TM Malaria HRP2 rapid diagnostic test (RDT) targeting Plasmodium falciparum was used to test a group of 500 asymptomatic children aged 1-15 years old (330 settled Bantu and 170 semi-nomadic BaAka Pygmies) inhabiting the villages in the Dzanga Sangha region (south-west CAR) in March 2020. In total, 32.4% of asymptomatic Bantu and 40.6% of asymptomatic Pygmy children had a positive result of malaria RDT. Our findings allowed us to demonstrate the high prevalence of asymptomatic malaria infections in south-west CAR. RDTs seem to be a useful tool for the detection of Plasmodium falciparum in areas with limited possibilities of using other diagnostic methods, such as light microscopy and molecular biology.According to the World Health Organization 94% of global malaria cases and 94% of global malaria deaths have been reported from Africa. Unfortunately, it is difficult to determine the exact prevalence of disease in some African countries due to a large number of asymptomatic cases. The aim of this study was to assess the prevalence of malaria infections in seemingly healthy children living in the Central African Republic (CAR). CareStart Malaria is a vector-borne parasitic disease. In humans, it is mainly caused by five protozoan species: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale , Plasmodium malariae, and Plasmodium knowlesi  ,2. ThankAccording to the latest World Health Organization’s (WHO) report on malaria prevalence, diagnostics, treatment, and prevention , a total of 229 million people globally fall ill with the disease each year and 94% of malaria cases are reported from Africa. The estimated number of malaria deaths is 409,000 per year, of which 94% occur in Africa . The WHOP. falciparum is responsible for 99.7% of all malaria cases in Africa and as much as 100% of cases in sub-Saharan Africa [P. falciparum are routinely used for the diagnosis of malaria in the region [P. falciparum species . In fact, the actual number of malaria cases in the CAR may be several times higher than the WHO suggests due to exceptionally high rates of asymptomatic cases [According to the WHO, n Africa . Therefoe region . Malariaic cases .The aim of the study was to assess the prevalence of asymptomatic malaria infections in sub-Saharan Africa on the example of seemingly healthy children living in the rural parts of the Dzanga Sangha region in South-West Central African Republic.The study was conducted in March 2020 (the end of dry/warm season with average temperatures ranging between 21 and 33 °C); it involved a group of 500 children of both sexes, aged 1–15 years (330 settled Bantu and 170 semi-nomadic BaAka Pygmies) living in the Monasao village and surroundings in the Dzanga Sangha region  in South-West Central African Republic .Most Bantu people are farmers who live in villages set up along the main communication routes in the region. Their livelihood largely depends on agriculture (growing crops) but some also work in gold or diamond mining industry . BaAka Pygmies, on the other hand, are a semi-nomadic people who inhabit local forests, or their outskirts, mostly in the vicinity of Bantu villages. Pygmies are often economically exploited by the Bantu people and are frequently used as cheap labour force.Monasao is a village 50 km north of the Bayanga village  and 60 km south of the Nola city , which lies at the altitude of 510 m above sea level. Initially, Monasao was inhabited entirely by the Pygmy tribes. Today, however, the village with its population of around 4000 people is inhabited by both the settled Bantus and the semi-nomadic BaAka Pygmies. The latter, however, tend to build their huts on the outskirts of the village. The Pygmies survive by hunting, fishing and gathering wild plants in local forests but they are also employed for field work by some Bantu people.Bantu and Pygmy children were recruited for the study at a health care facility run by the local catholic mission  and at a local primary school in Monasao. The inclusion criteria were: age ≤15 years and absence of clinical signs and symptoms of malaria (body temperature ≤37.5 °C). The exclusion criteria were presence of malaria signs and symptoms , anti-malarial treatment received in the past 28 days and difficulties in venipuncture procedure. All participants had the right to withdraw from the study at any stage. On behalf of the pediatric patients, the consent to participate in the study was given by their parents or legal guardians, who had first been informed of the study purpose and methods. The parents/guardians provided information on the age and sex of their children. The interviews were conducted in their native language, either in Sango (a language spoken by the Bantu) or Mbenzele (the language used by the BaAka Pygmies). Medical personnel responsible for carrying out the study procedures measured the participants’ body weight  and body temperature  using an electronic thermometer . Next, medical staff performed rapid diagnostic tests for malaria as well as hemoglobin measurements to identify any possible cases of anemia; hemoglobin level was measured using a portable analyzer.Plasmodium falciparum histidine-rich protein 2) was used for the detection of P. falciparum in vitro, in a whole blood sample . To perform the test a drop of capillary blood  is added into a sample well from which the specimen migrates through the nitrocellulose membrane (paper chromatography) by capillary action. Next, 2 drops of buffer assay attached to the test are added into the buffer well. The test pad is pre-coated with colloidal gold nanoparticles conjugated with specific antibodies. After the blood sample has been absorbed by the membrane, P. falciparum HRP2 antigens bind to the anti–HRP2 antibodies (if the test is positive). The reaction leads to the formation of antigen-antibody complexes which the test is able to detect. The interpretation of the test will depend on the presence or absence of colour bands in the control and test areas (the presence of the color line in the test area confirms the presence of antigens in the blood sample). Thus, the presence of two bands—in the control area and the test area indicates a positive result of the test. The presence of only one band in the control area within the result window indicates a negative result. The test is invalid and the interpretation of the test not possible if the line in the control area does not appear or it only appears in the test area. The results can be read after 20 min. The RDT sensitivity  is 98% and its specificity  is 97.5% (according to the manufacturer’s specifications) [Immuno-chromatographic rapid diagnostic test (RDT) targeting Pf HRP2 (cations) .P. falciparum on RDT test with no clinical signs of the disease .Asymptomatic malaria was defined as the presence of TM analyzer  by examining a peripheral blood sample from a finger prick. The definition of anemia was based on the WHO criteria, where hemoglobin level of ≥11 g/dL is regarded as normal, 10.0–10.9 g/dL as mild, 7.0–9.9 g/dL as moderate, and <7.0 g/dL is regarded as severe anemia.Hemoglobin level was measured using a portable DiaSpectwww.statsoft.com  and Microsoft Excel. Quantitative variables were characterized by the arithmetic mean, standard deviation, median, minimum and maximum value (range), and 95% CI . The variables of the qualitative type were presented in terms of counts and percentages (percentage). The Shapiro-Wilk test was used to examine if quantitative variables were normally distributed in a population. Levene’s test (the Brown-Forsythe test) was used to check the assumption of equal variances. The Student’s t-test or Mann-Whitney U test  were used to determine if the differences between the two study groups (subjects with a positive RDT result vs. subjects with a negative RDT result) were statistically significant. Chi-square tests of independence were used for qualitative variables . In all calculations, the level of significance was set at p = 0.05.The statistical analysis was performed using StatSoft Inc. (2014) STATISTICA  version 12.0. The research project was approved by the Ministry of Scientific Research and Technological Innovation, Bangui, Central African Republic (Research Authorization) and the Committee on Bioethics at the Military Institute of Medicine, Warsaw, Poland (Decision No. 22/WIM/2020) under the Declaration of Helsinki (1996) and the rules elaborated by the European Union “Good clinical practice for trials on medicinal products in the European Community. The rules governing medicinal products in the European Community” (1999) ratified by the Committee of Ethics in Poland (March 1993). The tests performed in the Central African Republic were carried out with the written consent of each participant and under the supervision of Father Wojciech Lula , with considerable help from the medical staff working at the healthcare center and the teaching staff from the Monasao primary school.n = 330), a total of 107 subjects (32.4%) tested positive on mRDT; the mean age of the children with mRDT (+) was 8.7 years and their mean body weight was 22.5 kg. The mean body temperature of the tested children, both with mRDT (+) and with mRDT (–) was 36.8 °C. Of all the Bantu children with mRDT (+), 45.8% were girls and 54.2% were boys. We have not observed any statistically significant correlations between age, body weight, body temperature, sex and the mRDT result (In the group of Bantu children (T result .n = 170), a total of 69 children (40.6%) tested positive on mRDTs; the mean age of the children with a positive result on mRDT was 7.0 years. Children with mRDT (+) were statistically significantly younger (p = 0.0139). The mean body weight of the children with mRDT (+) was 16.1 kg. Children with mRDT (+) had significantly lower body weight (p = 0.0038). The mean body temperature of the children with mRDT (+), as well as those with mRDT (–) was 36.8 °C. Of all the children with mRDT (+), 47.8% were girls and 52.2% were boys. No statistically significant correlation was found between the participants’ sex and their body temperature and the mRDT result (In the group of Pygmy children (T result .n = 500) was 35.2%; 32.4% in Bantu children (n = 330) and 40.6% in Pygmy children (n = 170).RDT results showed that the overall prevalence of asymptomatic malaria in the study group . Multivariate regression analysis demonstrated that the variables had no statistically significant effect on the probability of obtaining a positive mRDT result .n = 81), the mean age of the girls with mRDT (–) was 8.4 years, and of those with mRDT (+) 6.8 years. Females with a positive result on mRDT were statistically significantly younger (p = 0.0013). The mean body weight of Pygmy females with mRDT (−) was 19.8 kg, whereas of those with mRDT (+) it was 16.2 kg. Females with mRDT (+) had significantly lower body weight (p = 0.0017). No statistically significant correlation was found between temperature, hemoglobin level and the presence of asymptomatic malaria.In the group consisting of Pygmy females . 