Anopheles gambiae salivary gland antigen (gSG6-P1), in two communities in Ghana with similar blood stage malaria parasite prevalence.The human host elicits specific immune responses after exposure to various life stages of the malaria parasite as well as components of mosquito saliva injected into the host during a mosquito bite. This study describes differences in IgG responses against antigens derived from the sporozoite (PfCSP), asexual stage parasite (PfEBA175) and the gametocyte (Pfs230), in addition to an Plasmodium parasite density and species and DNA extraction from blood spots and P. falciparum confirmation was performed using PCR. This study used the stored plasma samples to determine IgG antibody levels to P. falciparum and Anopheles salivary antigens using indirect ELISA.This study used archived plasma samples collected from an earlier cross-sectional study that enrolled volunteers aged from 6 months to 70 years from Simiw, peri-urban community (N = 347) and Obom, rural community (N = 291). An archived thick and thin blood smear for microscopy was used for the estimation of Individuals from Simiw had significantly higher levels of IgG against mosquito gSG6-P1 [median (95%CI)] [2.590 (2.452–2.783) ng/mL] compared to those from Obom [2.119 (1.957–2.345) ng/mL], p < 0.0001. Both IgG responses against Pfs230proC (p = 0.0006), and PfCSP (p = 0.002) were significantly lower in volunteers from Simiw compared to the participants from Obom. The seroprevalence of PfEBA-175.5R (p = 0.8613), gSG6-P1 (p = 0.0704), PfCSP (p = 0.7798) IgG were all similar in Obom and Simiw. However, Pfs230 seroprevalence was significantly higher at Obom compared to Simiw (p = 0.0006). Spearman correlation analysis showed no significant association between IgG responses against gSG6-P1, PfCSP, Pfs230proC and PfEBA-175.5R and parasite density at both Obom and Simiw (p > 0.05).P. falciparum antibodies, PfCSP IgG and Pfs230proC IgG compared to participants from Obom.In conclusion, the study showed that participants from Simiw had higher concentrations of circulating gSG6-P1 IgG antibodies but lower concentrations of The online version contains supplementary material available at 10.1186/s12936-023-04557-8. Plasmodium parasites undergo a complex life cycle within the mosquito vector and the human host  [2.590 (2.452–2.783) ng/mL] than their counterparts in Obom [2.119 (1.957–2.345) ng/mL], p < 0.0001 , there was a significant difference between individuals with and without PCR detectable P. falciparum in Simiw (p = 0.0170)  was observed in the seroprevalence of gSG6-P1 IgG in Simiw [81.3% (282/347)] than Obom [65.5% (190/292)]  and Simiw (p = 0.3736). However, P. falciparum PCR-positive individuals in Obom had significantly (p = 0.0015) higher median anti-CSP IgG relative to counterparts in Simiw  ng/mL) compared to participants from Simiw   overall. Gender did not have any influence on the level and the seroprevalence of anti-PfEBA-175.5R IgG in Obom (p = 0.882) and Simiw (p = 0.982)  , there was a significant difference in Obom (p = 0.0071). Individuals with PCR confirmed infections in Obom had significantly higher Pfs230proC IgG levels than individuals with PCR confirmed infections in Simiw (p = 0.003)  higher IgG responses against Pfs230 were measured in participants from Obom [5993 5112–7296) ng/mL] compared to Simiw [4822 (4306–5328) ng/mL]   in Simiw had significantly lower IgG concentration of Pfs230 , and PfEBA-175  compared to individuals from Obom with similar gSG6-P1 IgG responses. However, IgG responses against PfCSP in individuals from Obom and Simiw were similar in people high levels (2–4.999 ng/mL) of anti-gSG6-P1 IgG  compared to individuals from Simiw with similar anti-gSG6-P1 IgG levels  gSG6-P1 IgG and anti-Pfs230 IgG levels in Simiw  but not in Obom . No other associations were identified between moderate and high gSG6-P1 IgG and the measured antiparasite IgG in Simiw and Obom , however, the insufficiencies associated with the traditional estimation of EIR using infected mosquitoes have led to the identification of some serological markers of exposure to malaria parasites as alternative and more effective measures of malaria transmission intensity in both high and low transmission settings [Several factors regulate the dynamics of settings , 18.Variations in host factors and exposures to malaria parasites can impact the immune responses generated by the host . Host-spThis study assessed antibody levels against the sporozoite stage of the malaria parasite, PfCSP, the asexual blood stage parasites, PfEBA175 and the sexual blood stage parasite Pfs230 in individuals with varying levels of exposure to the mosquito vector, in two communities of high malaria transmission in southern Ghana.Anopheles mosquito bites [The formulation of malaria control and elimination programmes are based on the intensity of malaria transmission within a community. The IgG to gSG6-P1 is a proxy to exposure and in some contexts has been associated to clinical outcomes , 18. It to bites , 20. Theto bites , 31. Theto bites . The higto bites –36, whento bites , 38 at OP. falciparum endemic communities, protect against erythrocytic stage invasion and symptomatic malaria [High IgG antibodies to PfEBA 175 in people living in  malaria , 13. TheP. falciparum.Gametocyte carriage is essential for malaria transmission however, exposure to gametocytes can reduce malaria transmission as some antibodies against gametocyte antigens, including antibodies against specific regions of Pfs230 serve as transmission blocking antibodies, which can prevent the development of the parasite in the mosquito , 38. TheP. falciparum sporozoites, indicating a higher prevalence of uninfected mosquitoes than Obom. Exposure to uninfected mosquito bites has been suggested to reduce liver stage hypnozoites as well as asexual stage parasite burden relative to exposure to malaria-infected mosquitoes [The study compared similar levels of exposure to mosquitoes in Obom and Simiw; the result showed that at a moderate concentration of gSGP1 IgG (concentration of 1–1.999 ng/mL), Simiw had lower exposure to squitoes , 41. A psquitoes . This stsquitoes , 41.P. falciparum antibodies, PfCSP IgG and Pfs230proC IgG compared to participants from Obom.The study showed that participants from Simiw had higher concentrations of circulating gSG6-P1 IgG antibodies but lower concentrations of Additional file 1: Figure S1. The overall Immunoglobulin G (IgG) antibody levels among the study communities. a Distribution of Anopheles salivary gland gSG6-P1; bPlasmodium falciparum CSP; cPlasmodium falciparum EBA 175; dPlasmodium falciparum Pfs230 immunoglobulin G (IgG) antibodies among the study communities. The data are represented in log10. The significance was tested using Mann Whitney U test; ns (p > 0.05) not significant; *(P < 0.05), significant; **(P < 0.01), significant; ***(p < 0.001), highly significant; ****(p < 0.0001), highly significant.Additional file 2: Figure S2. Immunoglobulin G (IgG) levels in male and female study participants. a The Anopheles salivary gland. b The P. falciparum CSP. c The P. falciparum erythrocyte binding antigen-175 (PfEBA-175). d The gametocyte surface antigen Pfs230 immunoglobulin G (IgG) antibodies concentration among male and female study participants between Obom (OB) and Simiw (SW). The data are represented in log10.Additional file 3: Figure S3.P. falciparum stage-specific IgG antibodies with similar mosquito exposures after excluding sub-groups expressing high Pfs230 IgG antibody. Association of IgG antibodies of PfCSP, Pfs230, PfEBA-175 between Obom and Simiw with a 2–5 ng/mL; b > 5 ng/mL gSG6-P1 IgG antibody exposure after excluding sub-groups expressing high Pfs230 IgG antibody at Obom. The data are represented in log10.Additional file 4: Figure S4. Correlation of P. falciparum stage-specific IgG antibodies across individuals with different levels of mosquito exposure. Correlation between a 1–2 ng/mL; b 2–5 ng/mL; c > 5 ng/mL gSG6-P1 IgG antibodies and PfCSP, Pfs230, and PfEBA-175 in Obom and Simiw.


