Plasmodium falciparum can cause a severe disease with high mortality. A major factor contributing to the increased virulence of P. falciparum, as compared to other human malarial parasites, is the sequestration of infected erythrocytes in the capillary beds of organs and tissues. This sequestration is due to the cytoadherence of infected erythrocytes to endothelial cells. Cytoadherence is primarily mediated by a parasite protein expressed on the surface of the infected erythrocyte called P. falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 is embedded in electron-dense protuberances on the surface of the infected erythrocytes called knobs. These knobs are assembled on the erythrocyte membrane via exported parasite proteins, and the knobs function as focal points for the cytoadherence of infected erythrocytes to endothelial cells. PfEMP1 is a member of the var gene family, and there are approximately 60 antigenically distinct PfEMP1 alleles per parasite genome. Var gene expression exhibits allelic exclusion, with only a single allele being expressed by an individual parasite. This results in sequential waves of antigenically distinct infected erythrocytes and this antigenic variation allows the parasite to establish long-term chronic infections. A wide range of endothelial cell receptors can bind to the various PfEMP1 alleles, and thus, antigenic variation also results in a change in the cytoadherence phenotype. The cytoadherence phenotype may result in infected erythrocytes sequestering in different tissues and this difference in sequestration may explain the wide range of possible clinical manifestations associated with severe falciparum malaria. Plasmodium and is transmitted by mosquitoes. The parasite has a complex life cycle involving specialized invasive forms and intracellular stages [P. falciparum, and other parasite species with 48-h replication cycles, the ring-stage lasts approximately 24 h. During the trophic period the parasite ingests host cell hemoglobin and produces hemozoin, also called the malarial pigment, as a waste product. Nuclear replication marks the beginning of the schizont stage and occurs approximately 36 h after merozoite invasion. During the schizont stage, there are multiple rounds of nuclear replication without cytoplasmic division. These newly replicated nuclei migrate to the parasite periphery and merozoites bud from the schizont. The infected erythrocyte is lysed following merozoite formation, and the released merozoites invade erythrocytes and repeat this blood-stage replication cycle. These repeated rounds of asexual replication in erythrocytes maintain the infection. Instead of repeating this asexual replicative cycle, some merozoites develop into sexual forms called gametocytes. Gametocytes are infective to mosquitoes and initiate sporogony in the mosquito. The sporozoites produced during sporogony migrate to the salivary glands of the mosquito and complete the life cycle. Malaria is caused by protozoan parasites of the genus r stages . The infPlasmodium species. This increased virulence associated with P. falciparum is also associated with higher parasitemia. One factor involved in the higher parasitemia of P. falciparum infection is the parasite’s ability to invade all erythrocytes, whereas P. vivax and P. ovale prefer immature erythrocytes and P. malariae prefer senescent erythrocytes [P. falciparum also has a greater replication potential since mature schizonts produce more merozoites than the schizonts of the other human malaria parasites. Disease manifestations associated with malaria are caused by the blood-stage replicative cycle, whereas the liver and gametocyte stages do not cause any significant pathology. The primary manifestations of malaria are periodic febrile paroxysms caused by the synchronous release of merozoites from infected erythrocytes. If untreated, these paroxysms generally last for a few weeks or months until immunity develops. Falciparum malaria exhibits higher morbidity and mortality than malaria caused by the other human hrocytes . P. falcP. falciparum virulence is the sequestration of infected erythrocytes in the deep tissues. This sequestration of infected erythrocytes is due to the cytoadherence of infected erythrocytes to endothelial cells of the capillaries, thereby decreasing blood perfusion, and causing localized inflammation. Cytoadherence is mediated by electron-dense protuberances on the surface of infected erythrocytes called knobs , the primary ligand associated with cytoadherence and severe disease [Another major parasite protein associated with knobs is  disease . PfEMP1 The ATS of PfEMP1 binds to both KAHRP and the spectrin lattice. The positively charged lysine and histidine rich repeat regions of KAHRP interact with the negatively charged ATS of PfEMP1  via elecPlasmodium helical interspersed sub-telomeric (PHIST) family. PHIST member PFE1605w, also known as lysine-rich membrane-associated PHISTb, binds to the ATS of PfEMP1 and localizes to knobs [There are also other parasite proteins that may be associated with knobs that have not been extensively characterized. These include knob associated heat shock protein 40 (HSP40), PfEMP3 , and a cto knobs ,27. The to knobs . Both PFto knobs . PFE1605to knobs . It is ato knobs . Proteomto knobs . P. falciparum, and erythrocytes infected with trophozoite and schizont stages are sequestered in the tissues. Starting at approximately 16 h post-merozoite invasion, knobs begin to appear on the surface of infected erythrocytes and increase in density as the parasite matures . The expPlasmodium export element (PEXEL), which is found in other apicomplexans and some stramenopiles [Although the timing of the expression of knobs and PfEMP1 on the erythrocyte membrane are well known, the mechanisms by which the parasite alters the erythrocyte are not completely understood. The malaria parasite exports numerous proteins into the host erythrocyte to better accommodate the parasite and facilitate its survival and replication. These proteins are exported into the host erythrocyte via trafficking pathways with unique features ,35,36. Fenopiles ,38. Bothenopiles . Howeverenopiles ,41. ThusThe host erythrocyte is devoid of organelles and the endomembranes of the endocytic and secretory pathways. Therefore, the malaria parasite cannot utilize the host pathways to modify the host cell, as is the case for many viruses and other pathogens. This raises questions about how the malarial parasite targets proteins to specific locations within the infected erythrocyte. Various membranous tubules and whorls originating from the parasitophorous vacuolar membrane (PVM) are observed in the cytoplasm of infected erythrocytes and these structures may be involved in the trafficking of exported parasite proteins within the host erythrocyte ,42. HoweP. falciparum-infected erythrocytes are the Maurer’s clefts [Another membranous structure found in the host cytoplasm of s clefts . The Maus clefts ,45. It is clefts .The submembrane cytoskeleton consists of a two-dimensional lattice of spectrin filaments on the cytoplasmic face of the erythrocyte membrane . The uniKAHRP does not appear to traffic via the Maurer’s clefts en route to the erythrocyte membrane . PresumaAt least eight genes have been identified that are involved in the export of PfEMP1 to the erythrocyte membrane and the formation of knobs . These pPfEMP1 is likely secreted from the parasite as a soluble protein that initially interacts with chaperone-containing complexes called J-dots . PfHSP70The direct binding of KAHRP to the submembrane cytoskeleton suggests that the knobs are assembled in situ at the erythrocyte membrane and the binding of the KAHRP to the submembrane cytoskeleton is the first step in knob formation. KAHRP is also known to self-associate into large complexes  and thisPfEMP1 is also incorporated into knobs after they have been at least partially formed . The delP. falciparum-infected erythrocytes -175 and the Duffy-binding proteins of P. vivax and P. knowlesi [PfEMP1 genes consist of two exons see . The firknowlesi . EBA-175Based on sequence phylogeny, the various DBL domains can be divided into seven distinct types designated as α, β, γ, δ, ε, ξ, or χ. CIDR domains are characterized by conserved cysteine-rich motifs  and exhiP. reichenowi [P. falciparum. The combination of DBL and CIDR domains in any given PfEMP1 protein is not completely random, but certain modules tend to occur together. For example, following an N-terminal sequence, more than 75% of the PfEMP1 alleles have a DBLα domain immediately followed by a CIDRα domain ,78. Thisichenowi , a malarP. falciparum to avoid antibody-mediated destruction of infected erythrocytes since the antibodies against one PfEMP1 allele do not recognize other PfEMP1 alleles. The intrinsic switch rate in var gene expression may occur approximately every 50 blood-stage generations [The expression of PfEMP1 exhibits an antigenic variation in which different PfEMP1 alleles are sequentially expressed. PfEMP1 alleles are antigenically distinct, and this sequential expression