Mass screening and treatment (MSaT) aims at reducing the spread of malaria in communities by identifying and treating infected persons regardless of the symptoms. This study was conducted to identify and treat asymptomatic cases using MSaT approaches in the community.Three rounds of MSaT using cluster combination approaches were carried out during September 2018 to December 2019 to identify and treat asymptomatic malaria cases in the community. All individuals who were present in the household were screened using RDT irrespective of malaria related symptoms. Simultaneously thick and thin blood smear and blood spot were collected for further analysis using microscopy and diagnostic PCR done in a subset of the samples.Logistic regression analysis revealed that asymptomatic malaria cases significantly less among the older age groups compared with < 5 years children , lowest in cluster 4 ; during third round of MSaT survey  and significantly higher in moderate to high endemic areas .Over the three rounds of MSaT, the number of asymptomatic cases were significantly less in the older age groups, and during third round. Similarly, the asymptomatic cases were significantly less in the low endemic area with API < 1 (cluster four). Therefore, the malaria elimination programme may consider the MSaT strategy to identify asymptomatic cases that would be otherwise missed by routine fever based surveillance. This MSaT strategy would help accomplish the malaria elimination goal in an expedited manner. India has committed to the global malaria elimination goal of 2030, which translates to achieving zero indigenous cases by 2027 . Over thThe Mass Screening and Treatment (MSaT) aims to detect and treat all malarial parasite infections, including asymptomatic parasite carriers within the community. The World Health Organization recommends MSaT as an intervention strategy targeting asymptomatic infections to reduce the prevalence . In anotIn the present study, three rounds of MSaT were conducted using RDTs, microscopy, and PCR to identify asymptomatic malaria-infected individuals. The MSaT was accompanied by vector control interventions such as LLIN and IRS, along with active fever surveillance and case management. These three round of MSaT were done during the pre-transmission, transmission, and post-transmission period for identifying and treating the asymptomatic cases in the community of district Mandla, Madhya Pradesh, India.This study was part of the Malaria Elimination Demonstration Project, which was a first-of-its-kind public–private-partnership between the Indian Council of Medical Research (ICMR) through the National Institute for Research in Tribal Health (NIRTH) Jabalpur, Government of Madhya Pradesh (GoMP), and the Foundation for Disease Elimination and Control of India .2, inhabited by ethnic tribal communities, mainly ‘Gonds’ and ‘Baiga’.The study was conducted in district Mandla, located between coordinates of 22°02′ and 23°22′ N latitudes and 80°18′ and 81°50′ E longitudes in the east-central region of the state of Madhya Pradesh (MP), India. The total geographical area of the district was 8771 kmA cross sectional MSaT was conducted during the malaria transmission season in September 2018, pre-transmission season in June 2019 and post-transmission season in December 2019 to assess the asymptomatic cases of malaria in the community. A multistage sampling method was adapted. In September 2018, for the first round of MSaT, a cluster sampling method was used in which all the villages of the district Mandla were divided into four clusters based on their Annual Parasite Incidence  during five preceding years (2013–2017) and accessibility to the study areas.The clusters were categorized as: (1) Hard-to-reach or inaccessible villages; (2) More than or equal to API 5; (3) API between 1 to 4.99; and (4) API between 0 and 0.99. The second round of the MSaT survey was conducted in June 2019 to validate the findings from the first round of MSaT and the entire study area was further stratified into moderate to high endemic (> 1 API) and low endemic (0–1 API) villages. Further, a third round of the MSaT survey was carried out in December 2019 in 50 houses surrounding the cryptic cases  diagnoseSample size was determined using the formula of simple random sampling for a finite population as given below:The sample size for the different clusters and malaria endemic areas was determined based on the lowest asymptomatic malaria prevalence reported by Wangchuk et al.  in BhutaDoor-to-door visits were done by trained Village Malaria Workers (VMWs) to screen the community for malaria parasites. The VMWs screened each consenting individual in the study area using bivalent malaria RDTs  per field, a blood slide was considered negative. All the samples having discordant results were re-checked by an independent WHO certified microscopist for quality assurance of light microscopy.Plasmodium species was determined using species specific nested PCR by targeting 18Sr RNA gene. To set up the primary PCR, 5 µL of genomic DNA as template was taken to amplify the 18S rRNA gene for the Plasmodium genus using forward and reverse primer [Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. The PCR product was analysed on 1.2% Agarose gel electrophoresis.Molecular analysis was done in about one-third of the randomly selected samples. Genomic DNA was isolated from the dried blood spots using the Chelex method [Chelex-100 Sodium form (50–100 Mesh) Himedia Laboratories . The pree primer . The priThe demographic  variables, clinical symptoms related to malaria, results of RDT, microscopy, and PCR were entered in Microsoft Excel 2007 worksheet, and the numerically coded data was exported in R 4.1.2 for Windows  for statistical analysis. Logistic regression analysis was used to examine the association of independent variable(s).A total of 88142 individuals, irrespective of malaria-related symptoms, were tested using RDTs during door-to-door visits. Out of which, 34694 (39.36%), 18550 (21.05%) and 34898 (39.59%) were screened in the first, second and third rounds of the MSaT surveys, respectively. In clusters one to four; 4437 (5.03%), 17616 (19.99%), 9709 (11.02%) and 56380 (63.96%) individuals were screened, respectively. About three-fourths of the individuals were adults with a mean age of 31.86 ± 20.07 years, and about 51% were female . Out of which, 0.20% (69/34694), 0.15% (28/18550), and 0.10% (35/34898) cases were recorded during the first, second and third rounds of MSaT, respectively, with a statistically significant declining trend (p = 0.003).The cluster-wise (one to four) prevalence of malaria was 1.19% (53/4437), 0.28% (50/17616), 0.29% (28/9709), and 0.002% (1/56380), respectively, which was statistically significant (p < 0.0001). Amongst the residents of low malaria endemic areas, the prevalence was 0.002% (1/56380). Whereas the prevalence in moderate-to-high malaria endemic areas was 0.41% (131/31762), which was significantly higher (p < 0.0001). The PCR based diagnosis was done in a sample of 24357, which was randomly selected from the total 88142 MSaT samples. Out of these 24357 cases tested by PCR, 23681 were asymptomatic, and the rest 676 were symptomatic. Amongst the samples tested by PCR, the malaria positive cases by RDT were 0.12% (30/24357), microscopy were 0.14% (35/ 24357), and PCR were 1.06% (258/24357).The prevalence of asymptomatic malaria infections in the first, second, and third rounds was 0.53% (179/34013), 0.57% 103/18202) and 0.06% (20/33797), respectively. Further, analysis revealed that the asymptomatic malaria prevalence significantly declined during the third round as compared to the first round . Similar declining trend was recorded in all the clusters (p < 0.0001). Neither asymptomatic nor symptomatic malaria cases was reported from clusters three (low endemic) and four (moderate endemic) during the second and third rounds of the MSaT survey  than first round 2.64% (18/681) of MSaT survey (p = 0.002), while it increased in the third round  but not statistically significant. A similar trend was reported in clusters one and two (hard to reach and high endemic areas). None of the symptomatic malaria cases was reported in cluster three and four during second and third round of MSaT survey , 0.48% (21/4348) and 0.89% (12/1347) in cluster one; 0.14% (24/1726), 0.10% (18/1726) and 1.33% (151/11348) in cluster two; 0.16% (15/9400), 0.08% (8/9400) and 2.72% (64/2349) in cluster three; and 0% (0/54995), 0.002% (1/54995) and 0.06% (5/8637) in cluster four, respectively. The analysis revealed that a significantly higher prevalence of asymptomatic malaria was reported using PCR diagnosis in all the clusters (p < 0.0001).This prevalence varied significantly over the MSaT rounds through all the clusters and diagnostic methods. Among all the diagnostic methods, the asymptomatic malaria prevalence significantly declined in the third MSaT round as compared to the first MSaT round (p < 0.001) except in cluster one, where the asymptomatic malaria prevalence was found to be significantly higher in the third round than the first round (p < 0.001). However, a higher prevalence of symptomatic malaria was found during the third round of MSaT as compared to the first round in all the diagnosis methods, and it was significant in cases diagnosed using RDT and PCR (p < 0.001) Table .Table 3CUnivariate logistic regression analysis revealed that asymptomatic malaria was significantly lower among the age groups 5–15 years , 15–25 years , 25–50 years  and above 50 years  in reference to the < 5 years children. Further analysis showed asymptomatic malaria was significantly lower in cluster four , during the third round of MSaT survey  and significantly higher in the moderate to high endemic areas  than their reference categories. However, the asymptomatic malaria cases did not significantly differ between age groups and gender (p > 0.05). Further, symptomatic malaria cases were significantly lower in the clusters two  and three  as compared to the cluster one.Symptomatic malaria cases did not differ significantly through different age groups, gender, endemicity and rounds of the MSaT surveys (p > 0.05). The total malaria prevalence in MSaT significantly decreased among the older age groups than < 5 years children (p < 0.05); in round three ; in cluster two  and cluster four  than their reference categories, while it was 104 times likely to be higher in moderate-to-high endemic areas than low endemic areas , 0.15% (second round), and 0.10% (third round). This observation was similar to a study done in Zambia that revealed 17% reduction in the incidence of clinical malaria after three rounds of MSaT . The groThe present study revealed a significantly higher prevalence of asymptomatic infections in moderate to high endemic areas (0.41%) as compared to the low endemic areas (0.0%). Compared to this, an opposite result was found in western Kenya, but the sample size was much lower .Durgama Anchalare Malaria Nirakarana (DAMaN)’ focussing on these hard-to-reach areas was conducted in Odisha and reported a significant reduction in malaria cases using MSaT, LLINs, and community mobilisation [The hard-to-reach areas remain a bottleneck of malaria elimination due to inadequate health care services/ service providers. The cluster wise analysis of the present study reported a prevalence of 1.19% (hard to reach area), 0.28% (API: > 5), 0.29% (API: 1–4.99), and 0.002% (API: 0–1) with a declining trend over different rounds (p < 0.0001). A study known as the ‘lisation .In another MSaT study conducted in Malawi in school children, a decrease in malaria transmission was reported , which iThe present study has revealed that the asymptomatic malaria infections were significantly reduced in the third round 0.06% of MSaT as compared to the first round (0.53%), which suggest that MSaT should be considered as a strategy along with other case management and vector control tools for effectively halting malaria transmission in the community.Another important observation of this study is the finding that PCR detects more than three times higher cases as compared to RDT / microscopy. The asymptomatic cases diagnosed by PCR were higher in all clusters than the conventional methods and were highest in moderate to high endemic areas. In a similar study from Zanzibar, it was found that MSaT detected more than 10 times higher cases using species-specific PCR (2.5%) as compared to RDT (0.2%) . This fiP. falciparum using PCR tools [The usefulness of a sensitive point-of-care PCR-based diagnostic tool for MSaT was highlighted by Von Seidlein L , which iCR tools , and repThe evidence generated by this Malaria Elimination Demonstration Project , 32–41 iThis study has demonstrated the significance of MSaT as an effective surveillance tool to detect and treat asymptomatic cases of malaria. Given the limitations of RDT and microscopy, it is recommended that national programmes and the WHO should implement a policy that recommends the use of nucleic acid-based detection tools, such as PCR, for detecting asymptomatic reservoirs of malaria during elimination phase.