Plasmodium falciparum among asymptomatic pregnant women on intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-Sp) in Osogbo, southwest Nigeria. Blood sample was obtained from consenting pregnant women attending antenatal clinics. Microscopy and Polymerase chain reaction (PCR) were employed to diagnose and analyse genetic diversity. Of the 301 samples, 53 (18%) and 83 (28%) were positive for P. falciparum by microscopy and PCR, respectively. Using the merozoite surface protein (msp)-1, msp-2, and glutamate-rich protein (glurp) genes of P. falciparum as polymorphic markers, the msp-1 gene showed nine alleles with R033 (66.7%) being predominant, followed by K1 (45.5%) and MAD20 (33.3%). The msp-2 gene had 16 alleles (eight each for FC27 and 3D7). The 3D7 alleles (82.1%) was significantly more than FC27 alleles (48.2%) (p = 0.0093). Nine alleles were detected with glurp gene, presenting with the highest monoclonal and the lowest polyclonal infection. The multiplicity of infection (MOI) of 1.5, 1.8, and 1.2 were obtained for msp-1, msp-2 and glurp genes. In light of the high P. falciparum genetic diversity among pregnant women on IPT-Sp in this study, additional strategies for preventing and controlling malaria in pregnancy might be required.This study investigated the genetic diversity of  Plasmodium falciparum is the dominant Plasmodium species and the leading cause of malaria in sub-Saharan AfricaPlasmodium falciparum has a complex genetic structure that undergoes adaptation and conformational changes in the face of drug pressure, environmental changes, and host immune responses. This genetic diversity influences antimalarial drugs' transmission intensity, pathogenesis, and treatment outcomeP. falciparum strains infecting an individual, is an appropriate malaria metric determined by genetic diversity to provide useful information about malaria transmission in an area. The molecular surveillance of P. falciparum's genetic profile has become the focus of biomedical research as control measures are more selectively targeted towards the genetic makeup of the parasitemsp-1 and msp-2, and glutamate-rich protein (glurp) are widely utilized biomarkers to elucidate the genetic structure of the parasite population in different settings. It is also used as a discriminatory tool to distinguish between recrudescent and new infections in drug efficacy trialsmsp-1, msp-2 and glurp genes are implicated in erythrocyte invasion and target immune response, making them candidates for blood-stage vaccine developmentP. falciparum genetic diversity has been reported in different regions and patient categoriesP. falciparum genetic diversity among pregnant women in Nigeria. This study examined the genetic diversity, allelic frequency, and MOI of P. falciparum in pregnant women attending antenatal clinics in Osogbo, Nigeria, utilizing the msp-1, msp-2 and glurp antigenic loci.Despite the endemicity of malaria and its significant toll on pregnant women, there is inadequate data on This cross-sectional study of pregnant women attending antenatal clinics in selected primary healthcare facilities was conducted in Osogbo, Osun-State in Southwestern Nigeria, from September 2015 to October 2016. Osogbo is the state capital of Osun State, and it represents a typical urban setting in Nigeria. Malaria transmission is seasonal, peaking during the rainy season from April to October. Osogbo shares boundaries with Ede, Ilesa and Obokun and is easily accessible from most of the state because of its central nature . The citAbout 2 ml of venous blood were collected for Giemsa-stained thick and thin blood film and spotted on filter paper for PCR analysis. Two glass slides for thick and thin film microscopy were labeled for each participant and allowed to dry before staining with 5% Giemsa for 30 minutes, examined microscopically for the presence of malaria parasite. Parasites were counted against 200 white blood cells (WBCs) from the thick film. As an indicator of anemia, packed cell volume (PCV) was estimated by centrifuging the blood collected into a heparinised capillary tube. Anemia in pregnancy was defined as a haemoglobin level of less than 11 g/L (haematocrit ≤33%)P. falciparum. Briefly, the Plasmodium genus was amplified using the 18S ribosomal RNA in the first reaction, and the secondary reaction amplified the falciparum species using specific primers. A template-free control and a known positive sample were used as negative and positive controls, respectively.Genomic DNA was extracted from whole blood using the QIAamp DNA mini kit , following the manufacturer's instructions. A previously described nested PCR protocolPlasmodium falciparum msp-1, msp-2, and glurp genes were amplified using nested specific primers as previously describedmsp-1, msp-2 and glurp genes, while the nested reaction primers targeted the allelic families of msp-1 , msp-2 (3D7 and FC27) and glurp (R2) region, respectively. The PCR amplification for each allelic family for the primary and nested reactions were performed separately. Amplified PCR products were analysed by gel electrophoresis on a 1.5% agarose stained with SYBR Green and visualized by UV trans-illuminator . Amplicon sizes were detected relative to the 100 bp molecular DNA ladder (New England Biolab).msp-1 and msp-2 genotypes by the number of positive samples for each marker. Isolates with more than one allelic family were considered a multiclonal infection, while the presence of a single allelic family was considered a monoclonal infection.The MOI, defined as the average number of genotypes per infected individual, was calculated by dividing the total number of distinct msp-1, msp-2 and glurp genes were calculated as the proportion of the allele detected for each allelic family out of the total alleles detected. The calculation of the frequency of multiclonal infection considered the number of samples harbouring more than one amplified clone out of the total sample. The proportionality assessment was performed using the Chi-square test, while Student's t-test was used to compare the MOI between allelic families. The level of statistical significance was considered at p < 0.05.Data were analysed using SPSS software version 20. The allelic frequencies of T/33) was received from the Osun State Research and Ethics Committee, State Ministry of Health Osogbo, Nigeria. At the same time, written informed consent was obtained from pregnant women before enrolment into the study.The study was conducted according to the standards of the 1964 Helsinki declaration. Ethical approval , with a geometric mean parasite density of 917.53 parasites/µl of blood (range 120 - 9520). In addition, 83 (27.6%) samples were PCR positive.The study recruited 301 pregnant women who gave informed consent and were on intermittent preventive treatment with sulfadoxine-pyrimethamine for malaria prophylaxis. The mean age of the women was 27.2 ±5.04 years, while the mean PCV, indicative of anemia, was 33.1 ± 5.37%. 32.2% of the women were primigravidae and 67.8% multigravida . Of the P. falciparum positivity rate (36.6%), followed by participants older than 35 years (31.0%), with the lowest (24.4%) among the age group, 25-34 years . Of the 83 samples positive by PCR for P. falciparum, 26 (26.8%) and 57 (27.9%) were primigravidae and multigravidae, respectively (p = 0.89). Anemia was present in 17 (32.1%) of the P. falciparum-positive women, while 66 (26.6%) of those positive did not have anemia (p = 0.49). On the use of insecticide-treated mosquito nets, 44 (25.9%) of women who reportedly slept under insecticide-treated bed nets were P. falciparum positive, while the remaining 126 (74.1%) were negative. Similarly, 39 (29.8%) of those who did not sleep under bed nets were also positive for P. falciparum , followed by K1 (45.5%) and MAD20 (33.3%). Fragment sizes ranged from 200 – 600 bp for K1-type, 200 – 300 bp for MAD20-type, and 150 – 200 bp for R033-type. Combined allelic frequency was more with K1 + R033 combination (21.2%) compared to other msp-1 allelic combinations (p = 0.002). For msp-2 gene, 16 alleles were detected, with 8 alleles each for FC27 and 3D7 families. Fragment size for FC27 ranged from 300 – 900 bp, while 3D7 was 200 – 900 bp. The predominant allelic frequency occurred in 3D7 (82.1%), with FC27 recording the least frequency (48.2%). There was a significant difference in the number of amplified alleles between FC27 and 3D7 (p = 0.009). Of the thirty-five isolates positive for the glurp gene, nine distinct alleles were identified, with fragment sizes ranging from 300 – 1100 base pairs. Overall, msp-2 was the predominant gene, while glurp had the least allelic frequency distribution  with 1.2 MOI. The msp-2 gene had the highest polyclonal infection of 44.6% among the three genes and 55.4% monoclonal infection, with an MOI of 1.8. Similarly, msp-1 recorded 60.6% monoclonal infections and 39.4% polyclonal infections with MOI of 1.5. There was no significant difference in the three genes between the mono- and polyclonal infections  followed by > 35 years and 25 – 34 years, although the differences were not statistically significant. Published reports from other African countries have consistently shown that the most significant risk of malaria infection and highest parasite densities among pregnant women were recorded among younger age groupsThe highest oup 17 –  years foA comparison of malaria infection with gravidity (primigravidae versus multigravidae) did not show any significant association in this study, agreeing with a previous studyP. falciparum genetic diversity is essential for the malaria vaccine development process and provides epidemiological information on transmission intensity in a given region. The genotyping of the three distinct genes encoding msp-1, msp-2 and glurp revealed a high degree of genetic diversity, with msp-2 recording the highest MOI (1.8). The observed high genetic diversity agrees with high malaria transmission in Nigeria and most of sub-Saharan Africa. In comparison, this study's relatively high MOI is consistent with a recent report that attributed this to the widespread use of insecticide-treated bed nets The msp-1 gene in the study. This observation was similar to our recent report in Ibadan, a neighbouring city to Osogbo msp-1 allelemsp-1 allelic families to clinical forms of malariaThe R033 family was the most predominant allele of the msp-2 gene, the FC27 allelic family was the most commonly observed allele, contrary to our previous report showing 3D7 as the most common msp-2 allele among children with uncomplicated malaria in this areamsp-1, the reported trends of the differential distribution of 3D7 and FC27 alleles concerning clinical status have yielded conflicting results making it difficult to establish the actual influence of the msp-2 gene allelic families on clinical course. The 3D7 allele is assumed to protect against the clinical disease since it is frequently encountered in asymptomatic malariaFor the glurp gene recorded the highest monoclonal and the lowest polyclonal parasite infection with nine distinct alleles and the least MOI among the three genes studied. Our previous report among children with uncomplicated malaria in this same locationThe A limitation of our study is the inability to supervise SP intake by pregnant women directly. The women were given the drugs freely to take home in some cases, and they confirm the use of the drug in the administered questionnaire, but their compliance cannot be independently verified.Plasmodium falciparum strains with different clones are infecting pregnant women without clinical symptoms in Nigeria. A high prevalence of polyclonal infections was observed for the three markers, with msp-2 allelic families showing the highest polyclonality and the highest genetic diversity. The high prevalence of asymptomatic P. falciparum and the genetic diversity in this study indicate sustained transmission. This study draws attention to the need to intensify efforts in identifying newer chemoprophylactic methods for malaria in pregnant women in Nigeria in addition to the existing ones. Also, rewards may be introduced to facilitate compliance with the use of SP for the intermittent preventive treatment of malaria in pregnancy.Genetically diverse


Dear Editor,Plasmodium falciparum and Plasmodium vivax, are known to cause serious problems for pregnant women and their unborn children. P. falciparum is the predominant species in sub-Saharan Africa, where a significant number of unfavorable birth outcomes are linked to malarial occurrence [Globally more than 125 million women are at risk of contracting malaria during pregnancy due to their weakened cell mediated immunity and glucose metabolism [currence .Congenital malaria, maternal anemia, stillbirth, preterm birth, maternal and neonatal mortality, and low birth weight (defined as birth weight <2.5 kg) are some of the adverse effects of malaria that harm both the mother and the newborn . AccordiP. Falciparum malaria in pregnancy can easily be reduced by integrating malarial prevention techniques with maternal and neonatal healthcare services, as over 70 % of women in Africa attend antenatal clinics at least once during pregnancy [Malaria remains a significant public health issue in East Africa due to challenges such as access to healthcare, infrastructure limitations, social beliefs, and lack of awareness. Hospital mortality due to severe malaria is high, and there are disparities in access to antenatal clinics (ANC) between urban and rural populations. regnancy . The WHOregnancy .Plasmodium falciparum malaria during pregnancy is a prominent contributor to maternal and newborn mortality rates in African regions. Despite significant progress in recent years, there remain several problems, shortcomings, and room for improvement in this field. These issues are of utmost importance and urgency in order to achieve the global standards set by the WHO for the eradication and prevention of malaria and to ensure improved healthcare quality for pregnant women specifically. Immediate, concerned and collaborative efforts are necessary to enhance preventive measures, raise awareness, eliminate misconceptions, and foster collective action in safeguarding the well-being of women and children, probably the most vulnerable sections of African society [The pillars of African society must work together to combat malaria. In situations when there are technical or budgetary constraints, collaborative efforts of government and NGOs are required for malaria prevention. Understanding a community's perception of febrile disease, its role in people's belief systems about sickness, and current behaviors that can help or impede preventive actions, is substantial . The suc society .Not applicable.Not applicable.Not applicable.The authors did not receive any financial support for this work. No funding has been received for the conduct of this study.: Kanza FARHAN and Burhan TARIQ, Design: Aymar AKILIMALI, Project administrator; Burhan TARIQ and Aliza AHMED, Supervisor: Kanza FARHAN, Funding acquisition; Kanza FARHAN, Investigation; Aymar AKILIMALI, Resources; Kanza FARHAN, Burhan TARIQ and Aliza AHMED, Validation; Aymar AKILIMALI, Visualization; Kanza FARHAN, Literature search: Aymar AKILIMALI and Kanza FARHAN, Manuscript preparation: Navira SAEED, Sufyan Raza KHAN, Manuscript editing; Aliza AHMED, Burhan TARIQ and Aymar AKILIMALI, Manuscript review: All authors, Final approval of manuscript: All Authors.ConceptionNot commissioned, externally peer reviewed.The authors declare that there no conflict of interest.


