Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory syndrome of inappropriate and excessive immune system activation. It often occurs in the setting of viral, bacterial, fungal, and parasitic infections. HLH associated with malaria is very rare, and literature on this association is limited. Significant overlap exists between these two conditions, which makes the diagnosis of HLH superimposed on malaria difficult. We present a case of a patient who recently traveled from Djibouti and was diagnosed with Plasmodium falciparum malaria. She had a transient improvement in response to antimalarial therapy followed by clinical deterioration. This prompted further investigations that revealed the diagnosis of HLH, which was confirmed by an elevated soluble interleukin-2 receptor CD25 (sCD25) level, a specific marker of HLH. Most patients recover with antimalarial therapy, supportive care, and monitoring, whereas some patients require immunosuppressive therapy. Maintaining a high index of suspicion for HLH-associated malaria in at-risk patients allows for early identification and management. Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of inappropriate and excessive immune system activation. Dysfunction of natural killer cells and cytotoxic lymphocytes allows for macrophage hyperactivation, causing excess cytokine release, tissue damage, and multiorgan failure . ConditiMalarial infections by Plasmodium parasites have been identified as a rare trigger of HLH; however, literature on this association is limited. The pathogenesis is unclear but likely involves an interplay between a patient's genetic susceptibility and a persistent and inappropriate immune response induced by the parasite. Diagnosing HLH in the setting of malaria is challenging as significant overlap exists between the clinical features and laboratory findings of these two entities. We present a case of HLH associated with Plasmodium falciparum malaria.A 32-year-old Yemeni woman presented to the hospital with complaints of persistent fever and generalized weakness for 10 days. She had myalgia, arthralgia, nausea, vomiting, and non-bloody diarrhea. She was mildly hypotensive with jaundice and marked splenomegaly. Her eldest son, an 11-year-old boy, was experiencing similar symptoms and had been admitted to another healthcare facility.The patient was born in Yemen and moved to Djibouti later in life. She spent five years in Djibouti, during which she applied for immigration to the United States of America. She had arrived in Michigan, USA, from Djibouti three weeks prior to her presentation. Yemen is a country in the Middle East that is located in the south of the Arabian Peninsula; its population has a low socioeconomic status, and access to healthcare is limited. Djibouti is a country in East Africa that shares borders with Eretria, Ethiopia, and Somalia. Malaria is endemic in both Yemen and Djibouti, and efforts to combat malaria in these countries have been unsuccessful; they continue to be regarded as areas with a high local malarial transmission -6.In the hospital, the patient's laboratory workup revealed a normal white cell count with anemia, thrombocytopenia, and elevated ferritin. Given the clinical picture and epidemiological risk factors, a malaria smear was ordered, which showed malaria parasites present with frequent ring forms and gametocytes consistent with Plasmodium falciparum species; parasitemia was estimated at 3%. Further infectious workup was unremarkable. She was diagnosed with uncomplicated malaria and started on antimalarial therapy with a three-day course of artemether 80 mg twice daily and lumefantrine 480 mg twice daily.The patient had a transient clinical recovery, after which she developed significant fatigue and endorsed left upper quadrant abdominal discomfort and swelling. Abdominal computed tomography (CT) scan demonstrated an enlarged spleen measuring 13.8 cm with a possible splenic infarct  level Table . DissemiAfter completion of her course of antimalarial therapy, our patient had marked clinical improvement, recovery of cell counts, and decline in inflammatory markers. Repeat malaria smear demonstrated a parasite burden of less than 0.1%.HLH is an inflammatory syndrome that occurs in the setting of pathologic immune system activation and manifests with a characteristic constellation of clinical features. It involves an interplay of genetic, environmental, and clinical factors and has a variable spectrum of disease and presentation.The pathogenesis of HLH remains poorly understood. Abnormal activation of the immune system is the cornerstone of this syndrome as it allows for excessive inflammation and destruction of tissue. Normally, natural killer (NK) cells and cytotoxic lymphocytes (CTLs) regulate the inflammatory response of macrophages through a negative feedback loop. In HLH, dysfunction of this regulatory pathway creates an environment in which macrophages are not eliminated; they persist in a state of hyperactivation -8. InappHLH is strongly associated with mutations in genes involved in the immune system. It can occur as an isolated genetic defect or as a component of primary immunodeficiency syndromes . In patiThe HLH-2004 diagnostic criteria have been established as the diagnostic standard for the syndrome. It was initially utilized in prospective treatment studies for children with HLH and was later extrapolated to adults and adopted for use in regular clinical practice ,12. The Significant overlap exists between features of malaria and HLH, which raises a diagnostic dilemma. Fever, splenomegaly, anemia, thrombocytopenia, and elevated inflammatory markers are often present in patients with malaria and can be explained by parasite-induced cytotoxicity, microvascular disease, and organ sequestration. However, high ferritin, high triglyceride, and low fibrinogen levels are uncommon in malaria and may be suggestive of superimposed HLH. The diagnosis can also be corroborated with immunological testing for sCD25, C-X-C motif chemokine ligand 9 (CXCL9), or NK cytotoxicity assay, which are specific for HLH.A review of the literature yielded 28 cases of HLH associated with malaria worldwide -41. MostA portion of patients with HLH improve with early identification and prompt treatment of the trigger. HLH-specific therapy, therefore, is reserved for patients with clinical deterioration or multiorgan failure. HLH-specific therapy typically consists of a combination of etoposide, glucocorticoids, methotrexate, and intravenous immunoglobulin. Among the 28 published cases of HLH associated with malaria, 18 patients (65%) had clinical improvement and resolution of abnormal laboratory findings after typical treatment courses with antimalarials alone -33. PatiHLH is a rare but life-threatening complication of severe infections. Its identification in the setting of malaria is uncommon, although this could partly be due to under-recognition and diagnostic difficulties. Our patient's clinical deterioration after an initial response to antimalarial therapy helped identify this syndrome; this prompted closer monitoring and supportive care, after which she had complete clinical recovery. This case of HLH associated with Plasmodium falciparum malaria serves to add to the existing literature on this association which will allow for a better understanding of this syndrome. Further investigation into this association is needed to help predict at-risk patients and to identify the most effective treatment regimen. Maintaining a high index of suspicion for HLH-associated malaria allows for early identification, close monitoring, and treatment of the underlying cause to improve patient outcomes.


