), Andhra Pradesh (Anantapur) and Tamil Nadu (Oddanchatram and Ambur) were included. The malaria prevalence was assessed by polymerase chain reaction (PCR), routine microscopy, and a rapid diagnostic test (RDT) with PCR as a reference method.P. falciparum single infection was detected in 46%, while 38% had P. vivax, 11% mixed infections with P. falciparum and P. vivax, and 5% P. malariae. Compared to PCR, microscopy had sensitivity of 29% and specificity of 98%, while the RDT had sensitivity of 24% and specificity of 99%.The malaria prevalence by PCR was 19% (268/1412) ranging from 6%  to 35% . Among malaria positive patients High malaria prevalence was identified by PCR in this cohort. Routine diagnostic methods had low sensitivity compared to PCR. The results suggest that malaria is underdiagnosed in rural India. However, low parasitaemia controlled by immunity may constitute a proportion of PCR positive cases, which calls for awareness of the fact that other pathogens could be responsible for the febrile disease in submicroscopic malaria. Malaria is one of the leading infectious causes of morbidity and death. The World Health Organization (WHO) estimated 214 million malaria cases and 438,000 malaria deaths globally in 2015 [Plasmodium species [Routine malaria diagnostic methods have several limitations compared to polymerase chain reaction (PCR). Accurate microscopy requires skilled personnel and high quality technical equipment , and com species , 7. In p species , 9. Furt species , 11.The primary objective of this study was to assess the proportion of malaria infections among patients with acute undifferentiated fever, presenting to secondary level community hospitals at multiple sites across India, using a highly sensitive and specific PCR targeting mitochondrial DNA as a reference method . The secThe present work was part of a multi-centre, observational, cross sectional study, investigating the proportion of acute undifferentiated fever attributable to different infections. From June 2011 to December 2012, 1564 patients admitted to seven hospitals in rural or semi-urban areas in six different states of India, were enrolled prospectively and consecutively. Inclusion criteria was inpatients aged ≥5 years with temperature ≥38°C for 2–14 days prior to admission, with no localized causes of fever. Patients received health care according to routines at the participating hospitals, and no additional interventions were performed as part of the study.The study sites were located in Tezpur , Raxaul , Mungeli , Ratnagiri , Anantapur , Oddanchatram and Ambur . Blood  was drawn into EDTA tubes and stored at -20°C at the study sites before they were bulk shipped on dry ice to the coordinating centre, where molecular and antigen testing for malaria was performed. Test analyses were performed blinded from other test results. Peripheral blood smears were prepared and examined for malaria parasites at the study sites as part of the routine microscopy work-up according to the hospitals procedures. Technicians were trained and involved in routine smear examination at the respective sites, and were retrained at the reference laboratory during the start of the study. Quality control slides were sent to the sites and reported back to the reference laboratory in a satisfactory manner.Plasmodium DNA by a genus-specific pan-malaria PCR assay targeting the mitochondrial genome. The assay was done as previously described [DNA for PCR analysis was extracted from 200 μl whole blood using QIAamp DNA Blood Mini Kit  according to the manufacturer’s instructions, and stored at -20°C prior to application. All samples were screened for presence of escribed , but witP. falciparum, P. vivax and P. malaria. A modified version of the original protocol by Padley et al. [P. ovale and P. knowlesi.All genus-specific PCR positive samples were further analysed by a species-specific PCR targeting the 18S of y et al.  was usedy et al. . Samplesy et al. . The seqAmplifications were done on AB Applied Biosystem veriti 96 well Thermal cycler , and products detected by electrophoresis on a 2% SeaKem agarose gel  stained with ethidium bromide.P. falciparum specific Histidine-Rich-Protein II and aldolase antigen of pan-malaria species . The test card has two regions, ‘Pf’ and ‘Pan’. A red band in the ‘Pf’ region alone indicates that the sample is reactive for P. falciparum . Red bands in both ‘Pf’ and ‘Pan’ region indicate either single infection by P. falciparum or a mixed infection of P. falciparum with P. vivax, P. ovale or P. malariae. Appearance of a red band in the ‘Pan’ region alone indicates that the sample is reactive for infection by a malaria species other than P. falciparum. The RDT kits were quality checked using known positive and negative controls.The EDTA-blood samples, stored at -20°C, were also tested with the RDT ParaHIT-Total Ver. 1.0 Device 55IC204-10  at the reference laboratory following manufacturer’s instructions. It detects Confidence intervals for tests’ sensitivities, specificities, positive and negative predictive values were calculated using the cii command in Stata 14 , and presented as exact 95% confidence intervals.th of August 2010) and by the Regional Ethics Committee of Norway (2010/2271-5). Written, informed consent was obtained from the patients.The study was approved by the Institutional Research Board at Christian Medical College, Vellore, Tamil Nadu (No. 7242 dated 11Among patients enrolled in the acute fever study (N = 1564), samples from 1412 patients were available for malaria PCR testing, and these patients were included in the analyses. Genus-specific PCR identified a malaria prevalence of 19% (268/1412) as shown in Seasonal variations in malaria prevalence is shown in P. falciparum single infection was detected in 46%, while 38% had P. vivax single infections, and 11% had mixed infections with P. falciparum and P. vivax. Species distribution at the sites is presented in P. falciparum dominated in Mungeli , Raxaul (East India) and Anantapur (South India). In Ambur (South India), P. vivax accounted for the majority. In Ratnagiri (West India), Oddanchatram (South India) and Tezpur (North-East India) the difference in P. vivax and P. falciparum distribution was less prominent. Mixed infections with P. falciparum and P. vivax were detected in a high number of patients in Anantapur, Ambur, and Tezpur. P. malariae was found at all sites except Mungeli and Raxaul. P. ovale was not detected in any of the patients.Species determination by species-specific PCR or sequencing was performed on samples from 251 patients . OverallAmong the 1412 samples analysed by PCR, 1168 samples were analysed with routine microscopy and 1407 with RDT .The total malaria prevalence detected by microscopy and confirmed positive by PCR was 6% (66/1168); at Ratnagiri 12% (29/267), Raxaul 2% (2/100), Mungeli 28% (2/5), Anantapur 8% (9/113), Tezpur 5% (14/276), Ambur 6% (7/117) and Oddanchatram 1% (3/318). Among these malaria patients, 92%  were adults (≥ 14 years old), and among PCR positive patients with negative microscopy, 85%  were adults. Compared to PCR the sensitivity of routine microscopy was 29% . Among fCompared to PCR the sensitivity of the RDT was as low as 24%, while only 1% (10/1407) was false positive . The totP. vivax than P. falciparum. None of the P. malariae infections were detected by microscopy.The species concordance between PCR and routine microscopy is presented in P. falciparum malaria infections as non-falciparum. None of the P. malariae single infections were detected by RDT.In this study, among hospitalized patients with acute undifferentiated fever in six states of India, we report a malaria prevalence as high as 19% using a sensitive PCR , compareThere are to our knowledge no previous hospital based malaria prevalence studies using PCR from the areas in the present study, and only a limited number of microscopy based reports. Two studies from Maharashtra (West India) among hospitalised fever patients reported a malaria prevalence of 10% (44/448) and 12% 144/1197) , 14, whi4/1197 , P. falciparum compared to P. vivax in India, which has been attributed to chloroquine resistance in P. falciparum [P. falciparum versus 38% single P. vivax infections. Predominance of P. falciparum is reported in West-, Central-, East- and North East India, and predominance of P. vivax in North- and South India [P. vivax (55%), and predominance of P. falciparum was found in the sites in Andhra Pradesh, Bihar, Chhattisgarh and Assam. However, also in Oddanchatram in Tamil Nadu the proportion of P. falciparum was higher than of P. vivax. Predominance of P. falciparum is expected among hospitalized malaria patients who are more prone to have severe disease, and species distribution in this study is not representative for malaria in the community population. However, the finding of P. falciparum predominance in the majority of the sites underlines the importance of considering P. falciparum aetiology in febrile patients in all parts of India. Furthermore, a high proportion of double infections (12%) were detected as supported by previous PCR studies in India [During the last 30 years there has been an increasing incidence of lciparum , 21. Ourin India , 22.The sensitivity detecting malaria infections by routine microscopy in the present study was only 29%, supported by several other field studies reporting low sensitivity of routine microscopy compared to PCR –25. One P. falciparum. The RDT ParaHIT-Total Device (Span Diagnostics) was chosen as it was widely used in routine diagnostics, easily available, reasonably priced, and could be stored at room temperature (25°C). In 2014 a study from India reported 70% sensitivity of this RDT compared to microscopy in detecting P. falciparum [P. falciparum specific RDT ParaHIT f (Span Diagnostics) compared to microscopy ranged from 11% and 30%, respectively, in two Tanzanian studies [P. falciparum parasites/μl with a detection score of 85% [In the present study RDT had a sensitivity as low as 24% compared to PCR, and >50% of the PCR positives which were negative by RDT were