Plasmodium falciparum (Pf) is the dominant malaria parasite in Nigeria though P. vivax (Pv), P. ovale (Po), and P. malariae (Pm) are also endemic. Blood samples  were collected from children aged 0-14 years during a 2018 nationwide HIV survey and assayed for Plasmodium antigenemia, Plasmodium DNA, and IgG against Plasmodium MSP1-19 antigens. Of all children, 6.6% were estimated to have Pm infection and 1.4% Po infection with no Pv infections detected. The highest household wealth quintile was strongly protective against infection with Pm  or Po . Overall Pm seroprevalence was 34.2% (95% CI: 33.3-35.2) with lower estimates for Po  and Pv . Pm seropositivity was detected throughout the country with several local government areas showing >50% seroprevalence. Serological and DNA indicators show widespread exposure of Nigerian children to Pm with lower rates to Po and Pv. Nigeria has a high burden of malaria which mostly results from Plasmodium falciparum infection, but other species are also endemic. Here, the authors estimate the prevalence of active infection and previous exposure to P malariae, P ovale, and P vivax in children in Nigeria in 2018 and determine risk factors. The 2020 World Health Organization (WHO) World Malaria Report estimated 61 million Plasmodium falciparum cases in Nigeria with no estimates provided for the other human malarias: P. vivax, P. ovale, or P. malariae1. Prevention and treatment efforts in Nigeria between 2000 and 2019 led to a considerable decrease in malaria-associated mortality by ~55%2. Malaria diagnosis in Nigeria is primarily through histidine-rich protein 2 (HRP2) rapid diagnostic tests (RDTs) which are specific for P. falciparum, accounted for 86.3% of all diagnostic tests provided in 2020 to patients with suspected malaria infection1. Furthermore, the most recent Nigerian Malaria Indicator Survey (MIS) in 2021 and Demographic Health Surveys (DHSs) in 2018 and 2021 utilized RDTs only detecting HRP2 antigen, though microscopy was also included3.As of the year 2020, Nigeria has the highest recorded malaria burden in the world, accounting for ~27% of all cases and 27% of all deathsP. falciparum prevalence is highest in northern and western Nigeria with holoendemic transmission occurring all year round4. Positive associations with increasing rainfall, proximity to bodies of water, and rural residence have been identified—all of which are indicative of more optimal Anopheline vector habitat6. Mass distribution of insecticide-treated bed nets (ITNs) are carried out approximately every 3 years in Nigeria, and ITNs are a primary tool for reduction of vector exposure and malaria burden in the population, though preventing mosquito bites would reduce risk for infection with any Plasmodium parasite. The current treatment of uncomplicated malaria in Nigeria is artemisinin combination therapy (ACT) with parenteral artesunate for severe malaria cases8, and these antimalarial treatments have broad spectrum activity against non-falciparum Plasmodium species as well10. However, hypnozoites formed by P. ovale and P. vivax parasites are not cleared by an ACT regimen and require an 8-aminoquinoline, such as primaquine or tafenoquine for successful anti-relapse therapy11.P. falciparum parasites mixed with another Plasmodium species13, which is parallel to findings from other African countries14. P. vivax infection has recently been reported in Nigeria16, adding to the growing evidence for ability of this parasite to sustain transmission cycles in human populations which are primarily Duffy antigen negative20. Infections with P. ovale have been reported multiple times from studies in Nigeria, but of the two subspecies of P. ovale , only P. ovale curtisi has been identified22. When compared to other human Plasmodium spp., P. malariae typically presents as a more benign infection and infection has rarely been identified in Nigeria22. This current study utilizes blood samples collected during the 2018 nationwide Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS) household survey to assess current infections at time of sampling and past exposure by serological data for P. vivax, P. ovale, and P. malariae were estimated in children under age 15 years.Non-falciparum malaria infections in Nigeria have typically been found to have As part of the Nigeria Multi-disease Serologic Surveillance using Stored Specimens (NMS4) project, of 45,462 eligible children (<15 years of age) eligible for enrollment in NAIIS, dried blood spot (DBS) analysis for multiplex antigen data was able to be collected on 31,234 (68.7%) samples showed an antigen profile positive for a non-HRP2 target, but with low/undetectable levels of HRP2 -PCR assays to 1213. Of these, 1204 (99.3%) were able to be retrieved for DNA extraction and subsequent PET-PCR speciation  were selected for PET-PCR assays for these estimates. As a validation exercise for the selection strategy for this specific Nigerian sample set, 200 DBS were selected for PCR assays that were found to be negative to all antigen targets. Of the 200 antigen negative specimens, zero were positive for P. ovale or P. vivax DNA, three (1.5%) were positive for P. malariae DNA, and nine (4.5%) were positive for P. falciparum DNA.Based on assay plate pass/fail criteria outlined in Methods and reported previouslyPlasmodium genus primers  being positive only for P. falciparum DNA, 13 (2.9%) positive for only P. malariae DNA, and four (0.9%) samples only positive for P. ovale DNA. Mixed infections were also common among this first selection subset with 68 (15.3% of 444 active infections) positive for both P. falciparum and P. malariae DNA, 15 (3.4%) P. falciparum/ P. ovale DNA positive, two (0.5%) P. malariae/P. ovale DNA positive, and three (0.7%) DNA positive to all three of these parasites: P. falciparum, P. malariae, P. ovale. From the second selection strategy , 596 DBS were retrieved (99.3% of the 600 selected). Of the 588 Plasmodium genus positive samples, all were P. falciparum DNA positive with 81 (13.8%) also mixed with only P. malariae, 15 (2.6%) mixed with only P. ovale, and three (0.5%) mixed infections with P. falciparum, P. malariae, and P. ovale DNA. None of the 1,032 Plasmodium DNA positive samples from the combination of the first and second selection strategies were positive for P. vivax DNA.From the first selection strategy (for low/no HRP2), 608 DBS were retrieved (99.2% of the 613 selected), and 164 (27.0%) of these were negative by Plasmodium PET-PCR positives) contained any P. malariae DNA, with 157 (92.4%) of these P. malariae infections mixed with either P. falciparum (87.6%), P. ovale (1.2%), or both (3.5%). In addition, 42 samples  contained any P. ovale DNA, with 38 of these (90.5%) mixed with either P. falciparum (71.4%), P. malariae (4.8%) or both (14.3%), and 2.91-fold increased odds of P. ovale infection (95% CI: 1.43–6.86). The oldest children aged 10–14 years did not show a significant difference in odds of P. malariae or P. ovale infection compared to the youngest children. Increasing wealth quintile showed successive decreases in odds of infection at time of sampling. Even comparing children from the middle wealth quintile to the lowest quintile showed a >50% reduction in odds of infection with either P. malariae  or P. ovale . Compared to the lowest wealth quintile, the highest wealth quintile had an 89% reduction in odds of active P. malariae infection  and 99% reduction in odds of P. ovale infection . Urban residence was found to be protective against P. malariae infection  but not P. ovale infection . Mosquito net coverage at the individual and community levels was not found to have an association with P. malariae or P. ovale infection at time of sampling. Among children with any Plasmodium infection, some significant associations were observed comparing infections with P. falciparum alone versus a mixed (or mono) infection containing P. malariae or P. ovale (or both) and the highest quintile .In total, 170 samples  obtained through the two selection strategies, 6.6% of all children enrolled were estimated to have P. malariae mono or mixed-infection, and 1.4% P. ovale mono or mixed infection. As no active P. vivax infections were identified by PET-PCR, no state or national estimates were available for that species. State-level estimates for all 36 Nigerian states plus federal capital territory (FCT) and are displayed in Fig. P. malariae prevalence in all children age <15 years ranged from 0.0 to 22.0%, and P. ovale prevalence ranged from 0.0 to 11.9%. For both Plasmodium species, infection prevalence was spread throughout the country in a heterogeneous manner.By extrapolating the proportion of PET-PCR confirmed Plasmodium spp. Data from 225 DBS  with MFI-bg reads >500 for the glutathione-S-transferase (GST) internal control were excluded from IgG analysis for quality assurance as explained in Methods. No correlation in IgG binding was observed in comparing the assay signals among all four merozoite surface protein 1 (MSP1) orthologous antigens  of all children with anti-PmMSP1 IgG, with state seroprevalence ranging from 5.2 to 57.6%  for children overall with state seroprevalence estimates ranging from 2.9 to 22.3%. P. vivax exposure by anti-PvMSP1 IgG seropositivity was lowest at 6.3% (95% CI: 6.0–6.7) for all children, with state ranges from 1.7 to 17.6%. Seroprevalence by LGA for these three antigens is displayed in Fig. A total of 31,234 DBS from children age <15 years were assessed for IgG to the MSP1-19kD (MSP1) antigens from all four human Indicating a greater amount of cumulative malaria exposure as Nigerian children age, increasing IgG levels were observed for all three non-falciparum MSP1 antigens with increasing age, though most accentuated for PmMSP1 Fig. . To modeReflecting results from the seroconversion analyses, unadjusted and multivariate logistic regression to investigate individual or demographic associations with non-falciparum MSP1 seropositivity found increasing age, rural residence, and decreasing SES to all be strong indicators of increased non-falciparum malaria exposure Table . Versus P. falciparum alone , but no observable trend for P. ovale (R2 = 0.00). The trend between P. malariae infection and exposure was more clearly elucidated as more infections and seropositive children were observed, and seroconversion estimates were able to be modelled for 86.5% (32/37) of states. However, for the lower prevalence of infections and observed seropositivity, the serocatalytic model for PoMSP1 converged for only 29.7% (11/37) of states, and this state-level correlation between infections and exposure by seropositivity was not observed.Non-significant increases were seen in median PmMSP1, PoMSP1, and PvMSP1 IgG levels for children actively infected with P. malariae at the time of sampling and 1.4% infected with P. ovale. However, mono-infections with either of these two species were rare, and the majority of detected P. malariae infections (91.2%) and P. ovale infections (85.7%) were mixed with P. falciparum parasites. A report outlining comprehensive analyses for P. falciparum infection and exposure among persons of all ages from NAIIS 2018 is forthcoming. Though P. falciparum is well-documented to be the dominant human malaria species in Nigeria3, reports of human exposure to the other three human malarias have also been produced over the past decades. Advances in PCR technologies have made sensitive detection of nucleic acids of these parasites more pragmatic, and IgG-detecting serological techniques can serve as a sensitive proxy for previous infection even after parasites have cleared from the host24. Though the clinical relevance of P. falciparum is predominant in Nigeria, it is evident from this current study that significant non-falciparum malaria parasite burden is also occurring—at least in the youngest segment of this population. Even as able to cause severe disease, P. malariae and P. ovale parasites typically present as a more benign malaria