Malaria in pregnancy (MiP) contributes significantly to infant mortality rates in sub-Saharan Africa and has consequences on survivors, such as preterm birth and low birth weight. However, its impact on long-term neurocognitive development in children remains unknown.Our prospective cohort included pregnant women and their live-born singletons from the Malaria in Pregnancy Preventive Alternative Drugs clinical trial. MiP was assessed using microscopy and real-time quantitative polymerase chain reaction (qPCR). Neurocognitive development in children was assessed using the Mullen Scales of Early Learning and the Kaufman Assessment Battery for Children, 2nd edition (KABC-II), at 1 and 6 years of age, respectively.Of 493 pregnant women, 196 (40%) were infected with malaria at least once: 121 (31%) with placental malaria diagnosed by qPCR. Multiple linear regression B-coefficients showed that impaired gross motor scores were associated with MiP at least once , placental malaria by qPCR , and high parasite density at delivery  after adjustment. Malaria and high parasite density at the second antenatal care visit were associated with lower KABC-II Non-Verbal Index scores at 6 years , respectively.This prospective cohort study provides evidence that MiP, particularly late term, could have important negative consequences on child development at 1 and 6 years of age. Mechanisms behind this association must be further investigated and diagnostic methods in low-income countries should be strengthened to provide adequate treatment.NCT00811421. This paper provides the first evidence of an association between late-term peripheral and placental malaria in pregnancy and impaired neurocognitive development in offspring at 1 and 6 years of age in Benin, sub-Saharan Africa. Plasmodium falciparum placental malaria at the time of delivery [Malaria remains one of the greatest contributors to morbidity and mortality in areas with stable transmission, particularly in sub-Saharan Africa (SSA). Of the 25 million pregnant women in SSA countries at risk of malaria infection, an estimated 25% will have evidence of delivery  and 50% delivery .While malaria in pregnancy (MiP) contributes to approximately 10 000 maternal and 200 000 fetal deaths , it can Ethical approval for all studies mentioned in this article was obtained by the institutional review boards of the Beninese Ethical Committee of the Faculté des Sciences de la Santé (FSS) and the Committee of Ethical Research of the Applied Biomedical Sciences Institute (CER-ISBA) in Benin, New York University in the United States (IRB #09-1253), and the Research Institute for Development’s (IRD) Consultative Ethics Committee in France. Written informed consent was obtained or thumbprints were provided for consent if women could not read or write.Fon) study 1 year after birth [Pregnant women in this prospective mother–child cohort were enrolled in the Malaria in Pregnancy Preventive Alternative Drugs (MiPPAD) clinical trial NCT00811421) during their second trimester. The objective of the trial was to compare the efficacy of 2 intermittent preventive treatments of MiP (IPTp), sulfadoxine-pyrimethamine (SP) and mefloquine (MQ), in 3 health centers in the Allada district in Benin . Information on inclusion and exclusion criteria is detailed in a preceding publication [11421 durer birth . SurviviP. falciparum using real-time quantitative polymerase chain reaction (qPCR) tests targeting 18S rDNA. Parasitemia was quantified by extrapolation of cycle thresholds, the value for which DNA is amplified, from a standard curve of P. falciparum ring-infected erythrocytes. Samples where no cycle thresholds were detected were considered negative, and a density of 2 parasites/μL was assigned for positive samples if amplification was lower than the standard curve of 5 parasites/μL [As part of the Benin pregnancy package, women were given IPTp (1500/75 mg SP per dose or 15 mg/kg MQ per dose) and iron and folic acid supplements during 3 antenatal care visits (ANVs) within the clinical trial, and mebendazole to treat helminth infection if present. Clinical malaria episodes were treated with oral quinine in the first trimester or with artemether-lumefantrine in subsequent trimesters for uncomplicated malaria; severe malaria was treated with parental quinine . Malariasites/μL .At 1 year of age, trained research nurses assessed cognitive function in infants using the Mullen Scales of Early Learning (MSEL) from April 2011 until November 2012. The MSEL is composed of 5 scales for gross motor, fine motor, visual reception, receptive language, and expressive language. Quality assurance of assessments, including interrater reliability, was established and the MSEL was adapted for use in this population in a pilot study . NormaliNeurocognitive development in children at 6 years was assessed using the Kaufman Assessment Battery for Children, 2nd edition (KABC-II), a psychological diagnostic tool that assesses processing ability, cognitive ability, and planning and learning capabilities. The French version of the KABC-II was orally translated into the local language by trained investigators and the construct validity for use in this population was verified during a pilot study . The MenAt recruitment, information on gravidity, iron deficiency, IPTp treatment, and sociodemographic characteristics such as maternal age, education, and socioeconomic status was collected. Soil-transmitted helminth (STH) infection was diagnosed in stool samples of women using the Kato-Katz technique. Prepregnancy body mass index (BMI) was calculated using the gestational age at inclusion, according to fundal height using manual palpation and estimated expected weight gain of women during pregnancy. At delivery, information on infant sex, gestational age according to the Ballard score, and birth weight in grams was collected. Risk factors for MiP and impaired child neurocognitive development were identified using direct acyclic graphs.Statistical analysis involved the description of sociodemographic and clinical characteristics of women during pregnancy and infant birth outcomes. Characteristics are shown for mother–child pairs included in analyses, those lost to follow-up, and deceased children. Univariate analyses and Kruskal-Wallis, Wilcoxon rank-sum, and chi-square tests were used to test the significance of common risk factors for exposures and outcomes. Linear regression models with bootstrapped 95% confidence intervals (95% CIs) tested associations between malaria diagnosed during pregnancy by microscopy and qPCR and neurocognitive development outcomes in children at 1 and 6 years. Sensitivity analyses were conducted to ensure the robustness of results, including the multiple imputation of missing variables. Analyses were conducted using STATA 16 . Statistical tests were 2-tailed with an ɑ risk of 5%.Of the 1183 pregnant women recruited into the MiPPAD clinical trial, the first 1005 women were asked to participate in the APEC nested-cohort study . At deliSociodemographic and clinical characteristics of mother–child pairs in our analyses during pregnancy and at birth are described and compared with those lost to follow-up and deceased before 6 years of age in Factors associated with MiP and cognitive development identified in the literature are shown in Linear regression results between MiP and child neurocognitive and motor development measured by the MSEL at 1 year are shown in Multiple imputation of missing variables (>20%) confirmed results seen in principal analyses ; howeverP. falciparum malaria has long-term consequences on neurocognitive development in surviving offspring in our cohort. Malaria in pregnancy at least once during pregnancy, placental malaria by qPCR, and high parasite density at delivery are associated with impaired gross motor development scores in children at 1 year. Malaria in pregnancy diagnosed by microscopy and high parasite density at the second ANV negatively impacted NVI scores of the KABC-II, and analyses showed a trend that infections occurring later in pregnancy more negatively impact KABC-II scores than earlier infections. Placental malaria by qPCR was borderline significantly associated with impaired KABC-II scores, and parasite density at delivery had a negative impact on scores after adjustment of risk factors.This paper reveals that prenatal exposure to The prospective nature of this mother–child cohort and the comprehensive biological and neurocognitive assessments carried out within this population are defining characteristics of this study. This is the first study to our knowledge to investigate the relationship between MiP and its long-term impacts on child neurocognitive development in such a large cohort from SSA. The validity of child development assessments was confirmed and assessments were adapted for use in this population previously , 14. TheThere are several limitations to our analyses, including the limited sample size in analyses for placental malaria and the multitude of tests conducted in this population, increasing the likelihood that results could be found by chance. The use of the Ballard score for gestational age at birth is also a limitation as this could have introduced measurement error when defining preterm birth. Pregnant women and children were followed extensively and treated promptly in the case of malaria infection, therefore potentially underestimating associations between MiP and child neurocognitive development. Due to the close follow-up, our study was also unable to investigate the impacts of severe malaria during pregnancy and childhood, known to have detrimental neurological consequences on survivors . HoweverResearch on the timing of maternal infections during pregnancy and its impact on fetal health remains equivocal , 23. OurPrevious studies have shown the effect of maternal infection during pregnancy on fetal neurodevelopmental abnormalities and risk of psychiatric disorders , with auPlacental malaria and parasite density at delivery, diagnosed by qPCR, were found to be particularly detrimental to development scores. One explanation for this could be that submicroscopic placental malaria infections may be particularly deleterious to fetus growth and development , 28, 29.P. falciparum infections in pregnant women. Further studies in large cohorts should be conducted to confirm our analyses and expand upon hypothesized mechanisms. Further research on clinical representations and diagnosis of neurocognitive outcomes and disability is also greatly needed in African cohorts; this population is being followed for 11 years postpartum in order to better interpret results from neurocognitive assessments and understand their norms and clinical representations.This research provides evidence of neurocognitive consequences on fetuses exposed to peripheral and placental malaria during pregnancy and expands on existing epidemiological knowledge of malaria in pregnancy in endemic areas. Submicroscopic, asymptomatic malarial infections pose a high risk to pregnant women in malaria-endemic countries like Benin, and sufficient IPTp during antenatal care, as well as in early childhood , is vitaClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciab569_suppl_Supplementary_Figure_S1Click here for additional data file.ciab569_suppl_Supplementary_Table_S1Click here for additional data file.ciab569_suppl_Supplementary_Table_S2Click here for additional data file.