lciparum . The sen studies , 29, to The low sensitivity of microscopy and RDT compared to PCR may have two potential causes with impact on case management: True low sensitivity of routine diagnostics or asymptomatic parasitaemia in patients with other infections. Challenges regarding routine diagnostics in resource poor settings has been described above. A challenge using PCR in clinical diagnosis is that the method potentially also detects low parasitaemia in semi-immune individuals and not the true cause of fever. The pathogen actually causing the febrile disease may thereby be overlooked. In the present study, submicroscopic malaria was detected by PCR in 71% (162/228), and the level of asymptomatic malaria, and another infection responsible for the febrile disease, among these is unknown. Submicroscopic and asymptomatic malaria is not restricted to high endemic regions; increased prevalence has been reported from areas of low transmission intensity –34, and In addition to the probability of parasitaemia controlled by immunity, a proportion of the patients in this cohort potentially suffering from a nonmalarial cause of fever, could have positive malaria PCR explained by other mechanisms. PCR may remain positive for several weeks after effective malaria treatment, which is probably explained by residual asexual parasites and/or gametocytaemia . The mitAlthough there is a risk for over-diagnosing malaria as the cause of febrile disease by PCR, the fact that the method detects low density parasitaemia, undetected by microscopy and RDT, underlines the need for accessible molecular tools for better diagnosis and estimations of the true malaria burden in India. Further, in control and elimination strategies, PCR is essential for detection of submicroscopic and asymptomatic malaria which contribute to the infectious reservoir of the disease , 39, 40.A potential limitation regarding the distribution of malaria across the sites is that a proportion of the patients may have contracted malaria in another area than they were hospitalised. Further, occurrence of selection bias in the inclusion of patients and samples in the favour of clinically suspected malaria cannot be ruled out. Ineffective adherence to routines in a PCR laboratory can result in false positivity due to contamination. Contamination can occur in all steps of the procedure, but it is especially important to limit accumulation of amplification products in the laboratory environment . SubstanThis multi-centre study from secondary level community hospitals in six states in India, reports a high prevalence of malaria among patients admitted with acute undifferentiated fever, applying PCR as the reference method. Routine microscopy and RDT had low sensitivity and species-specificity compared to PCR. As PCR is often not available or feasible in routine diagnostics, RDT and microscopy remains the mainstay in work-up of fever patients. The results of this study calls for awareness of the importance of quality assurance of malaria routine diagnostics. In work up of hospitalised febrile patients it should also be taken into consideration that patients with low parasitaemia might serve as a reservoir for transmission rather than suffering from clinical malaria, and other potential causes of fever should be ruled out.S1 Dataset(SAV)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


P. falciparum-specific IgG, but the extent and factors influencing maternal-foetal transfer in low transmission areas co-endemic for both P. falciparum and P. vivax are unknown. Pregnant women were screened weekly for Plasmodium infection. Mother-neonate paired serum samples at delivery were tested for IgG to antigens from P. falciparum, P. vivax and other infectious diseases. Antibodies to malarial and non-malarial antigens were highly correlated between maternal and neonatal samples (median [range] spearman ρ = 0.78 [0.57–0.93]), although Plasmodium spp. antibodies tended to be lower in neonates than mothers. Estimated gestational age at last P. falciparum infection, but not P. vivax infection, was positively associated with antibody levels in the neonate . Maternal-foetal transfer of anti-malarial IgG to Plasmodium spp. antigens occurs in low transmission settings. P. vivax IgG acquisition is not associated with recent exposure unlike P. falciparum IgG, suggesting a difference in acquisition of antibodies. IgG transfer is greatest in the final weeks of pregnancy which has implications for the timing of future malaria vaccination strategies in pregnant women.During pregnancy immunolglobulin G (IgG) antibodies are transferred from mother to neonate across the placenta. Studies in high transmission areas have shown transfer of  Plasmodium falciparum and P. vivax malaria infections are a major cause of mortality and morbidity, resulting in an estimated 584,000 deaths and 198 million clinical cases each year, predominantly young children under the age of five2The blood stage of Plasmodium spp. infections. Immunity acts by reducing parasite densities and associated clinical symptoms rather than protecting against Plasmodium spp. infection per se516727923P. falciparum and P. vivax densities, compared to non-pregnant adults4P. falciparum that accumulate in the placenta56P. falciparum-IEs in the placenta is mediated in part by PfVAR2CSA, a specific variant of P. falciparum erythrocyte membrane protein (PfEMP1) expressed on the surface of P. falciparum-IEsPfVAR2CSA antibodies which have been associated with a decrease in the rates and density of placental infection13Naturally acquired immunity develops in individuals living in malaria endemic areas after repeated exposure to P. falciparum IgG has been shown to correlate between maternal and cord samples, and detectable IgG titres and P. falciparum antigen-specific antibodies have been demonstrated in newborns living in high transmission areas of Africa and Papua New Guinea20212223P. falciparum in low transmission settings and even fewer studies addressing the transfer of P. vivax antibodies. Importantly there are few studies comparing the maternal-foetal transfer of antibodies to Plasmodium spp. compared to other pathogens and vaccine-preventable diseases. In addition, very little is known about factors that influence infant antibody levels and, importantly, that influence the rate of maternal-foetal antibody transfer. Previous studies have shown that P. falciparum placental infection, HIV, gestational age at birth and hypergammaglobulinemia can reduce transplacental transfer of maternal antibodies252627Although foetuses are capable of synthesising immunoglobulin from the twelfth week of gestation, the majority of foetal immunoglobulin is of maternal origin1618P. falciparum and P. vivax antigens representing different life-cycle stages in maternal, umbilical cord, and neonatal samples at delivery, in Karen women attending antenatal clinics at the Thai-Myanmar border. In this setting both P. falciparum and P. vivax transmission is low and placental infection is relatively rare as is the presence of HIV (<0.2%)P. falciparum and P. vivax merozoite antigens, and antigens on the surface of Pf-IEs and gametocytes as well as to tetanus, measles and cytomegalovirus (CMV). We investigated how factors such as species-specific Plasmodium exposure (and timing of exposure), gravidity, chemoprophylaxis and gestational age influenced maternal-foetal transfer and neonatal antibody levels.In this study we determined antibodies to a panel of Plasmodium spp. infection by light microscopy using a finger prick blood sample, and every second week for anaemia by haematocrit. All women are invited to deliver at SMRU although Karen women traditionally deliver at home. The epidemiology of malaria in this area, and the effects of P. falciparum and P. vivax malaria during pregnancy and on birth outcomes, have been described in detail previously3031This study took place in the antenatal clinics (ANCs) of the Shoklo Malaria Research Unit (SMRU) in north-west Thailand from November 1998 to January 2000. More than 90% of pregnant women in the camps attended SMRU ANCs on a weekly basisPlasmodium spp. immunity, nested in a placebo-controlled trial of chloroquine prophylaxis33et al., 2007Plasmodium spp. parasitaemia detected by light microscopy at any time during pregnancy) and 111/331 controls (women with no parasitaemia at any time during pregnancy). The controls represented a subset, selected on IgG response to schizont extract at enrolment for determination of antibody responses during pregnancyMother-neonate pairs at delivery were selected from women included in a case-control study of Written informed consent was obtained from all participants. The study was performed in accordance with the guidelines approved by the Ethics Committee of the Faculty of Tropical Medicine of Mahidol University, Thailand; the Ethics Committee of the London School of Hygiene & Tropical Medicine, UK; and the Walter and Eliza Hall Institute of Medical Research, and the Alfred Hospital, Australia.P. falciparum and P. vivax that represented both biomarkers of exposure and protective immunity9P. falciparum  merozoite antigens apical membrane antigen 1, (PfAMA1), erythrocyte binding antigen 175 (PfEBA175) regions III-V, merozoite surface protein (MSP) 2 (PfMSP2), PfMSP3; infected erythrocyte antigen PfVAR2CSA (Duffy binding like (DBL) 5ε domain as it is recognized across geographically diverse isolates compared to other domains which are highly strain-specificP. vivax merozoite antigen (PvAMA1) was measured by high-throughput ELISA as previously describedTotal IgG was determined to antigens representing a range of life-cycle stages from both P. falciparum merozoite antigens EBA 140 regions II and III-V (PfEBA140-RII and PfEBA140-RIII-V)PfRh2)P. vivax merozoite antigens PvMSP119PvDBP)P. falciparum-IE surface antigen PfDBLα (IT4var14 NTSB3-DBLa00.23)P. falciparum sporozoite protein (PfCSP)Pfs230)PfDBLα, PfDBL5ε, Pfs230, PvAMA1 and measles at 1:250, PfMSP2, PfMSP3, PfEBA140-RII, PfEBA140-RIII-V, PfEBA175, PfRh2, PfCSP, tetanus toxoid and CMV at 1:500, PvMSP119 and PvDBP at 