Malaria is one of the most prominent illnesses affecting children, ranking as one of the key development concerns for many low- and middle-income countries (LMICs). There is not much information available on the use of anti-malarial drugs in LMICs in children under five. The study aimed to investigate disparities in anti-malarial drug consumption for malaria among children under the age of five in LMICs.This study used recent available cross-sectional data from the Malaria Indicator Survey (MIS) datasets across five LMICs , which covered a portion of sub-Saharan Africa. The study was carried out between January 2, 2023, and April 15, 2023, and included children under the age of five who had taken an anti-malarial drug for malaria 2 weeks before the survey date. The outcome variable was anti-malarial drug consumption, which was classified into two groups: those who had taken anti-malarial drugs and those who had not.In the study of LMICs, 32,397 children under five were observed, and among them, 44.1% had received anti-malarial drugs. Of the five LMICs, Kenya had the lowest (9.2%) and Mali had the highest (70.5%) percentages of anti-malarial drug consumption. Children under five with malaria are more likely to receive anti-malarial drugs if they are over 1 year old, live in rural areas, have mothers with higher education levels, and come from wealthier families.The study emphasizes the importance of developing universal coverage strategies for anti-malarial drug consumption at both the national and local levels. The study also recommends that improving availability and access to anti-malarial drugs may be necessary, as the consumption of these drugs for treating malaria in children under the age of five is shockingly low in some LMICs.The online version contains supplementary material available at 10.1186/s12936-023-04805-x. Plasmodium vivax is the leading cause of morbidity and death in children under 5 years of age [In 2017, there was an estimated 219 million cases of malaria, marking a 5% rise from the previous year. Moreover, over 60% of fatalities were concentrated among children under the age of five in 91 different countries . MalariaAnopheles mosquitoes, which act as carriers of Plasmodium parasites, is the source of malaria. Among the 5 parasite species that cause an acute febrile illness in the form of malaria, two species—Plasmodium falciparum and P. vivax—create the utmost risk, but all the species can infect children [The sting of infected female children –10. ThesUnconsciousness and seizure known as cerebral malaria, acute anaemia that requires urgent life-saving dissemination, heavy breathing caused by acute metabolic acidosis, jaundice, and diarrhoea are the frequent lethal symptoms for under-five children with malaria in Africa . The mosP. falciparum species, chloroquine did remain the main form of medication for uncomplicated P. falciparum malaria for a major bulk of sub-Saharan countries until after 2000 [Quinoline derivatives, antifolate, and artemisinin derivatives are the three major groupings of anti-malarial medications. Artemisinin and its derivatives are advocated in combination with other anti-malarial agents owing to their short half-life and slow absorbability . Currentter 2000 . In thister 2000 .This study aimed to ascertain the disparities in anti-malarial drug consumption among children under the age of five in five malaria-endemic countries using a region-based geographic information system (GIS) map. Moreover, the study examined the socioeconomic factors that are associated with anti-malarial drug consumption in children under the age of five across LMICs.In this cross-sectional study, five malaria-endemic countries, namely Guinea, Kenya, Mali, Nigeria, and Sierra Leone, utilizing the most recent malaria survey data available from 2020 to 2021. This survey was based on the MIS , a household survey framework developed by the Monitoring and Evaluation Working Group (MERG) as part of Roll Back Malaria, an international partnership dedicated to coordinating global malaria control efforts . The MISThe MIS survey employs a two-stage stratified cluster sampling technique. The initial phase was selecting certain regions or clusters. The second stage comprises methodically selecting each cluster or family within an enumeration area (EA). To facilitate cross-country comparisons, MIS surveys follow a standardized set of operating protocols encompassing sampling, questionnaire design, data collection, data cleaning, coding, and analysis.The MIS program collects data on various key malaria indicators, including the ownership and usage of insecticide-treated mosquito nets, intermittent prenatal care, blood tests for diagnosing fever in children under the age of five, and indoor residual pesticide spraying, among other widely recognized indicators related to malaria. The MIS also collects data on home demographics and possession of things like radios, bicycles, indoor plumbing, and electricity. Anaemia and malaria parasite counts can be assessed as part of the MIS for the most vulnerable family members, such as small children and pregnant women. Participants who meet the eligibility criteria and provide their consent are requested to offer a small blood sample, typically a few drops. Trained interviewers then conduct immediate on-site testing for anaemia. The prevalence of malaria has been assessed in more than 60 DHS and MIS surveys, as well as multiple DHS surveys, and many more MIS surveys .https://dhsprogram.com/ website. To conduct the research, "Children Recode (KR)" datasets were employed, as the study exclusively focused on children under the age of five. Approximately among the 30 LMICs where MIS surveys are typically undertaken, five LIMCs were ultimately chosen . These countries were chosen because they delivered recent MIS data and satisfied the criteria for inclusion  within a stratum. Missing data is just one of several factors that can lead to a single PSU in a stratum, among other possible causes. The inability to compute standard errors is just one of several problems this creates when analysing the data. To handle single PSUs within each stratum, a scaled singleton method was employed. This involved scaling the singleton (certainty) for each stratum by computing the average of variances obtained from different sample units within those strata. Additionally, the authors ensured that categorical explanatory variable levels were accurately defined to enhance the clarity of the analysis. Following the exclusion of research variables, datasets from each country were subsequently combined.The study revealed that, despite the existence of nationally representative surveys with a focus on malaria, such as the MIS, there is currently insufficient data to rate the effectiveness of the various treatment cascades for the disease. Consequently, malaria surveys may not comprehensively capture the entirety of the quality of care provided for malaria.Second, the MIS surveys omitted information that would have been crucial for enhancing access to care for malaria, including the reasons people chose not to seek treatment. Information on blood tests was unavailable for four countries with endemic malaria. Just five of the 25 countries included in the study provided data on malaria test results that might establish the best course of therapy for febrile children under five.Third, the data were based on mothers' accounts of the anti-malarial medications for their children received, rather than the actual prescriptions from healthcare professionals. Consequently, this might not provide an entirely accurate representation of the treatment process. Lastly, in future surveys, it could be beneficial to monitor children not only during the treatment process but also after the completion of treatment to assess the long-term impact on the quality of malaria care provided to the children. Another absence from most surveys is patient history, which should be made available because the recurrence of malaria may affect how a healthcare provider administers therapy for the disease.Given these data gaps, the findings, which are presented in the following sections, estimate anti-malarial drug consumption patterns where data are most complete and plausible assumptions can be made to provide the essential proof of whether an anti-malarial was administered for a recent fever in each region across five LMICs.The MIS survey classified explanatory variables into two primary categories: the first category involves individual-level variables, while the second category pertains to community-level variables. Level one variables include the child's age, sex, the mother's highest level of education, the number of children under the age of five, and the wealth index. Level two variables cover the type of place of residence and the individual's home country. These variables were documented and then pre-coded based on the available data and the most probable causal connections. A comprehensive description of each variable can be found in Table The outcome variable, labelled "Anti-malarial Drug," is a binary variable with categories "Yes = 1/No = 0". Initially, inquiries were made regarding recent experiences of fever among the children. If the response was affirmative, subsequent questions were posed to determine whether they had received anti-malarial medication for the treatment of the fever  . A backwThis study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement—a checklist of items that should be included in the reporting of cross-sectional studies  children under the age of five had received anti-malarial drugs  percentage of anti-malarial consumption in children under five among the five LMICs, whereas Mali has the highest (70.5%). Guinea, Nigeria, and Senegal had anti-malarial consumption of 66.3%, 31.6%, and 12.5%, respectively. Senegal has the lowest (12.9%) percentage of malaria, and Nigeria has the highest (46.1%). Children who recently experienced a fever have a high prevalence of malaria, except for those in Senegal  region and the lowest observed in the North-Eastern and Eastern regions (approximately zero percent) Fig. . HoweverIn Mali, the percentage of anti-malarial consumption in children under five was very high (above 60%) in all regions, except for Kidal 21.44%). The highest percentages of anti-malarial drug consumption were observed in the Koulikor (83.8%) and Gao (81.4%) regions, while the lowest was observed in Kidal (21.44%). The prevalence of malaria was quite high (above 30%) in all regions of Mali, except for Bamako (20.4%). However, the prevalence of malaria varied significantly across different regions of the country Fig. . The hig1.44%. Th , Kogi (85.8%), and Benue (83.8%) regions, while the lowest was observed in the Borno region (approximately zero percent) Fig. . Most reIn Senegal, the percentages of anti-malarial drug consumption in children under five were very low in most regions, with the highest percentages observed in Saint Louis (34.0%) and Sedhiou (30.0%) regions, while the lowest was observed in Kaffrine, Fatick, Louga, Thies, and Kaolack regions (approximately zero percent) Fig. . Most reTable About 44.3% of children who are male have taken anti-malarial drugs, whereas 43.8% of children who are female have taken anti-malarial drugs. The percentage of