allows erations . Therefoerations ,81. Partvar gene is expressed by an individual parasite and this monoallelic expression, also called mutually exclusive expression, is a complex process that is not completely understood [Only a single derstood ,83. By dderstood . There aderstood ,86. Chromatin can exist in two states called euchromatin and heterochromation. Heterochromatin is highly condensed and transcriptionally inactive, whereas euchromatin is more open and capable of being transcribed . The travar genes are highly conserved between alleles and the intron regulates the expression of PfEMP1 on two levels. By mechanisms that are not completely understood, there is a pairing of a PfEMP1 intron and its promoter at the nuclear expression site that results in transcription silencing [var genes has been identified. There is still a lot to be learned about the mechanisms that maintain monoallelic expression, the activation of expression from the repressed state, and the coordination to ensure that the activation of one allele coincides with the silencing of the previously active allele.The introns of ilencing . In addiilencing . The senilencing ,92. HoweP. falciparum-infected erythrocytes in the capillary beds of various organs [Proteins or glycosaminoglycans expressed on the surface of endothelial or other cells serve as cytoadherence receptors, and these receptors are responsible for the sequestration of s organs . Numerouvar gene family. Initially, the large number of potential receptors and the strain specificity of receptor binding were a bit of an enigma since receptor–ligand interactions are generally viewed as highly specific. The realization that PfEMP1 was part of the multi-gene family helped clarify this enigma. The various PfEMP1 alleles have different binding phenotypes in that the different PfEMP1 alleles recognize different ECR. Therefore, as the parasite undergoes antigenic variation, there is a corresponding change in the specific receptor that is recognized. This means that each PfEMP1 allele potentially has a unique cytoadherence phenotype. Many of these potential receptors were identified before the description of PfEMP1 as the major cytoadherence ligand and the discovery of the Many of the potential ECRs are adhesion molecules that participate in cell–cell interactions, especially interactions between leukocytes and endothelial cells, which occur during inflammation. For example, ECRs include several members of the immunoglobulin-like superfamily, selectins, integrin, complement receptor 1, gC1qR, and fractalkine. These proteins participate in adhesive phenomenon and are often associated with inflammation see . In addiThe better characterized ECRs are CD36, EPCR, and intercellular adhesion molecule-1 (ICAM1). CD36 was one of the first described ECRs and has been extensively characterized . This reThe various alleles of PfEMP1 exhibit different phenotypes in regard to the endothelial cell receptors that they recognize. This cytoadherence phenotype is largely due to the subtypes of DBL and CIDR adhesion domains that make up a specific PfEMP1 allele ,20,122. Similarly, a CIDRα1 module in conjunction with DC8  or DC13 Another well characterized cytoadherence receptor is chondroitin sulfate A (CSA), which is a glycosaminoglycan found in the placenta ,118. GlyA single PfEMP1 allele, called var2csa, is the ligand that binds CSA ,139 and Two other phenomena that are linked to PfEMP1-mediated cytoadherence are rosetting and platelet-mediated clumping. Rosetting refers to the adherence of uninfected erythrocytes to infected erythrocytes to form a relatively large clump of cells with an infected erythrocyte in the center that is surrounded by uninfected erythrocytes . PfEMP1 P. falciparum infection [Platelets have also been implicated in the formation of erythrocyte clumps during nfection . Infectenfection . Platelenfection . Three pnfection ,144, gC1nfection , and P-snfection . Other pnfection . Disseminfection . Clinical manifestations associated with malaria can range from asymptomatic carriage to a febrile illness to severe organ dysfunction resulting in death . FebrileP. falciparum, which all can affect parasitemia. It is widely accepted that hyperparasitemia is associated with a poor prognosis and the total parasite biomass correlates with severe disease [Several factors are involved in the development of severe malaria . These i disease . However disease . General disease . There iThe three most common manifestations of severe falciparum malaria are cerebral malaria, respiratory distress, and severe anemia in children . CerebraSevere anemia is due to both the increased destruction of erythrocytes and decreased production of new erythrocytes . In addiThe cytoadherence of parasitized erythrocytes to the capillary endothelium of vital organs, such as brain, bone marrow, lungs, kidneys, or intestines, certainly plays a major role in complicated falciparum malaria. The sequestration of parasitized erythrocytes results in microvascular obstruction, localized inflammation, increased vascular permeability, coagulation disorders, and ultimately organ dysfunction . The adherence of infected erythrocytes in the capillaries obstructs blood flow and deprives tissues of nutrients and oxygen. This ischemia may be enhanced by rosettes  or plateIn addition to the obstruction of capillaries and ischemia, cytoadherence causes increased production of inflammatory cytokines—especially TNF-α—and endothelium inflammation is a major feature of severe malaria ,162. ActA logical inference from the wide range of endothelial cell receptors recognized by PfEMP1 is that different PfEMP1 alleles may be responsible for the organ specificity of the various complications associated with severe falciparum malaria ,151. IndSeveral studies have shown that the expression of specific PfEMP1 alleles is correlated with the risk of developing severe disease ,156,158.A good example of a specific PfEMP1 allele that is correlated with a specific disease manifestation is the association of var2csa expression with placental malaria. The expression of var2csa is significantly upregulated following the selection for adhesion to CSA in vitro . FurtherP. falciparum-infected erythrocytes, and PfEMP1 mediates cytoadherence. Cytoadherence to endothelial cells results in the sequestration of infected erythrocytes in capillary beds and diminishes the clearance of infected erythrocytes by the spleen. PfEMP1 is encoded by the var gene family, which includes approximately 60 alleles per genome. The expression of PfEMP1 also exhibits antigenic variation in which only a single PfEMP1 allele is expressed at a time. The mechanism of antigenic variation involves histone modifications, cis-DNA elements, non-coding transcripts, and nuclear membrane expression sites. Antigenic variation circumvents elimination of infected erythrocytes via antibody-mediated mechanisms directed at PfEMP1. PfEMP1 is a variable protein that is expressed on knobs at the surface of PfEMP1 also functions as the cytoadherence ligand and binds to receptors expressed on the surface of endothelial cells. The various PfEMP1 alleles exhibit distinct cytoadherence phenotypes in regard to the specific endothelial cell receptors that are recognized. Therefore, a switch in the expressed PfEMP1 allele may be accompanied by a switch in the endothelial cell receptor that is recognized. The various endothelial cell receptors recognized by PfEMP1 alleles are differentially distributed in the various organs. This means that a change in the receptor-binding phenotype may also direct sequestration to different organs. A change in sequestration can influence clinical manifestations and the severity of malaria. Pathology associated with sequestration includes ischemia related phenomenon—such as localized hypoglycemia and acidosis—due to the obstruction of blood vessels, localized inflammation of the endothelium, and endothelium dysfunction leading to vascular leakage and edema. Decreased perfusion, acidosis, and vascular leakage all contribute to the higher mortality and morbidity associated with severe falciparum malaria.A better understanding of the biology of cytoadherence and sequestration may lead to the development of therapeutic interventions. Drugs or other therapeutics that interfere with knob formation, expression of PfEMP1 on the erythrocyte surface, binding of PfEMP1 to ECR, or antigenic variation have the potential to reduce the severity of falciparum malaria. Much progress has been made in describing the molecular interactions involved in the knob formation and the receptor–ligand interactions mediating cytoadherence. Understanding the specifics of these interactions opens up the possibility of developing low molecular weight molecules that interfere with these processes. Detailed knowledge about the molecules involved in cytoadherence may also lead to the development of therapeutic vaccines. Furthermore, understanding how cytoadherence mediates the pathology associated with severe malaria can lead to better supportive care in patients with severe malaria.