Insecticide-treated nets (ITNs) are the most widely used interventions for malaria control in Africa. The aim of this study was to assess the ownership and utilization of ITNs and the knowledge of malaria and their effects on malariometric and haematological indices in children living in the Mount Cameroon area.A community-based cross-sectional study involving a total of 405 children aged between 6 months and 14 years living in Batoke–Limbe was carried out between July and October 2017. A semi-structured questionnaire was used to document demographic status, knowledge on malaria and ITN ownership and usage. Venous blood sample was collected from each child to determine the prevalence and intensity of parasitaemia by Giemsa-stained microscopy and full blood count by auto haematology analysis to obtain white blood cell (WBC) and red blood cell (RBC) counts, haemoglobin (Hb) level, haematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). A multilinear regression model was used to determine the relationship between haematological parameter as dependent variable and the independent variables.The overall prevalence of parasitaemia, anaemia, knowledge about malaria, ITN ownership, usage and effective usage was 46.7%, 54.7%, 40.7%, 78.8%, 50.9% and 29.9%, respectively. The prevalence of parasitaemia was significantly higher (P < 0.001) in children who ineffectively utilized ITNs (54.9%) than effective users (27.3%). Having knowledge of malaria, negatively correlated with WBC counts (P = 0.005), but positively correlated with Hb levels (P < 0.001), RBC counts (P < 0.001), Hct (P < 0.001), MCV (P < 0.001) and MCH (P < 0.001). ITN use positively correlated with WBC counts (P = 0.005) but negatively with Hb levels (P = 0.004), RBC counts (P = 0.006), and MCH (P < 0.001). Meanwhile, parasitaemia negatively correlated with Hb levels (P = 0.004), RBC counts (P = 0.01), Hct (P = 0.04) and MCHC (P = 0.015).There is need for more sensitization on the benefits of using the ITNs to meet up with the intended and expected impact of the free distribution of ITNs.The online version contains supplementary material available at 10.1186/s12936-021-03860-6. Anopheles vector, thereby considerably reducing their longevity and entomological inoculation rate [. [Globally, malaria is still a public health concern as 228 million cases of malaria occurred worldwide in 2018 compared with 251 million cases in 2010 and 231 million cases in 2017, with Cameroon accounting for 3% of the total number . At high rate [. , reporte rate [. .In 2018, the World Health Organization (WHO) . Yet, prAnopheles gambiae, Anopheles arabiensis, Anopheles funestus, Anopheles nili and Anopheles moucheti) [Irrespective of the increased ownership of ITNs/LLINs, a decrease in malaria transmission or morbidity is still to be appreciated especially as Cameroon is reported to harbour the five most efficient and common malaria vectors (oucheti) . Moreoveoucheti) , 23. In . [The study was conducted in Batoke–Limbe a coastal community in the Fako Division of the South West Region of Cameroon. The area is subjected to a Cameroonian-type equatorial climate characterized by fairly constant temperatures and two seasons. Batoke–Limbe is situated at the foot of Mount Cameroon and is bounded to the west by the Atlantic Ocean. The area has been described in detailed by Teh et al. .Fishing, oil palm plantation agriculture and small-scale peasant farming are the main agricultural practices in this community. The main house type is mostly plank, although block houses are also common. Some of the plank houses are quite old with many crevices on the walls and many others have no ceiling or a window mosquito mesh as seen in Fig. All children ≤ 14 years in Batoke–Limbe whose parents/caregivers signed the consent/assent forms and had resided in the area for at least a month, were recruited in the study. Children who presented with high fever or any other medical conditions requiring intensive care hospitalization, were excluded from the study.The community-based cross-sectional study was conducted between the months of July to October 2017, during the peak malaria transmission period in the Mount Cameroon area . ParticiThe sample size was calculated using the following formula :\documenudy area . The minSemi-structured questionnaires, with questions on ITN usage and its state, derived from Sumbele et al.  were adm. [Relating to the knowledge and scoring of malaria; the questions relevant to knowledge of malaria in the questionnaire were derived from Kimbi et al. . For anaThe axillary body temperature of each child was measured using an electronic thermometer and fever was classified as body temperature ≥ 37.5 °C.Out of the 4 ml of blood collected by venepuncture using a sterile 5 ml syringe, 6 and 3 µl were dispensed immediately on the same slide for the preparation of thick and thin blood films, respectively. The films were Giemsa-stained and examined in the laboratory following standard procedures . The malBriefly, the blood samples were placed on a multi mixer rotator for uniform mixing. A complete blood count was run following the manufacturer’s instructions using an auto-haematology analyser (MINRAY 2800 BC) to obtain haemoglobin concentration (Hb), haematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH), and red blood cell distribution width coefficient of variation (RDW-CV). Anaemia was defined as Hb < 11 g/dl and further classified as mild , moderate  and severe (Hb < 7 g/dl) .2). Malaria parasite counts were log transformed before analysis. The geometric mean parasite densities (GMPDs) were used to compare the intensity of infection in the study population and differences were compared using Mann–Whitney U test and Kruskal–Wallis test where appropriate. The combined level of knowledge of malaria was classified according to each respondent’s score. The mean knowledge score was 5. Appropriate knowledge corresponded to a score greater than or equal to 5, while inappropriate knowledge corresponded to a score less than 5. A multilinear regression model was used to determine the relationship of each haematological parameter as dependent variable and age, gender, knowledge about malaria, malaria parasite status, ITN use, and effective use of ITN as independent variables. Significant levels were measured at 95% confidence interval (CI) with significant differences designated at P < 0.05.Data was entered into log books and later entered into spread sheets using Microsoft Excel. After data cleansing, analysis was performed using the Statistical Package for Social Sciences (SPSS) version 20  and Epi–Info version 7 software. Data was summarized into means and standard deviations (SD), and percentages were used in the evaluation of the descriptive statistics. The comparison between malaria parasite, anaemia, ITN (ownership and usage), as dependent variables and demographic and clinical variables as independent variables were evaluated using the Chi-square test  following an administrative clearance from the South West Regional Delegation of Public Health, Cameroon. Informed consent/assent forms were read, given and explained to parents or caregivers of the children at presentation. The role of the participants was well explained and only participants who gave written and/or verbal consent or assent took part in the study. Even though participation was voluntary, parents or caregivers were free at any point to terminate the participation of the child/children in the study.A total of 405 children with a mean (SD) age of 6.31(3.54) years, and of both sexes; males  and females  residing in Batoke–Limbe participated in the study. The ITN ownership (N) was 78.8% (319) with a utilization of 50.9% (206). Most of the parents/ guardians of the children had a primary level of education  with a few  having no formal education. The proportion of children who effectively used a mosquito net was 29.9% (121), with a great proportion of the nets in good condition  in the study population was 46.7% (189/405) and majority of the children had a low  followed by moderate 20.0%, 38) and high parasitaemia . As shown in Table 0.0%, 38 2 = 7.17, P = 0.03) than their negative counterparts. Similarly, the proportion of afebrile children with moderate and mild anaemia was significantly higher  than in the febrile children as shown in Table 3The overall prevalence of anaemia was 54.6%. The prevalence of anaemia decreased significantly (P < 0.001) with an increase in age group. Contrarily, the youngest age group (< 5 years) had the highest prevalence of severe anaemia (5.2%), than those older (5–9 and 10–14 years) although the difference was not significant as shown in Table As shown in Fig. 2 = 26.08, P < 0.001) in children who were malaria parasite positive (54.9%) than negative (27.3%) as shown in Fig. The proportion of ineffective users was significantly higher (χative 27.% as showThe mean parasite density was comparable between ITN users (2.63 parasite/µl of blood) and non ITN users (2.67 parasite/µl of blood). However, the mean parasite density was lower  among children who effectively used ITN (2.24 parasite/µl of blood) compared to those who didn’t (2.74 parasite/µl of blood) as seen in Fig. The overall appropriate knowledge on malaria was 40.7%. As shown in Table The effect of malaria parasite on haematological indices is presented in Additional file Using a multilinear regression model with each haematological variable as the dependent variable, to examine the influence of age, gender, knowledge on malaria, ITN use, effective ITN use and parasitaemia status on each haematological variable; it was observed that age negatively correlated with WBC (P < 0.001) and positively correlated with Hb (P = 0.013), MCV (P < 0.001) and MCH (P < 0.001). Malaria knowledge negatively correlated with WBC P = 0.005) but positively correlated with Hb levels (P < 0.001), RBC counts (P < 0.001), Hct (P < 0.001), MCV (P < 0.001) and MCH (P < 0.001). Similarly, ITN use positively correlated with WBC counts (P = 0.005) but negatively correlated with Hb levels (P = 0.004), RBC counts (P = 0.006), and MCH (P < 0.001). In addition, a significant negative correlation of effective ITN use with WBC (P = 0.006) was observed. Furthermore, MP negatively correlated with Hb levels (P = 0.004), RBC counts (P = 0.01), Hct (P = 0.04) and MCHC (P = 0.015) as shown in  in the study area is very low. Similar results were obtained by Birhanu et al.  in Ethio.  who repo. [.  reported. [. . Consequnfection .. [The prevalence of anaemia 54.6%) in children less than 15 years underscores the high burden of anaemia among the population in this area. However, this prevalence is lower compared to the 72.7% reported by Teh et al.  and the .6% in ch.  respecti.  in 16 suPlasmodium density observed in this age group. Similar findings were also reported by Sumbele et al. [Children of the < 5 years age group had a significantly higher occurrence of anaemia compared to their counterparts. The higher prevalence could be linked to the higher e et al. . Udoh ete et al.  reportedThe proportion of bed net possession was 78.8% among the children. The coverage rate was slightly lower than the WHO recommended 80% for acceptable protection. This ownership frequency is higher than the 63.5% obtained by Fokam et al. . With reAnopheles mosquito thus preventing malaria transmission [Observations from the study demonstrated significantly lower prevalence of malaria parasitaemia in children who effectively utilized the ITNs. This finding corroborates those of Ntonifor and Veyufambom, . Insectismission .Plasmodium parasite infection in children. Although malaria parasitaemia was not significantly associated with WBCs. The mean WBC counts in malaria infected and negative groups were normal and is in line with other studies [. [This study confirms that haematological changes including RBC and its indices, are common complications encountered in  studies . Neverthudies [.  who indiudies [.  and Sumbudies [. , reporteAnopheles vector thereby preventing them from contracting the Plasmodium infection, which has been proven in several studies [. [Children who used ITN had significant higher mean Hb levels, RBC count, MCHC, RDW-CV and Plt count compared to those who did not use ITNs. Based on the results of the linear regression model, it was observed that ITN use had a significant influence on WBC, Hb, RBC, Hct, MCHC, RDW-CV and Plt. The significantly higher Hb, RBC, MCHC, RDW-CV and Plt count in ITN users than non-users, may be as a result of the protective efficacy of the ITNs that prevents the ITN users from the bite of the  studies , 61, 62 udies [.  who repoudies [. . Further. [The age of the children had a significant influence on MCV, MCH, RDW-CV and Plt counts. Likewise, the gender had a significant influence on the WBC counts, Hb and RDW-CV of the children. The results are in line with those of Onwurah et al.  who repo.  and, the. .As a limitation, the study did not explore other personal and behavioural factors such as attitude, beliefs and perception regarding the role of ITN and health care-seeking which have been reported to influence behaviours.Plasmodium transmission but reducing morbidity as well.Findings of this study revealed that malaria remains prevalent and is a major cause of morbidity in children less than 15 years of age in Batoke–Limbe, Mount Cameroon area. Despite the efforts put in place by the government of Cameroon to scale up ITN distribution so that universal coverage can be attained, coverage remains lower than the 80% recommended by the WHO. Effective utilization of ITN is low and is significantly associated with a decrease in parasite prevalence hence nation-wide campaigns to educate the population on proper use of ITN should be carried out. The overall knowledge of malaria in the study area is very low and improved malaria knowledge is associated with higher level of education hence, the higher prevalence of malaria parasite among children whose parents/ caregivers were less educated. Furthermore, ITN users had a higher Hb, RBC, MCHC, RDW-CV and Plt count than non-users, demonstrating a protective efficacy of the ITNs by not only preventing Additional file 1. Mean haematological indices as affected by malaria parasite status.Additional file 2. Mean haematological indices as affected by ITNs use.