infection25, and since most of these infections were mixed with P. falciparum, it is likely antimalarial treatment for P. falciparum malaria will also clear these other parasites. In human populations largely negative for the Duffy red blood cell antigen, such as in Nigeria26, P. vivax infections are now well-documented to occur in some populations, though at very low prevalence rates20. Overall findings from this current study emphasize the heterogeneous nature of non-falciparum species distribution in Nigeria. While P. falciparum transmission appears to follow a strong north-south gradient with higher transmission in the northern states3, this finding was not observed for children’s exposure to the non-falciparum species, and no discernable geospatial pattern was noted in this current Nigeria study.This current study from the NAIIS 2018 investigated exposure of Nigerian children aged <15 years to non-falciparum malaria by detecting infections through antigen and DNA presence and estimating previous exposure by anti-malarial IgG presence. The findings presented here estimated that 6.6% of Nigerian children enrolled in NAIIS 2018 were infected with P. malariae infection prevalence higher than P. ovale and most infections with either of these two species also containing P. falciparum. A survey of two towns in southwest Nigeria enrolling non-treatment-seeking persons and detecting infections by PCRs found 11.7% of participants with P. malariae infections and 6.8% with P. ovale infections, but >97% of these infections also contained P. falciparum27. Another recent study of asymptomatic persons age <20 years in southwestern Nigeria found 34.1% of persons with P. malariae infection, and 15.7% with P. ovale, with 81.5% of these infections including P. falciparum parasites6. Health facility surveys of treatment-seeking persons in Nigeria have observed similar findings with P. malariae as the most frequently identified non-falciparum parasite followed by P. ovale28. However, since nearly all of these infections include P. falciparum, it is unclear how much these non-falciparum species contribute to clinical disease in Nigeria. Though active P. vivax infections were not observed in this current study, recent reports have identified endemic16, and imported29, P. vivax infections from Nigeria and provided evidence for this parasite being present in the country.Previous MISs (utilizing participant enrollment at households), as well as studies in limited areas of the country, had observed similar trends with P. malariae and P. ovale prevalence among children age 5–9 years compared to other children as observed in this current study has also been seen in other countries in sub-Saharan Africa31, and reflects different P. falciparum infection rates seen among children in some studies34. Even when considering P. falciparum mono-infections as the referent, P. ovale infections were still significantly higher in this age category. It is unclear why children in this age group would be more susceptible to malaria infection, but it may reflect behavioral changes (including outdoor activities and net usage34) and a lack of acquired immunity which would occur through multiple prior exposures. Odds of P. malariae or P. ovale infection at the time of study enrollment were lower for children living in urban areas, though this was only statistically significant for P. malariae. This finding has been consistently observed for studies investigating P. falciparum prevalence and is likely a factor of improved housing structures and less-optimal Anopheline habitat35. The most striking association with reduced odds of P. malariae and P. ovale infections was observed with increasing SES as measured by wealth quintiles. When modeling controlled for other covariates, a consistent negative trend for infection prevalence was seen with increasing wealth quintile with the highest quintile showing an 89% reduction in odds of P. malariae infection and 99% reduction in odds of P. ovale infection compared with children included in the lowest quintile. Even children in the second lowest wealth quintile showed lower odds of P. malariae (reduced by 15%) and P. ovale (reduced by 55%) when compared to children in the lowest SES category. These same prominent associations with malaria infection and wealth quintile had also been observed by the 2010 and 2015 Nigerian MISs, though asymptomatic parasite prevalence was only reported for P. falciparum3. In addition, infections with these two non-falciparum species had also been seen to be higher among pregnant woman of lower SES in western Africa36. These consistent protective associations with higher SES are multifactorial with access to better nutrition, improved housing, and a variety of other aspects afforded by those with higher SES. Known protective factors against vector exposure would inherently lead to protection against establishment of Plasmodium infection. Indeed, in our bivariate analyses, correlates of wealth  were all significantly protective against exposure to all three non-falciparum malaria parasites.The trend of higher Plasmodium exposure (at time of sampling or previously in life) to non-falciparum species through IgG serology found several LGAs throughout the country with >50% P. malariae seropositivity rate, with much lower estimates for IgG against P. ovale or P. vivax. In utilizing the Plasmodium spp. MSP1-19kD IgG targets in this study, previous data had shown the limited degree of IgG cross-binding among these antigens37, and we did not observe any significant correlation in the magnitude of IgG binding among these antigens in this Nigerian population  had a complete absence of PvMSP1 seropositive children. Seroprevalence estimates by individual LGAs where the RSEs are >30% should be interpreted with caution. Specifically, the overall lower IgG seroprevalence to the P. ovale and P. vivax MSP1 targets led to higher RSEs by LGA with most exceeding an RSE of 30%. Overall, this study estimates that there is moderate transmission of P. malariae occurring throughout Nigeria, and by age 15 years, children have approximately a 40% likelihood of have been infected at some point in their lives. The SCR estimate for PmMSP1 was also significantly higher than PoMSP1 and PvMSP1, and estimated by year of life, a child in Nigeria has a 9.6% likelihood of seroconverting to PmMSP1, but much lower likelihood for PoMSP1 (3.4%) or PvMSP1 (1.5%). A previous study in two states in southern Nigeria enrolling persons of all ages found high P. malariae seroprevalence of >70% for each of the five P. malariae antigens tested12, and in neighboring Benin, high seroprevalence to PmMSP1 (68%) and PoMSP1 (57%) was detected among microscopy-negative adults38. As this current study only sampled children <15 y old, the seropositivity estimates and SCRs for P. malariae and P. ovale are lower; though the same trend of higher P. malariae seroprevalence compared to P. ovale and P. vivax was observed.P. vivax. Lifetime exposure to all three non-falciparum malarias trended significantly lower with increasing SES, and this significant reduction in odds was seen even in comparing the second wealth quintile to the lowest wealth quintile for both P. malariae and P. vivax exposure. Urban residence was found to be a strong protective factor against exposure to all three non-falciparum species, and this protection appears to be initiated very early in life as PmMSP1, PoMSP1, and PvMSP1 seroprevalence curves are completely separated by urban/rural residence by 1 year of age. Similarly, separation of seroprevalence curves by SES quintiles was observed by approximately 4 years of age. Individual mosquito net coverage was not found to have a protective effect against P. malariae, P. ovale or P. vivax infection or exposure. The community net coverage indicator showed a positive association with having antibodies against P. malariae MSP1 antigens, possibly showing the connection between higher malaria transmission and greater awareness of needing to sleep under a net. However, the net coverage associations should be interpreted cautiously as this variable only indicates whether there was at least 1 net per 1.8 persons living in the household and does not indicate the actual frequency of net usage for a child.Increased household wealth and residing in an urban area were found to have a protective effect against lifetime exposure to all three non-falciparum species, and this finding was consistent with the PET-PCR active infection estimates from this study. Due to greater numbers of seropositive children (versus children with active infections), greater statistical power was allowed by the IgG data to assess non-falciparum exposure and include estimates for n = 1204) was too small to permit generation of representative infection prevalence estimates by LGA.This study has several important limitations. The cross-sectional design and timeframe of the study (during the rainy season) prevents assessing the effects of temporality, such as seasonality on active infection. The household NAIIS sampling design captured people more likely to be asymptomatic or mildly symptomatic, so the epidemiology of malaria species among persons with symptomatic malaria was unable to be assessed. In addition, very low density infections for these non-falciparum infections would likely be missed by antigen detection and/or PCR assays, so the absolute estimates for prevalence of infections with these species are certainly an underestimate of the true prevalence. Though sampling design was based on Nigerian census data, the NAIIS survey was initially designed to generate state and national estimates of HIV prevalence. Also, the number of samples tested by PCR  project was approved by the National Health Research Ethics Committee of Nigeria (NHREC/01/01/2007) and determined to not involve human research by the Centers for Disease Control and Prevention Human Subjects office (project 0900f3eb819d4c63).40. The sampling design utilized a stratified two-stage cluster sample with 37 strata . Enumeration areas (EAs) were selected within each state in the first stage with probabilities proportionate to estimated population size according to the projected 2018 number of households (based on the 2006 census data). When calculating the number of EAs to be selected per state, state variability of household size was considered. This sampling frame produced 662,855 EAs, of which 4035 were selected for NAIIS. For the second stage, a household listing exercise was carried out in all selected EAs, and a random sample of 28 households per EA were chosen for inclusion. The resulting sample included 101,580 households to be approached for enrollment of household participants into NAIIS.NAIIS 2018 was a national HIV population-based household survey led by the Government of Nigeria under the Federal Ministry of Health, and survey methods have been previously reportedThis report from the NMS4 project is inclusive of data from all children (persons <15 years of age) who provided a DBS for the NAIIS survey. Priority was given this this subset of samples as children represent the segment of the population most susceptible to adverse outcomes from malaria infection. Collection of data for persons >15 years of age is still ongoing, and an upcoming report will outline those findings. All multiplex antigen and IgG detection assays were performed at the National Reference Laboratory (NRL) of the Nigeria Centre for Disease Control (NCDC) in Gaduwa, Nigeria; DNA assays were performed at at the US Centers for Disease Control and Prevention in Atlanta, GA. Multiplex antigen and IgG detection were conducted for all available DBS specimens for children <15 years of age, and DNA assays performed for only samples selected as described below.Escherichia coli extract)41. Elutions were stored at 4 °C until testing. As described previously23, samples were assayed in singlet at the 1:40 whole blood dilution for histidine-rich protein 2 (HRP2), pan-Plasmodium