Plasmodium vivax multi-drug resistance protein-1 (pvmdr-1) gene to monitor the risk of CQR.Malaria chemoprophylaxis using chloroquine (CQ) and primaquine (PQ) has been administered to resident soldiers in the 3rd Army of Republic of Korea (ROK) to prevent malaria infection since the year 1997. Due to mass chemoprophylaxis against malaria, concern exists about the occurrence of chloroquine resistance (CQR). This study aimed to investigate the single nucleotide polymorphisms (SNPs) of the pvmdr-1 gene were analysed in 73 soldiers of the 3rd Army of ROK diagnosed with infection by P. vivax.SNPs of the P. vivax malaria-endemic areas in ROK, at a frequency of 1.3% (1/73). In addition, synonymous mutations were detected at positions K44 , L493 , T529 , and E1233 . Based on these SNPs, pvmdr-1 sequences of ROK were classified into 6 haplotypes. The phylogenetic analysis closed to the type of North Korean showed that P. vivax malaria of ROK could be a reason of influx from North Korea.Quintuple mutations  were detected in 73 soldiers. A newly identified non-synonymous mutation in the Y541C position had been introduced into pvmdr-1 were observed in the malaria chemoprophylaxis-executed regions of ROK from 2016 to 2017. Based on the rapid transition of pvmdr-1 SNPs, continuous surveillance for SNPs of pvmdr-1 related to CQR in the malaria-endemic regions of ROK is essential.This study showed that synonymous and non-synonymous mutations of The online version contains supplementary material available at 10.1186/s12936-022-04214-6. Plasmodium parasites, endangers about 40% of the world's population [P. vivax malaria, it decreased from about 7% in 2000 to 3% in 2019 [P. vivax malaria-endemic regions are localized near the DMZ  [Malaria, a life-threatening disease caused by pulation , 2. Depe in 2019 . Accordi in 2019 . In ROK,a, DPRK) . The detP. vivax in asexual blood stages and gametocytes, and primaquine (PQ) is responsible for killing the hypnozoite form of P. vivax in the liver stage. Due to the risk of chloroquine resistance (CQR) by massive and long-term use of chemoprophylaxis for prevention of malaria [pvmdr-1 [pvcrt-o and pvmdr-1 was detected by CQ treatment [pvmdr-1 .CQ is effective to eradicate  malaria , the arm[pvmdr-1 . Subsequ[pvmdr-1 . To date[pvmdr-1 –19. Ther[pvmdr-1 . Recent reatment –23. ThusThis study was approved by the ethics committee of the Armed Forces Medical Command . An approval form was used to obtain written informed consent and permission from each participant for providing a 5 ml blood sample.18S rRNA nested PCR.Blood samples were collected from malaria patients who agreed to the study. Positive patients with a rapid diagnostic test  or blood smear test with microscopy, or malaria-suspected patients  were enrolled this study. Most patients have a history of fever in the previous 48 h without malaria chemoprophylaxis compliance. All blood samples collected from 3 military hospitals , during 2016–2017 were screened using 141 venous blood samples (Whole blood-EDTA samples) from male patients infected with malaria or malaria suspected patients with fever were collected in the three Armed Forces Hospitals  near the DMZ located in northern Gyeonggi Province and northwest region of the ROK from 2016 to 2017. The Armed Forces Hospitals performed a rapid diagnostic test , and a blood smear test with microscopy for all participants.P. vivax infection, purified DNA samples from all patients were diagnosed by nested polymerase chain reaction (PCR) targeting P. vivax 18S rRNA. 1100 bp or 120 bp was detected by 2nd nested PCR [Genomic DNA was extracted from 200 ul Whole blood-EDTA using DNeasy Blood and Tissue Kit  as recommended by the manufacturer. For screening sted PCR .pvmdr-1 gene was amplified using the indicated primer sets of nested PCR  by electrophoresis . Direct sequencing of PCR products was performed by using Big Dye™ Terminator v3.1 Cycle Sequencing Kit  and the products were resolved on ABI 3730XL Genetic Analyzer .After the amplification, sequence analysis was performed by specific sequencing primers as shown in Table pvmdr-1 was used to perform phylogenetic relationship analysis. The sequences of Sal I strain (GenBank Accession# AY571984) and global parasites in PlasmoDB [pvmdr-1 sequences of 73 P. vivax clinical samples isolated from military personnel of the ROK army in 2016 and 2017 were analysed using BioEdit Sequence Alignment Editor. The phylogenetic analysis was constructed by 1000 bootstrap replications [Nearly full-length sequence  of PlasmoDB  were useications  using thications .P. vivax infected specimen through nested PCR analysis targeting 18S rRNA . The neighbourhood types of 6 haplotypes were identified as China_NB-16 and Papua New Guinea-PNG58/Thailand_VKBT-106  of pvmdr-1 SNP, Suwanarusk et al. revealed that the Y976F mutation was linked to CQR based on an increase in the IC50 value [In the reports investigating the relationship between CQR and 50 value . In anot50 value . This stP. vivax malaria was introduced from North Korea, rather than as an influx from foreign countries.Phylogenetic analysis showed that Type 4  was significantly related to the Type identified for North Koreans. In addition, a review by Chai indicated that a mosquito with blood from a malaria patient in North Korea near the DMZ flew south and infected Korean soldiers [P. vivax genes have been examined by the Armed forces Medical Command (AFMC) and the Armed forces Medical Research Institute (AFMRI) of ROK. This study focused SNPs of pvmdr-1 against malaria-infected soldiers near chemoprophylaxis-executed malaria-endemic regions. However, for a more practical resistance analysis and effectiveness of malaria chemoprophylaxis, in vivo or ex vivo resistance studies via analysing malaria infection ratio or CQ metabolite under malaria chemoprophylaxis are needed via further research. Therefore, it is currently planned to perform these studies in cooperation with AFMC and KDCA (Korea Disease Control Agency).Since 2016, to evaluate the risk from malaria chemoprophylaxis in the ROK, G6PD deficiency prevalence has been investigated , and to pvmdr-1. Thus, it inferred that quintuple mutation  was a prevalent background mutation in malaria-infected military personnel of the ROK. Phylogenetic analysis indicated that the malaria type was close to the type seen in North Korean, indicating that P. vivax malaria in the ROK could have come from North Korea. Furthermore, consistent monitoring of chemoprophylaxis-linked pvmdr-1 polymorphisms should be conducted to detect rapidly changed SNP profiling against P. vivax malaria in the ROK.In this study, there were clinical samples that possessed various SNPs of Additional file 1: Fig. S1. Alignment and mapping data of pvmdr-1 wild-type and mutant-type sequences in ROK army in 2016-2017. After the amplification of pvmdr-1using 73 P. vivax clinical samples, sequencing of PCR products was performed by using Big Dye™ Terminator v3.1 Cycle Sequencing Kit and ABI 3730XL Genetic Analyzer. Sequence analysis was performed using BioEdit Sequence Alignment Editor. The red box indicates the changed nucleotide in the alignment of pvmdr-1 SNPs for 20 and 53 specimens in 2016 and 2017. MDRF-WT is used as a reference sequence of pvmdr-1 gene (Gene Accession# AY571984).


Plasmodium falciparum–specific antibodies in highly endemic areas provides valuable information about malaria status and parasite exposure in the population. Although serological evidence of Plasmodium exposure is commonly determined by Plasmodium-specific immunoglobulin G (IgG) levels; IgM and IgA are likely markers of malaria status that remain relatively unexplored. Previous studies on IgM and IgA responses have been based on their affinity for single antigens with shortage of immune responses analysis against the whole Plasmodium proteome. Here, we provide evidence of how P. falciparum infection triggers the production of specific IgM and IgA in plasma and its relationship with parasite density and changes in hematological parameters. A total of 201 individuals attending a hospital in Breman Asikuma, Ghana, were recruited into this study. Total and P. falciparum–specific IgM, IgA, and IgG were assessed by ELISA and examined in relation to age ; infection ; pregnancy and hematological parameters. Well-known IgG response was used as baseline control. P. falciparum–specific IgM and IgA levels increased in the population with the age, similarly to IgG. These data confirm that acquired humoral immunity develops by repeated infections through the years endorsing IgM and IgA as exposure markers in endemic malaria regions. High levels of specific IgA and IgM in children were associated with microscopic malaria and worse prognosis, because most of them showed severe anemia. This new finding shows that IgM and IgA may be used as diagnostic markers in this age group. We also found an extremely high prevalence of submicroscopic malaria (46.27% on average) accompanied by IgM and IgA levels indistinguishable from those of uninfected individuals. These data, together with the observed lack of sensitivity of rapid diagnostic tests (RDTs) compared to PCR, invoke the urgent need to implement diagnostic markers for submicroscopic malaria. Overall, this study opens the potential use of P. falciparum–specific IgM and IgA as new serological markers to predict malaria status in children and parasite exposure in endemic populations. The difficulties in finding markers of submicroscopic malaria are highlighted, emphasizing the need to explore this field in depth.Assessment of serological  Plasmodium falciparum infection through memory B cells, which upon primo-infection are activated and persist for long periods of time to rapidly respond to persistent malaria re-infections  and the Ethical and Protocol Review Committee of University of Ghana’s College of Health Sciences (Ghana). The study was conducted according to the guidelines laid down by the Helsinki Declaration, with written informed consent obtained for each adult participant or, in the case of children, a parent or a guardian of the child participant provided written informed consent on their behalf or the child’s assent. The samples were specifically obtained for this study including complementary analysis of polymorphisms and immunomics in connection with malaria immunity that is not yet published. Volunteer information was anonymized prior to analysis.P. falciparum . Samples were collected from patients at Our Lady of Grace Hospital (Breman-Asikuma) in the Central Region