anti-malarial consumption seems to increase as the age of children increases , except for 5 years with 46.1%. Children who live in urban areas received 46.0% of the anti-malarial drugs, compared to 43.6% in rural areas. Among the children, 63.9% of those whose parents had higher education took anti-malarial drugs, while only 40.2% of children whose parents had no education received such treatment. The percentage of anti-malarial drug usage varied with the number of children in the household, as follows: one child, 45.3%; two children, 42.0%; three children, 42.0%; four children, 42.5%; and five children, 50.1%. Furthermore, there appears to be an increasing trend in the percentage of children taking anti-malarials based on the wealth index of the family, with the percentages rising in ascending order from the poorest (33.4%), poorer (36.1%), middle (45.4%), richer (54.5%), to the richest (61.0%) . They have limited access to reliable malaria diagnosis and treatment, which is another factor. Artemisinin-based combination therapy (ACT), the usual treatment for uncomplicated malaria, is very efficient in curing the disease. Consequently, those patients have repeatedly taken anti-malarials for recent fever since they have limited access to ACT due to weak healthcare facilities and resources , 35. The percent.Anopheles mosquito, responsible for transmitting the malaria parasite, are widespread in rural regions of sub-Saharan Africa, where many malaria cases are recorded. In addition to having restricted access to preventative measures like insecticide-treated bed nets and indoor residual spraying, rural regions often have worse housing conditions, which might make them more attractive mosquito breeding grounds. Furthermore, compared to urban areas, rural communities frequently have less developed healthcare infrastructure and resources. As a result, it could be more difficult for people in rural areas to receive an appropriate diagnosis and a malaria treatment that works. Community health workers or other non-medical employees may be the primary point of contact for people looking for healthcare services in many rural locations. Because of the lack of diagnostic resources and the need to administer rapid treatment for malaria, these individuals may be more inclined to offer anti-malarial medication to people presenting with malaria symptoms. Finally, due to their livelihoods and everyday activities, those who reside in rural regions may be more exposed to malaria. Rural dwellers could be more inclined to work outside or spend more time outside, which would increase their exposure to mosquito bites and malaria risk. The incidence of malaria may rise because of this increased exposure, increasing the requirement for anti-malarial medication [Children in rural areas typically receive more anti-malarials for malaria than those in urban areas. There are several reasons for the high anti-malarial consumption in rural areas. The World Health Organization has reported that the species of dication –39.Mothers' education was found to have a significant impact on their children’s anti-malarial consumption. Children whose mothers had higher education tend to receive more anti-malarial drugs for malaria than children whose mothers had no formal education. This is not surprising, as educated people tend to have better health-seeking abilities. They tend to go themselves and take their children to healthcare facilities more often than less educated ones , 40. EduChildren's age was found to have a significant impact on their anti-malarial consumption for malaria. Compared to newborns under 1 year old, children between the ages of 2 and 5 are more likely to get anti-malarial medications due to a higher risk of having severe malaria. Because their immune systems are still growing, children in this age range are less able to fend off the malaria parasite. The natural immunity to malaria that older children and adults have established from repeated exposure to the disease may not have been formed in young children in this age range. Moreover, antibodies developed from mothers during pregnancy still frequently provide protection for infants under the age of one. When a child gets older and the antibodies start to wear off, this protection may diminish, leaving them more vulnerable to malaria infection. Hence, unless they are showing signs of malaria, young children who have not yet lost this protection might not need anti-malarial medication , 45.According to this study, several LMICs  have very poor anti-malarial drug consumption for children under the age of five who have malaria. Anti-malarial drugs should be accessible and available at all levels of the healthcare system, according to nations. Effective supply chain management is required for this, including forecasting, purchasing, and distribution. To support vulnerable groups, such as children aged 2 to 5 and those with illiterate mothers, it is essential to implement targeted interventions. Possible interventions encompass health education programs, the provision of healthcare services without cost, and improving accessibility to ACT. Furthermore, raising public awareness about appropriate anti-malarial drugs can be accomplished through community outreach efforts and public health campaigns.This study represents a pioneering endeavour in investigating anti-malarial drug consumption in children under the age of five. Advanced geospatial analysis was utilized to develop a detailed and comprehensive portrayal of anti-malarial drug consumption in each of the five Low- and Middle-Income Countries (LMICs) where endemic malaria is prevalent, and data on malaria test results were available. This innovative approach provides a valuable resource, allowing local and national policymakers to gain timely access to a comprehensive perspective on anti-malarial drug consumption through region-specific GIS maps.This study has several limitations. First, more country studies would be preferable, but only five countries malaria test results are available. Second, specific MIS datasets contained incredibly few observations, which produced some surprising findings. Finally, the study only looked at the anti-malarial drugs taken for malaria in children under five. Further research is required for all age groups.The study reveals that while some Low- and Middle-Income Countries (LMICs) exhibit a high prevalence of anti-malarial drug usage for malaria treatment , others receive lower dosages . Anti-malarial drug consumption in children under five is influenced by various factors, including the type of residence, the child's age, the mother's highest level of education, and the wealth index. The analysis recommends putting in place different anti-malarial medicine checks and balances as well as a more robust health infrastructure. It is also recommended to alert the local authority to this terrible scenario and suggest offering support services based on individual needs.Additional file1. STROBE Statement—Checklist of items that should be included in reports of cross-sectional studies.Additional file 2: Table S1. Weighted Descriptive Statistics for Guinea. Table S2. Weighted Descriptive Statistics for Kenya. Table S3. Weighted Descriptive Statistics for Mali. Table S4. Weighted Descriptive Statistics for Nigeria. Table S5. Weighted Descriptive Statistics for Senegal.


P. vivax outbreaks. Molecular analysis of two highly polymorphic genes Pvmsp-1 F2 and Pvmsp-3α was performed for 49 samples, collected during October 2019 through September 2021 from four different regions with varying malaria transmission risks. RFLP-profiling revealed that outbreak samples from three indigenous villages, almost exclusively, harbored a single clonal type, matching the “Palumeu” lineage previously described in 2019, despite multiple relapses and drug pressure exerted by mass drug administration events, suggesting a limited P. vivax hypnozoite reservoir in Suriname. In contrast, isolates originating from Sophie, a mining area in neighboring French Guiana displayed a highly heterogeneous parasite population consistent with its endemic malaria status, demonstrating the differentiating capacity and thus the usefulness of PCR-RFLP for P. vivax genetic diversity studies. Outbreak reconstruction emphasized the impact of undetected human movement and relapses on reintroduction and resurgence of P. vivax malaria and PCR-RFLP monitoring of circulating parasites guided the roll-out of targeted interventions. PCR-RFLP seems a suitable molecular alternative in low-endemic areas with restricted resources for outbreak analysis, for monitoring the spread or containment of circulating strains and for identification of imported cases or potential foci.The road to malaria elimination for low- and middle-income countries is paved with obstacles, including the complexity and high costs of advanced molecular methods for genomic analysis. The usefulness of PCR-RFLP as less complex and affordable molecular surveillance tool in low-endemic malaria regions was assessed in a cross-sectional study conducted in Suriname, currently striving for malaria elimination, but plagued by recent  Plasmodium vivax malaria. Suriname is a South American country striving for malaria elimination and Plasmodium vivax malaria is the main impediment, mostly due to the parasite’s ability to stay dormant in the liver and relapse within months/years after the initial infection, triggering new outbreaks.The global malaria landscape has changed considerably since 2000, with a drastic reduction of overall malaria cases, but a regional increase of P. vivax populations. We report the successful application of the PCR-RFLP technique in determining genetic signatures of Plasmodium vivax isolates, obtained from four different regions with varying malaria transmission risks. RFLP-profiling revealed that outbreak samples from three indigenous villages, almost exclusively, harbored a single clonal type, suggesting a limited P. vivax hypnozoite reservoir in Suriname. In contrast, isolates originating from Sophie, a mining area in neighboring French Guiana displayed a heterogeneous parasite population, consistent with its endemic malaria status. We demonstrated the usefulness of PCR-RFLP for assessments of P. vivax genetic diversity, outbreak analysis and identification of imported cases in a low-transmission setting, where resources are limited.As most low- and middle-income countries we are confronted with the lack of effective and affordable molecular tools to assess and monitor the genetic diversity of circulating  The global malaria landscape has changed considerably since 2000, with a drastic reduction of overall malaria cases, but still accounting for more than 241 million malaria cases worldwide in 2020  were considered heterologous infections, while identical restriction profiles [identical code] were designated as homologous infections. Heterologous infections are represented in The RFLP patterns were translated into a six-digit code to facilitate the comparative analysis of isolates. The first three characters reflected the pattern for the Pvmsp-1 F2 and Pvmsp-3α) among the sample sets from the different villages.Fisher’s Exact Test (R version 4.2.2) was applied to compare genetic differences for each gene (Lilliefors-corrected Kolmogorov-Smirnov test (R