13.1% of the study participants with mRDT (+) vs. 19.7% with mRDT (–) had normal hemoglobin level, which confirms a high prevalence of mild to moderate anemia in the region, regardless of the mRDT result  had a significantly lower hemoglobin level (T result .The mean hemoglobin level in Pygmy children with mRDT (+) was 9.9 g/dL, whereas in those with mRDT (–) it was 10.2 g/dL. No statistically significant correlation between hemoglobin level and the result of mRDT was found. Only 5.8% study participants with mRDT (+) vs. 13.9% with mRDT (–) had normal hemoglobin level. Similarly as in Bantu children, the prevalence of mild to moderate anemia was high among the Pygmy children, regardless of the mRDT result .P. falciparum histidine-rich-protein 2 /Pf HRP2/ or the enzyme called Plasmodium lactate dehydrogenase /pLDH/). RDTs have their limitations as they cannot provide quantitative results and can stay positive months after infection with P. falciparum, or are, if testing for pLDH, positive only while there are living parasites in the blood [Plasmodium falciparum infections, especially in areas where other diagnostic methods, such as light microscopy or molecular biology techniques are not available. The accuracy of RDTs for the diagnosis of uncomplicated P. falciparum infection is equal or superior to routine microscopy (but inferior to expert microscopy) [P. falciparum in vitro, in a whole blood sample . The fact that WHO recommends simplified diagnostic procedures, i.e., the use of RDTs capable of detecting only one Plasmodium species  may become a major obstacle to effective malaria control in Africa. According to WHO, P. falciparum is the etiological factor of up to 100% malaria cases in sub-Saharan Africa, but it must be emphasized that a large proportion of cases that occur in the region are never diagnosed because of widespread asymptomatic carriage of Plasmodium. Unpublished findings of a study which was carried out by the authors in the Bayanga subprefecture (in the same region where the present study was conducted) in 2018, using molecular methods (PCR), in a group of 540 Pygmies (aged 1–75 years) presenting with clinical signs of malaria, confirmed the presence of P. falciparum in 94.8% patients, but also found genetic material of P. malariae in 11.1%, P. ovale in 9.8%, and P. vivax in 0.7% study subjects. The results obtained indicate that there are different species of Plasmodium in Central Africa, which is a significant finding with respect to implementing effective diagnostic procedures and treatment protocols .The study aimed to determine the occurrence of asymptomatic malaria infections in children living in the Central African Republic (CAR). According to the official statistics malaria affects approximately 8% of the country’s population each year. However, the WHO has indicated that the actual number of malaria cases in the CAR is grossly underestimated and in fact it might be even several times higher than the reports suggest. This is largely due to the fact that a significant proportion of malaria cases in the region are asymptomatic . The epihe blood  but theyhe blood . RDTs seroscopy) . Many reroscopy) ,24,25. Troscopy) . The prepfhrp2 gene deletions /Pf HRP2-detecting RDTs/ and cross-reactions between Plasmodium antigens and detection antibodies), as well as the possibility of false-positive results caused by other infections [One should also be aware of numerous limitations of RDTs , the possibility of false negative results  and malaria diagnosis remains a significant challenge for local health care professionals. The most vulnerable groups affected by malaria in high-transmission areas, such as sub-Saharan Africa, are children . In malaptomatic . The asysquitoes . Accordisquitoes ,30. Convsquitoes .Wuchereria bancrofti) in Central Africa, which have not been studied in the local population, should also be considered. The present study is the first screening ever conducted in asymptomatic patients with malaria infection in the Dzanga Sangha region, in South- West CAR.The results of this study, which was aimed to assess the prevalence of asymptomatic malaria infections in a sample of 500 children, aged 1–15 years, living in the rural areas of the Dzanga Sangha region in the south-west of the CAR, revealed 35.2% of asymptomatic malaria cases: 32.4% in Bantu and 40.6% in Pygmy children. A total of 516 children entered the study (339 Bantu and 177 Pygmy), of which 16 were excluded because of fever ≥37.6 °C . Of the 16 children with fever ≥37.6 °C (9 Bantu and 7 Pygmies) who were excluded from the study, a majority presented with symptoms of a respiratory infection ; the possibility of other febrile illnesses, that are epidemic (dengue) or endemic  were significantly younger, we also found that older age and higher body weight were associated with a lower probability of a positive result on mRDT in this ethnic group. Similar findings have been reported by Ndamukong-Nyanga et al. , where tWe have not found any statistically significant correlation between sex and the result of the mRDT in our study sample. In the group of Bantu children, boys accounted for 54.2% of children with mRDT (+) and for 54.7% of those with a negative result on mRDT; in the group of Pygmy children, boys accounted for 52.2% of those with a positive result on mRDT and 52.5% of the children with mRDT (–). A study by Teh et al.  and KimbThe strength of the presented study was the assessment of malaria prevalence in asymptomatic children aged 1–15 years old living in remote and hard-to-reach parts of the Central African Republic. Because asymptomatic individuals are not usually tested for malaria, it is impossible to assess the actual malaria morbidity in the country. The study had its limitations as well. Participants were recruited from among the children population living in the Monasao village or in its close vicinity, which limited the sample size. Further, limiting the sample to pediatric population only  prevented the assessment of malaria prevalence in the adult population, especially in pregnant women and patients with underlying conditions.Plasmodium falciparum infections, especially in areas where the access to medical care is limited and where other diagnostic methods, such as light microscopy or molecular biology are unavailable. The WHO has warned that there may be an increase in malaria-related morbidity and mortality in Africa in the next few years, because the resources which are at the disposal of national health care systems will, in the first place, be allocated to finance the COVID-19 control strategies, rather than managing the spread of tropical diseases. Given the fact that the Bayanga sub-prefecture in the south-west of the Central African Republic is one of the most attractive tourist destinations in Africa , the knowledge of the exact occurrence rates of malaria in sub-Saharan Africa seems to be as important for the local health care providers as for the centers for travel and tropical medicine operating in developed countries on other continents.The overall occurrence of asymptomatic malaria in children living in the south-western parts of the Central African Republic is very high. RDTs seem to be an effective method for detecting


Background: Anaemia is a major public health concern especially in African children living in malaria-endemic regions. Interferon-gamma (IFN-γ) is elevated during malaria infection and is thought to influence erythropoiesis and iron status. Genetic variants in the IFN-γ gene(IFNG) are associated with increased IFN-γ production. We investigated putative functional single nucleotide polymorphisms (SNPs) and haplotypes ofIFNG in relation to nutritional iron status and anaemia in Gambian children over a malaria season.Methods: We used previously available data from Gambian family trios to determine informative SNPs and then used the Agena Bioscience MassArray platform to type five SNPs from theIFNG gene in a cohort of 780 Gambian children aged 2-6 years. We also measured haemoglobin and biomarkers of iron status and inflammation at the start and end of a malaria season.Results: We identified fiveIFNG haplotype-tagging SNPs . TheIFNG+2200C [rs1861493] allele was associated with reduced haemoglobin concentrations  and a trend towards iron deficiency compared to wild-type at the end of the malaria season in multivariable models adjusted for potential confounders. A haplotype uniquely identified byIFNG+2200C was similarly associated with reduced haemoglobin levels and trends towards iron deficiency, anaemia and iron deficiency anaemia at the end of the malaria season in models adjusted for age, sex, village, inflammation and malaria parasitaemia.Conclusion: We found limited statistical evidence linkingIFNG polymorphisms with a risk of developing iron deficiency and anaemia in Gambian children. More definitive studies are needed to investigate the effects of genetically influenced IFN-γ levels on the risk of iron deficiency and anaemia in children living in malaria-endemic areas. In this region, iron deficiency (ID) and anaemia are highly prevalent4, and may lead to impaired brain development6, while iron deficiency anaemia (IDA) is a leading cause of years lived with disability in African children7. Increasing evidence suggests that malaria may be contributing to ID and IDA9. Previous studies reported that the prevalence of ID and IDA increased over the malaria season in Gambian and Kenyan children11, and decreased with the interruption of malaria transmission in the Kenyan highlands12.Malaria and iron deficiency are major public health problems for children living in sub-Saharan Africa. The majority (94%) of the 405,000 global deaths due to malaria in 2018 occurred in sub-Saharan Africa, where up to 24% of the population have malaria parasitaemia at any given time13. Among other type 1 responses, IFN-γ is involved in regulating erythropoiesis16 and iron-regulatory proteins20. IFN-γ has also been reported to increase the expression of hepcidin17, and divalent metal transporter 1 (DMT1)19, while suppressing ferroportin19, ferritin20, and transferrin receptors20. This regulation of iron proteins may be aimed at starving invading pathogens of iron, a critical nutrient for pathogen growth, but could also play an important role in the pathogenesis of ID and IDA. Indeed, higher IFN-γ levels have been reported in Kenyan children with severe malarial anaemia21.Multiple factors may contribute towards the development of ID and IDA following a malaria infection. One such factor is interferon-gamma (IFN-γ), which is induced during acute and persistent malaria infectionIFNG) on chromosome 12q14 have been associated with increased production of IFN-γ23, and with susceptibility to severe malaria25. Despite evidence that malaria induces the production of IFN-γ13 and that this cytokine influences iron regulation20, it is not known whether variation in theIFNG gene influences the risk of ID and IDA among children in malaria-endemic areas. We investigated SNPs and haplotypes in theIFNG gene locus in relation to nutritional iron status and anaemia in a cohort of 780 Gambian children prior to and at the end of a malaria season, using an approach based on informative SNPs and Agena Bioscience MassArray platform typing.Single nucleotide polymorphisms (SNPs) in the IFN-γ gene  and end (December 2001/January 2002) of a malaria season, as previously described26. All children had a clinical examination and a blood sample collected in the morning between 6 and 11 am for full blood count, malaria film, and biomarkers of iron status and inflammation at the start and end of the malaria season. Children with pyrexia (temperature ≥37.5°C) had appropriate clinical investigations, clinical treatment and a blood sample taken 2 weeks later after recovery from illness. All children received a 3-day course of mebendazole for possible hookworm infection at recruitment.We used previously collected data from a cohort of 780 children aged two to six years, recruited at the start of a malaria season as previously described1-antichymotrypsin  were assayed according to the manufacturers’ instructions from plasma samples stored at -80°C. Transferrin saturation (TSAT) was calculated from plasma iron and UIBC (TSAT = [plasma iron/ (UIBC + plasma iron)] X 100)27. Hepcidin/ferritin and TSAT/hepcidin ratios were also calculated28. Giemsa-stained thick and thin blood films were examined forPlasmodium falciparum and otherPlasmodium species at the start and end of the malaria season.Haemoglobin (Medonic CA 530 Haemoglobinometer) and zinc protoporphyrin (ZnPP) levels  were measured within 24 hours of sample collection. Hepcidin , ferritin , soluble