In sub-Saharan Africa (SSA), malaria remains a public health problem despite recent reports of declining incidence. Severe malaria is a multiorgan disease with wide-ranging clinical spectra and outcomes that have been reported to vary by age, geographical location, transmission intensity over time. There are reports of recent malaria epidemics or resurgences, but few data, if any, focus on the clinical spectrum of severe malaria during epidemics. This describes the clinical spectrum and outcomes of childhood severe malaria during the disease epidemic in Eastern Uganda.® 10 SG, SIEMENS, Ref.2300) at the bedside was done. Data were analysed using STATA V15.0. The study had prior ethical approval.This prospective cohort study from October 1, 2021, to September 7, 2022, was nested within the ‘Malaria Epidemiological, Pathophysiological and Intervention studies in Highly Endemic Eastern Uganda’ (TMA2016SF-1514-MEPIE Study) at Mbale Regional Referral Hospital, Uganda. Children aged 60 days to 12 years who at admission tested positive for malaria and fulfilled the clinical WHO criteria for surveillance of severe malaria were enrolled on the study. Follow-up was performed until day 28. Data were collected using a customized proforma on social demographic characteristics, clinical presentation, treatment, and outcomes. Laboratory analyses included complete blood counts, malaria RDT  and blood slide, lactate, glucose, blood gases and electrolytes. In addition, urinalysis using dipsticks . Day 28 follow-up was achieved in 247/300 (82.3%).A total of 300 participants were recruited. The median age was 4.6 years, mean of 57.2 months and IQR of 44.5 months. Many children, 164/300 (54.7%) were under 5 years, and 171/300 (57.0%) were males. The common clinical features were prostration 236/300 (78.7%), jaundice in 205/300 (68.3%), severe malarial anaemia in 158/300 (52.7%), black water fever 158/300 (52.7%) and multiple convulsions 51/300 (17.0%), impaired consciousness 50/300(16.0%), acidosis 41/300(13.7%), respiratory distress 26/300(6.7%) and coma in 18/300(6.0%). Prolonged hospitalization was found in 56/251 (22.3%) and was associated with acidosis, During the malaria epidemic in Eastern Uganda, severe malaria affected much older children and the spectrum had more of prostration, jaundice severe malarial anaemia, black water fever and multiple convulsions with less of earlier reported respiratory distress and cerebral malaria. Plasmodium species; in sub-Saharan Africa (SSA), Plasmodium falciparum is responsible for most of the malaria morbidity, including severe disease, which is often fatal. This species is also responsible for epidemics, but the clinical spectrum of severe malaria during epidemics is poorly described. The 2020 World Malaria Report of the World Health Organization (WHO) reported an increase in the estimated number of malaria cases from 227 million in 2019 to 241 million in 2020 in 85 malaria-endemic countries . A history of the illness was taken, and qualified medical officers also trained on the study protocol did a clinical examination. Blood and urine samples were then taken for bedside as well as laboratory tests. In the ward, some of the bedside procedures included screening for malaria using RDT SD—Bioline™ malaria—Ag-pf/pan , random blood sugar assays (RBS) (mmol/L) , lactate (mmol/L)  and urine dipsticks . In addition, i-STAT CHEM8 +  was used to measure blood sodium, potassium, chloride, total carbon dioxide, anion gap, ionized calcium, glucose, urea nitrogen, creatinine, lactate, haematocrit and haemoglobin. Qualified and certified laboratory staff carried out blood test analyses using a Beckman Coulter DxH-500 haematology analyser , while biochemistry was performed using a Cobas c111 analyser . Blood slides were examined at MCRI laboratory with quality control undertaken by experienced microscopists. Haemoglobin was obtained using HemoCue™ Hb 301 . Lastly, urine dipstick results were obtained using Siemens multistix10 SG reagent strips .The study procedures were conducted in the ACU ward of Mbale RRH and the laboratory procedures at Mbale Clinical Research Institute [MCRI . The database was designed according to the outline and sections of the questionnaire for easy data entry and resolution of data queries. Data were captured and entered using Redcap V12.2.1  by GCP-trained data assistants. Data were presented using frequency tables and proportions. Data analysis was in three stages, the univariate included frequencies, percentages and continuous variables reported as measures of central tendency as mean, median and interquartile range (IQR) depending on the distribution. Bivariate analysis we reported cross tabulations between the dependent variable  against the independent variables  and reported the chi-square. At multivariate table, a logistic regression reporting odds ratios and respective p-values.The study had ethical approval by Mbale Regional Referral Hospital Research Ethics committee (MRRH-REC) REC #MRRH-2021-76 prior to the data collection.Research assistants were trained in data collection methods. All data from this study were cleaned and analysed using STATAA total of 300 eligible participants aged 60 days to 12 years were enrolled from October 1, 2021, to August 2, 2022.The median age in this study was 4.6 years, mean of 57.2 months and IQR of 44.5 months. In total, 164/300 (54.7%) were under 5 years. More than half, 171/300 (57.0%), were males. Many of the participants were from areas where the malaria epidemic was reported by the Uganda Ministry of Health, including the districts of; Budaka 88/300 (29.3%), Mbale 68/300 (22.7%), Butaleja 28/300 (9.3%), Butebo 23/300 (7.7%), Kibuku 26/300 (8.7%), Namutumba 12/300 (4.0%), Bukedea 12/300 (4.0%), Sironko 10/300 (3.3%), Tororo 4/300 (1.3%) and Pallisa (12/300 (4.0%); in Eastern Uganda.Fever was present in 295/300 (98.3%). Prostration was the most commonly observed presentation in 236/300 (78.7%), of whom 134/236 (55.0%) had severe anaemia, and 76/134 (56.7%) received a blood transfusion immediately at admission.Jaundice was the second commonest clinical feature and was observed in 205/300 (68.3%) participants. Of these, 124/205 (60.5%) had severe malaria anaemia, 144/205 (70.2%) had reported abdominal pain and 152/205 (74.1%) had reported vomiting. The majority of those with jaundice, 42/205 (20.5%), had an elevated LDH, 95/197(48.2%) leukocytosis, and 50/197 (25.3%) had thrombocytopenia.Severe malaria anaemia was observed in 158/300 (52.7%) participants, while 104/158 (65.8%) had reported abdominal pain, 107/158 (67.7%) had vomiting, and 62/158 (39.2%) were reported to have passed dark urine. Tachycardia was present in 64/75 (85.3%), 17/158 (10.8%) had respiratory distress, and 16/158 (10.1%) had a weak pulse.Haemoglobinuria was observed in 158/300 (52.7%) participants, the majority of whom were males 99/158(62.7%), and those 5 years or more were 95/158 (60.1%). A sizeable number—116/158 (73.4%) had reported abdominal pain, and 126/158 (79.7%) reported vomiting. Over half 118/158 (74.7%), had two or more admissions in the previous year. In this group with haemoglobinuria, 139/158 (88.0%) had jaundice and 86/158 (54.4%) had severe anaemia.Multiple convulsions were observed in 51/300 (17.0%) participants, in whom a majority 41/51 (80.3%) were less than 5 years old. More than half 35/51 (68.6%) were febrile, 25/51 (49.0%) had impaired consciousness, and 6/51(11.8%) had respiratory distress. LDH was elevated in 17/51 (33.3%) of the participants, 47/51 (92.2%) had hyperlactataemia and 2/51(3.9%) hypoglycaemia.Impaired consciousness was found in 50/300 (16.7%) participants. More than half—34/50 (68.0%) had reported vomiting, with 25/50 (50.0%) having had convulsions. A number of these patients had the following laboratory features: leukocytosis 26/50 (52%), hyperlactatemia 42/50(84%), hyperglycaemia 41/50 (82%), hypoglycaemia 5/50 (10%), and hyperpyrexia was in 6/50 (12.0%). Acidosis (lactic acidosis) was noted in 41/300 (13.7%) participants; the majority, 36/41 (87.8%), had severe pallor and 33/41(80.5%) jaundice. In addition, leukocytosis was observed in 26/41 (63.0%) and 06/41 (14.6%) had elevated lactic dehydrogenase (LDH).Hyperparasitaemia was observed in 26/300 (8.7%) participants; 19/26 (73.1%) were below 5 years of age. All participants 26/26 (100%) presented with fever.Respiratory distress was a finding in 20/300 (6.7%) participants, 8/20 (40%) of these had hyperlactatemia, 17/20 (85.0%) had impaired consciousness, and hypoxaemia was observed in 3/20 (15%). Only one patient had pneumonia as a co-infection.Cerebral malaria was recorded in 18/300 (6.0%) participants and, out of these, only one (1/18) was hypoglycaemic. However, for technical reasons, we did not carry out lumber punctures to rule out any other causes of coma.Impaired renal function was observed in 15/300 (5.0%) participants. Six out of 15 (40%) were reported to have passed dark urine. Jaundice, severe pallor and profound anaemia were present in all 15 participants. All had elevated BUN > 20 mg/dL and raised creatinine levels on laboratory evaluation. One progressed to acute kidney injury with anuria and electrolyte derangement. However, all recovered without progression to full-blown renal failure. Hypoglycaemia (random blood sugar < 2.2 mmol/L) was an uncommon feature in this study, observed in only 14/300 (4.7%).Spontaneous bleeding was in only 9/300 (3.0%) participants who had reported spontaneous bleeding. All these participants had normal lactate and blood sugar levels, and none had thrombocytopenia. Only one out of nine had raised creatinine, elevated liver enzymes and the presence of respiratory distress. The bleeding episodes included epistaxis (2/9), haematemesis in 2/9 participants, and 5/9 had melaena. None had prolonged bleeding from injection sites.The hypovolemic shock was in only two 2/300(0.7%) participants. This was the least common of all features. Both had jaundice with severe conjunctival pallor, temperature gradient, tachycardia and capillary refill time > 3 s. Laboratory parameters indicated severe malaria anaemia, leukocytosis, and normal platelet count with elevated LDH and bilirubin levels.Some of the participants had multiple severe malaria features. Figure 3/ul, glucose 5.0 mmol/L, blood lactate 2.8 mmol/L, BUN( blood urea nitrogen) 16.75 mg/dL, temperature 37.8 ℃, and Blantyre coma scale had a median of 5.In addition, the mean of the haematological, biochemical and vital results were as follows; haemoglobin 5.8 g/dL, white blood cells 10.2 × 10P = 0.016) and acidosis (P = 0.016) were