Malaria parasite has been observed to be a common infection in Human Immunodeficiency virus (HIV), an increase malaria infection in adults.This experimental study is sets to determine the prevalence and density of malaria parasitaemia in Warri community, South-Southern Nigeria.A total of 600 participants were screened for Human immunodeficiency virus and malaria parasite using WHO systems two and Geimsa staining technique for thick and thin blood films and absolute parasite counts done respectively. The prevalence rate of 38% and 39% were obtained for malaria parasite infection among HIVSP and HIV/span>SN respectively. The difference in malaria parasite infection was not statistically significant (P>0.05) between HIVSP and HIVSN. However, the mean parasite density in HIVSP was significant (P<0.05) when compared with HIVSN. The mean parasite densities of 2384 ± 747 and 1883 ± 645 were recorded for HIVSP and HIVSN respectively. The mean parasite densities of 2385 ± 782 and 2383 ± 717 observed for males and females respectively showed no statistical significant difference (P<0.05).This study has shown a high prevalence of malaria parasite among the HIV infected subjects. Malaria is a parasitic disease that is transmitted from one individual to another through the bite of the female anopheles mosquito8P. falciparum are modified by HIV and further increase the potential reservoir in the patient thereby increasing the number of chemokine receptor positive macrophages. This response stimulates HIV-1 replication through the production of cytokines by activated lymphocytesIn respect to geographical overlap between malaria and HIV/AIDS, a reasonable number of coinfections occurA study in Guinea Bissau found no difference in HIV-2 progression as measured by viral load between adults with parasitaemia and those without21. This is an indication that malaria is still a compound factor in HIV infection. Thus, this study was aimed at determining the prevalence and density of Malaria parasitaemia among HIV infected patients to provide information to improve the management of HIV and malaria inBoth HIV and malaria are probable factors in the modification of each other's development, disease severity, and disease progression rateThis study was conducted amongst inhabitants of Warri, in Central Hospital, Nigeria for a period of 2016 to 2018. Prospective comparative study was carried at Central Hospital, Nigeria. A total of 600 subjects were recruited into the study.Samples were collected from participants based on the approved ethical procedures of Central Hospital, Warri research and ethics committee with No. CHW/HA/45/2018.Blood samples were collected from the subjects into EDTA containers and transported to the laboratory for analysis.This was carried out using two enzymes linked immunosorbent assay rapid screening kits based on WHO systems twoThis was determined by Geimsa staining technique of thick and thin blood films16. Malaria parasite count was done using WHO standard method of absolute parasite count (counting parasites number/microlitre of blood)Data obtained were analyzed using statistical package for social sciences confidence level.Out of the 600 subjects examined, 300 were HIV seropositive (HIVSP), comprising of 143 males and 157 females while 300 were HIV-seronegative (HIVSN).. The prevalence rate of 38% and 39% were obtained for malaria parasite infection amongst HIVSP and HIVSN subjects respectively. The malaria parasite infection was not statistically significant (P>0.05) among HIVSP and HIVSN .This study has demonstrated that malaria parasite infection was not significantly different among HIV seropositive and HIV seronegtive (p=3.843)This confirms the earlier report14 that there was no significant difference between HIV infection in adults with parasitaemiaor without to malaria parasite which might be associated with failure to prevent infection as earlier reportedThe overall prevalence rate of 77% observed for malaria parasite infection in this study is far higher than the prevalence rate of 57.9% reported6 in part of Cross River State. This difference may be due to the timing of the study and seasonal variation. The high mean parasite density observed in HIVSP subjects was significantly higher than in HIV and agrees with the earlier report Meanwhile, the high prevalence rate of malaria parasite infection among HIVSP subjects in this study was far higher than the 7.3% earlier reportedThis study has shown that there was no significant difference among malaria parasite and HIVSP subjects in the studied area. Therefore, HIV infected patients should be encouraged to avoid malaria infection by ensuring that mosquitoes are adequately prevented by sleeping under insecticide treated net (ITN) as recommended by World Health Organization (WHO) so as to reduce the morbidity and mortality associated with malaria and HIV infections.


Plasmodium genus are responsible for causing malaria in humans. Plasmodium species have a complex life cycle that requires post-translational modifications (PTMs) to control cellular activities temporally and spatially and regulate the levels of critical proteins and cellular mechanisms for maintaining an efficient infection and immune evasion. SUMOylation is a PTM formed by the covalent linkage of a small ubiquitin-like modifier protein to the lysine residues on the protein substrate. This PTM is reversible and is triggered by the sequential action of three enzymes: E1-activating, E2-conjugating, and E3 ligase. On the other end, ubiquitin-like-protein-specific proteases in yeast and sentrin-specific proteases in mammals are responsible for processing SUMO peptides and for deconjugating SUMOylated moieties. Further studies are necessary to comprehend the molecular mechanisms and cellular functions of SUMO in Plasmodium. The emergence of drug-resistant malaria parasites prompts the discovery of new targets and antimalarial drugs with novel mechanisms of action. In this scenario, the conserved biological processes regulated by SUMOylation in the malaria parasites such as gene expression regulation, oxidative stress response, ubiquitylation, and proteasome pathways, suggest PfSUMO as a new potential drug target. This mini-review focuses on the current understanding of the mechanism of action of the PfSUMO during the coordinated multi-step life cycle of Plasmodium and discusses them as attractive new target proteins for the development of parasite-specific inhibitors and therapeutic intervention toward malaria disease.Malaria is a parasitic disease that represents a public health problem worldwide. Protozoans of the  Plasmodium, being the majority of recurrence due to five species: Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi , causes the most virulent form of human malaria  reports that in 2019, there were 229 million cases and an estimated number of malaria deaths at 409,000, worldwide, of the estimated victims, the most vulnerable group affected by the disease are children below five years old. It must be emphasized that Africa holds 94% of these cases and deaths. Malaria in humans is caused by apicomplexan parasites of the genus knowlesi . It is n malaria .Plasmodium has a multistage life cycle that comprises specialized asexual form, it establishes infection and proliferation within the vertebrate host and sexual form, that occurs in the mosquito vector , which provides a way for spatiotemporal control of cellular activity. Several PTMs are described in Plasmodium ssp. such as SUMOylation , ubiquitination, phosphorylation, acetylation, nitrosylation, lipidation, and methylation  modulates a wide variety of biological and molecular processes such as protein-protein interactions  across sPlasmodium ssp. Further studies are necessary to precisely understand the molecular and cellular functions of PfSUMO. In this minireview, we consider the potential of the SUMOylation pathway as a target for malaria disease intervention and highlight the current knowledge about the SUMO mechanism during Plasmodium’s life cycle.Novel treatments and, consequently, new therapeutic targets must be ready to overcome divergent biology associated with the complex life cycle stages of Plasmodium encodes a single SUMO paralogue so far revealed in P. falciparum, namely PfSUMO  and the E2-conjugating enzyme (Ubc9 or PfSUMO-E2) are functionally active in P. falciparum  PfSENP1 and PfSENP2 , which highlights that this might be a challenging drug target  through the grant 301524/2019-0.The authors acknowledge the financial support provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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.


Plasmodium falciparum malaria. Mechanisms that drive this protection remain unclear due to a poor understanding of malaria clinical phenotypes during infancy.Infants are protected against We enrolled a birth cohort of 678 infants in Busia, Uganda, an area of high malaria transmission. We followed infants through 12 months of age and quantified protection against parasitemia and clinical disease.P < .001), but age modified this relationship (Pint = <0.001), with nonlinear protection that waned between 0 and 9 months of age before increasing. Increasing age was associated with higher parasite densities at the time of infection and, in infants with HbAS, a reduced ability to tolerate high parasite densities without fever.Symptomatic malaria incidence increased from 1.2 to 2.6 episodes per person-year between 0 and <6 months and between 6 and 12 months of age, while the monthly probability of asymptomatic parasitemia given infection decreased from 32% to 21%. Sickle cell trait (HbAS) was protective against symptomatic malaria (incidence rate ratio  = 0.57 comparing HbAS vs hemoglobin AA (HbAA); 95% confidence interval, 0.44–0.74; Age-dependent changes in HbAS protective efficacy in infancy were accompanied by differential loss of antiparasite and antidisease protection among HbAS and HbAA infants. This provides a framework for investigating the mechanisms that underlie infant protection against malaria.NCT02793622. In 678 Ugandan infants followed through 12 months of age, 1131 malaria episodes were observed. Age-dependent changes in sickle cell trait (HbAS) protective efficacy were accompanied by differential loss of antiparasite and antidisease protection among HbAS and HbAA infants. Plasmodium falciparum [Malaria is responsible for 228 million clinical cases and 405 000 deaths annually, with morbidity and mortality highest in young African children due to lciparum . Infantslciparum , though Plasmodium infection [Epidemiologic studies suggest that detection of microscopic parasitemia may be uncommon in the first 6 months of life . This hanfection , 11, andnfection , althougnfection , 13. Furnfection , modify To better understand malaria clinical phenotypes among infants, we followed a birth cohort of infants living in a high transmission setting in southeastern Uganda. We quantified age-related changes in malaria incidence and the probability of symptoms given infection in infancy and determined the relative protective effects of both individual and household-level factors. We then focused on 2 specific types of protection: antiparasite  and antidisease (the ability to tolerate higher parasite densities without developing fever), as these have been defined as independent components of acquired antimalarial immunity , 18.This study was conducted from September 2016 to December 2018 in Busia district, Uganda, an area of high malaria transmission intensity. Liveborn infants included in this study were born to women enrolled in a trial of intermittent preventive treatment of malaria in pregnancy (IPTp); inclusion and exclusion criteria for the parent trial have been published . BrieflyAll women were provided long-lasting insecticide-treated bednets at enrollment, and a household survey was conducted to collect socioeconomic and house construction data . HandhelMothers were encouraged to bring their infants to a study clinic for all care. Routine assessments were conducted every 4 weeks, including collection of blood for detection of malaria parasites. Infants who presented with a history of fever in the past 24 hours or were found to have a tympanic temperature ≥38.0°C on clinical assessment had a blood smear performed for detection of malaria parasites. If the smear was positive, infants were treated for malaria according to the Uganda Ministry of Health guidelines. For uncomplicated malaria, infants were treated with artemether-lumefantrine. For complicated malaria  and severe malaria, infants were treated with intravenous artesunate. Asymptomatic parasitemia was not treated, and children diagnosed with sickle cell disease were provided weekly chloroquine prophylaxis, per Ugandan guidelines. Infants were followed to aged 12 months. Criteria for