transferrin receptor , serum iron, unsaturated iron binding capacity , and α25. Details of the primer sequences and assays are given inExtended datafiles 1 and229. The most informative haplotype-tagging SNPs (htSNPs) to type in Gambian subjects were identified by analysing the pattern of linkage disequilibrium (LD) in theIFNG gene loci using previously available data from 32 Gambian family trios30. The PHASE program (http://stephenslab.uchicago.edu/software.html) version 2.1 was used to infer haplotypes from the genotypes of the study population and estimate the frequency of each inferred haplotype31. The entropy maximization method was used to identify htSNPs that described >90% of the observed haplotypic diversity in this gene region30. The HaploXT program (http://www.sph.umich.edu/csg/abecasis/GOLD/docs/haploxt.html) was used to estimate pairwise LD statistics. Sickle cell  and glucose-6-phosphate deficiency (G6PD) deficiency (rs1050828 and rs1050829) were also genotyped using the Agena Bioscience MassArray platform.Genotypes were determined on whole-genome amplified DNA (primer extension pre-amplification) by the Agena Bioscience MassArray platform (formerly SEQUENOM) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described32, anaemia as Hb<11.0 g/dL (or Hb <11.5 g/dL in children ≥5 years) and IDA as ID plus anaemia33.Inflammation was defined as ACT >0.6 g/L. ID was defined as ferritin <12 µg/L or <30 µg/L in the presence of inflammation or <15 µg/L in children ≥5 yearsStatistical analyses were conducted using STATA 15.1 . Categorical data were expressed as proportions with corresponding percentages. Pearson chi-squared test was used to compare the prevalence of malaria and iron status  at the start and end of the malaria season. Changes in haemoglobin levels and markers of iron status over the malaria season were assessed using the paired t-test. Biological data that were not normally distributed were log-transformed, and geometric means were calculated from original untransformed values.34 was applied when the five SNPs and six haplotypes were considered individually as independent factors. For multivariable analyses, P values are noted as adj. P for non-Bonferroni corrected analyses and as Bonferroni adj. P for multivariable analyses that are Bonferroni corrected, and for univariable models P values are similarly presented as P or Bonferroni P if Bonferroni corrected. All analyses were considered statistically significant at P <0.05.Log-transformed markers of iron status and risk of ID, IDA and anaemia were analysed using univariable and multivariable linear and logistic regression models, as appropriate. Multivariable regression models were adjusted for age (grouped by year), sex, village , malaria parasitaemia and ACT at the start and end of the malaria season. The Bonferroni correction for multiple testingIndividual written informed consent was obtained from children’s parents or guardians and the study was approved by The Gambian Government and the Medical Research Council Ethics Review Committee (874/830).Extended datafile 329. At the start of the malaria season we found little difference in iron status in children with HbAS and G6PD deficiency compared to those with wild-type. At the end of the malaria season, children carrying HbAS had lower zinc protoporphyrin levels  compared to those with HbAA  and children with G6PD deficiency had lower haemoglobin levels  than those with G6PD wild-type genotype . The prevalence of ID and IDA increased over the malaria season , as previously reported26. Individual markers of iron status also reflected an increase in ID over the malaria season. We found that hepcidin and hepcidin/ferritin ratio decreased while the TSAT/hepcidin ratio increased across the malaria season in keeping with the need for increased erythropoiesis and increased rates of iron absorption at the end of the malaria season.A total of 756 children, including 403 males (53%) and 353 females (47%), were followed up to the end of the malaria season. Most of the children were from the Mandinka ethnic group . A total of 99/751 (13.2%) children carried sickle cell trait (HbAS) and 136/683 (19.9%) children G6PD deficiency and the effects of these polymorphisms on iron status and anaemia are shown inIFNG haplotype tagging SNPs at positionsIFNG-1616 (rs2069705),IFNG+874 (rs2430561),IFNG+2200 (rs1861493),IFNG+3234 (rs2069718) andIFNG+5612 (rs2069728) relative to the ATG start codon (Extended datafile 129). All of theIFNG SNPs were in Hardy Weinberg equilibrium. TheIFNG+2200C  allele was associated with a reduced risk of malaria parasitaemia at the end of the malaria season  but not at the start. The otherIFNG SNPs were not associated with malaria parasitaemia at either time point (Extended datafile 429). TheIFNG SNPs were not associated with HbAS or G6PD genotypes following Bonferroni correction in adjusted models. TheIFNG+874T andIFNG+3234C alleles were associated with the Fulani ethnic group . The minor allele frequencies by ethnic group are presented inExtended datafile 129.We identified fiveIFNG SNPs were not associated with ID, IDA or anaemia at the start of the malaria season in multivariable logistic regression analyses adjusted for age, sex, village, ACT and malaria parasitaemia following Bonferroni adjustment (Extended datafile 529).We found that theIFNG SNPs were similarly not significantly associated with ID, IDA or anaemia at the end of the malaria season after Bonferroni correction. TheIFNG+2200C allele (rs1861493) was associated with trends towards increased risk of anaemia , IDA , and ID  in multivariable logistic regression models, but not after Bonferroni correction  compared to those with theIFNG+2200 TT genotype , as well as a trend towards ID in other markers of iron status at the end of the malaria season in adjusted linear regression models (Therection . Childre models .IFNG gene locus (IFNG-CATTC) was present at a frequency of 35% in the Gambian children, while haplotype 6 (IFNG-CACTC), uniquely identified by theIFNG+2200 SNP, was present at a frequency of 7%. Using haplotype 1 as the reference, haplotype 6 was associated with a trend towards increased risk of ID , IDA  and anaemia  at the end of the malaria season  resolved by SNPs at nucleotide positions -1616, +874, +2200, +3234 and +5612 in thee locus . Six hap season . Haploty season .13, might play a role in influencing the risk of ID and anaemia in children exposed to malaria. In addition to directly reducing erythrocyte half-life15, evidence suggests that IFN-γ induces hepcidin and inhibits ferroportin, hence reducing iron absorption and promoting sequestration of iron in macrophages19. Consequently, high levels of IFN-γ induced during malaria infections may concomitantly lead to ID and anaemia.In this study we observed an increase in the prevalence of ID, IDA and anaemia across the malaria season in Gambian children. Dietary iron insufficiency may be an important cause since the malaria season also coincides with the ‘hungry season’ in The Gambia when there is a scarcity of staple foods. We hypothesized that IFN-γ, a pro-inflammatory cytokine induced during malaria infectionIFNG+2200C (rs1861493) allele, located at intron 3 of theIFNG gene, was associated with reduced haemoglobin levels and a trend towards ID, IDA and anaemia at the end of a malaria season in multivariable analyses adjusting for potential confounders. We then constructed haplotypes to increase the probability of capturing functional mutations which might reside within a given haplotype. Haplotype 6 , was associated with reduced haemoglobin levels and TSAT and increased ZnPP levels in keeping with iron deficiency compared to the wild-type haplotype. Haplotype 6 was similarly associated with trends towards increased risk of ID, IDA and anaemia at the end of the malaria season.We found that theIFNG+2200C genotype and a haplotype uniquely defined by this genotype potentially lead to reduced haemoglobin levels and ID at the end of the malaria season? A possible explanation may be through increasingIFNG gene expression and IFN-γ levels. TheIFNG+2200C allele was associated with increased IFN-γ levels in Kawasaki disease patients23, although another study in patients with ankylosing spondylitis found no difference in IFN-γ levels byIFNG+2200 genotype35. Elevated IFN-γ levels promote dyserythropoiesis, anaemia and iron dysregulation. IFN-γ inhibits proliferation of erythroid progenitor cells by disrupting lineage differentiation, blocking renal production of erythropoietin, inhibiting renal iron reabsorption, and reducing red blood cell half-life36. As a type 1 immune response, IFN-γ also induces defensive transcriptional programs within enterocytes resulting in reduced dietary iron absorption37. Additionally, IFN-γ promotes iron sequestration in macrophages either directly or through its influence on hepcidin, ferroportin, and DMT120. These responses reduce circulating transferrin-bound iron, which is required byPlasmodium parasites for metabolism and proliferation38. In keeping with this, we observed decreased ferritin levels and TSAT and increased ZnPP levels in children carrying theIFNG+2200C haplotype. Lower haemoglobin levels may also translate into reduced amino acid availability forPlasmodium parasites39 and hence protection against blood-stage parasitaemia. Indeed, we observed that theIFNG+2200C SNP was associated with protection against malaria parasitaemia at the end of the malaria season .So how might theIFNG+2200C allele on haemoglobin and iron status was only observed at the end of the malaria season. We hypothesized that the effects of this SNP may be most marked when expression ofIFNG is upregulated, such as during malaria infections24. This also highlights the influence of gene-environment interactions in promoting disease, in this case ID, IDA and anaemia. It is unlikely that the decreased haemoglobin levels observed in individuals carrying theIFNG+2200C allele was due to increased malaria since these children had reduced prevalence of malaria parasitaemia at the end of the malaria season. It is possible that higher IFN-γ levels, putatively produced byIFNG+2200C carriers, may induce a protective proinflammatory response against malaria40, but at the expense of iron homeostasis. TheIFNG+874TT (rs2430561) genotype, located at the first intron coinciding with the NFkB binding region, has also been associated with higher production of IFN-γ41. However, studies have found no association betweenIFNG+874TT and malaria25 or aplastic anaemia42, and in our study we observed a trend towards a decreased risk of ID in these individuals. Further investigations are required on a cellular level to explore putative functional effects ofIFNG genotypes on IFN-γ levels and iron status.The influence of theIFNG gene polymorphisms in relation to iron status. We found that theIFNG+2200C (rs1861493) allele, and a haplotype defined by this allele, were associated with reduced haemoglobin levels and a trend towards ID at the end of the malaria season, a finding that may be due to increased IFN-γ levels23. However, our study had a number of important limitations and our findings should be viewed with considerable caution. The study was conducted in a single site and had relatively small numbers (n = 756). Additionally, many of our findings were of marginal significance with wide confidence intervals and lost statistical significance after correction for multiple testing with Bonferroni adjustment. It is also unclear if our findings have clinical relevance at an individual level. TheIFNG+2200C SNP may also be in linkage disequilibrium (LD) with another genetic variant within the haplotype that might influence IFN-γ levels and / or measures of iron status in Gambian populations. Finally, we did not measure IFN-γ levels to determine if they differed between genotypes at the end of the malaria season. Thus, our findings need to be examined in larger population-based studies, in other malaria-exposed populations, and functional assays are needed to identify whether genetic variation in theIFNG gene influences iron status. However, our study supports the hypothesis that preventing and treating malaria infection may improve haemoglobin levels and iron status in African children8.To our knowledge, this is the first study examining the role ofhttps://doi.org/10.7910/DVN/2NKJID43. This project contains the following underlying data:Havard Dataverse: Replication Data for: Interferon-gamma polymorphisms and risk of iron deficiency and anaemia in Gambian children,IFNG_final_data_v2 (dataset containing demographic information and results of laboratory assays for participants included in the study).IFNG_analysis_KM .KMokaya_IFNG_Codebook (contains variable description and labels).Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of theIFNG polymorphisms in Gambian children and risk of iron deficiency and anaemia,https://doi.org/10.6084/m9.figshare.11807277.v629.Figshare:This project contains the following extended data:IFNG single nucleotide polymorphismsExtended datafile 1. Assayed® primer-extension definitions data for the fiveIFNG polymorphismsExtended datafile 2. Agena Biosciences (formerly SEQUENOM) MassARRAYExtended datafile 3. Markers of iron status at the end of the malaria season in children with sickle cell trait and glucose-6-phosphate dehydrogenase deficiencyIFNG single nucleotide polymorphisms and risk of malaria parasitemia at the start and end of the malaria seasonExtended datafile 4.IFNG genotypes and risk of iron deficiency, iron deficiency anaemia and anaemia at the start of the malaria seasonExtended datafile 5.Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the In this paper Abuga et al investigate the role of IFNG SNPs in influencing iron deficiency (ID) and iron deficiency anemia (IDA) in Gambian children over a malaria season. They identify a SNP, IFNG+2200C, located in intron 3, associated with a reduced risk of malaria parasitemia, reduced Hb, and a trend towards ID and IDA. The authors conclude that although more studies are needed to assess the role of IFNG SNPs in ID and IDA, this represents the first study that investigates the association of IFNG genetic variants with iron status.1).Serum hepcidin levels are influenced by circadian rhythm and serum/iron stores. Please indicate at what time of the day blood was drawn. To “normalize” serum hepcidin to body iron concentration, the hepcidin/ferritin or hepcidin/TSAT ratio should be shown in Table 1 and Table 3  related to these children? The study is well conducted and of interest. I have only minor comments:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Laura Silvestri has long-term expertise in the characterization of molecular mechanisms responsible for the pathogenesis of iron/hepcidin-related disorders as Hereditary Hemochromatosis, beta-thalassemia, and iron refractory iron deficiency anemia (IRIDA).I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. a) Serum hepcidin levels are influenced by circadian rhythm and serum/iron stores. Please indicate at what time of the day blood was drawn.Response: Thank you, Blood was drawn from the participants in the morning hours (between 6 and 11AM). We have updated the study design section to indicate the time of sample collection.Thank you for your helpful comments. Methods, under Study design: “All children had a clinical examination and a blood sample collected in the morning between 6 and 11 am for full blood count, malaria film, and biomarkers of iron status and inflammation at the start and end of the malaria season.”b) To “normalize” serum hepcidin to body iron concentration, the hepcidin/ferritin or hepcidin/TSAT ratio should be shown in Table 1 and Table 3 .Response: Thank you for the suggestion. We have added hepcidin/ferritin and transferrin saturation (TSAT)/hepcidin ratios to tables 1 and 3. We have also made the following changes:Methods section under laboratory methods: Hepcidin/ferritin and TSAT/hepcidin ratios were also calculated.1Results section under Characteristics of participants: We found that hepcidin and hepcidin/ferritin ratio decreased while the TSAT/hepcidin ratio increased across the malaria season in keeping with the need for increased erythropoiesis and increased rates of iron absorption at the end of the malaria season.2. In the M&M section, the authors claim that some children are carriers of the HbAS, and G6PD deficiency. Is it possible to present the haematological and iron data (in Table 1 and Table 3) related to these children?Response: Thank you for this suggestion. We have now added haemoglobin and iron biomarker data for children with HbAS and G6PD deficiency as a Supplementary file (Extended datafile 3) that can be accessed on Figshare (https://doi.org/10.6084/m9.figshare.11807277.v6). We have also made the following changes to the Results section:Results under Characteristics of participants: “A total of 99/751 (13.2%) children carried sickle cell trait (HbAS) and 136/683 (19.9%) children G6PD deficiency and the effects of these polymorphisms on iron status and anaemia are shown in Extended datafile 3.2 At the start of the malaria season we found little difference in iron status in children with HbAS and G6PD deficiency compared to those with wild-type. At the end of the malaria season, children carrying HbAS had lower zinc protoporphyrin levels  compared to those with HbAA  and children with G6PD deficiency had lower haemoglobin levels  than those with G6PD wild-type genotype .”ReferencesPediatr Blood Cancer 2020;67(3): e28038. 1.         Donker AE, Galesloot TE, Laarakkers CM, Klaver SM, Bakkeren DL, Swinkels DW. Standardized serum hepcidin values in Dutch children: Set point relative to body iron changes during childhood.https://doi.org/10.6084/m9.figshare.11807277. 2.         Abuga KM, Robert KA, Muriuki JM, et al. IFNG polymorphisms in Gambian children and risk of iron deficiency and anaemia. 2020. Abstract:  It would be good if the authors provided the age range of children enrolled in the study (2 – 6 years).  This could be added in the Methods section at the end of the sentence ‘….780 Gambian children.’ The last sentence at the end of the Results section mentions a number of covariates that were adjusted for in the models. One such covariate is ‘malaria parasitaemia’. Was malaria parasitaemia adjusted for only at the beginning and at the end of the malaria season or did it capture all the malaria cases during the study period? Does ‘malaria parasitaemia’ mean malaria episodes during the study period?  If it only captures the two time points, what would the effect of ‘additional episodes’ during the study period have on the results? In malaria endemic areas, repeated infections are more likely to result in anaemia than just the patent infection.  The authors need to clarify on this and if there were no malaria cases in-between the two time-points they need to indicate so.    Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank the reviewer for his helpful comments, which we have addressed point-by-point below:Reviewer #1Abstract: It would be good if the authors provided the age range of children enrolled in the study (2 – 6 years).This could be added in the Methods section at the end of the sentence ‘….780 Gambian children.’Response: Thank you for the suggestion. We have added the age range (2-6 years) in the methods section of the abstract.Abstract, Methods: “We used previously available data from Gambian family trios to determine informative SNPs and then used the Agena Bioscience MassArray platform to type five SNPs from the IFNG gene in a cohort of 780 Gambian children aged 2-6 years”.2. The last sentence at the end of the Results section mentions a number of covariates that were adjusted for in the models. One such covariate is ‘malaria parasitaemia’. a) Was malaria parasitaemia adjusted for only at the beginning and at the end of the malaria season or did it capture all the malaria cases during the study period?Response: In the current analysis, we adjusted for malaria parasitaemia detected on blood film at the beginning and at the end of the malaria season, at the same timepoints when iron biomarkers were measured since malaria parasitaemia influences markers of iron status. We did not monitor for malaria cases during the malaria season. Methods, under Statistical analyses: “Multivariable regression models were adjusted for age (grouped by year), sex, village , malaria parasitaemia and ACT at the start and end of the malaria season.”b)  Does ‘malaria parasitaemia’ mean malaria episodes during the study period?Response: Malaria parasitaemia referred to the identification of Plasmodium parasites on the blood film taken from the study participants at either of the two cross-sectional timepoints, i.e. at the beginning and / or end of the malaria season. It does not refer to malaria episodes during the study period.c) If it only captures the two time points, what would the effect of ‘additional episodes’ during the study period have on the results? In malaria endemic areas, repeated infections are more likely to result in anaemia than just the patent infection. The authors need to clarify on this and if there were no malaria cases in-between the two time-points they need to indicate so.Response: We agree that repeated malaria episodes would increase the risk of anaemia. Based on published data we estimated that the majority of the children would be likely to have had one or more episodes of malaria over the malaria season during the study period.1 Studies indicate that malaria increases IFN-g levels2 and that IFN-g regulates erythropoiesis and iron-regulatory proteins including hepcidin.3-6We therefore hypothesized that IFNG SNPs, which might alter IFNG expression, would influence iron status at the end of the malaria season. Thus, we would expect that the effect of ‘additional episodes’ of malaria would be to increase malaria-induced IFN-g  levels and IFN-g -induced iron deficiency and anaemia. To clarify we have updated the Methods section as follows: Methods, under Laboratory methods: “Giemsa-stained thick and thin blood films were examined for Plasmodium falciparum and other Plasmodium species at the start and end of the malaria season.”  We thank the reviewer for his helpful comments, which we have addressed point-by-point below:Abstract: It would be good if the authors provided the age range of children enrolled in the study (2 – 6 years).This could be added in the Methods section at the end of the sentence ‘….780 Gambian children.’    