independently associated with mortality.Out of 300, 251 (83.7%) stayed up to discharge, and these were analysed for prolonged hospitalization, 34 (11.3%) were self-discharges (runaways) and in-hospital mortality was14 (4.6%) and 28 day mortality was 19 (6.3%). Table Anopheles stephensi mosquito in SSA [Malaria incidence (i.e. cases per 1000 population at risk) reduced from 80 in 2000 to 58 in 2015 and 57 in 2019 globally . The emeo in SSA , has poso in SSA , Tanzanio in SSA , and UgaAnopheles mosquito and enhanced efficiency for its disease transmission capabilities in the last decade [There are also changing characteristics of the host. In recent studies, whereas children remain vulnerable to malaria, the age strata and clinical disease picture have changed –18. Furtt decade –21. A coA total of 300 children, randomly selected among children being recruited into a longitudinal malaria study. This is a much larger sample size compared to recent similar studies for the work we report here . TherefoP = 0.041. More than half 154/300 (51%) of the study participants had already been treated with either oral or parental anti-malarial. Our data show that the proportion of patients with each clinical feature has changed with some increasing tremendously compared to similar data reported in the same setting a decade ago [In this study, the common clinical features reported were prostration 236/300 (78.7%), jaundice 205/300(68.3%), severe malarial anaemia 158/300(52.7%), black water fever (BWF) 158/300 (52.7%), and multiple convulsions 51/300 (17.0%). Prolonged hospitalization was found in 56/251(22.3%). Participants with acidosis were more likely to have prolonged hospitalization cade ago  to a higcade ago , but thicade ago , the patConversely, BWF associated with cerebral malaria , 24 is aOther clinical observations in our current study may suggest that earlier treatment with antimalarials may be responsible for the variation in the clinical spectrum and outcomes observed. Over one-half of the study participants had taken anti-malarial before admission. In SSA, there is a paucity of data on the role of ACT in the causation or prevention of BWF. Earlier data reported that BWF was associated with quinine, the first-line drug for treating severe malaria two decades ago  or where. [In this study, the median age was 4.6 years and a proportion of children below 5 years of 164/300 (54.7%). This demonstrates a shift towards older children and a lower proportion of children under the age of 5 years compared to a similar study in the same settings by Olupot-Olupot et alIn most malaria series, low rates of clinical jaundice have been reported. In Eastern Uganda, for instance, in 2012 Olupot-Olupot and colleagues  reportedIn Eastern Uganda, cerebral malaria has consistently remained low . In the P = 0.041), were more likely to have prolonged hospitalization. The overall in hospital mortality was 4.6%, similar to the 4.6% reported in Tanzania [. in the same settings [On the outcomes, 52/251 (22.3%) study participants had prolonged hospitalization, similar to other series previously reported, participants with acidosis  , the morP = 0.016) and impaired consciousness (P = 0.016). These same factors have been reportedly associated with poor outcomes in African children with severe malaria [Overall, the factors that were independently associated with mortality in this study were acidosis ( malaria –33. In aThe limitations were; short duration, small sample size, lumbar puncture/CSF studies and blood cultures were not done. Adherence to anti-malarial therapy could not be closely tracked and neurological sequelae of severe malaria were not looked for and documented.These findings indicate a changing trend in the clinical spectrum of severe malaria associated with the disease epidemic and older age. There were more children with prostration, BWF and jaundice. Pre-referral treatment may play a role in both the clinical spectrum of disease and outcomes. Further research is needed in other populations to see if these findings are more widespread.


Malaria is a parasitic disease that is spread by the bite of an Anopheles mosquito carrying the infection. Microscopic analysis of thick and thin Giemsa-stained smears is the gold standard for diagnosis. If the initial test is negative, but clinical suspicion is high, further smears are required. A 25-year-old man presented with abdominal distension, cough, and a seven-day fever. In addition, the patient developed pleural effusions and ascites. The thick and thin smear tests for malaria and all other fever testing came out negative. Plasmodium vivax was later identified by reverse transcription polymerase chain reaction (RT-PCR). There was a considerable improvement once the anti-malarial medicine was started. It was difficult to diagnose him because pleural effusion and ascites are unusual for someone with malaria. Furthermore, several Giemsa stain smears and malaria quick diagnostic tests were negative, and only a few labs in our country performed RT-PCR. Malaria is a parasitic disease transmitted via the bite of an infected Anopheles mosquito that may lead to life-threatening illness . It contThe gold standard for malaria diagnosis is a microscopic examination of Giemsa-stained thick and thin smears. To avoid low-level parasitemia, examinations must be performed with oil immersion at 100 and 1000 times magnification . A negatA 25-year-old male presented to the emergency department with the chief complaint of fever for seven days, cough with occasional whitish sputum, and a distended abdomen for three days. The maximum recorded temperature was 102 °F with an evening rise and was not associated with chills or rigor. He had no prior history of malaria or travel history to malaria-endemic areas.Initially, the patient was treated at a nearby medical clinic with a combination of azithromycin 500 mg for five days and cefixime 200 mg for seven days, as well as antipyretics and cough suppressants. As his symptoms were persistent, he visited our institution for further management.On presentation, the patient looked ill, conscious, and oriented to time, place, and person, with a temperature of 102 °F, a pulse of 110 beats per minute (bpm), a blood pressure of 110/80 mmHg, a respiratory rate of 24 breaths per minute, and a SpO2 of 96% at room air. On examination, there was no pallor, icterus, or pedal oedema, but there was decreased air entry over the right lower hemithorax with bilateral basilar crepitation. The abdominal examination was positive for a fluid thrill. There was no significant lymphadenopathy, and the rest of the examination was unremarkable.Investigation Table  revealedChest X-ray Figure  showed rAn initial ultrasonogram of the abdomen and pelvis revealed moderate right and mild left pleural effusions and gross ascites. Diagnostic and therapeutic ascitic and pleural tapping was done.The serum ascites albumin gradient (SAAG) was found to be 0.5 mg/dl , indicating an exudative etiology. Pleural tapping, according to Light's criteria, was likewise indicative of an exudative cause Table .We also found gross ascites with diffuse omental thickening and nodularity, a thickened and enhancing peritoneal fold, a mild thickening and enhancing wall of the terminal ileum, a few enhancing necrotic mesenteric lymph nodes, moderate right and mild left pleural effusions, partial collapse/consolidation of the right middle lobe, and enlarged subcarinal and hepatic hilum lymph nodes. However, the possibility of underlying metastatic disease must be ruled out.Mycobacterium tuberculosis, anti-tubercular therapy was started with dosing as per directly-observed therapy, short-course. Despite starting anti-tuberculosis therapy, the patient didn't improve clinically. Ascitic and pleural tapping were repeated for therapeutic and diagnostic purposes. Cytology and cell block for malignant cells, AFB and gene Xpert, Gram stain and culture, and fever panel serology  were performed on both fluid samples. All the reports were unremarkable.On the basis of the above results, although there was no evidence of Plasmodium vivax. The patient was given antimalarial medication (a combination of artemether and lumefantrine). Surprisingly, his fever began to subside after 72 hours. Clinically, the patient improved significantly. Gradually, his ascites and pleural effusion subsided. He was discharged after the chest tube drain was removed (Figure A sample was sent to the central lab for RT-PCR for malaria, and it tested positive for The most common clinical features of malaria include fever with chills and rigor, headache, joint pain, and fatigue . The twoPlasmodium vivax. As demonstrated in our case, the PCR test is useful for low-level parasitemia. In a case series of 17 patients published by Alexandre et al. [Plasmodium vivax malaria, once thought to be a benign disease, is now causing increasingly severe malaria [The gold standard for malaria diagnosis remains microscopic examination of Giemsa-stained thick and thin smears. To avoid low-level parasitemia, examinations are performed at 100 and 1000 times magnification with oil immersion. A negative smear does not rule out malaria, and if clinical suspicion is high and microscopy is negative, smears must be repeated in 12 and 24 hours. Similarly, in our cases, we sent smears for Giemsa stain multiple times; however, they all came back negative. Moreover, the patient was started on antitubercular therapy on an experimental basis. In our country, only a few labs perform RT-PCR for malaria in non-endemic zones. As the patient was not improving clinically and the fever was persistent, our team decided to send RT-PCR for malaria to the central lab, the only lab in our district with a PCR facility for malaria. We were working diligently to get a diagnosis in every way possible. Finally, we sent a malaria RT-PCR sample to a central lab, and the sample tested positive for e et al. , Plasmod malaria . It has  malaria .Severe malaria is a life-threatening but very treatable disease. Nonspecific and uncommon clinical symptoms , as in oAlthough fever is the principal symptom of malaria, its clinical symptoms can vary and are sometimes unusual, which makes it difficult to make a timely diagnosis and treat it appropriately. Microscopic evaluation of Giemsa-stained thick and thin smears remains the gold standard for malaria diagnosis; however, the results could be negative in the initial phase. If clinical suspicion is high, PCR can be a useful modality for the early diagnosis of patients with suspected malaria and patients with low-level parasitemia.