Plasmodium parasites binding to RBCs at different steps of host infection. The formation of a tight junction between EBL merozoite ligands and glycophorin receptors is the crucial interaction in ensuring merozoite entry into RBCs. It was proposed that sialic acid residues of O/N-linked glycans form clusters on a human glycophorins polypeptide chain, which facilitates the binding. Therefore, specific carbohydrate drugs have been suggested as possible malaria treatments. It was shown that the sugar moieties of N-acetylneuraminyl-N-acetate-lactosamine and 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which is its structural analog, can inhibit P. falciparum EBA-175-GPA interaction. Moreover, heparin-like molecules might be used as antimalarial drugs with some modifications to overcome their anticoagulant properties. Assuming that the principal interactions of Plasmodium merozoites and host cells are mediated by carbohydrates or glycan moieties, glycobiology-based approaches may lead to new malaria therapeutic targets.The key element in developing a successful malaria treatment is a good understanding of molecular mechanisms engaged in human host infection. It is assumed that oligosaccharides play a significant role in  Malaria remains a major global health problem, being responsible for approximately 500,000 deaths each year globally, mostly among children and pregnant women in sub-Saharan Africa . Long-tePlasmodium, which are transmitted via bites of infected female Anopheles mosquitoes [Plasmodium species can infect not only humans but apes as well. Humans are infected by Plasmodium falciparum; three other species infect chimpanzees  while the other three infect gorillas  [Malaria is caused by apicomplexan parasites of the genus squitoes . The Pla adleri) ,5.Plasmodium parasites multiply in the liver and then are released in form of merozoites, which infect red blood cells (RBCs) [Plasmodium merozoites can be divided into multiple steps in which several protein ligands expressed by merozoites are involved [P. falciparum EBL ligands were identified [After a bite by an infected mosquito, s (RBCs) . This ass (RBCs) ,8. The iinvolved ,10,11,12involved . Among tinvolved . Four hoentified  , carrying sialylated O-glycans and/or N-glycans . Most GP kDa, carHBB) [P. falciparum proteins following malaria infection; thus, a therapeutic blockade of this pathway has been proposed to shorten the life cycle of the Plasmodium [In addition, alternations of the O-glycosylation pathway in beta-thalassemic RBCs were identified as an increase in O-GlcNAcylation of serine and threonine residues . This haHBB) . HeterozHBB)  and was asmodium . This obPlasmodium host specificity.In this review, we present the role of sugars as major mediators of merozoite–ligands interaction and suggest that they may determine Sialic acids are terminal carbohydrate moieties on cell glycocalyx. Most of them are linked to glycoproteins, except for human neuronal cells, where sialic acids are linked to sphingolipids (gangliosides) . As termN-5-acetylneuraminic acid (Neu5Ac) and N-5-glycolylneuraminic acid (Neu5Gc) (CMAH gene) [CMAH gene is nonfunctional in New World monkeys, so European hedgehog, Mustelidae, Procyonidae, some bats, sperm whale, white-tailed deer, and platypus [CMAH gene ceased to encode active protein some 2 million years ago [Sialic acids are derivatives of neuraminic acid (nonulosonic acid) . There a(Neu5Gc)  [35,36,3AH gene) ,42. The platypus ,44 do noears ago ,46,47,48N-acetylgalactosamine and α in polysialic acids. These bonds are created by specific sialyltransferases and polysialyltransferases, respectively [The linkage configurations of sialic acids are α or α with galactose or ectively . The α-gectively ,51.Plasmodium merozoites binding to RBCs [P. falciparum EBA-175 ligand was resolved, giving insight into the molecular mechanisms underlying the GPA binding [It was shown previously that EBA merozoite ligands do not bind to RBCs treated with neuraminidase, which cleaves terminal α-linked sialic acids from oligosaccharide chains . Since t to RBCs . The cry binding . The dim binding  , while P. falciparum EBA-175 and EBA-140 bound to Neu5Ac [P. falciparum EBA-175 ligand revealed the strong binding of EBA-175 to Neu5Gc monosaccharide as well [The ape homologues of the EBA P. falciparum ligands, including chimpanzee entified ,62. It wo Neu5Ac . Thus, io Neu5Ac ,64. Howe as well ,66,67. TP. falciparum [P. reichenowi EBA-165 bound only to glycans containing Neu5Gc [EBA-165, another homologous merozoite protein, is present in all ape-infective species, but due to a 5′ frameshift in genes, which truncates the transcript before the RII binding domain, is not expressed in human-infecting lciparum . It was g Neu5Gc .P. falciparum shows the specificity towards both Neu5Ac and Neu5Gc. It suggests that P. falciparum has gained the ability to bind Neu5Ac without losing the binding specificity for Neu5Gc. In conclusion, there is a clear preference of the binding to Neu5Gc in all studied ape species, while human It is generally assumed that individuals with sickle cell disease, which is caused by a point mutation in the β-globin gene and manifests by chronic anemia, hemolysis, and inflammation, are more resistant to malaria than non-sicklers . It has Plasmodium malaria merozoites exploit the diversity of sialic acids on the surface of RBC to invade their hosts.Thus, it may argued that ABO blood group antigens are expressed not only on the RBCs but also on epithelial and endothelial cells. ABO blood group phenotype is determined by a presence on the RBCs surface of the A, B, H blood group antigens, respectively. These antigens are trisaccharides, which consist of N-acetylgalactosamine, galactose, and fucose residues . It has 1 and A2 in RBCs was also found [1 subgroup express approximately four to five times more A antigens than those of the A2 subgroup [1 phenotype were larger and less susceptible to anti-PfEMP1 antibodies and the damaging effect of heparin than A2-phenotype RBC rosettes [Rosetting is the adhesion of infected RBCs to other uninfected peripheral RBCs . It was so found ,81. The subgroup . Thereforosettes . Moreoverosettes ,84. Howerosettes ,83.Sequestration is the adhering of infected RBCs to endothelial cells, which occurs when young parasites reach the trophozoite stage . It reduThe genes belonging to two multigene families encode RIFIN and STEVOR proteins that are expressed on the surface of the infected RBCs, which implies that they may serve as potential cytoadherence-mediating virulence factors ,87. RIFIGlycosaminoglycans are components of mammalian cells extracellular matrix . HeparinP. falciparum merozoite surface protein 1 (MSP-1), the most abundant merozoite surface protein, is responsible for the initial contact with RBCs [Full-length ith RBCs . MSP-1 aith RBCs .P. falciparum EBA-140 ligand contains several potential GAG-binding motifs and blockages of these motifs by soluble heparin or HS induce an inhibition of this ligand binding and the merozoite invasion [invasion . HS was P. falciparum circumsporozoite protein (CSP), mediating the transmission of parasite to liver cells [Moreover, HS on hepatocytes was suggested to bind to er cells ,97.P. falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on parasite-infected RBCs, mediating the binding of infected RBC to the vascular endothelial cells or other RBCs [Additionally, HS is a receptor for her RBCs ,99.Although the role of HS as an RBC rosetting receptor requires confirmation, it was shown that heparin and other GAGs, due to their rosette-disrupting effects have clear potential as adjunctive therapies for severe malaria .The first approach in using heparin in malaria therapy was attempted half a century ago , but theN-Acetyl-D-galactosamineβ(1-4)glucuronic acid units (Plasmodium merozoites binding to chondroitin sulfate A (CSA) in the placenta may lead to the accumulation of parasites causing severe symptoms called placental malaria [Chondroitin sulfate (CS) is a linear GAG polysaccharide, consisting of β(1-3)id units . Plasmod malaria . This is malaria . The int malaria ,107. Moreover, cytoadhesion and invasion inhibitory effects were also reported for natural HLMs, fucosylated chondroitin sulphate (FucCS), isolated from sea cucumber . The prePlasmodium merozoites use host oligosaccharides in the invasion of RBCs. It was shown that sugars are the significant mediators of the merozoite ligands binding to RBC receptors at different steps of host infection. The key interaction ensuring merozoite entry into RBCs is the formation of the tight junction between the EBA ligands and glycophorin receptors. It was suggested that sialic acid residues of O/N-linked glycosaccharides form a conformation-dependent cluster on human glycophorins polypeptide chains, which facilitates the binding. Therefore, specific approaches with carbohydrate drugs have been proposed as possible malaria treatments [In conclusion, it is now generally assumed that human and ape eatments .N-acetylneuraminyl-N-acetate-lactosamine and 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which is its structural analog, can inhibit P. falciparum EBA-175-GPA interaction in 81% and 84% at 100 uM, respectively [N-acetylneuraminic acid (from 25 to 68% at 100 uM), in comparison with full GPA (88%). Moreover, it was shown that DANA is able to inhibit P. falciparum RBCs invasion (50–60% at 5000 µM). Thus, such compounds might be used as part of drug cocktails to reduce disease severity.It was shown that the sugar moieties of ectively . ModeratP. falciparum severe and complicated malaria.Considering the emerging problem of the antimalarial drug resistance, including quinolines, chloroquine, antifolate, and most recently artemisinin ,113, glyPlasmodium binding to placenta [P. falciparum protein responsible for binding to the placenta via CSA. PAMVAC vaccine formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies in healthy malaria-naive adults. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area.Another approach was using a vaccine for the inhibition of placenta . The vacPlasmodium merozoites and host cells are mediated by carbohydrates or glycan moieties [In summary, a fresh approach, based on glycobiology, may lead to new malaria therapeutic targets, since the principal interactions of moieties ,116.