premature study withdrawal included movement out of the study area, inability to be located for >60 days, withdrawal of consent, and death.Blood smears were stained with 2% Giemsa and read by experienced microscopists. Blood smears were considered negative when the examination of 100 high-power fields did not reveal asexual parasites. All slides were read by a second microscopist, and a third reader settled any discrepancies. Hemoglobin (Hb) genotype was ascertained by polymerase chain reaction (PCR)–based detection  using DNAnalyses were conducted using Stata version 14 and R version 3.6.1. Incident outcomes included symptomatic malaria  and complicated malaria . For monthly prevalence measures, we considered a 28-day window around each routine visit for the presence/absence of malaria parasitemia. If parasitemic, monthly periods were further characterized as symptomatic malaria (from 21 days prior to 7 days after the routine visit) or asymptomatic parasitemia . We also considered the peak parasite density and objective temperature measured while parasitemic during this 4-week window.Exposure variables included infant characteristics at birth  and household characteristics . Principal components analysis was used to generate a wealth index based on ownership of common household items , with hoP values  < .05 were considered statistically significant.Follow-up started at birth and ended on the day the infant was aged 12 months or prematurely withdrawn. Incident outcomes were compared using negative binomial regression models. Repeated prevalence measures were compared using log-binomial, log-Poisson, or Gaussian models with robust standard errors and generalized estimating equations to adjust for clustering. Exposure variables found to be significant on univariate analyses were included in multivariate models; these models also included maternal IPTp regimen. We assessed for interaction between exposure variables and age. In all analyses, 2-tailed Generalized additive models were used to model and visualize associations between age, parasite density, and probability of symptoms if infected . For antBetween December 2016 and December 2017, 678 live infants were born to 666 mothers. Maternal households ranged from 0.1 to 26 km from the study clinic . The majHb genotype was ascertained in 650 of 678 (95.5%) infants with available DNA for testing, of which 79.2% were HbAA, 18.8% sickle cell trait (HbAS), and 2.0% HbSS (with sickle cell disease). Pediatric characteristics at birth were similar between Hb genotype groups . Of 97 iint = 0.001; P = .002) and 51% less symptomatic malaria between 3 and 6 months of age  compared with HbAA infants. Between 6 and 9 months of age, there was no significant difference between HbAS vs HbAA infants . From 9 to 12 months of age, infants with HbAS again had 49% less symptomatic malaria than HbAA infants . Infants with sickle cell disease (HbSS) had 69% less symptomatic malaria than HbAA infants  without significant interaction with age (Pint = 0.26), although this may have been due (in part) to these infants receiving chloroquine prophylaxis, given evidence of a return of parasite susceptibility to chloroquine in Uganda [Overall, 1131 incident episodes of symptomatic malaria were observed; incidence increased from 1.15 episodes per person-year (ppy) between 0 and <6 months of age to 2.58 episodes ppy between 6 and 12 months of age . Infantsn Uganda . Other iP < .001). Infants living in houses constructed with modern materials had 27% less symptomatic malaria than infants living in traditional households . No significant interactions were observed between infant age, symptomatic malaria, and maternal education (Pint = 0.92) or house type (Pint = 0.40).Regarding maternal and household characteristics, infants born to mothers with O level of education or higher had 38% less symptomatic malaria than infants born to mothers with none or primary level education , with no significant interaction observed between age and Hb genotype (Pint = 0.27). Other infant characteristics were not significantly associated with the incidence of complicated malaria . Infants living closer (<5 km) to the study clinic had 57% less complicated malaria compared with those living ≥ 5km from the clinic . Neither maternal education nor house type were associated with protection against complicated malaria , without significant interaction with age  and was significantly higher in infants with HbAS ; however, from 9 to 12 months of age, infants with HbAS again had a significantly higher probability that parasitemia was asymptomatic .The probability that parasitemia was asymptomatic decreased from 32.1% between 0 and 6 months of age to 20.7% between 6 and 12 months of age  vs loss of the ability to tolerate higher parasite densities without fever .10 parasites/μL from 0 to <3 months of age to 4.1 log10 parasites/μL from 6 to <12 months of age , which an study . In the Our study had limitations. We did not capture episodes of complicated and severe malaria occurring after age 1 year. However, prior reports suggest that the risk of severe malaria peaks within the first year of life . InfantsBy distinguishing between the risk of infection and risk of disease once infected and assessing interactions between age and sickle cell trait, our study provides a framework for investigating the mechanisms that underlie protection against malaria in infants. Defining these mechanisms, determine why they wane, and determine how they differ between infants with and without sickle cell trait may spur the development of novel vaccine and/or antibody-based preventive strategies for malaria.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciab245_suppl_Supplementary_MaterialsClick here for additional data file.


Plasmodium vivax infects an estimated 7 million people every year. Previously, vivax malaria was perceived as a benign condition, particularly when compared to falciparum malaria. Reports of the severe clinical impacts of vivax malaria have been increasing over the last decade.P. vivax infection in pregnancy is associated with maternal anemia, prematurity, fetal loss, and low birth weight. More than 11,658 patients with severe vivax malaria have been reported since 1929, with 15,954 manifestations of severe malaria, of which only 7,157 (45%) conformed to the World Health Organization (WHO) diagnostic criteria. Out of 423 articles, 311 (74%) were published since 2010. In a random-effects meta-analysis of 85 studies, 68 of which were in hospitalized patients with vivax malaria, we estimated the proportion of patients with WHO-defined severe disease as 0.7% [95% confidence interval (CI) 0.19% to 2.57%] in all patients with vivax malaria and 7.11% [95% CI 4.30% to 11.55%] in hospitalized patients. We estimated the mortality from vivax malaria as 0.01% [95% CI 0.00% to 0.07%] in all patients and 0.56% [95% CI 0.35% to 0.92%] in hospital settings. WHO-defined cerebral, respiratory, and renal severe complications were generally estimated to occur in fewer than 0.5% patients in all included studies. Limitations of this review include the observational nature and small size of most of the studies of severe vivax malaria, high heterogeneity of included studies which were predominantly in hospitalized patients (who were therefore more likely to be severely unwell), and high risk of bias including small study effects.We describe the main clinical impacts of vivax malaria, incorporating a rapid systematic review of severe disease with meta-analysis of data from studies with clearly defined denominators, stratified by hospitalization status. Severe anemia is a serious consequence of relapsing infections in children in endemic areas, in whom vivax malaria causes increased morbidity and mortality and impaired school performance. Young children and pregnant women are particularly vulnerable to adverse clinical impacts of vivax malaria, and preventing infections and relapse in this groups is a priority. Substantial evidence of severe presentations of vivax malaria has accrued over the last 10 years, but reporting is inconsistent. There are major knowledge gaps, for example, limited understanding of the underlying pathophysiology and the reason for the heterogenous geographical distribution of reported complications. An adapted case definition of severe vivax malaria would facilitate surveillance and future research to better understand this condition. Elizabeth A Ashley and colleagues describe the main clinical impacts of vivax malaria globally. Plasmodium vivax has been understudied and reported compared to falciparum malaria.Historically, the clinical impact of the relapsing malaria caused by We reviewed the literature on the clinical impact of vivax malaria focusing on children and pregnant women and performed a rapid systematic review of published evidence for severe disease and meta-analysis of selected studies.Vivax malaria in young children is associated with severe anemia and increased mortality. Infections in pregnancy lead to maternal anemia, prematurity, fetal loss, and low birth weight. We estimated the proportion of patients with World Health Organization (WHO)-defined severe disease as 0.7% [95% confidence interval (CI) 0.19% to 2.57%] in all patients with vivax malaria and the mortality from vivax malaria as 0.01% [95% CI 0.00% to 0.07%] in all patients and 0.56% [95% CI 0.35% to 0.92%] in hospital settings.Reporting of severe vivax malaria has increased dramatically over the last 10 years, but different case definitions have been applied.Limitations of our analysis include high risk of bias of published studies, the majority of which are in hospitalized patients (who are therefore more likely to be severely unwell), while most vivax malaria is managed in primary care. As a result, estimates should be interpreted with caution.Prospective cohort studies using an adapted case definition of severe vivax malaria would improve estimates of incidence.Preventing vivax malaria and subsequent relapses in children and pregnant women is a priority to reduce morbidity and mortality. Plasmodium falciparum, the Plasmodium vivax parasite has an intraerythrocytic life cycle lasting approximately 48 hours from red cell invasion by merozoites to schizont rupture. In synchronous infections, schizogony triggers a febrile paroxysm every 3 days, hence the term “tertian fever” given to early descriptions of patients with vivax or falciparum malaria. Malaria caused by P. vivax was termed benign tertian malaria because the clinical course was generally considered to be mild compared to falciparum malaria even though occasional descriptions of severe, including cerebral, presentations of vivax malaria have been reported since 1902 ) . The relp-value range: <0.001 to 0.138).Data from the same cohort were used to assess the risk of preterm birth and small for gestational age (SGA) infants . Having P. vivax infection of 108 g (95% CI 17.5 to 199) was reported. P. vivax infection was also associated with an increased risk of moderate, but not severe, maternal anemia compared to no malaria with a reduction of Hb concentration of 0.4 g/dl (95% CI 0.1 to 0.7 g/dL).Between 2004 and 2006, a cross sectional study in Papua, Indonesia examined the relationship between malaria at delivery and pregnancy outcomes in 2601 women . A decreP. vivax), showed that malaria episodes were associated with decreased birth weight and length and maternal anemia  among hospitalized patients and 0.02% [95% CI 0.00% to 0.13%] among patients from studies that included all patients diagnosed with vivax malaria (inpatient and outpatient) .We found 8 (0.07%) reports of PMNS: 4 cases of acute disseminated encephalomyelitis (ADEM), 2 of Guillain–Barré syndrome, 1 report of a patient with behavior change, and 1 of myelitis.2 saturations <92% with respiratory rate more than 30/min); 330 (26.6%) were reported to have ARDS; and 593 (48.6%) had other respiratory complications such as respiratory distress (not otherwise specified), pleural effusion, and pneumothorax  fulfilled WHO criteria for pulmonary edema  met WHO criteria for AKI , 233 13. From thn = 20), with a second diagnosis reported in 9 cases; cortical necrosis was reported in 16, thrombotic microangiopathy in 12, and mesangiocapillary glomerulonephritis in 7 patients.We found reports of renal biopsy results from 45 patients with renal impairment and vivax malaria . The mosA total of 553 deaths were associated with severe vivax malaria . Out of n = 7), coexisting with splenic rupture in 2 patients. The other causes of death were multiple organ dysfunction , primaquine-induced hemolytic anemia in the context of glucose-6-phosphate dehydrogenase (G6PD) deficiency (n = 2), and isolated splenic rupture (n = 1). A total of 7 patients had comorbidities such as hepatic cirrhosis or pulmonary emphysema. Postmortem