Plasmodium species infections is essential for any country heading towards elimination. Odisha, one of the malaria-endemic states of India is targeting elimination of malaria by 2030. To support decision-making regarding targeted intervention, the distribution of Plasmodium species infections was investigated in hard-to-reach areas where a special malaria elimination drive, namely Durgama Anchalare Malaria Nirakaran (DAMaN) began in 2017.Information on the foci of Plasmodium species. The species were identified by polymerase chain reaction (PCR) followed by sequencing, in case of Plasmodium ovale.A cross-sectional survey was conducted in 2228 households during July to November 2019 in six districts, to evaluate the occurrence of P. ovale spp. and 10.3% were Plasmodium malariae infections. The majority of P. ovale spp. (75.8%) infections were mixed with either Plasmodium falciparum and/or Plasmodium vivax and found to be distributed in three geophysical regions  of the State, while P. malariae has been found in Northern-plateau and Eastern Ghat regions. Speciation revealed occurrence of both Plasmodium ovale curtisi (classic type) and Plasmodium ovale wallikeri (variant type).Of the 3557 blood specimens tested, malaria infection was detected in 282 (7.8%) specimens by PCR. Of the total positive samples, 14.1% were P. ovale spp. and P. malariae were detected in a wide geographical areas of Odisha State, which contributes around 40% of the country’s total malaria burden. For successful elimination of malaria within the framework of national programme, P. ovale spp. along with P. malariae needs to be incorporated in surveillance system, especially when P. falciparum and P. vivax spp. are in rapid decline.In the present study a considerable number of  Plasmodium species causing malaria in humans Plasmodium falciparum is most virulent and responsible for most fatalities [Plasmodium vivax, once known to cause relapse, has recently been found to be associated with severe clinical presentations and deaths as well [Plasmodium malariae, an established cause of nephrotic syndrome, can lead to progressive renal failure, particularly in adolescents or young adults [Plasmodium knowlesi, a simian parasite, has recently been detected in humans, causing severe disease and fatalities [Plasmodium ovale causes clinically mild disease but possesses the ability to cause relapse, like P. vivax [Plasmodium ovale wallikeri and Plasmodium ovale curtisi [P. vivax was the predominant species causing malaria in India (> 53%), but in the recent past the proportion P. falciparum has increased to 64%, while P. malariae remained around 2% [Plasmodium ovale spp. has been formally reported on only two occasions from India [Plasmodium knowlesi has not been reported from India so far, but P. knowlesi-specific gene sequences have been reported in the malaria vector Anopheles sundaicus in Andaman and Nicobar Islands [Malaria is one of the most important global public health challenges that contributes to a high burden of mortality and morbidity , 2. Accotalities . Plasmod as well . Plasmodg adults . Plasmodtalities . PlasmodP. vivax . Molecul curtisi . Historiround 2% , 11. Plaom India , 13. Pla Islands .P. falciparum and 8.5% by non-falciparum species [Durgama Anchalare Malaria Nirakaran , in short DAMaN.With 4% of the total population of India, the state of Odisha, with its humid and hot climatic conditions and forested low-hill topography, is highly conducive to malaria transmission, and contributes to around 40% of the country’s total burden of malaria, 91.5% of which is caused by  species . The Nat species . RealisiP. falciparum or P. vivax, but other species are often unrecognized or underestimated, due to low parasite densities, technical difficulties and observational error [DAMaN targets high-transmission, recalcitrant malaria ‘pockets’, which were identified in hard-to-reach areas of the state, spread over 23 districts. DAMaN comprised half-yearly mass malaria screening of populations living in villages and hamlets in hard-to-reach areas with historically high malaria burden, less access to routine malaria programme, and complete treatment of malaria positive cases. It also comprised distribution of long-lasting insecticidal nets (LLIN) to large sections of the state population and actively promoted their use. The details of DAMaN are described elsewhere . Althougal error . RoutineP. malariae and P. ovale spp. in areas of declining P. falciparum transmission [. As a part of the DAMaN assessment study, this current study aimed to identify the presence of different types of malaria species in the communities covered by DAMaN.Recently a study conducted in Tanzania has revealed persistent transmission of smission . WhetherDAMaN is being implemented in 23 districts out of 30 administrative districts of the State of Odisha . The curA survey was conducted at 2,228 households in those six districts between August 2019 and February 2020. One out of six sampled households had pregnant women and one out of two had children under 5 years of age. The average distance of these villages from ICMR-RMRC, Bhubaneswar laboratory is around 300 km. Blood samples were obtained from 3557 consenting individuals who were present at the time of the survey in the selected households.P. falciparum and/or other Plasmodium infections from a finger prick blood. At least 500 μl blood was taken into a microcuvette and post-centrifugation, the blood components were separated and stored at − 80 ℃. DNA was extracted from the red blood cells (RBCs). Laboratory-confirmed Plasmodium infections were those with parasites in peripheral blood as detected via RDT and/or polymerase chain reaction (PCR).During this survey 1 ml of venous blood sample was collected in BD Vacutainer ® EDTA tubes. The blood samples collected from the field sites were preserved at 4 ℃ at the local hospital and transported to the ICMR-RMRC laboratory within 24 h. The Pf-PAN RDT  was used for point-of-care detection of P. falciparum, P. vivax, P. malariae, and P. ovale spp. were amplified in two successive rounds of PCR as described by Snounou [P. ovale , partial sequence of small subunit ribosomal (18 S rRNA) gene was amplified by using two-step PCR and confirmed by DNA sequencing. PCR products were visualized under UV light following electrophoresis on 1.2% (w/v) ethidium bromide-stained agarose gel and images captured using a gel documentation system.Genomic DNA was extracted from blood using the QIAamp® DNA Blood Mini Kit  and eluted in 50 μl of distilled water. The species-specific nucleotide sequences of the 18S ribosomal RNA (18SrRNA) genes of  Snounou . For dif2 . The reaction was allowed to proceed for 35 cycles after an initial denaturation at 94 ℃ for 1 min, annealing at 50 ℃ for 1 min and extension at 72 ℃ for 1 min; final extension was at 72 ℃ for 10 min in a Thermal Cycler . The nested PCR reaction condition was same as primary PCR except the annealing temperature which was 55 ℃.Briefly, primary PCR was performed using PO1: CTACTTGACATTTCTACT TACA and PO2: CGTTCTTGATTAATGGAAGTAT and nested PCR was carried out using PO3: GCTGTAGCTAATACTTGCTTTA and PO4: TTCACCTCTGACATCTGAATC primers. The primary PCR was performed in a 25 μL reaction mixture that contained 0.2 U of Taq DNA polymerase , 0.2 mM each dNTP , 0.4 mM each primer  and 2.0 mM MgClP. ovale spp.-positive samples were purified using QIA quick gel extraction kit  following the protocol provided by the manufacturer and then subjected to DNA sequencing. 18 S rRNA gene was sequenced partially from both directions using the above-mentioned primers (PO3 and PO4) in a DNA Sequence Analyser . The consensus sequences were created after editing the DNA sequences using MultAlin . The sequences thus obtained were analysed by aligning with the available sequence at the National Centre for Biotechnology Information (NCBI) database for P. o. wallikeri (KF219558.1) and P. o. curtisi (KF696373.1).PCR product of Of the 3557 respondents from whom blood samples could be collected, 1,127 (31.7%) were male and 2,430 (68.3%) were female. The age of the study population ranged from 1 to 90 years. Overall, the median age was 24 years (IQR = 26 years) and the mean age was 23.12 years (± SD = 17.09). Amongst the individuals from whom blood samples were collected, 339 (9.5%) were pregnant and 724 (20.4%) were under five years old.Plasmodium DNA in 282 (7.9%) samples  were carrying mono-infection and 114 (40.4%) mixed infections. The majority of the cases with mono-infection were P. vivax, accounting for 40.8%. The P. falciparum mono-infection was present in 15.6% (44/282), P. malariae in 2.5% (7/282) and P. ovale spp. 0.7% (2/282) of the cases. Amongst the mixed infections 29.1% (82/282) were double infection, 10.3% (29/282) were triple infection, and 1.1% (3/282) quadruple infection. Out of 82 mixed infections P. ovale spp. was found in 27 (32.9%) of the cases and the majority  were mixed with either P. falciparum and/or P. vivax  were clinically symptomatic, while 17 (58.6%) were clinically asymptomatic. Overall, the median age of the individuals positive for P. ovale spp. was 8 years (IQR = 22.5 years) and the majority were female , two were pregnant/lactating. Asymptomatic malaria in this study was classified as a malaria infection (PCR positive) found in participants with axillary temperature < 37.5 °C at the time of the survey with absence of fever within the previous two days. Participants with asymptomatic malaria accounted for 55.67% (157/282) of the study population, and symptomatic 44.3% (125/282).Of the 3557 blood samples, 170 (4.8%) were found to be RDT-positive for malaria parasites, whereas PCR successfully amplified P. ovale spp. malaria was detected in five community health centres (CHCs) belonging to four districts and three geophysical regions. Prevalence estimates ranged from a maximum of 12.9% (43 out of 331) in Chandrapur CHC of Raygada district to a minimum of 0.74% (2 out of 268) in Chhendipada CHC of Anugul district. Region-wise distribution indicated high prevalence of P. ovale spp. in Eastern ghat , Central table land (Anugul) and Northern plateau (Sundargarh) regions. However, P. malariae has been detected in three districts belonging to Eastern ghat  and Northern plateau (Sundargarh) regions.The presence of P. ovale spp., 5 (17.2%) belong to P. o. wallikeri, 11 (37.9%) P. o. curtisi and 13 (44.8%) to both P. o. wallikeri and P. o. curtisi. DNA sequencing was carried out using PCR product of seven P. ovale spp.-positive samples, which were of sufficient quality and quantity for the detection of distinct Plasmodium spp. 18S haplotypes in an ABI-3730 XL DNA Analyser (Applied Biosystems). Obtained DNA sequences after editing were analysed by aligning with the sequence available at the NCBI Gene Bank for P. o. curtisi and P. o. wallikeri. The sequence data have been submitted to the NCBI data base and the GenBank Accession No of P. o. curtisi is MW426407/MW 426,410 /MW426419/MW426434/W426443 and P. o. wallikeri is MW295851/MW295941 . PCR assay has been used to obtain a high sensitivity and specificity in detection of both mixed-species and non-falciparum infections.This study has demonstrated, the presence of P. ovale spp. and P. malariae have the potential to cause significant morbidity. Recently reports of P. ovale spp. infection with acute respiratory distress syndrome (ARDS) [Plasmodium ovale spp. has limited distribution, with endemic transmission traditionally limited to areas of tropical Africa, New Guinea, the eastern parts of Indonesia and the Philippines. Infections with P. ovale spp. however, have also been reported in the Middle East and different parts of Southeast Asia [P. ovale spp. has been reported from Bastar Division of Madhya Pradesh in 2017 [P. ovale spp. infections during the current survey, using PCR in a wide geographical area (covering north to south) of a state that has been experiencing substantial reduction of P. falciparum infection lately, might be one of the risk factor in persistence of malaria in the future. Furthermore, prevalence of P. ovale spp. infections almost exclusively as mixed species infections with P. falciparum and/or P. vivax rather than mono-infections, illustrates that the transmission of P. ovale spp. followed those of P. falciparum and/or P. vivax. Identification of the sibling species P. o. curtisi and P. o. wallikeri of P. ovale alone and in combination during the present study can conclude that there is no general physical or temporal barrier preventing recombination of these related parasite species, but rather that P. o. curtisi