aldolase (pAldolase), pan-Plasmodium lactate dehydrogenase (pLDH), and P. vivax LDH (PvLDH). Assay plates were read on MAGPIX™ instruments , and with a target of at least 50 beads/region, the median fluorescence intensity (MFI) was generated for each analyte by Luminex xPonent® software version 4.2 (Luminex Corp). For each assay plate, the MFI signal for sample dilution buffer (buffer background) was subtracted for each target’s MFI signal to provide a MFI-bg assay signal for analysis. Plate-to-plate variation was accounted for by assessment of controls on each plate, inclusive of antigen negative blood (at 1:40 dilution) and a 4-point titration curve of a recombinant antigen positive pool. As described previously23, assay plates ‘failed’ if the negative controls showed a positive signal, or if the positive controls for both pLDH and HRP2 were both 2 standard deviations outside of the cumulative moving average of the MFI-bg signal for assay plates. Previous analysis found 1.2% of initial antigen detection plates failed23, and failed plates were re-run to obtain valid data. To determine the assay signal threshold for antigen positivity, a finite mixture model approach was employed to estimate the ‘antigen negative’ population within these data and then establishing a cutoff using the mean + 2 standard deviations from this distribution42.To rehydrate blood samples from DBS, a 6 mm punch (~10 µL whole blood) was taken from each DBS sample and blood eluted to a 1:40 concentration in blocking buffer  pH 7.2, 0.5% Bovine Serum Albumin (BSA), 0.05% Tween 20, 0.02% sodium azide, 0.5% polyvinyl alcohol, 0.8% polyvinylpyrrolidone and 3 µg/mL 41, and included targets for a variety of infectious diseases, as well as vaccine-preventable disease targets. Assay plates were run on same machines utilizing the same software as the antigen detection assay. From the DBS elution described above, an approximate final serum dilution of 1:400 was used for the assay with samples run in singlet. Analysis in this current study was restricted to the Schistosoma japonicum glutathione-S-transferase (GST) internal control antigen and the four Plasmodium merozoite surface protein 1, 19kD (MSP1) antigens from the human malarias: P. falciparum, P. malariae, P. ovale, and P. vivax37. Plate-to-plate variation was accounted for by assessment of controls included on each assay plate, and a similar ‘pass/fail’ structure as listed above for the multiplex antigen assay. Assay controls were inclusive of a 1:400 dilution of a sera pool from U.S. residents , and a positive control pool of persons residing in various P. falciparum transmission settings. In a similar manner as described previously43, Levey-Jennings charts were generated for the positive control to track deviations in assay signals over time, and an assay plate ‘failed’ if negative controls showed a positive signal, or positive controls were 2 standard deviations outside of the cumulative moving average. Of 523 IgG assay plates, 31 (0.6%) failed and needed to be repeated to obtain valid data.The IgG-detecting multiplex bead assay (MBA) was performed as described previouslyPlasmodium antigen data to select samples for further DNA assays, and to provide a representative estimate of the burden of non-falciparum malaria in Nigeria. Results from both sampling designs for DNA testing were considered when presenting national and state-level active infection estimates. Estimates are presented either as non-falciparum single-species infection, or a P. falciparum infection mixed with another species, or as a combination of these two .A two-part selection strategy was utilized based on the 46, and illustrated in Supplementary Fig. Plasmodium targets. If any specimen was positive to a non-HRP2 target, but found to have a low or negative HRP2 signal relative to other Plasmodium antigens by visual identification, this sample was selected for suspicion of non-P. falciparum malaria infection 46. This selection strategy would favor the identification of single species non-P. falciparum infections, or infections where P. falciparum was not the dominant Plasmodium species present.As described in previous studiesP. falciparum infection. For each of the six zones in Nigeria  a range of HRP2 levels (100–1000 ng/mL) was chosen from DBS from children in each zone, with 100 DBS randomly selected to geographically represent P. falciparum infections for each zone (target sample size of 600), and ultimately the country as a whole. This selection strategy allowed assessment by DNA assays of P. falciparum infections that may also harbor another Plasmodium species (mixed infections). As only P. falciparum infections were selected from the second selection strategy, the percentages of mixed infections were calculated for each Nigerian state and extrapolated to the total number of samples with antigen data from that Nigerian state.The second selection criteria involved specimens positive to HRP2, which would indicate active Plasmodium genus and for P. falciparum as described previously47. PET-PCR primer targets for the P. ovale reticulocyte binding protein 2 (rbp2) gene have been published previously48, as well as the PET-PCR dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene for P. malariae and P. vivax49. Primer sequences for all targets listed above are shown in Supplementary Data P. falciparum HEX-labelled primer which was used at a 62.5 nM. Each sample was run with 2 μL of DNA in the PCR reaction with the following cycling parameters: initial hot-start at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 40 s. Fluorescence channels were selected to detect each fluorescently labelled primer-set and cycle threshold (CT) values recorded at the end of each annealing step. All assays were performed using Agilent Mx3005pro thermocyclers  with Agilent Aria 2.0 analytical software. The PET-PCR criteria for DNA positivity for any primer set was a CT value < 40.0.One 6 mm DBS punch was placed into a 1.5 mL tube for processing according to manufacturer’s instructions. The QIAamp DNA blood mini kit  was used to elute whole DNA in 150 µL of elution buffer, aliquoted, and stored at −20 °C until use. The PET-PCR reaction was performed with primer targets for the 18 S ribosomal RNA gene for S-transferase (GST) MFI-bg reads above 500 (indicating non-specific binding) were excluded from the analysis. To dichotomize seropositivity to each species MSP1 antigen, a 2-component finite mixture model (FMM) of the log10-transformed MFI-bg signal was fit for each IgG response37, and the positivity cutoff was determined by the lognormal mean of the first component (presumed seronegative) plus 2 standard deviations42. A 6-component model was created for PfMSP1 to more accurately define the seronegative population. FMM plots were generated in SAS version 9.4.Survey questionnaire data cleaning was conducted using Census and Survey Processing System (CSPro) version 7.7.2  and SAS version 9.4 . For the IgG analysis, DBS with glutathione-survey package50, and data were weighted using normalized survey weights. Demographic, socioeconomic, and behavioral risk factors were considered in relation to malaria infection and exposure, including sex, age group, wealth quintile, and living in a household with at least 1 mosquito net per 1.8 household members. Since there was no data collected on mosquito net usage per child, a proxy net coverage variable was created using the ratio of nets to household members, based on the WHO criteria recommended for procurement purposes to ensure universal net coverage in households51. Cluster level covariates included: place of residence  and community net coverage. Community net coverage was defined as the proportion of individuals in the cluster with adequate household net coverage (1 net per 1.8 household members). Individual net coverage was normalized by subtracting the average community net coverage from the individual-level variable52. Three multivariate, mixed-effects logistic regression models were fit to determine factors associated with malaria exposure  and included a random intercept for the cluster variable. For the malaria infection risk factor analysis, multivariate logistic regression models were fit without random effects since there were only 1204 observations with over 800 clusters represented (average of 1.4 persons per cluster). Firth’s penalized-likelihood logistic regression was performed for the P. malariae and P. ovale infection models to reduce the bias due to small sample size and quasi-complete and complete separation of the wealth covariate for the P. malariae and P. ovale models, respectively53.Remaining analyses were conducted in R version 4.1.1  or Microsoft® Excel® version 2208. Estimation of malaria exposure was adjusted while taking into account the complex sample design of the NAIIS using the R 55. The model estimated the seroconversion rate (SCR or λ) which is the mean annual rate of conversion from any individual in the population from seronegative to seropositive, and the seroreversion rate (SRR or ρ) which is the mean annual rate of reversion from seropositive to seronegative. Children under age 1 year were excluded to remove the potential effect of maternally-derived anti-malarial antibodies. The parameters were estimated using Markov Chain Monte Carlo (MCMC) with 10,000 MCMC iterations. R code to fit these models was utilized from the code provided by Dr. Michael White, Serology Github Repository56. The geospatial analysis was conducted in R version 4.1.1 and QGIS version 3.16.3, and spatially smoothed sero-prevalence maps were created using local Empirical Bayes spatial smoothing. All code is available at: https://github.com/cleonard297/nonPf_seroPCR_code.To model the rate of change from seronegative to seropositive for each of the three antigens , serocatalytic conversion models were fit to the seropositivity by age data for each antigenFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Platelet parameter alteration such as platelet count and platelet indices are more common than in other blood cell lines due to diverse causative pathophysiological mechanisms in severe malaria infection. In malaria patients, no more studies evaluated platelet indices in relation to disease severity and prognosis. Therefore, this review assessed the current scientific knowledge on the potential role of platelet indices for the diagnostic marker of severe malaria infection. 9/L, MPV ≥ 9.05 fL, and PDW ≥ 14.550% as well as significantly higher P-LCR and decrease in PCT are shown significant sensitivity and specificity as they are used as diagnostic and prognostic values in severe malaria infection.  Hence, after reviewing recent literatures, elevation of mean platelet volume and platelet distribution width in addition to decreased plateletcrit and platelet counts is the known potential risk factor associated with warning signs of severe malaria. Thus, thrombocytopenia < 150 × 10 Platelet indices are useful predictors of malaria severity. Immature platelet fraction (IPF%) is raised in the case of severe malaria, and it was significantly more useful than MPV. Advanced research will further investigate the platelet index abnormality associated with specific age and gender among specific malaria species. Plasmodium vivax (PV) is more predominant in Asia and Plasmodium falciparum (PF) in Africa [Plasmodium knowlesi and PV may also cause severe illness and result in death. An extremely high number of severe malaria illnesses and deaths occur especially in children under 5 years, elderly, and pregnant women before visiting a health service [Malaria is a blood parasitic disease caused by protozoans of five plasmodium species and transmitted by infected female anopheles mosquito. The different plasmodia are not uniformly distributed throughout the world. n Africa . Severe  service –4. Sever service .