1:1000 and PfAMA1 at 1:2000 , gravidity , chloroquine prophylaxis  and EGA at delivery in weeks (continuous). In addition a new variable was created from case-control status to represent species-specific exposure. We defined an “exposed case” as a woman who had been exposed during pregnancy to the species representing the antigen, so that P. falciparum antibodies could be related to P. falciparum exposure and P. vivax antibodies to P. vivax exposure. Each species-specific case may also have had infection with the other parasite species. In addition controls were divided into high and low responders which were selection criteria for inclusion in the immunological studyPlasmodium spp. exposure, gestational age at delivery, gravidity or chloroquine prophylaxis are associated  with neonatal IgG levels. The second model included maternal IgG levels to investigate whether variables of interest were associated with transfer of maternal antibodies to the foetus. To investigate whether gestational age at delivery, gravidity or chloroquine prophylaxis modified the transfer of maternal antibodies to the foetus, an interaction term with maternal IgG levels and estimated gestational age , or gravidity, or chloroquine prophylaxis were investigated. These multivariate analyses were also conducted on the ratio of neonatal/maternal antibodies in seropositive women. Sensitivity analysis was performed by running the model including and excluding the 7 preterm deliveries in the <40 weeks category. Coefficients were less than 10% different between each model therefore preterm deliveries data were included in the <40 weeks group rather than a separate category containing few observations. Final models included maternal IgG levels together with an interaction term with estimated gestational age with adjustments for gravidity, chloroquine prophylaxis and Plasmodium spp. exposure. Analyses were conducted with Stata version 13.0 .Spearman’s rank correlation coefficients (ρ) were calculated to measure the association between antibody levels derived from maternal, cord and neonatal blood, and for those mothers with a malaria infection during pregnancy, between antibody levels and estimated gestational age of last exposure to malaria infection. To investigate potential predictors of neonatal antibody levels we used multivariable robust linear regression (to reduce the influence of multivariate model outliers) on logP. falciparum infection, and 57.9% experienced a P. vivax infection during pregnancy, the median number of infections for both species was one  and neonatal samples  and P. vivax (n = 33). We examined the correlation of antibody levels in the neonate at delivery with estimated gestational age of the last exposure . EGA at last P. falciparum infection was positively associated with antibody levels in the neonate, with higher levels seen in neonates whose mothers had been exposed later on in pregnancy . There was no association between P. vivax antibody responses and EGA at the last P. vivax infection (ρ 0.19 [0.09–0.3]). There was no correlation between the time since maternal tetanus vaccination and neonatal tetanus antibodies at delivery (ρ −0.08).Due to the weekly parasitological assessment, we were able to assess how the timing of infections during pregnancy influenced the levels of neonatal antibodies and maternal transfer of antibodies in a subset of women who had been exposed to To identify factors that modified the maternal-foetal transfer of antibody levels, log transformed maternal antibody levels, together with interaction terms between variables of interest, were added to the model predicting neonate antibody levels. Gravidity and chloroquine prophylaxis were not shown to modify the relationship between maternal and neonatal antibody levels (data not shown), but EGA was an effect modifier of the maternal-foetal transfer relationship. The increase in neonatal antibody levels per unit increase in maternal antibody levels were typically moderately greater in neonates born after 40 weeks gestation compared to neonates born before 40 weeks gestation, with varying levels of magnitude of this effect depending on antigen . AnalysiP. falciparum and P. vivax antibody responses in an endemic low transmission area of South-East Asia, we demonstrated transfer of a broad range of both P. falciparum and P. vivax antibodies across the placenta during pregnancy. Anti-malarial IgG was slightly lower in neonates compared to mothers, but reached higher levels when gestation was longer. P. falciparum specific IgG was positively associated with recent P. falciparum infection, however P. vivax IgG was not as strongly linked with infection.In a comprehensive study of the maternal-foetal transfer of 3. Saturation of Fc receptors may be caused by maternal hypergammaglobulinemia (IgG > 15 g/L)3 which has been associated with reduced levels of anti-measles and anti-viral, but not tetanus, antibodies in the cord blood compared to the mother in several studies272849Plasmodium spp. antibody also appears impaired. Despite this impairment, our data would suggest that even in low transmission areas with prompt treatment of malaria, maternal IgG to malarial antigens is high and antimalarial antibodies are transferred to neonates at maximum capacity. Maternal-foetal transfer was dependent on EGA at delivery, with more maternal antibody transfer occurring at later stages of pregnancy. In healthy pregnancies (both term and preterm), neonatal total IgG is directly related to length of gestation31851Antimalarial IgG levels were lower in neonates compared to mothers, a finding which has been reported in other malaria studies in high transmission areas21244647Plasmodium spp. during pregnancy. Increases in P. falciparum-specific neonatal antibody levels were strongly associated with P. falciparum maternal infection during pregnancy. However increases in P. vivax antibody levels, with respect to P. vivax maternal exposure, were of lower magnitude. We have previously demonstrated in these women that P. falciparum merozoite and pregnancy-specific responses are boosted with each successive P. falciparum infection in pregnancy but P. vivax responses are not boosted in response to P. vivax infectionP. vivax infections during pregnancy in this cohort which may not be detected with microscopyPlasmodium spp. exposure during pregnancy has a knock-on effect on neonatal antibody levels, but how differential species-specific antibody responses translates to differential species-specific risk in the first six months is yet to be determined.The only maternal factor associated with neonatal antibody levels was exposure to Plasmodium spp. occurred during pregnancy, and investigate the association between timing of infection and neonatal antibody levels as previous studies of infant antibodies have only examined parasitaemia at delivery2646Plasmodium spp. antibodies. We have previously shown that antibody half-life is shorter for Plasmodium spp. antibodies (1–2 years) compared to tetanus antibodies (10–12 years) in this cohort335152The weekly sampling for the presence of parasites also enabled us to accurately classify when exposure to Plasmodium spp. antigens. In the trial chloroquine prophylaxis successfully prevented P. vivax, but not P. falciparum infection during pregnancy56Antibody transfer may be affected by drug regimens designed to protect women from malaria during pregnancy. Women included in the current study were participating in a randomised controlled trial of chloroquine prophylaxisPlasmodium in a blood smear. Rapid treatment upon diagnosis of Plasmodium spp. infection may mean that high parasitaemias and placental malaria are reached less often and consequently lower levels of antibody are observed in our study population than in both high transmission settings and populations in low transmission settings who do not receive intensive screening and treatment. In this setting there are low numbers of premature births , and low prevalence of placental malaria )58Plasmodium spp. antigens are IgG1 and IgG3 that mediate reductions in parasitemia by a number of mechanisms , but different Plasmodium spp. antigens have different IgG1/IgG3 biasPfEMP1 antibodies are transferred may also suggest an ability for transferred IgG to protect the neonate from severe malaria potentially via parasite clearance or adherence inhibition6263This study was designed to assess anti-malarial antibody transfer in women in a low transmission setting. These women are also quickly treated on the presence of P. falciparum antibodies in the first year of life221264765666869P. falciparum led to increased levels of P. falciparum antibodies in the infant, but the effect of P. vivax exposure on P. vivax antibody levels was not as marked. How this translates to the differential risk of P. falciparum and P. vivax during the first six months of life is unknown and further studies are warranted.The clinical relevance of the antimalarial antibodies detected in infants is debated with conflicting evidence on the protective effects of passively transferred P. falciparum and P. vivax antibodies in treated malaria episodes. Infant antibody levels were influenced by maternal exposure to infection, and timing of those exposures. Functional assays are needed to determine the validity of the passive immunity hypothesis that maternal transfer of antibodies leads to protection from disease in infants in the first months of life. If this is indeed the case this work indicates that gestational age is important for antibody transfer, and timing of vaccination during pregnancy would also be important. If protective immunity can be gained from the mother, studies to determine the length of time that infants remain protected will inform the optimal timing of vaccination for infants.In a low transmission area in Thailand, we demonstrated the effective maternal-foetal transfer of both How to cite this article: Charnaud, S. C. et al. Maternal-foetal transfer of Plasmodium falciparum and Plasmodium vivax antibodies in a low transmission setting. Sci. Rep.6, 20859; doi: 10.1038/srep20859 (2016).