Response: Thank you for the suggestion. We have added the age range (2-6 years) in the methods section of the abstract.Abstract, Methods: “We used previously available data from Gambian family trios to determine informative SNPs and then used the Agena Bioscience MassArray platform to type five SNPs from the IFNG gene in a cohort of 780 Gambian children aged 2-6 years”.2. The last sentence at the end of the Results section mentions a number of covariates that were adjusted for in the models. One such covariate is ‘malaria parasitaemia’. a) Was malaria parasitaemia adjusted for only at the beginning and at the end of the malaria season or did it capture all the malaria cases during the study period?Response: In the current analysis, we adjusted for malaria parasitaemia detected on blood film at the beginning and at the end of the malaria season, at the same timepoints when iron biomarkers were measured since malaria parasitaemia influences markers of iron status. We did not monitor for malaria cases during the malaria season. Methods, under Statistical analyses: “Multivariable regression models were adjusted for age (grouped by year), sex, village , malaria parasitaemia and ACT at the start and end of the malaria season.”b) Does ‘malaria parasitaemia’ mean malaria episodes during the study period?Response: Malaria parasitaemia referred to the identification of Plasmodium parasites on the blood film taken from the study participants at either of the two cross-sectional timepoints, i.e. at the beginning and / or end of the malaria season. It does not refer to malaria episodes during the study period.c) If it only captures the two time points, what would the effect of ‘additional episodes’ during the study period have on the results? In malaria endemic areas, repeated infections are more likely to result in anaemia than just the patent infection. The authors need to clarify on this and if there were no malaria cases in-between the two time-points they need to indicate so.Response: We agree that repeated malaria episodes would increase the risk of anaemia. Based on published data we estimated that the majority of the children would be likely to have had one or more episodes of malaria over the malaria season during the study period.1 Studies indicate that malaria increases IFN-g levels2 and that IFN-g regulates erythropoiesis and iron-regulatory proteins including hepcidin.3-6We therefore hypothesized that IFNG SNPs, which might alter IFNG expression, would influence iron status at the end of the malaria season. Thus, we would expect that the effect of ‘additional episodes’ of malaria would be to increase malaria-induced IFN-g  levels and IFN-g -induced iron deficiency and anaemia. To clarify we have updated the Methods section as follows: Methods, under Laboratory methods: “Giemsa-stained thick and thin blood films were examined for Plasmodium falciparum and other Plasmodium species at the start and end of the malaria season.” ReferencesLancet  2008;372(9649): 1545-54. 1.         Ceesay SJ, Casals-Pascual C, Erskine J, et al. Changes in malaria indices between 1999 and 2007 in The Gambia: a retrospective analysis.Journal of leukocyte biology 2010;88(6): 1131-43. 2.         McCall MB, Sauerwein RW. Interferon-gamma--central mediator of protective immune responses against the pre-erythrocytic and blood stage of malaria.Respiratory research 2011;12(1): 100. 3.         Frazier MD, Mamo LB, Ghio AJ, Turi JL. Hepcidin expression in human airway epithelial cells is regulated by interferon-γ.European journal of immunology 2008;38(7): 1923-36. 4.         Nairz M, Fritsche G, Brunner P, Talasz H, Hantke K, Weiss G. Interferon-gamma limits the availability of iron for intramacrophage Salmonella typhimurium.Blood 2003;101(10): 4148-54. 5.         Ludwiczek S, Aigner E, Theurl I, Weiss G. Cytokine-mediated regulation of iron transport in human monocytic cells.The Journal of clinical investigation 1993;91(3): 969-76. 6.         Byrd TF, Horwitz MA. Regulation of transferrin receptor expression and ferritin content in human mononuclear phagocytes. Coordinate upregulation by iron transferrin and downregulation by interferon gamma.


P. falciparum infections are important surrogate indicators for assessing malaria transmission intensity in different regions of endemicity. Determination of MOI and diversity of P. falciparum among asymptomatic carriers will enhance our understanding of parasite biology and transmission to mosquito vectors. This study examined the MOI and genetic diversity of P. falciparum parasite populations circulating in Mbita, a region characterized as one of the malaria hotspots in Kenya. The genetic diversity and multiplicity of P. falciparum infections in 95 asymptomatic school children (age 5–15 yrs.) residing in Mbita, western Kenya were assessed using 10 polymorphic microsatellite markers. An average of 79.69% (Range: 54.84–95.74%) of the isolates analysed in this study were polyclonal infections as detected in at least one locus. A high mean MOI of 3.39 (Range: 2.24–4.72) and expected heterozygosity (He) of 0.81 (Range: 0.57–0.95) was reported in the study population. The analysed samples were extensively polyclonal infections leading to circulation of highly genetically diverse parasite populations in the study area. These findings correlated with the expectations of high malaria transmission intensity despite scaling up malaria interventions in the area thereby indicating the need for a robust malaria interventions particularly against asymptomatic carriers in order to attain elimination in the region.Multiplicity of infection (MOI) and genetic diversity of  An increase in the global incidence of malaria cases from 219 million in 2017 to 228 million in 2018 with a staggering 405 000 related deaths was registered2. Sub-Saharan Africa bears the greatest malaria cases and death rates compared to other regions like South-East Asia, Eastern Mediterranean, Western Pacific, and the Americas with an unprecedented 213 million malaria cases1.Malaria continues to be a major public health problem in many parts of the world despite numerous national and international efforts in combatting the ongoing disease transmission. Although enormous success in the fight against the disease has been registered, reports have indicated an insignificant progress in the reduction of global malaria cases for the period 2014–20184. Children registered the highest prevalence of the disease and recorded an average of 8 percent and 15 percent among younger (<5 years) and older (5–15 years) age categories respectively5. Older children living in endemic environments around lake shores recorded a much higher prevalence of about 38 percent6. In a bid to attain its goal of a “Malaria-free Kenya”, the country has implemented many malaria control and eradication interventions including; the use of Artemisinin based Combination Therapy (ACT) as a first line of treatment, Long-Lasting Insecticidal Net (LLIN), Intermittent Presumptive Treatment in pregnancy (IPTp), Indoor Residual Spraying (IRS) and Rapid Diagnostic Test (RDT) for efficient parasite diagnosis8.In Kenya, about 74 percent of the total population are reported to be at risk of malaria infection and regions such as the coastal areas and western part of the country records the highest transmission intensity11. The management and epidemiology of malaria in school-age children as a group has initially received little attention in comparison to the pregnant women and children under the age of five years groups14. In many endemic settings, malaria contributes immensely to the annual school absenteeism and poor academic performance among school children14. Older children in high malaria transmission areas develop parasite specific immunity against Plasmodium parasite and this contributes to reduction in clinical malaria episodes and high prevalence of asymptomatic parasite carriers17. As a result, malaria asymptomatic school children might not be absent from school due to clinical malaria episodes but may serve as major reservoir for the transmission of the disease19.A larger proportion of malaria morbidity and mortality studies were centred on preschool-aged children (2–4 years of age). This group contributes a substantial number of incidence to the global malaria burden each year23. However, the high prevalence of asymptomatic carriers especially among older children28, high recombination rates among distinct P. falciparum clones in endemic settings leading to emergence of highly diverse parasite isolates, rapid emergence and distribution of drug resistant P. falciparum parasite strains33, and prevalence of infections characterized by multiple genetically distinct parasite strains are some of the major contributing factors hindering the global malaria control and elimination35. In highly endemic environments, many individuals carry multiple parasite clones38. This may have both positive and negative implications in the fight against malaria. The carriage of multiple distinct parasite clones by an individual is reported to enhance the development of multiple strain specific anti-parasite immunity. However, due to an intense intra-host competition, harbouring multiple distinct parasite clones is also implicated in high gametocyte production and emergence of highly virulent and drug resistant parasite strains43.Overall, progress towards malaria control and elimination has stalled across all World Health Organisation (WHO) regions. The stagnancy in the global fight against the disease is partly due to decreased funding from international donorsP. falciparum infection are important surrogate tools for the determination of malaria transmission intensity in different regions of endemicity46. These indices of P. falciparum parasite infections are also used in determining the impact of some key malaria interventions like drug and vaccine efficacy studies48. Numerous studies about multiplicity and diversity of P. falciparum infections have been carried out in various WHO regions using different tools including size-polymorphic antigens and microsatellite markers followed by agarose gel and capillary electrophoresis respectively, single nucleotide polymorphism genotyping, amplicon ultra-deep sequencing and whole genome sequencing49. The use of molecular genotyping of polymorphic antigenic markers like merozoite surface protein (MSP) 1 and 2, glutamate rich protein (Glurp) for parasite diversity studies are faced with some criticisms due to the fact that these genes are implicated to be under strong immune selection52. Amplicon ultra-deep sequencing and whole genome sequencing techniques have high sensitivity and specificity particularly to detect minority clones in case of multiclonal infections52. However, the costs and time factors attached to the use of these techniques are relatively high thereby limiting their application especially in resource scarce settings. Microsatellite markers are highly polymorphic, widely distributed in the Plasmodium genome, perceived to be selection neutral and readily amplifiable using cheap methods like PCR54. These criteria have made them an ideal tool for the estimation of parasite genetic diversity and MOI48.Multiplicity of infection (MOI) and genetic diversity of Plasmodium parasite carriage among school children have been reported55, studies on diversity level and multiplicity of P. falciparum infections among this group still remained scanty. This study was carried out to investigate the diversity and multiplicity of P. falciparum infections among asymptomatic school children (age 5–15 yrs.) in one of the highly malaria endemic regions of Kenya in order to provide an up-to-date critical information for monitoring parasite transmission dynamics and effective evaluation of the impact of currently implemented malaria control interventions in the area.While studies about asymptomatic The Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU) granted approval for the original study (KEMRI/RES/7/3/1). Consent for sample reuse was part of the original consent form. Study participants were only enrolled after obtaining a written informed consent from their parents or legal guardians. Participants above 12 years also provided assent in addition to parental consent. All experiments were performed in accordance with the relevant guidelines and regulations.2 and is approximately 400 km west of the capital city of Kenya, Nairobi. Mbita sub-county has a population of about 115,896 and the major economic activities include subsistence farming, fishing, small scale businesses and domestication of animals like sheep, goat, chicken, pig and cattle56. Majority of the residents live in a compound system which comprises of one or more households together. The housing structures range from traditional mud with grass thatched huts to modern concrete and corrugated iron buildings56. The region experiences two periods of rainfall, from March to June and then October to November each year57 and is characterized as one of the HIV