of Ghana. This region is categorized as having a high transmission intensity throughout the year with 100% of the reported malaria infections caused by lciparum . Samples® Rapid Test for P. falciparum malaria (Viola Diagnostic Systems) and, if positive, they were confirmed by thick blood smear microscopy. Slides were air-dried and stained for 10 min in Giemsa diluted 1:9 in distilled water. The number of parasites per WBC was counted and the density of parasites per microliter of blood was calculated for each donor. When not available, an average count of 8,000 WBCs/µl was used. A SYSMEX KX-21 hematology analyzer was used to count WBCs, hemoglobin, platelets, and percentages of neutrophils, lymphocytes, and monocytes. Anemia degree defined throughout this study  follows WHO criteria  assay. DNA from dried blood spots (DBS) of FTA cards was extracted with the InstaGene Whole Blood Kit (Bio-Rad) following the described modification  and useduplicate . Cyclinguplicate  and spotP. falciparum isolate UCM7  during 30 min on ice. Resuspension was subjected to four freeze-thaw cycles and centrifuged at 20,000g for 10 min at 4°C to obtain a supernatant rich in P. falciparum proteins that were kept at −20°C. Protein concentration was determined using the Bradford protein assay (Merck).Clinical ate UCM7  from Wesate UCM7 . When cuAntibodies were eluted from cards following a previously described protocol . BrieflyTotal IgM, IgA, and IgG were quantified using the corresponding human IgM, IgA, and IgG Quantification ELISA kits (Bethyl Laboratories) following the manufacturer’s instructions, using 100 µl of diluted human serum  with 1:75,000 dilution for the secondary antibody reaction. A standard curve was included at each assay following the manufacturer’s instructions using human reference serum (Bethyl Laboratories).Pf-specific antibodies using a modified ELISA procedure  per well overnight at 4°C. Plates were washed five times with PBS-Tween 20 (0.05%) and subsequently blocked for 30 min with BSA 1% in tris-buffered saline solution at room temperature. Then, plates were washed and incubated for 1 h at room temperature with 100 µl of diluted human serum . After five washing steps, 100 µl of diluted horseradish peroxidase (HRP)–labeled secondary antibody (Bethyl Laboratories)  was added to each well for 1 h at room temperature. A standard curve was included using for coating 1 µg of the corresponding primary anti-Ig subtype diluted in 100 µl of coating buffer. Upon identical washing and blocking steps, 100 µl of human reference serum (Bethyl Laboratories) at several known Ig concentrations was added. After five washing steps, 100 µl of diluted HRP-labeled secondary antibody (Bethyl Laboratories) was added to each well in identical dilutions and conditions than patients’ sera samples.Serum samples were also screened for rocedure  using tom lysate . BrieflyFinally, plates were washed and incubated with 100 µl of TMB Substrate Solution (Thermo Fisher Scientific) and optical densities were recorded at 652 nm in a Varian Cary 50 Bio spectrophotometer (Agilent Technologies). A four-parameter logistic  model was fit to the data using SigmaPlot software.Sera used as a panel of negative control from individuals never exposed to malaria  rendered consistent and significant lower IgM binding values (average: 1.41 mg/dl) in comparison with those from the Ghana population (average: 4.41 mg/dl).2-transformed with a pseudo-count of 1 for data normalization and standardized into z-scores. Normalized and standardized data were used for both statistical analyses and graphical representations. Unsupervised principal components analysis (PCA) was conducted to explore major sources of variation in data accordingly to meta-information of participants . Statistical differences between groups were analyzed by non-parametric unpaired Wilcoxon rank-sum tests. Spearman’s tests were also applied to study the correlation between different variables and Fisher’s tests to assess whether the proportion of infected individuals changed between the age groups established. Statistical significance was considered at p-value ≤ 0.05.All data analyses, statistics, and graphics were performed in R v3.5.2 environment. When indicated, raw data were logA total of 201 participants, 158 (78.6%) females and 43 (21.4%) males, with an age average of 27.2 y.o., were included in this study. Experimental groups were established according to age, pregnancy, and parasitemia. Four age groups were defined: children under 5 y.o. , young between 6 and 13 y.o. , young adults between 14 and 49 y.o. classified into pregnant  and the rest of adults , and people older than 50 y.o. . Of the 123 women in reproductive age, 88 (71.5%) were pregnant. The 6–13-year group was included in whole population studies but discarded for age group analyses due to its low sample size.Plasmodium by microscopy was defined as 150 parasites/µl based on the average LOD described in the literature  parasitemia potentially detectable by microscopy  accounting for 14.93% of all samples; (ii) parasitemia at submicroscopic infection level  that was the largest group (46.27%); and (iii) no parasitemia (38.81%). The prevalence of P. falciparum infection in the whole population was 61.19%, being slightly higher in young and old adults  than in children (53%). Furthermore, overall prevalence of submicroscopic malaria among age groups was higher in older adults (57%) compared to young adults between 14 and 49 y.o. (47%) or children (28%) (The limit of detection (LOD) of terature . Once blen (28%) Table 1.P. falciparum infection in comparison to non-pregnant women  and accordingly to parasitemia level . There were 30 donors without RDT diagnostics, so they were discarded from the comparison. The global prevalence of malaria substantially increased from 8.86% by local RDT diagnosis to 61.39% by qPCR Table 2.The accuracy of the parasitemia values obtained between the microscopic examinations performed by the local technicians and those obtained by qPCR was compared in those samples that tested positive by RDT. Given that outliers were present in the data, a Spearman’s correlation was performed between the two parasitemia values as this type of test minimizes the effect of outliers on the correlation. The parasitemia values provided by both techniques were similar, with a strong correlation of parasite density values detected by microscopy and qPCR   to observe possible clustering of individuals and identify possible correlations between variables. The results revealed that under-5-y.o. children with submicroscopic malaria or no infection clustered together and separated from the rest of the population including children with microscopic malaria, who behaved like adults  were lower than Pf-IgG (median 22.62 mg/dl) and the three were present in all patients, whereas healthy donors not previously exposed to malaria lacked specific Ig (data not shown).We begin by describing the differences caused by age within each infection group. In the healthy population, adults (age groups 14–49 and ≥ 50) showed a significant increase in the level of both In submicroscopic malaria infections, these differences between adult and children groups were repeated for the specific antibodies Pf-IgG and Pf-IgA, whereas Pf-IgM was only increased in those over 50 years of age compared to children  reaching levels similar to those of adults with high parasitemia. Microscopic and submicroscopic infections did not influence total IgM and IgA levels within each age group, but in children, the infection with high parasitemia resulted in an increase in total IgG relative to uninfected children . It was particularly important that, in children with submicroscopic infection, the percentage of specific IgM was the only serological value that changed comparing to healthy children , independently of the infection status. Thus, there is a general anemia background in this population, as individuals without malaria infection also presented varying degrees of anemia, ranging from 76.92% in children to 40% in older adults  and/or severe anemia correlated with Pf-specific antibody levels . Odds ratios showed that Pf-IgA levels had the highest potential to predict both, whether a child has microscopic malaria or severe anemia , followed by Pf-IgM levels.Because children were the only age group in which y levels Table 4.Pf-specific Ig isotypes were significantly correlated between them within both groups, and the highest coefficient of correlation was found between Pf-IgG and Pf-IgM . Although all Pf-specific Igs were strongly associated with their total amounts in infected patients , but such association was not found between IgA and parasitemia  in parallel to IgG, Pf-specific IgM and IgA increase over the years and are strongly correlated with each other; (ii) baseline (lowest) levels of Pf-specific IgM and IgA (as well as IgG) were observed in infants under 5 years of age, reflecting their weak immune protection and lower exposition; (iii) all three isotypes increased significantly at young ages in response to early life infections; (iv) in adults, levels of all three Pf-specific Ig isotypes were consistently elevated and did not show increases upon re-infection, suggesting that, once they have reached peak levels, they may not increase even after successive exposures to the parasite; and (v) Pf-specific IgM had the highest correlation with parasitemia as compared to other antibody isotypes, and its elevated concentration was highly associated with microscopic malaria and severe anemia in children, which could be considered as an additional marker. Indeed, Pf-specific IgM levels should be considered confident serological markers of malaria exposure. Although IgM has been associated by other authors with some protection against malaria given its effector role in complement fixation  hemoglobin levels were not only significantly lower in malaria-infected patients but also negatively correlated with parasite density. Because Pf-specific IgM (and IgG) showed a strong indirect correlation with hemoglobin concentration, they could be used as predictors of malaria-associated anemia. Malaria-associated anemia is a leading cause of death (ii) Circulating platelet counts were associated with hemoglobin levels and thus decreased together in infected patients. This may be associated with the role of platelets in parasite clearance directly through platelet-erythrocyte complexes, which are a high percentage of the total platelet pool in malaria infection