P. falciparum HRP2, and to a lesser extent for pLDH and aldolase ; a trait that was independent of geographical origin. Possible reasons for this include the duration of infection at the time of blood collection and principally, differences in antigen expression between different isolates and stages of parasites. It is critical that performance evaluation panels are standardised at a parasite density being not only clinically relevant but also being close to the test detection limit to detect differences in test performance. Given the significant fluctuation in antigen concentrations at a given parasitaemia, appropriate sample sizes are required to cover that range and challenge the RDTs at the low end of this range of antigen concentrations. Antigen concentrations that are relevant for improvements in target sensitivity for RDTs are still to be determined for samples with parasite densities below 200 p/μl.Malaria rapid diagnostic tests (RDTs) are now considered an acceptable alternative for parasite based diagnosis in the absence of adequate quality microscopy. Selection of the most appropriate RDT among the sheer number of tests available commercially has long been a challenge, raising the need to objectively assess and compare test performance. In order to facilitate RDT procurement and stimulate improvement of the quality of RDTs, methods were developed for the collection and characterization of geographically diverse, malaria infected clinical blood specimens to set up a specimen bank for the evaluation of malaria RDTs, as part of the WHO Malaria RDT Product Testing programme. Parasite positive samples were diluted to low (200 p/ul) and higher (2000 p/ul) parasite densities and used to provide comparative data on the performance of RDT lots submitted by manufacturers. Only samples containing a single species and producing consistent antigen quantification results were eligible for inclusion in the WHO Malaria Specimen Bank. At both 200 and 2000 p/μl, a large variation in antigen concentrations was found, especially for


Malaria remains endemic in Sub-Saharan Africa. Hematological changes that occur have been suggested as potential predictors of malaria. This study was aimed at evaluating the diagnostic relevance of hematological parameters in predicting malaria.A cross-sectional study involving 370 patients with signs and symptoms of malaria was conducted at Mulago Hospital, Kampala, from May, 2012 to February, 2013. Thin and thick blood films were prepared for each patient and stained with Giemsa to aid the detection of malaria parasites. Patients’ hematological parameters were determined.P. falciparum malaria parasitemic patients and nonparasitemic patients were only observed in mean (±SD) of the differential monocyte count  and the platelet count (172.43 (± 80.41) ×103 cells/μl versus 217.82 ± (95.96) ×103 cells/μl p = 0.00). The mean (±SD) values of the red blood cell indices , the differential neutrophil and lymphocyte counts, and the mean platelet volume (MPV) did not significantly differ between the two groups.Out of the 370 patients, 61 (16.5%) had malaria. Significant differences in the hematological parameters between Plasmodium falciparum malaria.Hematological changes are unreliable laboratory indicators of malaria in acute uncomplicated  The last decade has witnessed a massive scale up of malaria prevention efforts, primarily through the use of long-lasting insecticide treated bed nets, indoor residual spraying with insecticides, and an increase in accessibility to malaria diagnostic facilities. These measures, among others, are said to have saved an estimate of more than 735,000 lives in 34 African countries over the last 10 years. Yet malaria remains an enormous public health problem, responsible for 781,000 deaths in a year, most of which are African children of less than five years of age .Plasmodium. P. falciparum, P. ovale, P. vivax, and P. malariae are the most common species, with P. falciparum being the most virulent. Hematological alterations that are thought to characterize malaria may be related to the overt biochemical changes that occur during the asexual stage of the life cycle of the malaria parasite. Entry of P. falciparum into erythrocytes usually leads to a marked increase in secretion of inflammatory cytokines , endothelial cell activation , activation of the coagulation cascade , and sequestration of parasitized RBCs . ThesThe World Health Organization recommends that all persons of all ages in all epidemiological settings with suspected malaria should receive a parasitological confirmation of diagnosis . MicroscP. falciparum malaria have been clearly mentioned from prior studies, there is still a lack of evidence regarding their diagnostic relevancy in uncomplicated P. falciparum malaria. This paper therefore intends to bridge the knowledge gap by providing a report from a study that set out to determine the hematological parameters in patients with acute uncomplicated malaria attending the Mulago hospital outpatients’ clinic, in a malaria endemic area of Kampala (Uganda) so as to ascertain their diagnostic relevance or reliability in predicting malaria.Therefore, during the past decades, various efforts to replace the traditional blood film for the diagnosis of malaria have revived interests in the possibility of using routine hematological blood parameters to aid the presumptive diagnosis of malaria infection . AlteratA prospective cross-sectional study was conducted at the Mulago National Referral Hospital in Kampala during and after the rainy season, a period between the month of May 2012 and February 2013.Plasmodium falciparum, leading to the burden of endemic malaria.Mulago hospital is a 1,500-bed hospital serving both as the Uganda national referral as well as the Makerere University college of Health sciences teaching hospital. It is located in the Central division of Kampala, Uganda’s capital. Kampala lies in the south central region of Uganda, which is endemic to malaria. This region has two rainy seasons: February to April and August to November, the rest of the months being relatively dry. In addition to urban and suburban areas, Kampala city has several swampy and unpopular slum areas with poor drainage and waste disposal facilities. These in turn provide fertile breeding grounds for the female anopheles mosquito, the vector for A total of 370 patients with signs and symptoms highly suggestive of malaria were recruited. For each subject, a written informed consent was obtained. A detailed clinical history was obtained and a thorough examination was done for every subject enrolled for the study. Patients with clear signs or symptoms of a localized infection, for example, an upper respiratory tract infection, urinary tract infection, or other infections, and patients who gave a clear history of receiving antimalarial treatment before presentation to the clinic were excluded.μL.A sample of 3mLs of venous blood was collected from each participant into ethylene diamine tetra-acetic acid (EDTA) bottles and promptly analyzed for routine hematological parameters which included total white blood cell count (TWBC), differential WBC count percentages for neutrophils, basophils, eosinophils, and monocytes, Hemoglobin level (Hb), red blood cell count (RBC), hematocrit (Hct), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelet count (PC), and mean platelet volume (MPV). The hematological parameters were determined using a Coulter A-T Pierce hematology analyzer . Daily quality assurance checks were performed and recorded, and commercial standards were used in accordance with the manufacturer’s instructions. In this study, anemia was defined as a hemoglobin concentration of <10 g/dL for both male and female subjects, whereas thrombocytopenia was defined as a platelet count of <150,000 cells/μL of blood).Two slides for microscopy were promptly prepared after specimen collection, each with a thin and thick blood smear. Blood smear slides were stained with Giemsa. Two experienced microscopists examined every slide from a study participant independently, and a consensus was reached in recording the final result. Presence or absence of plasmodia parasites, the plasmodia species, and the number of asexual parasites per 200 WBCs were determined. Parasite density was calculated as the ratio of parasites to WBCs permicroliter of blood using a thick blood film . A P value of <0.05 was considered statistically significant for t-test comparisons, where, as a value under Welch’s F  = 10.95, P < 0.001 was statistically significant for comparisons done with ANOVA.Data was entered in Microsoft Excel (2010) software program and doubly checked for errors before being exported to IBM SPSS Statistics 19 software for analysis. Patients were categorized based on microscopy results into parasitemic  and nonparasitemic  groups. Data for the different hematological parameters was expressed as mean (±SD). Multivariate linear regression models were used to assess for any sex differences in the hematological parameters in either parasitemic or nonparasitemic groups. Hematological parameters between different groups were compared using the Student’s Written informed consent was obtained from all patients. The study was conducted following approval from the Ethical Review Boards of the School of Biomedical Sciences, Makerere University College of Health Sciences, Mulago Hospital, and the Uganda National Council of Science and Technology.n = 61) had P. falciparum malaria confirmed by microscopy, whereas the rest tested negative (n = 309). The mean age of the parasitemic and nonparasitemic patients was 26.31 (±9.94) years and 28.25 (±10.14) years, respectively. Therefore, this close age range shows that age was not a confounding factor in our analysis.The study involved 370 suspected cases of malaria infection. The mean age of the participants was 28 years, whereas the male (108) to female (262) ratio was approximately 1 to 2.5 (1 : 2.5). Out of the total number of participants, 16.5%  are shown in P > 0.05). However, fever plus headache, or generalized body weakness, or joint pains were the commonest presentations in both the parasitemic and nonparasitemic groups.On presentation to the clinicians, most of the patients had been ill for about 3–5 days before the first visit. The various symptoms, their frequency, and level of significance (t-test as shown in The mean (±SD) values of the different hematological parameters, stratified for sex, were determined for both malaria parasitemic and nonparasitemic groups. Multivariate linear regression analysis showed no significant sex differences in the different hematological parameters in either the parasitemic or nonparasitemic groups. Hematological parameters for the malaria parasitemic group were then compared with those of the nonparasitemic group using the Student’s 3 cells/μL, and 37.38 (±18.54)%) compared with the nonparasitemic group (217.82 (±95.96) × 103 cells/μL and 42.42 ± (15.82)%). Conversely, the differential percentage of monocytes was significantly higher in the parasitemic group (10.89 (±6.23)%) compared to the nonparasitemic group (8.98 (±5.02)%).The respective mean (±SD) values of the total WBC count, lymphocytes, neutrophils, eosinophils, monocytes, RBC count, hematocrit, hemoglobin, RDW, platelets, and MPV in malaria parasitemic patients versus nonparasitemic patients are shown in Although there were mild differences in the means of the remaining hematological parameters, the differences observed were not statistically significant.12/L), MCV < 80 (fl), RDW > 15 (%), PLT < 150 (×109/L) and MPV < 8 fl had been singled out as the most likely predictors of malaria infection from prior studies [To determine the diagnostic value of the different hematological parameters, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and likelihood ratio (LR) were determined after comparison between parasitemic and nonparasitemic groups as shown in T < 150 ×9/L and MμL. Higher parasite densities were associated with lower lymphocyte and monocyte counts.As shown in For malaria patients, a prompt and accurate diagnosis is key to effective disease management . CurrentThe newer serological and molecular based malaria diagnostic approaches not only provide superior sensitivity and specificity, but also do so at a huge cost, in terms of equipment, infrastructure, and personnel. This makes most of the newer diagnostic methods inapplicable to many areas in Sub-Saharan Africa, where malaria is highly prevalent. Some of the hospitals in the region, however, can afford to carry out a complete blood count for hematological parameters in patients suspected to have an infection. Because microscopy is still considered by many as an imperfect gold standard, efforts have been made to examine the role of hematological parameters in the diagnosis of malaria infection.P. falciparum malaria are unreliable indicators for the presence of disease. Previous studies had revealed significant morphological and numerical changes in all the blood cell lines in malaria [The results from this study show that hematological parameters of patients with uncomplicated  malaria , 15–19.  