Delayed capillary refill time , hypotension , anemia , low tissue oxygen delivery , high parasite density , and acute kidney injury (p = 0.00047) were associated with higher lactate levels. In children with malaria and RD, bedside lactate may be a useful triage tool, predictive of mortality.Respiratory distress (RD) in pediatric malaria portends a grave prognosis. Lactic acidosis is a biomarker of severe disease. We investigated whether lactate, measured at admission using a handheld device among children hospitalized with malaria and RD, was predictive of subsequent mortality. We performed a pooled analysis of Ugandan children under five years of age hospitalized with malaria and RD from three past studies. In total, 1324 children with malaria and RD  from 21 health facilities were included. Median lactate level at admission was 4.6 mmol/L (IQR 2.6–8.5) and 586 patients (44%) had hyperlactatemia (lactate > 5 mmol/L). The mortality was 84/1324 (6.3%). In a mixed-effects Cox proportional hazard model adjusting for age, sex, clinical severity score (fixed effects), study, and site (random effects), hyperlactatemia was associated with a 3-fold increased hazard of death (aHR 3.0, 95%CI 1.8–5.3,  Plasmodium falciparum infection in children under the age of five in sub-Saharan Africa. Of the human malaria-causing species, P. falciparum is the most virulent, owing largely to its extensive antigenic variation and ability to sequester in the microvasculature of host tissues [In 2021, malaria killed an estimated 619,000 people worldwide [ tissues . Individ tissues . HoweverSevere falciparum malaria is a complex disease that affects multiple organ systems, with symptoms varying widely depending on multiple host and parasite factors. Clinical presentations of severe malaria include cerebral malaria (CM), severe malaria anemia (SMA), and malaria with respiratory distress (RD) . In chilHyperlactatemia at admission is an important predictor of mortality in pediatric malaria patients ,15,16,17Plasmodium parasites, as well as impaired renal and hepatic clearance of lactate. While hypotension leading to systemic tissue hypoperfusion is thought to be a major contributor to hyperlactatemia in sepsis [Plasmodium parasites produce lactate as a metabolic by-product [Lactate accumulation may result from increased lactate production or impaired clearance. In malaria, hyperlactatemia has been attributed to lactate production by hypoxic host tissues and n sepsis , hypotenn sepsis . Howevern sepsis . A uniqun sepsis ,25,26. T-product . This so-product . Acute k-product ,30,31,32-product ,34. End The objective of this study was to examine whether POC measurement of blood lactate can provide a simple, accurate means of risk-stratifying pediatric patients with malaria and RD in a variety of clinical settings . As a secondary objective, we explored associations between the lactate level and other clinical and laboratory parameters to develop hypotheses about the relative contribution of different potential mechanisms of lactate buildup in malaria with RD.n = 180) [n = 2502) [n = 2405) [P. falciparum by malaria rapid diagnostic test (mRDT) or by microscopy; (5) had objective signs of respiratory distress; and (6) had a POC blood lactate measurement recorded at admission.We carried out a secondary analysis of children hospitalized with malaria and RD, combining data from three different studies in Uganda. Study 1 was a randomized controlled trial of inhaled nitric oxide as adjunctive treatment for severe malaria  . In this = 2502) . Study 3 = 2405) . PatientP. falciparum and is estimated to account for 5% of malaria cases globally [P. falciparum infection in different regions ranging from 7.9 to 52.3 cases per 1000 per month [Uganda is a high transmission setting for globally . There ier month . There ier month .A sample size calculation indicated that we would need at least 740 patients for our primary analysis. To estimate this sample size, we used package gsDesign  for a tiP. falciparum antigen histidine-rich protein 2 (HRP2) and the pan-Plasmodium antigen parasite lactate dehydrogenase (pLDH), according to manufacturer’s guidelines. A test was considered positive if either the HRP2 or the pLDH band were positive. Microscopy was not performed for all patients included in this study. However, among patients with a microscopy result available, semiquantitative parasitemia estimates were determined by analysis of thick smears, prepared using Field’s stain. For Study 1, parasite density was quantified using the ratio of trophozoites to leukocytes on the thick smear, assuming 8000 leukocytes/µL. For Studies 2 and 3, parasite density was graded by the clinical laboratory at each site, according to the “plus” scale: + (1 to 9 trophozoites in 100 fields); ++ (1 to 10 trophozoites in 10 fields); +++ (1 to 10 trophozoites per field); ++++ (>10 trophozoites per field). The grade of parasitemia was used to estimate the order of magnitude of parasite density: +  =  50 parasites/µL; ++  =  500 parasites/µL, +++  =  5000 parasites/µL; ++++ = 50,000 parasites/µL [Participants were diagnosed with malaria using mRDTs targeting the sites/µL . Patientsites/µL . For Stu2) less than 90% as measured using a fingertip pulse oximeter. Respiratory distress was defined as the presence of any of the following signs: nasal flaring, intercostal or subcostal retractions, or abnormally deep (acidotic) breathing . HyperlaSBP) below the lower limit of the normal range for age (5th percentile at 50th height percentile), using the clinical formula [Hypotension was defined as a systolic blood pressure . Odds ratios and the corresponding 95% confidence intervals were calculated in the R statistical environment [U test) were used to examine associations between variables for continuous data and categorical data were analyzed using Pearson’s two-tailed chi-square or Fisher’s exact test, as appropriate. For nonparametric rank correlation coefficients between variables, we used Kendall’s tau (τ) as a robust method that did not require normal distribution of data and was permissive for tied ranks [Descriptive data were summarized using the median and interquartile range (IQR) for continuous variables or the number (%) for binary variables. Following dichotomization based on lactate level at admission, Kaplan–Meier curves were used to compare survival between the normal and high lactate populations. To determine if hyperlactatemia was an independent predictor of mortality, we used a linear mixed-effects Cox proportional hazard model, with adjustment for age, sex, and clinical severity (LODS) as fixed effects. Because the lactate was assessed across 3 studies and 21 sites, we adjusted for study and site as random effects in the model. We used package coxme  in the Rcoxme  for the ironment . Nonparaed ranks . Analysied ranks .Written informed consent was obtained from the parent or guardian of all study participants prior to enrolment in the primary study. The primary studies were approved by Makerere University School of Medicine Research Ethics Board, Kampala, Uganda (REC Protocol 2010-107 and 2011-255), the Makerere University School of Biomedical Sciences Research Ethics Committee, Kampala, Uganda (REC Protocol SBS 644), the University Health Network Research Ethics Board, Toronto, Canada (REB Number 10-0607-B and 12-0039-AE), and the University of Alberta Health Research Ethics Board (Pro00084784).We included 1324 children with malaria and RD  hospitalized across 21 centres in Uganda from February 2012 to November 2021. We included 49 patients from Study 1, 603 patients from Study 2, and 680 patients from Study 3 that met the inclusion criteria . Clinicap < 0.0001). Hyperlactatemia was associated with a crude HR of mortality of 5.3 . Subgroup analysis by study is shown in The median lactate level at admission was 4.6 mmol/L (IQR 2.6–8.5) and 586 patients (44%) had hyperlactatemia (lactate > 5 mmol/L). Mortality in Studies 1, 2, and 3 was 12.0%, 9.0%, and 3.7%, respectively, and the pooled mortality was 84/1324 (6.3%). There were 67 deaths (11%) among children with hyperlactatemia at admission and 17 deaths (2.3%) among children with normal lactate (p < 0.0001). The optimal cut-off (threshold of maximum discrimination) using the Youden index for the ROC curve was 5.7 mmol/L . Lactate had similar discriminatory power to the LODS, a composite clinical severity score . With respect to other clinical signs, SpO2 was not a statistically significant predictor of subsequent mortality . Eight patients (4.9%) had a rising lactate level and 90 (55%) had slow lactate clearance, based on a first-order decay half-life greater than 10.8 h [p = 0.39). Among 586 patients