findings consistent with ARDS were reported from one fatal case in India ) of patients with P. vivax infection were bacteremic   to 0.56% [0.35% to 0.92%] in studies of hospitalized patients only. These results were similar to a case fatality rate estimate of 0.3%  from a fixed-effect meta-analysis in another systematic review, which used a modified WHO definition of severe malaria and included thrombocytopenia [Our estimate of mortality resulting from ytopenia . HoweverDetailed studies from a small number of research groups so far have not identified a common pathophysiological process underlying different complications of vivax malaria. Sequestration of parasitized red blood cells in the microcirculation, which is the pathophysiological hallmark of severe falciparum malaria, has been not been demonstrated.Plasmodium species causing malaria in humans. Incidence rates of ARDS in severe falciparum malaria have been shown to vary between 2% and 25% and are more likely to lead to a fatal outcome than ARDS with P. vivax [ARDS has been reported with infection from all P. vivax ,96.P. vivax infections in patients presenting to a referral hospital with severe thrombocytopenia to be 1.5% (25/1650) below 50 × 109/L and 3.6% (6/168) below 20 × 109/L in patients with P. vivax and proposed the latter threshold as a severity criterion [The need for an adapted case definition for severe vivax malaria has been recognized for over a decade. Reporting by different groups is inconsistent, impeding gathering reliable incidence data or conducting research. A Brazilian study looking at predictors of intensive care unit (ICU) admission in patients with vivax malaria found that many of the criteria in the severe falciparum malaria definition were predictive, with the exception of hyperbilirubinemia . The impriterion . The utiriterion . Conversriterion . Very feP. vivax infection to those reported here [Limitations of our rapid systematic review include limiting our search to the English language and omitting to search other databases such as the Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS) database. A systematic review of the Brazilian published and gray literature in 2012 described a similar array of complications from ted here . The litted here .Vivax malaria has emerged from the shadow of falciparum malaria over the last decade with improved recognition of the negative clinical impacts associated with this relapsing infection.Preventing severe anemia associated with relapsing vivax malaria, particularly in very young children, is a priority to reduce morbidity and mortality in this group. Progress has been slowed by low uptake of antihypnozoiticidal treatment with 8-aminoquinoline drugs due to fears of hemolysis in patients with G6PD deficiency, compounded by lack of access to G6PD testing .P. vivax in pregnancy may need to target young women preconception in order to prevent the risk of relapse during pregnancy and the consequent negative impacts of maternal anemia, increased fetal loss, and low birth weight [Prevention of infection with h weight . Severe S1 FigPRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TablePRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


Understanding the prevalence of signs of severity identified in the Thai population with malaria could aid clinical management and disease control efforts, decrease mortality, and promote malaria elimination in Thailand. This systematic review aimed to collate the evidence regarding signs of severity identified in the Thai population with malaria. MEDLINE, Web of Science, and Scopus were searched for potentially relevant studies. The quality of the included studies was assessed using the Joanna Briggs Institute critical appraisal tools. The pooled prevalence of signs of severity among patients with severe malaria and the pooled proportion of each sign of severity among all signs of severity were estimated using random-effects models. Heterogeneity among included studies was assessed using Cochran’s Q test. A subgroup analysis was performed to evaluate whether differences in pooled estimates between different study sites. Publication bias was assessed by visualizing funnel plot asymmetry and using Egger’s test. Among 741 studies identified by literature searching, 12 studies of a total of 2900 patients with severe malaria, in 7 Thai hospitals, met the eligibility criteria. Results of meta-analyses showed that the signs of the severity of malaria with the highest prevalence in Thailand were jaundice (54%), hyperparasitemia (47%), impaired consciousness/coma (21%), acidosis (18%), renal impairment (13%), shock (10%), convulsions (9%), severe anemia (8%), pulmonary edema/acute respiratory distress syndrome (ARDS) (8%), hypoglycemia (4%), and bleeding/disseminated intravascular coagulation (DIC) (2%). The signs of the severity of malaria that made up the highest proportion of all signs of severity identified in the Thai population with malaria were hyperparasitemia (33%), jaundice (33%), impaired consciousness/coma (12%), acidosis (9%), renal impairment (7%), severe anemia (6%), convulsions (5%), shock (5%), pulmonary edema/ARDS (3%), bleeding/DIC (1%), and hypoglycemia (1%). The present study revealed the prevalence of signs of severity identified in the Thai population with malaria. Jaundice, hyperparasitemia, and impaired consciousness/coma were the most common signs of severity identified. These results may inform the management of patients with severe malaria and promote malaria-elimination efforts in Thailand. Plasmodium that are transmitted to humans via the bites of infected female Anopheles mosquitoes [Plasmodium species  most commonly infect humans [P. cynomolgi—a parasite that primarily infects non-human primates—is being detected more frequently in humans [Plasmodium parasites can lead to different clinical outcomes depending on pre-existing partial immunity to malaria. In moderate- to high-transmission areas, clinical disease is confined mainly to young children who have no immunity to malaria, whereas adolescents and adults with partial immunity have less severe clinical outcomes. In contrast, in low-transmission areas, both children and adults alike suffer from malaria symptoms and are at risk of severe malaria if left untreated [Malaria is a life-threatening disease caused by members of the protozoan genus squitoes . Globallsquitoes . Five Plt humans ,3. In son humans . Infectintreated .P. falciparum asexual parasitemia with one or more of the following signs of severity: impaired consciousness, prostration, multiple convulsions, acidosis, hypoglycemia, severe malarial anemia, renal impairment, jaundice, pulmonary edema, significant bleeding, shock/hypotension, and hyperparasitemia  [P. falciparum, fewer patients develop severe malaria following P. knowlesi, P. vivax, P. malariae, and P. ovale infection [Plasmodium species, the degree of vital organ dysfunction, age, background immunity, co-morbidities, and co-infections [According to the recent World Health Organization (WHO) guidelines for malaria, severe malaria is defined as the presence of a > 10%) . Althougnfection ,6,7,8. Snfection . Neverthnfection . Moreovefections ,10,11.P. falciparum malaria [Plasmodium parasites in blood samples  was associated with male sex and staying outdoors at night [Thailand is one of the six countries of the Greater Mekong subregion and is bordered by Myanmar, Laos, Cambodia, and Malaysia. Malaria cases and deaths in Thailand have continuously decreased from 81,692 cases and 625 deaths in 2000 to 3538 cases and 13 deaths in 2019 [ malaria . Based o malaria . A previ malaria . Another malaria . A recenat night .A better understanding of the signs of severity identified in the Thai population with malaria could inform the clinical management of patients with severe malaria and decrease mortality. To the best of our knowledge, a systematic review of the characteristics of signs of severity of malaria has not been conducted in Thailand. Therefore, the present systematic review aimed to collate the evidence regarding signs of severity identified in the Thai population with malaria. The results of this study will help to better understand the signs of severity in the Thai population with malaria and will serve as a guide for clinicians to recognize and treat these signs of severity. These data may also contribute toward better management and control of malaria, with the eventual goal of malaria elimination in Thailand.The reporting of this systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . The proMEDLINE, Web of Science, and Scopus were searched for relevant studies using a combination of search terms “ AND (Thai OR Thailand OR Siam)” from inception to 8 August 2021 . AdditioP. falciparum malaria in all age groups according to WHO guidelines [P. falciparum malaria; (2) studies must be conducted in Thailand; (3) any study design was considered; and (4) studies reported signs of severity among patients with malaria. The exclusion criteria were as follows: (1) duplicate studies using the same groups of participants; (2) case reports of severe malaria; (3) studies of uncomplicated malaria; (4) studies that do not provide data on signs of severity; (5) short reports, (6) studies with no full-text available; and (7) review articles.Eligibility criteria were based on a PICo  framework. Participants were patients with severe idelines . The outPotentially relevant studies were selected according to the eligibility criteria. After duplicate studies were removed, the titles and abstracts of the remaining studies were screened, and irrelevant studies were excluded. Next, the full texts of the remaining studies were examined for eligibility, and studies that did not meet the eligibility criteria were excluded. The reason for exclusion was noted. The studies that met the eligibility criteria were included for further analysis. Study selection was performed by two authors independently. Disagreements between authors in study selection were resolved by discussion.The following data were extracted from each study into a standardized spreadsheet: name of the first author, publication year, year the study was conducted, study sites, study design, number of participants, age groups included, percentage of male participants, number of patients who died, number of patients with each sign of severity, and the total number of signs of severity. Data were extracted by two authors. Disagreements between authors in data extraction were resolved by discussion.The quality of the included studies was assessed using the Joanna Briggs Institute (JBI) critical appraisal tools . For clip < 0.01 or I2 more than 50% indicated substantial heterogeneity among the included studies. If the outcome of the included studies was homogenous , data were pooled using fixed-effects models. A subgroup analysis was performed to evaluate differences in pooled estimates between different study sites. Publication bias was assessed by visualizing funnel plot asymmetry and using Egger’s test. If the funnel plot was asymmetrical or Egger’s test returned a value of p < 0.05, contour-enhanced funnel plots were evaluated to identify potential sources of funnel plot asymmetry. All analyses were performed using Stata version 14.2 .The pooled prevalence of signs of severity identified in the Thai population with malaria and the pooled proportion of each sign of severity among all signs of severity were estimated using random-effects models assuming that the outcomes of the included studies were heterogeneous. Heterogeneity among the included studies was assessed using Cochran’s Q test. Values of A total of 741 studies were identified from the 3 databases: 299 from MEDLINE, 142 from Scopus, and 300 from Web of Science. After 326 duplicates were removed, the titles and abstracts of the 415 remaining studies were screened, and 351 irrelevant studies were excluded. The full texts of 64 studies were examined, and 53 studies were excluded for the following reasons: 30 studies used the same group of participants, 7 studies were case reports of severe malaria, 5 studies were of uncomplicated malaria, 5 studies did not report data on signs of severity, 3 studies were conducted in other countries, 1 short report did not provide data on signs of severity, 1 study had no full-text available, and 1 study was a review article. Eleven studies ,26,27,28P. falciparum malaria in adult patients , whereas the remaining studies examined severe malaria in both children and adults [P. falciparum malaria were included in the meta-analysis.The characteristics of the included studies are shown in d adults ,27,29 ord adults . IncludeFor clinical trials, one study was a high-quality study, while three studies were moderate- or low-quality studies ,23,27. F2: 99.02%). The lowest prevalence of jaundice  