and P. o. wallikeri maintain their distinct genetic identity through yet unknown biological mechanisms [P. malariae infections have been observed in all major malaria-endemic regions of the world [P. malariae  [P. ovale spp. that was currently observed in the DAMaN assessment survey.Although generally considered benign, e (ARDS) , acute re (ARDS)  and splee (ARDS)  have draast Asia . With re in 2017  and thre in 2017 . Howeverchanisms . Moreovehe world  includin. vivax)  in compaP. ovale spp. and P. malariae can be done by microscopy but is often confused with P. vivax. RDTs are even less sensitive for detection of non-falciparum malaria (NFM) as compared to microscopy. However, PCR, being most sensitive for such purpose, has been recognized as a valuable tool for confirming or diagnosing NFM, especially in less-endemic regions [P. ovale spp. infections [P. falciparum in national programmes, is also highly efficacious against asexual stages of both P. malariae and P. ovale spp. However, to prevent relapses of malaria from these two rarer/neglected species, radical treatment with primaquine is necessary, or else they will remain in circulation and continue to fuel the malaria problem. This can only be undertaken when the programme can detect the rarer/neglected species routinely, the modalities of which may be studied through an implementation research framework. However, currently there is no provision for this, and rightly so, because the rarer/neglected species are yet to caste a considerable burden on malaria scenario in the country. The current study demonstrates that as India gears up to eliminate malaria by 2030, the burden of malaria due to rarer/neglected species can assume substantial proportions in the face of dwindling rates of falciparum and vivax malaria. This study informs the national programme of what might be a future challenge, so that pre-emptive measures can be instituted.Diagnosis of  regions . Accordifections . The firP. ovale spp and P. malariae in a geographical area where the prevalence of P. falciparum has declined may support previously raised concerns that strategies designed for reducing transmission of P. falciparum may be less effective in reducing transmission of the non-falciparum species of Plasmodium [P. falciparum and P. vivax are as effective with P. ovale spp. and P. malariae or if additional strategies will be necessary to eliminate these broadly distributed and chronically under-detected rarer/neglected species of malaria. The current malaria programme of India may have to be redesigned with newer diagnostic and surveillance tools and treatment protocols to address rarer/neglected malaria species, especially when P. falciparum and P. vivax spp. are in rapid decline. Microscopy and currently available RDTs have their merits and demerits in diagnosis of P. ovale spp. and P. malariae. However, molecular-based diagnostic approach is a highly sensitive and specific method to determine the molecular signature of all human Plasmodium spp. During the COVID-19 pandemic all district headquarter hospitals (DHHs) have established molecular diagnostic facility and deployed trained human resources for diagnosis and reporting of SARS-CoV-2 infection in resource-prone states such as Odisha. These laboratories can be utilized as a central facility in future for diagnosis and confirmation of malaria species, particularly rare species, in those high-burden malaria districts. In the absence of proper diagnosis of malaria species, the ambitious goal of malaria elimination may otherwise be derailed by a surge in these rarer species.The findings highlight some of the key challenges that need to be addressed if malaria elimination is to be achieved in the Indian sub-continent. The observed high and wide prevalence of asmodium . FurtherP. ovale spp. and P. malariae in different geophysical regions of the state of Odisha. This finding may spur incorporation of appropriate surveillance strategies by the national malaria programme to detect such rarer/neglected species in the community. That may further lead to institution of newer treatment modalities to combat these parasites within the framework of the programme, so that comprehensive malaria elimination can be achieved in India.The study presents to the malaria control community the wide distribution of


Understanding the age patterns of disease is necessary to target interventions to maximise cost-effective impact. New malaria chemoprevention and vaccine initiatives target young children attending routine immunisation services. Here we explore the relationships between age and severity of malaria hospitalisation versus malaria transmission intensity.PfPR2–10). Admission with all-cause malaria, severe malaria anaemia (SMA), respiratory distress (RD) and cerebral malaria (CM) were analysed as means and predicted probabilities from Bayesian generalised mixed models.Clinical data from 21 surveillance hospitals in East Africa were reviewed. Malaria admissions aged 1 month to 14 years from discrete administrative areas since 2006 were identified. Each site-time period was matched to a model estimated community-based age-corrected parasite prevalence to provide predictions of prevalence in childhood (PfPR2–10 varied from < 1 to 48.7%. Twelve site-time periods were described as low transmission (PfPR2–10 < 5%), five low-moderate transmission (PfPR2–10 5–9%), 20 moderate transmission (PfPR2–10 10–29%) and 12 high transmission (PfPR2–10 ≥ 30%). The majority of malaria admissions were below 5 years of age (69–85%) and rare among children aged 10–14 years (0.7–5.4%) across all transmission settings. The mean age of all-cause malaria hospitalisation was 49.5 months  under low transmission compared with 34.1 months  at high transmission, with similar trends for each severe malaria phenotype. CM presented among older children at a mean of 48.7 months compared with 39.0 months and 33.7 months for SMA and RD, respectively. In moderate and high transmission settings, 34% and 42% of the children were aged between 2 and 23 months and so within the age range targeted by chemoprevention or vaccines.52,684 malaria admissions aged 1 month to 14 years were described at 21 hospitals from 49 site-time locations where Targeting chemoprevention or vaccination programmes to areas where community-based parasite prevalence is ≥10% is likely to match the age ranges covered by interventions (e.g. intermittent presumptive treatment in infancy to children aged 2–23 months and current vaccine age eligibility and duration of efficacy) and the age ranges of highest disease burden.The online version contains supplementary material available at 10.1186/s12916-021-02224-w. Plasmodium falciparum. Exposure to bites from infected mosquitoes results in human infection which can be asymptomatic or develop into mild symptoms including fever, and a minority of infections progress to severe complications that might result in death [Malaria is a vector-borne disease endemic to large parts of sub-Saharan Africa (SSA), where the predominant parasite that infects humans is in death , 2. Immuin death , 4. The in death , 5, 6 buReliable information on the age-specific patterns of malaria mortality are difficult to obtain in SSA  where moPlasmodium falciparum parasite prevalence is measured in households or schools among asymptomatic individuals providing a quantity of the intensity of malaria transmission in a given locality and traditionally standardised to a single age group 2–10 years (PfPR2–10) [Community-based parasite prevalence continues to be one important measure of malaria used in the epidemiological sub-national stratification for intervention decision-making , 17. ThefPR2–10) .Twenty years ago, hospitalised malaria events were used to provide epidemiological descriptions of severe malaria across different malaria transmission ecologies in SSA –31 durinThe absence of contemporary empirical data on the age patterns of malaria mortality and severe disease across the range of transmission settings that characterise Africa today limits our ability to target novel interventions to maximise impact. Here we present a description of age and clinical epidemiology of malaria hospitalisation among children 1 month to 14 years of age presenting to 21 hospitals in East Africa that serve communities of varying parasite prevalence common to the sub-region since the introduction of artemisinin-based combination therapy and expanded vector control.A secondary analysis was performed on data from hospitals that maintained continuous paediatric ward surveillance including malaria parasitology, purposively designed case record forms and documentation of patient residential addresses between 2006 and 2021. These were sentinel hospital sites for randomised controlled trials or sites for intensive clinical surveillance , generated from Bayesian model-based spatiotemporal geostatistical analysis of > 18,000 empirical geocoded point surveys undertaken across East Africa since 2010 [PfPR2–10 at unsampled locations. Data were corrected to standardise surveys using rapid diagnostic tests (RDT) to microscopy using a Bayesian probit regression [PfPR2–10 at 1 × 1 km spatial resolutions for each year from 2010 and aggregated to the spatial extents of each administrative, hospital polygon and over the surveillance period of each hospital-catchment period. For sites pre-2010, similar, published geostatistical modelled approaches were used to obtain predicted PfPR2–10 but these models did not include covariate priors nor correct RDT to microscopy in Kenya [PfPR2–10 < 5%, low-moderate for PfPR2–10 5–9%, moderate for PfPR2–10 10–29%, and high for PfPR2–10 ≥ 30%.Parasite prevalence among residents of areas served by the 21 hospitals provided a categorical quantity of malaria exposure in each location. We have used temporally and spatially  matched predicted estimates of nce 2010 . In briegression . Computain Kenya  and Tanzin Kenya . We havein Kenya , 68, 69:Children were included in the analysis if they were aged 1 month to 14 years, were resident in selected administrative areas and had evidence of laboratory-confirmed malaria infection on admission and a final discharge diagnosis of malaria following review of all available clinical and laboratory findings by hospital clinicians. At admission, information was documented for each patient on severe disease phenotypes including measured levels of consciousness, respiratory distress and anaemia . Uncertainty was reported as 95% credible intervals (CIs). A detailed description of the model structure and selection procedure can be found in Additional file A Bayesian generalised linear mixed model (GLMM) was developed to analyse how the age distribution of all-cause malaria admissions changed with parasite prevalence. Age was assumed to follow a Gamma distribution, with the average of this distribution modelled with a log-linear function of parasite prevalence. The random effect accounted for site-time deviations from the predicted mean. We compared a model with and without the log-linear effect of parasite prevalence to determine the statistical support for the inclusion of this term in the model using the widely applicable information criterion, WAIC . AdditioIn the pilot implementation study in Ghana, Malawi and Kenya, the first dose of RTS,S vaccine is recommended in children aged 5–6 months, subsequent doses 2 and 3 given at respective infant EPI schedules and a 4th dose at 22–24 months . IPTi isPfPR2–10 , five were defined as low-moderate transmission (5–9% PfPR2–10), 20 were moderate transmission (10–29% PfPR2–10) and 12 were defined as high transmission (≥30% PfPR2–10). When admissions were adjusted for the number of months of surveillance per transmission category, malaria hospital admission burdens were considerably lower in the low and low-to-moderate parasite transmission intensity settings compared to moderate and high transmission settings  at low transmission (< 5% PfPR2–10) to 34.1 months  in the highest transmission classification (≥30% PfPR2–10).Including the effect of parasite prevalence in the age distribution model was supported by GLMM model selection WAIC = 2.6) and the model fit for the individual site-time periods is shown in Additional file .6 and thCI 64.0, 73.8) in the lowest transmission category (PfPR2–10 < 5%) to 85.2%  in the highest transmission category (PfPR2–10 ≥ 30%) as predicted from the cumulative probability function of the Gamma distribution. Furthermore, across all transmission settings, the probability of admitted children being aged 10 years and above was very low at 