μL), is a common hematological alteration during malaria infections. Thrombocytopenia seems to occur primarily by peripheral destruction, bone marrow alterations, excessive removal of platelets by the spleen, platelet consumption by the process of disseminated intravascular coagulopathy [Hematological abnormalities are the most common complications during severe malaria infections and play a major role in the fatality. Level of malaria endemicity, background hemoglobinopathy, demographic factors, nutritional status, and malaria immunity are factors for hematological alterations associated with malaria infection , 8. Throulopathy , and pseulopathy , 11. In ulopathy –14. Thesulopathy . Not onlulopathy , 16. Thrulopathy , 18.falciparum parasites [The gold standard method for malaria diagnosis is microscopic examination by identifying and confirming parasite species. But there are major causes of blood film negativity in severe malaria, such as recent treatment initiation, inadequate examination of blood films, expertise of microscopist, and identification of non-arasites , 19. Duearasites , 21. In The primary function of platelet is regulating hemostasis, but it also plays a secondary role in the innate immune response to infection and the pathogenesis of malaria. This is accomplished by direct killing of the plasmodium parasite and enhancing the sequestration of infected red blood cells (iRBCs) in the vasculature . PlateleAs malaria is a hematological disease, all its clinical complications are probably due to its erythrocyte life stage, and consequently, it may interact with the platelets. It was known that platelets are involved in all stages but exceptionally at the hepatic stage of malaria infection. Preferentially, platelets were bound to be infected more than uninfected erythrocytes in the bloodstream and then killing of intraerythrocytic parasites of each plasmodium species specifically mature stages of PV. The spread of parasite pigments is the typical characteristic of dying of platelet-bounded iRBCs –25. The 3/μL) was the most common manifestation of “severe” PV than PF infection and uncomplicated malaria [falciparum malaria, whereas the mild and moderate degree of thrombocytopenia was in PV malaria [3/μL) was more characterized by falciparum (40%) as compared to vivax case (11.1%). This reflection indicates that there was a significant association between species of malaria and degree of thrombocytopenia [Thrombocytopenia, a quantitative alteration in platelets, causes great morbidity. The possible mechanism could explain either peripheral destruction and/or hypoproliferative thrombocytopenia. However, malaria thrombocytopenia is usually due to peripheral destruction of platelets , 30. Mos malaria , 32–34.  malaria . A studyytopenia . Thus, tytopenia . A betteytopenia , 38.falciparum malaria-infected patients whereas it was seen in about 85% of the patients with uncomplicated malaria. Many studies have been done to support this finding that 80% of patients of either PV or PF malaria develop thrombocytopenia during their infection. In such study, thrombocytopenia has a sensitivity of 83% and a specificity of 68% on malaria-infected individuals [Severe malaria infection may increase thrombocytopenia by 12–15 times compared to uncomplicated malaria. Thrombocytopenia was detected in all severe ividuals , 40. A rividuals .Platelet indices are biomarkers of platelet activation and could be useful for the diagnosis of malaria. They allow extensive clinical investigations focusing on the diagnostic and prognostic values in a variety of settings without bringing extra costs. Platelet indices including PCT, MPV, P-LCR, and PDW are a group of platelet parameters determined by automatic complete blood count profiles, and they are related to platelet morphology and proliferation kinetics . AlteratμL; MPV: 8-12.4 fL; PDW: 9–14 fL; PCT: 0.22-0.24%; P-LCR: 15-35%, respectively [The normal ranges of MPV, PDW, PCT, and P-LCR for analyzer were as follows: platelet count: 150.00‐450.00 × 103/ectively –44.Mean platelet volume is the measure of platelet volume and the most extensively studied markers of platelet activation. It is an indirect measurement of platelet function and activation. Larger platelet size correlates with enzymatically active platelets. Reduced fragmentation of megakaryocytes in the bone marrow and splenic release of larger platelets (increased demand) may increase MPV. However, a decrease in platelet release from the marrow reduces MPV. Platelets change their shape from discoid to spherical and form pseudopodia. This pronounced pseudopod formation leads to increase MPV during platelet activation . PlateleDetermination of MPV was found to be extremely useful in ascertaining the etiology of hyperdestructive thrombocytopenia especially malaria. According to this study, a cut-off MPV value of 8.5 fL showed the maximum sensitivity (92.4%) and specificity (100%). The positive predictive value, negative predictive value, and diagnostic accuracy were 100%, 77.78%, and 94%, respectively. Therefore, findings from this study showed that the MPV value of ≥8.5 fL can be used for diagnosing thrombocytopenia cases due to hyperdestructive etiology . This isPlateletcrit, which is similar to hematocrit for erythrocytes, is the volume occupied by platelets in the blood as a percentage and calculated according to the formula . The normal range for PCT is 0.22–0.24% , 46. It PF and PV malaria infection. The higher PDW value in malaria could be due to by bone marrow formation of megakaryocytes to compensate for the low absolute platelet count during acute malaria infection. In malaria patients, the production of a significantly higher level of the key platelet growth factor such as thrombopoietin has been reported. Furthermore, excess secretion of dense granules and platelet sensitivity to ADP is increased by the parasitized RBCs [falciparum (82.6%) than vivax (69.5%) malaria [Platelet distribution width is an indicator of variability of volume in the platelet size and is increased in the presence of platelet anisocytosis. It is used to directly measure variability in platelet size, changes with platelet activation, and reflects the heterogeneity of platelet morphology. Under a normal physiological condition, there is a direct relationship between PDW and MPV. Meanwhile, the relationship between platelet volume and platelet count is not concluded, which suggests that they are affected by different mechanisms . High PDzed RBCs , 56. Larzed RBCs . Then, rzed RBCs . This is malaria .Platelet large cell ratio, the proportion of platelets that have MPV above the upper limit of normal (12 fL), is indicative of the presence of megathrombocytes in circulation . It is tImmature platelet fraction indicates the percentage of immature platelets, as a percentage of the total platelet population measured in the reticulocyte platelet channel of the hematology analyzer by a flow cytometer. The IPF percentage increases as the production of platelets increases, and low values indicate suppressed thrombopoiesis . ReticulAlthough platelet indices are easy to perform, inexpensive, and involved in routine hematological examinations, they are considered to be relatively less important by clinicians for predicting malaria severity. The present study is menacing that determination of platelet count and platelet indices had significant sensitivity and specificity to identifying malaria severity. Elevation of MPV and PDW with decreased PCT and platelet counts is a useful predictor of malaria severity. The IPF% is raised in conditions with increased peripheral destruction such as in the case of severe malaria, and it was significantly more useful than the MPV. Further studies are needed to explore and validate the utility of platelet indices as a marker of malaria severity prior to the use of these indices.


Plasmodium falciparum and non-falciparum malaria infections. A laboratory evaluation was performed to assess the performance of the mini-dbPCR-NALFIA for diagnosis of pan-Plasmodium and P. falciparum infections in whole blood.Point-of-care diagnosis of malaria is currently based on microscopy and rapid diagnostic tests. However, both techniques have their constraints, including poor sensitivity for low parasitaemias. Hence, more accurate diagnostic tests for field use and routine clinical settings are warranted. The miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative, easy-to-use molecular assay for diagnosis of malaria in resource-limited settings. Unlike traditional molecular methods, mini-dbPCR-NALFIA does not require DNA extraction and makes use of a handheld, portable thermal cycler that can run on a solar-charged power pack. Result read-out is done using a rapid lateral flow strip enabling differentiation of Plasmodium-positive blood samples from returned travellers (n = 29), and Plasmodium-negative blood samples from travellers with suspected malaria (n = 23), the Dutch Blood Bank (n = 19) and intensive care patients at the Amsterdam University Medical Centers (n = 16). Alethia Malaria (LAMP) with microscopy for species differentiation were used as reference. Limit of detection for P. falciparum was determined by 23 measurements of a dilution series of a P. falciparum culture. A fixed sample set was tested three times by the same operator to evaluate the repeatability, and once by five different operators to assess the reproducibility.Diagnostic accuracy of the mini-dbPCR-NALFIA was determined by testing a set of P. falciparum was 10 parasites per microlitre of blood. The repeatability of the assay was 93.7%  and reproducibility was 84.6% .Overall sensitivity and specificity of the mini-dbPCR-NALFIA were 96.6%  and 98.3% . Limit of detection for Plasmodium infections in whole blood and differentiation of P. falciparum. Incorporation of a miniature thermal cycler makes the assay well-adapted to resource-limited settings. A phase-3 field trial is currently being conducted to evaluate the potential implementation of this tool in different malaria transmission areas.Mini-dbPCR-NALFIA is a sensitive, specific and robust method for molecular diagnosis of  Plasmodium species, its sensitivity for low parasite densities is limited and adequate slide reading requires extensive training and experience [P. falciparum strains with a genetic deletion for the antigen targeted by RDTs, histidine-rich protein 2 (HRP2). Over the past decade, this genotype has become widespread in South America, and increasing prevalence has now been reported for African and Asian countries as well [Correct and timely diagnosis of malaria is key in the management and control of this disease. Traditionally, microscopy of Giemsa-stained thick and thin blood film has been the standard diagnostic technique applied in endemic settings. Although it is able to differentiate the causative perience –4. The dperience . Howeverperience –8. False as well –12. Conv as well , 14.Plasmodium species differentiation [The limitations of microscopy and RDTs can be overcome by the use of nucleic acid amplification techniques (NAATs) . Examplentiation .Plasmodium species. An innovative assay has recently been developed to meet these requirements: the miniature direct-on-blood PCR nucleic acid lateral flow immunoassay . It is a hand-held, portable device that can be programmed with a smartphone or laptop application, either through USB cable or Bluetooth connection. The latest model, mini16, has an affordable price of approximately 800 USD  and can process 16 samples per run. The mini16 can run on mains power, but also on a portable and solar-chargeable power pack, making the system completely autonomous and suitable for rural or emergency settings with unstable or no electricity supply. Finally, the result of the dbPCR is easily and rapidly read out with NALFIA, an immunochromatographic flow strip that can detect labelled PCR amplicons [Consequently, there is still a need for a highly sensitive, user-friendly and field-deployable diagnostic test for malaria that can discriminate mplicons –25. A NAP. falciparum infections of 1 parasite per microlitre (p/μL) of blood [Plasmodium and P. falciparum infections in blood.Earlier prototypes of the dbPCR-NALFIA assay have shown promising results in field evaluations, with sensitivity and specificity results up to 97.2% and 95.5%, respectively, using light microscopy as reference standard, and a detection limit for of blood , 23. In Plasmodium 18S rRNA gene: one that is highly conserved in the genus Plasmodium (the pan-Plasmodium target), and a second that is specific for P. falciparum [The dbPCR is a duplex reaction targeting two regions in the lciparum , 27. By lciparum . The dbPP. falciparum-infected EDTA blood sample and a Plasmodium-negative EDTA blood sample. As a first step, the samples were lysed at 98 °C for 10 min on the mini16 thermal cycler , a miniature endpoint PCR device  which can also be used for heat block protocols. The miniPCR smartphone application was used to programme the lysis protocol on the mini16 device through Bluetooth connection. After the lysis of the EDTA blood templates, 22.5 μL of the dbPCR reagent mix was added to each . The dbPCR was also run on the mini16 thermal cycler. Its protocol consisted of an initial activation step of 1 min at 98 °C, followed by 10 cycles of 5 s at 98 °C, 15 s at 61 °C and 30 s at 72 °C; next, 28 cycles of 5 s at 98 °C, 15 s at 58 °C and 30 s at 72 °C; and a final extension step of 72 °C for 2 min.The template format for the dbPCR is 2.5 μL of EDTA-anticoagulated blood. Every mini-dbPCR-NALFIA run includes controls, which are a Plasmodium amplicon was positive, it indicated the presence of Plasmodium infection. If the second anti-FITC test line for the fluorescein amidite (FAM)-labelled P. falciparum amplicon was also positive, the sample was infected specifically with P. falciparum . A sample with a positive pan-Plasmodium line and an absent P. falciparum line was classified positive for a non-falciparum malaria species, i.e. Plasmodium vivax, Plasmodium malariae, Plasmodium ovale or Plasmodium knowlesi. When only the P. falciparum line was visible, this result was interpreted to be positive for this species. A NALFIA test was considered invalid when the flow control line was absent.Read-out of the results was done with NALFIA . The test strip consists of a sample absorption pad, a conjugate pad with neutravidin-labelled carbon binding to the amplicons’ biotin label, and a nitrocellulose membrane coated with anti-digoxigenin (Dig) and anti-fluorescein isothiocyanate (FITC) antibody lines detecting and visualizing the amplicon-carbon complex. A third line on the membrane functions as a flow control Fig. . After cPlasmodium and P. falciparum targets was determined by testing 23 aliquots of a tenfold dilution series of a FCR3 ring-stage P. falciparum culture. The parasite density of the culture was determined by light microscopy. Dilutions were made in Plasmodium-negative EDTA blood from the Dutch blood bank. Tested parasite densities ranged from 1000 to 0.1 p/μL. LoD was defined as the lowest parasite density that was detected with 90% confidence (≥ 21 of 23 runs).The limit of detection (LoD) for the pan-P. falciparum and 0.1 p/µL for P. vivax [Plasmodium species had been determined with expert microscopy. This set included 23 P. falciparum, 3 P. vivax, 2 P. ovale and 1 P. malariae infections. The P. falciparum samples had been quantified microscopically and ranged from 106 to 102 p/μL; the parasite counts of the non-falciparum malaria samples had not been determined at the time of microscopic examination. The 58 Plasmodium-negative samples comprised 19 samples from the Dutch blood donors, 16 samples from intensive care unit patients and 23 samples from malaria-suspected returned travellers with a negative Alethia diagnosis. The operator that tested all samples with mini-dbPCR-NALFIA was blinded to the reference test outcomes.To determine the laboratory sensitivity and specificity of the mini-dbPCR-NALFIA, a set of 87 blood specimen was tested, including samples from returned Dutch travellers with suspected malaria infection, Dutch blood donors, and intensive care unit patients from the Academic Medical Centre . All samples were derived from a pre-established Biobank at the Laboratory for Experimental Parasitology at the Academic Medical Centre. Both blood donors and intensive care unit patients did not travel to malaria-endemic areas in the 6 months before blood collection. The malaria status of all samples had been determined previously using the Alethia Malaria assay , a highly sensitive LAMP-based method for diagnosing malaria in non-endemic settings with a detection limit of 2 p/µL for P. vivax , 29. ForP. falciparum culture and five Plasmodium-negative blood samples. For the accordance assessment, one operator tested three sets of aliquots with mini-dbPCR-NALFIA on three consecutive days, using the same equipment and dbPCR reagent batch numbers. To determine the concordance of the mini-dbPCR-NALFIA, five different operators from the same laboratory each tested a set of sample aliquots once. All five operators were blinded to the nature of the samples and used the same equipment and dbPCR reagent batch numbers.Accordance and concordance are measures to express, respectively, the repeatability (intra-operator variability) and reproducibility (inter-operator variability) of qualitative tests , 31. To Plasmodium target, the P. falciparum target and the overall assay. The Clopper-Pearson Exact method was used to calculate the 95% confidence interval (CI) of the sensitivity and specificity. Accordance and concordance were calculated in random framework, using the formulae proposed by Van der Voet and Van Raamsdonk (2004): 0 the proportion of negative results, p1 the proportion of pan-Plasmodium single positive results (i.e. only the pan line), p2 the proportion of P. falciparum single positive results  and p3 the proportion of double positive results , for a particular sample i. For the random concordance, the following formula was used: 0,i, p1,i, p2,i and p3,i represent the proportion of negative, pan single positive, P. falciparum single positive and double positive results for a particular operator i [Sensitivity and specificity were calculated for the pan-erator i . The 95%erator i , 32.P. falciparum culture dilution series testing are displayed in Table Plasmodium test line and 10 p/µL for the P. falciparum line.The results of the Plasmodium samples, 28 tested positive for the pan-Plasmodium line in the mini-dbPCR-NALFIA, while 1 P. vivax sample was false-negative for this line. All 23 P. falciparum samples also showed the P. falciparum test line. 57 Plasmodium-negative blood samples were negative for both test lines with mini-dbPCR-NALFIA; 1 sample from a Dutch blood donor was false-positive for the P. falciparum line. None of the test samples had an invalid NALFIA result.Of the 29 Plasmodium line. The sensitivity of the P. falciparum test line was calculated to be 100% , and its specificity 98.4% . When the results of the two NALFIA test lines were combined, there were three possible outcomes: a non-falciparum infection, a P. falciparum infection and Plasmodium-negative. This approach resulted in an overall sensitivity of 96.6%  and specificity of 98.3%  of the mini-dbPCR-NALFIA.This resulted in a sensitivity of 96.6%  and a specificity of 100%  for the pan-. The overall accordance of all tested samples in a random framework was 93.7% . An overview of the accordance test results for the mini-dbPCR-NALFIA is shown in Table 3Table This study demonstrates that the mini-dbPCR-NALFIA is a robust, highly sensitive and specific tool for molecular diagnosis of malaria. It has a simpler workflow than traditional NAATs and requires much less resources. By incorporating the mini16 as portable, battery-powered thermal cycler, the mini-dbPCR-NALFIA can be used even in remote healthcare settings without an extensive laboratory infrastructure or stable power supply.P. vivax sample gave a false-negative result. This may have been due to a low parasite density, which is common in P. vivax infections [Plasmodium-negative sample could have been the result of carry-over contamination from a Plasmodium-positive sample during the preparation of the dbPCR or NALFIA.With an excellent overall sensitivity of 96.6% and specificity of 98.3%, the diagnostic accuracy of the mini-dbPCR-NALFIA is similar to that of traditional molecular techniques for malaria diagnosis, such as conventional PCR, qPCR and nested PCR . One P. fections . UnfortuPlasmodium line and 10 p/μL for the P. falciparum line demonstrate the high sensitivity of the mini-dbPCR-NALFIA for low falciparum parasite densities. Although the LoD of extremely sensitive nested and qPCR techniques can go as low as 0.1 p/μL [falciparum parasitaemias than light microscopy and RDTs, which generally fail to detect infections below 50 to 200 p/µL [falciparum cases with sub-microscopic infections [falciparum samples were available for this study, additional evaluation of the LoD of the mini-dbPCR-NALFIA for these other Plasmodium species is warranted.The LoDs of 100 p/μL for the pan-0.1 p/μL –37, most200 p/µL . As such200 p/µL –41. On tfections . As no qP. falciparum blood dilution series and five malaria-negative blood samples, the mini-PCR-NALFIA showed a high accordance of 93.7%, demonstrating the robustness of the method. Discordant results were mainly observed for parasite densities < 10 p/μL, which are close to the LoD of the test. At such low Plasmodium DNA concentrations, stochastic variations tend to have a more prominent influence on the assay’s outcome. This phenomenon was also believed to be the main reason for the concordance being 84.6%. The laboratory experience of the different operators in the concordance evaluation ranged from basic to proficient. They were only given written and oral instructions, which was sufficient for them to correctly perform the mini-dbPCR-NALFIA. This observation underlined its simplicity and user-friendliness.When analysing a falciparum infections [Plasmodium species in one reaction. This issue is not encountered with mini-dbPCR-NALFIA, a duplex assay that can distinguish falciparum malaria from infections with other Plasmodium species. To further evaluate the performance of the mini-dbPCR-NALFIA for diagnosis of (submicroscopic) infections with P. vivax, P. malariae and P. ovale., additional research is required, since this study tested only a limited number of non-falciparum malaria blood samples.Compared to other molecular methods for malaria diagnosis, mini-dbPCR-NALFIA shares some characteristics with LAMP, which also has a simplified protocol with easy read-out and high accuracy for diagnosing malaria, including low density fections , 44. HowThe adaptation of the assay described by Roth et al.  to operafalciparum malaria species and identify mixed infections. Although the vast majority of malaria cases in Africa is caused by P. falciparum, the relative contribution of P. vivax, P. malariae and P. ovale infections in this region appears to be increasing [P. falciparum and a P. vivax test line. In the same way, the mini-dbPCR-NALFIA also has the potential to be modified to detect other blood-borne pathogens.A limitation of the current mini-dbPCR-NALFIA is its inability to differentiate between the non-creasing –48. FortP. falciparum HRP2 antigen persistence in the blood after clearance of the parasites [Plasmodium DNA in blood can remain positive up to seven weeks after curative malaria treatment [In areas with high malaria transmission, the mini-dbPCR-NALFIA could be a valuable alternative to RDTs, which are likely to suffer from false-positive results due to arasites , 49, 50.reatment , 52. ThiP. falciparum and non-falciparum infections is possible thanks to its duplex format. A handheld miniature thermal cycler makes the assay well-adapted to resource-poor conditions in malaria endemic regions. The high diagnostic accuracy and low LoD of the mini-dbPCR-NALFIA could make it a valuable tool in many malaria control programmes, especially for detection of asymptomatic and low-density cases in near-elimination areas. A phase-3 field trial is currently being conducted to evaluate the potential of the mini-dbPCR-NALFIA in different epidemiological settings.The mini-dbPCR-NALFIA is an easy-to-use method for sensitive and specific diagnosis of malaria. Compared to other simplified molecular diagnostics, it has the advantages that there is no need of prior sample processing and that differentiation of