Recent research has established that acute kidney injury (AKI) is a common problem in severe paediatric malaria. Limited access to kidney diagnostic studies in the low resources settings where malaria is common has constrained research on this important problem.Enrolment data from an ongoing clinical trial of antipyretics in children with central nervous system (CNS) malaria, CNS malaria being malaria with seizures or coma, was used to identify risk factors for AKI at presentation. Children 2–11 years old with CNS malaria underwent screening and enrollment assessments which included demographic and anthropomorphic data, clinical details regarding the acute illness, and laboratory studies including creatinine (Cr), quantitative parasite count (qPC), quantitative histidine rich protein 2 (HRP2), lactate, and bilirubin levels. Children with a screening Cr > 106 µmol/l were excluded from the study due to the potential nephrotoxic effects of the study drug. To identify risk factors for AKI at the time of admission, children who were enrolled in the study were categorized as having AKI using estimates of their baseline (i.e. before this acute illness) kidney function and creatinine at enrollment applying the Kidney Disease: Improving Global Outcome (KDIGO) 2012 guidelines. Logistic regressions and a multivariate model were used to identify clinical and demographic risk factors for AKI at presentation among those children enrolled in the study.465 children were screened, 377 were age-appropriate with CNS malaria, 22 (5.8%) were excluded due to Cr > 106 µmol/l, and 209 were enrolled. Among the 209, AKI using KDIGO criteria was observed in 134 (64.1%). One child required dialysis during recovery. Risk factors for AKI in both the logistic regression and multivariate models included: hyperpyrexia  and age with older children being less likely to have AKI .AKI is extremely common among children presenting with CNS malaria. Hyperpyrexia with associated dehydration may contribute to the AKI or may simply be a marker for a more inflammatory systemic response that is also affecting the kidney. Appropriate fluid management in children with CNS malaria and AKI may be challenging since generous hydration to support kidney recovery could worsen malaria-induced cerebral oedema in this critically ill population.Trial registrationhttps://clinicaltrials.gov/ct2/show/NCT03399318 Over the past decade, there has been increased appreciation that acute kidney injury (AKI) is a common complication of severe malaria in children , 2. AvaiThe AKI guidelines from the Kidney Disease: Improving Global Outcome (KDIGO) consortium uses change in creatinine and urine output to define AKI . Baselinhttps://clinicaltrials.gov/ct2/show/NCT03399318).A retrospective observational study was conducted using screening and admission data from an ongoing clinical trial of antipyretics in CNS malaria being conducted in Zambia at Chipata Central Hospital (CCH) and Malawi at Queen Elizabeth Central Hospital (QECH).  ≤ 2) or seizures. Because part of the clinical trial intervention includes ibuprofen, children were excluded if they presented with a Cr > 106 µmol/l. Other exclusion criteria were vomiting, circulatory collapse, jaundice or a total serum bilirubin > 265 µmol/l, having an allergy to paracetamol or nonsteroidal anti-inflammatory drugs, a contraindication to nasogastric tube insertion, or a history of liver disease, gastric ulcers, thrombocytopenia or haematological disorders.Inclusion criteria for the clinical trial includes age 2–11 years old, having malaria based upon a positive rapid diagnostic test or Among eligible children, written informed consent from the parent/guardian was required. At the time of admission to the study site, enrolled children had extensive data collected including demographic characteristics, anthropomorphic data, clinical data regarding the acute infection including seizures prior to enrollment and administration of antipyretics, if applicable. Time of coma onset relative to presentation for care was captured only in Malawi. Laboratory data collected during the admission on enrolled children included quantitative parasite count (qPC), quantitative histidine rich protein 2 (HRP2), lactate, bilirubin, and packed cell volume (PCV). Admission data and blood samples were collected concurrent with treatment initiation and resuscitation, if needed. Fundoscopy to determine whether the malaria retinopathy was present was completed on admission at the Malawi site only , 9. DiscCr was determined based upon analysis of whole blood captured via finger prick using a STAT Sensor Creatinine Meter . QuantitAmong admission characteristics, temperature was measured using a TempTraq® device placed in the axilla for measuring temperature every 2–4 min. Temperature was categorized as hypothermic < 36° Celsius (C), normothermic (36.0–37.0 °C), febrile  or hyperpyrexic (≥ 38.5 °C). These categories were utilized rather than temperature as a continuous variable since hypothermia is uncommon but a risk factor for death  and hypeChildren enrolled in the clinical trial were categorized as having AKI using the KDIGO 2012 consortium guidelines which defines AKI based upon a change in Cr from baseline. For this analysis, the change in creatinine was determined by the difference between the admission creatinine and an estimated baseline creatinine, which was back-calculated using the Pottel age-based equation, where estimated glomerular filtration rate (GFR) = 107.3/(SCr/Q), (Q = 0.0270 * age + 0.2329) assuming a normal GFR of 120 mL/min per 1.73m2 as validated by recent studies in Ugandan children [For continuous variables, normally distributed variables were compared using an independent t-test and non-normal data were compared using the Mann–Whitney U tests. Chi-square, or Fisher’s exact tests were used when a cell count was less than 5, to identify differences in categorical variables. Bivariate logistic regressions were run to identify potential risk factors for AKI. Variables were included in a multivariate logistic regression if the information was captured at all study sites and the p-values from the bivariate logistic regression was less than or equal to 0.10. All statistical analyses were conducted using STATA Version 16.From January 7, 2019 to November 18, 2021, 465 children were screened, 377 were age-appropriate with CNS malaria or CM, 254 met eligibility criteria for clinical trial enrolment, and 210 were enrolled. Records for one child were lost during an infection control intervention on the research ward in Malawi so data from 209 children were available for analysis. See Fig. Among age-appropriate children with CNS malaria screened for the clinical trial, 22 (5.8%) were excluded due to Cr > 106 µmol/l. All children excluded from the clinical trial for Cr > 106 µmol/l would have had AKI by the KDIGO criteria. See Table Table Among 377 children aged 2–11 years with CNS malaria or CM who were screened for enrollment in the clinical trial, 156 (41.4%) had evidence of AKI at presentation based upon screening Cr > 106 µmol/l OR applying the KDIGO criteria for AKI using estimates of baseline kidney function compared to admission kidney function among those enrolled in the clinical trial. This is similar to other recent reports on AKI in African children with severe malaria  and/or tThe primary limitation of this study is that the inclusion criteria for the clinical trial and thus the analytic sample for AKI risk factor analyses excluded children with Cr > 106 µmol/l, elevated bilirubin and/or vomiting. As such, the population included in the risk factor analyses is not fully representative of all children with CNS malaria. Nevertheless, the risk factor analysis provides some insights. Potential pathophysiologic mechanisms for AKI in malaria include haemoglobinuria, parasite sequestration, volume depletion, endothelial activation, microcirculatory dysfunction, inflammation and immune activation , 20, 21.In resource limited malaria endemic areas, children with CNS malaria at highest risk of AKI need to be identified for opportune supportive care as kidney replacement therapy is not easily accessible , 34. Unf


Using a descriptive survey design, the prevalence and management practices of malaria and malaria- typhoid co-infection in Unwana South East Nigeria was determined. Two hundred and thirty-six (236) febrile volunteers comprising 104 males and 132 females attending the Medical Centre of Akanu Ibiam Federal polytechnic Unwana, Afikpo Ebonyi state Nigeria participated in this study. Using thick film microscopy and Widal antigen-based agglutination test, one hundred and thirty-seven participants were diagnosed with malaria mono infection while ninety-nine were diagnosed with malaria-typhoid co-infection. Structured questionnaire was used to obtain data on the management practices and attitudes that constitute risk factors to increased incidence of treatment failure of malaria and malaria- typhoid co-infection. The dataset  Specifications Table•The data provided are of value as it describes the incidence, prevalence as well as the knowledge attitude and practices associated with management of malaria and its co-infection with typhoid in the study population.•BioMedical or clinical researchers interested in epidemiology of malaria and malaria typhoid can use the data as baseline further epidemiological survey.•This dataset provides information on the prevalence of malaria and malaria-typhoid co-infection and management practices in south east part of Nigeria which may be useful to health policy makers in the region as well as the entire the country.1This section consists of analysed data on parasitaemia and bacteraemia determined from blood samples obtained by vein puncture function of 236 febrile volunteers comprising 104 males and 132 females attending the Medical Centre of Akanu Ibiam Federal polytechnic Unwana, Afikpo Ebonyi state Nigeria. The analysed data of incidence of malaria in the past (2013–2018) is shown in 2The protocol for data collection is based on a descriptive cross-sectional design. The main criterion for the classification of the participants into the two groups of malaria and malaria-typhoid co-infection is clinical laboratory diagnosis based on Giemsa thick blood film microscopy and Widal antigen agglutination test.2.10.53N and longitude 70.56E. Afikpo has a population of about 156,611 The dataset was deduced from responses to validated structured questionnaire on knowledge, attitude practices between June and November, 2019 at the Akanu Ibiam Federal Polytechnic Unwana Medical Centre located in Afikpo North Local Government Area of Ebonyi state, Nigeria. The Medical Centre serves the Medical needs of the entire polytechnic community and patients living in and around the polytechnic community. Afikpo is the second largest city in Ebonyi state and is located at latitude 52.2Participants were adults of both sexes between 18 and 60 years of age presenting symptoms like fever, head ache, vomiting, and weakness of the body, body pains and other symptoms indicating uncomplicated malaria and malaria-typhoid infection. The research goal and objectives were explained to the participants and they gave written consent before participating. Measures were taken by the researchers to ensure confidentiality to all individuals who participated. The researchers assigned codes as identification to all collected samples and the filled questionnaires.2.3Two milli-Litres (2 ml) of blood sample used for determination of prevalence of malaria and the co-infection with typhoid were collected from participants by vein puncture