and, consequently, linked to thrombocytopenia ((iii) Children with submicroscopic malaria had a higher percentage of monocytes compared with those infected at the microscopic level, in agreement with the inverse correlation existent between monocytes and severe malaria (Pf-specific IgM levels (and at some extent IgG) had an inverse relationship with monocyte percentage which, together with its relationship with anemia (see above), could be a marker of malaria severity development.The routine hematology tests that can be done in rural hospitals in malaria-endemic regions may help to assess clinical risk and guide therapy. Thus, in our study, we explored these data to find possible associations to changes in Ig levels. Three connections of potential prognostic value can be highlighted: (of death . In agreytopenia . (iii) C malaria . FurtherFinally, we would like to address some limitations that we consider our study has. First, all individuals tested, including the youngest children, may have been exposed to previous malaria infections, so it is not possible to establish a baseline level of humoral response prior to infection. Negative ELISA controls were Spanish individuals never exposed to malaria, but this is not necessarily comparable to the status of a hyperendemic region. Second, cases of submicroscopic infection, although statistically consistent, may include cases of onset of infection with low parasite densities but later, along the disease progresses, may reach microscopic densities. Moreover, although we consider submicroscopic malaria as an indication of the individual’s protective status, the sample reflects only a time point, lacking the clinical history of antibody responses associated with malaria risk and functional immunity. Last, this study has a limited number of samples which, while offering in many cases sufficient statistical power, suggests that the study could be extended to larger and different hyperendemic populations. This would provide a more general population screening and greater statistical power to identify further correlates of protection.P. falciparum. Children are more likely to develop microscopic malaria, whereas adults have a higher prevalence of submicroscopic infection, in agreement with the lower concentration of Pf-specific Igs in children than in adults in a basal state. Detection of IgM, IgA, and IgG levels against P. falciparum blood-stage antigens does not help to reveal submicroscopic malaria infections, which, to make matters worse, are barely detected by RDTs. However, in our dataset, it is possible to establish a cutoff value in the concentration of Pf-specific IgM and IgA to serologically determine those cases of microscopic malaria and severe anemia in children, the human population most vulnerable to malaria. It remains to be established whether these cutoff values could be extrapolated to datasets from other populations. The susceptibility of pregnant women, who are the group with the highest risk of developing malaria infection among adults, unfortunately does not correlate with their levels of Pf-specific Ig isotypes. Finally, we must emphasize the need to search for new serological markers for the detection of submicroscopic malaria carriers, because, despite being the most common type of malaria in hyperendemic regions, it is not detectable by local diagnostic systems or serology.In conclusion, this study conducted in a malaria-hyperendemic region supports the accepted idea that age is key for the development of humoral immunity to The original contributions presented in the study are included in the article/This study was reviewed and approved by Ethical Review Board at Research Institute Hospital 12 de Octubre, Madrid (Spain) and the Ethical and Protocol Review Committee of University of Ghana’s College of Health Sciences (Ghana). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JB and IA designed and conceptualized research. PA, MH, AH, JF and JB contributed to sample and data collection on site and analysed haematological parameters. PA, PM-G, AD, AP, AR-P, IA and JB performed research. PA, JF, AD, AP, AR-P, IA and JB analysed data. PA and AR-P constructed graphics and figures. JB and IA supervised research. PA, IA and JB wrote the manuscript; and JB, AR-P and AP acquired financial support for the project leading to this publication. All authors contributed to the article and approved the submitted version.This work was supported by Spanish-MINECO grant BIO2016-77430R, a research fellowship to PA from the Universidad Complutense de Madrid CT42/18-CT43/18 and the Regional Program of Research and Technological Innovation for Young Doctors of the Comunidad de Madrid grant PR65/19- 22460.We thank all the donors and parents who participated in this study for their contribution. We also thank to Maria L. Rupérez, Edwige Gaba and Juana Garrido and all the Breman-Asikuma Community of the Sisters of Charity of St. Anne for strategic support and logistics, and Abraham Mensah, Samuel Baah, Alexander Cobbina, Stephen Apprey, and all the laboratory staff at Our Lady of Grace Hospital for excellent technical assistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malaria and HIV, 2 of the world's deadliest diseases, share a lot of territory in sub-Saharan Africa.This study seeks to investigate the effect of HIV on the immune response to malaria infection among pregnant women in Kumba in the southwest region (SWR) of Cameroon. The study aims to determine the prevalence of malaria infection, assess the occurrence of Plasmodium falciparum genetic diversity, and evaluate the antibody  and cytokine  response to malaria infection among pregnant women with and without HIV in Kumba.P. falciparum will also be performed. Furthermore, sequencing will be performed to study the nucleotide sequence of different alleles, and the genetic diversity of the alleles responsible for malaria infection among pregnant women will be assessed. A multiplex assay will be conducted to analyze the peripheral blood plasma and cord blood plasma for the cytokine and total antibody response to malaria infection among pregnant women with and without HIV. The questionnaire for data collection will be pretested at the Kumba District Hospital, and ethical clearance will be obtained from the University of Buea and the Regional Delegation of Public Health for the SWR. Data will be analyzed using SPSS Statistics and STATA. All P values <.05 will be considered statistically significant. BioEdit 7.0.0 software will be used to align the nucleotide sequences of different genes after sequencing. Phylogenetic tree searching will be conducted using the maximum-likelihood (ML) method in MEGA V6.0.The study will be a hospital-based cross-sectional design that will run from March 2022 to February 2023. It will recruit pregnant women with and without HIV who are in their third trimester of pregnancy. The study will be carried out in 5 health institutions in Kumba: General Hospital Kumba, Presbyterian Hospital Kumba, District Hospital Kumba-town, Kossala Integrated Health Center Kumba, and Catholic Hospital Kumba. About 3 mL of the mother’s venous blood, placental blood, and baby cord blood will be collected from each pregnant women at the point of delivery. Microscopy, rapid diagnostic tests (RDTs), and nested polymerase chain reaction (PCR) will be performed to identify the malaria parasite in all the samples, and nested PCR targeting the different genetic diversity markers for P. falciparum genetic diversity among pregnant women in Kumba, (2) the maternal and neonatal immune response to MSP1, MSP2, and EBA175 IgG antibody response to P. falciparum–caused malaria infection among pregnant women, and (3) the maternal and neonatal pro-inflammatory and anti-inflammatory cytokine response to malaria infection.The project started in March 2022 and will end in February 2023. Presently, three-fourth of the project funding has been disbursed to date. A total of 218 participants have been enrolled: 193 (88.5%) women without HIV and 25 (11.5%) women with HIV. Between February 2023 and March 2024, the following results will be ready for publication: maternal-neonatal malaria prevalence among pregnant women and babies in Kumba, the effect of HIV on (1) P. falciparum, with MSP1 alleles being the most prevalent. HIV infection also reduces the antibody response to malaria infection, as well as altering the level of pro-inflammatory and anti-inflammatory responses to malaria infection.HIV infection increases the prevalence of malaria infection among pregnant women and also influences the genetic diversity of DERR1-10.2196/38213 Plasmodium falciparum being the most virulent of them all ) primers for the detection of yd et al .P. falciparum at the level of placental and cord blood. The 3 polymorphism loci MSP1, MSP2, and EBA175 will be amplified using specific primers targeting the alleles of P. falciparum in nested PCR. Primary amplification followed by secondary PCR using specific primers for K1, Mad20, and RO33 (for MSP1) and 3D7 and FC27 (for MSP2) allelic families will be performed [A portion of the DNA extract from placental and cord blood will be used to determine the genetic diversity for samples that test positive for erformed . Primer erformed . For theerformed .MSP1, MSP2, MSP3, and EBA175 at the level of placental and cord blood will be randomly selected and sent for analysis to the Inqaba Biotechnical Industry in Nigeria for sequencing to identify and study the DNA sequences of the alleles responsible for placental and transplacental infection.A total of 10 samples positive for The stored plasma will be used to determine levels of cytokines IL-10, TNF-α, and IFNγ using a magnetic multiplex screening assay (Luminex) with the help of a Human Multi-Analyte Premixed Kit  . A portiData will be entered into Microsoft Excel, cleaned, and transformed for the different analyses to be performed. SPSS Statistics (IBM Corp) and STATA will be used for statistical analysis. A normality test will be performed to determine the different parametric and nonparametric tests to be used for the different analyses.t-test/Mann-Whitney U test will be performed to analyze continuous and categorical variables. The Kruskal-Wallis test/ANOVA will be conducted to analyze continuous and more than 2 categorical variables, while linear regression/Spearman correlation will be performed for multivariate analysis to determine the strength of association between different factors of the study and our dependent variables. All P values <.05 will be