version 4.2.2) was used to test for uniform distribution of gene profiles.The statistical significance level was set at p<0.05.The median age of the study participants (n = 49) was 21 years with a range from 1–80 years. The male to female ratio in the study sample was 1.13:1.Pvmsp-1 F2 and Pvmsp-3α gene. Pvmsp-1 F2 genotyping based on size polymorphisms rendered the two types A (1150 base pairs [bp]) and B (ca 1087 bp), corresponding to size variants described earlier [Pvmsp-1 F2 gene based on sequence polymorphisms yielded 7 and 8 different restriction profiles for each of the restriction enzymes AluI and MnlI respectively. The combination of size variants with both restriction patterns of Pvmsp-1 F2 polymorphisms yielded 10 allelic types.All DBS from field isolates could be successfully extracted and amplified for the  earlier . FurtherPvmsp-3α gene revealed two discrete size variants Type A (1.9 kilobases [kb]) and Type B (1.5 kb), corresponding with the allele sizes, occurring worldwide. The most common allelic variant was type A, consistent with the global occurrence [The PCR amplicons of the currence . On the Pvmsp-3α sequence polymorphisms rendered 7 allelic types for AluI and 7 allelic types for HhaI. The combination of both restriction patterns yielded 7 haplotypes and inclusion of size variants did not augment the results.Genotyping based on size and sequence polymorphisms of both genes allowed us to distinguish 12 haplotypes among the 49 isolates.The results of the PCR-RFLP profile determination are visualized in Pvmsp-1 F2 profile on the left side and the Pvmsp-3α profile on the right side. The total number of cases diagnosed per region in the presented period is shown as national trend lines for Palumeu, Apetina and Pelele Tepoe . No trend line was added for Sophie (French Guiana), since this study did not include malaria cases, diagnosed in French Guiana.Parasite isolates are depicted as circles or squares divided in two colored halves, representing the The outbreak and post-outbreak samples from Palumeu matched the variant Aa1-Aa1, which was earlier observed as single clonal lineage during the outbreak and designated as the “Palumeu haplotype” . In addiMoreover, even the majority (87%) of the investigated samples from the Pelele Tepoe outbreak were regarded as homologous infections also harboring this “Palumeu haplotype”.In order to gain some insight in the evolution of genetic signatures with multiplication in the liver, paired isolates from two patients with recurring infections in Pelele Tepoe within the study period were included. As both patients were either pregnant or breastfeeding, relapses were suspected due to the non- use of Primaquine. The paired samples indeed harbored the “Palumeu haplotype” .Interestingly, the only three samples in Pelele Tepoe with diverging variants were collected from patients with a cross border (Brazil) travel history  did not reveal the “Palumeu haplotype” and no less than 9 variants could be demonstrated .Pvmsp-1 F2 gene (p<0.001) or just the Pvmsp-3α gene (p<0.001) would be considered.The utilized PCR-RFLP displayed adequate discriminating capacity as the strains from Sophie were significantly different from the strains collected in Palumeu, Apetina and Pelele Tepoe (p<0.001), even if only the Pvmsp-1 F2 showed a higher degree of diversity than Pvmsp-3α.It should be noted that Pvmsp-1 F2 gene (p<0.001), with a high prevalence for one specific RFLP-profile and also for the Pvmsp-3α gene, two more common RFLP-profiles could be observed in the non-uniform distribution, although borderline significant (p = 0.046).Interestingly, samples from Sophie exhibited a non-uniform distribution for the Complementary RFLP analysis was performed on paired isolates from previous episodes from two patients from Sophie  from Pelele Tepoe.The malaria cases observed in Apetina, the other indigenous village, also harbored the “Palumeu haplotype” implicating unregistered migration of people during an active malaria outbreak as cause for these individual cases.The comparison of PCR/RFLP profiles of specimens throughout the lengthy outbreak in Pelele Tepoe, depicted as trend line in These results corroborated the registered travel history, as no less than 60% of the first fifteen diagnosed cases, including the first diagnosed case in Pelele Tepoe had travelled to Palumeu.RFLP results in conjunction with the epidemiological data, especially travel history obtained during testing or treating also pointed to undetected human movement during an active malaria outbreak as trigger for this subsequent Pelele Tepoe outbreak with locally acquired secondary cases, contracted via primary index case(s) imported via Palumeu.It could be hypothesized that the prolongation of the outbreak with a second peak , despiteThis hypothesis is substantiated by the homologous character of the RFLP profiles of the paired isolates from the breastfeeding/pregnant women with recurring infections, the absence of indications for drug resistance and the finding that 54% of recurrent malaria episode(s) in the Pelele Tepoe outbreak constituted of persons not receiving Primaquine.However, it should be noted that even with correctly administered Primaquine treatment, relapse rates can range from 2.8–19.0% ,22.After the occurrence of the second peak in Pelele Tepoe, national precautions such as strict follow-up of pregnant women with biweekly microscopic monitoring till delivery and continued subsequent screening during breastfeeding were implemented in order to address reintroduction of malaria via possible relapses.The introduction of the additional measures may have been instrumental in the success of fully containing the further spread of the “divergent” allelic variants, presumably imported from Brazil, as none of the other survey samples displayed the “divergent” haplotypes even though one of the returning travelers did not receive Primaquine, due to a pregnancy. The efficient suppression of the small-scale post-outbreak in Palumeu in May/June 2021, also involving two pregnant/breastfeeding women, provided further support for this notion.The use of RFLP as molecular tool enabled reconstruction of the outbreak in Pelele Tepoe, elucidated the correlation between parasite strains circulating in the three indigenous villages and aided in the determination of the effectivity of measures implemented during the outbreak.P. vivax malaria in the indigenous villages, thus requiring even more stringent malaria control and monitoring in all villages consisting of restricted travel during outbreaks and strict visitor-screening (including returning villagers), a strong awareness and monitoring system of symptoms in the community, especially for pregnant and breastfeeding women and early molecular characterization to guide the roll-out of interventions.Our findings also implicated unregistered human movement and relapses as triggers for the reintroduction and resurgence of The unexpected phenomenon of a seemingly stable single-clone, throughout a period of more than two years with circulation in three indigenous villages, infecting and multiplicating in more than 250 persons, demonstrated significant inbreeding and infrequent or lack of recombination between heterologous parasite genotypes.P. vivax hypnozoite reservoir in Suriname, in contrast to the observations of maintained high levels of genetic diversity in other low-endemic areas as Sri Lanka [Even multiple relapses in different individuals and drug pressure exerted by several MDA events presumably did not add to the gene pool of the parasite, suggesting a limited ri Lanka .P. vivax hypnozoites through repeated P. vivax inoculations.The highly heterogeneous parasite population in the Sophie area in French Guiana  was in sThe findings in the indigenous villages on one hand and Sophie on the other hand also corroborated the common perception that declining transmission leads to a decreased genetic diversity, most likely due to diminished recombination between genetically distinct clones.A more detailed spatial clustering of allelic variants in Sophie was prohibited, due to the limited number of samples from Sophie and the unreliable travel histories from miners.Reduced genotyping accuracy as the common limitation of RFLP genotyping in areas of high- transmission with a high parasite density and high multiplicity of infection  was not Notwithstanding the use of only two loci in the investigation, the findings from this study further substantiated the usefulness of PCR-RFLP as alternative for parasite genotyping analyses in hypo-endemic settings.Pvmsp-1 F2 and Pvmsp-3α genes.The results from this study clearly demonstrated the differentiating capacity and therefore the usefulness of the utilized PCR-RFLP method with the P. vivax genetic diversity and for identification of imported cases or potential foci.Therefore, we recommend the use of RFLP as molecular alternative for low endemic areas with restricted resource settings, especially for outbreak investigations, for monitoring of the spread or containment of circulating strains, for assessments of the degree of P. vivax malaria in hypo-endemic settings and we strongly advocate for stringent and timely malaria prevention, control and monitoring measures during outbreaks, including early molecular characterization. Implementation of such measures will not only limit or prevent the reintroduction of malaria in near elimination areas, but will also restrict overall genetic diversity and impede the global spread of drug resistance.Our findings highlighted the impact of human movement and relapses on the reintroduction and resurgence of S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Plasmodium falciparum infections. However, its diagnostic performance has not been evaluated in the Democratic Republic of Congo (DRC). This study aims to determine the performance of the usRDT in malaria diagnosis in asymptomatic individuals under field condition in Kisangani, Northeast of DRC.Ultrasensitive rapid diagnostic test (usRDT) was recently developed to improve the detection of low-density Plasmodium histidine rich protein 2 (HRP2) antigen as a proxy for the presence of P. falciparum. The diagnostic performance of the usRDT was compared with cRDT, microscopy and PCR.A community-based cross-sectional study was carried out from June to August 2022 on 312 asymptomatic individuals residing in the city of Kisangani. Capillary blood samples were collected by finger prick for microscopic examination of thick and thin blood film, RDTs, and nested polymerase chain reaction (PCR). Alere™ Malaria Ag P.f usRDT and conventional RDT  kits were used for the detection of P. falciparum malaria was 40.4%, 42.0%, 47.1% and 54.2% by cRDT, microscopy, usRDT and PCR, respectively. By using PCR as a reference, usRDT had sensitivity and specificity of 87.0% (95% CI 81.4–91.7) and 100.0% (95% CI 97.5–100.0), respectively, whereas the cRDT had sensitivity and specificity of 74.6% (95% CI 67.3–80.9) and 100% (95% CI 97.1–100.0), respectively. By using microscopy as a reference, usRDT had sensitivity and specificity of 96.9% (95% CI 92.4–99.2) and 89.0% (95% CI 83.5–93.1), respectively, while the cRDT had sensitivity and specificity of 96.2% (95% CI 92.3–98.7) and 100% (95% CI 97.9–100.0), respectively.The prevalence of asymptomatic The usRDT