at the laboratory. This was used for through thick film microscopy as well as for Widal antigen-based agglutination tests for diagnosis of Typhoid. Structured questionnaire was validated by experts and the reliability found to be 87.34% using the test-retest method. The researchers assigned codes as identification to all the filled questionnaires.2.4P. falciparum parasitaemia was in microliter (μl) of thick blood film preparation and graded as: low + (1 to 999/μl), moderate ++ (1000 to 9999/μl) and high +++  WHO The determination of malaria parasite density was by Giemsa-stained thick blood films. The thick films were prepared, fixed in methanol stained with Giemsa stain and examined under the microscope using × 100 oil immersion objective lens. Determination of the level of 2.5Salmonella typhi was used as a baseline To determine the prevalence of typhoid, the Widal agglutination test was performed on all blood samples by the rapid slide titration method using commercial antigen suspension to detect somatic (O) and flagella (H) antigens. About 50 μl of the blood serum was placed on eight rows of circles on the test tiles and a drop of positive and negative serum suspension were placed beside each sample on the test slide. Antibody titer value ≥1: 80 against O and H antigen of 2.6P < 0.05) was taken as statistically significantData were processed using Microsoft Excel software and analyzed using Statistical Package for the Social Sciences (SPSS version 17.0) software. Descriptive statistics such as frequency and percentages were used to summarize participants’ demographic characteristics as well as the the responses of the participants to questionnaire variables. Data on prevalence of malaria and its co-infection with typhoid was analyzed for disparity between the sexes and malaria and malaria-typhoid co-infection groups using Chi-squared statistics. Institutional ethical clearance (AIFPU/REG/OR/102/VOL.4/416) was obtained after a review of the research protocol by the AIFPU Medical Centre board and the polytechnic Research and Ethics Committee. The institution Ethics committee directed the Medical Centre doctors, nurses and laboratory scientist to attend to participants and help collect data. Consent forms were distributed to consenting patients attending the Medical Centre out-patient department after the objectives and benefits of the study had been relayed to them. The signed or thumb-printed informed consent forms were then retrieved from the volunteer study subjects and tallied in a record book before inclusion in the study. Therefater, the participants were given the questionare to fill before seeing their physician.We acknowledged the funding by the Nigeria Tertiary Education Trust Fund (TETfund) Institution Based Research (IBR) grant (TETFUND/DRSS/POLY/UWANA-AFIKPO/2014/RP/VOL1). The funder had no role in design, data collection and interpretation of the findings.Segun Solomon Ogundapo: Conceptualization, Methodology, Data curation, Writing – original draft. Soniran Olajoju Temidayo: Writing – review & editing, Methodology. Karian Chigozie Ngobidi: Methodology. Ibukun Caroline Vining-Ogu: Methodology. Nwogo Ajuka Obasi: . Victor Uzochukwu Olugbue: Methodology, Writing – review & editing. Lawrence Olusegun Ajala: Methodology, Writing – review & editing. Benneth Nnanyelugo Enemchukwu: Methodology, Writing – review & editing. Kalu Udo Kalu: Methodology. Nnanna Oji Okoro: Methodology. Ifeoma Obasi Otu: Methodology. Racheal Iniobong Okon: Methodology. Emelda Chinedu Ezugwu: Methodology. Peace Nkeiruka Okoro: Methodology. Jessica Chinagorom Onyeamachi: Methodology. Ocha Udu Arua: Methodology.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


ObjectiveTo compare the WHO cut-off of the mid-upper arm circumference (MUAC) with the weight for height z-score (WHZ) in different age groups of children (6 months to 59 months of age) with acute malnutrition in Pakistan.MethodologyA cross-sectional study was carried out in the pediatric unit of Ziauddin Medical University and Hospital on malnourished children from six to 59 months of age to compare two different indices of malnutrition, MUAC and WHZ. A total of 450 children with WHZ of <-2SD and <-3SD were included in the study after excluding children with failure to thrive due to chronic illness, congenital defects, and immune deficiencies/malabsorption.ResultsThe study revealed a significant mean difference in weight, height, and MUAC among the participants . The sensitivity of MUAC at <11.5 cm was highest in the 12-24-month age group with a decline at 24-48 months while specificity was highest at six to 12 months of age, which shows a mixed response.ConclusionThe result revealed variation in the cut-off value of MUAC in different age groups; the best specificity of MUAC was found at six to 12 months of age and the best sensitivity at 12-24 months of age. Malnutrition is a common and major health concern of communities in the developing world and malnourished children with complications are at a high risk of mortality . There aMoreover, negligence like lack of knowledge of breastfeeding, lack of a healthy diet rich in macro and micronutrients, poor hygiene, and poverty are some of the many causes of malnutrition.Thus, the screening of malnutrition in children under five years of age at the community level is extremely important. The two indices used as diagnostic criteria of acute malnutrition are mid-upper arm circumference (MUAC) and weight for height z-score (WHZ) . ChildreThe main objective of this study was to compare the WHO cut-off of MUAC with WHZ in different age groups of children with acute malnutrition in Karachi, Pakistan.www.OpenEpi.com). Children of age six to 59 months with WHZ <-2SD and <-3SD were included in the study and children with failure to thrive due to chronic illness, congenital defects, and immune-deficiencies/malabsorption were excluded from the study. The final sample of the study was 450 after necessary exclusion.A cross-sectional study was carried out at the pediatric unit of Ziauddin Medical University and Hospital. This study was conducted on malnourished children from 6-59 months of age to compare the WHO cut-off of MUAC with WHZ over one year after approval of the institutional review board. The sample size was calculated to be 536 at the 95% confidence interval using Open Epi v 3.0 (Children of ages six to 59 were recruited after informed consent and divided into four categories: category A (6-12 months), category B (12-24 months), category C (24-48 months), and category D (48-59 months). Ages were confirmed by birth date, calculated in completed months. Additionally, anthropometric measurements, such as weight, MUAC, and height of children, were taken. Standard guidelines were followed while taking anthropometric measurements. WHO provided the non-stretch tape, which was used for the measurement of MUAC (to the nearest 1 mm). Moreover, weight was recorded using a digital scale (weighed to the nearest 0.1 kg) while an infantometer and stadiometer were used to calculate length and height for children <2 years and >2 years, respectively. The data were plotted on the WHO WHZ chart separate for boys and girls, and the results of both indices (MUAC and WHZ) were calculated accordingly. Information about gender, demographic profile, socioeconomic status, and feeding practices was noted. Moreover, the status of education of the mother was determined and the number of children under the age of five was recorded.The data were analyzed using the SPSS 26 software package . Mean, median, and mode were calculated to identify the normality of data. The chi-square test was utilized to develop the comparison of categorical variables. To compare the continuous variables, a student t-test was used and less than 5% of probability  was considered a significant value. Additionally, a curve analysis, known as receiver operating characteristics (ROC), was utilized to select the best cut-off value of MUAC in the examination of SAM. Moreover, the area under the curve (AUC) and its standard error were calculated.Table Figure Table Figure The present study demonstrated that there were several variations found in the diagnosis of SAM through the MUAC and WHZ of the infants. There was a significant mean difference found in weight, height, and MUAC among the participants with a p-value of 0.030, 0.053, and 0.02, respectively. This statement shows that weight, height, and MUAC were all found to help detect the degree of malnutrition present in infants. Furthermore, there was a difference in height/length (cm) in males and females. Our study showed an overall percentage of 42.2% of infants diagnosed with SAM while 57.8% were in the moderate category of malnutrition. The sensitivity of MUAC at <11.5 cm was highest at the age of 12-24 months, with a decline at 24-48 months while specificity was highest at 6-12 months of age, which shows a mixed response.Marshall et al. found the mean data for height (cm), weight (kg), and MUAC (cm) of infants in their research consistent with our readings   showed sIn our study, the AUC values of 0.331 in 6-12 months and 0.462 in 12-24 month infants were analyzed from the ROC curve of MUAC (cm), which shows the poor diagnostic ability of MUAC. On the other hand, in our study, an AUC value of 0.684 at 24-48 months and 0.829 at 48-60 months were analyzed from the ROC curve of MUAC (cm), showing good prognostic ability with increasing age. On the contrary, this paper found literature with differing findings of AUC from the ROC curve of 0.94 and 0.95 in 6-12 months and 12-24 month infants showing excellent MUAC diagnostic ability . One stuOur research has found a positive relationship between the MUAC and Z-scores , which means that both diagnostic tools can be used to detect SAM. Marshall et al. also studied a strong positive linear relationship between WHZ and MUAC . In anotThere was countless literature that gave results that were inconsistent with our research findings. MUAC was stated as a low-sensitivity diagnostic tool with 17.5% (sensitivity) . The creThe differences in the results could be due to multiple reasons according to the area, environmental conditions, hygiene issues, poverty status, and nutritional status. The present study tried to highlight as much about the diagnostic use of both WHZ scores and MUAC for SAM but conflicting results can be because this is the only study that is specifying MUAC /WHZ according to different age groups. There were comorbidities involved in the current study, which could have affected the preciseness, as it was not known how long they were present and their origin.This study has a few limitations. For example, due to the coronavirus (COVID) pandemic, a target sample size couldn’t be collected in the given period. Further, this study was conducted in only one center, which might have caused selection biases. More prospective multi-centric studies at the community level need to be conducted for future recommendations regarding WHZ and MAUC.This study has shown that wasting, edema, WHZ, and MUAC are independent criteria for screening for malnutrition and are recommended by WHO for decades. The universal value of MUAC < 12.5 cm for the diagnosis of malnutrition is compared by the gold-standard WHZ in this study. The result revealed variation in the cut-off value of MUAC in different age groups; the best specificity of MUAC was found at 6-12 months of age and the best sensitivity at 12-24 months of age. As this was a single-center study, for the generalization of findings at the community level, large multicenter studies need to be done for changing recommendations.