was reported in a study conducted at the Hospital for Tropical Diseases in 1994 [The meta-analysis of the results of nine studies that reported jaundice as a sign of severity, including a total of 2233 patients, showed that the pooled prevalence of jaundice among patients with severe malaria in Thailand was 54% : 36–72%, I in 1994  . A lower pooled prevalence of jaundice was observed in Tak/Kanchanaburi  and Tak   .2: 97.07%). The lowest proportion of jaundice  was reported in a study conducted at Mae Sot Hospital from 2003 to 2005 [Meta-analysis of the results of nine studies, including a total of 3552 signs of severity, showed that the pooled proportion of jaundice among all signs of severity was 33% . Lower pooled proportions of jaundice were observed in Tak/Kanchanaburi  and Tak   .2: 95.07%). Subgroup analyses of hyperparasitemia among patients with severe malaria defined using cutoffs of >2%, >4%, and >10% showed a pooled prevalence of 42% , 55% , and 41% , respectively . Subgroup analysis of the proportion of hyperparasitemia using different cutoffs  was performed. The pooled proportion of hyperparasitemia among all signs of severity using cutoffs of >2%, >4%, and >10% was 32% , 42% , and 23% , respectively . The lowest prevalence of impaired consciousness was reported in a study conducted at the Hospital for Tropical Diseases in 1999  [Meta-analysis of the results of 12 studies, including a total of 2900 patients, showed that the pooled prevalence of impaired consciousness among patients with severe malaria in Thailand was 21%  . The hig2: 95.84%). A lower pooled prevalence of impaired consciousness was observed in Tak/Kanchanaburi  and Bangkok   .2: 96.55%). The lowest proportion of impaired consciousness was reported in a study conducted at the Hospital for Tropical Diseases in 1999  [Meta-analysis of the results of 12 studies, including a total of 4324 among all signs of severity, showed that the pooled proportion of impaired consciousness among all signs of severity was 12%  . The higI: 0–3%)  (Supplem2: 54.42%). The lower pooled proportions of impaired consciousness were observed in Tak/Kanchanaburi  and Bangkok   .2: 95.62%). The lowest prevalence of acidosis was reported in a study conducted at Somdejt Prachaotaksin Maharaj Hospital from 2003 to 2006  [Meta-analysis of the results of five studies, including a total of 997 patients, showed that the pooled prevalence of acidosis among patients with severe malaria in Thailand was 18%  . The hig: 3–19%)  . The lowest proportion of acidosis was reported in a study conducted at Somdejt Prachaotaksin Maharaj Hospital from 2003 to 2006  [Meta-analysis of the results of five studies, including a total of 1966 among all signs of severity, showed that the pooled proportion of acidosis among all signs of severity was 9%  . The hig: 8–31%)  (Supplem2: 95.81%). The lowest prevalence of renal impairment was reported in a study conducted at the Hospital for Tropical Diseases in 1999  [Meta-analysis of the results of 11 studies, including a total of 2291 patients, showed that the pooled prevalence of renal impairment among patients with severe malaria in Thailand was 13%   . The lower pooled prevalence of renal impairment was observed in Tak/Kanchanaburi  and Bangkok   .2: 90.32%). The lowest proportion of renal impairment was reported in a study conducted at the Hospital for Tropical Diseases in 1999  [Meta-analysis of the results of 11 studies, including a total of 3630 signs of severity, showed that the pooled proportion of renal impairment among all signs of severity was 7%   (Supplem2: 84.14%). Lower pooled proportions of renal impairment were observed in Tak/Kanchanaburi  and Bangkok   .2: 94.26%). The lowest pooled prevalence of severe anemia was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals in Sangklaburi (1994 to1995), and hospitals in Mae Sot  [Meta-analysis of the results of nine studies, including a total of 2726 patients, showed that the pooled prevalence of severe anemia among patients with severe malaria in Thailand was 8%  . The higI: 1–4%)  . The lower pooled prevalence of severe anemia was observed in Bangkok  and Tak/Kanchanaburi   .2: 95.57%). The lowest proportion of severe anemia was reported in a study conducted at the Hospital for Tropical Diseases from 2006 to 2021  [Meta-analysis of the results of nine studies, including a total of 4026 signs of severity, showed that the pooled proportion of severe anemia among all signs of severity was 6%   (Supplem2: 96.06%). Lower pooled proportions of severe anemia were observed in Bangkok  and Tak/Kanchanaburi   .2: 98.41%). The lowest pooled prevalence of convulsions was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals at Sangklaburi (1994 to 1995), and hospitals in Mae Sot  [Meta-analysis of the results of three studies, including a total of 1479 patients, showed that the pooled prevalence of convulsions among patients with severe malaria in Thailand was 9%  . The hig2: 98.32%). The lowest proportion of convulsions was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals at Sangklaburi (1994 to 1995), and hospitals in Mae Sot (1995 to 2002)  [Meta-analysis of the results of three studies, including a total of 2355 signs of severity, showed that the pooled proportion of convulsion among all signs of severity was 5%  . The higI: 0–1%)  (Supplem2: 96.47%). The lowest pooled prevalence of shock was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals in Sangklaburi (1994 to 1995), and hospitals in Mae Sot  [Meta-analysis of the results of seven studies, including a total of 1291 patients, showed that the pooled prevalence of shock among patients with severe malaria in Thailand was 10%  . The higI: 0–1%)  . The lowest proportion of shock was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals in Sangklaburi (1994 to 1995), and hospitals in Mae Sot (1995 to 2002)  . The hig2: 88.15%). The lowest pooled prevalence of pulmonary edema/ARDS was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals in Sangklaburi (1994 to 1995), and hospitals in Mae Sot  [Meta-analysis of the results of seven studies, including a total of 1141 patients, showed that the pooled prevalence of pulmonary edema/ARDS among patients with severe malaria in Thailand was 8%  . The higI: 1–3%)  . The lowest proportion of pulmonary edema/ARDS was reported in a study conducted at the Hospital for Tropical Diseases in 1991  [Meta-analysis of the results of seven studies, including a total of 1141 signs of severity, showed that the pooled proportion of pulmonary edema/ARDS among all signs of severity was 3%  . The higI: 0–3%)  (Supplem2: 74.38%). The lowest pooled prevalence of bleeding/DIC was reported in a study conducted at the Hospital for Tropical Diseases from 2006 to 2012  [Meta-analysis of the results of four studies, including a total of 950 patients with severe malaria, showed that the pooled prevalence of bleeding/DIC among patients with severe malaria in Thailand was 2%  . The higI: 0–5%)  . The lowest proportion of bleeding/DIC was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals in Sangklaburi (1994 to 1995), and hospitals in Mae Sot (1995 to 2002)  [Meta-analysis of the results of four studies, including a total of 1913 signs of severity, showed that the pooled proportion of bleeding/DIC among all signs of severity was 1%  . The hig 1995 to 02 . The lowest pooled prevalence of hypoglycemia was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals in Sangklaburi (1994 to 1995), and hospitals in Mae Sot (1995 to 2002)  [Meta-analysis of the results of three studies, including a total of 933 patients, showed that the pooled prevalence of hypoglycemia among patients with severe malaria in Thailand was 4%  . The higI: 0–2%)  . The lowest proportion of hypoglycemia was reported in a study conducted at hospitals in Kanchanaburi (1986 to 1993), hospitals in Sangklaburi (1994 to 1995), and hospitals in Mae Sot (1995 to 2002)  . The higProstration was reported in two studies conducted at Somdejt Prachaotaksin Maharaj Hospital from 2003 to 2006   and Mae Of all patients with severe malaria, 54% of patients had jaundice, 47% had hyperparasitemia, 21% had impaired consciousness/coma, 18% had acidosis, 13% had renal impairment, 10% had shock, 9% had convulsions, 8% had severe anemia, 8% had pulmonary edema/ARDS, 4% had hypoglycemia, and 2% had bleeding/DIC . Of all 2: 94.06%). Subgroup analysis by province showed that the highest pooled prevalence of mortality among patients with severe malaria was observed in Tak/Kanchanaburi  and Tak . A lower pooled prevalence of mortality was observed in Bangkok   .p = 0.014), indicating that the asymmetrical distribution of the funnel plot was related to small-study effects. A contour-enhanced funnel plot indicated that the asymmetrical distribution of the included studies arose from the heterogeneity of the included studies  and P. falciparum (4–5%) were the most common causes of malaria in Thailand in the year 2020 [Although ear 2020 , neverthear 2020 . The NMEear 2020 . Under t9/mL, presence of schizonts or gametocytemia in peripheral blood smears, and albumin levels of < 3.5 g/dL were associated with increased risks of severe malaria in Thailand [The most recent study in Thailand that reported patients with severe malaria was conducted between 2006 and 2012, and those patients were likely to have been referred for treatment to the Hospital for Tropical Diseases in Bangkok . The HosThailand ,33,34. FThailand .P. falciparum malaria had jaundice (58.90%) [P. falciparum malaria in India could be explained by the difference in the age groups of patients. Signs of severity such as jaundice and acute renal failure were common in adults, whereas severe anemia was a predominant sign of severity in children [P. falciparum malaria, whereas shock (50%) and DIC (37.5%) were commonly found in P. vivax malaria [P. knowlesi (29%), P. vivax (16%), and P. falciparum (11%) [P. knowlesi infections, acute kidney injuries (AKI) (45.6%), jaundice (42%), and hyperparasitaemia (32.5%) were commonly observed [P. falciparum malaria, whereas hypotension (71%) and jaundice (29%) were frequently observed in P. vivax infections [P. falciparum  included prostration (68%), impaired consciousness (65%), and respiratory distress (48%) [P. falciparum, respectively [P. falciparum malaria (69%) in Malaysia [The present study showed that jaundice was the most frequent sign of severity in patients with severe malaria (54%), followed by hyperparasitemia (47%) and impaired consciousness (21%). In South Asian countries, such as India, jaundice (44%) and severe anemia (20%) were reported as predominant signs of severity ,36. The (58.90%) . The difchildren . For sev malaria . In Malaum (11%) . In patifections . In Cambss (48%) . In Myanss (48%) . Meanwhijaundice %, and hyectively . TherefoMalaysia  and MyanMalaysia .P. vivax is the most common Plasmodium species found in Thailand, patients with severe P. vivax in Thailand were rarely reported [P. vivax in Thailand are not available for inclusion in the present meta-analysis. In Africa, where malaria is endemic, such as Ethiopia, severe anemia (42%) and hyperpyrexia (21%) were the most common signs of severity in patients with P. vivax malaria [P. vivax malaria [P. vivax malaria demonstrated that severe anemia, jaundice, and respiratory distress were frequently observed [Although reported . Therefo malaria . Another malaria . In Asia malaria . The metobserved .The present study had several limitations. First, the prevalence and proportions of each sign of severity were heterogeneous among the included studies. Therefore, random-effect models were applied for all meta-analyses. With the limitation of the high heterogeneity between studies, careful interpretations of the meta-analysis results were recommended. Second, the period under analysis ends in 2012 due to the limited number of studies reporting signs of the severity of malaria in Thailand because the prevalence of malaria in Thailand has continuously decreased in recent decades. In addition, no severe malaria in Thailand has been reported since 2014 . Third, The present study revealed that jaundice, hyperparasitemia, and impaired consciousness/coma were the most common signs of severity identified in the Thai population with malaria. Data from the meta-analysis results are essential in managing patients with severe malaria in Thailand, with particular attention to the management of signs of severity, such as monitoring and managing jaundice, monitoring patients’ parasite status, or giving parenteral antimalarial treatment for patients with hyperparasitemia, and close observation of unconscious patients. The effective management of patients with malaria will reduce the signs of severity and prevent mortality which promotes malaria-elimination efforts in Thailand.