5.4%  in the lowest transmission category and 0.7%  in the highest transmission category  of admitted children were predicted to be aged 2–11 months and 27.5%  to be children aged 2–23 months. In the low-moderate, the corresponding probabilities of admission were 11.1%  for 2–11 months and 29.2%  for 2–23 months. At levels of moderate transmission intensity, the probability of admitted children being aged 2–11 months was 13.2%  and 33.8%  for the age range 2–23 months. These values rose to 17.4%  and 42.0% , respectively, in the highest transmission intensity of PfPR2–10 ≥ 30%.Across all transmission settings, admissions were concentrated in children under 5 years old Figs. B and 2B,ry PfPR2–0 < 5% toPfPR2–10, SMA was the most common phenotype, accounting for 74.3% (4642/6244) of all severe malaria admissions and in 76.3% and 78.6% in the moderate and high transmission settings, respectively  in low transmission areas to 31.1 months  in areas of highest transmission  of SMA cases were reported below 5 years of age and 88.6%  in the high transmission areas  in the lowest transmission category to 27.9 months  in areas of high transmission  and 39.1 months  in the highest transmission category. Across all severe phenotypes and all transmission classifications, the majority of admissions continued to occur among children under-5 years  of all SMA cases in the low transmission category to 45.9%  in the highest transmission category. Similarly, RD had a higher proportion of cases in the 2–23-month age group in the high transmission setting (50.9%) compared to the low transmission setting (34.8%). CM was rarer among children aged 2–23 months in the low transmission setting (22.8%) compared to the highest transmission setting (35.8%) , ranging from 3.2% in the lowest transmission category where overall disease burdens were low to 1.0% in the highest transmission, high burden category , RD  and CM  (Table p = 0.070). Similarly, the case fatality for RD was highest in the lowest transmission category (12.9%) although there were no differences across the four categories of PfPR2–10 (p = 0.076). Case fatality for CM was high (13.3–14.4%) and similar across all the endemicity classes (p = 0.997)  individuals where guardians discharged patients against medical advice, 272 (0.5%) were transferred to another facility and 1431 (2.7%) where no details were documented. Among 48370 patients where the discharge outcome was documented, 744 (1.6%) children died. There were differences in case fatalities between transmission settings (PfPR2–10) compared to those admitted from areas with lower transmission intensity (< 10% PfPR2–10)  of East Africa are IPTi and RTS,S/R21 vaccination. Both interventions are aimed at operationally capitalising on successful existing EPI programme strategies (IPTi) or adapted EPI . Here we show that malaria hospitalisation among all admissions below 15 years occurring between 2 and 23 months was 34% in areas where predicted PfPR2–10 was 10–29% rising to 42% where predicted PfPR2–10 ≥ 30%. The probability of an admitted child being aged 2–11 months was 13% rising to 17% when predicted PfPR2–10 was 10–29% and ≥ 30%, respectively, in support of the approach of targeting young children aged 2–23 months in transmission settings where PfPR2–10 is ≥10%. In 2019, there were 125/366 (34%) decentralised administrative areas in East Africa that would benefit from additional personal protection offered through expanded EPI services, where predicted PfPR2–10 was ≥10%, covering 2.3 million children aged less than 2 years [Two individual-level protection strategies that have the potential to augment the current emphasis on vector control in high transmission areas   Table . CM tendMortality among malaria hospitalisation in East Africa ranged from 1% in the high transmission, high admission burden settings, to 3.2% in the lowest transmission, low admission burden settings (Table Unlike our work defining hospital admission rates that purposively chose rural communities within easy reach of the hospital and no competing local admission facilities , the preSites included in this analysis were those with stable perennial transmission in East Africa, as such we did not include traditionally acutely seasonal transmission settings more common in semi-arid areas of West Africa , 30, 88.We consider properly resourced, staff training, laboratory support and electronic data capture tools as essential to defining the clinical epidemiology of severe malaria and may serve as barometers of community-level transmission intensity to target interventions and ultimately used to monitor impact , 86, 89.Targeting chemoprevention or vaccination programmes to areas where community-based parasite prevalence is ≥10% is likely to cover the highest hospital burden. Extending chemoprevention e.g intermittent presumptive treatment in infancy to children aged 2–23 months and current vaccine age eligibility would target the most vulnerable age groups for malaria hospitalisation and the dominant severe disease phenotype of SMA.Additional file 1. Supplementary figures.Additional file 2. Supplementary tables.Additional file 3. Supplementary methods.


Pathogens may change the odor and odor-related biting behavior of the vector and host to enhance pathogen transmission. In recent years, volatile biomarker investigations have emerged to identify odors that are differentially and specifically released by pathogens and plants, or the pathogen-infected or even cancer patients. Several studies have reported odors or volatile biomarkers specifically detected from the breath and skin of malaria-infected individuals. This review will discuss the potential use of these odors or volatile biomarkers for the diagnosis of malaria. This approach not only allows for the non-invasive mean of sample collection but also opens up the opportunity to develop a biosensor for malaria diagnosis in low-resource settings. Volatile organic compound (VOC)-based diagnostics have tremendous potential in becoming the next generation screening approaches for pathogen identification and infectious disease management. VOCs refer to low molecular weight metabolic compounds that easily evaporate at ambient temperatures due to their high vapor pressures and low boiling points. VOCs comprise a diverse group of carbon-based molecules, including alcohols, ketones, aldehydes, hydrocarbons, isocyanates, amines, terpenes and sulfides . As a reIncreasing evidence shows that particular VOCs, or profiles of VOCs, may be unique to certain diseases or disease states. Pathogens can change both the quantity and composition of VOCs produced by patients during infections, and the VOCs detected in the headspace of infected cell cultures grown in vitro have proven that different pathogenic species may produce characteristic profiles of VOCs based on their distinct metabolisms ,14,15,16Non-invasive diagnosis of disease via detection and measurement of volatile biomarkers has long been of great interest in healthcare applications. Any interference in the normal functions of an organ or body system, or distribution of normal flora due to infections or diseases will cause the production of disease biomarkers deep in the body that eventually circulate in the blood and are excreted through several pathways, such as urine, saliva, sweat, sputum, breath and skin. The excreted biomarkers that are volatile create inspiration and the prospect of developing non-invasive methods for disease diagnosis .Helicobacter pylori [With the goal of increasing sensitivity and discrimination, the use of dogs for various infectious and non-infectious diseases like r pylori , differer pylori ,23, hypor pylori , epileptr pylori  and COVIr pylori ,27 has br pylori ,29. Accor pylori ,30. HoweIn the case of malaria, several studies did show the differential emission of odors or VOCs by malaria-infected individuals compared to those uninfected (Tables 1–3). Lindsay et al., attempted malaria diagnosis using two trained dogs, sniffing socks harboring foot odors of malaria-infected and -uninfected school children . The twoP. falciparum relative to the uninfected RBCs, the results of proton transfer reaction time of flight mass spectrometry (PTR-ToF-MS) were substantially reproduced by a gas sensor array, which was claimed as an E-nose, despite the fact that E-nose was unable to differentiate between asexual stage P. falciparum-infected and uninfected RBCs [The recent introduction of a new versatile diagnostic tool known as the electronic nose (E-nose) has shown significant promise in realizing the diagnosis of various diseases using VOCs for point-of-care settings . It is ated RBCs .Plasmodium parasites or its interaction with RBCs, and (ii) indirect manipulation through alterations in skin microbial composition that eventually affect the host’s skin odor profile [Anopheles gambiae compared to sweat incubated with skin bacteria [An. gambiae compared to those from other parts of the body such as the hands and sweat [Plasmodium parasites or changes in skin microbiota may serve as potential biomarkers for the development of the volatile-based diagnosis of malaria. There are two possible routes in which malaria parasites can manipulate host odor: (i) direct signal emission from  profile . Freshly profile  and onlybacteria ,40. Furtbacteria ,42,43,44P. falciparum) and -negative children, with a classification accuracy of 83%, specificity of 94% and sensitivity of 71% [P. falciparum-infected individuals not only complicates the use of these thioesters as volatile biomarkers but also limits their use for detection of other Plasmodium species [P. vivax and P. falciparum using breath terpenes were up to 91% and 93.5%, respectively [VOCs are normally identified and analyzed using gas chromatography-mass spectrometry (GC/MS). Limited studies can be found on describing the VOCs emitted from malaria patients. In a study involving children infected with malaria in Malawi, some breath volatiles were identified and a cumulative abundance of as few as six VOCs was able to differentiate between malaria-positive  of malaria-infected individuals, as well as significant divergence in volatile profiles between symptomatic and asymptomatic infections . They usPlasmodium parasites or malaria patients as mosquito attractants are relatively more extensively investigated and reported. Plasmodium-infected children are shown to draw in more mosquitoes than parasite-free children [Anopheles and enhance transmission of Plasmodium parasites. The study involving Malawian children infected with malaria also found significantly increased breath levels of mosquito-attractant terpenes, α-pinene and 3-carene [An. coluzzii antennae [P. falciparum-infected cohort in the Netherlands showed significant differential emission of 2-ethyl hexanoic acid, 2-methylbutanal, 3-methylbutanal, 3-hydroxy-2-butanone, 6-methyl-5-hepten-2-one, 1-dodecene, dodecanal, sesquiterpene and methyl dodecanoate either before, during or after the infection was induced, in which increased emissions of 2- and 3-methylbutanal and 3-hydroxy-2-butanone are known to be produced by skin bacteria, suggesting that changes in skin microflora are a factor [Anopheles mosquitoes’ differential attractiveness to P. falciparum-infected humans.VOCs released by children . Malaria3-carene  -4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which was found to be both directly and indirectly manipulated by vector behavior, was hypothesized to be emitted from gametocyte-infected persons [An. gambiae sensu stricto to RBCs. A short distance attractiveness of the mosquito An. darlingi particularly in patients carrying P. vivax gametocytes was also observed [patients ,53. In tpatients   was found to be commonly present on extracellular vesicles from infected cultures, despite the parasitemia of the cultures [VOCs emitted by gifolene   and can be inactivated (overloaded or poisoned) by certain highly polar compounds ,68,70,71Given that the investigation of the feasibility of volatile biomarkers for malaria diagnosis is still at its premature stage compared to other diseases, extensive studies are required to address the challenges in the rigorous identification and validation of volatile biomarkers that specifically and accurately distinguish individuals with and without malaria, as well as those with different infection statuses. With the rapid development and improvement of the sensitivity of analytical instruments, we envision the use of volatile biomarkers for malaria diagnosis in the field, not only for early treatment but also for monitoring disease epidemiology.