Malaria vaccines are urgently needed in the fight against this devastating disease that is responsible for almost half a million deaths each year. Here, we discuss recent clinical advances in vaccine development and highlight ongoing challenges for the future. Kurtovic et al. highlight some of the recent advances in the development and clinical evaluation of malaria vaccines. The authors outline key vaccine strategies and clinical trials, and discuss priorities for research into the development of an efficacious malaria vaccine. Malaria is one of the most devastating infectious diseases in humans, responsible for >200 million cases and almost half a million deaths annually. Control programs have significantly reduced disease burden since 2000, but numbers have plateaued since 2015. The disruption caused by the ongoing COVID-19 pandemic will further unwind these efforts and is predicted to increase the number of malaria deaths. New control measures are urgently needed, and lessons learned from emerging diseases such as COVID-19 highlight the enormous potential of effective vaccines. This has been recognized by the World Health Organization (WHO) and key stakeholders who recommend that, within the next decade, a vaccine should be developed that is at least 75% efficacious against malaria over 2 years  and also reduces malaria transmission.This is an exciting time for malaria vaccine development, with a number of promising clinical trials recently completed and others currently underway…Plasmodium falciparum causes the majority of malaria burden and has been the primary focus of vaccine development, and Plasmodiumvivax is the second major cause of malaria. There are several vaccine strategies that target different developmental stages of the malaria-causing parasite 2. Another approach is to combine replication–intact sporozoites with antimalarial drug prophylaxis to prevent malarial illness, known as PfSPZ-CVac. PfSPZ-CVac can induce sterilizing immunity in malaria-naive volunteers, but there is no published data in malaria-endemic populations3. A third approach is to genetically attenuate sporozoites to prevent intrahepatic development. This includes the triple-gene knockout vaccine, GAP3KO, which recently completed an efficacy trial in malaria-naive volunteers (NCT03168854). Although sporozoite-based vaccines are promising, major obstacles include the fact that currently sporozoites cannot be cultured in vitro and are therefore isolated from infected mosquito salivary glands, and they currently require direct venous inoculation of vaccine doses and storage and transportation of vaccines using liquid nitrogen.Vaccines can target sporozoites to prevent or impair infection of the liver, and therefore aim to induce immunity whereby an individual is protected against infection and disease. Live-attenuated sporozoite-based vaccines have the capacity to induce sterilizing immunity (no detectable parasitemia) against CHMI in malaria-naive volunteers. This includes the PfSPZ vaccine that contains radiation-attenuated sporozoites incapable of replicating. It was recently shown that an optimized PfSPZ vaccine dosage induced sterilizing immunity against CHMI in six Tanzanian adults4. Additional trials are underway in African children to evaluate a modified regimen where a fraction of the third dose is administered months later, which appeared to be moderately more efficacious in malaria-naive volunteers (NCT03276962)5. There is also interest in whether RTS,S can synergize with seasonal malaria chemoprevention (NCT03143218).Subunit vaccines aim to target a key antigen, or sub-region, and have the advantage of using existing production technologies and delivery using established infrastructure and systems. The most successful is the RTS,S vaccine based on a truncated form of the major sporozoite surface antigen, circumsporozoite protein (CSP). This is expressed as a virus-like particle and administered with an adjuvant (AS01), which is used to enhance the immune response to vaccination. Promising findings from a phase 3 clinical trial in >15,000 African children led to pilot implementation of a four-dose booster regimen in children aged 5 months in Ghana, Kenya, and Malawi to further evaluate vaccine safety and efficacy, which is ongoing6. Initial results from a pediatric phase 2 clinical trial in Burkina Faso demonstrated 76% efficacy against malaria over 12 months6. Although this was considerably higher than vaccine efficacy achieved in previous RTS,S trials4, R21 was administered prior to the peak malaria season and most malaria occurred in the first 6 months6, making direct comparisons with RTS,S efficacy difficult to interpret. Furthermore, antibody levels had declined markedly by 12 months. Participants are still being monitored to determine vaccine efficacy over 2 years, and children are currently being enrolled in phase 3 clinical trials across multiple African sites (NCT04704830).A recent advance was the development of the R21 malaria vaccine, a virus-like particle that is similar to RTS,S, but has higher density of the CSP antigen on the particle surface and is formulated with Matrix-M adjuvant7. The most recent was RH5.1 comprised of full-length RH5 protein, an indispensable merozoite invasion ligand. In a phase 1/2a clinical trial in malaria-naive volunteers, RH5.1 with AS01 adjuvant was highly immunogenic but only resulted in a 1-day delay until threshold parasitemia levels were reached following CHMI compared with that of the unvaccinated control group8. An upcoming phase 1 clinical trial of RH5.1 will further evaluate safety and immunogenicity in Tanzanian adults and children (NCT04318002).Blood-stage vaccines target the merozoite form or infected red blood cells and aim to control parasitemia and prevent clinical illness. There has been considerably less progress in achieving good efficacy for blood-stage vaccines, with only a handful of candidates evaluated in phase 2 clinical trials, and none having progressed further7. Several vaccines, including RH5.1, have generated substantial GIA activity but shown limited efficacy8. Achieving higher efficacy with merozoite vaccines may require harnessing multiple immunologic mechanisms. Recent studies found that antibodies can activate complement and mediate Fcγ-receptor effector functions against merozoites, which were associated with clinical protection in malaria-endemic population studies9. This creates a new opportunity to evaluate the targets of functional antibody responses and potentially identify new vaccine candidate antigens using these novel approaches9.Merozoites express a multitude of surface proteins and invasion ligands, including many that are polymorphic, which makes it difficult to identify candidate antigens for subunit vaccines. In addition, there are major knowledge gaps on specific mechanisms that mediate protective antimalarial immunity. Antibodies play a critical role, but the current reference growth inhibition assay (GIA) that measures antibody inhibitory activity has neither strongly nor consistently been associated with protection against malaria10. Further studies are needed on this particular vaccine approach, including improvements in vaccine production and formulation to avoid immune responses to the red blood cell membrane.Given the lack of success in developing blood-stage subunit vaccines, there is interest in whole parasite blood-stage vaccines, which present a large antigen repertoire to the immune system and may overcome the challenge of antigen-specific polymorphisms. The first clinical study of chemically attenuated parasite-infected red blood cells promisingly induced cellular immune responses in malaria-naive volunteers, but parasite-specific antibodies were not detected12.Once a merozoite infects a red blood cell and matures, the host cell undergoes extensive changes and expresses parasite-derived proteins on the red blood cell surface. One such protein, VAR2CSA, facilitates the accumulation and sequestration of parasite-infected red blood cells in the placenta. Malaria during pregnancy is, therefore, a major concern and can lead to adverse outcomes such as maternal anemia, preterm delivery, stillbirth, and low birthweight babies. Two VAR2CSA-based vaccines (PAMVAC and PRIMVAC) aimed at inhibiting placental malaria have recently completed phase 1 clinical trials and were immunogenic and had acceptable safety profiles, but are yet to be evaluated for vaccine efficacy13.Vaccines that target the gametocyte and gamete forms aim to prevent parasite transmission to the mosquito and thereby block malaria transmission throughout a population. Although this vaccine approach does not offer any direct clinical protection in humans, blocking malaria transmission is an essential component of effective control and elimination strategies. There has been limited progress for transmission-blocking vaccines, with one candidate based on the gametocyte surface antigen, Pfs230 (Pfs230D1M), showing promise in early clinical trials and a phase 2 clinical trial is ongoing in Mali (NCT03917654)These vaccine approaches targeting different stages of the parasite life cycle have the ability to afford protection against infection, disease, and/or prevent parasite transmission among a population. Although each approach is of value, there may be additional benefits to developing multi-stage subunit vaccines that incorporate antigens expressed at different developmental stages.6. The immunological factors underlying these vaccine limitations remain poorly understood but will be crucial for achieving higher efficacy and wider vaccine implementation in endemic populations. Furthermore, very little progress has been made toward the development of vaccines against P. vivax, which needs to be accelerated.This is an exciting time for malaria vaccine development, with a number of promising clinical trials recently completed and others currently underway, although there are still considerable challenges that stand in the way of achieving vaccine goals within the next decade with several priority areas for research Fig. . Malaria14. There has also been continued interest in heterologous prime-boost with ChAd43 and Modified Vaccinia Ankara viral vectors encoding different malaria antigens alone or in combinations 15.Recent advances in vaccine technologies, including those used in licensed COVID-19 vaccines, have opened new opportunities for malaria vaccine development. This includes mRNA-based vaccine platforms of the CSP malaria antigen, which has already shown encouraging results in animal modelsFurther research on these areas will greatly advance the development of effective malaria vaccines in endemic populations.