showed better diagnostic performance with higher sensitivity than the cRDT which is currently in use as point-of-care test. Further research is necessary to assess the access and cost-effectiveness of the usRDTs to use for malaria surveillance. Plasmodium falciparum is the predominant cause of malaria in the country [Malaria remains a global public health concern, affecting hundreds of millions of people each year. In 2021 alone, an estimated 247 million cases and 619,000 deaths due to malaria were reported globally. Most of the cases (95%) and deaths (96%) were in the African region [ country .Vector control using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), and prompt detection and treatment of symptomatic malaria cases are the key strategies widely used for malaria prevention and control in Africa . The scaOne of the major challenges to malaria control and elimination efforts in sub-Saharan Africa is asymptomatic malaria, which is highly prevalent in many African settings including DRC –12. AsymP. falciparum infections [However, the effectiveness of ACD is limited due to the inability of the commonly used malaria diagnostic tools to detect low density asymptomatic infections –20. Micrfections –24. Molefections –27. Howefections , 28.P. falciparum usRDT) has recently been developed for the detection of P. falciparum infections [P. falciparum infections in earlier days than the cRDT [To overcome the limitation of the cRDTs, ultra-sensitive RDT  and perennial transmission of malaria in the area [A community-based cross-sectional study was carried out in selected communes of Kisangani from June to August, 2022. Kisangani is a capital city of the province of Tshopo in the Northeast of the DRC Fig. . It is l1602,144 . The cli. [The sample size was calculated using the method of Buderer et al. . First, zambique . The nexThe city of Kisangani is administratively subdivided into six communes. This study covered three communes selected randomly: Kabondo, Makiso and Kisangani. The sample size was proportionally allocated to each commune based on their population size. A systematic sampling technique was used to select 312 households, and one individual was randomly selected per household /LLINs the previous night. Prior to commencement of the study, a pretest was conducted in a neighboring commune on five percent of the total sample size, and the questionnaire was further modified after the pretest. The axillary temperature was measured using an electronic thermometer before each blood sample collection and fever was defined as a temperature ≥ 37.5 °C. Finger-prick blood sample was collected from each study participant for RDTs, microscopic examination of thick and thin blood film, and PCR. For the PCR, 2–3 dry blood spots (DBS), each with 50µL of blood, was prepared on Whatman 3 MM filter paper for each study participant. Blood sample collection from all study participants was done by experienced laboratory technologists.P. falciparum HRP2 according to the manufacturer’s instructions. For both RDT kits, 5 µL of capillary whole blood was applied to sample well, followed by addition of four drops of assay buffer. The test results were read after 15 min for the cRDT and after 20 min for the usRDT. A positive signal (red color) on both control and test lines was considered as a positive test result for P. falciparum, and a positive control line without signal on the test line was interpreted as a negative result. Any test result without a signal on the control line was considered an invalid test and the test was repeated in this case. All individuals found positive for malaria by RDTs were advised to visit the nearest health facility for treatment.Both cRDT  and usRDT  were used to test for Thick and thin blood smears were made on the same slide, and the thin smears were fixed by absolute methanol. The slides were then transported to the Kisangani provincial laboratory and stained with 10% Giemsa following standard operating procedures . All sliP. falciparum by PCR. DNA was extracted from the DBS using the QIAamp DNA Mini Kit , following the manufacturer’s instructions.The collected DBS were transported to the Institut National de Recherche Biomédicale (INRB) in Kinshasa to test for the presence of P. falciparum as previously described elsewhere [P. falciparum (rFAL1: 5′-TTAAACTGGTTTGGGAAAACCAAATATATT-3′ and rFAL2: 5′-ACACAATGAACTCAATCATGACTACCCGTC-3′) were used [Plasmodium falciparum infections detected by PCR but not by microscopy were classified as sub-microscopic infections [A nested PCR was used to amplify species-specific sequences of the small sub-unit ribosomal ribonucleic acid (18S SSU rRNA) genes of lsewhere , 49. In fections .Data were checked for completeness and consistency, coded and entered into Epi-info version 7.0, and exported into the Statistical Package for Social Science version 25  and JASP 0.16  for further analysis. The basic characteristics of the study participants were summarized using descriptive statistics.Sensitivity, specificity, positive predictive value, and negative predictive value of cRDT and usRDT were determined using microscopy and PCR as reference methods . The 95%Most of the study participants (94.2%) were over the age of 15 years, with mean age of 25.4. The majority (54.5%) of the participants were females. Study participants from Makiso, Kabondo and Kisangani represent 34.4%, 34.3% and 31.3%, respectively. Most (94%) of the study participants reported that they had at least one LLIN in their house and 79% of them reported to have used LLINs the previous night. Only 4.2% of the study participants reported that IRS was applied to their houses in the past 6 months. Malaria prevalence by cRDT, microscopy, usRDT and PCR was 40.4%, 42.0%, 47.1% and 54.2%, respectively  and 89.0% (95% CI 83.5–93.1), respectively. The cRDT had sensitivity and specificity values of 96.2% (95% CI 91.3–98.7) and 100% (95% CI 97.9–100.0), respectively. The level of agreement between microscopy and both RDT types was significant, with a strong agreement documented between usRDT and microscopy , and almost perfect agreement between cRDT and microscopy   had low parasitaemia whereas 3.0% of them had moderate parasitaemia. The mean parasitaemia was 541.30 (± 300.32) p/µL for cRDT positive results with minimum and maximum values of 78 p/µl and 2678 p/µL, respectively. Regarding the usRDT positive results, the mean parasitaemia was 467.62 (± 334.62) p/µL with a minimum parasitaemia of 0 p/µL and a maximum parasitaemia of 2678 p/µL.The performance of the two RDT kits in terms of sensitivity was higher in people with moderate parasitaemia compared to people with low parasitaemia. For the usRDT, the sensitivity was 100% and 86.6% (95% CI  80.4–91.4) for moderate and low parasitaemia cases, respectively, whereas for the cRDT, the sensitivity was 100% and 73.8% (95% CI  66.4–80.3) for moderate and low parasitaemia cases, respectively . The false negative results recorded by both usRDT and cRDT might be due to the presence of low parasite density below their respective limit of detection. The presence of parasites with  results –57, alth results .P. falciparum can persist in the circulation for weeks after an infection is cleared by treatment, and this can give false positive RDT results even in the absence of active infection [Studies have shown that HRP2 antigen of nfection , 60. In nfection . The lownfection , 61, 62 P. falciparum in samples with parasitaemia of up to 183 p/uL [P. falciparum infections than the cRDT. This suggests that the usRDT could be a potential diagnostic tool for screening asymptomatic and submicroscopic Plasmodium infections for prompt case treatment especially in settings targeted for malaria elimination.The relative sensitivity of the two RDT types varied based on parasite density. The usRDT exhibited significantly higher sensitivity than the cRDT in detecting low parasitaemia infections, whereas both usRDT and cRDT showed 100% sensitivity for moderate parasitaemia. Other studies have also documented similar findings , 29. In 183 p/uL , confirmP. falciparum infection was high, around 54.2% by PCR, in the study area despite high LLIN ownership. A multi-center study conducted in 2016 in DRC by Mvumbi et al. has also reported an overall malaria prevalence of 43% using PCR, and a prevalence rate of over 63% in Maniema region near Tshopo district, where the city of Kisangani is located [P. falciparum infections was 12.5% in the study area. This implies that malaria surveillance system should be strengthened in the area using sensitive diagnostic tools such as usRDT in order to accurately identify malaria hotspot areas to prioritize control intervention based on evidence.It is worth mentioning that the prevalence of asymptomatic  located . Thus, t located . The higThe limitation of this study is that parasitaemia was quantified using microscopy and parasite density was not determined for PCR positive samples which were negative by microscopy.P. falciparum infections among asymptomatic individuals in a high malaria transmission setting of Kisangani. This is the first study that evaluated the performance of usRDT in DRC and generated evidence to guide policy and the program on the implementation of this tool to diagnose asymptomatic individuals. This study also documented high prevalence of asymptomatic P. falciparum infections among communities residing in the city of Kisangani. Further research is necessary to assess the impact and cost-effectiveness of the usRDTs to be used in the health care system.In this study, usRDT showed higher sensitivity than cRDT in detecting


Subgroup analyses indicated that regional disparities, especially between Africa and Asia, and participant age groups may influence these outcomes. While a trend of decreased calcium levels in malaria patients was observed, the meta-analytical results suggest regional and age-related variations. Further investigations should emphasize these differences to better guide clinical management, prognostic applications, and the crafting of policies concerning malaria’s metabolic effects.Despite several studies examining the relationship between calcium levels and malaria, inconsistencies and varied results remain in the literature. This study aimed to synthesize the evidence on the association between blood calcium levels and malaria severity. A systematic literature search was conducted in the Embase, Scopus, PubMed, Ovid, and Google Scholar databases. The studies that investigated calcium levels in participants with malaria were reviewed and included for synthesis. The quality of included studies was assessed based on a standardized checklist by the Joanna Briggs Institute (JBI) critical appraisal checklists. The thematic synthesis had been used for qualitative synthesis. For the quantitative synthesis, the meta-analysis was performed to estimate the pooled effect sizes for differences in calcium levels between groups of participants using a random effect model using Hedge’s g as a measure of effect size. Out of the 4574 identified records, 14 studies were reviewed. The thematic synthesis across these studies noted a consistent theme: reduced calcium levels in malaria patients compared to uninfected controls. However, the meta-analysis encompassing three specific analyses—comparing calcium levels between