considered statistically significant. BioEdit 7.0.0 software will be used to align the nucleotide sequences of different genes after sequencing. Phylogenetic tree searching will be performed using the maximum-likelihood (ML) method in MEGA V6.0.Descriptive analysis of the general study population will be conducted, and both the mean and median of each parameter will be considered. The chi-square test will be performed to analyze categorical variables, and the Student P. falciparum genetic diversity of MSP1, MSP2, MSP3, and EBA175; and the total antibody  and cytokine  response to malaria among pregnant women with and without HIV will be evaluated.The prevalence of malaria infection; the occurrence of The first part of the project funding was in December 2021, the second part was in May 2022, and the third part was in December 2022. Currently, three-fourth of the project funds have been released. Data and sample collection started in March 2022 and will end in February 2023. A total of 218 participants have been enrolled: 193 (88.5%) pregnant women without HIV and 25 (11.5%) pregnant women with HIV see .P. falciparum genetic diversity, and evaluate the antibody  and cytokine  response to malaria infection among pregnant women with and without HIV in Kumba.Immune development during pregnancy is important as it determines the resistance of pregnant women to malaria transmission. Few studies have shown that women develop protective immunity against pregnancy-associated malaria (PAM), which is thought to be due to the acquisition of antibodies to the parasite variant surface antigen 2 chondroitin sulphate A (VAR2CSA) . This imIn Nigeria, the prevalence of malaria infection among pregnant mothers with HIV was 81% compared to 75% among pregnant women without HIV . When coP. falciparum, the most virulent species, has a wide genetic variety at all degrees of endemicity, which is not surprising, given that genetic diversity has been shown to be a function of transmission in specific regions [FC27 allele type in pregnant women isolates is higher (33.3%) than that in nonpregnant women (29.2%) when looking at the genetic diversity of P. falciparum (MSP1 and MSP2) infection among pregnant women in Lagos, Southwest Nigeria [P. falciparum among pregnant women with and without HIV as 2 distinct entities, a couple of studies have evaluated the diversity of P. falciparum among individuals with HIV. In Mutengene, Cameroon, it was reported that the MSP1 allelic variants detected among patients with HIV were KI (65.22%), MAD20 (27.54%), and RO33 (27.54%), while the MSP2 allelic variants were FC27 (30.43%) and 3D7/IC (31.88%), which was high and associated with anemia [P. falciparum isolates from patients with malaria and concurrent malaria-arbovirus in Kedougou [P. falciparum isolates from patients with malaria [P. falciparum population dominance is expected to be observed in regions with declining endemicity as a result of genetic drift caused by scale-up interventions [Studies have shown that  regions ,48. Oboh regions  discover Nigeria . This fi Nigeria . Although anemia . A studyKedougou . However malaria . The fre malaria . Neverthventions . In otheventions ,54. As aventions .PfAMA1, PfEBA175, PfMSP2, and PfVAR2CSA. Most controls’ antibody profiles for PfMSP3 and PvAMA1 remained low throughout gestation regardless of malaria exposure, whereas the antibody response varied more among the cases and the control group of pregnant women with and without infection [In addition, the protective role of antibodies to malaria cannot be overemphasized, as studies have shown that the antibody response plays a vital role in the protection of pregnant women against PAM infection ,56. A senfection . This stnfection . This confection  even demnfection .In contrast, studies have shown that HIV infection amplifies the effect of malaria, especially among pregnant women ,43,44. OCytokines and chemokines are elevated in peripheral blood during an acute malaria infection and contribute to parasite clearance, but they are also likely to be responsible for many of the symptoms and pathological changes seen during malaria ,62. The P. falciparum coinfection [P. falciparum infection and a potential pathogenic role [Among pregnant women with HIV, it was discovered that cytokine activity during pregnancy, as measured by plasma cytokine concentrations, varies with antiretroviral therapy and nfection . In Mozanic role . Anothernic role . This goP. falciparum resistance to SP-IPT as well as a longitudinal study on children born to mothers with HIV in order to understand why these children are more vulnerable to infections.There is a need for research into the prevalence of malaria among pregnant women in Kumba, considering all trimesters. It is also necessary to conduct a study on P. falciparum MSP1 alleles are associated with malaria infection among individuals with HIV and that genetic diversity is a function of malaria transmission in specific regions. In addition, HIV infection lowers the level of IgG response of antibodies to malaria infection for both mother and newborn babies. The likelihood of children with HIV to develop an immune response to AMA1 is low. HIV infection also alters the levels of the pro- and anti-inflammatory cytokine response, contributing to the lack of immune parasite clearance and parasite pathogenicity.The results of previous studies depict that HIV affects the prevalence of malaria among pregnant women irrespective of gravidity; malaria prevalence turns out to be much higher among pregnant women with than pregnant women without HIV. It was also revealed that more The findings of this work will be published in peer review journals and will be presented in seminars as well as conferences. Copies of the work will be submitted at the Pan-African University of Life and Earth Sciences Institute, University of Ibadan, Nigeria; the University of Buea; and the hospitals where the samples were collected. A copy will also be sent to the Union for African Population Studies.


Plasmodium falciparum has been studied to assess local malaria transmission genetic profile using evidence-based intervention measures. However, there are no known previous reports of P. falciparum polymorphism in Badewacho and Boset districts, Southern Ethiopia. The purpose of this study was to determine the genetic diversity of the merozoite surface protein-1 and -2 (msp-1 and msp-2) allelic families in P. falciparum isolates from an asymptomatic populations.  The genetic variation of msp-1 and msp-2.  This study was conducted from finger-prick blood samples spotted on 3 mm Whatman filter paper collected during a community-based cross-sectional study. Nested polymerase chain reaction amplification was used to type the allelic variants of P. falciparum were included for molecular analysis. Of 50 positive samples, 43 P. falciparum isolates were successfully amplified for the msp-1 and msp-2 allelic families. A total of twelve different allele sizes (75–250 bp) were identified within the three allelic families of msp-1, whereas ten different allele sizes (250–500 bp) were detected within the two allelic families of msp-2. MAD20 had a higher allelic proportion, 65% among allelic families of msp-1, whereas the 3D7 allelic family 90.7% was higher in msp-2. A slightly higher frequency of polyclonal infection 53.5% was found in msp-2 allelic family, whereas a low proportion polyclonal infection 46.5% was found in msp-1 allelic family. The overall mean multiplicity of infection (MOI) for msp-1 and msp-2 was identical (MOI = 1.56). Correspondingly, the expected heterozygosity (He) value for msp-1 (He = 0.23) and msp-2 (He = 0.22) was almost similar.  From 669 asymptomatic study participants, a total of 50 samples positive for msp-1 and msp-2 allelic families in P. falciparum isolates. However, continued monitoring status of the local genetic diversity profile in the P. falciparum population is required to support current malaria control and elimination strategies. The findings of this study revealed low genetic diversity of the  Malaria remains a major public health problem in the world. According to the World Malaria Report, an increase in malaria cases was observed between two consecutive years, and it is estimated that 241 million cases of malaria occurred worldwide in 2020, whereas 227 million cases in 2019. In 2020, with high burden of malaria, 95% of malaria cases and deaths originated from the World Health Organization (WHO) African Region [Plasmodium falciparum is characterized by high genetic variation in several populations of malaria parasite isolates [P. falciparum causes life-threatening disease and challenges the development of effective drugs, diagnostic tools, and vaccines in the global community [P. falciparum could support the current malaria control and elimination effort in the world [P. falciparum isolates, such as the merozoite surface protein-1 and -2 (msp-1 and msp-2) genes, the glutamate-rich protein (glurp) gene, and the circumsporozoite protein (csp) gene [P. falciparum isolates, the msp-1 and msp-2 genes are widely used to assess allelic diversity and play a vital role in determining the extent of malaria transmission in a given community [Among human malaria parasites, isolates . As a reommunity . Thus, uhe world . There asp) gene . Among tommunity .P. falciparum isolates with a size of 190 kDa, encoded by the msp-1 gene found on chromosome 9. Among the seven variable blocks in msp-1 gene, block 2 is the most polymorphic region that occurs in three allelic families, such as K1, MDA20, and R033 [P. falciparum isolates, encoded by the msp-2 gene found on chromosome 2. Of the five variable blocks, block 3 is the most polymorphic central region with two allelic families, namely, FC27 and 3D7 [Merozoite surface protein-1 (MSP-1) is a surface protein in and R033 , 9. Mero and 3D7 , 11.P. falciparum by genotyping msp-1 and msp-2 is essential to monitor the extent of intervention in different scenarios of malaria [P. falciparum in symptomatic malaria patients in the world [P. falciparum from asymptomatic individuals in Boset and East Badewacho districts in Southern Ethiopia. This study aimed to assess the genetic variation of msp-1 and msp-2 in P. falciparum from asymptomatic individuals from Boset and East Badewacho districts, Southern Ethiopia.In the era of malaria control and elimination in the world, the continuous assessment of the genetic variation of  malaria –13. In lhe world –18. Howehe world , 19–21. This study was carried out from samples