Plasmodium falciparum infection, but have not been studied in clinical malaria.Circulating myeloid-derived-suppressor-cells (MDSC) with immunosuppressive function are increased in human experimental Plasmodium vivax (n = 8) and uncomplicated (n = 4) and severe (n = 16) falciparum malaria from Papua, Indonesia.Using flow-cytometry, circulating polymorphonuclear-MDSC were evaluated in cryopreserved samples from patients with uncomplicated The absolute number of circulating polymorphonuclear-MDSC were significantly elevated in severe falciparum malaria patients compared to controls (n = 10). Polymorphonuclear-MDSC levels in uncomplicated vivax malaria were also elevated to levels comparable to that seen in severe falciparum malaria.Control of expansion of immunosuppressive MDSC may be important for development of effective immune responses in falciparum and vivax malaria. Plasmodium falciparum is responsible for over 97% of global malaria cases and nearly all malaria deaths [Plasmodium vivax also causes significant morbidity [P. falciparum parasite biomass, including delayed treatment [Plasmodium infection remains incompletely understood, although failure to control Plasmodium infection and clinical disease are associated with an immunoregulatory immune response [Malaria remains a major life-threatening infectious disease in tropical and subtropical countries. The World Health Organization reported an increase in malaria cases and deaths in 2020, with an estimated 241 million malaria cases and 627,000 deaths worldwide [reatment . The wayresponse .P. falciparum sporozoites report expansion of PMN-MDSC in individuals developing blood-stage parasitaemia with suppression of CD4+ and CD8+ T cell proliferation [Myeloid-derived-suppressor-cells (MDSC) are a heterogenous group of polymorphonuclear-type (PMN-MDSC) or monocytic-type MDSC with strong immunosuppressive activity against T-cells, that can regulate the function of other immune cells. MDSC are emerging as key pathological mediators of disease including in cancer , sepsis feration . MDSC exferation –18. DespP. falciparum and P. vivax malaria, including patients with severe disease, and compared their levels to uninfected individuals residing in the same endemic area.During peripheral blood separation by density gradient centrifugation, low-density PMN-MDSC co-purify with peripheral blood mononuclear cells (PBMC) , enablinP. falciparum and P. vivax are similar [P. falciparum or P. vivax attending Rumah Sakit Mitra Masyarakat Hospital, and had PBMC cryopreserved. Definitions for enrolment of uncomplicated and severe malaria cases  conjugated to fluorescein isothiocyanate, anti-CD14 (clone MφP9) conjugated to allophycocyanin , CD11b (clone LM2) conjugated to phycoerythrin and anti-CD15 (clone W6D3) conjugated to peridinin chlorophyll protein complex (PerCP) . To provide insight into immunosuppressive function, we stained one million PBMC with anti-CD4 (clone RPA-T4) conjugated to PerCP to count CD4+ T cells in the same samples. Stained cells were acquired on a portable BD Accuri C6 flow cytometer (BD Biosciences).Flow cytometric data were analysed on FlowJo v10  and statistical analysis was conducted using Graphpad Prism v9 . PMN-MDSC levels were reported as absolute numbers per microlitre of blood, calculated from the number of PMN-MDSC events relative to events of mononuclear cells for which automated cell counts were available. More specifically, the number of PMN-MDSC events was divided by the number of PBMC events (gated on lymphocytes and monocytes) and multiplied by the absolute number of PBMC (sum of automated lymphocyte and monocyte counts). The Kruskall–Wallis test with Dunn’s multiple comparison was used for comparison of continuous variables between groups. The chi-square test was used for comparison of categorical variables. The Mann–Whitney test was used for comparisons of severity criteria in severe malaria cases. Correlations were evaluated using the Spearman test .The work was approved by the Human Research Ethics Committees of Gadjah Mada University (KE/FK/763/EC and KE/FK/544/EC) and Menzies School of Health Research (HREC 10-1397 and 10-1434). Written informed consent was obtained from all participants.P. vivax, 20 had P. falciparum infection (4 uncomplicated and 16 severe) and 10 were PCR-negative healthy controls  properties and cell surface markers. After gating on SCClls Fig. A and tholls Fig. B; the anlls Fig. C, D.s= − 0.50, p = 0.22; Fig. s = 0.12, p = 0.68; Fig. + T cells and lymphocyte counts were examined to gain insight into MDSC immunosuppressive function. In accordance with previous reports from the same study area [s = 0.21, p = 0.66 and rs = 0.20, p = 0.48, respectively). Further, PMN-MDSC levels were not correlated with automated lymphocyte counts for both species . Lastly, it was determined if circulating PMN-MDSC levels were associated with parasitaemia or patient age. While no relationships were observed with parasitaemia , an inverse association with age was observed in P. vivax infection .Whether circulating PMN-MDSC levels were associated with automated neutrophil counts was assessed. Despite sharing surface markers and ancestry , there wudy area , loss ofP. vivax, P. falciparum and severe falciparum malaria display increased PMN-MDSC in circulating blood. Circulating PMN-MDSC were at comparably high levels in uncomplicated P. vivax and severe falciparum malaria. PMN-MDSC may contribute to the greater immunoregulatory response previously observed in P. vivax infections [P. falciparum infections is consistent with elevated PMN-MDSC reported in CHMI developing P. falciparum parasitaemia [This is the first study evaluating MDSC in clinical malaria. Patients with uncomplicated fections . Severe sitaemia . With th+ T cells was not associated with PMN-MDSC levels in peripheral blood, nor were there any correlations between PMN-MDSC and lymphocyte counts. Larger studies directly assessing the immunosuppressive activity of circulating PMN-MDSC in clinical malaria are warranted. Analysis of severe falciparum malaria as a single group indicated similar PMN-MDSC levels to uncomplicated malaria, though the sample size was underpowered to confirm this observation. The greater expansion of PMN-MDSC in severely ill patients with jaundice compared to those without was not statistically significant but is plausible, and is consistent with a previous study reporting increased PMN-MDSC in cancer patients with related jaundice [PMN-MDSC mediate T cell dysfunction and have been shown to suppress ex-vivo T cell proliferation in CHMI volunteers . In the jaundice . Conversjaundice , potentijaundice . HoweverP. vivax may be affecting the age-related alterations in MDSC homeostasis, though the data supporting this should be interpreted with caution given the small sample size in our cohort.PMN-MDSC are pathologically activated neutrophils distinguished from classical neutrophils by density gradient separation . In the P. vivax infections, PMN-MDSC concentrations were influenced by age and were at least as high as levels in severe P. falciparum infections. In severe disease, the level of PMN-MDSC expansion may contribute to different severe manifestations. Larger studies on the kinetics of PMN-MDSC (as well as monocytic-MDSC) in clinical disease and their role in asymptomatic infections are warranted to evaluate MDSC as a potential control target to improve immune responsiveness for both species.Circulating PMN-MDSC expand in clinical vivax and severe falciparum malaria, and may contribute to the immunosuppression previously observed in clinical disease from both species. In uncomplicated


Immunoglobulin G (IgG) subclasses have been suggested to confer naturally acquired immunity to Plasmodium falciparum malaria. Cytophilic IgG1 and IgG3 with their potential for opsonization, phagocytosis, and antibody-dependent cellular inhibition in association with monocytes have been suggested to have a critical role in malaria. The potential for production of antibodies is influenced by micronutrient status. This study aimed at exploring the effect of micronutrients, particularly zinc status, on the profiles of IgG subclasses in 304 Tanzanian children aged ≤ 5 years. An enzyme-linked immunosorbent assay was performed using whole asexual blood stage malaria antigens to determine plasma malaria-specific antibody titers. This baseline cross-sectional study was done from 2005 – 2010 prior to the larger randomized control trial of the Micronutrient and Child Health (MACH) Study. Plasma concentrations of zinc and magnesium were measured by inductively coupled plasma atomic emission spectrometry and results correlated with plasma IgG subclass levels. The findings reveal zinc deficiency to possibly influence the production of IgM, total IgG, and several IgG subclasses in a malaria status-dependent manner. Among IgG subclasses, IgG3 and partly IgG2 displayed a remarkable association with zinc deficiency, particularly IgG3 which was predominant in children with malaria. Nevertheless, zinc, magnesium, and malaria status did not influence the association between IgG3 and IgG4. The study leads to the conclusion that, under conditions of micronutrient deficiency and malaria status, an imbalance in IgG subclass production may occur leading to predominantly higher levels of IgG3 and IgG2 that may not confer sufficient protection from infection. The profile of both cytophilic and non-cytophilic IgG subclasses has been shown to be variably influenced by zinc status; the effects vary with age at least in under-fives. These results provide insight for inclusion of micronutrients, particularly precise amounts of zinc, in future malaria interventional programs in endemic areas. Plasmodium falciparum blood-stage surface antigens and is vital in limiting parasite multiplication and the conferral of protection to clinical malaria. In malaria endemic areas, the development of naturally acquired immunity to severe disease takes place in children at an age range of 1 – 5 years  cells  cells , 5. The h) cells , 7. Studh) cells  reportedh) cells .P. falciparum antigens is thought to be critical for the generation of an effective immune response  and National Ethics Review Committee were 094 and NIMR/HQ/R.8a/VolIX/540, respectively). Informed consent was obtained from community leaders and local government officials, and from parents or guardians.The cross-sectional study was conducted in the period of May 2005 – July 2006 in a lowland area around Segera village , Handeni District of Tanga, north-eastern Tanzania. Malaria is highly endemic in this area, with virtually all infections being due to A census list was made with all resident children aged 6−72 months in the study area. Using this list, 16 children were randomly selected from each of 19 communities, resulting in a total of 304 children. Children were eligible when they had no fever, and showed no signs of other severe disease or severe malnutrition (weight-for-height z-score below−3 SD). Further details are provided elsewhere .All children were examined by clinical officers, who also measured axillary temperature by electronic thermometer. Information on the state of health such as malaria, fever, sickness, and