malaria patients and controls, severe and non-severe malaria cases, and fatal cases versus survivors—showed no significant difference in calcium levels. The statistics were as follows: (1)  Plasmodium and is transmitted through the bite of an infected female Anopheles mosquito [P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi—each presents varying severity and clinical manifestations [P. falciparum is the leading cause of severe malaria and related deaths. However, other species can occasionally cause severe forms of the disease [Malaria remains a significant global health challenge, with 247 million cases and 625,000 deaths in 2021, predominantly in sub-Saharan Africa [ disease ,4. UnderCalcium is a vital mineral with diverse physiological roles, from contributing to bone health to facilitating intracellular signaling, blood clotting, muscle contraction, and nerve function ,6,7. ItsWhile several studies have explored the relationship between calcium levels and malaria, inconsistencies and varied results persist in the literature ,21,22. TThe systematic review has been registered with PROSPERO under the code CRD42023464711. A systematic literature review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [The outcomes of the study were the differences in blood calcium levels between: (i) patients with malaria and uninfected individuals; (ii) patients with severe malaria and those with non-severe malaria; and (iii) fatal malaria cases and survivors.Plasmodium infection, severe, or fatal malaria; ‘C’ denoted uninfected participants, those with less severe malaria, or survivors; and ‘O’ corresponded to blood calcium levels.The Population, Exposure, Comparator, Outcome (PECO) framework was used to develop the systematic review question . ‘P’ repA systematic literature search was conducted across four primary databases: Embase, Scopus, PubMed, and Ovid. The search strategy, which included the terms ‘calcium’ AND ‘malaria’ OR ‘Plasmodium’ OR ‘Plasmodium Infection’ OR ‘Remittent Fever’ OR ‘Marsh Fever’ OR ‘Paludism’, was employed to identify studies investigating the relationship between calcium levels and malaria. The search terms and their synonyms were identified using Medical Subject Headings (MeSH). The search strategy varied slightly across different databases see . StudiesStudies were eligible for inclusion if they (i) investigated calcium levels in participants with malaria; (ii) compared calcium levels between patients with malaria and uninfected individuals or investigated the relationship between calcium levels and severity or type of malaria; (iii) were cross-sectional, case-control, cohort, or clinical trials. Reports were excluded if they were in vitro, animal, proteomic, in silico/docking, computational, genomic studies, reviews, case reports, lacked relevant data or if the data were impossible to extract.Plasmodium species studied, participant demographics, symptomatology, severity of malaria, methods for Plasmodium identification, and the blood sample used for calcium measurements. Two independent reviewers selected studies based on eligibility criteria and extracted data from the included studies, with any disagreements being resolved through discussion.Study selection began with the removal of duplicates, and then titles and abstracts of articles were screened. Full-text articles of relevant articles were assessed, leading to the exclusion of non-relevant articles for specific reasons. Extracted information encompassed study characteristics like publication year, design, geographical location, The quality of included studies was assessed based on a standardized checklist by the Joanna Briggs Institute (JBI) critical appraisal checklists . For croPlasmodium species, age groups, and disease severity. For the quantitative synthesis, calcium levels and their variability measures  were extracted. When these values were not directly provided, they were calculated or estimated using available data as described previously [I2 statistic, which describes the proportion of the total variation across studies that is due to heterogeneity rather than chance [I2 statistic, where values exceeding 50% suggest significant heterogeneity [Plasmodium species, symptoms, methods for Plasmodium identification, and blood samples for calcium measurement were assessed in the meta-regression analyses. Subgroup analyses were performed to explore potential effect modifiers. A leave-one-out meta-analysis approach was employed to understand the impact of each individual study on the overall pooled effect estimate [p-values less than 0.05 considered statistically significant.The thematic synthesis  had beeneviously . For staeviously . Hedge’sn chance . The hetogeneity . The metestimate . This anA total of 4574 records from databases were identified: 1622 from Embase, 1695 from Scopus, 937 from PubMed, and 320 from Ovid. Before screening, 2355 duplicate records were removed, leaving 2219 records for screening. Upon screening, 1783 records were excluded for specific reasons: 1084 were not related to the participants of interest, and 699 were not related to the outcome of interest. This led to 436 reports being sought for retrieval. Out of these, 3 reports could not be retrieved, resulting in 433 reports assessed for eligibility. Among the assessed reports, 423 were excluded for the following reasons: 270 were in vitro studies, 46 were reviews, 44 were animal studies, 17 were proteomic studies, 14 had no information on calcium, 13 were in silico/docking studies, 6 were computational models, 4 were genomic studies, 4 were case reports, 3 had data on calcium that were impossible to extract, and 2 lacked calcium data in comparison groups. Besides the 10 reports from the main databases that met the eligibility criteria, 4 reports were found from Google Scholar. In total, 14 studies were included in the review ,39,40,41Plasmodium species, a significant majority (71.43%) focused on P. falciparum, 14.29% on mixed infections of P. falciparum and P. vivax, 7.14% on both P. falciparum and P. vivax, and 7.14% did not specify the species. The participant demographics were predominantly adults at 50%, children made up 35.71%, and a combination of children and adults represented 14.29%. Symptomatic cases constituted 78.57% of the studies, while the severity of the malaria was distributed among severe malaria (21.43%), a combination of severe and non-severe malaria (42.86%), non-severe cases such as uncomplicated, mild, or asymptomatic malaria (7.14%), and 28.57% did not specify the severity. The methods of Plasmodium identification primarily utilized the microscopic method at 64.29%, with a combination of microscopic and RDT in 14.29%, and microscopic combined with quantitative buffy coat in 7.14%. The blood samples used for calcium were mainly serum (64.29%) and plasma (35.71%)  were cross-sectional, 28.57% were case-control studies, and both the cohort studies and clinical trials each made up 7.14%. Geographically, the studies were equally divided between Asia and Africa, representing 50% each. In Asia, the breakdown was as follows: India (21.43%), Vietnam (14.29%), Thailand (7.14%), and China (7.14%). For the African region, Nigeria was the most represented at 21.43%, followed by Cameroon at 14.29%, with Sudan and Kenya each contributing 7.14%. According to (35.71%) .For eight cross-sectional studies, one study had certain ambiguities regarding confounding factors but was generally clear in other areas . Two stuP. falciparum as the causative Plasmodium species in malaria. The studies in Asia, such as in Thailand [P. falciparum and P. vivax infections, with some mixed infections. For the difference in calcium levels between patients with malaria and uninfected controls, a notable theme across many studies, irrespective of continent, was the significant decrease in calcium levels in malaria patients compared to uninfected controls. This observation was consistent in both children and adults in a group of studies [For geographical patterns, the studies in Africa, such as in Sudan , NigeriaThailand , China [Thailand , VietnamThailand ,41, and Thailand ,39,40, o studies ,31,32,41 studies ,35 foundPlasmodium species.For the disease severity and calcium levels, the study from Nigeria showed a correlation between disease severity and calcium levels, with the latter decreasing with increasing severity from mild to severe malaria . Contrarp = 0.15, Hedge’s g: −1.00, 95% CI: −2.37–0.38, I2: 98.97, 9 studies, Using data from nine studies that reported mean/median and standard deviation/range of calcium levels ,36,38,41Plasmodium species, clinical status, diagnostic method for Plasmodium species, and blood samples for calcium measurements as covariates. The results indicated that both age group and Plasmodium species influenced the pooled effect estimate, suggesting these factors play a role in varying calcium levels in patients with malaria . A similar non-significance was observed across different study designs (p > 0.05). Geographically, studies from Africa displayed a significant pooled effect estimate (p = 0.02), whereas studies from Asia did not show any significant difference (p = 0.76). Regarding age groups, a significant pooled effect was observed in children (p = 0.02), but not in adults (p = 0.55). Finally, there was no significant difference in calcium levels among studies using various methods for Plasmodium identification or different blood samples for calcium measurement (p > 0.05, Subgroup analyses of calcium levels in patients with malaria, compared to uninfected controls, showed varied results across categories. For publication years, there was no significant difference in calcium levels across all year ranges (p = 0.35, Hedge’s g: −0.33, 95% CI: −1.02–0.36, I2: 81.61, 3 studies, Using data from three studies that reported on the mean/median and standard deviation/range of calcium levels ,38,40, tp = 0.71, Hedge’s g: −0.14, 95% CI: −0.91–0.62, I2: 87.05, 3 studies, Using data from three studies that reported on the mean/median and standard deviation/range of calcium levels ,35,37, tThe leave-one-out meta-analysis approach was employed to assess the influence of each individual study on the overall pooled effect estimate. By systematically excluding one study at a time and recalculating the pooled estimate, the results indicated variability in the pooled effect estimate, revealing the sensitivity of the meta-analysis to the exclusion of individual studies . The leaPlasmodium species appeared to significantly influence the pooled effect estimates, underscoring their potential role in dictating calcium dynamics in the context of malaria.The systematic review and meta-analysis aimed to explore the relationship between calcium levels and malaria, a relationship which has hitherto been underexplored in the literature. The review exposed distinct patterns, intricacies, and consistencies surrounding this relationship. A consistent theme emerged across studies from different continents; malaria patients, both children and adults, generally displayed decreased calcium levels compared to uninfected controls. This decrease may hold potential implications for patient management, although its clinical significance remains to be elucidated further. However, the uniformity in this observation was punctuated by a couple of