Plasmodium falciparum commonly detect histidine-rich protein 2 (HRP-2), but HRP-2 deletions are increasingly recognized. We evaluated a prototype test detecting parasite lactate dehydrogenase (pLDH) and compared it to commercially available RDTs at a health facility in Uganda, using quantitative polymerase chain reaction as a gold standard. The prototype pLDH test had a high sensitivity for infections with at least 100 parasites/µL (98%), comparable to HRP-2, and greater than an existing pLDH RDT (89%). Specificity for the prototype test was 99.5%, which is greater than the HRP-2 tests (93–95%). Therefore, the prototype pLDH test may be an attractive alternative malaria diagnostic.Rapid diagnostic tests (RDTs) for  In 2019, malaria cases numbered 229 million worldwide, an annual estimate that has remained nearly unchanged for the past 4 years.Plasmodium falciparum malaria perform effectively in case management scenarios, detecting malaria infections at a threshold of approximately 100 parasites/µL, which is roughly equivalent to microscopy.P. falciparum.P. falciparum infection in some populations. The first problem is that HRP-2 gene deletions in parasite populations have become more prevalent, leading to false-negative test results. This issue has long been recognized in the Amazon of South America,Most currently available malaria RDTs detect histidine-rich protein 2 (HRP-2), parasite lactate dehydrogenase (pLDH), or aldolase as a marker of infection. HRP-2-based RDTs for P. falciparum pLDH-detecting RDTs. The University of California at San Francisco and the Infectious Disease Research Collaboration evaluated clinical performance of the test in febrile patients presenting to a health facility in Uganda. The objective of this study was to evaluate the performance of the pLDH prototype test against other RDTs, including the best-in-class commercial pLDH test and the standard-of-care HRP-2 test. Microscopy, qPCR, and quantitative detection of pLDH using the Quansys platform were all used as references to compare sensitivity, specificity, and level of detection of the point-of-care tests.To overcome these limitations, Global Health Laboratories, alongside Access Bio, designed a more sensitive We performed a prospective cross-sectional study of patients seeking care at the Masufu Hospital in Busia, Uganda. Patients eligible for inclusion were those age 0.5 to 75 years presenting to the clinic with suspected malaria based on a history of fever within the past 24 hours or objective fever (temperature > 38.0°C tympanic). We excluded patients with symptoms of complicated malaria or those who reported receiving any antimalarial medication in the previous month.Study personnel conducted the informed consent discussion in the appropriate language. For children younger than 18 years of age, written informed consent was obtained from the parent/guardian. In addition, for children 8 to 17 years, assent was obtained.For microscopy, thick blood smears were stained with 2% Giemsa stain. Parasite densities were calculated by counting the number of asexual parasites per 200 leukocytes, assuming a leukocyte count of 8,000 leukocytes/µL.For assessment of the RDT results, the two independent operators assessed each test for the presence or absence of test and control lines. The pLDH prototype used 5 µL of whole blood and 75 µL of buffer, with a 20-minute read time. Instructions for use were followed for all commercial tests. These tests included the CareStart™ Malaria Pf (HRP2) Ag RDT  (standard of care in Uganda) and the SD Bioline Malaria Ag Pf (HRP2/pLDH) .var gene acidic terminal sequence (varATS) qPCR assay with a lower limit of detection of 0.05 parasites/µL.For qPCR, DNA was extracted from 200 µL of whole blood using the PureLink Genomic DNA Mini Kit (Invitrogen). Parasitemia was quantified using an ultrasensitive The Q-Plex™ Human Malaria Array kit  was used to measure the concentration of HRP-2 and pLDH in frozen whole blood samples per manufacturer instructions. Frozen blood samples were diluted 1:4 and 1:100 from the stock. Sample concentrations were determined using Quansys-provided Q-View software.Sample size was determined by the prevalence of malaria and to ensure that the sensitivity and specificity of the pLDH test was within 20% margin of error of expected values .Table 1). Eight percent of participants had an objective fever measured at the time of presentation to the clinic. Approximately half of participants had P. falciparum detected by qPCR, but only 11% had parasite densities of at least 1,000 parasites/µL as measured by qPCR.A total of 395 individuals participated in this study . These discordant samples tended to have very low concentrations of pLDH detected , and all except one were negative by microscopy, suggesting they may represent false-positive tests as a result of persistent pLDH or as a result of the Quansys assay from cross-contamination or matrix effects from whole blood. Microscopy and qPCR had an expected relationship, with microscopy detecting nearly all infections with qPCR parasite densities greater than 100 parasites/µL, but with very limited sensitivity at less than 10 parasites/µL .pLDH concentration in the blood correlated strongly with estimates of parasite density from qPCR (Spearman’s rho = 0.88), but there was still a variation of ≈30 fold in pLDH concentration for a given parasite density . The majority of qPCR-positive samples were also positive by pLDH, but more than half of the qPCR-negative samples had pLDH detected at some level (Supplemental P = 0.03, Table We evaluated the performance of the RDTs compared with these reference standards. The pLDH prototype RDT had a sensitivity of 98% for infections with at least 100 parasites/µL, comparable to both HRP-2 RDTs and significantly greater than the commercial pLDH-based test, which had a sensitivity of 89% for these infections (TM HRP-2 RDT, but not the pLDH prototype, had a very consistent relationship with pLDH concentration despite variation in parasite densities as measured by qPCR.We compared RDT results to pLDH concentration and parasite densities obtained from qPCR Figure . DetectaThe prototype pLDH RDT evaluated here demonstrated excellent sensitivity for infections likely to cause illness and generally detectable by microscopy, comparable to existing HRP-2-based RDTs and better than the best commercial pLDH-based test. Although the sensitivity of this new test appears sufficient for the diagnosis of malaria in this initial study, sensitivity in low-density infections was less than that of HRP-2-based RDTs, with potential implications for screening asymptomatic populations for parasitemia. On the other hand, specificity of the pLDH RDT was much greater than HRP-2-based RDTs, as might be expected given the shorter half-life of pLDH versus HRP-2 in the blood. Quantitative measurement of pLDH confirmed that the prototype RDTs are performing as expected, with pLDH levels matching visual results.There are limitations to this study that may not make it generalizable. Malaria transmission was very high in the Busia District of Uganda when this evaluation occurred, potentially resulting in a larger proportion of low-density infections resulting from acquired immunity and/or many of these infections being “older” with lower parasite density and not necessarily being the cause of the acute illness. However, this distribution of parasite density allowed for the evaluation of sensitivity near the limit of detection.Evaluating this test more widely, particularly in settings were HRP-2 deletion is present, would demonstrate its use case more effectively and should be pursued given the lack of suitable alternative diagnostics.Supplemental materials


Plasmodium vivax–specific consensus repeat sequence (CRS)-based and Plasmodium falciparum–specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp™ Malaria Detection  kit was investigated using nested PCR confirmed mono- and co-infections of P. vivax and P. falciparum positive (n = 149) and negative (n = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min  and Pf LAMP assay in 30 min (LOD 2 parasites/µL). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% . The LoopampTM Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas LoopampTM Pv showed a sensitivity of 98.36%  and specificity of 100% . The developed two-tube LAMP assay is highly sensitive (LOD ≤ 2 parasite/µL), demonstrating comparable results with the commercial Loopamp™ Malaria Detection (Pf/pan) kit, and was superior in detecting the P. vivax co-infection that remained undetected by the Loopamp™ Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both P. falciparum and P. vivax malaria.To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings.  Plasmodium, which affects 3.4 billion of the world’s population. In 2019, around 229 million malaria cases were reported worldwide, causing a toll of 409,000 deaths [Malaria is a mosquito-borne acute febrile infection caused by the protozoan parasite P. vivax (primarily in plain areas) and P. falciparum . P. ovale and P. malariae infections are also reported in some parts of the country with milder symptoms [P. vivax and P. falciparum makes species-level identification a critical step for patient care and transmission control. In India, the most prevalent species associated with human morbidity and mortality is symptoms ,4. A nonIn the elimination phase, the number of cases decreases substantially, with each case detection becoming essential to prevent perpetual transmission. Though the conventional method for the diagnosis of malaria by light microscopy (sensitivity of 50–500 parasites/μL) remains the gold standard in most clinical settings ,7,8,9. TAn indigenous, highly sensitive field-ready assay, detecting foci of infection in a way timely enough to enable treatment as well as benefit antimalarial drug efficacy monitoring, vaccine studies and screening of vulnerable populations, is perceived as a major priority for malaria elimination. The Loop-mediated isothermal amplification (LAMP) provides an alternative for rapid, sensitive, simple, cost-effective and point-of-care (POC) diagnostic assay with minimal need for trained individuals in malaria-endemic areas. Several LAMP assays have been developed for genus and species-specific identification of malaria infections . The devTM Malaria and Illumigene® have been applied in different settings, such as low-transmission areas, and used for screening of imported malaria in non-endemic settings and malaria in pregnancy [TM Malaria Pan/Pf/Pv Detection kit is an in vitro diagnostic test for the qualitative DNA detection of Plasmodium genus , P. falciparum  and P. vivax . The LoopampTM Malaria Detection kit has been recognized worldwide and is included in the WHO policy brief on the diagnostics of malaria in low-transmission settings. The applicability