reports on fever in the past 14 days was diligently recorded. A project phlebotomist collected fasting venous blood samples (6 mL) in containers (vacutainer tubes) suitable for mineral element analysis with sodium heparin as the anticoagulant . Immediately upon collection, the cap was sprayed with ethanol and allowed to dry; approximately 1.3 mL of blood was then drawn in a sterile manner from the collected blood into a different tube for different measurements including peripheral blood mononuclear cells (PBMCs), plasma for ELISA, parasite count, etc. Children were treated for common childhood infections and anemia according to the guidelines of the Tanzanian Ministry of Health.g) at ambient temperatures for 10 min and plasma obtained from the centrifuged blood (1.2 mL) was stored in liquid nitrogen, and subsequently transported on dry ice to the Netherlands to measure additional biochemical indicators of micronutrient status and inflammation  was used to coat the plates, and these were covered and incubated overnight at 40C. The parasite extract was obtained as a kind gift from Professor Robert Sauerwein from the Medical Parasitology Laboratory, Radboud University Medical Center Nijmegen, the Netherlands. The plates were blocked with 150 μL of 1% w/v bovine serum albumin, (BSA)  in phosphate-buffered saline (PBS). After blocking, the plates were washed three times with an extensive volume of 0.05% Tween 20 (v/v)  in PBS. Plasma samples were diluted five  or 20  times in 0.2% w/v BSA and 0.05% Tween 20 in PBS. Plasma samples were added to the wells at 50 μL and malaria-positive and negative samples were added as controls. The positive control consisted of a pooled sample of 25 highly immune Tanzanian individuals. Addition of plasma to the wells was followed by incubation under cover for 3 h at room temperature. After incubation, the plates were washed three times in 0.05% Tween 20 in an extensive volume. Thereafter, 50 μL of horseradish peroxidise (HRP) conjugated sheep anti human antibody  was added and incubated at room temperature in a shaker in the dark for 1.5 h. The following concentrations were used: 1 μg/ml for IgG total (AP003) and IgG1 (AP006) and 2 μg/ml for IgM (AP012), IgG2 (AP007), IgG3 (AP008), and IgG4 (AP009). Plates were emptied and washed six times in 0.05% Tween 20 and finally 100 μL of substrate  and ABPlasma samples were diluted 20 times in milliQ  and concp-value < 0.05). A generalized linear model (GLM) was utilized to determine whether the effect of zinc deficiency on plasma IgG subclasses was age-dependent. Lots were used as scaling weight variables to account for the diversity in malaria prevalence among lots. The analysis compared the differences among age groups in P. falciparum -specific antibodies plasma reactivity. Three age groups were established: 0.5 – 1.5 years, 1.5 – 3 years, and 3 – 5 years.The data were entered and analyzed using SPSS for Windows . The previously defined zinc and magnesium deficiencies  were useThis study was carried out in a population of children consisting of 135 boys and 169 girls with similar age distributions. Details of population characteristics and other relevant information are revealed in previous similar reports –55.Initially the possible difference in levels of IgM and total IgG class , 2 relatP < 0.05) in zinc-replete children with asymptomatic malaria and those who were sick and had malaria infection. In these subjects, the changes in IgG3 and IgG4 levels were almost the same with IgG4 being slightly higher than IgG3 in children reported sick with malaria and those who were reported sick with malaria infection and a report of fever in the previous 24 h. With zinc deficiency (P < 0.05). On the other hand, IgG2 predominantly showed increased levels in children reported sick with malarial infection (P < 0.05). There was a marked difference with respect to zinc status in which only IgG3 and partly IgG2 predominated in zinc deficiency which may imply that these IgG subclasses remain as critical weapons to fight against malaria in nutrient deficiencies. The insignificant change in IgG subclasses in children reported sick without malaria infection strengthens the notion that the antibodies studied are specific for malaria antigens.Evaluation of the general plasma changes in IgG subclasses in different malaria situations is shown in ficiency , only thPlasmodium falciparum-specific IgG subclasses in plasma were assessed to determine whether there were differences within individual IgG subclasses with respect to zinc status  and in children who were reported sick and found to be malaria-positive by dipstick . There was no significantly high levels of IgG1 in the zinc-deficient group. Increase in IgG2 levels followed a similar trend to IgG1 in the zinc-replete group, but in the zinc-deficient group, the IgG2 levels were significantly higher in the children reported sick and found positive for malaria . Levels of IgG3 were significantly higher in both zinc-replete and zinc-deficient groups (P < 0.05) relative to healthy individuals, except in children reported sick without malaria  and those reported sick with malaria and a history of fever in the past 24 h  were significantly and relatively higher than their counterparts in the zinc sufficient group. With the exception of children who were reported sick without malaria, IgG4 levels were higher in the zinc-replete group and the higher levels of IgG4 in the zinc-deficient group in all cases were statistically insignificant  and, secondly, the report that IgG4 is more associated with disease than protection . Howeveric areas .Having assessed the unit change and levels of primary response by IgM and total IgG plasma levels and the unit change in IgG subclasses, the levels of individual IgG subclasses with regard to zinc status were compared . Resultset al.  and UN Children's Fund (UNICEF).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plasmodium knowlesi has been detected in humans throughout South East Asia. The highest risk groups for this infection are males, adults and those performing forest-related work. Furthermore, asymptomatic cases of P. knowlesi malaria have been reported including among women and children.In the last decade P. knowlesi malaria published between 2010 and 2020 were searched. A systematic literature review was conducted to identify studies reporting the prevalence and incidence of laboratory confirmed asymptomatic P. knowlesi cases in humans, their clinical and demographic characteristics, and methods used to diagnose these cases.Pubmed, Scopus and the Web of Science databases for literature describing asymptomatic P. knowlesi infections have been detected in 0.03%–4.0% of the population depending on region, and infections have been described in children as young as 2 years old. Various different diagnostic methods were used to detect P. knowlesi cases and there were differing definitions of asymptomatic cases in these studies. The literature indicates that regionally-differing immune-related mechanisms may play a part on the prevalence of asymptomatic P. knowlesi.By analysing over 102 papers, thirteen were eligible for this review. Asymptomatic P. knowlesi malaria in different regions reinforces the need to further investigate disease transmission mechanics. Effective public health responses to changes in P. knowlesi epidemiology require proactive intervention and multisectoral collaboration.Differing epidemiological characteristics of asymptomatic The online version contains supplementary material available at 10.1186/s12936-022-04339-8. Plasmodium knowlesi is an emerging public health challenge in South East Asia  and gray literatures. The CoCoPop framework was used to address the aim of the review relevant to prevalence or incidence . ‘Conditsubjects . ArticleP. knowlesi cases. Exclusion criteria were any asymptomatic P. knowlesi study with no related context including case reports and case series, editorial, animal studies, drug studies, proceedings, short communications and experimental studies. Further excluded were articles on malaria in pregnancy, co-infection with other infection (besides other Plasmodium species), and review articles.The inclusion criteria were any study published in English that reported laboratory confirmed asymptomatic To reduce the risk of bias, studies were assessed by three authors  independently to identify relevant articles based on the title and abstract. All articles were reviewed by NA, MRH, and RH. All three authors, searched references for additional relevant studies. In case of discrepancies, it was resolved by consensus by a third (KA), fourth reviewer (MSJ), and fifth reviewer (RC). The data set of this systematic review, including the name of the author (s), publication year, study design, country of study (distribution), study period, diagnostic method, the prevalence or incidence, sociodemographic factor, other relevant factors, were extracted into Microsoft Excel , for further analysis.P. knowlesi infection were based on the definition by the World Health Organization (WHO), in which asymptomatic parasitaemia is defined as the presence of asexual parasites in the blood without symptoms of illness [P. knowlesi infection, it was accepted for this review.The criteria for asymptomatic  illness . HoweverTo assess the quality of the observational studies, the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist was used, and independently assessed by three reviewers. It has the total of 22 items. Each question based on STROBE was categorized as 'yes' (meet criteria), 'no' (did not meet criteria), or 'not applicable.' A 'partly' option was not included to avoid it being used to negate having to choose between yes and no . The quaPlasmodium species studies, opinion paper and reviews  and the rainy season (May) in Thailand . A studyPlasmodium, but afebrile within the past 48 h [P. knowlesi by polymerase chain reaction (PCR) [Plasmodium Loop-Mediated Isothermal Amplification (LAMP) [P. knowlesi was identified by nested PCR.In Thailand, Shimizu et al. (2020) defined asymptomatic malaria as those who were positive for ast 48 h . In Cambon (PCR) , howeveron (PCR) . Otherwin (LAMP) . In anotn (LAMP) . In all Cases were presented as both absolute numbers and as percentages. The majority of the studies used univariate and multivariate logistic regression analyses to identify potential risk factors  asymptomatic cases detected in Aceh Besar, Indonesia by rapid active case detection (RACD) . HoweverP. knowlesi cases [P. vivax were also detected [In Matunggong district of Sabah, 0.03%  and 0.3% (20 positive out of 1147 blood samples) of individuals were detected as asymptomatic si cases  in two sdetected .P. knowlesi cases out of 3002 blood samples [A study done among the people residing in longhouses in Betong, Sarawak, detected seven 0.23%) asymptomatic % asymptoP. knowlesi from 14,732 samples taken from 23 villages [Plasmodium cynomolgi in 11 samples. All samples were analysed using nested PCR and positive samples were confirmed by nucleotide sequencing. In Surat Thani Thailand, in January 2019, one case was detected among 7034 subjects, and in May 2019, two cases among 8671 subjects. In both months, cases