malaria , disease malaria   within the normal range [3/μL, which is in agreement with results from prior studies [P. vivax, P. falciparum, or dual infection in which a significant decrease in the mean TLC in the parasitemic group was also observed [Commonly, majority of patients with acute uncomplicated al range , 22. Howal range . The mea studies . Neverthobserved .P. vivax infection in Panama [P. vivax) [P. vivax, or P. vivax, and P. falciparum dual infection [In addition, according to previous studies, leukopenia does not appear to be parasite specific as exemplified in a study on patients with n Panama , in Turk. vivax) , and in nfection  where a P = 0.03) in P. falciparum parasitemic patients as opposed to nonparasitemic controls (42.42 (±15.82)%). The transient malaria lymphopenia, particularly observed in T lymphocytes, is usually attributed to the tissue redistribution of lymphocytes [P. falciparum infection , the absolute and the differential WBC counts are low [Overall, the changes seen in the total lymphocyte count in malaria parasitemic patients are usually attributed either to an increase or a decrease in the differential white blood cell (WBC) lines. In consideration of lymphocytes for instance, there have been varying reports from different studies on whether the differential lymphocyte count remains normal, increased, or decreased in an acute malaria infection. Pre-1970s literature had indicated that lymphocyte count remains normal during an acute malaria infection . Howeverphocytes  from thephocytes , or lympphocytes . Because are low . This co are low .P = 0.01) mild monocytosis was observed in parasitemic patients (10.89 (±6.23)%) compared to the nonparasitemic patients (8.98 (±5.02)%).Part of the body’s innate immune response to blood borne pathogens usually involves activation of effector cells which are either phagocytes (neutrophils and macrophages) or Natural killer (NK) cells. It is not surprising therefore that reticuloendothelial hyperplasia, involving macrophages, is one of the most important early pathological hallmarks in malaria . Hence, The mean neutrophil count was normal for both parasitemic (46.28 (±18.30)%) and nonparasitemic patients (42.87 ±15.77)%) in this study. These findings are similar to those from two studies: one involving 400 cases in a malaria endemic region of India, in which about 85% of the patients had normal neutrophil counts  and anot.77%) in P = 0.72). A few other studies that looked at eosinophils in malaria found low levels (eosinopenia) in majority of patients [The eosinophil count was not significantly different between the parasitemic and nonparasitemic patients in this study  × 103/μL) was normal, it was significantly (P = 0.00) lower than that of the nonparasitemic group (217.82 (±95.96) × 103/μL). This only reiterates the fact that acute uncomplicated malaria is not associated with a marked reduction in platelets, as compared to severe malaria.Platelets and coagulation factors are vital components of the extraordinary complex environment that surrounds flowing or sequestered parasitized RBCs and the enclosing tubular vascular endothelium . BecauseIn an attempt to compensate for the low absolute platelet count, the bone marrow increases the formation of megakaryocytes, which usually escape from the bone marrow as mega platelets during an acute malaria infection. Evidence to support this hypothesis comes from a study by Kreil et al.  that fouThe pathogenesis of thrombocytopenia is thought to involve a constellation of processes, some of which include splenic pooling of platelets, antibody (IgG) mediated platelet destruction, adenosine diphosphate (ADP) release following the hemolysis of parasitized RBCs, dysmegakaryopoiesis, platelet aggregation and activation, parasite invasion of platelets, platelet phagocytosis, platelet adhesion to erythrocytes, and oxidative stress , 38. TheFor continued survival and reproduction, plasmodium parasites need to infect the red blood cells of their human host. Consequently, changes in the red blood cell indices are some of the commonest observations seen in malaria. Anemia, which is a fall in hemoglobin level below the normal range for age, sex, race, or pregnancy status, is the most frequent outward manifestation of such changes. Malaria is the most common cause of severe anemia in endemic areas .α)) [Anemia in malaria is believed to occur due to hemolysis of parasitized and nonparasitized RBCs, peripheral removal/sequestration of RBCs, and ineffective erythropoiesis (due to high circulating tissue necrotic factor (TNFα)) , 22. In As observed elsewhere , the meaP. falciparum malaria infection [Previous studies involving patients with complicated malaria had demonstrated that a reduced platelet count, reduced white blood cell counts, and decreased red blood cell indices had relatively good sensitivities and specificities in predicting the presence of malaria infection , 11. Hownfection , 11. NevP. falciparum uncomplicated malaria does not produce significant changes in the total WBC count, differential WBC  count, and RBC indices . However, mild changes were observed in the levels of platelets and monocytes, with the platelet count being inversely proportional to the level of parasitemia, although the differences were not diagnostically relevant because of very low sensitivities. These observations could possibly be explained by a milder biochemical reaction  that is thought to characterize uncomplicated malaria. The major limitation of this study was the smaller number of patients with deranged hematological parameters especially in the parasitemic group, which created a slightly higher margin of error in the determination of the diagnostic relevance of the deranged parameters. Nevertheless, generally, hematological parameters were found to be unreliable predictors of disease in patients with uncomplicated malaria in a malaria endemic area like Kampala.In conclusion,


Plasmodium falciparum malaria.Mast cells (MCs) play an important role in the immune response and inflammatory processes. Generally, MCs can be stimulated to degranulate and release histamine upon binding to immunoglobulin E (IgE). In malaria, MCs have been linked to immunoglobulin (Ig) E-anti-malarial antibodies. This study investigated the response of MCs in the skin of patients with P. falciparum malaria cases. Normal skin tissues from 29 cases served as controls. Pre- and post-treatment tissues were included. Histopathological changes of the skin were evaluated using haematoxylin and eosin stain. MCs were investigated using toluidine blue staining. The percentage of MC degranulation was compared among groups and correlated with clinical data.Skin tissue samples were examined from ten uncomplicated and 20 complicated P. falciparum (43.72% ± 1.44) group than the uncomplicated P. falciparum (31.35% ± 3.29) (p <0.05) and control groups (18.38% ± 1.75), (p <0.0001). MC degranulation correlated significantly with the degree of parasitaemia . Associated pathological features, including extravasation of red blood cells, perivascular oedema and leukocyte infiltration were significantly increased in the malaria groups compared with the control group .MC degranulation was significantly higher in the complicated MCs in the skin dermis are activated during malaria infection, and the degree of MC degranulation correlates with parasitaemia and disease severity. Plasmodium falciparum malaria include cerebral malaria, pulmonary oedema, acute kidney injury, severe anaemia, bleeding, acidosis and hypoglycaemia [Malaria is the most serious and widespread parasitic disease in humans, occurring widely throughout the tropical areas of the world. In 2012, 207 million cases of malaria were reported, with 627,000 deaths ,4. The mlycaemia . These cde novo-synthesized mediators such as histamine, proteases, leukotrienes, and various cytokines [P. falciparum infection, the significant elevation of the blood concentrations of IgE, IgE-anti-malarial antibodies and histamine have been associated with disease severity in human [P. falciparum malaria.Mast cells (MCs) are pivotal effector cells in allergic diseases, and play a role in the body’s inflammatory response and immunity. These immune cells are abundant in tissues exposed to the external environment, including the skin ,8. MCs cytokines . MCs areytokines . In P. fin human  and animin human . MorpholP. falciparum malaria patients were classified into two groups, uncomplicated (ten cases) and complicated (20 cases) P. falciparum malaria cases, according to the available history in the clinical chart, based on WHO criteria [P. falciparum malaria patients enrolled had no history of underlying disease. Normal skin tissues served as control cases (29 cases). All tissues were retrieved from paraffin-embedded specimens collected at the Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University. The use of leftover specimens was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (MUTM 2013-044-01).Skin biopsied specimens from criteria . All P. et al. [. Briefly, after deparaffinization and hydration, the sections were immersed in 0.1% toluidine blue working solution for 5–10 min and washed in distilled water, with three changes. The tissues were then dehydrated quickly through 90%, and two changes of absolute ethanol. Sections were then cleared in xylene, and mounted with mounting medium.The collected skin tissues in paraffin blocks were re-embedded, sectioned at 4 μm thickness and stained with routine haematoxylin and eosin (H&E) stain for histopathological examination. MCs were evaluated using toluidine blue stain modified from Mills et al. . BrieflyMCs (both intact and degranulated forms) were quantified in two areas, the upper dermis (papillary layer) and the lower dermis (reticular and subcutaneous fatty layers). MCs were counted in 15 fields each, at the areas adjacent to the blood vessels, and in the connective tissues, under high-power magnification (400×). For specimens with a small counting area, all MCs were evaluated before the percentage of degranulated MCs was calculated. Parenchymatous changes, including extravasation of RBCs, perivascular oedema, and the presence of leukocytes, were quantified and presented as percentages.U test was used to compare between malaria groups, and between pre- and post- treatment data. The Statistical Package for the Social Sciences (SPSS) version 11.0 software  was used, and a value of p <0.05 was deemed statistically significant.Differences among the three