with hyperlactatemia at admission, a subgroup of 164 (28%) had a second lactate measurement obtained at 8–12 h after admission, allowing for the early lactate clearance to be computed. Of note, a second lactate measurement was available in only 5 (7.4%) fatal cases, compared to 159 (31%) nonfatal cases , subcostal retractions , and deep (acidotic) respirations  A. The sap < 0.0001, p = 0.00049). Lactate was higher among patients with hypotension  compared to patients without hypotension .Delayed capillary refill time is a clinical sign of impaired tissue perfusion. Lactate levels rose monotonically with increasing capillary refill time  were hypoxemic (SpO2 < 90%). We did not observe a statistically significant correlation between lactate levels and peripheral oxygen saturation .Hemoglobin is the major carrier protein for the bulk transfer of oxygen from the lungs to the tissues. In a subset of 810 patients in whom a hemoglobin measurement was performed, 448 (55%) were severely anemic (hemoglobin < 50 g/L). The lactate level was inversely correlated with the hemoglobin . Patients with AKI had higher levels of lactate  than patients with normal kidney function , p = 0.00047, Lactate is mainly cleared through metabolic pathways in both the liver and kidney . We did n = 1324) and the multiple health facilities from which they were recruited increases the generalizability of our findings. RD is associated with high mortality in patients with malaria  and riskPatients with hyperlactatemia at admission had a significantly higher hazard of death compared to those with normal lactate levels. In a multivariable analysis adjusting for age, sex, disease severity, and site, the hazard of death was threefold higher in patients with hyperlactatemia at admission. The estimate of the HR was similar between the 3 primary studies included in this secondary analysis B, despitP. falciparum parasitemia may be an incidental finding and not the primary etiology of respiratory distress, as wheezing is more commonly a symptom of asthma or viral bronchiolitis. Our study informs clinical practice by providing evidence for signs that healthcare workers can use in low-resource settings to identify patients at increased risk of hyperlactatemia and death.Respiratory distress in pediatric malaria is associated with metabolic acidosis and hyperlactatemia . We founLactate clearance rate has been proposed as a prognostic indicator of mortality in pediatric malaria patients . AlthougThere are several potential use cases for POC lactate in clinical settings in LMICs. First, lactate may be useful as a stand-alone triage tool. In our study, lactate was as accurate as LODS for the prediction of mortality . ClinicaKey advantages of POC lactate include objectivity of the measurements, simplicity of use, and minimal training requirements. The Lactate Scout analyzer is simple to operate and uses a finger prick blood sample to measure lactate levels, making it accessible in community settings. The device is designed to be used by nonexperts, as it is marketed to athletes to monitor blood lactate levels during training. Thus, nurses at rural health facilities in our study were able to collect accurate lactate measurements with minimal training. POC lactate measurements obtained by multiple users across different settings were equally predictive of mortality B, demonsPlasmodium parasites, and impaired lactate clearance [As a secondary objective, we explored associations between the lactate level and other clinical and laboratory parameters . Based olearance . We founAnaerobic cellular respiration resulting in lactate as a metabolic by-product may occur when tissue oxygen demands exceed the circulatory supply. In severe systemic inflammatory diseases, distributive shock may lead to tissue hypoxia through impaired systemic perfusion. In our study, we used capillary refill time as a clinical surrogate of global tissue perfusion. We found a statistically significant association between increased lactate levels and prolonged capillary refill time. Consistent with this, median lactate levels were significantly higher in the small number of patients (2.4%) with overt systolic hypotension (decompensated shock). Delayed capillary refill may also be observed in hypovolemic shock due to dehydration. Children with hyperlactatemia were more likely to present with symptoms suggestive of dehydration including vomiting and being unable to drink or breastfeed. Although distributive and hypovolemic shock contribute to hyperlactatemia in patients with bacterial sepsis, their role in severe malaria is controversial (reviewed in ). AccordIn addition to impaired perfusion, inadequate tissue oxygenation may be related to low oxygen-carrying capacity due to anemia. In our study of children with malaria and RD, comorbid SMA was common (55%). Lactate levels were inversely correlated with hemoglobin concentrations and those with SMA had 3.4-fold higher odds of having hyperlactatemia. These findings are consistent with those reported in the literature; however, there is evidence that the association between hemoglobin and lactate levels differs, depending on the clinical syndrome. While most studies have found an inverse relationship between hemoglobin and lactate in malaria ,62, a stIn malaria with RD, tissue hypoxia and lactate production may occur when blood oxygen content falls due to impaired gas exchange at the level of the lungs (hypoxemia). It is common for adult malaria patients to develop noncardiogenic pulmonary edema, leading to impaired lung function . In our Global oxygen delivery (put (Qt) . In our put (Qt) .Lactate production may be attributable to specific end organs, such as the central nervous system. Altered mental status and lethargy were significantly higher in children with hyperlactemia . Brain tBesides systemic factors, local tissue hypoxia, caused by sequestration of parasitized erythrocytes, may contribute to increased lactate production in severe malaria ,25,26. LPlasmodium parasites themselves may contribute to the pool of circulating lactate [ lactate . Using p lactate , as was  lactate . Our cal lactate . We also lactate . On the  lactate , may hav lactate .Decreased hepatic and renal clearance of lactate may represent another mechanism of hyperlactatemia. We did not collect data on liver function, but we were able to assess kidney function in a subset of 224 patients. Lactate clearance by the kidney occurs via metabolic conversion of lactate to pyruvate (gluconeogenesis) and urine excretion. Approximately 30% of the lactate is removed via gluconeogenesis in the proximal tubules of the renal cortex under physiologic conditions . UrinaryAdditional mechanisms of hyperlactatemia, not assessed in our study, may warrant further research. For example, malaria is associated with reduced gut barrier integrity, as evidenced by increased levels of intestinal injury markers in children with malaria ,68. A reThrough systematic analysis of clinical and laboratory factors associated with hyperlactatemia, our study adds to the current understanding of the underlying mechanisms that lead to elevated lactate levels. This was a secondary analysis of individual patient data from three previous studies in Ugandan children. These studies were not designed to address our specific research question; nonetheless, similarities between the patients enrolled in the three studies allowed us to assemble a large, pooled cohort of patients with well-defined malaria and RD, all of whom had measurements for lactate at admission and prospective follow-up for subsequent mortality. We addressed potential heterogeneity in the three studies with a subgroup analysis B and adjHere, we demonstrated that POC lactate measurement at admission predicts mortality in pediatric patients with malaria and RD across a range of studies  and settings . POC lactate testing offers a simple and objective means by which to risk-stratify patients, particularly in locations without access to a central laboratory. Furthermore, in the 20 rural health facilities, nurses performed POC lactate measurements with little oversight from study staff, demonstrating the feasibility of implementing POC blood lactate testing in a wide range of low-resource settings. The addition of this measurement to current triage algorithms in resource-limited settings could improve the efficiency of healthcare resource allocation by identifying children at highest risk of mortality. In areas such as sub-Saharan Africa, where malaria is a leading cause of death in children under five years of age, combining POC lactate measurements with existing triage protocols may offer a strategy to reduce childhood mortality.