were analysed by nested PCR using capillary blood samples collected by finger pricking in EDTA tubes [A study in Cambodia reported eight (0.05%) asymptomatic villages . The stuTA tubes  rather tTA tubes . Thus, tP. knowlesi infected patients are assumed to have a degree of naturally acquired immunity against the parasite, presumably through previous exposure [P. falciparum and Plasmodium vivax might confer cross-reacting immunity against P. knowlesi. There is an understanding gap of many of the factors surrounding the involvement of immunity in asymptomatic P. knowlesi [Asymptomatic exposure , 22, 28.knowlesi .P. falciparum, P. vivax and P. knowlesi antigens among the population residing in rural areas [P. knowlesi antigen, with the highest prevalence in Limbuak, Pulau Banggi  followed by Matunggong  and Bacungan  [A study from Sabah, Malaysian Borneo, and Palawan, the Philippines, used enzyme-linked immunosorbent assay to detect antibodies against al areas . The stu; 6/546) .P. knowlesi infection. The mean geometric parasite density was 52,488 parasites/ml [Not all studies used microscopy for confirmation of malaria. In these cases, either PCR , 28, 29 sites/ml .P. knowlesi patients, which involved microscopic diagnostic screening of all household members of an index case and the neighbours living close to the index case [Moreover, two studies from Indonesia used a RACD policy for close contacts of confirmed dex case , 28.Plasmodium DNA. The majority of studies used nested PCR to amplify P. knowlesi specific 18S ribosomal DNA. Imwong et al. used whole genome amplification to characterize the Plasmodium species in samples with insufficient DNA volumes [P. knowlesi infection [The type of samples used in these studies were either whole blood collected in EDTA containing tubes , 26, 28, volumes . In addi volumes . The majnfection , 24, 29.Plasmodium species [P. knowlesi cases in Aceh Besar, Indonesia [Shimizu et al. used quantitative PCR (qPCR) able to detect down to 10 copies of the target gene per reaction. All positive samples were tested by nested PCR to determine  species . LAMP wandonesia , and a sndonesia .P. knowlesi cases. The most significant characteristic of asymptomatic P. knowlesi malaria is that it predominantly affected males [In the majority of studies, questionnaires were used to assess the demographic, socioeconomic and environmental characteristics of ed males , 22, 26.ed males , 13. A med males . Howevered males .P. knowlesi by PCR, but not by microscopy [P. knowlesi cases were in children between 2 and 15 years old [P. knowlesi infection, and 51.18% (130/254) of the cases were among those above 15 years old [P. knowlesi [P. knowlesi than any other [Interestingly, asymptomatic children as young as two were found to be infected with croscopy , 29. In ears old , and in ears old . These fears old . Howeverears old . These fears old . Again td 51.18% 0/254 of ny other .P. knowlesi infection [P. knowlesi [P. knowlesi exposure based on model analysis [Those who work in the plantation sector and farmers are at particular risk of asymptomatic nfection , 23, 27 knowlesi . In Belaknowlesi . Some caknowlesi , 22, 27.analysis .P. knowlesi infection [Individuals residing within 1 km of the forest, and those living within 500 m of cleared areas have increased risk of nfection . Exposurnfection , 22, 28.nfection , 22, 28.P. knowlesi exposure [A study from Surat Thani, Thailand demonstrated that the use of bed nets and indoor residual spray (IRS) for four months acts as a preventive method against malaria infection. People who practiced self-protection, such as repellent usage and wearing protective clothing were protected from mosquito bites . These rexposure .P. knowlesi exposure [In ecological studies, individuals residing in houses less than one meter from ground level and individuals residing at higher geographical elevations had lower risks of exposure .P. knowlesi is widespread and worrying. Although currently there are no report of human-mosquito-human transmission, the possibility remains that this occurs. The P. knowlesi Evidence Review Group has stated that the exponential rise in P. knowlesi cases warrants further genetic, laboratory, and entomological studies on the transmission dynamics of zoonotic malaria [P. knowlesi malaria must perceive the threat of the infection, and seek medical treatment appropriately [P. knowlesi cases were detected among all age groups and both genders, including children, women, and those not related to forest related occupations. A major risk factor is habitation proximity to forested areas. Currently limited number of studies addressing asymptomatic P. knowlesi malaria and, therefore, more studies are required.Despite clear anti-malaria guidelines and widespread control efforts, the prevalence of asymptomatic  malaria . Cross-s malaria . As asym malaria , and thepriately . AsymptoP. knowlesi, P. falciparum and Plasmodium malariae at certain stages of their life cycle may contribute to misdiagnosis during case detection. To avoid this, molecular diagnostic methods are commonly utilized, and these can lead to improved detection of asymptomatic infections [P. knowlesi-specific schizont-infected cell agglutination variant antigens (SICAvar) PCR assay used by Lubis et al. identified an unexpectedly large number of P. knowlesi infections compared to a similar study from Acheh, Indonesia [P. knowlesi with other Plasmodium species, for example P. vivax demonstrates the need for specific assays for P. knowlesi to overcome possible cross-amplification of (for example) P. vivax, which can occur with some 18 s RNA based assays. In addition, SICAvar based PCR is suggested to be more specific and sensitive compared with PCRs that target the cytochrome B gene [P. knowlesi and not from the other Plasmodium species tested in the study . The assay is sensitive as it was estimated, to detect as low as 0.1 parasite per µL of whole blood [P. knowlesi reference genome [P. knowlesi infections [Asymptomatic malaria cases are rarely detected by light microscopy due to relatively low levels of parasitaemia, leading to an underestimation of the prevalence of these types of infections , 17, 27.fections . The P. ndonesia . The dete B gene . The SICle blood . The robe genome . LAMP hafections . Next-gefections .The underestimation of asymptomatic cases skews epidemiological parameters such as incidence rate, and this can lead to suboptimal guideline design. Thus, sub-optimal diagnostic methodology can hamper efforts to determine disease incidence and are detrimental to public health.P. knowlesi malaria due to work-related exposure, the infection also affects women and children, indicating that a further understanding of the risk factors for exposure is required. In Vietnam, children were found to harbour the parasite a year after an initial positive result. As the children did not receive treatment after the initial screening, it is possible that these infections were chronic and persistent, although re-infection cannot be ruled out. Future studies could incorporate genotyping assays to differentiate between infections [Although males and adults are usually at higher risk of fections .P. knowlesi infection. Particularly, it is unclear why P. knowlesi infection in certain cases remains asymptomatic while others develop clinical symptoms. Furthermore, immunity related factors have been described to play a role in contributing to the P. knowlesi malaria disease outcome [P. knowlesi endemic regions, focusing on the inflammatory and immune responses of different genders and age groups. The presence of severe and fatal cases of P. knowlesi indicates that additional studies are required to determine whether age, gender or genetic predisposition contributes to disease outcome.Several gaps remain in the understanding of asymptomatic  outcome , 26. In  outcome . FurtherP. knowlesi cases, including children, there are implications for control and awareness programs in schools and public areas. There can be different comprehension levels in the population which might lead to differing perspectives regarding the infection. Thus, malaria prevention approaches must be customized to target different groups [Given that the risk of asymptomatic cases is present in the communities living in region with high t groups , 33. ChiPlasmodium knowlesi exposure is high among households that reside in areas surrounded by forested areas [P. knowlesi cases has been detected for the first time among febrile individuals in Kalimantan, Borneo, and Jambi, Sumatra, Indonesia [ed areas . Public ed areas . Studiesed areas . Amid coed areas . Deforesed areas , 36. Fored areas , as comped areas  was suggndonesia , 38. As ndonesia . Surveilndonesia , and thendonesia .. suggested that P. knowlesi vector mosquitoes bite exclusively outdoors, during the early evening around 1800 to 2200 h—thus suggested the usage of bed nets are not an effective method to control P. knowlesi infection [Umbrosus group were found to carry Plasmodium and these species bite earlier, at noon [Poor housing increases malaria risk, and this is also true of zoonotic malaria . Howevernfection . Further at noon . Therefo at noon .P. knowlesi are required.Malaria is a complex disease in which the causative agent, the malaria parasite, is highly genetically polymorphic . This poP. knowlesi are detectable in people living in endemic regions, longitudinal studies may be designed to determine whether the occurrence of these antibodies are associated with disease outcomes, i.e., whether they may be predictive of the development of asymptomatic rather than acute malaria.As antibodies against One of the limitations of this review is that the study was limited to articles published in English therefore, it could not capture studies published in other languages. The other limitation is that literature search was done from 2000 through 2020, therefore, studies done afterwards are not included. However, to ensure that the systematic review is free from bias and produce a reliable finding, the search strategy was carefully planned, the quality of included articles was checked using quality appraisal tool (STROBE checklist), and reviewed by the authors of this review article.Plasmodium knowlesi infection is a complex zoonotic disease. Several reports reviewed here reveal parasite carriage in relatively large numbers of people living in endemic areas. This situation warrants further extensive cross-sectional studies to assess the prevalence of asymptomatic P. knowlesi parasite carriage, and, perhaps more importantly, other research methodology to better inform understanding of the transmission dynamics of the disease.P. knowlesi due to the zoonotic nature of the parasite, but there is an urgent need to improve control measures by including P. knowlesi malaria into national malaria control programmes [It will be extremely difficult to eliminate ogrammes .Additional file 1. Table S1. The quality assessment of the included studies using STROBE checklist.Additional file 2. Figure S1. The PRISMA flowchart for systmatic review on asymptomatic Plasmodium knowlesi cases.Additional file 3. Table S2. The characteristic of asymptomatic Plasmodium knowlesi cases.