groups were analysed using the Kruskal-Wallis test, and expressed as geometric means ± standard error of the mean (SEM). The Mann–Whitney P. falciparum patients usually presented with mild symptoms including fever, headache, myalgia, chills, and nausea. Seventy five percent of complicated malaria cases were associated with cerebral malaria, 30% with acute kidney injury, 45% with hyperparasitaemia, 35% with schizontaemia and 35% with severe anaemia. The clinical details of the malaria patients are shown in Table p <0.05). Artemisinin derivatives were used for the treatment of both uncomplicated and complicated P. falciparum malaria patients.Uncomplicated P. falciparum (43.72% ± 1.44) group than the uncomplicated P. falciparum  or control group  . No difference was found between pre- and post-treatment in both malaria groups. The mean number of MCs per high power field (HPF) was 2.54 ± 0.10 in the control group, 1.83 ± 0.24 in the uncomplicated P. falciparum group, and 2.69 ± 0.22 in the complicated P. falciparum group. At seven days post-treatment, total percentage MC degranulation remained elevated in both malaria groups, similar to pre-treatment (p >0.05). MC degranulation percentage correlated significantly with parasitaemia  . In complicated P. falciparum malaria, RBC extravasation was significantly increased (by 23.76%) in post-treatment, compared with pre-treatment (p <0.0001). However, there was no change in RBC extravasation between pre- and post-treatment in the uncomplicated malaria group . In the post-treatment group, perivascular oedema was significantly decreased by 36.05% in uncomplicated P. falciparum malaria, compared with pre-treatment (p <0.0001); however, this was not observed in complicated P. falciparum malaria  , but there was no change in uncomplicated P. falciparum malaria.The skin epidermis of all groups appeared similar and unremarkable. Within the dermis, RBC extravasation, perivascular oedema, and leukocytic infiltration were significant features of complicated a Figure . Extravaa Figure A. Extravd by 23.7% in postd by 23.7% in postd by 23.7% in postP. falciparum malaria-infected patients. MCs were localized mostly in the papillary layer, which correlated with blood vessel density and contributed to an immediate defense against stimuli [MCs have been implicated in the inflammatory response and allergic process by releasing various cytokines, associated with IgE-mediated immediate-type hypersensitivity reactions ,15. This stimuli . MC degr stimuli . General stimuli . In mala stimuli ,20. In a stimuli  and inte stimuli . These c stimuli . Increas stimuli , interst stimuli , gastroi stimuli  and myoc stimuli . The chain vivo. Quinine and tetracycline have also been reported to cause MC degranulation [Artemisinin derivatives were the drugs of choice used to treat all malaria cases. A previous report has documented that artemisinin can decrease MC degranulation by blocking IgE-induced MC degranulation in an anaphylactic animal model . Howevernulation . Mefloqunulation . The effnulation .rs = 0.66, p <0.0001), indicating that MCs are activated in severe P. falciparum malaria. Normally, MCs can undergo repeated rounds of degranulation and regranulation [in vitro human lung MC culture showed late recovery (18–48 hr) from IgE-induced MC degranulation [p = 0.091) and complicated P. falciparum  remained elevated. The process of MC regranulation at day 7 was not observed in the study, indicating that MC regranulation in the skin of P. falciparum is a delayed response (over seven days).Changes in the number of MCs have been reported in wound healing , keloid nulation -38. An inulation . In addinulation . Howevernulation . At day Parenchymatous changes, including RBC extravasation, perivascular oedema and leukocytic infiltration were significantly increased in the malaria groups. It can be speculated that RBC extravasation and perivascular oedema are secondary to endothelial cell damage, following cyto-adhesion and sequestration of PRBCs, and the local effects of MC cytoplasmic granules, such as TNF, histamine, heparin, and proteases . In addiP. falciparum malaria patients. In addition, the number of MCs and the degree of degranulation correlate with parasitaemia and disease severity. Associated histopathological changes in the dermis during malaria infection include extravasation of RBCs, perivascular oedema and leukocyte infiltration around the blood vessels.MC activation and degranulation are observed in the skin dermis of severe


Plasmodium vivax and Plasmodium falciparum infections. The present study aimed at investigating for the first time in Honduras the frequency of the two most common G6PDd variants.The frequency of deficient variants of glucose-6-phosphate dehydrogenase (G6PDd) is particularly high in areas where malaria is endemic. The administration of antirelapse drugs, such as primaquine, has the potential to trigger an oxidative event in G6PD-deficient individuals. According to Honduras´ national scheme, malaria treatment requires the administration of chloroquine and primaquine for both P. vivax, P. falciparum, or both. The most common allelic variants of G6PD: G6PD A+376G and G6PD A−376G/202A were assessed by two molecular methods .This was a descriptive study utilizing 398 archival DNA samples of patients that had been diagnosed with malaria due to The overall frequency of G6PD deficient genotypes was 16.08%. The frequency of the “African” genotype A− (Class III) was 11.9% . A high frequency of G6PDd alleles was observed in samples from malaria patients residing in endemic regions of Northern Honduras. One case of Santamaria mutation (376G/542T) was detected.Compared to other studies in the Americas, as well as to data from predictive models, the present study identified a higher-than expected frequency of genotype A− in Honduras. Considering that the national standard of malaria treatment in the country includes primaquine, further research is necessary to ascertain the risk of PQ-triggered haemolytic reactions in sectors of the population more likely to carry G6PD mutations. Additionally, consideration should be given to utilizing point of care technologies to detect this genetic disorder prior administration of 8-aminoquinoline drugs, either primaquine or any new drug available in the near future.The online version of this article (doi:10.1186/s12936-015-0823-z) contains supplementary material, which is available to authorized users. Plasmodium vivax and Plasmodium falciparum, with P. vivax being responsible for 79% of symptomatic cases, which represent more than 4,000 cases a year [Honduras is a malaria-endemic country where the two circulating parasite species are s a year . Honduras a year  and are P. vivax malaria includes the 8-aminoquinoline primaquine (PQ), to prevent relapses due to dormant liver stages [P. vivax relapse [Treatment of r stages . Nationa relapse . However relapse , 6.The geographical distribution of this genetic disorder extends through Africa, Asia, Southeast Asia and Latin America and overlaps with that of malaria . Recent The G6PD locus is sex-linked, and the deficiency of this enzyme is one of the most prevalent polymorphisms causing hereditary enzymopathies in humans, particularly among males who are hemizygous for this trait –12. Nume202A with 12% of enzymatic activity is classified as class III, and individuals with such variant experience clinical manifestations after specific triggers [Due to the large number of variants and differences in enzymatic activity, creating a classification system with corresponding public health and clinical significance has been challenging. A recent proposal, as reviewed by Howes et al.  suggeststriggers . In turntriggers –23.It is widely recognized that deficient variants of G6PD are more common in African, Mediterranean, and Asian populations . HoweverThe present study aimed at investigating the frequency of the most common G6PDd variants in a subset of Honduran malaria patients. This preliminary information will be useful to determine the need for larger population-based prevalence studies to assess the potential risk of haemolytic anaemia due to PQ administration in populations living in malaria-endemic areas.® cards . Patients had been routinely assessed for malaria by the Honduran Ministry of Health laboratory network both by standard microscopy and subsequent confirmation by molecular analysis. Cards had been stored in the molecular biology laboratory at the National Autonomous University of Honduras (UNAH), and archived by malaria species diagnosis and patient’s provenance, but devoid of patients’ personal information. Other than malaria infection, no other clinical information  was associated with the samples. According to the place of origin, samples were from 22 municipalities located in five malaria-endemic departments  of Honduras: Atlántida (n = 35), Colón (n = 45), Gracias a Dios (n = 133), Islas de la Bahía (n = 107), and Olancho (n = 78) .Plasmodium-positive filter paper blood spots using a Chelex-based method [Plasmodium sp. DNA was performed using an 18S rRNA nested PCR approach, according to Singh et al. [P. vivax, 66 (16.58%) for P. falciparum, and 10 (2.51%) for both species (mixed infections) , 4  pmol of each primer, and 40  ng of DNA. PCR conditions were as follow: 2 min at 94°C and 35 cycles of 94°C for 30 s, 66°C for 30 s and 72°C for 30 s, and a final extension of 15 min at 72°C. PCR products were visualized on 2% agarose gels.Two DNA fragments of the G6PD gene, corresponding to exons IV  and V  were PCR amplified independently according to protocols previously described , 35, witG6PD genotyping was carried out by RFLP of the PCR products in order to detect two of the most common African variants, G6PD A+ and G6PD A− . The 376The expected pattern of digestion for all genotypes Table  was confIn addition to the PCR–RFLP analysis, all samples showing a G6PD deficient genotype were screened using DiaPlexC™ G6PD Genotyping Kit, African type , which enables to detect 6 different G6PD variants by a one-step multi-allelic specific PCR. The six variants amplify PCR products of different sizes, as follows: G6PD A− (202) ; G6PD A+ ; G6PD Santamaria ; G6PD A− (680) , G6PD A− (968) Betica, Selma, Guantanamo , G6PD Mediterranean, Dallas, Panama, “Sassari” . Each PCR reaction was confirmed by an internal control (947pb).For further confirmation of results, a set of 8 random samples with G6PD A+ (n = 4) and G6PD A− (n = 4) genotypes was sequenced. The PCR products were purified and sequenced at the Macrogen facilities (macrogenusa.com) using both forward and reverse primers. Chromas Pro and Mega5 software were used for sequence analysis. Positions 202 and 376 of exons IV and V were evaluated searching for the confirmatory SNP.