collected during a community-based cross-sectional study in Boset and East Badewacho districts from March to June 2020, Southern Ethiopia. Boset district is located in the East Shewa Zone in the Oromia Region of Southeastern Ethiopia. The altitude of the district varies from 1100 to 2700 m above sea level and receives an annual average of rainfall that varies between 700 and 800 mmHg. The district is characterized by a hot and dry climate with an average annual temperature that varies between 25 and 30°C for the tropical and between 15 and 20°C for the subtropical. It is an agricultural area, and Nura Hera of upper Awash agro-industry is found here, where extensive agriculture is carried out in Ethiopia through the irrigation of the Awash River. Badewacho district is located in Hadiya Zone in southern nation nationality population region, South Ethiopia. It is found 225 km from the capital city, Addis Ababa. The altitude of the district ranges from 1501 to 2500 m above sea level and receives a mean annual rainfall of 801–1400 mmHg. The average annual temperature ranges from 17.6 to 22.5°C. Agriculture is the principal source of livelihood for the rural population. Both districts are known to be malarial with decreased malaria transmission due to the current intervention measure.P. falciparum samples, 37 dried blood spot samples from Badewacho district, and 13 from Boset district were included for molecular analysis of msp-1 and msp-2 allelic families in this study. Three to four drops of finger-pricked blood were spotted on 3 mm Whatman filter paper, dried, and placed in airtight plastic bags with desiccant and stored at −20°C in the Parasitology Research Laboratory at Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia.This study was carried out from samples collected during community-based studies in East Badewacho district (415 study participants) and Boset district (254 study participants). A total of 50 microscopy and polymerase chain reaction (PCR) confirmed μl), low (100–499 parasites/μl), moderate (500–4999 parasites/μl), high , and very high .The level of parasitemia was taken from microscopy blood film examination. The thin smear was used for malaria parasite species identification, whereas the thick smear was used to estimate parasite density according to the WHO protocol . ParasitGenomic DNA was extracted from dried blood spots using the Chelex–saponin method, and the final extracted genomic DNA samples were stored at −20°C until used for PCR amplification as described previously .P. falcipaurm msp-1 (block 2) and msp-2 (block 3). Two round PCR amplifications were performed as described previously by Hamid et al. [msp-1 (block 2) and msp-2 (block 3), whereas the secondary/nested PCR reactions target the family specific alleles of msp-1  and msp-2  was performed using the average number of PCR fragments per infected individual. Genetic diversity of Plasmodium falciparum isolates was measured using expected heterozygosity (He). He was calculated using formula, He = n/(n − 1)(1−∑Pi2), where n is the number of isolates analyzed, and Pi is the frequency of each different allele at a locus. P-values ≤0.05 were considered to indicate statistical significance.Data were entered and analyzed using SPSS version 25. The proportion of P. falciparum-positive isolates collected during the community-based study were included in this study. Of the 43 study participants, 55.8% (24/43) more men than women were enrolled. The mean age of the participants was 26.88 years (±15.78 SD), with a range of 2–80 years. The characteristics of the study participants are indicated in A total of 43 microscopy and PCR-confirmed P. falciparum-positive samples collected from the asymptomatic population, 43 P. falciparum isolates were successfully amplified for genetic diversity of P. falciparum msp-1 and msp-2 genes. Out of 43 P. falciparum isolates  based on the size of the allelic fragments  in a set of two or three specific allelic combinations. The overall mean MOI and He index for the msp-1 genotype were 1.56 and 0.23, respectively. Similarly, from 43 P. falciparum isolates, 10 different allele sizes (250–500 bp) were detected within the two allelic families of msp-2 (FC27 and 3D7). The frequency of 3D7 allelic family 90.7% (39/43) was highest compared with FC27 allelic family 62.8% (27/43) in msp-2. A lower frequency of monoclonal infection 46.5% (20/43) was detected in msp-2 allelic families compared with polyclonal infection 53.5% (23/43) in a set two alleles (FC27 and 3D7). The overall mean MOI and the He index for the msp-1 genotype were 1.56 and 0.22, respectively.Of the 50 microscopy and PCR-confirmed isolates , a totalragments . MAD20 hmsp-1  and msp-2 (FC27 and 3D7) were identified among the isolates at the two study sites  was observed between study sites only for the msp-1 allelic family. The distribution of both msp-1 and msp-2 specific allelic families with respect to age group and sex of the study participants showed a slight difference, but no statistically significant variation (P > 0.05) was observed.All reported allelic families of dy sites . The dismsp-1 was higher in the younger and older age group, whereas the MOI for msp-2 showed a slight increase with increasing age, but no statistical difference was observed in both msp-1 (P = 0.268) and msp-2 (P = 0.607) allelic families. The MOI for the allelic families of msp-1 and msp-2 was higher in females, but the results did not show a significant variation in both msp-1 (P = 0.970) and msp-2 (P = 0.325). The MOI was higher in Bedawach district than Boset district; however, no statistical differences were observed in the allelic families of msp-1 (P = 0.436) and msp-2 (P = 0.385). Regarding parasite density, a decrease in MOI was shown as parasite density increased  and msp-2 (P = 0.576).The MOI for ncreased , except msp-1 and msp-2 genes in P. falciparum isolates collected from the asymptomatic population. In the present study, a low degree of genetic diversity, msp-1 (12 genotype) and msp-2 (10 genotype) allelic variants were found in P. falciparum population in Boset and East Badewacho districts, Southern Ethiopia.This study was carried out to assess the genetic diversity of the msp-1 , MAD20 was found to be the predominant polymorphic allele type. This pattern is consistent with studies from Adama in Ethiopia [msp-2 allelic families, 3D7 were the most predominant compared with FC27 allelic families in this study. This is in line with reports from northeast Ethiopia [msp-1 and msp-2 [In this study, of the three allelic families of Ethiopia , East AfEthiopia , and CamEthiopia . On the Ethiopia , NigeriaEthiopia , and MalEthiopia . RegardiEthiopia , southweEthiopia , BurkinaEthiopia , and CamEthiopia . In contEthiopia  and centEthiopia . This dind msp-2 , 31. Mormsp-1 allelic families, the overall mean MOI for both msp-1 and msp-2 (MOI = 1.56), and the expected He value for msp-1 (He = 0.23) and msp-2 (He = 0.23) were slightly lower in Boset and Badewacho districts. This finding is consistent with a report from Adama and its surroundings in Ethiopia [In the present study, polyclonal infections for Ethiopia , DjiboutEthiopia , and YemEthiopia . In contEthiopia , northweEthiopia , Sudan [Ethiopia , southweEthiopia , and CôtEthiopia . The lowEthiopia , 36.msp-1 and msp-2 allelic families, even if a slight difference were observed in the younger and older age group. This finding is similar to previous reports from northeast Ethiopia [msp-1 and msp-2 allelic families was higher in females, but without statistical significant variation. In this study, a decline in MOI was detected when parasite density increased, but no statistical differences were observed in both msp-1 and msp-2. This is compatible with studies from northwest Ethiopia [In the present study, no statistical association was observed between age group and MOI for both Ethiopia , Sudan [Ethiopia , and CôtEthiopia . In contEthiopia , NigeriaEthiopia , and SudEthiopia . LikewisEthiopia  and NigeEthiopia . In contEthiopia , SenegalEthiopia , and ConEthiopia . This diEthiopia , 43, 44.msp-2 was observed in Badewacho district. This could be explained partly by the difference in the sample size and degree of local malaria transmission. The use of small sample size and less sensitive molecular methods to differentiate minor fragments is the limitation of this study. However, as far as we know, the present study is the first action in Boset and Badewacho districts, which generates valuable information about the genetic diversity of polymorphic region of msp-1 and msp-2 allelic families in P. falciparum isolate.Regarding the study sites, although no statistical differences were found between Badewacho and Boset districts, higher distribution of allelic families and MOI for both msp-1 and msp-1 and msp-2 allelic families in terms of MOI, He, and multiclonal infections was found in P. falciparum population in Boset and Badewacho districts, Southern Ethiopia. This study can be used as baseline data to assess the intensity of malaria transmission and evaluate the current malaria control and elimination programs in Ethiopia. Further study with a large sample size between these two study regions/sites will be required to analyze the genetic similarity and difference of circulating polymorphic marker of the msp-1 and msp-2 allelic families in P. falciparum isolates. In addition, investigation to differentiate minor fragments of msp-1 and msp-2 should consider the use of sensitive molecular laboratory methods, such as capillary electrophoresis, single nucleotide polymorphism, and next-generation sequencing.In this study, lower genetic diversity of