hotspots with some of the poorest health indicators in Kenya. Due to the biannual rainfall pattern and the proximity to the shores of Lake Victoria, the region experiences a perennial transmission of malaria thereby making the disease the leading cause of morbidity and mortality among children in the region58.The study was conducted in Mbita sub-county, situated in the shores of Lake Victoria in Homa Bay County formerly Nyanza Province in western Kenya. The district is bordered from the north, west and south by Lake Victoria and located between latitudes 0° 21′ and 0° 32′ South and longitudes 34° 04′ and 34° 24′ East. The area of the district is about 163.28 kmPlasmodium parasite carriage using SD Bioline malaria Ag Pf/Pan (HRP-II/pLDH) Rapid Diagnostic Test (RDT) and microscopy. A drop of blood from each participant was also obtained for the preparation of thick and thin blood films, stained with 10% Giemsa for 10 minutes and used for the microscopy diagnosis. Two drops of blood were collected from each individual on a filter paper , for genomic DNA extraction. The filter paper dry blood spots (DBS) samples were air dried and sealed in airtight plastic bags containing silica gel and were stored at −20 °C until usage. The blood smear slides were read by a well-trained microscopists and any slide with no observable parasites in 100 microscopic fields were declared negative; gametocytes were counted against 500 white blood cells while the asexual parasites were counted against 200 white blood cells. Slides that were declared negative were all double-read and confirmed to be negative if no parasites are observed in 200 microscopic fields. A second microscopist re-read all the slides for the purpose of quality control. A total of 95 RDT and microscopy positive filter paper dry blood spots (DBS) samples were randomly selected for this study.The study used archived samples that were previously collected from asymptomatic school children in Mbita, western Kenya. The participants were recruited from various primary schools in the region and enrolled between December 2016 and October 2018 as part of a study to evaluate symbiotic microbes and mosquito vector competence using membrane feeding assays in a bid to isolate and characterize potential malaria transmission blocking endosymbiont candidates. Blood samples were obtained from school children aged between 5–15 years and were screened for 59.Single-hole Kangaro punch  was used to cut out six circles (3 mm in diameter) from each filter paper dry blood spots (DBS) sample and the punched DBS were placed in 1.5 ml Eppendorf tubes. In order to ensure that there is no cross contamination between samples, the punch was sterilized using sodium hypochlorite (NaClO) and 100% ethanolDNA was extracted using the QIAamp DNA Mini Kit  following the manufacturer’s protocol. The quality and concentration of each extracted genomic DNA sample was assessed using a Nanodrop 2000C  and samples were stored at −20 °C.Plasmodium falciparum DNA extracted from each DBS sample was genotyped using ten polymorphic microsatellite markers60. Primer sets targeting specific sequences flanking (TAA)n repeat regions in the P. falciparum genome were used to amplify each locus using a slightly modified published hemi-nested PCR protocol in a SimpliAmp Thermal Cycler 60. The primers were dye labelled using 6-FAM for locus TA1, ARA2, PfPK2, TA87, and TA109; HEX for TA81, TA42, Polyα, TA60 and Pfg377. The hemi-one amplifications were performed in a total PCR reaction volume of 20 µL containing 1× FIREPol Master Mix , 0.3 µM of each primer  and 3 µL (10 ng/µL) of DNA template. The cycling conditions for nest-one PCR were; initial denaturation for 2 min at 94 °C; 30 cycles of 30 sec at 94 °C, 30 sec at 42 °C, 30 sec at 40 °C and 30 sec at 65 °C; final elongation for 5 min at 65 °C. The total reaction volume of hemi-two PCR was also 20 µL containing 1× FIREPol Master Mix , 0.4 µM of each primer   genetic Analyser using GeneScan 400HD ROX Size Standard . Allele sizes scoring and peak height quantification was carried out using GeneMarker V3.0.1 software .The PCR amplification for the different loci were confirmed by fragment separation in 2% agarose gels and visualized using ethidium bromide . The amplified products were pooled together as described elsewherePlasmodium falciparum parasite stages found in human blood are haploid, the presence of more than one allele at a particular locus per isolate was interpreted as polyclonal or multiple clone infections. That is, samples with more than one allele at any given locus are termed as co-infection with genetically distinct parasite clones. Isolates with minor allele peak heights that are ≥25% of the predominant allele per locus were scored as multiple alleles. DNA samples with only one allele detected at all the genotyped gene loci were scored as monoclonal infections. Multiplicity of P. falciparum infection (MOI) is defined as the number of genetically distinct clones co-infecting a host at any given time. In this study, MOI and average MOI were estimated per locus. The average MOI of all the samples included in this study was estimated as the ratio of the total number of distinct fragments  scored for a given gene loci in relation to the number of samples successfully amplified by that marker locus35.The fact that 61. The predominant allele in case of multiple alleles or the only allele in the case of single allele at each locus was used to estimate the expected heterozygosity (He) per locus as a measure of genetic diversity using a formula, He = [n/(n − 1)][1 −Σpi2], where n and pi represents the number of isolates analysed and frequency of the ith allele in a given population. Expected heterozygosity (He) values range between 0 and 1 for no genetic diversity and high genetic diversity, respectively60.Descriptive statistics were computed using IBM SPSS Statistics for Windows, version 20 . Allelic frequency, expected heterozygosity, number of alleles and number of effective alleles of the study population were determined using a population genetic software, GenAIEx 6.5P. falciparum positive samples detected by RDT and microscopy was included in this study. The ages of the study participants ranged from 5 to 15 years. In Table P. falciparum while 18 (18.9%) of the subjects were infected with P. falciparum plus another Plasmodium species  and 50.5% carried the sexual stage of the parasite (gametocyte).A total of 95 P. falciparum infections [3.39 (Range: 2.24–4.72)] detected at each of the genotyped genetic locus. More than six alleles were found at almost all the genotyped genetic loci (except in the Pfg377 loci) in the study samples indicating an overall complex infections.The amplification of the 10 polymorphic microsatellite marker loci was successful in 99% of the isolates. Overall, the Pfg377 locus was the marker with the highest number of missing data in which 3 (3%) of the isolates had a missing data. The full genotype profile and allelic frequencies of the 95 samples were presented as Supplementary Tables Overall, based on genotyping of the 10 polymorphic microsatellite marker loci, there were variations in the number of isolates with polyclonal infections detected by each marker locus with TA1 (54.84%) and TA42 (95.74%) markers detecting the lowest and highest number of polyclonal isolates  per locus ranging from 8 (for locus Pfg377) to 23 (for locus Polyα) and the number of effective alleles (Ne) varied between 2.3 and 15.3. The least and most polymorphic markers were Pfg377 and Polyα respectively. Overall, Na values were much higher than the Ne values indicating that majority of the alleles were present at low frequency Table .Table 3GHe) defined as the probability that two randomly selected clones from a population will carry distinct alleles at each marker loci was used to estimate the allelic diversity per loci based on allelic frequencies computed (See Supplementary files: Table (He) values per locus in the study population varied from 0.57 (TA109) to 0.95 (Polyα) with an average score of 0.81, thereby depicting a high genetic diversity among the isolates.The expected heterozygosity 68, Gambia (2.1), Senegal (2.2), Guinea Bissau (2.6), Guinea (4.0)54, Tanzania (3.5)62, Uganda (3.0–3.7)65 and Madagascar (3.72–3.73)66 but higher than results from low malaria transmission regions like Djibouti (1.0–1.4), Dakar (1.0–1.5), Niamey (1.0–1.8)67 and Sudan69. The high MOI found in this study maybe due to the boosted parasite specific immunity in people residing in high malaria transmission regions and the study population as children were reported to carry higher MOI infections compared to adults70.The current study revealed a high level of multiplicity of infections and genetic diversity among the study population. This is illustrated by high level of polyclonal infections and high proportions of locus scores with multiple genotypes. The 79.69% prevalence of polyclonal infections found in the study population is consistent with results from other studies carried out in malaria endemic and high transmission intensity regions where multi-clonal infection rates varied from 50% to 100%, respectivelyP. falciparum recorded at each of the 10 polymorphic microsatellite marker loci in our study are indications of high genetic diversity among P. falciparum parasites circulating in the study area. This finding could be due to the high malaria transmission and endemicity reported in the area71. Another contributing factor for the high genetic diversity maybe due to the efficient land and water transport networks in the study area facilitating human movements from other malaria endemic regions leading to the introduction of foreign strains to the local parasites gene pool68. These results corroborates the findings of previous studies conducted in other parts of western Kenya66 but different from those of low malaria transmission intensity areas like lowland coastal area of Kenya 64, Philippines72 and other regions of South America75. The high mean He value (0.81) observed in our study was similar to those obtained in high malaria endemic and transmission settings of Africa: Kenya (0.81)64, Congo DRC (0.71)75, Ghana (0.67–0.69)35, Nigeria (0.65–0.79)76, The Gambia (0.75), Senegal (0.72), Guinea Bissau (0.78), Guinea Conakry (0.78)54, Asia: Vietnam (0.52–0.91) and Oceania: Papua New Guinea (0.79)73. However, He values from studies conducted in low malaria transmission intensity regions such as Djibouti (0.41)67, Brazil (0.39–0.52)77, Honduras (0.35) and Nicaragua (0.38)78 were lower than that of our study.The high number of alleles and high expected heterozygosity of P. falciparum infections among malaria asymptomatic school children (age 5–15 yrs.) in Mbita, western Kenya using 10 polymorphic microsatellite marker loci reveal a high level of multi-clonal infections and high genetic diversity of P. falciparum parasites circulating in the study area. These findings correlated with the expectations of high malaria transmission intensity and endemic region despite the scaling up of malaria control interventions by the government. Therefore, in order to attain ultimate malaria elimination in the region, there is a need for the implementation of more robust and holistic interventions against the parasite particularly the asymptomatic carriers. Furthermore, the findings from our study have pinpointed the need for periodic Plasmodium parasite molecular genetic surveillance studies in the area. This will help maintain up-to-date information about parasite transmission dynamics for effective impact assessment of the currently implemented malaria control interventions. Additionally, it will further guide in the introduction of new malaria control programs whenever needed.The analysis of genetic diversity and multiplicity of Supplementary Information.Supplementary Information 2.