® and exported to SPSS version 21.0  for statistical analysis. Frequency distributions and percentages were calculated for sex, malaria infecting species and G6PD variants. The measure of agreement between PCR–RFLP and DiaPlexCTM G6PD Genotyping Kit  analysis was assessed by the Cohen’s kappa coefficient. A p value <0.05 was considered statistically significant. Strengthening the reporting of observational studies in epidemiology (STROBE) guidelines were followed whenever appropriate in reporting this study  and 184 (46.23%) were from female and male individuals, respectively. The frequency of two G6PDd allelic variants (A+ and A−) was determined in all samples. Overall 16% (64/398) of the samples analyzed had a G6PD A genotype. The distribution of the frequencies of G6PD A genotypes among the participants are shown in Tables 3.77% andAll 398 samples were genotyped by PCR–RFLP for the G6PDd A− allele (with two mutations in positions 376 A → G and 202 G → A). Sixteen (4.02%) were A− males, and 31 (7.79%) were A− females. For the G6PDd A+ allele (376A → G mutation), 4 (1.01%) were A+ males and 13 (3.27%) were A+ females Table . The remOf the 398 individuals genotyped by PCR–RFLP, 17 4.27%) showed the G6PDd A+ allele, of which 4 (23.53%) were A+ hemizygous males, 1 (5.88%) was an A+ homozygous female, and 12 (70.8%) were A+ heterozygous females. In addition, 47 of 398 (11.81%) samples exhibited the G6PD A− allele, of which 16 (34.04%) were A− hemizygous males, 6 (12.76%) were A− homozygous females, and 25 (53.1%) were A− heterozygous females. The remaining 334 (83.92%) individuals, −170 (42.71%) females and 164 (41.21%) males—did not carry either mutation  +  = 16/6 + (25 × 0.1%) = 1:1.88), based on previous studies describing this calculation and reviewed by Howes et al. .As shown in Table k = 0.959).To confirm the results obtained by PCR–RFLP, all 64 samples showing a G6PD A+ or G6PD A− genotype were analyzed using the commercial kit DiaPlexC™ G6PD Genotyping Kit (African type). The kit enables the detection of six variants of the G6PD gene, including those revealed through PCR–RFLP. Concordant results were obtained in all 47 samples with an A− genotype and 15 of the 17 samples with A+ genotype by PCR–RFLP. The discordant results revealed that one sample had an A− genotype whereas the other was a Santamaria mutation (376 A → G/542 A → T) (Table Of the eight samples with G6PD A+ (n = 4) and A− (n = 4) restriction patterns selected randomly for sequence analysis, all eight had the expected mutation in position 376. Conversely, the A− genotype could not be demonstrated in any of the four G6PD A− samples because of insufficient quality in the obtained sequences. A figure summarizing the overall study findings can be found as an additional file  cannot be ruled out.These findings are consistent with a recent review of the literature from Latin America and the Caribbean, which concluded that the predominant genetic variant was A− (81.1% of the surveyed population) ; the samIn the present study, the overall frequency of the deficient “African” genotype A− is greater than previous reports from highly prevalent areas in the Latin American subcontinent. These findings suggests that, as observed in other tropical regions of Latin America, G6PD deficiency is a common condition in Honduras, especially in areas that are inhabited by peoples of African descent.In terms of the distribution of G6DPd by sex, the findings of the present study are in agreement with current global estimates. Recently, Howes et al. , appliedIn terms of the proportion of hemizygous males compared with deficient females (either homozygous or heterozygous), both Howes et al.  and Mont202A/376G genotype, while the remaining 20% had A−376 G/968C or Mediterranean B− genotypes [202A [Unfortunately, the lack of published data from most Central American countries prevents us from establishing sub-regional comparisons. A single publication from Panama reports the analysis of 75 children with G6PDd detected by neonatal screening. Eighty percent of these children had an A−enotypes . Anotheres [202A . AltogetThe distribution of G6PDd variants in malaria endemic areas of Honduras shows two departments with higher allele frequencies: Gracias a Dios 38/133, 28.6%) and Islas de la Bahía 13/107, 12.2%). In contrast with the rest of the country, where the population is predominantly Mestizo, Islas de la Bahía has a higher proportion of Afro descendant people (the English-speaking and Garifuna communities); whereas Gracias a Dios is largely inhabited by the Miskitos, an ethnic group with distant African Ancestry but nowadays considered Amerindian, an indigenous community 8.6% and . These d, 12.2%. Without information on the ethnicity of the study samples, explaining the high proportion of the A− genotype found is difficult. However, based on current population demographics originating from colonization and post-colonization periods, it could be expected that a countrywide G6PDd survey would confirm higher prevalence of the “African” genotype in the same departments.Plasmodium spp. infection in non-immune adults [The high frequency of the G6PD A− variant among malaria patients in Honduras lends support to the well-known hypothesis correlating their geographical distribution –43. Sevee adults , 44, 45.e adults –49. Basee adults . NaturalFuture prevalence studies of the general population should also endeavor to include samples from both malaria-negative individuals and individuals residing in non-endemic areas of the country. Lacking these reference groups is an important limitation of the present study. Notwithstanding, our results suggest that a significant sector of the Honduran population might potentially be at risk of adverse effects due to PQ treatment.The use of 8-aminoquinolines in individuals with G6PDd has the potential to trigger adverse clinical effects, and therefore it would not be reasonable nor responsible to give one of these drugs before analyzing the patient’s G6PD status . HoweverP. vivax hypnozoites (and thus prevent relapses). In terms of PQ, the Honduran national regulation for malaria control establishes that treatment should consist of 0.25 mg/kg/day for 14 days to patients with uncomplicated vivax malaria, and 0.75 mg/kg as single dose the first day of treatment for a P. falciparum infection [Four percent of the individuals analysed here were G6PDd A− hemizygous males while 1% of them were homozygous females. These individuals could be potentially harmed by the administration of PQ, the only drug available in Honduras to eliminate liver nfection . This trBased on the importance that malaria elimination programs have acquired , MonteirWhile the number of complicated cases triggered by PQ could be low, the risk of haemolysis in individuals with a class II or III deficiency might override the benefits of malaria treatment. In turn, failure to treat malaria may put the patient at risk of serious clinical disease . Hence, The diagnosis and management of G6PDd is currently limited by cost, infrastructure and logistics. Fast, low-cost, and easy techniques are urgently needed for malaria-endemic countries such as Honduras. This is particularly true for remote, high-transmission areas where disadvantaged populations of African descent reside, as they might be more at risk of developing PQ-triggered haemolytic events , 61. NatThis study documented for the first time the frequencies of the G6PDd A+ and A− genotypes through two genotypic approaches among malaria-infected individuals in Honduras. These frequencies were higher than the average expected for Latin America. Although ethnic groups potentially carrying genetic mutations in the G6PDd gene might have been overrepresented in this study, these populations reside in areas where malaria transmission is the highest in the country. Therefore, this report highlights the need for immediate research to determine whether these populations are at risk of PQ-triggered haemolytic complications. Additionally, our findings contribute to filling the knowledge gaps regarding the distribution and diversity of G6PD variants in Latin America.


Plasmodium falciparum is acquired after only a few infections, whereas longitudinal studies show that some children experience multiple episodes of severe disease, suggesting that immunity may not be acquired so quickly. We fitted a mathematical model for the acquisition and loss of immunity to severe disease to the age distribution of severe malaria cases stratified by symptoms from a range of transmission settings in Tanzania, combined with data from several African countries on the age distribution and overall incidence of severe malaria. We found that immunity to severe disease was acquired more gradually with exposure than previously thought. The model also suggests that physiological changes, rather than exposure, may alter the symptoms of disease with increasing age, suggesting that a later age at infection would be associated with a higher proportion of cases presenting with cerebral malaria regardless of exposure. This has consequences for the expected pattern of severe disease as transmission changes. Careful monitoring of the decline in immunity associated with reduced transmission will therefore be needed to ensure rebound epidemics of severe and fatal malaria are avoided.Previous analyses have suggested that immunity to non-cerebral severe malaria due to  Immunitng study . Manifesng study –9, and ing study , suggestng study . Howeverng study ,13, suggame area .A shift in the burden of severe malaria from younger to older ages with decreasing malaria transmission intensity is also associated with changes in the dominant manifestations of disease. In high transmission settings, severe malaria is concentrated among young children over the age of six months who most commonly present with severe malarial anaemia. At intermediate and low transmission settings, cases present in older children and adults with an increasing proportion of cerebral malaria ,9,15–17,Few studies have quantified the rate of development of immunity to the different manifestations of severe malaria. Quantifying these processes is important to allow the prediction of the likely impact of interventions which are designed to reduce exposure during early years, including seasonal malaria chemoprophylaxis (SMC), use of insecticide-treated nets (ITNs) and vaccination via the Expanded Programme for Immunization (EPI), but which may leave older individuals at risk. A better understanding of the rate at which immunity is acquired is additionally likely to promote a better understanding of the pathophysiology of severe malaria.et al. used a mathematical model fitted to the age distribution of cerebral and non-cerebral malaria incidence across different transmission settings in Kenya and Gambia to estimate the rate of acquisition of immunity to these syndromes [In 1999, Gupta yndromes ,31. Thisyndromes ,32.In an attempt to resolve some of these contradictions, we estimate the rate of acquisition of immunity to different forms of severe malaria by extending a published mathematical model of malaria transmission ,34 to in2.(a)Full details of the Tanzanian study are published elsewhere  but are P. falciparum transmission intensity, which depend on the altitude from sea level to above 1800 m [The population in this area is culturally and ethnically homogeneous, but exposed to highly variable levels of e 1800 m . Childree 1800 m . In totae 1800 m ) and knoP. falciparum parasitaemia (of any density) with haemoglobin concentration (Hb) less than 5 g dl−1 . Cerebral malaria was defined as P. falciparum parasitaemia (of any density) with Blantyre coma score (BCS) less than 4 and, if there was reduced response to pain, blood glucose level greater than 38 mg dl−1 (2.1 mmol l−1), no convulsions within 1 h of diagnosis and no anticonvulsants administered within 6 h of diagnosis. Malaria with respiratory distress was defined as parasitaemia (of any density) with lower chest wall inspiratory recession or abnormally deep respiration. Patients who did not meet any of these criteria but who met the criteria for inclusion in the study reported in [Severe malarial anaemia was defined as Each ward was classified into one of six groups of differing transmission intensity: below 600 m, 600–1200 m and more than 1200 m.a.s.l. in each of Kilimanjaro and Tanga regions. The median journey time to the hospital that patients reported was also used in the analysis.