Plasmodium falciparum malaria infections.Most previous studies support a direct link between total parasite load and the clinical severity of P. falciparum parasite loads in 3 groups of children with malaria infections of differing severity: (1) children with World Health Organization–defined severe malaria (n = 1544), (2) children admitted with malaria but without features of severity (n = 200), and (3) children in the community with asymptomatic parasitemia (n = 33).We estimated P. falciparum histidine-rich protein 2 (PfHRP2) values (95% CI) increased with severity, being 7 (4–12) ng/mL in asymptomatic malaria, 843 (655–1084) ng/mL in uncomplicated malaria, and 1369 (1244–1506) ng/mL in severe malaria. PfHRP2 concentrations were markedly lower in the subgroup of patients with severe malaria and concomitant invasive bacterial infections of blood or cerebrospinal fluid  than in those without such infections .Peripheral parasitemias were highest in those with uncomplicated malaria , almost 3 times higher than in those with severe malaria  and >100 times higher than in those with asymptomatic malaria . However, the GM P. falciparum parasites. A quantitative test for plasma concentrations of PfHRP2 could be useful in identifying children at the greatest clinical risk and identifying critically ill children in whom malaria is not the primary cause.The clinical severity of malaria infections related strongly to the total burden of  Plasmodium falciparum histidine-rich protein 2, a marker of total body parasite load, are strongly correlated with the severity of P. falciparum infections among children in Kilifi, Kenya.Through a clinical surveillance study, we show that plasma levels of  Plasmodium falciparum histidine-rich protein 2 (PfHRP2) is a 30-kDa molecule that is produced by most strains of P. falciparum malaria parasites .arasites  and is i rupture , and PfHic tests , 6. PrevPfHRP2 and disease severity in a number of previous studies  × (1 – hematocrit) × body weight [kg] × 1013, (2) andPtot/Pcirc.(3) SI = t or Mann-Whitney tests as appropriate, and proportions were compared using the χ 2 test. Nonnormal data were log-transformed before analysis. Linear regression was performed both with and without adjustment for ethnicity, HbS phenotype and α-thalassemia genotype. Children displaying multiple features of severe malaria were included in multiple categories. All data were analyzed using Stata software, version 15.1 (StataCorp).Continuous data were compared using Student’s Written informed consent was provided by the parents of all participants. Ethical approval was granted by the Kenya Medical Research Institute/National Ethical Review Committee in Nairobi, Kenya, and the Oxford Tropical Research Ethics Committee in Oxford, United Kingdom.PfHRP2 was undetectable in 30 (1.7%) of these samples , leaving 1747 contributing data to the final analysis.The study flow is summarized in P < .001). Conversely, the mortality rate was comparatively lower (4.8%) in those with none of the main features but who instead displayed other features such as prostration, hypoglycemia, and hyperparasitemia  seen in those with severe malaria  than in those without . This difference was not seen in the subset of 11 infections with nontyphoidal Salmonella species , being confined to the 39 other gram-negative and gram-positive infections  , along with 240 patients without IBI. Hence 23 of 263 patients (8.7%) with a plasma PfHRP2 level <200 ng/mL had an IBI, compared with only 16 of 1258 (1.3%) with levels above this threshold (an approximately 7-fold difference).One child with uncomplicated malaria and 50 with severe malaria had concomitant invasive bacterial infections (IBIs) diagnosed by culture of blood or cerebrospinal fluid (see  < .001) . Of thesPfHRP2 levels exceeded 1000 ng/mL in 1007 of 1521 children (66.2%) with severe malaria and 104 of 198 (52.5%) with uncomplicated malaria but was <1000 ng/mL  in all children with asymptomatic malaria. The parameter that most markedly differed between groups was the SI. The GM SI (95% CI) was only 4.0 (1.8–8.8) and 4.9 (3.5–6.8) in children with asymptomatic or uncomplicated malaria, respectively, but was 40.6 (35.7–46.0) in those with severe malaria (P < .001). Overall, after adjustment for HbS, α-thalassemia, and ethnicity, the GM SI was 8.3  times higher in children admitted with severe malaria than in those admitted with uncomplicated malaria. Within those with severe malaria only, parasite densities were significantly lower in the subgroup who died in the hospital, although a trend was seen toward an increased SI  times higher and the SI 18.2  times higher than in children with uncomplicated malaria.Finally, we calculated the various measures of parasite biomass in children with specific severe malaria phenotypes and compared them with values from children in the uncomplicated group . Among tP. falciparum parasites in children with malaria of differing severity in Kilifi County, Kenya. We found no significant differences in the SI between those with asymptomatic and uncomplicated P. falciparum malaria infections, but the SI was approximately 8 times higher in those with severe malaria, being particularly high in those with severe malarial anemia. Our study supports the hypothesis that malaria severity is proportionate to total parasite load, an observation that could be helpful in directing care to those at greatest need [We have estimated both the peripheral and sequestered burdens of est need , 10.PfHRP2-based methods have supported this general conclusion. In the first study of that kind, a strong relationship was seen with total parasite load in adults admitted to hospital on the Thai-Burmese border [A direct relationship between parasite load and malaria severity was first suggested in the late 19th century , and mose border . Loads we border .PfHRP2 concentration as a marker of intracerebral parasite sequestration, identifying children with cerebral malaria confirmed by either autopsy or malarial retinopathy. Later studies have confirmed the strong relationship between total parasite burden and malaria severity in African children [PfHRP2 concentrations were found in the majority of children while levels were significantly higher in those who died than in those who survived. Of particular interest, a U-shaped association was seen between PfHRP2 concentration and death, potentially explained by the misclassification to malaria of the subgroup with the lowest concentrations [Children presenting in coma with a positive malaria blood film result in Africa are generally assumed to have cerebral malaria. However, asymptomatic parasitemia is common during childhood, and one postmortem study showed that many such children have an alternative diagnosis . Subsequchildren , 26. Firtrations .PfHRP2 level was 19 (15–23) ng/mL in asymptomatic carriers, 163 (137–194) ng/mL in children with uncomplicated malaria, detected in the community, and 1510 (1180–1933) and 1746 (1577–1934) ng/mL among children with severe malaria admitted during 2 different time periods [PfHRP2 levels were substantially lower in the subgroup of children with severe malaria who also had a concomitant IBI. In a subsequent study conducted in Tanzanian children, the plasma  periods . A conceSalmonella species, because previous studies have shown that IBIs due to Salmonella can complicate malaria via a mechanism involving gut barrier dysfunction [PfHRP2 levels were approximately 7.5 times lower in children with other gram-negative as well as gram-positive bloodstream infections, indicating that these IBIs were likely to have been the main agents of severe illness in many of these children. In our study, plasma PfHRP2 levels were <200 ng/mL in only one-sixth of severe cases but identified 23 of the 39 children with non-Salmonella IBIs: that is, approximately 9% of children with these low PfHRP2 levels had an IBI. The 16 other children with such IBIs were among 1258 with a PfHRP2 level above this cutoff (1.3%). Hence, the 200-ng/mL threshold enriched for children with non–malaria-associated IBIs by approximately 7-fold. As expected, this was not the case for the subset whose cultures were positive for nontyphoidal function . ComparePfHRP2 concentration and distinct categories of IBI was less clear cut than in the current study. PfHRP2 levels have also been correlated with childhood cerebral malaria in a number of smaller studies conducted in Tanzania [PfHRP2. The development of such tests in the future would be of major potential benefit.These findings are similar to those of Hendriksen and colleagues , althougTanzania , Malawi Tanzania , and UgaTanzania , and witTanzania . At presP. falciparum infection will progress to become severe or fatal probably depends on a wide range of genetic, immunological, physiological, and behavioral characteristics of both the host and the infecting parasite [The pathogenesis of severe falciparum malaria is complex, but a compromised microcirculation in vital organs caused by the sequestration of cytoadhered parasitized red blood cells to the vascular endothelium is central, compounded by endothelial dysfunction, reduced red blood cell deformability , and rosparasite . P. falciparum are directly related to the sequestered parasite load.With specific reference to our current study, the central role played by sequestration is a question that has been long debated. Although florid sequestration of parasites in the small vessels of multiple organs is a consistent feature of postmortem studies and is supported by observations of microvascular blockage in the retina and rectal mucosa in living patients with severe falciparum malaria , it is lMost previous studies of severe malaria have been too small to investigate differences in sequestration between children with different clinical phenotypes. However, the relatively large sample size allowed this in our current study. We found that the SI was high across a range of different phenotypes but particularly high in severe malarial anemia. As with most complications, the etiology of severe anemia is multifactorial, involving both hemolysis and an inappropriately low erythropoietic response . NeverthPfHRP2 value of >1000 ng/mL, a value that has been proposed as a threshold for identifying children with “true” severe malaria [PfHRP2 of >1000 ng/mL, again perhaps reflecting a sicker population, although the median level was still less than half that in children with severe malaria.The children with “uncomplicated” malaria who we recruited to our current study were significantly sicker than those in many previous studies, including the Tanzanian study described above , because malaria , 26. The malaria  also fouPfHRP2 concentrations between the uncomplicated and severe malaria groups, but the SI was more discriminatory, being approximately 8-fold higher in the severe than in the uncomplicated malaria group, in which the SI was similar to that in the group with asymptomatic malaria. The SI of approximately 40 is close to estimates based on the postmortem examination of brain tissue from Southeast Asian patients with cerebral malaria [In our current study there was considerable overlap in plasma  malaria , 35. The malaria , 26. Als malaria . UnfortuPfHRP2 and malaria severity has been found in the majority of studies, this has not been universal. In one study from Papua New Guinea, no significant difference in PfHRP2 was found between children with severe versus uncomplicated malaria [PfHRP2 concentrations in children defined as having severe malaria were considerably lower than in other studies. The same profile of low mortality rate and low PfHRP2 concentrations was reported in a second study, conducted in the Gambia [PfHRP2 and severity. The lack of agreement between these studies and our own might thus be explained by different definitions for severe malaria.Although a positive correlation between plasma  malaria . In agre malaria , case fae Gambia , in whicPfHRP2 levels for predicting prognosis and directing care to those at greatest risk. First is the recent recognition that deletions in the genes encoding pfhrp2 (and its homolog pfhrp3) mean that some clones of P. falciparum parasites do not express PfHRP2 [PfHRP2 levels going forward. Furthermore, our observations cannot be extended to nonfalciparum infections, which are common in parts of Ethiopia and Eritrea [There are 2 potential caveats to the use of s PfHRP2 . Althougs PfHRP2 , 40; rats PfHRP2  . This iPfHRP2 that could potentially reduce the levels measurable in plasma [PfHRP2 was undetectable despite the presence of P. falciparum parasites in peripheral blood in only 1.7% of the children, suggesting that despite the above caveats, PfHRP2-based risk-assessment methods remain currently useful.The second caveat relates to the recognition that some individuals produce antibodies to n plasma . Althougn plasma . In the P. falciparum parasite load, as estimated through measurement of plasma PfHRP2, is strongly related to the severity of clinical malaria in children on the Kenyan coast, and that a low plasma PfHRP2 level suggests an alternative pathological mechanism. This does not mean that severely ill children with a positive blood film result but a low PfHRP2 level should be denied appropriate antimalarial treatment, nor that antibiotics should be withheld from those with a high PfHRP2 level. Both should be treated empirically, as currently recommended in the World Health Organization guidelines [P. falciparum parasites.In summary, we have found that the idelines , but cliidelines , might bClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciaa1141_suppl_Supplementary_Figure_S1Click here for additional data file.