Parasitological diagnosis generates data to assist malaria-endemic countries determine their status within the malaria elimination continuum and also inform the deployment of proven interventions to yield maximum impact. This study determined prevalence of malaria parasitaemia and mRDT performances among febrile patients in selected health care facilities across Ghana.This study was a cross-sectional survey conducted in the previously 10 regions of Ghana from May to August 2018. Each patient suspected to have uncomplicated malaria was tested using microscopy and two malaria rapid diagnostic tests (mRDTs): routinely used CareStart™ Malaria HRP2 (Pf) and SD Bioline Malaria Ag Pf (HRP2/pLDH). Main outcome variables were malaria slide and CareStart™ Malaria HRP2 (Pf) positivity rates; and diagnostic accuracy of CareStart™ Malaria HRP2 (Pf) and SD Bioline Malaria Ag Pf (HRP2/pLDH) using microscopy as “gold standard”.Plasmodium falciparum mono-infection accounting for 98.0% of all infections. The odds of parasitaemia by microscopy was significantly lower among female patients compared with males , and among patients with history of previous antimalarial intake compared with those with no such history . Overall sensitivity of CareStart™ Malaria HRP2 (Pf) was statistically similar to that of the HRP2 band of SD Bioline Malaria Ag Pf (HRP2/pLDH) combo kit  but significantly higher than the pLDH band . The same pattern was observed for negative predictive value.Overall parasite positivity rates were 32.3% (6266/19402) by mRDT and 16.0% (2984/18616) by microscopy, with Malaria control interventions should be targeted at the general population, and history of antimalarial intake considered a key predictor of malaria slide negativity. Furthermore, HRP2-based mRDTs remain effective diagnostic tool in the management of suspected uncomplicated malaria in the country.The online version contains supplementary material available at 10.1186/s12889-021-10290-1. Malaria remains one of the major public health problems in Ghana as in other sub-Saharan African countries. The WHO African region accounted for 93.0% of the 228 million global cases and 94.0% of the 405,000 global deaths that occurred in 2018 [The objective of uncomplicated malaria case management is to cure the infection as rapidly as possible in order to prevent progression to severe disease and reduce transmission to others. Key to this is prompt and accurate parasitological diagnosis, which is also critical in assessing impact of preventive strategies such as long lasting insecticidal nets (LLINs), indoor residual spraying (IRS), larviciding/larval source management, seasonal malaria chemoprevention (SMC), and intermittent preventive treatment in pregnancy (IPTp) , 4. Parad-1)*SI [d is the facility/cluster number.A total of 10 health care facilities were selected in each of the then 10 regions of Ghana *SI , where dPf positive cases, by microscopy, was generated for each region based on estimated pfHRP2 gene deletion prevalence of 3.2% at 95% confidence interval, 0.022 margin of error, and a design effect of 1.5 using 10 clusters per region. A total of 20,000 suspected malaria cases (2000 per region) were to be screened to get the minimum sample size of 370 based on an estimated average malaria slide positivity rate of 20.0% (unpublished data).A minimum sample size of 370 pfhrp2/pfhrp3 gene deletions using the 2018 WHO protocol [The study was a cross-sectional survey among all symptomatic individuals seeking care in selected health care facilities in the previously 10 regions of Ghana during the early phase of the peak transmission season of 2018 (May – August). This was part of a study to estimate the prevalence of protocol .Trained Nurses used the KoBoCollect application to administer standard questionnaires to all suspected uncomplicated malaria patients visiting the outpatient department of the selected health care facilities. Prior to this, informed consent (for adult participants), parental consent (for children under 18 years), and assent (for children aged 12–17 years) were obtained for all participants. Data collected included participant’s age, gender, educational background, marital status, and antimalarial intake during the 2-week period preceding survey  was performed by trained laboratory Technicians / Technologists according to manufacturer’s instructions using the standard test kit in use routinely ) as well as a stocked test kit ). All patients were exposed to the two mRDTs. Thick and thin blood smears for malaria microscopy were also prepared. The thin film was fixed in methanol, and both thick and thin films stained with 3.0% Giemsa stain for approximately 45 min and air-dried. All slides were stored in plastic slide boxes for later examination by WHO-certified expert microscopists.The thick film was used for parasite quantification per white blood cells (WBCs) whilst the thin film was used for species identification. As per WHO guidelines, the thin film was used for parasite quantification per parasitized red blood cells (RBCs) in cases where ≥100 parasites were observed in each thick film field under × 100 objective. Parasite quantifications per WBCs were computed per microliter (μL) of blood assuming 8000 WBCs/μL whilst counts per parasitized RBCs were computed per μL of blood assuming an average of 5,000,000 RBCs per μL . A bloodTwo main datasets (questionnaire and microscopy) were captured separately using KoBoCollect. The datasets were extracted and cleaned separately in IBM SPSS Statistics version 21 using data quality tabulations and source data (e.g. facility laboratory record books), where necessary. The 2 datasets were subsequently merged after matching the individual IDs for analyses. Main outcome variables analyzed were malaria slide and mRDT positivity rates ); and mRDT  and SD Bioline Malaria Ag Pf (HRP2/pLDH)) sensitivity (SEN) and specificity (SPEC) as well as positive predictive value (PPV) and negative predictive value (NPV) using microscopy as the “gold standard”. The following formulae were applied:p < 0.05). Variables showing significant associations were subsequently used in a multivariable logistic analysis to determine their independent effects. To account for clustering of measurements of individuals within the same health care facility and region, the Generalized Estimating Equation (GEE) approach in R was used to assess the associations between explanatory variables and malaria slide (microscopy) and mRDT positivity rates (significant at p < 0.05). Taking cognizance of the fact that GEE takes into account the dependency of observations by specifying a working correlation structure, four working correlation structures were identified: independence, exchangeable, auto regressive and unstructured. These parameter estimates were still valid even when the working correlation structures were mis-specified [Explanatory variables for malaria slide and mRDT positivity rates were participant’s age, gender, educational background, marital status, and history of antimalarial intake within the two-week period preceding survey. Analysis of educational level was controlled for patients aged < 5 years because official age for basic education in Ghana is 6 years . Similarpecified , 17. FolA total of 19,787 suspected malaria cases from the 10 regions of Ghana were screened during the study period Fig. . MajoritParasite positivity rates, by CareStart™ Malaria HRP2 (Pf), ranged between 21.4% (in the Greater Accra and Upper East regions) and 41.5% in the Brong Ahafo region yielding a national rate of 32.3% whilst parasite positivity rates by microscopy ranged between 8.0% (in the Eastern region) and 23.4% (in the Ashanti region) yielding a national rate of 16.0%. Generally, majority of parasite densities ranged between 10,000 and 99,999 per μL, with the Upper West region showing the highest geometric mean parasite density of 57,739 per μL  mono-infection was the most prevalent infection type (98.0%), ranging between 92.4% (in the Volta region) and 99.5% (in the Greater Accra and Upper East regions). Prevalence of Plasmodium malariae (Pm) mono-infection ranged between 0.2% (in the Ashanti region) and 1.9% (in the Upper West region) yielding a national prevalence of 0.7%. Plasmodium ovale (Po) mono-infection occurred in four regions with prevalence ranging between 0.3% (in the Brong Ahafo region) and 1.7% in the Volta region. Regions with mixed infection prevalence of over 1.0% were Northern (2.4%) and Volta (1.3%) for Pf + Pm infection type and Eastern (1.4%) and Volta (3.0%) for Pf + Po infection type. There were no Plasmodium vivax (Pv) infections  were 31.2% (95% CI: 30.6–31.9) for HRP2 and 25.3% (95% CI: 24.7–25.9) for pLDH.Univariate analysis showed that generally, malaria parasitaemia by microscopy and CareStart™ Malaria HRP2 (Pf) was associated with gender, age, education, and marital status. Malaria parasitaemia by microscopy was further associated with history of antimalarial intake 2 weeks preceding survey Table .Table 3Multivariable GEE analysis, which accounted for clustering and controlled for patients aged < 15 years, showed that malaria parasitaemia by microscopy was significantly associated with gender, age, and previous antimalarial intake whilst parasitaemia by CareStart™ Malaria HRP2 (Pf) was significantly associated with gender, age, and education. The odds of parasitaemia by microscopy was significantly lower among female patients compared with males ; significantly lower among patients aged ≥20 years compared with those aged 15–19 years ; and significantly lower among patients with history of previous antimalarial intake prior to the survey compared with those with no such history   was significantly lower among females compared with males ; significantly lower among patients aged ≥20 years compared with those aged 15–19 years ; and significantly lower among patients with tertiary education compared with those with no formal education  (Table p = 0.093) but significantly higher than pLDH band . The pLDH band of the SD Bioline Malaria Ag Pf (HRP2/pLDH) combo kit showed the highest specificity compared with the HRP2 band  and CareStart™ Malaria HRP2 (Pf)   and CareStart™ Malaria HRP2 (Pf) . On the contrary, positive predictive value for the pLDH band of SD Bioline Malaria Ag Pf (HRP2/pLDH) combo kit was significantly the highest compared with the HRP2 band and CareStart™ Malaria HRP2 (Pf)  was statistically similar to that of HRP2 band of SD Bioline Malaria Ag Pf (HRP2/pLDH) combo kit  among symptomatic individuals reporting at health care facilities was found to be significantly associated with gender and age. Parasitaemia by microscopy was further associated with reported history of antimalarial intake prior to visit to the health care facility whilst parasitaemia by CareStart™ Malaria HRP2 (Pf) was further associated with patient’s educational level.Female patients were less likely to be parasitaemic either by microscopy (22.0%) or CareStart™ Malaria HRP2 (Pf) (30.0%) compared with males. This finding suggest that women benefit from malaria interventions targeted at children under 5 years by virtue of the fact that they are the primary caretakers of this vulnerable group in most endemic countries . This stPatients who had a history of previous antimalarial intake during the two-week period before the survey were about 28.0% less likely to be parasitaemic by microscopy compared with those with no previous history of antimalarial intake. This finding compares well with a previous study in the northern region of the country, where children with a reported history of antimalarial intake were over 50.0% less likely to have a positive microscopy result . HistoryThe risk of parasitaemia by mRDT, which is a measure of exposure and not only acute infections, was significantly lower among patients with tertiary educational background compared with those with no formal education. Generally, the attainment of higher educational levels translates into higher knowledge levels which affect behavior modification and subsequently incidence and prevalence of malaria and other health events , 22. ProWith regards to diagnostic accuracy using microscopy as standard, CareStart™ Malaria HRP2 (Pf) kit showed over 90.0% SEN and NPV in all the regions of the country, which compares well with previous studies showing 95.1–98.2% SEN and 95.8–97.7% NPV in parts of the country , 24. SinPlasmodium falciparum. To facilitate Ghana’s progress from control to pre-elimination phase, the general population should benefit from malaria control interventions. Uptake of these interventions will be enhanced by the promotion of higher level education, particularly tertiary. History of antimalarial intake within the two-week period preceding visit to the clinic is a key predictor of malaria slide negativity, and should therefore guide management of symptomatic individuals. Furthermore, HRP2-based mRDTs remain effective diagnostic tool in the management of suspected uncomplicated malaria in the country.Ghana remains in the control phase of the malaria control to elimination continuum, with over 90.0% of disease across the country caused by Additional file 1. QuestionnaireAdditional file 2. mRDT diagnostic accuracy by type and region.