Anopheles albimanus salivary gland extract (SGE) and four Plasmodium falciparum antigens  in 11,541 children enrolled at 350 schools across Haiti in 2016. Logistic regression estimated odds of an above-median anti-SGE IgG response adjusting for individual- and environmental-level covariates. Spatial analysis detected statistically significant clusters of schools with students having high anti-SGE IgG levels, and spatial interpolation estimated anti-SGE IgG levels in unsampled locations. Boys had 11%  lower odds of high anti-SGE IgG compared to girls, and children seropositive for PfMSP1 had 53%  higher odds compared to PfMSP1 seronegatives. Compared to the lowest elevation, quartiles 2-4 of higher elevation were associated with successively lower odds  of high anti-SGE IgG. Seven significant clusters of schools were detected in Haiti, while spatially interpolated results provided a comprehensive picture of anti-SGE IgG levels in the study area. Exposure to malaria vectors by IgG serology with SGE is a proxy to approximate vector biting in children and identify risk factors for vector exposure.IgG serology can be utilized to estimate exposure to Anopheline malaria vectors and the Plasmodium species they transmit. A multiplex bead-based assay simultaneously detected IgG to  Plasmodium parasite infection in humans and is transmitted by mosquitoes in the genus Anopheles. In 2020, there were an estimated 241 million malaria infections globally, 0.26% of which were estimated to result in patient death  detects multiple analytes from the same specimen simultaneously  and has nfection . Malariaulations . In partrculates , and hasrculates . This curculates , and assChildren were enrolled and samples were collected in 2016 as part of lymphatic filariasis (LF) transmission assessment surveys in Haiti, with integration of malaria RDTs and microscopy for soil-transmitted helminths in stool specimens . IndividSurveys were conducted in evaluation units (EUs) that had met World Health Organization (WHO) criteria to conduct an LF TAS, with the current WHO recommendation to conduct a school-based TAS in areas where the net primary-school enrollment rate is ≥75%. Haitian school enrollment data for 2014 were utilized along with population census data to determine the sampling approach employed in each EU, which are program defined and dependent on baseline LF prevalence found during initial mapping surveys . Per WHOAn. albimanus mosquitoes (lab strain STECLA) and frozen for later use. Whole salivary glands were homogenized with glass tissue grinder in phosphate buffered saline  and freeze-thawed twice for further protein dissociation. Total protein concentration of this SGE homogenate was determined by BCA assay .As described previously , whole sSchistosoma japonicum glutathione-S-transferase (GST) was utilized as a non-binding internal well control and was coupled at pH 5.0 at 20 μg/mL. The malaria antigens in the multiplex panel have all been reported before: the P. falciparum merozoite surface protein 1 19kD fragment , P. falciparum apical membrane antigen 1 N-terminal region , P. falciparum circumsporozoite protein (NANP)5 peptide fused to GST , and Pl1043 epitope from P. falciparum liver stage antigen 1 (LSA-1)  were coupled with antigens for IgG capture and subsequent detection. Recombinant  (LSA-1) . The homE. coli extract to prevent non-specific binding) and diluted to a final concentration of 1:200, which is approximately a 1:400 serum dilution. Diluted blood samples were stored at 4°C until assayed.Participant whole blood was eluted from a single tab of the TropBio filter wheels to provide sample for the IgG detection assay. A single DBS tab (10 μL whole blood) was rehydrated in blocking buffer , 0.5% polyvinylpyrrolidine (SigmaAldrich), 0.1% casein , 0.5% bovine serum albumin (SigmaAldrich), 0.3% Tween 20, 0.05% sodium azide, and 0.01% 3. Positive and negative controls, which had been determined by preliminary assay runs as high or low responders, were included on each IgG detection plate to ensure appropriate assay performance. The bead mix including all bead regions contained approximately 1,000 beads/region added to each assay well. Samples (50 μL of 1:200 dilution of eluted whole blood) were incubated with beads for 90 min at room temperature under gentle shaking protected from light in MultiScreen-BV filter plates (SigmaAldrich). After three washes  following incubation, beads were incubated with 50 μL biotinylated detection antibody  for 45 min. After three washes, 50 μL streptavidin-phycoerythrin  were added to all wells (1:250x of 1 mg/mL) for a 30-min incubation. After three washes, samples beads were incubated with 50 μL reagent buffer for 30 min, washed once, and resuspended in 100 μL PBS. Assay plates were briefly shaken and read on a Bio-Plex 200 machine (BioRad) by generating the median fluorescence intensity (MFI) for a target of 50 beads. The final measure, denoted as MFI minus background (MFI-bg), was reported by subtracting MFI values of beads on each plate only exposed to sample diluent during the first incubation step. The MFI-bg threshold for true positive IgG assay signal against Plasmodium antigens was ascertained if the sample MFI-bg was higher than the mean + 3SD of the MFI-bg signal of a panel of 92 known anti-malaria IgG negative DBS samples.All IgG assay reagents were diluted in buffer containing PBS, 0.05% Tween 20, 0.5% bovine serum albumin (SigmaAldrich), and 0.02% NaNP. falciparum antigens, PfAMA1, CSP, LSA-1, and PfMSP1. Environmental covariates by school GPS coordinate included elevation, normalized difference vegetation index (NDVI), population density, rainfall, distance to the nearest water body , and air temperature . Figures were produced using R Statistical Software  and GraphPad Prism . Individual covariates included sex; rapid diagnostic test ; II3FRC30, Premier Medical Corporation, New Jersey) result; and seropositivity to four perature Table S1Spatial analysis was conducted in SaTScan v10.0.2 to detect statistically significant clusters of schools with students who had elevated anti-SGE IgG responses using a Bernoulli probability model. Due to a lack of sampling in all regions of the country, clusters were detected separately in northern  and southern (Grand’Anse and Sud departments) regions of Haiti. Outputs were overlayed onto geospatial surfaces of Haiti using QGIS. Spatial interpolation of anti-SGE IgG levels to unsampled locations within the TAS study area was conducted in QGIS using inverse distance weighting; select environmental variables were depicted for visual comparison with the spatially interpolated results.P. falciparum malaria infection by RDT, while 919 (8.0%) had a positive IgG response against any P. falciparum antigen. Overall, seropositivity to PfAMA1 (3.8%) and PfMSP1 (4.9%) was more common than seropositivity to CSP (0.8%) and LSA-1 (0.4%). Compared to other departments, Grand’Anse had the highest proportion of participants who were RDT positive  and had past malaria exposure . The median elevation of schools was 208 m (IQR: 62-346 m); the median distance to the nearest water body was 2.2 km (IQR: 0.8-4.5 km); and the median normalized difference vegetation index (NDVI) was 0.6 (IQR: 0.5-0.7), indicating relatively dense vegetation throughout the study area  compared to those without     compared to those who were seronegative to all P. falciparum antigens  .The mean log-transformed MFI-bg value for anti-SGE IgG was 6.07 (SD = 1.36) in the study population. The mean anti-SGE IgG level was significantly higher among six-year-olds  compared to seven-year-olds      and distance to the nearest body of water . Rainfall , population density , and temperature  were positively correlated with anti-SGE IgG.Estimated mean values of anti-SGE IgG from children enrolled in different schools varied from 4.2 to 8.4. A negative relationship was observed between elevation and anti-SGE IgG by school ; aPORQ4: 1.52 ] compared to the lowest quartile.In the adjusted model, sex and seropositivity to PfMSP1 were the only individual factors found to be significantly associated with high anti-SGE IgG levels Table S3In total, seven statistically significant clusters were identified in the study area with children who had elevated anti-SGE IgG levels Table S4Plasmodium sporozoites within mosquito saliva during a blood meal, and estimating exposure to malaria vectors represents an indirect assessment of malaria risk where Plasmodium parasites are endemic  antigen  was insufficiently large to explore associations by infection status, it was nonetheless an expected finding in Haiti’s low-transmission setting. This points to the importance of using other markers of exposure to assess malaria risk when infection is rare; while 0.2% of the study population had a positive RDT, 8% of study participants—and 20% of participants in Grand’Anse department—were IgG positive to Anopheles IgG supports these previous findings. Indeed, at lower elevations, there are more possibilities for standing water sites where these mosquitoes can complete their life cycles (These data link associations between vector exposure (through the anti-SGE IgG biomarker) and a variety of environmental factors. Anopheline mosquitoes prefer breeding sites below 500 m in elevation , and thee cycles . While de cycles . Howevere cycles , and stuP. falciparum is not prevalent in the human population. Visual comparison between spatially interpolated results and a map of Haiti’s elevation shows that highest levels of anti-SGE IgG matched well to areas of low elevation across the TAS study area; this also corresponds with the strong relationships observed between elevation and anti-SGE IgG in both bivariate and multivariate analyses. Because the other environmental variables used in this study were temporally averaged, whereas elevation has static values, this may not only account for the weak correlations observed in bivariate analyses, but also for the lack of a clear relationship with anti-SGE IgG levels in spatial analysis. The low resolution of rainfall data relative to other environmental variables also makes comparison difficult with interpolated anti-SGE IgG. More complex geostatistical analyses could further evaluate these relationships.The regions of Haiti included as school sampling sites have high heterogeneity in malaria disease burden and vector density . High leAn. albimanus bites was occurring, as bites could have occurred anywhere outside the school where children enrolled. However, it is reasonable to assume that most children’s households were likely within a few kilometers of the school they were attending. Another limitation is the lack of data on entomological measures such as human biting rate (HBR) and entomological inoculation rate (EIR), which would have been useful for validating anti-SGE IgG as a biomarker for both vector exposure and malaria transmission; future directions would include collection of these data for analysis. Moreover, while the RDT was the most practical tool available to indicate active infection in this study, RDTs may not have captured all of the very-low-density P. falciparum infections in this non-treatment-seeking population. It is thus possible that a more-sensitive diagnostic tool such as polymerase chain reaction (PCR) could have detected more infections in this setting, allowing for a more robust analysis by infection status .