studies which did not find significant differences based on disease severity or survival outcomes, emphasizing that factors beyond geographical location might influence this association. Interestingly, while individual studies showcased associations between disease severity and calcium levels, the meta-analysis results did not establish significant differences in calcium levels between patients with malaria and uninfected controls, or even between different severities of malaria or survival outcomes. The high heterogeneity observed across the studies could account for these findings. Factors such as age group and Plasmodium infection can lead to an increase in the urinary excretion of minerals, especially calcium [Plasmodium trophozoites are known to sequester calcium within their internal compartments for metabolic activities, potentially contributing to the observed hypocalcemia in patients with malaria [The decline in calcium levels may be linked to the clinical manifestations of malaria, which impact neuromuscular excitability, nerve transmission, and muscle contraction . The ren calcium . A signi calcium . Interes calcium . Moreove malaria . This ob malaria . Another malaria . Support malaria . Additio malaria . In rela malaria ,40, suggP. falciparum, which might be more prevalent in Africa than in Asia, leading to distinct clinical outcomes and influences on calcium metabolism. The second factor was the coexisting nutritional deficiencies in Africa, such as differences in the dietary intake of calcium or Vitamin D, could make certain populations more susceptible to hypocalcemia when infected with malaria [Subgroup analyses further refined the understanding of results obtained through the meta-analysis. While studies across different publication years and designs did not show significant variances in calcium levels, geographical distinction emerged as a pivotal factor. African studies revealed a significant reduction in calcium levels in patients with malaria, implying a more pronounced relationship between malaria and calcium levels in this region compared to Asia. Several factors could contribute to the more pronounced reduction in calcium levels in patients with malaria from one region compared to another, such as Africa versus Asia. The first factor was  malaria ,46,47. A malaria ,49,50. IThe subgroup analysis of age group showed that calcium levels seemed to be differentially influenced by age, with children showing a significant pooled effect compared to adults. The influence of age on calcium levels was observed in children who were infected with malaria parasites. The differential impact of malaria on calcium levels between children and adults is not fully understood. Calcium metabolism in children is characterized by rapid bone growth, high rates of bone turnover, and the demands of developing and maintaining healthy skeletal structures . During The sensitivity of our meta-analysis, as evidenced by the leave-one-out approach, underscores the influence of individual studies on the overall effect estimate. While this is a routine facet of meta-analyses, it also prompts a degree of caution in interpreting results. While the review offered a comprehensive outlook, it is essential to recognize the inherent limitations. The pronounced heterogeneity among the studies may obscure some finer nuances. Furthermore, it is important to recognize that another limitation of this study was the lack of uniformity in the specific methods used to evaluate hypocalcemia in children across the included studies. This variability in diagnostic approaches could introduce some heterogeneity into our findings, emphasizing the need for future research to standardize these methods to enable more robust comparisons. Given the variations in observations among individual studies and the absence of statistical significance in the meta-analysis results, future research may benefit from a more detailed exploration, potentially at a regional or population subgroup level.This systematic review and meta-analysis shed light on the relationship between calcium levels and malaria, revealing a consistent decline in calcium levels among malaria patients across various regions, with children being particularly susceptible. The findings highlighted the potential for calcium monitoring in clinical management and hint at its use as a prognostic biomarker for disease recovery. Notably, geographical differences suggested region-specific strategies might be required in managing malaria. Given the children’s unique vulnerability to hypocalcemia, specialized interventions might be necessary in pediatric populations. Despite the insights provided, marked heterogeneity among the studies underscores the need for further granular research, especially considering regional and age-related nuances. This work served as a foundational step, emphasizing the importance of exploring malaria’s metabolic and clinical impacts, which can guide future research and public health policies.Plasmodium species and nutritional aspects. The uniquely heightened vulnerability in children suggests potential developmental and metabolic intricacies that warrant further exploration. Future research should delve deeper into regional, age-based, and severity-associated nuances to better inform clinical management strategies, potential prognostic applications, and public health policies centered around malaria’s metabolic repercussions.This systematic review and meta-analysis have provided crucial insights into the previously underexplored relationship between calcium levels and malaria. Across multiple geographical regions, a discernible trend was identified: a decrease in calcium levels among malaria-infected individuals, with children exhibiting a pronounced susceptibility. The distinct regional variances, especially between Africa and Asia, hint at the complex interplay of factors, including the prevalence of certain


Plasmodium falciparum lysate was used to estimate age-specific seroprevalence among residents of southern Zambia, where malaria transmission has declined to pre-elimination levels during the past two decades. Plasma was eluted from 3,362 dried blood spots collected during five cross-sectional surveys conducted between 2009 and 2012, and again in 2018. Annual seroconversion rates (SCRs), an estimate of the force of infection, were calculated using a reversible catalytic model. The SCR decreased by two thirds from a level of approximately 0.15/year in 2009 and 2010 to approximately 0.05/year in 2011 and 2012, and then decreased 5-fold to 0.01/year by 2018, demonstrating the utility of serology in documenting progress toward elimination.Obtaining accurate malaria surveillance data is challenging in low-transmission settings because large sample sizes are required to estimate incidence and prevalence precisely. Serology is an additional tool to document progress toward malaria elimination. An enzyme immunoassay to  Malaria remains a threat to global health, with an estimated 247 million cases and 619,000 deaths attributed to malaria in 2021.Plasmodium falciparum schizont crude extract, and annual seroconversion rates (SCRs)—the rate at which seronegative individuals become seropositive—were calculated. A strong, positive correlation was observed between SCRs and entomological inoculation rates, demonstrating the utility of serological data in assessing malaria transmission intensity in low-transmission settings.Serological surveillance may offer an additional tool to estimate malaria transmission intensity and document progress toward malaria elimination in low-transmission settings.,Zambia has set ambitious goals for malaria elimination, originally by 2021.Anopheles arabiensis,Anopheles squamosus also contributing to transmission.The catchment area of Macha Hospital is located approximately 70 km north of the provincial capital of Choma and is mainly inhabited by subsistence farmers. The area is characterized by Miombo woodland, with a rainy season that lasts from early December through the end of March followed by a cool, dry season from April to July and a hot, dry season from August to November.P. falciparum histidine-rich protein 2 (PfHRP2)–based RDT , and blood samples were collected on Whatman 903 protein saver cards . Blood samples were dried overnight, packaged individually with desiccant, and stored at −20°C as dried blood spots.Between 2009 and 2012, 533 households in the catchment area of Macha Hospital in Choma District, Southern Province, Zambia, participated in serial cross-sectional, community-based surveys conducted every other month . Households were selected randomly without replacement, and thus households and individuals participated only once. All household residents were eligible for inclusion. Data were aggregated by calendar year as the unit of analysis. The sampling frame was based on satellite imagery as described previously.P. falciparum were measured using an enzyme immunoassay (EIA) to asexual parasite lysate of P. falciparum NF54. Plasma was extracted by soaking the dried blood spots in 5% skim milk and phosphate-buffered saline containing 0.05% Tween 20 for 1 hour at room temperature. IgG antibody levels to whole P. falciparum lysate were measured at 405 nm and expressed as the optical density (OD).P. falciparum–endemic areas.IgG antibodies to A previously reported reversible catalytic model was fitted to the age-specific seroprevalence data using Stata version 16  to estimate annual SCRs.–,,–Demographic characteristics of the study participants and parasite prevalence by PfHRP2-based RDT by year of enrollment are summarized in Table 2Anopheles funestus abundance dramatically.Reasons for the sharp decline in SCRs are not entirely clear, but likely are a consequence of malaria control interventions,P. falciparum–specific antigens such as merozoite surface protein 1 and apical membrane antigen 1.P. falciparum–specific IgG antibodies. Whole parasite lysate was prepared in-house, and the EIA was a relatively low-cost and easy-to-implement assay to measure changes in age-specific seroprevalence in this low-transmission setting. A serological assay that measures antibodies to a broad range of parasite antigens may be a sensitive measure of exposure to P. falciparum. A serological survey conducted in the same study area in southern Zambia from 2013 to 2015 using a protein microarray revealed the breadth and depth of IgG antibody responses to 500 different P. falciparum peptides,P. falciparum in low-transmission settings, an EIA using whole parasite lysate may be sufficient.Many serological studies of malaria used a selected set of As malaria transmission declines, commonly used diagnostic tests such as RDTs or microscopy fail to detect prevalent infections. The use of polymerase chain reaction testing becomes cost prohibitive because of the large sample size requirements for precision. Unlike polymerase chain reaction testing, which detects active infections, serosurveys can detect past infections, alleviating the issue of sample size. Serosurveys using an EIA described in this article could be performed readily by national malaria control programs, and information from serial cross-sectional serosurveys can provide an additional quantitative measure of declining malaria transmission intensity in pre-elimination settings.