Plasmodium falciparum malaria remains one of the world’s major infectious diseases that cause most morbidity and mortality, particularly in children. In Ghana, most children below the ages of 5 years depending on the severity of the infection often lose their lives. However, it is still debatable why infection with falciparum malaria contributes to thrombocytopenia.This study sought to investigate the expression of the various platelet indices and activation markers in children with falciparum malaria. Platelet indices  and platelet surface membrane glycoproteins  expressions were determined in children with falciparum malaria (cases) and healthy children (controls) using automated blood cell analysis and flow cytometry techniques, respectively.falciparum malaria with the mean fluorescence intensity of PAC1  and CD36 . Moreover, the percentage expression of CD62p  was also observed to be probably associated with increased risk of falciparum malaria although not statistically significant (p > 0.05).Except for P-LCR, all the other platelet indices  were significantly lower in the cases than the controls (P < 0.05). Also, it was observed that the level of expression of the activation markers; PAC 1 and CD62p showed a significant (P < 0.05) decreased before and after activation in falciparum malaria cases than in the controls. On the contrary, CD36 expression in the controls did not differ significantly (p > 0.05) from the malaria cases. Platelet activation markers were known to be associated with increased risk of Plasmodium falciparum malaria has been known to be associated with platelet activation markers, which probably contributes to thrombocytopenia. Plasmodium falciparum is the most prevalent malaria parasites on the African continent, responsible for most malaria deaths globally. It contributes to most childhood parasitic infection and presents with signs and symptoms such as recurrent fever, fatigue, and body joint pains. Other symptoms may include headache, nausea, chills, sweating, pallor and body weakness. Children with severe malaria frequently develop one or more of the following symptoms: severe anemia, respiratory distress, cerebral malaria ). Sodium citrate blood samples were used for flow cytometry analysis of the platelet membrane glycoproteins. Venous blood samples collected into anticoagulant EDTA- K2 sample tubes were also analysed for the assessment of the various platelet indices.Six millilitres of whole blood were withdrawn from the study participants. A volume of 3.6 ml of blood was dispensed in the sodium citrate vacutainer tube and the remaining in the ethylenediamine tetraacetic acid (KPlatelet membrane glycoproteins were detected following the protocol described by Becton Dickson (BD) . Sodium Ten microlitres of platelet-specific antibodies , USA]) were used in the detection of the platelet membrane glycoproteins . PAC1- FITC and CD62p-PE were double stained while CD36- PE was single stained. 5μL Arginylglycylaspartic acid (RGD)  solution was added in the staining mixture of PAC-1-FITC and CD62p-PE to demonstrate specific PAC-1 binding.Equal volumes (2.5 μL) of inactivated and activated blood samples were stained with the antibodies and incubated for 15 min at room temperature in the dark. 500 μL of the cold 1% paraformaldehyde solution  was used to fix the stained cells for flow cytometry analysis.Acquisition and analysis of the processed samples were performed on scatter gating using CellQuestPro software of the FasCalibur (BD). The flow cytometry settings or acquisition of platelets were optimized by changing the linear scale to logarithmic signal amplification in all five detectors. FacsComp was used in daily CaliBrite beads calibration. A total of 10,000 events in a log side scatter versus forward scatter were collected for each sample. The analysis of the platelet activation markers PAC1- FITC, CD62p- PE and CD36-PE were based on the gated population enclosing platelets as defined by forward scatter (FSC) and side scatter (SSC) characteristics  blood samples were analysed using the Sysmex XT4000i haematology analyzer to assess the various platelet indices.Ethylendiaminetetraacetic acid . Bleeding score was later summarized into bleeding and no bleeding, the type of bleeding and its corresponding score. The bleeding symptoms was scored from grade 0 (absence or no symptoms) to grade 3  and the overall bleeding score was achieved by adding the scores for all of the bleeding symptoms .Plasmodium falciparum species was first performed using the first response rapid diagnostic test kit (RDT), and finally confirmed with microscopy. Five microlitres of EDTA- K2 blood samples were placed in the sample wells using the pipettes. The assay diluent was completely emptied by dispensing it to the diluent wells and the results were read within 20 min. Thin and thick blood films were prepared and flooded with Giemsa. The stained slides were washed under running tap water after 10 min. The stained blood films were allowed to air dry and viewed microscopically using × 100 (oil immersion) objective lens.The identification of Sodium citrate anticoagulant blood samples were spun for 15 min after sample preparation for flow cytometry. Fifty microlitres of plasma were used for the measurement of PT and APTT using CoaRad 2A .t test (Mann–Whitney). The effect of the categorical variables on the possible disease outcome was estimated using the Odds ratio (OR) with 95% confidence intervals (CI) for multivariate and univariate analysis. The various proportions were assessed using Fisher’s-exact or X2 − test. A p value < 0.05 was considered statistically significant.The data obtained in this study was analysed using EPI v7.2.0.1 info (CDC) and Graphpad prism 5. All the categorical variables and continuous variables were summarized by descriptive analysis. Continuous data was expressed as median (25th and 75th percentile range and interquartile ranges) for skewed data and the difference between the various groups was tested using the student Table falciparum malaria had bleeding history which was graded as minor type (grade 1–2). None of the participants had a bleeding history of grade 3–4. The commonest bleeding type reported in the cases include oral bleeding, haematuria and epistaxis .Three children infected with P.  grade 1–. None ofThe median (25th and 75th percentiles) expressions of the platelet indices  in children with falciparum malaria were lower compared to the controls. However, with the exception of the P- LCR, all the other platelet indices recorded in the cases differed statistically significant from the controls Table .Table 3EThe percentage expression of PAC -1 was significantly (p < 0.05) lower in children with falciparum malaria (9.66 [2.81–12.86]%) than the controls (20.94 [9.34–33.48]%) in both the resting and activation phase. However, there were an increase expression of PAC-1 in both the malaria group (20.56 [20.19–22.74]%) and the controls (33.62 [24.66–38.86]%) during the activation phase. But the expression still remained significantly (p < 0.05) higher in the controls than in the cases  than the healthy children (24.48 [11.05–39.10]%) during the resting phase  and the controls (31.92 [25.10–52.71]%) during the resting phase  and CD36   molecules which has plasmocidal activity [From the study, most children with falciparum malaria had low platelet counts compared to the controls. PLT is a dynamic indicator of peripheral blood platelet production and destruction. This was used in this study to assess thrombocytopenia in children with falciparum malaria. The significant decrease in PLT counts Table  in the mactivity , 15. As To assess platelet functionality, flow cytometry analysis was performed using PAC-1, CD62p and CD36 monoclonal antibodies for the detection of GPIIb/IIIa, P-selectin and GPIV, respectively. Except for CD36, the expression of the other platelet markers in malaria cases differed significantly from the controls. Also, a decreased expression of the platelet membrane glycoproteins was observed in the malaria cases than in the controls. This may be due to the low level of circulatory platelets as a result of immune-mediated destruction of the platelets. Alternatively, increased splenic removal of platelet clumped PRBC or increased release of platelet factor 4 (PF4) molecules may also be a contributory factor. The increased expression of the membrane glycoproteins  were observed to be significantly p > 0.05) associated with increased risk of falciparum malaria (Table  associat malaria . This wa malaria  who repoIn summary, falciparum malaria has been known to be associated with the various platelet activation markers which possibly contribute to thrombocytopenia. Severe malaria contributes to decrease platelet surface membrane glycoproteins due to excessive adhesion of platelets to parasitized red blood cells. Hence, further studies are recommended in establishing the mechanism of the various platelet membrane glycoproteins in the pathogenesis of falciparum malaria.