Plasmodium falciparum malaria.Iron deficiency (ID) and malaria are common causes of ill-health and disability among children living in sub-Saharan Africa. Although iron is critical for the acquisition of humoral immunity, little is known about the effects of ID on antibody responses to P. falciparum antigens including apical merozoite antigen 1 (anti-AMA-1) and merozoite surface antigen 1 (anti-MSP-1) in cross-sectional and longitudinal studies.The study included 1794 Kenyan and Ugandan children aged 0–7 years. We measured biomarkers of iron and inflammation, and antibodies to P. falciparum parasitemia  −0.46; 95 confidence interval [CI], −.66, −.25 P < .0001; β −0.33; 95 CI, −.50, −.16 P < .0001, respectively). Additional covariates for malaria exposure index, previous malaria episodes, and time since last malaria episode were available for individual cohorts. Meta-analysis was used to allow for these adjustments giving β −0.34; −0.52, −0.16 for anti-AMA-1 antibodies and β −0.26; −0.41, −0.11 for anti-MSP-1 antibodies. Low transferrin saturation was similarly associated with reduced anti-AMA-1 antibody levels. Lower AMA-1 and MSP-1-specific antibody levels persisted over time in iron-deficient children.The overall prevalence of ID was 31%. ID was associated with lower anti-AMA-1 and anti-MSP-1 antibody levels in pooled analyses adjusted for age, sex, study site, inflammation, and P. falciparum-specific antibodies in iron-deficient children might reflect impaired acquisition of immunity to malaria and/or reduced malaria exposure. Strategies to prevent and treat ID may influence antibody responses to malaria for children living in sub-Saharan Africa.Reduced levels of  P falciparum-specific antibody levels. Ferritin, transferrin saturation, hepcidin, and soluble transferrin receptor levels were associated with malaria-specific antibody levels. Iron deficiency in early life may influence subsequent malaria antibody levels.Iron deficiency is associated with reduced  Iron deficiency (ID) is highly prevalent among young children living in sub-Saharan Africa , and iroIron is important for the development of humoral immunity and antibody production. ID impairs B-cell proliferation and antibody production , and a mP. falciparum schizont extract [P. falciparum antigens in 1794 Kenyan and Ugandan children. We evaluated antibodies to 2 major merozoite antigens, anti-AMA-1 and anti-MSP-1, which are targets of acquired immunity, and antibodies to these antigens have previously been associated with protective immunity to malaria in our study population [Although ID is highly prevalent among African children and is known to influence immune responses little is known about the effect of ID on the acquisition of immunity to malaria. We previously observed that ID was associated with decreased total immunoglobulin G (IgG) and immunoglobulin E (IgE) levels to  extract  and that extract , in smalpulation , 22.Ethical approval was provided by the Scientific Ethics Review Unit of the Kenya Medical Research Institute (KEMRI/SERU/CGMR-C/046/3257/2983), by the Uganda Virus Research Institute (reference GC/127/12/07/32), the Uganda National Council for Science and Technology (MV625), and in the United Kingdom by the London School of Hygiene & Tropical Medicine Ethics Committee (A340) and the Oxford Tropical Research Ethics Committee .We used data from community-based cohorts of children in Kilifi, Kenya, and Entebbe, Uganda.Kenya: The Kenyan children included two community-based cohorts exposed to varying levels of malaria transmission, Junju and RTS,S. Junju is a surveillance cohort evaluating immunity to malaria as described elsewhere [lsewhere . The RTSlsewhere . Both coUganda: The Entebbe Mother and Baby Study (EMaBS) is a prospective birth cohort that was originally designed as a randomized double-blind placebo-controlled trial to determine whether anthelmintic treatment during pregnancy and early childhood was associated with differential responses to vaccination or incidence of infections such as malaria, pneumonia and diarrhea [diarrhea . ChildreP. falciparum parasitemia was determined at the time of malaria antibody measurement using Giemsa-stained thick and thin blood smears.The measured biomarkers of iron status and inflammation were iron , ferritin, transferrin , soluble transferrin receptor , hepcidin , transferrin  hemoglobin , and C-reactive protein . In Uganda, hemoglobin concentrations were adjusted for an altitude of > 1000 m above sea level (by subtracting 0.2 g/dL) . P. falc42, 3D7 sequence of P falciparum antigens were measured from plasma samples by enzyme-linked immunosorbent assays (ELISAs) according to standard protocols as previously described for the RTS,S, Junju [Antibodies against the AMA1 3D7 sequence and MSP1S, Junju  and EMaBS, Junju . A pool z-score < −2 using the WHO Growth Reference Standards [Inflammation was defined as CRP > 5 mg/L. ID was defined as plasma ferritin < 12 µg/L or < 30 µg/L in the presence of inflammation in children < 5 years or < 15 µg/L in children ≥ 5 years as defined by the World Health Organization (WHO) . Low tratandards . Hemoglot-tests and univariable linear regression models. Multivariable linear regression models were used to estimate the association between ID and malaria antibody levels, pooling data from all cohorts and adjusting for study site, age, sex, inflammation, and P. falciparum parasitemia at the time of antibody measurement. The linear regressions were run on log transformed data; hence the beta values returned reflect changes on a log scale. To transform these to fold differences that could be applied to the linear scale, we used the formula 10^Beta. The significance of possible interactions was estimated from the Wald test. Because not all indexes of previous malaria exposure were available for all cohorts, a meta-analysis of results from multivariable models for individual cohorts was fitted, additionally adjusting for malaria exposure index in the Kenyan cohorts, malaria vaccination status in the RTS,S cohort, and prior malaria incidence, time since last malaria episode, and time between iron and antibody measurements in the EMaBS cohort. In longitudinal analyses EMaBS children were further grouped by those that had iron measurements 0–2 or 2–4 years before antibody measurement. A P-value of < .05 was considered significant.Data analysis was performed using STATA 13.0 . Non-normally distributed iron and inflammation biomarkers, and malaria antibodies were normalized by natural-log transformation. Crude differences in means of log antibody levels between iron-deficient and iron-replete children were determined using 2-tailed Student P. falciparum parasitemia varied by study cohort from 34.70% in Junju to 1.97% in the RTS,S cohort   . ID remactively) .To account more fully for the effects of previous malaria on antibody levels, we conducted a meta-analysis of individual cohorts with further adjustments for additional covariates including malaria exposure index , incidenConsidering other iron biomarkers, we found that ID defined by TSAT < 10% was associated with reduced anti-AMA-1 but not anti-MSP-1 antibody levels in multivariable analyses and meta-analyses . IncreasWe tested the hypothesis that ID might influence subsequent malaria antibody levels for a prolonged period of time in the EMaBS birth cohort. ID was associated with lower anti-AMA-1 and anti-MSP-1 antibody levels up to 2 years after iron status measurements and only with lower anti-AMA-1 antibody levels 2–4 years after iron measurements .P. falciparum antibodies in 1794 Kenyan and Ugandan children. We found that ID was associated with reduced levels of anti-AMA-1 and anti-MSP-1 antibodies, even after adjustment for potential confounders including previous malaria exposure. TSAT < 10% was similarly associated with reduced levels of anti-AMA-1 antibody levels. A range of individual iron markers, including ferritin, TSAT, hepcidin, and sTfR levels were positively associated with malaria antibody levels. ID remained associated with reduced malaria antibody levels for up to 4 years.We have investigated the association between iron status and anti-P. falciparum antibody levels was most marked among children with the highest malaria exposure and antibody levels, as seen in the Junju cohort, although little difference by ID was observed in children with very low levels of malaria exposure and antibody levels, as seen in the RTS,S cohort. In agreement with our findings, Nyakeriga et al reported that total IgG, IgG2, and IgE antibody levels were lower in iron-deficient compared to iron-replete Kenyan children [We found that ID was associated with lower anti-AMA-1 and anti-MSP-1 malaria antibody levels, even after adjustment for potential confounders including previous malaria exposure. The relationship between malaria, iron parameters, and antibody levels differed between study sites. The effect of ID on anti-children . We founWe further investigated the effects of a range of iron markers on malaria-specific antibody levels. TSAT, an indicator of low levels of circulating iron, may more accurately reflect what iron status would be in the absence of malaria and inflammation compared to ferritin , 34. TSAHow might ID lead to reduced malaria-specific antibody levels? One explanation is that iron may play a critical role in humoral immunity and particularly in antigen-specific antibody production as suggested by recent studies , 8, 11. Reduced malaria-specific antibodies in iron deficient children may also be explained by the complex relationship between iron status and malaria. ID has some protective effect against malaria infection in children , and thuStrengths of our study included its large sample size of 1794 children from cohorts of varying malaria intensity in Kenya and Uganda. We also measured specific malaria antibodies known to contribute to immunity to clinical malaria , 22, assIn summary, we found that ID was associated with lower levels of anti-AMA-1 and anti-MSP-1 malaria antibodies, known to be important in antibody-mediated immunity to clinical malaria in African children , 6, 22. Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciaa728_suppl_Supplementary_Figure_1Click here for additional data file.ciaa728_suppl_Supplementary_Figure_2Click here for additional data file.ciaa728_suppl_Supplementary_Figure_3Click here for additional data file.ciaa728_suppl_Supplementary_MaterialClick here for additional data file.