Schistosoma infections and the risk of anaemia among school children in the Tono irrigation area in the Kassena Nankana East Municipal (KNEM) in the Upper East Region of Northern Ghana.Malaria and schistosomiasis persist as major public health challenge in sub-Saharan Africa. These infections have independently and also in polyparasitic infection been implicated in anaemia and nutritional deficiencies. This study aimed at assessing asymptomatic malaria, intestinal Schistosoma eggs in stool. Finger-prick capillary blood sample was used for the estimation of haemoglobin (Hb) concentration and blood smear for malaria parasite detection by microscopy.A cross sectional survey of 326 school children was conducted in the KNEM. Kato Katz technique was used to detect Schistosoma mansoni was 32.0% (n = 104) and 25.2% (n = 82), respectively with 7.7% (n = 25) coinfection. The prevalence of anaemia in the cohort was 40.5% (95%CI [35.3–45.9]), of which 44.4% harboured at least one of the parasites. The prevalence of anaemia in malaria parasite spp or S. mansoni mono-infections was 41.8% and 38.6%, respectively and 64.0% in coinfections. There was no statistically significant difference in the odds of being anaemic in mono-infection with malaria  or S. mansoni  compared to those with no infection. However, the odds of being anaemic and coinfected with malaria parasite species and S. mansoni was 3.03 times higher compared to those with no infection .The average age and Hb concentration were 10.9 years  and 11.2 g/dl (SD: ± 1.39) respectively with 58.9% (n = 192) being females. The overall prevalence of infection with any of the parasites (single or coinfection) was 49.4% . The prevalence of malaria parasite species or ConclusionS. mansoni infection and anaemia among school children in the irrigation communities. The risk of anaemia was exacerbated by coinfections with malaria parasite(s) and S. mansoni. Targeted integrated interventions are recommended in this focal area of KNEM.The data show a high burden of malaria,  Plasmodium falciparum, is prevalent across Ghana at varied levels of endemicity. In the Kassena Nankana East Municipal in the Upper East Region of Northern Ghana, malaria transmission is highly seasonal [Malaria and schistosomiasis both constitute a serious public health challenge and are the topmost parasitic infections causing a significant amount of morbidity and mortality globally –4. In suseasonal , 14.Schistosoma infections are also common and widespread with an estimated country-wide schistosomiasis prevalence of 23.3% and focal prevalence levels exceeding 50% in some areas [Schistosoma parasites revolves around freshwater snails as an intermediate host, which releases the infective form  into fresh water, leading to human infection through contact with the contaminated water. Schistosoma infection in most parts of SSA and in Ghana is either intestinal, caused by Schistosoma mansoni or urogenital caused by Schistosoma haematobium [S. mansoni and S. haematobium have been previously reported [S. mansoni was found to be 54.2% in the south-western corridor of the district where irrigation canals with intermittent reservoirs supply water from the Tono dam to irrigate farmlands downstream in communities close to the irrigation scheme, with childhood activities making it easy for contact with snail infested stagnant waters and increasing the risk of infections.In Ghana, me areas . The lifmatobium , 15. In reported . The preMalaria and schistosomiasis collectively and independently have been implicated in nutritional stress among the people they infect –21. SuchMalaria leads to lower concentration of Hb levels and anaemia through mechanisms such as the destruction of infected red blood cells (RBCs), reduction in non-infected RBCs life span and suppression of bone marrow resulting in decreased erythropoiesis –32. SchiSchistosoma infection control has been implemented since 2008 mainly among school-age children (SAC) with water, sanitation and hygiene (WASH) campaigns to improve hygiene practices [To mitigate the effect of these parasitic infections, in Ghana, malaria control programmes are implemented including the use of insecticide-treated bed nets (ITNs), indoor residual spraying (IRS), and a test and treat policy for febrile illnesses with artemisinin-based combination therapy , 35. Masractices . Despiteractices . This stS. mansoni infection, and had low haemoglobin were referred to the C. K. Tedam University Hospital, Navrongo for further evaluation and management. According to the current Ghana Health Service guidelines, asymptomatic infections are not eligible for treatment. However, children who tested positive for malaria were advised to visit the nearest Health Centre for further evaluation and treatment as per Ghana Health Service protocols if they go on to develop fever.Ethical approval for the use of human participants in this study was obtained from the Institutional Review Board of the Navrongo Health Research Centre. Written informed consent was obtained from parents' or guardians of student participants attending the selected schools. Additionally, for children who were ≥ 10 years, an additional assent was obtained. Permission was obtained from the District Education Office and from the Headteachers of the selected schools. Children who tested positive for Between September and October 2022, a cross sectional survey was conducted in schools located in five communities  in the Kassena Nankana East Municipal (KNEM) located either close to the Tono dam or irrigated lands Fig. . School-Participants were recruited from all five primary schools within the KNEM located in communities along the Tono irrigation scheme. Each time a school was visited, the study protocol and rationale were explained and only children resident in the area for at least the past six months were eligible for recruitment. Informed consent was obtained from the parent/guardian and prior to sample collection and recruitment. Participants’ recruitment and enrolment is illustrated by Fig. Demographic information, sanitation and water contact activities  were collected using a structured questionnaire. The SAC were also asked if they saw blood in their stool in the past days to few weeks. All completed data forms were manually checked for completeness and inconsistencies. The data was then entered into EpiData version 3.1.Haemoglobin (Hb) concentration was determined in the field using a portable Urit-12 haemoglobin meter  as per the manufacturer’s instructions. In brief, a drop of capillary blood obtained from a finger prick was collected to fill the microcuvette and inserted into a well-calibrated photometer. The haemoglobin concentration was measured in g/dl and recorded instantly.In addition, thin and thick blood films were also prepared from the finger prick capillary blood and allowed to air dry. The prepared slides were transported to the C. K. Tedam University Hospital laboratory. The thin film was fixed with methanol for a few seconds. Both blood films were then stained with 10% Giemsa stain for 15 minutes after which they were gently rinsed with buffered tap water for about 10 seconds and allowed to air dry . The sta., 1972 [S. mansoni eggs and other intestinal parasites.Stool samples collected were transported to the CKT-UTAS university hospital laboratory, Navrongo, in a pre-chilled cold box with ice packs (2–8 °C) for analysis. From each sample, Kato-Katz thick smear was prepared according to the techniques previously described by Katz et al., 1972 . In brieTo ensure quality control, 10% of the Kato-Katz thick smear slides and those of malaria thin and thick smears were crossed examined by two independent experienced microscopists from the Navrongo Health Research Centre.S. mansoni eggs per gram (Epg) of stool, the egg counts were multiplied by 24 standard constants. The intensity of S. mansoni infection was considered light, moderate, and heavy if the egg load was 1–99 Epg, 100–399 Epg, and ≥ 400 Epg, respectively [S. mansoni only; and (iv) coinfection of malaria parasite species and S. mansoni.Data analysis was done using GraphPad Prism (version 9.0.0) and STATA (version 17.0) statistical software. Malaria parasite counts were converted to parasite density/μL of blood assuming 8000 WBCs/μL of blood. Malaria parasite intensity was classified as light infection (1–499 parasites/μl of blood), moderate infection (500–1999 parasites/μl of blood) and heavy infection (≥ 2000 parasites/μl of blood) [ectively . Infectiχ2) or Fisher Exact test was used to compare proportions of categorical variables such as sociodemographic factors, prevalence of malaria infection, S. mansoni infection and anaemia. To allow for estimations of the odds ratio between the infection groups and anaemia, haemoglobin concentrations were categorised into normal, moderate and severe anaemia. To investigate predictors of anaemia, a logistic regression model was fitted. To evaluate the association of SAC’s activities and demographic factors with the risk of S. mansoni infections, a separate logistic regression model adjusting for the location of schools was implemented. Estimates were considered statistically significant with an alpha value less than 0.05.The Chi-square test . The basic demographic characteristics of the study participants are presented in Table χ2 test, p < 0.001), with the majority being farmers . The source of drinking water for most of the participants was tap or borehole water . Open bush defecation was prevalent  in the area and similar across the sites.A total of 326 primary school children were screened in this study for malaria and Overall, 65.5% (n = 213) of the children in the area had access to the canals or used the dam for activities, such as bathing/swimming , domestic purposes  and farming . Among the participants, 17.8% (n = 58) knew about schistosomiasis and its prevention measures, and 32.2% (n = 105) and 9.2% (n = 30) reported having previously seen blood in their stool or urine respectively from days to few weeks prior to the study.Plasmodium falciparum (98%) being the predominant malaria parasite species detected. In few cases, Plasmodium malariae (1%) and mixed infection of P. falciparum and P. malariae (1%) were also detected. The prevalence of malaria parasite carriage was higher in males, 37.3% compared to females, 28.3% but the difference was not statistically significant (p = 0.085)  carry heavy (81.8%) infections compared to older children (14.8%).Malaria parasite carriage was 32.0% (95% CI 27.1–37.3) with Schistosoma mansoni infection prevalence was 25.2% (95% CI 20.5–30.2) with male participants having significantly high prevalence compared to their female counterparts   as shown by Fig. 16) Fig. A. Althou16) Fig. B. The vaS. mansoni  and 13–17 years  compared to those aged 5–8 years old. The differences were, however, not statistically