Similarly, CMD patients had a much higher prevalence of diabetes than those without CMD . At the one-year follow-up, the LVEF in the CMD group had decreased to significantly lower levels than those in the non-CMD group , whereas it had been higher in the CMD group at baseline . Similarly, during the follow-up, the CMD group had a greater incidence of AF . In the adjusted multivariable analysis, the IMR and TMAO were associated with increased odds of AF development , and , respectively. Similarly, elevated levels of IMR and TMAO were linked with decreased odds of LVEF improvement, while higher CFR values are related to a greater likelihood of LVEF improvement. Conclusions: CMD and elevated TMAO levels were highly prevalent three months after STEMI. Patients with CMD had an increased incidence of AF and a lower LVEF 12 months after STEMI.Introduction: Persistent coronary microcirculatory dysfunction (CMD) and elevated trimethylamine N-oxide (TMAO) levels after ST-elevation myocardial infarction (STEMI) may drive negative structural and electrical cardiac remodeling, resulting in new-onset atrial fibrillation (AF) and a decrease in left ventricular ejection fraction (LVEF). Aims: TMAO and CMD are investigated as potential predictors of new-onset AF and left ventricular remodeling following STEMI. Methods: This prospective study included STEMI patients who had primary percutaneous coronary intervention (PCI) followed by staged PCI three months later. Cardiac ultrasound images were obtained at baseline and after 12 months to assess LVEF. Coronary flow reserve (CFR), and index of microvascular resistance (IMR) were assessed using the coronary pressure wire during the staged PCI. Microcirculatory dysfunction was defined as having an IMR value ≥25 U and CFR value <2.5 U. Results: A total of 200 patients were included in the study. Patients were categorized according to whether or not they had CMD. Neither group differed from the other with regards to known risk factors. Despite making up only 40.5% of the study population, females represented 67.4% of the CMD group  New-onset atrial fibrillation (AF) and left ventricular systolic function deterioration remains a prevalent consequence of ST-elevation myocardial infarction (STEMI) . DespiteStructural and electrical cardiac remodeling plays a crucial role in the development of AF in patients who have had an acute myocardial infarction (AMI) . Left atTrimethylamine N-oxide (TMAO), a novel biomarker, may also play a role in cardiac electric remodeling. Trimethylamine is produced from the breakdown of dietary L-carnitine, choline, and lecithin in the human gut and is then oxidized in the liver to form TMAO . AtherosThe current study aims to investigate the role of coronary microcirculatory dysfunction and TMAO in predicting new-onset AF and left ventricular systolic function in patients who have had a STEMI.This was a prospective single-center cohort study conducted in the Hospital of the Lithuanian University of Health Sciences Kaunas Clinics which covers four out of ten administrative regions in the Republic of Lithuania. The study was carried out in agreement with the declaration of Helsinki and approved by the Kaunas Regional Biomedical Research Ethics Committee ; informed consent was obtained from all study participants.The study included patients aged 40 years or older with STEMI who received dual antiplatelet therapy  and successfully underwent primary percutaneous coronary intervention (PCI) of the culprit vessel, followed by a staged PCI for non-culprit disease three months later.Patients were excluded from the study if they had a history of atrial fibrillation/flutter, a concomitant illness affecting blood counts or inflammatory biomarkers, or significant comorbid disease . Patients with severe valvular heart disease were excluded due to the risk of ventricular fibrillation with hyperemia and those with coronary artery bypass grafts were excluded due to altered coronary circulation . PatientVenous blood was collected from all study participants at admission, and at a three-month follow-up. Complete blood counts, serial troponin I levels, Erythrocyte sedimentation rate (ESR), B-type natriuretic peptide (BNP), high sensitivity reactive protein, and lipid levels were obtained as part of routine clinical care. The blood samples were centrifuged appropriately to obtain plasma samples, which were stored at −80 °C. TMAO ELISA (enzyme-linked immunosorbent assay) kit  was utilized to measure plasma TMAO levels at 3-month follow-up.Coronary flow reserve (CFR), fractional flow reserve (FFR) and index of microvascular resistance (IMR) were assessed using the CoroFlow system . After undergoing staged PCI, nitroglycerin was administered through the intracoronary catheter and a calibrated coronary physiology wire  was equalized to the guide catheter pressure at the sinus of the aorta. The coronary physiology wire was then advanced to the distal two-thirds of the left anterior descending artery. Three milliliters of normal saline was administered through the guiding catheter in triplicates during rest and during maximal hyperemia, which was induced by injecting intracoronary adenosine.Data collected included patient demographics, medical history, clinical course, laboratory values, angiographic characteristics and follow-up data were recorded. Linear measurements of the left atrial chamber, left atrial volume (LAV), LAVI, and LVEF were obtained according to the recommendations of the European Association of Cardiovascular Imaging (EACVI), which was evaluated through obtaining two-dimensional and three-dimensional images using an ultrasound device  over a 24-h period, and with a one-year follow-up .The primary endpoint was the existence of atrial fibrillation twelve months following an ST-elevation myocardial infarction. The secondary endpoint was the recognition of improved left ventricular systolic function following a year of echocardiographic monitoring. Any interactions between CMD, new-onset AF and change in LVEF were also examined.STEMI was defined according to the fourth universal definition of myocardial infarction . Door-top < α  was taken as statistically significant. Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 29.0 Continuous variables were skewed and are presented as median . Categorical variables are presented as frequency and percentage. Differences in patient and clinical characteristics between those with confirmed CMD versus the non-CMD were assessed via the Wilcoxon rank sum test and chi-square or Fisher’s exact test, as appropriate. Differences in the outcomes between both study groups were assessed via chi-square or Fisher’s exact test, as appropriate. We used stepwise selection (backward and forward selection) to create multivariable logistic regression models to investigate factors identified as having a significant association with primary and secondary outcomes. We adjusted for potential confounders  in the multivariable regression model designed to investigate independent predictors of AF. A probability level of p < 0.001). Similarly, diabetic patients were more prevalent in the CMD group than in the non-CMD group   had CMD, while 154 patients (77%) did not. There were no differences in age, BMI, STEMI location, or risk factors between the two groups . Despite< 0.001) .p = 0.001)  between the two groups, except for creatinine clearance which was lower in the CMD group  .p = 0.021) and ESR  were higher in the CMD group after three months. Similarly, the CMD group had higher levels of BNP and TMAO at three months than the non-CMD group.  and , respectively , nonetheless, this was short-lived, as the LVEF in the CMD group deteriorated at one year compared to the non-CMD group   or echocardiographic  parameters . In cont< 0.001) .p < 0.001). However, those in the CMD group had lower rates of LVEF improvement   .p = 0.007), , and , respectively (p = 0.029)  area under the curve (AUC) of 0.763 and 0.669 respectively, indicating good predictive ability for new-onset AF . In the ectively . On the = 0.029) .p < 0.001) or an elevated TMAO  (p = 0.035)  . Convers= 0.035) .Two hundred patients presenting with STEMI who underwent primary PCI were enrolled in this study. At three months, the presence of CMD was assessed and at 12 months the incidence of new-onset AF and change in LVEF were assessed. Patients with CMD were more likely to have worse renal function on admission, higher BNP and higher TMAO at three months, and lower LVEF at 12 months. In addition to well-established factors such as LVEF and LAVI, this study found that the presence of CMD and a higher TMAO at three months correlated with an increased risk for new-onset AF at 12 months.CMD is a poorly understood syndrome that ultimately leads to myocardial ischemia. There are multiple proposed etiologies including oxidative stress leading to a chronic inflammatory response, microthrombi formation, endothelial and vascular smooth muscle dysfunction leading to inappropriate coronary microvascular constriction and/or impaired dilatation . FurtherWe found that the presence of CMD was also associated with higher BNP and lower creatinine clearance. Chronic kidney disease is associated with microvascular dysfunction in multiple organ systems, including the eye and the brain . The Womp = 0.02) [A lower LVEF and higher LAVI correlated with an increased risk of new-onset AF, which is consistent with prior studies . We foun = 0.02) .Similarly, research has suggested that patients with STEMI are more likely to have CMD and new-onset AF than those without STEMI, although the exact reasons for this are not yet fully understood . CMD is CMD has been linked to heart failure with preserved ejection fraction in recent years ,32,33. HThe study’s primary limitation is that it was conducted at a single institution. Additionally, despite the small size of the study’s sample (200 patients), it is still one of the largest studies involving invasive testing of CMD. Further, since coronary physiology measurements were only obtained after the STEMI event, we were unable to disqualify patients with CMD prior to the event. Furthermore, we were unable to determine if our findings also apply to single-vessel diseased patients because the study recruited only those with a coronary multivessel disease with the intent of obtaining the coronary physiology measurements during the staged PCI. The LVEF was used as a metric for cardiac structural remodeling in this study, but other echocardiographic parameters, such as LV strain and diastolic parameters, were not accounted for. Further, we were unable to control for the ischemia time or the myocardial volume at risk which likely differ across patients and would influence the post-PCI LVEF.Patients with STEMI have a relatively high prevalence of CMD when assessed three months after their event. They also have a higher TMAO levels, an emerging biomarker that may play an important role in microvascular disease. Patients with CMD exhibited an increased incidence of new-onset AF at 12 months compared to patients without CMD. Further, the LVEF was lower at 12 months in patients with CMD compared to their post-PCI LVEF, suggesting that the chronic ischemia and adverse cardiac remodeling propagate systolic dysfunction in this population. While this is one of the largest prospective cohorts examining CMD to date, further research is needed to better understand how CMD impacts myocardial recovery in acute myocardial infarction patients and which therapies can ameliorate it deleterious effects.