Clinical manifestation of reported malaria cases in Aceh varied widely from asymptomatic, mild uncomplicated to severe and fatal complications. The present study aims to explore the allelic diversity of merozoite surface protein 1 gene  amplified for the msp1 and msp2 allelic subfamilies.Screening of over 500 malaria cases admitted to the hospitals in 11 districts hospital within Aceh Province during 2013–2015, identified 90 cases of msp1 gene revealed the presence of K1 allele subfamily in 34 subjects, MAD20 in 42 subjects, RO33 in 1 subject, and mixed allelic of K1 + MAD20 in 5 subjects, K1 + RO33 in 4 subjects, and MAD20 + RO33 in 4 subjects. Analysis of msp2 gene revealed 34 subjects carried the FC27 allelic subfamily, 37 subjects carried the 3D7 and 19 subjects carried the mixed FC27 + 3D7. Analysis of multiplicity of infection revealed that msp1 alleles is slightly higher than msp2 with the mean of MOI were 2.69 and 2.27, respectively. Statistical analysis to determine the association between each clinical manifestation and msp1 and msp2 alleles revealed that liver function abnormal value was associated with the msp2 mixed alleles (odds ratio (OR):0.13; 95%CI: 0.03–0.53). Mixed msp1 of K1 + RO33 was associated with severe malaria .Analysis of clinical manifestation revealed that fever-chill is the most frequent symptom. Based on WHO criteria showed 19 cases were classified as severe and 71 as mild malaria. Analysis of msp1 mixed alleles of K1 and RO33. The liver function abnormal value associated with the msp2 mixed allelic subfamilies. Further study in different geographic areas is recommended.This study found a strong association between severe malaria in Aceh with subjects carrying the  Malaria remains a major public health problem in Indonesia despite a success in bringing down the incidence within the last few decades. Since the implementation of the malaria elimination programme in 2009, the malaria cases in Indonesia dropped significantly from 418,439 to 261,617 and the vast majority of the cases occurred in 5 provinces of the eastern parts of country  and msp2 genes [M2-OF 5′- ATGAAGGTAATTAAAACATTGTCTATTATA -3′ and M2-OR 5′- CTTTGTTACCATCGGTACATTCTT -3′] with a total of 25 µl volume was used for all reactions [msp1 , and msp2 (IC/3D7 and FC27); K1 = F-5: AAA-TGA-AGA-AGA-AAT-TAC-TAC-AAA-AGG-TGC-3 and R-5: GCT-TGC-ATC-AGC-TGG-AGG-GCT-TGC-ACC-AGA-3; MAD20 = F-5: AAA-TGA-AGG-AAG-AAC-TGG-AAC-AGC-TGT-TG-3 and R-5: ATC-TGA-AGG-ATT-TGT-ACG-TCT-TGA-ATT-ACC-3; RO33 = F-5: TAA-AGG-ATG-GAG-CAA-ATA-CTC-AGT-TGT-TG-3 and F-5: CAT-CTG-AAG-TTG-GAT-CAG-CAC-CTG-GAG-ATC-3; 3D7 = F-5: AGA-AGT-ATG-GCA-GAA-AGT-AAK-CCT-YCT-ACT-3 and R-5: GAT-TGT-AAT-TCG-GGG-GAT-TCA-GTT-TGT-TCG-3; FC 27 F-5: ACT-AAG AAT-AGT-GTA-GGT-GCA-RAT-GCT-CCT-3 and R-5: TTT-TAT-TTG-GTG-CAT-TGC-CAG-AAC-TTG-AAC-3F [msp1  or msp2 (IC/3D7 and FC27). Reactions for each set of primary and nested primers were performed separately.The parasite DNA was extracted from the filter paper using Chelex-100 ion exchanger method as described previously . The DNAeactions . FurtherG-AAC-3F . The secmsp1 and msp2 genotypes by the number of positive samples for each marker. The mean MOI was calculated by dividing the total number of alleles detected in both msp1 and msp2 by the total number of positive samples for both markers. Samples were considered single infected when harbouring only one allele at each of the genotyped loci. Multiclonal infections were defined as infections with more than one allele in at least one locus.MOIs were calculated by dividing the total number of distinct msp1 and msp2 was calculated as the proportion of the allele detected for each allelic family out of the total of alleles detected. The frequency of polyclonal infection was calculated using number of samples with more than one amplified fragment out of the total samples. The mean MOI was determined by dividing the total number of alleles detected in both msp1 and msp2 by the total number of positive samples for both markers. The Chi-square test was used to analyse the association of the clinical manifestations and the allelic subfamilies of the msp1 and msp2. Statistical significance was defined as p < 0.05.All data were collected with Epidata and analysed by R version 3.4.0. The allelic subfamily frequency of P. falciparum. Further validation by PCR revealed 90 subjects with P. falciparum mono infection. Among the 90 study subjects, 57.7% of them were males and 42.3% were females with most subjects 46.7% aged between 21 and 30 years old , MAD20 (46.7%), and RO33 (1.1%) subfamily, either as single or mixed allelic subfamilies. Mixed allelic subfamilies infections between K1 and MAD20, K1 and R033 and MAD20 with RO33 were also found in less frequency  were found to carry multiple allelic subfamilies infection of msp1, whereas for msp2, 19 subjects (21.1%) were found. The MOIs for both msp1 and msp2 were 2.27 and 2.69, respectively. The MOI for msp2 was slightly higher than msp1 . All others clinical manifestations showed no significant correlation with allelic subfamilies of msp1 and msp2 (p > 0.05)   Tables , 5. Base0) Table . Other aAnalysis of the clinical manifestation of the malaria cases admitted to the hospitals in Aceh revealed that the classical symptoms, such as fever with chill is still the primary symptom experienced by the patients, followed by spleen enlargement and dyspnoea. Majority of the malaria cases are classified as mild, uncomplicated malaria but over 20% of which are severe according to WHO criteria . The finmsp1 and msp2 revealed a value of 2.27 and 2.67 for the msp1 and msp2, respectively. This finding is slightly higher than the MOI data from Myanmar [Analysis of the MOI based on the allelic diversity of  Myanmar  and Sout Myanmar , but low Myanmar –23. The  Myanmar –26. In t Myanmar  but othe Myanmar , 30. Low Myanmar . With reP. falciparum with the malaria clinical manifestation may provide useful information about parasite characteristics to design specific intervention strategies targeting the virulence factors [msp1 and msp2 alleles of P. falciparum among the hospitalized malaria cases. Analysis to determine the association between allelic subfamilies of the msp1 and msp2 with malaria severity revealed a strong association between mixed msp1 K1 + RO33 with severe malaria. The finding is slightly different with that of Ariey et al. [var gene overexpression associated with severe malaria. Other findings reported a high proportion of subjects carrying the FC27 allele subfamily among the uncomplicated malaria patients [P. falciparum isolates, which was conducted in Libreville, Gabon. In the study, extensive genetic polymorphism within msp1 allelic families  has been observed. This is consistent with the diversity found in Bakoumba  in 1999, Senegal  in 1995, and in Mauritania  in 2010 [msp2 allele subfamilies showed the presence of two allelic subtypes in the study area, which is similar to the results of study conducted by Kang et al. in Myanmar where the geographical areas are alike. Their study also found only two allelic subtypes in Myanmar, which are the FC27 and 3D7. The difference between their study and our study is that the number of patients infected by mixed allelic subtypes is higher than patients affected by single infections; while our study has demonstrated contrasted finding where single infection, either by FC27 or 3D7 alleles is higher [msp2 infection. This finding has never been analysed in previous studies except for the severe malarial anaemia [Analysis on the genetic diversity profiles of  factors  as well  factors . This sty et al.  where K1patients . This dipatients . Another in 2010 , 37. This higher . This st anaemia . Despitemsp1 and msp2 genes among the hospitalized uncomplicated and severe malaria cases in Aceh have been analysed. Association between liver function abnormal value with the mixed allelic type of msp2 was observed. Mixed allelic infection of msp1 K1 and RO33 is strongly associated with severe malaria. This study has several limitations such as analyzing symptomatic malaria cases and only few severe cases. Further study to explore more subjects in different geographic setting and different clinical manifestation is recommended.Allelic subfamilies analysis of the