Malaria affects a significant portion of the global population, with 247 million cases in 2021, primarily in Africa. However, certain hemoglobinopathies, such as sickle cell trait (SCT), have been linked to lower mortality rates in malaria patients. Hemoglobin (Hb) mutations, including HbS and HbC, can cause sickle cell disease (SCD) when both alleles are inherited (HbSS and HbSC). In SCT, one allele is inherited and paired with a normal allele . The high prevalence of these alleles in Africa may be attributed to their protective effect against malaria. Biomarkers are crucial for SCD and malaria diagnosis and prognosis. Studies indicate that miRNAs, specifically miR-451a and let-7i-5p, are differentially expressed in HbSS and HbAS compared to controls. Our research examined the levels of exosomal miR-451a and let-7i-5p in red blood cells (RBCs) and infected red blood cells (iRBCs) from multiple sickle Hb genotypes and their impact on parasite growth. We assessed exosomal miR-451a and let-7i-5p levels in vitro in RBC and iRBC supernatants. Exosomal miRNAs exhibited distinct expression patterns in iRBCs from individuals with different sickle Hb genotypes. Additionally, we discovered a correlation between let-7i-5p levels and trophozoite count. Exosomal miR-451a and let-7i-5p could modulate SCD and malaria severity and serve as potential biomarkers for malaria vaccines and therapies. Plasmodium falciparum (Pf) infections [In 2021, malaria affected 247 million people, with the vast majority 95%) of cases occurring in Africa [fections , genetic% of casefections . SCT andfections ,9. Indivfections ,11. NoneMicroRNAs (miRNAs) are small, non-coding RNAs that endogenously up- or down-regulate targeted gene expression post-transcriptionally . MiRNAs Plasmodium parasites enter the human body through a female anopheles mosquito bite and release sporozoites that travel to the liver. In the liver stage, schizonts are formed and break open, releasing merozoites into the bloodstream, which causes the symptoms of malaria. During the blood stage, immature trophozoites develop into mature trophozoites or gametocytes, which have five phases and can be transmitted to the female mosquito during a blood meal [ood meal .Plasmodium lacks the enzymatic machinery necessary for miRNA production [Plasmodium parasites. Previous studies have shown that miR-451a and let-7i-5p levels were elevated in HbSS and HbAS erythrocytes and negatively regulated parasite growth in vitro [Plasmodium vivax [Interestingly, oduction . Neverthoduction . Therefoin vitro ,24. Addium vivax . In our P. falciparum infection, and there were four participants per genotype  (p = 0.03). We completed post hoc multiple comparison tests to determine group differences while controlling for Type I error inflation [Blood samples were collected from 24 participants without S, HbCC) . ParasitS, HbCC) . Area unnflation . Data from daily parasite counts were analyzed on days 3, 8, 9, and 16 . On day It is interesting to note that the parasite count for HbSS was the lowest on days three, eight, and nine. HbCC had the lowest parasite count on day 16 , while HPf cultures so that only the ring stage was present at the beginning of the experiment. To understand parasite phase distribution, we synchronized p < 0.05) differences between sickle Hb genotypes (p = 0.01), HbSS , and HbCC  genotypes. Additionally, we examined whether parasite growth was correlated with sickle Hb genotype over time (p < 0.006) correlated with trophozoite counts. Moreover, there was a significant (p < 0.05) correlation between all sickle Hb genotypes and phases four and five gametocytes, indicating that sickle Hb genotypes influence the development of parasites, specifically for trophozoites and gametocytes at phases four and five.Comparison of parasite numbers by phase based on genotype for days 3, 8, 9, and 16  showed tenotypes . In HbAAenotypes , and thever time . InteresPlasmodium infection of RBCs resulted in lower let-7i-5p levels in HbAS+, HbAC+, HbSS+, and HbSC+ sickle Hb genotypes, while HbAA+ had the lowest levels of let-7i-5p  and non-infected RBCs at different time points  during a 16 day culture (as shown in p = 0.02). Furthermore, miRNA levels were compared between RBCs and iRBCs for each genotype on days 3, 8, 9, and 16 (p = 0.03) higher for HbSS+ compared to HbSS-, and exosomal let-7i-5p levels at day eight were significantly (p = 0.04) higher for HbAC+ compared to HbAC- , 8 , 9 , and 16 . However, on day eight, we found that trophozoite counts were negatively correlated with exosomal let-7i-5p levels , indicating that higher trophozoite counts were associated with lower exosomal let-7i-5p levels (data not shown). Ultimately, while exosomal miR-451a and let-7i-5p levels were positively correlated in RBCs, this correlation was not observed when the RBCs were infected.Exosomal miR-451a and let-7i-5p were compared on days 3, 8, 9, and 16 to assess their correlation . We obsePf parasite growth in relation to exosomal miR-451a and let-7i-5p levels in different sickle Hb genotypes. By examining exosomal miRNA levels in iRBCs in vitro instead of an in vivo model, we gain insight into what the miRNA levels are in RBCs regardless of the influence of other tissues, such as WBCs, vascular cells, and other blood components. This helps us understand how exosomes found in RBCs can influence Pf and vice versa. To our knowledge, this is the first study to examine Other studies have shown that SCA patients have a higher mortality rate from malaria than HbAA or SCT patients . AdditioIn this study, we observed 16 days of parasite growth in vitro in different sickle Hb genotypes. Based on AUC curves, HbAA had a significantly higher parasite count than HbSS and other sickle Hb genotypes over 16 days. HbCC had the lowest parasite count overall in both AUC and ANOVA tests. AUC data assessed parasite growth over time, while the one-way ANOVA multiple comparison test compared the means of the groups independently, revealing that the HbCC group had significantly lower overall parasite counts H,I. AUC Pf growth is dependent on the sickle Hb genotype. Days 3, 8, 9, and 16 were selected to analyze exosomal miRNAs. The rationale behind choosing those days is that previous parasite count data showed that Pf growth peaked around days eight and nine before decreasing again. To ensure a comprehensive assessment of the entire experimental period, we chose one day at the beginning, two days during the peak, and one day at the end for analysis.When we looked at specific days, HbAA did not consistently have the most parasites. We saw that HbAC and HbSC had the most parasites on day three and that overall parasite count peaked on day eight for HbCC, on day nine for HbAA, HbAS, HbAC, and HbSC, while HbSS had the lowest parasite count overall . Based oNext, each genotype was examined for gametocyte count. In humans, gametocytes appear 10 to 12 days after infection . The firPf replication through modification of the molecular machinery found in the parasite. We found that exosomal miRNA levels differed between sickle Hb genotypes for iRBCs and RBCs  Institutional Review Boards, the University of Ghana’s (UG) College of Health Sciences at the Korle-Bu Teaching Hospital , and the Noguchi Memorial Institute for Medical Research . All participants provided informed written consent, and they were recruited from the Greater Accra Region in Ghana during June 2017 and June 2019 as part of an ongoing SCD and malaria study funded by the National Institutes of Health (NIH) and conducted in collaboration with UG at MSM. Experiments were carried out at NMIMR, and each participant was assigned a numerical anonymized code.Inclusion criteria were individuals without malaria, aged > 16 years, HIV negative, with no high fetal Hb (HbF), a detailed complete blood count (CBC), involving 6 groups of sickle Hb genotypes, HbAA, HbAS, HbAC, HbSS, HbSC, and HbCC and both sexes. Samples were sex and aged grouped ranging from 25 to 35 years old. Individuals with malaria or HIV-positive, high HbF, or partial CBC data were excluded. A total of 24 participants (4 per group) met all the inclusion criteria . Pf-specific protein HRP2 and Pan (Pan LDH), which can detect multiple Plasmodium species. The absence of parasites in blood samples was confirmed using thin smear microscopy [Blood samples were collected using Vacutainer ACD tubes . Sickle cell status was determined using a cellulose acetate membrane electrophoresis method with HbAA and HbSS as controls ,41. PartPf clone 3D7 is widely used [Pf parasites, RBCs were isolated from whole blood, washed to remove residual plasma and exosomes, and cultured following the WHO-FIND-CDC Malaria Methods Manual 2018, SOP 4.1 Preparation of Reagents and Media for Culture of Malaria Parasites [Pf [2, 5% O2, and 90% N2.For malaria in vitro studies, ely used ,23,42. Earasites . AlbuMAXites [Pf . Generalg for 30 min to eliminate debris. Subsequently, 0.5 volume of Total Exosome Isolation (from cell culture media) was added to the sample, and the mixture was vortexed and left overnight at 2–8 °C. The next day, the sample was centrifuged at 10,000× g for one hour at 2–8 °C. The supernatant was then removed, and the exosomes were resuspended in 1XPBS.Exosomes were extracted from the supernatant of RBCs using the Total Exosome Isolation kit (from cell culture media)  in accordance with the manufacturer’s guidelines . To begiThe first concentrate exosome pellet was suspended in 200 µL of 1XPBS, followed by RNA isolation from exosomes using the Total Exosome RNA/Protein Isolation kit  in accordance with the manufacturer’s instructions for sections “1-Isolate RNA-Organic extraction, 2-Isolate RNA- Purify the RNA, 3-Bind the RNA, 4-Wash the RNA, and 5-Elute the RNA”. To elute RNA, 50 µL of Elution Solution was used, and the concentration and purity of RNA were determined using the Thermo NanoDrop 1000 Spectrophotometer (ThermoFisher). The samples were then stored at −80 °C until further analysis.The RNA was reverse transcribed using the miRCURY LNT RT kit (Cat #: 339340) and miRCURY LNA SYBR Green PCR Kit (Cat #: 33936) by Qiagen . RT-qPCR was carried out using Roche LightCycler 480  and CFX Real-Time PCR Detection Systems . Primers for miR-451a, let-7i-5p, and U6 snRNA  were obtained from Qiagen.t-test or one-way ANOVA with Tukey’s multiple comparison test.The relative fold change in miRNA expression was evaluated using the ΔCT method and normalized to U6 snRNA expression. The Livak method was used to calculate ΔCt values in MS Excel , with U6 snRNA as an endogenous control . The datp < 0.05 was considered for all analyses. For data that passed the normality test, either one-way ANOVA and Tukey’s multiple comparison test for comparisons between more than two groups or an unpaired t-test for comparisons between two groups were used to test for significance. For data that did not pass the normality test , a Kruskal–Wallis test and a Dunn’s multiple comparison test or a Mann–Whitney test were used. Additional analyses included chi-squared, linear regression, Pearson correlation, receiver operating characteristic (ROC) curves, and the calculation of the area under the curve (AUC).All statistical analyses were performed using GraphPad Prism version 9.4.0 for Windows  unless otherwise specified. The normality of the data was assessed using the D’Agostino–Pearson normality test. If the data did not pass the normality test and could not be transformed, nonparametric tests were used. A significance level of