significant. Children who visit the canal or dam were at 3.07 times  increased risk of being infected with S. mansoni compared to children who do not visit the stream. Childhood activities performed in the stream such as farming , bathing or swimming in the stream  and performing domestic chores  all increased the risk of children getting infected with S. mansoni.Compared to girls, boys had 1.98  increased risk of being infected with p = 0.163). Moderate anaemia was detected across all the school communities except Nangalikinia where 1.4% of severe anaemia was detected. The prevalence of anaemia across the schools was not significantly different (p = 0.69).The average haemoglobin (Hb) concentration was 11.16 g/dl . The proportion of anaemia cases among the children was 40.5% (95% CI 35.3–45.9); 40.2% were moderate and 0.3% were severe anaemia  was 49.4%  with 7.7% (n = 25) coinfection. The prevalence of anaemia among children with no infection was 36.7% (95%CI 29.4–44.6) and 44.4% (95%: 36.5–52.4) among children with infection   compared to those with no infection but this did not reach statistical significance. However, the risk of anaemia among children that were co-infected with the malaria parasite(s) and S. mansoni was 3.03  compared to those with no infection.The risk of anaemia was 22% higher  in males compared to females although it was not statistically significant  nearly half (49.4%) of the school children screened asymptomatically harboured at least one of the parasites or both (ii) malaria, intestinal schistosomiasis and anaemia were highly prevalent in the area (iii) comorbidity of malaria and intestinal schistosomiasis was common and (iv) comorbidity was associated with increased risk of anaemia among school children in the communities surveyed.The area is hyper-endemic for malaria and one out of 3 (32.0%) children harboured the malaria parasites but were largely asymptomatic. This is similar to what was reported in the Kassena Nankana West District, an adjourning district in 2022 but differs from elsewhere in the country , 11, 42.S. mansoni. This is not surprising because open defecation was widely practiced and over 65% of them had access to stagnant water in the irrigation canals and reservoirs. This is coupled with a poor knowledge level of the disease. The area is endemic to S. mansoni. According to the World Health Organization, areas with Schistosoma infection ≥ 10% and < 50% are considered to be moderately endemic and require preventive control measures such as mass drug administration (MDA), water, sanitation and hygiene (WASH) and vector control [S. mansoni in this irrigated area was previously reported to be 54.2% in 2014 [In addition to the asymptomatic carriage of malaria infections among the school children, over a quarter (25.2%) of the children were infected with  control . In Ghan control . MDA wit in 2014  suggesti in 2014 , 16. DueS. mansoni infections did not independently increase the risk of anaemia significantly in the study populations similar to previous observation with malaria and S. haematobium in an adjourning district [S. mansoni infections [S. mansoni. Notwithstanding the lack of association between mono-infections with malaria parasite or S. mansoni, children with concurrent infection of the asymptomatic malaria parasite(s) and S. mansoni were at three-time high risk of anaemia. This finding was corroborated by other studies that indicate that the interaction between multiple parasitic infections can increase the risk of anaemia [S. mansoni infections [The prevalence of anaemia among the children was high with a number of them moderately anaemic. This is similar to other studies of similar age groups elsewhere , 20. Thidistrict . This fifections , 29, 51.fections , 52–54.  anaemia , 19–22.  anaemia –59. The  anaemia . Quite rfections .Anaemia has effects on growth and development and cognitive abilities, which requires urgent attention , 61, 62.S. mansoni infections were common among school children attending school in communities located along the Tono irrigation area. The study further revealed that anaemia was prevalent among school children mediated by concurrent infection of malaria parasites and S. mansoni. This suggests that children presenting with anaemia in this area may require more than just a malaria diagnosis to ensure children are free from the debilitating consequence of anaemia. The high level of anaemia among the children in this study is also suggestive of poor nutrition and poor health in the area. Therefore, in addition, MDA with PZQ campaigns should integrate malaria control and nutritional interventions to help reduce the current prevalence of asymptomatic malaria carriage and anaemia.Overall, the findings of this study reveal that asymptomatic carriage of malaria parasites and, S. mansoni infection and anaemia include: (i) the effect of other parasitic infections such as soil-transmitted helminths (STH) that was not assessed; (ii) non-infectious aetiology of anaemia such as malnutrition was not evaluated; (iii) information on the cytokine profile of the infected participant was not assessed. Therefore, a firm conclusion could not be made on the correlation between concurrent malaria and Schistosoma infections and the risk of anaemia; (iv) the scope of the current study is limited to school children aged 5–17 years and may not be adequate for use to plan community interventions; (v) although malaria microscopy and Kato Katz techniques are the gold standards for malaria and S. mansoni diagnosis respectively, PCR technology could increase the sensitivity of detection since all the participants were asymptomatic and parasite load could be below the threshold for microscopy but PCR was not used due to resource constraints.Key limitations of this study which investigated asymptomatic malaria parasite carriage,


Plasmodium falciparum and, to some extent, in people who were exposed to medications like quinine and mefloquine. The exact pathogenesis of classic BWF remains unclear. The mechanism leading to damage to the red blood cells (RBCs) can be immunologic nonimmunologic, leading to massive intravascular hemolysis. Case Presentation. We present a case of classic blackwater fever in a 24-year-old otherwise previously healthy male without any history of antimalarial prophylaxis use, returning from recent travel to Sierra Leone. He was detected to have P. falciparum malaria in the peripheral smear test. He was treated with artemether/lumefantrine combination therapy. Unfortunately, his presentation was complicated by renal failure and was managed with plasmapheresis and renal replacement therapy.  Blackwater fever (BWF) is a severe clinical syndrome occurring as a complication of malarial infection characterized by intravascular hemolysis, hemoglobinuria, and acute renal failure in people exposed to P. falciparum, are even more uncommon. Care should be taken to retain a high level of suspicion to consider the diagnosis, especially in returning travelers from endemic areas. Malaria continues to be a parasitic disease that can have devastating effects and continues to be a challenge globally. Although cases of malaria in the United States are rare and cases of severe malaria, mainly attributed to  Plasmodium is a rare disease in the United States, with most cases imported from travel to endemic malarial regions [Malaria is the infection caused by obligate intraerythrocytic protozoa of the genus  regions . Accordi regions . In 2015P. falciparum malaria and any one of the following: Plasmodium falciparum parasitemia >10%, signs of organ dysfunction, including renal impairment (creatinine > 3 mg/dl), severe anemia (hemoglobin <6 g/dl), recurrent bleeding and bleeding disorders, acidosis, impaired consciousness, convulsions with two or more episodes in 24 hours, hypoglycemia (<40 mg/dl), pulmonary edema, and shock [Severe malaria is defined as the presence of We are reporting the case of a previously healthy 24-year-old African American male with no significant past medical history who presented to the emergency department with six days of fever, headache, fatigue, and myalgia. Travel history was notable for a 3-week trip to Sierra Leone, West Africa, from where he returned one week before the presentation. He was in his usual state of health before and during his trip and denied insect bites or sick contacts. In addition, he denied any history of immunodeficiency, splenectomy, or hematologic disorder. A review of systems was negative for diarrhea, dysuria, cough, runny nose, neck rigidity, nausea, or vomiting. He did not receive pretravel prophylaxis against malaria, hepatitis A, or yellow fever. On examination, he was ill-appearing and lethargic but oriented to time, place, and person. On abdominal examination, he had epigastric and right upper quadrant tenderness. The rest of the physical examination was unremarkable.Plasmodium falciparum with a blood parasitemia percentage of 1% , inspired by the French word “fievre bilieuse melanurique,” is a severe clinical syndrome occurring as a complication of malarial infection characterized by intravascular hemolysis, hemoglobinuria, and acute renal failure in people exposed to lciparum , 4, 7–9.fections . Plasmod malaria –13. The  feeding . Apart f feeding .P. falciparum malaria to other antimalarial drugs [st line treatment replacing quinine during the 2nd World War and that patients showed resurgence in the 1990s after quinine and other amino-alcohol drugs resurfaced in response to chloroquine resistance [The exact pathogenesis of classic BWF remains unclear; however, some postulated mechanisms leading to damage to the red blood cells (RBCs) are immunologic and nonimmunologic, leading to massive intravascular hemolysis . Antibodal drugs , 8–10. Lsistance , 4, 9. Ssistance .Acute renal failure (ARF) has been reported in severe forms of malaria, such as blackwater fever (BWF), which is associated with high mortality rates of 15–45% if not managed promptly and correctly . Acute rApart from the above complications, different malarial gastrointestinal complications have been reported in other studies, one of which is acute acalculous cholecystitis (AAC), as seen in our patient . It is sP. falciparum, are even more uncommon, care should be taken to retain a high level of suspicion to consider the diagnosis, especially in people returning from recent trips and prolonged stays in endemic areas. Physicians need to be aware of the various rare and severe presentations of malarial disease to ensure prompt diagnosis and appropriate treatment, especially since the treatment is particular [Malaria continues to be a parasitic disease that can have devastating effects and continues to be a challenge globally. Although cases of malaria in the United States are rare, and cases of severe malaria, mainly attributed to rticular , 7, 8. Mrticular .