(b)et al. [P. falciparum where no other detectable cause for the clinical presentation could be identified. Some of the data were summarized in [To add information from areas with intermediate transmission, which were not represented in the Tanzanian study, we also used data on incidence of severe malaria from nine sites across Africa collected together by Marsh & Snow , togetheet al. . In thesrized in , in whic(c)We used a published transmission model , which hIVA. At birth, individuals have some level of maternally derived immunity, IVM, which is also determined by exposure of mothers. Mathematically, this can be written as the level of this immunity in individuals of age a in an area with EIR ɛ, increasing with exposure asΛ is the force of infection at age a, uV represents a period during which immunity cannot be boosted following a previous boost and dV governs the duration of immunity, representing the assumption that this immunity would decline in the absence of exposure. Maternal immunity IVM is assumed to be at birth a proportion PVM of the acquired immunity of a 20-year-old in the same transmission setting and to decay exponentially with rate 1/dVM.Here, we extended the model by adding another immunity function to capture the peak incidence of severe disease at younger ages than the peak incidence of uncomplicated malaria. As individuals are repeatedly infected with malaria, they acquire this immunity, measured by the variable θ0 is the proportion in naive individuals, θ0θ1 is the proportion in a maximally immune individual, and IV,0 and κV are scale and shape parameters. fV(a) is a purely age-dependent function that allows the effect of immunity to differ by age, given by fV(a) = 1−(1−fV0)/(1+(a/aV)γv) at age a, with parameters fV0, aV and γV. Hence the incidence of severe malaria is θΛ.The proportion of new infections that develop into severe disease declines as this level of immunity increases, with a sigmoidal function given byθ on either exposure or age. The other model parameters not related to severe disease were fixed at the values estimated by Griffin et al. [The parameters allow for a flexible functional form for the dependence of the proportion n et al. .For each incident case of severe malaria the proportion of severe cases presenting with any individual symptom  was assumed to be age-dependent. The proportion of severe cases presenting with each symptom was assumed to be independent of the others and therefore any combination of the three symptoms occurs with a probability given by the product of the individual symptoms.a isq0, q1 and r for each symptom. The case fatality ratio was estimated for each combination of symptoms, with no age dependence (other than in the proportion of cases presenting with each symptom).The proportion of severe malaria cases presenting with each symptom at age We included a journey-time-dependent probability of cases arriving in hospital as a number of studies have demonstrated that longer journey times to hospital are associated with a lower probability of arrival at a healthcare facility –39.The differences in definition of the population at risk in the Tanzania study compared with the pooled summary studies from elsewhere in Africa give rise to markedly different scales for the overall incidence of disease for a similar age profile of disease , and we accounted for these differences by introducing scaling factors .The model was fitted to the data on incidence of different types of severe disease by age. The EIR in each setting was primarily informed by the parasite prevalence in that setting as the modelled relationship between EIR and parasite prevalence had previously been validated against a large number of settings ,34. Furt3.a), with hospitals placed in areas of highest population density (cd). By far the highest incidence of severe malaria was seen in the low-altitude area in the southwest, near the sea, in Tanga (b). Just under half (47%) of the severe malaria cases, and 78% of cases who died, presented with at least one of cerebral malaria, severe malarial anaemia or respiratory distress, and 8% of cases were admitted with more than one of these syndromes. The combination of respiratory distress and anaemia accounted for 60% of all cases with more than one syndrome. In the highest transmission area , the majority of admissions were in the youngest age groups (43% aged below 1 year), the most common manifestation was severe malarial anaemia (47% of cases) and 3% of cases presented with cerebral malaria.People in the Tanzanian study lived at a wide range of altitudes, leading to a high degree of variability in malaria risk a, with h density cd. By fain Tanga b. Just uet al. [Figure 3a shows the estimated proportion of infections that develop into severe malaria, plotted against the expected number of infections experienced so far. Overall, the proportion of infections resulting in severe disease is very low. In high transmission settings this proportion rises at first , before gradually falling. At an EIR of two infectious bites per person per year, the proportion of infections resulting in severe disease falls more rapidly, presumably because of age-related gains in immune system competence , but still takes several infections to fall below 1/10 of the initial proportion. The presentation of different symptoms was estimated to be modified by age, with the proportion of cases presenting with severe anaemia declining from 55% (95% CI: 49–62) at birth to 21% (18–25) at age 10 years (b). The proportion of cases presenting with cerebral malaria increased from 2% (0.5–4) at birth to 14% (11–16) by age 10 years. The proportion of cases presenting with respiratory distress was approximately constant after the first year of life at 10%, consistent with previous analyses of these data, which found that the variable most closely associated with respiratory distress was travel time to hospital [c). The combination of age-related symptoms and symptom-related mortality gives an overall case fatality ratio of the fitted model between 6% and 7%, increasing gradually with age, although there is some indication that the fitted value underestimates the case fatality ratio at older ages (d). As shown in electronic supplementary material, figure S3, the assumption that the occurrence of each symptom depends only on age may not be valid as there were some differences in symptoms seen between two areas of differing transmission intensity.The best-fitting mathematical model captured the differences in admission patterns by age  across tet al. , these d10 years b. The pr 95% CI: 9–62 at bder ages d. As shoIn the fitted mathematical model, the incidence of severe malaria increases with parasite prevalence, reaching a plateau when the prevalence in 2–10-year-olds is approximately 50% . A sligh4.risk of severe disease varies with force of infection, we were able to explain patterns of severe disease syndromes using a model of a solely physiological, age-dependent process. In particular, the proportion of the first few infections that lead to severe disease is highly dependent on the EIR (a). In a high-transmission setting, the first 30 infections are experienced very rapidly, and each individual infection has a low probability of progressing to severe disease episodes as initially infants are protected by maternal immunity, and then immunity is acquired rapidly. In a lower-transmission setting (e.g. EIR = 2), infections are experienced more slowly, and therefore maternal immunity is less important, but age-related gains in immune system competence decrease the proportion of infections that result in severe malaria. This interaction between acquisition of immunity to severe disease and age is a complex dynamic process, and our model provides a mechanism for investigating how disease patterns may change as transmission reduces.Our analysis demonstrates that variations in patterns of severe malaria with age and transmission intensity can be explained by exposure- and age-driven acquisition of immunity, which determine the incidence of severe malaria, coupled with age-dependent physiological changes, which determine the dominant clinical syndrome of severe malaria. In accordance with previous studies ,31, our  the EIR a. In a hper se with exposure and a declining proportion of severe cases developing severe malarial anaemia with age. The increased proportion of severe cases developing cerebral malaria with increasing age, although real, is likely to have a smaller effect on the overall pattern of disease. The balance between acquired immunity and exposure results in an increasing incidence of severe malaria with increasing EIR, which appears to tail off slightly at very high EIR, as suggested by Snow et al. [et al. [In addition to the dynamics of severe disease overall, we have modelled the effect of age on symptoms. The proportion of severe cases presenting with severe malarial anaemia is estimated to decline steadily with age, whereas the proportion of cases presenting with cerebral malaria increases, and the proportion of cases presenting with respiratory distress was estimated to be independent of age. These observations suggest that the observed variation in severe disease presentation with age across a wide range of transmission settings may be driven by a combination of variation in the rate of acquisition of immunity to severe disease w et al. . This cow et al.  and Lang [et al. , showingThere are, of course, limitations to the use of hospital admission data for studies of severe illness. First, whether people with severe malaria come to the hospital at all, and how severely ill they are when they arrive, will be affected by access, both spatial and economic, and treatment-seeking behaviour . HospitaFurthermore, previous or subsequent episodes of mild or severe disease were not recorded for these cases. Ideally we would test our model predictions in longitudinal studies incorporating severe and uncomplicated malaria and asymptomatic exposure into the model framework. However, the resulting close monitoring and active case detection of malaria are likely to result in prompt and effective treatment of non-severe malaria such that severe malaria becomes less likely.It should also be stressed that the majority of these data come from a small number of sites within a small number of countries. Studies such as this should be repeated in many other countries across Africa before the model can be reliably used to predict the patterns and absolute burden of severe malaria elsewhere. Factors that could affect the patterns of severe disease include nutritional status, socio-economic status, human and parasite genetics and seasonality. Furthermore, health systems vary widely across Africa, as does access to ACTs, which will affect the incidence of severe disease .There are also limitations to this modelling approach. We have assumed that immunity to severe disease is acquired as a direct consequence of exposure but do not consider the biological mechanisms by which this immunity is acquired. Strain-specific immunity is one mechanism that can reproduce the observed relationship between age and disease risk –47. HoweGiven the progressive declines in transmission that have been observed in many settings in the last decade, it is likely that both the incidence and pattern of severe disease will continue to change in the coming years. If transmission continues to decline, reduced acquisition of immunity in young children coupled with gradual loss of immunity in older children and adults will leave a larger proportion of the population susceptible to severe disease. Although the absolute risk of severe disease—at any age—is expected to decline and remain low as long as transmission continues to decline, a very large at-risk population will emerge that is highly vulnerable should malaria transmission start to rise again. This at-risk population will include a large proportion of older children and adults, and this analysis suggests that severe disease is likely to present as cerebral malaria, with an associated high case fatality ratio. Careful monitoring of the decline in immunity associated with reduced transmission will therefore be needed to ensure rebound epidemics of severe and fatal malaria are avoided.