of these kits in various settings, the type of samples assessed and comparative diagnostic accuracy have been reviewed recently [P. falciparum and P. vivax and compared against the commercially available LoopampTM Malaria Pan/Pf/Pv Detection kit.The commercially available malaria LAMP kits Loopampregnancy ,36,37. Trecently . The widrecently ; Table 1P. vivax (n = 117); P. falciparum (n = 28) and cases of mixed infection of P. falciparum and P. vivax (n = 4); total N = 149) and 67 microscopy confirmed malaria parasite negative cases (comprising of typhoid (n = 34), visceral leishmaniasis (n = 25), tuberculosis (n = 2) and healthy control (n = 6) samples) were analyzed.The study was conducted according to a detailed protocol that conforms to the STARD (Standards for the Reporting of Diagnostic Accuracy Studies) guidelines. Ethics clearance was obtained from the institutional ethics committees of (a) VMMC and Safdarjung Hospital, New Delhi, India (IEC/VMMC/SJH/Project/January/2018/1029) and (b) ICMR—National Institute of Malaria Research, New Delhi, India (ECR/NIMR/EC/2019/253). Patients with febrile illness reporting at OPD, Safdarjung Hospital, New Delhi, India, and at fever clinic, National Institute of Malaria Research, New Delhi, India, were involved in the study. Venous blood was collected in tubes containing anticoagulant heparin for routine diagnosis of malaria during 2018 and 2019. In this study, a total of 216 blood samples from 149 microscopy confirmed malaria cases . Five microliters of nest-1 PCR amplification product was used as DNA template for each 25 µL of nest-2 PCR amplification reaction containing species-specific primers. The amplified products of nest-2 PCR were visualized by gel electrophoresis in ethidium bromide stained 2% agarose gel. Reaction with no DNA template having nuclease-free water served as negative control. The samples with positive amplification in nested PCR were considered as true positive, and samples without amplification were considered as true negative [DNA was extracted from 200 µL of blood samples using a QIAamp DNA Blood Mini Kit  as per the manufacturer’s protocol. For the current study, nested polymerase chain reaction (nested PCR) was considered as the gold standard test and performed on 216 clinical samples as described previously ,39. Brienegative ,39. http://primerexplorer.jp/e/), a program for designing LAMP primers. The LAMP primers were designed corresponding to consensus repeat sequence (CRS) Pvr47 of P. vivax, present in 14 copies per parasite. The CRS Pvr47 was selected as the target gene as it exhibited higher sensitivity compared to 18S rRNA-based conventional and multiplex PCR assay. The CRS Pvr47 is a sub-telomeric >300 bp repeat with a minimum distance of 100 bp between the multiple copies, bypassing any potential complications in PCR amplification [P. falciparum, LAMP primer sets targeting the 18S rRNA gene, previously reported by Han et al., were selected [The species-specific LAMP primer sets were designed using the Primer Explorer tool  and verified by Sanger sequencing. Recombinant plasmid with 4)2SO4, 8 mM MgSO4, 0.1% Triton X-100, 8 units of warm start Bst DNA polymerase , and 3 μL of DNA sample, independently for P. vivax and P. falciparum. One μL of 1:10 diluted SYBR™ Green I  was placed on the inner side of the tube lid, and closed tubes were incubated in a dry bath for the amplification reaction. At the end of the reaction, the tubes were allowed to cool down to room temperature, and a brief spin was given to allow the mixing of SYBR Green I with the amplified product. The reaction mixture with positive amplification instantaneously turned green, while the negative without amplified DNA remained orange. The reaction conditions were standardized for primer concentrations, incubation time (20–60 min) and temperature (60–65 °C).The closed-tube LAMP assay was performed as described previously . BrieflyThe results were read by two independent interpreters; discordance, if any in interpretation, was resolved by the assessment of a third interpreter.8 to 10 copies of plasmid/µL) of recombinant plasmids containing the target sequence of P. falciparum and P. vivax was prepared and tested by SYBR™ Green I based Pf LAMP and Pv LAMP assay to determine the analytical sensitivity. For analyzing the specificity of the P. vivax specific LAMP assay, other species malaria parasite DNA, other disease control samples  and healthy human control DNA were used. Specificity of 18SrRNA based Pf LAMP have been reported previously [Tenfold serial dilution -specific primers, designed specifically to detect the mitochondrial DNA of the four most widespread Plasmodium species . The LoopampTM Malaria Pf Detection kit and LoopampTM Malaria Pv Detection kit contain Pf-specific and Pv-specific primers that detect the target mitochondrial DNA specific for P. falciparum and P. vivax sequences, respectively. The cap of the reaction tube is equipped with the strand displacement Bst DNA polymerase, dNTPs, Calcein, reaction buffers and Malaria Pan(genus)-specific primers/P. falciparum (Pf)-specific primers/P. vivax (Pv)-specific primers in vacuum-dried form. The assay was performed in a batch of 24 reactions comprising 22 test samples, with PC MALARIA (provided in the kit) as positive control and NC MALARIA (nuclease-free water) as a negative control. For the assay, 30 µL of diluted DNA (1:6) extracted from the patient blood sample was transferred into reaction tubes containing dMALPan, dMALPf or dMALPv dried reagents. Sealed reaction tubes were flicked and allowed to stand upside down for 2 min to reconstitute the vacuum-dried reagents. After a short spin to collect the reaction mixture, tubes were incubated at 65 °C for 40 min for DNA amplification. Post amplification tubes were observed in illuminator and in-house UV light detection units. Tubes with light green fluorescence were interpreted as having positive amplification and tubes with no fluorescence as having no amplification. Prototypical STARD diagram to report flow of participants through the study for Loopamp™ Pan/Pf kit and Loopamp™ Pv kit is shown in The LoopampTM Malaria (Pan/Pf/Pv) Detection kit were calculated as follows:The percentage specificity and sensitivity of Pv LAMP, Pf LAMP and Loopamp®.In addition, 95% Confidence Intervals (95% CI), positive predictive values and negative predictive values were calculated using MEDCALCP. vivax samples were identified as P. vivax mono-infection, 22 of 28 of P. falciparum as P. falciparum mono-infection; 12 samples were identified as P. falciparum and P. vivax co-infection against four co-infection confirmed by microscopy. No amplification specific to P. falciparum and P. vivax sequences was observed in visceral leishmaniasis (n = 25), typhoid (n = 34), tuberculosis (n = 2) and healthy control (n = 6) samples.Among the 149 malaria-infected samples tested, 115 of 117 microscopy confirmed P. vivax were successfully designed, and the one that performed better during standardization was used. For P. falciparum, previously reported primer targeting 18S rRNA sequence was used [P. vivax specific CRS target sequence and P. falciparum specific 18S rRNA sequence were successfully cloned in pGEM-T easy vector and used as controls for the optimization of Pv LAMP and Pf LAMP assay. The Pv LAMP assay showed positive amplification at 30 min of reaction incubation at 65 °C with primer concentration of F3/B3 0.2 µM, FIB/BIP 1.3 µM and LF/BF 0.66 µM. For Pf LAMP assay amplification was observed at 40 min of reaction incubation at 65 °C with primer concentration of F3/B3 0.25 µM, FIB/BIP 2 µM and LF/BF 1 µM. Further, for the two-tube based LAMP assay, reactions were performed at 65 °C for 40 min.Two sets of LAMP primers specific to consensus repeat sequence of was used . P. viva8 to 10 copies/µL) containing the targeted CRS region of P. vivax. The assay showed positive results up to 10 copies/µL, equivalent to 0.71 parasites. The Pf LAMP assay showed positive amplification up to 10 plasmids/µL, corresponding to 2 parasites  infection samples, giving it a sensitivity of 100% . No fluorescence was observed when Pv LAMP assay was performed with DNA samples of P. falciparum (n = 22), visceral leishmaniasis (n = 25), typhoid (n = 34), tuberculosis (n = 2) and healthy control (n = 6), giving a specificity of 100% .The Pf LAMP and Pv LAMP assay were evaluated against clinical samples, and the results are summarized in P. falciparum and 12 mixed-infection samples, presenting a sensitivity of 100% . No amplification was observed in 115 P. vivax and 67 non-malaria DNA samples, giving a specificity of 100% .For the Pf LAMP reaction, a positive fluorescence was noted in 22 TM Malaria Pan Detection kit was tested against 44 samples , P. vivax (n = 15), co-infection (n = 8) and other disease (n = 6)) . No amplification was observed in 15 P. vivax and six other disease samples, giving the LoopampTM Malaria Pf Detection kit a specificity of 100% . Of 88 samples tested using the LoopampTM Malaria Pv LAMP Detection kit , 53 samples were detected as true P. vivax positive. However, among the eight mixed-infection samples, only seven samples had positive amplification, giving the kit a sensitivity of 98.36% . No fluorescence was observed among the P. falciparum (n = 15), visceral leishmaniasis (n = 3), typhoid (n = 3) and healthy control (n = 6) DNA samples, giving the LoopampTM Malaria Pv Detection kit a specificity of 100% . Cross-tabulation of index tests  results by the results of reference standard test (Nested PCR) for the diagnosis of malaria  is given in Loopamp(n = 6)) . A positPlasmodium parasite detection is a potential point-of-care diagnostic test, as it fulfills all the criteria for a complete commercial sample processing system when special features such as vacuum-dried reaction mixtures sealed within reaction tubes and long-term stability are taken into account. The test is simple, rapid, user friendly and less sophisticated; hence, it can be performed easily in field conditions in low-resource settings. The Global Technical Strategy for Malaria Elimination 2016–2030 set up by the WHO and the Roll Back Malaria Partnership emphasizes the "high burden to high impact (HBHI)" approach to intensify support for countries with a high burden of malaria. India’s National Vector Borne Disease Control Program (NVBDCP), in collaboration with the WHO, has adopted HBHI approaches in the different states of India for situation analysis. India is progressing towards attaining malaria-free status by 2027 and eliminating the disease by 2030 [P. falciparum and P. vivax malaria infections. The LOD for Pf LAMP was 2 parasites/µL, and for Pv LAMP, LOD was 0.7 parasites/µL, presenting a detection tool for very low parasitaemia cases. The parasite count of clinical malaria samples tested in this study ranged from 912 to 345,600 