Plasmodium falciparum, are even more uncommon. With the coronavirus disease 2019 (COVID-19) pandemic, there have been distractions in evaluation and diagnosis leading to a rise in cases and deaths. We present a case of autoimmune dysregulation and blackwater fever secondary to severe malaria, requiring multiple courses of antimalarial therapy. Careful travel history and prompt recognition and treatment facilitates improved patient survival and recovery.Despite advances in treatment and prevention, malaria still carries significant morbidity and mortality. Cases of malaria in the United States are rare and cases of severe malaria, mostly attributable to  Plasmodium falciparum and cases are defined by one or more of the following symptoms: jaundice, >5% parasitemia, neurological symptoms, renal failure, acute respiratory distress syndrome, or severe anemia [Plasmodium species cause malaria infection in humans, with P. falciparum and Plasmodium vivax being the most common [P. falciparum infection and quinine use with a possible genetic link to glucose-6-phosphate dehydrogenase deficiency [In 2020, there were 241 million new malaria infections worldwide. Of those infections, 627,000 cases caused mortality [e anemia . Six PlaP. falciparum malaria was confirmed by polymerase chain reaction (PCR) of whole blood and by whole blood peripheral smear using QuantStudio 12K Flex Real-Time PCR system . COVID-19 PCR test was negative.A 62-year-old male with a past medical history of hypertension, hyperlipidemia, and diabetes presented to the emergency department complaining of dehydration, headache, and chills. He took two coronavirus disease 2019 (COVID-19) tests at home, the most recent test being three days prior to admission. The first test indicated positive and the second showed negative. He received both doses of the Pfizer vaccine in March 2021. Home medications included metformin, amlodipine, losartan, simvastatin, aspirin, and ibuprofen. Initial vital signs were notable for a temperature of 37.8°C and a heart rate of 105 beats per minute. Respiratory rate, blood pressure, and oxygen saturation in room air were within normal limits. The physical exam was unremarkable. Laboratory tests were significant for lactic acid of 3.6 mmol/L, creatinine of 2.1 mg/dL, total bilirubin of 5.1 mg/dL, and platelet count of 37,000/uL. Urinalysis was positive for protein, ketones, nitrites, bilirubin, urobilinogen, mucus, and bacteria. He was admitted for management of sepsis and presumed COVID-19 infected. Infectious Disease was consulted for severe sepsis. He initially reported recent travel to London, United Kingdom. Later, his wife reported his travel to Nigeria to visit family during the same 12-day trip. He returned to the United States 11 days prior to admission. The next day he became confused and computed tomography of the head showed no acute process. There was a concern for cerebral malaria with his new impaired mental status. Peripheral smear obtained in light of travel history showed greater than 30% parasitemia consistent with malaria along with classic rings and head-phoned rings Figures , 1B. TheThe CDC was contacted and he was started on artesunate. On day three, he was transferred to the intensive care unit for further management of decompensating mental status, renal function, and liver function. During his prolonged hospital stay, he developed acute renal failure secondary to acute tubular necrosis requiring dialysis and acute hemolytic anemia requiring blood transfusion. The patient had relapsing fevers up to 39.4˚C with waxing and waning mentation. After traumatic nasogastric tube insertion, he developed diffuse nasal bleeding unrelieved by nasal packing. He was intubated for concern of risk of hemorrhagic aspiration. Bleeding was successfully controlled with bilateral nasal tampons. Esophagogastroduodenoscopy performed for melena showed no acute signs of bleeding. A percutaneous endoscopic gastrostomy (PEG) tube was placed on day 24 for tube feeding. There was concern regarding hemophagocytic lymphohistiocytosis (HLH) given his cytopenia, fever, hypertriglyceridemia (>354mg/dL) off of propofol, ferritin greater than 6000 ng/dL, hepatomegaly, and significantly increased IL-2 level of 4031 units/mL. Using the HScore for reactive hemophagocytic syndrome, he had a >99% probability of HLH with a score of 247. However, improving thrombocytopenia and lack of persistent fever, hypofibrinogenemia, or splenomegaly went against a diagnosis of HLH. Total bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) increased to 34 mg/dL, 649 unit/L, and 259 unit/L, respectively, suggesting liver failure. The serology panel showed positive Epstein Barr virus IgM, IgG, and nuclear antigen antibody. Anti-smooth muscle antibody was positive and liver biopsy was recommended, but the patient’s family declined. Pertinent serology and immunology tests are summarized in Table Parasite percentage eventually decreased to 0%; however, he required two courses of artesunate, artemether/lumefantrine, and artemisinin for persistent parasitemia Table . The patSevere malaria is rare in the United States and blackwater fever is even more uncommon. In the current case, the patient had jaundice, >5% parasitemia, cerebral malaria, renal failure, and severe anemia consistent with severe malaria. Diagnostic challenges in this patient included delay in diagnosis due to the relative rarity of malaria in the United States as well as initial presumed COVID-19 diagnosis. During the COVID-19 pandemic, there have been distractions in evaluation and diagnosis leading to a rise in cases and deaths. In 2019, there were 227 million global malaria cases. In contrast, in 2020 there were 241 million new malaria cases worldwide [P. falciparum antigens can induce lytic activation of previously latent EBV B cell infection during acute malarial infection [Unique points of this case include autoimmune dysfunction likely secondary to cytokine upregulation and inflammation. Although artemisinin derivatives have been documented to cause mild (1-4%) elevation of serum aminotransferase, autoantibodies have not been described . Thus, pnfection . Similarnfection . Malarianfection . The patnfection ,13. AlthDespite the advances of medicine, malaria still carries significant morbidity and mortality if not promptly recognized and treated. In particular, there is a mortality rate of 15-20% in cerebral malaria even when treated . PrecautMalaria is still a serious and potentially fatal disease worldwide despite the approval of the first malaria vaccine for children in 2021 and the availability of prophylactic anti-malarials. Severe and uncommon complications such as autoimmune dysregulation and blackwater fever may result. With the focus on the COVID-19 pandemic for the past few years, education on the prevention and treatment of malaria has been lagging. It is important to promptly recognize risk factors and treat malaria symptoms.