for P. falciparum and 362 to 79,053 for P. vivax. Both the Pf LAMP and Pv LAMP assay had 100% specificity, presenting no cross-reactivity with other diseased patient samples. Our new LAMP assay has a detection time of 40 min. The new consensus repeat based Pv LAMP test developed in this study exhibited sensitivity and specificity of 100% for detection of P. vivax, an improvement over other targets both in terms of sensitivity and specificity, and turnaround time of less than one hour. The diagnostic accuracy and sensitivity of the new malaria LAMP assay in this study were higher than those of microscopy and equal to those of nested PCR, with the added advantage of reduced assay time and ease of operation.This study describes the development of a two-tube lamp assay for the differential diagnosis of TM Malaria Pan/Pf Detection kit has been evaluated by various groups from different countries. A diagnostic accuracy study carried out on febrile returned travelers with this kit reported a sensitivity and specificity of 97.0% and 99.2%, respectively, for the LoopampTM Malaria Pan test and 98.4% and 98.1%, respectively, for the LoopampTM Malaria Pf test [TM Malaria Pf test using samples with a P. falciparum qPCR titer of ≥2 parasites/μL [TM Malaria Pan and LoopampTM Malaria Pf was observed as 83.3% and 60%, respectively, with specificity of 99.7% [The WHO-recognized, commercially available Loopamp Pf test . At the sites/μL . Howeverof 99.7% . These rTM Malaria Pan Detection kit demonstrated a sensitivity and specificity of 100%. Against the same batch of 44 samples, LoopampTM Malaria Pf Detection kit reactions showed a sensitivity of 100% and specificity of 100%. For the 88 samples tested by the LoopampTM Malaria Pv Detection kit, a specificity of 100% and a sensitivity of 98.36% was observed, where all the 53 true P. vivax samples were detected. However, only seven samples had a positive amplification among the eight co-infection  samples. The undermined sample was a mixed infection case confirmed by the nested PCR that went undetected by microscopy, probably due to difficulty in detecting low P. vivax parasitemia, as the P. vivax often has a lower parasite density  than P. falciparum [P. vivax samples and eight co-infection  samples. The cases of high-density infections show symptoms, and the patients approach health facilities for treatment. However, low-density infections in the community develop very few symptoms; thus, patients seek no treatment, and the infection goes undetected. These low-density infections, below the detection threshold of microscopy and RDTs, continue to infect the vector flies and sustain transmission [In this study, the Loopamplciparum . On the smission ,44. The The different LAMP assays developed using different genus or species-specific gene targets presented sensitivity in the range of 83.3–100% and a specificity of 85–100% . When peP. falciparum) and CRS (P. vivax) as targets, demonstrated high sensitivity (100%) and specificity (100%). It successfully detected P. vivax in a co-infection case that remained undetected using the commercially available LoopampTM Malaria Pv Detection kit. The CRS Pvr47 target deployed in Pv LAMP of our assay is a non-coding, sub-telomeric and highly conserved repeat sequence, without any internal tandem repeats that could potentially interfere with the PCR amplification [P. falciparum and P. vivax will enable easy handling for simultaneous differential diagnosis of P. falciparum and P. vivax species. Malaria LAMP provides several advantages, including ease of operation and requirement of minimally trained personnel. The diagnostic test requires only a small blood volume (30–60 µL) and is patient-friendly. The Pv and Pf LAMP reactions (for a batch of 24 samples) were completed within 90 min by a single staff member when the DNA template was available beforehand. The need for a simple isothermal heating block, lower start-up costs, and significantly reduced assay time gives LAMP an edge over nested PCR, which requires three to four operational hours. Though the expenses of LAMP reagents are close to those used for nested PCR, a comparison of both techniques in terms of equipment and labor costs reveals that LAMP would be a more affordable option for laboratories in malaria-endemic countries. The frequently observed contamination issue, giving false-positive results, was prevented in the current assay development by following the closed-tube method and using a separate hood for reaction mixture preparation and addition of DNA sample, respectively. The SYBR Green I based two-tube test developed here, using 18S rRNA (fication . After fWith an adaptable high-throughput 96-well-plate format, the assay could be deployed for country-wide surveillance, antenatal screening and drug efficacy monitoring ,45,46. MP. falciparum and P. vivax supporting targeted treatment, has advantages over other molecular tests in terms of robustness, applicability and sensitivity. The current test format with certain improvisations may potentially replace other nucleic acid– based methods for the diagnosis of malaria in endemic countries. The test will enable parasite identification in asymptomatic and very low parasite density individuals in field settings for mass screening, case management and transmission control. In conclusion, the current challenge of malaria elimination, including the complication of developing resistance, requires simple diagnostics for prompt and accurate detection and treatment of malaria parasites. The applicability of this assay remains to be evaluated under field conditions. The integration of the cost-effective and straightforward DNA template preparation method, ideally avoiding pieces of equipment, into the assay pipeline will make it better suited for mass surveillance studies. The two-tube malaria LAMP test developed here for the primary diagnosis of malaria, with differential diagnoses of


Plasmodium infection is thought to represent the majority of infections, though they are not diagnosed nor treated. Therefore, there might be an underestimation of the malaria reservoir, resulting in inadequate control strategies. In addition, these untreated asymptomatic Plasmodium infections maintain transmission, making it difficult or impossible to reach malaria elimination goals. Thus, the aim of this study was to determine the prevalence of asymptomatic Plasmodium infections in southeastern Senegal.Malaria control and elimination strategies are based on levels of transmission that are usually determined by data collected from health facilities. In endemic areas, asymptomatic A cross sectional study was conducted among asymptomatic individuals (N = 122) living in the village of Andiel located in Bandafassi, Kédougou, which consisted of about 200 inhabitants during the malaria transmission season in late October 2019. For each individual without malaria-related symptoms and who consented to participate, a rapid diagnostic test (RDT) was performed in the field. Results were confirmed in the laboratory with photo-induced electron transfer (PET-PCR).Plasmodium. Plasmodium falciparum was predominant and represented 90.2% of the infections, while 6.5% were due to Plasmodium ovale and 3.3% to Plasmodium malariae. 59.4% of children targeted for SMC (zero to ten years old) were infected.Malaria prevalence was 70.3% by PET-PCR and 41.8% by RDT. During the same period, the health post of the area reported 49. 1% test positivity rate by RDT. The majority of the infected study population, 92.9%, was infected with a single species and 7.1% had two or three species of Plasmodium infections at the community level. The results suggest that this area could be eligible for mass drug administration. Moreover, non-falciparum species could be more common and its prevalence should be determined countrywide.In southeastern Senegal, where the transmission is the highest, malaria control strategies should address asymptomatic  Plasmodium infections are not detected as in endemic areas individuals can carry the Plasmodium parasites without any symptoms. There is overwhelming evidence indicating that a significant proportion of Plasmodium infections are asymptomatic in endemic areas. It has been shown that over 25% of individuals in sub-Saharan Africa  presented sub-microscopic gametocytes and were capable of infecting mosquitoes [Plasmodium species [Malaria is an endemic parasitic disease, with an estimated 229 million cases worldwide in 2020 , and mossquitoes . Likewis species . These aPlasmodium infections are a serious threat to elimination and should be addressed by the National Malaria Control Programmes in their policy implementation for malaria elimination. In endemic areas, the parasite density among subjects with asymptomatic Plasmodium infections is usually low, and generally not detected by classical methods, such as rapid diagnostic test (RDT) and light microscopy, which have low sensitivity compared to molecular techniques [Asymptomatic chniques .Plasmodium falciparum is the primary species responsible for malaria, but the prevalence of other species of Plasmodium remains unknown. However, in the context of malaria elimination, the prevalence of other Plasmodium species that may cause asymptomatic Plasmodium infections with low density infection or relapses must be determined. The circulation of non-falciparum species has been previously reported in Senegal; Plasmodium ovale and Plasmodium malariae have been found in Dakar and Kedougou [Plasmodium vivax in southeast Senegal [P. vivax is debated.In Senegal, Kedougou –9, as we Senegal . HoweverPlasmodium spp. infections are usually not diagnosed as people are not likely to seek care in the absence of symptoms. In remote villages of high transmission zones, such as southeastern Senegal, asymptomatic Plasmodium infections may be more prevalent than symptomatic malaria cases detected in the health facilities. The aim of this study was to investigate the prevalence of asymptomatic Plasmodium infections in the village of Andiel in Kédougou, southeastern Senegal.Asymptomatic CareStart™ Malaria Pf/Pan,  and blood was collected from the finger prick and spotted on filter paper to create a dried blood spot (DBS), and stored at room temperature. All samples were tested with PET-PCR at the laboratory for confirmation and Plasmodium species identification. Non-consenting individuals or those with symptoms were referred to the HCP serving the village for a malaria rapid diagnostic test. All residents with a positive test received first line treatment according to the National Malaria Control Programme (NMCP) policy. Data from the health post of Bandafassi which oversees Andiel village was collected through the NMCP database. The study was approved by the ethical committee of the Ministry of Health of Senegal (00000171/MSAS/DPRS/CNERS).The study was a cross-sectional survey. In the village, after sensitization of the community by the local health team and community leaders, the inhabitants were asked if they were experiencing symptoms related to malaria; if the answer was negative, they were invited to participate in the study. Consenting participants who met the inclusion criteria received a malaria rapid diagnostic test using the P. falciparum, P. ovale, P. malariae, have been reported in the region and P. falciparum is the predominant species [The study was conducted in the region of Kédougou. It is bordered on the west by the hills of Bassari country and Mount Assirik, which dominates the Niokolo-Koba National Park. Kédougou is one of the wettest regions in the country, with at least 1300 mm of rainfall/year. The rainy season lasts about six months, from May to October, followed by a long dry season. However, this rainfall is characterized by great spatio-temporal variability, the months of August and September being the rainiest . Tempera species . Since 2The study was carried out on apparently healthy participants in the village of Andiel during the malaria transmission season at the end of October 2019. Date of sampling and demographic data such as age and sex were collected for participants who were enrolled in the study. No information related to test result or epidemiological data were collected for non-consenting inhabitants and individuals with symptoms.All residents older than 6 months who did not present any malaria-related symptoms such as fever or history of fever, headaches, nauseas vomiting, aches and pains were eligible. Residents who presented symptoms consistent with malaria or reported being treated for malaria the last two weeks were ineligible.CareStart™ Malaria Pf/Pan, Accessbio. This two-band RDT detects the P. falciparum histidine rich protein 2 (PfHRP2) specific to P. falciparum and pan-Plasmodium parasite lactate dehydrogenase (p-LDH). This allows the RDT to distinguish P. falciparum from the other Plasmodium species in mono-infections or in mixed infections.The RDT used was the Plasmodium species was also performed for all samples using the photo-induced electron transfer (PET)-PCR assay [P. falciparum strain) control. Samples with a cycle threshold (CT) of 40 or less were scored as positive [P. malariae, P. vivax, and P. ovale was performed in a 20 ml reaction containing 2X TaqMan Environmental MasterMix 2.0 (Applied BioSystems), 250 nM each forward and reverse primer, and 5 μl of DNA template. All reactions were performed in a total volume of 20 μl containing: 5 μl of DNA, 10 μl of 2X ABI TaqMan buffer and 250 μM of each forward and reverse primers. The reactions were performed under the following cycling parameters: initial hot-start at 95 °C for 15 min, followed by 45 cycles of denaturation at 95 °C for 20 s and annealing at 60 °C for 40 s.Molecular characterization of CR assay  on a Rocpositive , 13. TheThe health post of Bandafassi oversees twenty (20) villages, including Andiel, for a total of 7944 inhabitants. The number of malaria cases reported by the health post of Bandafassi which oversees Andiel village was obtained through the NMCP database . Malaria cases were diagnosed among patients seeking care at the health post and who presented fever during the consultation or a history of fever during the preceding 48 h, using SD Bioline Malaria antigen Pf® RDT. The test positivity rate was calculated for all the patients attending the health post  and for specific groups of patients  during the malaria transmission season (from July to December).Data were collected into Excel and analyzed. Statistical analysis was performed using GraphPad prism version 5.01. Differences were considered significant with a p-value < 0.05. The non-parametric Mann–Whitney U test was used to calculate the p-value.Plasmodium infections” for infected individuals who did not present any symptoms and “Plasmodium infections” when referring to all infections . “Participants” was used for the individuals without malaria symptoms and who consented to participate in the study. Data were not collected from people with symptoms consistent with malaria. Asymptomatic Plasmodium infection was defined as the absence of fever or history of fever, headaches, nausea, vomiting, or aches and pains, with a positive biological malaria test (RDT or PCR).In this manuscript “symptomatic malaria cases” was used strictly for symptomatic malaria episodes, “asymptomatic The village of Andiel is composed of approximately 200 inhabitants, among whom one hundred twenty-two 122) met the inclusion criteria and were enrolled. Twenty-six (26) individuals did not present any malaria related symptoms, but did not consent for the study. Fifty-two (52) of the inhabitants did not meet the inclusion criteria: thirteen (13) reported malaria treatment during the two weeks before the study, nineteen (19) presented malaria related symptoms, and twenty (20) were not present during the period of the study  were female and 45.1% (55) were male. The participants' ages ranged from 11 months to 90 years with the mean (SD) of 27.2 (22.69) years and a median of 19 years (Q1–Q3: 10–43). Age distribution is summarized in Table RDT was performed for all the 122 participants at the community level in the village; 121 samples were tested using PET-PCR in the laboratory (there was one missing sample). The prevalence of malaria was by PET-PCR was 70.2% (95% CI 29.2–53.9) and by RDT was 41.0% (95% CI 54.4–86.6).The PET-PCR and RDT were both positive in 33.9% (41) of samples and both negative in 22.3% (27) of samples, while 7.4% (9) were positive by RDT and negative by PET-PCR, and 36.4% (44) were detected with the PET-PCR but not RDT. The Ct for the samples detected by PET-PCR but negative with the Pan/RDT were higher than the Ct for the samples positive for both techniques , though the sample size was small. Among the children aged 0 to ten years old (N = 32), who were targeted for SMC, 53.1% (N = 17) had asymptomatic Plasmodium species and prevalence of mixed infection were determined using PET-PCR. Among positive samples (85), single infections (caused by one Plasmodium species) represented 92.9% , while 7.1% were mixed infections . Plasmodium vivax was not detected.P. falciparum bands in 94.1% (48/51) of the RDT positive samples, and P. falciparum and pan bands, suggesting the presence of non-falciparum species, in 5.9% (3/51 samples). Among the three samples with positive pan bands on RDT, two were mixed infections with P. falciparum and P. ovale and one was a P. falciparum single infection. Conversely, one P. malariae infection, one P. ovale infection, one mixed P. falciparum + P. malariae infection, and two mixed P. falciparum + P. ovale infection were not detected by RDT.The RDT identified The test positivity rate (TPR) at the health post of Bandafassi using RDT  was determined during the transmission season at the same period (October) and was 40.7% (598/1468). While children 3 months to 10 years are targeted for SMC, the TPR from July to December 2019 among patients with febrile illness who attended the health post and received an RDT was lowest among children under five years old, while children aged 5 to 10 years had a TPR more comparable to older age groups during the same period   of 49.1% was recorded at the health post of Bandafassi using PfHRP2 RDT. The high Ct value of samples detected by PET-PCR but not by RDT  suggests that the parasite density is low for those samples. The low detection of asymptomatic Plasmodium infections by Pan/RDT compared to PET-PCR shows the importance of utilizing sensitive techniques such as real time PCR for malaria diagnosis during surveys.This community-based study conducted in southeast Senegal reports the prevalence of asymptomatic Plasmodium by PET-PCR. While the highest proportion and overall numbers of asymptomatic Plasmodium infection were detected by PET-PCR were in those 10 years and older, asymptomatic malaria infection remained startlingly high among those targeted for SMC, particularly among children 5–9 years old. Similarly, the test positivity rate at the health post was highest among children 5–9 years of age. This finding is concerning for the effectiveness of SMC not only in Andiel, but among children in this catchment zone, whether due to lower uptake, declining efficacy, or lower coverage of co-interventions, such as bed net use.Overall, 53.1% of children up to ten years old, the age group targeted for seasonal malaria chemoprevention, were infected with P. falciparum was the main species identified, P. ovale and P. malariae were detected mostly as mixed infections with P. falciparum, which have been reported in several regions of the country [P. falciparum is predominant in sub-Saharan Africa, more attention should be given to the other species as their prevalence often becomes higher when the prevalence of P. falciparum decreases [Plasmodium vivax was not identified in this study, however, some studies in Senegal have reported its presence in some parts of the country [P. vivax circulation.Although  country –10, 14 a country –17. Whilecreases . Moreove country . More inThere were several important limitations to this study. It was conducted in a small village, and as such the findings are not representative and cannot be generalized. Non-inclusion of symptomatic malaria cases during the sampling at the community makes it impossible to directly compare to data from the health facility. It was also a missed opportunity to collect information about bed net availability and use and SMC coverage, which might have further illuminated the results.Plasmodium infection in a high transmission setting, the discrepancy between PET-PCR and RDT-based parasite prevalence, which suggests that parasite prevalence may be higher in other regions of the country than currently thought. Molecular techniques such as PET-PCR should be integrated into cross sectional surveys in order to obtain a more accurate understanding of parasite prevalence and species differentiation.However, this study gives important information about the frequency of asymptomatic Plasmodium infections at the community level. Therefore, it becomes necessary to put in place a surveillance strategy for these infections that can serve as a reservoir for malaria transmission in a country such as Senegal, which is working towards malaria elimination in 2030. In Senegal, malaria surveillance became an intervention in low transmission settings, however it should be extended to the intermediate and high transmission areas such as Kédougou. The prevalence of non-falciparum species should be monitored nationally as these will become relatively more prevalent as P. falciparum decreases.The high prevalence of asymptomatic malaria observed in this study suggests that malaria control strategies should address asymptomatic