Plasmodium falciparum parasitaemia forms a reservoir for the transmission of malaria disease in West Africa. Certain haemoglobin variants are known to protect against severe malaria infection. However, data on the potential roles of haemoglobin variants and nongenetic factors in asymptomatic malaria infection is scarce and controversial. Therefore, this study investigated the associations of iron homeostasis, inflammation, nutrition, and haemoglobin mutations with parasitaemia in an asymptomatic cohort from a P. falciparum-endemic region during the high transmission season.Asymptomatic HBA and 22 HBB mutations. Demographics, inflammatory markers , nutritional status (mid upper-arm circumference and body mass index), and anaemia  were assessed as potential predictors through logistic regression.A sub-study population of 688 asymptomatic individuals  from rural Burkina Faso previously recruited by the NOVAC trial (NCT03176719) between June and October 2017 was analysed. Parasitaemia was quantified with conventional haemocytometry. The haemoglobin genotype was determined by reverse hybridization assays targeting a selection of 21 95: 2.45–19.4, p < 0.001). Furthermore, statistically significant associations (p < 0.05) with age, cytokines, hepcidin and heterozygous haemoglobin S were observed.Malaria parasites were detected in 56% of subjects. Parasitaemia was associated most strongly with malnutrition. The effect size increased with malnutrition severity (OR = 6.26, CIAccording to these findings, asymptomatic parasitaemia is attenuated by haemoglobin S, but not by any of the other detected genotypes. Aside from evidence for slight iron imbalance, overall undernutrition was found to predict parasitaemia; thus, further investigations are required to elucidate causality and inform strategies for interventions.The online version contains supplementary material available at 10.1186/s12936-023-04686-0. Malaria is still a large public health concern and a burden on health economies, especially in West Africa. Despite continuing advances in malaria control and elimination , 2, suchThe spread of malaria disease by mosquitoes is dependent on environmental, parasite- and host-related factors such as the amount of precipitation, parasite genotype or host red blood cell variants and immunological control of parasites –6. TransHBB) mutations, such as haemoglobin C (AC) or haemoglobin S (AS), and have demonstrated a geographic correlation with these mutations [HBA) mutations, such as the prevalent 3.7 kb deletion (-a/aa), have been demonstrated to offer some protection against severe Plasmodium falciparum malaria infection [Numerous factors influencing asymptomatic parasite carriage are poorly understood. For instance, certain mutations are overrepresented in malaria-endemic regions compared to where malaria infection is less prevalent , 10. In utations , 5, 9. Infection . HoweverOn the other hand, nongenetic factors play an important role, albeit direct or indirect. For example, iron parameters, such as ferritin or hepcidin, are affected by malaria parasite carriage  and cytoP. falciparum hyper-endemic rural region Nanoro in Burkina Faso was used to clarify the impact of several possible influencing factors on parasitaemia and parasite density. Among these factors, haemoglobin genotypes as well as several surrogate markers for iron homeostasis , inflammation , and nutrition were investigated. Furthermore, the abundance of P. falciparum parasitaemia is reported in these asymptomatic individuals in this region and its predictors are described.A large and well-characterised cohort of asymptomatic non-febrile individuals in the For this study, the cohort of the Nonspecific Effects of Vaccination on Mortality and Morbidity (NOVAC) trial  was investigated. This large cross-sectional study included healthy volunteers randomly selected from the Nanoro health and demographic surveillance system  during tParticipants were included when neither signs nor symptoms were noticed by the study physician. Eligibility was further tested with the following exclusion criteria: current febrile illness, known chronic illness, HIV, tuberculosis, renal failure, or cardiac disease. Furthermore, individuals who had participated in the concurrent vaccination trial from Glaxo-Smith-Kline  at the same inclusion site were excluded.The study protocols were approved by the national ethics committee of Burkina Faso (ref 2016-01-006). Written informed consent was obtained from all participants or their parents/legal guardians. Assent was obtained from all participants aged 7 to 20 years according to the local requirements.Molecular genetic analyses were performed at the Institute of Laboratory Medicine  using the α-Globin StripAssay and β-Globin StripAssay MED kits  to identify 21 and 22 haemoglobin mutations, respectively  and body temperature were measured upon inclusion. Malnutrition categorization was performed by calculating z scores for the MUAC-for-age with the “zscorer” package  for indiHaemocytometry, including malaria-infected red blood cell and gametocyte quantification, was performed on an XN-30 automated haematology analyser . Anaemia was defined by age-specific cutoffs for total haemoglobin concentrations published by the WHO . ParasitMeasurement of inflammatory biomarkers  and iron biomarkers  was conducted by multiplex immunoassay  and quantitative sandwich immunoassay (Quantakine and Thermo Scientific), respectively.The Wilcoxon–Mann–Whitney U test was employed for comparisons of location parameters when parametric assumptions were not met. The t test was used for comparison of means after log transforming the data, and the subsequent test for departure from normality was not significant. Univariate and multivariable analyses for the assessment of possible predictors of a binary response (presence of parasitaemia) were carried out through logistic regression using odds ratios to quantify the relationship. 95% confidence intervals for proportions were calculated per category based on binomial distribution theory. Multiple logistic regression was performed on a subset of the dataset where measurements for all the covariates were present. The chi-squared test for trend was used for the assessment of the existence of a linear trend for ordered categorical data vs. a binary outcome. Statistical analyses were performed using R version 4.3.0 . The testwise alpha level was set at 0.05. P values less than 0.05 were considered statistically significant.HBA (n = 41) or HBB (n = 1) genotyping results and missing values for anthropometry (n = 6), haemocytometry (n = 50), iron or inflammation biomarkers (n = 68). Characteristics of the included study population (n = 688) according to age ranges are given in Table In total, 1012 asymptomatic individuals were included in the original NOVAC study. For the current study, genotyping was performed in 847 individuals with sufficient sample volume for genomic DNA extraction. Several further samples had to be excluded from subsequent analysis due to inconclusive HBA combined with wild type HBB (-a/aa with AA).Haemoglobin genotyping was performed to identify thalassaemic and nonthalassaemic variants at the molecular level. This revealed a heterogeneous distribution, as shown in Table Regarding exclusively the alpha genes, 70.6% (n = 417) wild type, 27.7% (n = 223) -a/aa and 1% (n = 8) anti-3.7 kb triplications were observed. Furthermore, two individuals with a 4.2 kb deletion were integrated into the respective 3.7 kb deletion group for further analysis, as they are considered phenotypically equivalent. The remaining 0.4% (n = 4) of the alpha mutations  were found only in single individuals each. One subject with the combination of 3.7 deletion and anti-3.7 triplication was included in the wild-type group for statistical analysis since this combination appears as a normal alpha phenotype.Regarding exclusively the beta gene, 74.3% were wild type, 18.1% (n = 146) AC, 5.6% (n = 45) AS, 1.2% (n = 10) homozygous haemoglobin C (CC) and 0.7% (n = 6) compound heterozygous haemoglobin SC (SC).Moreover, coinheritance of mutations in both genes occurs frequently for -a/aa with either AC  or AS . More complex mutations were observed at low frequencies (n < 10) and therefore grouped together into a rarer traits category. Further details and frequencies for each screened mutation are given in Additional file Univariate and multivariable logistic regression were performed in a subset of the dataset (n = 688) where values for all the covariates were present to assess the effect of various possible predictors Fig. .95: 0.10–0.74, p = 0.013). A marginally nonsignificant  effect was observed for the -a/aa with AS group. Furthermore, for these two groups, lower proportions of parasite positives were counted  and parasite density. Figure 95: 1.02–1.86, p = 0.038) but was not when adjusted for possible confounders in the multivariable model .In the univariate analysis, younger age was a significant predictor of parasitaemia  and anaemia categories  were significantly associated with parasitaemia  and microcyte percentage , were not associated with the outcome.In the univariate analysis, haemoglobin concentration . However, the multivariable model-adjusted odds ratio for hepcidin  was marginally significantly elevated. Concerning inflammatory parameters, both IL-6  and IL-10  showed significant effects in the adjusted model, while when fitted on its own, IL-6 had a borderline nonsignificant effect . Gametocytaemia was not significantly associated with parasitaemia in the univariate analysis . Further details are shown in Additional file Biomarkers of iron status, ferritin and soluble transferrin receptor, were not associated with parasitaemia in the multivariable analysis , moderate , and severe , were strong predictors of parasitaemia. The proportions of parasitaemia-positive individuals increased per category of malnutrition severity  of otherwise healthy individuals (n = 688) from an endemic rural region in Burkina Faso during the high transmission (wet) season. Asymptomatic parasitaemia was attenuated in carriers of AS (aa/aa with AS and -a/aa with AS) mutations only but not in other haemoglobin genotypes . Furthermore, it is showen that asymptomatic parasite carriage was strongly associated with malnutrition, and the effect size increased linearly with the severity of energy deficiency. To a minor extent, parasitaemia was also associated with other parameters, such as age and inflammation.These findings complement the current knowledge on asymptomatic malaria parasitaemia in general and in haemoglobin variant carriers specifically.Moreover, the screened haemoglobin genotypes displayed a similar distribution as has been reported previously from Burkina Faso –30, althHBA gene [A considerable effect on malaria parasitaemia by common haemoglobin variants in asymptomatic subjects was expected, such as higher frequencies for individuals with aa/aa with AC and -a/aa with AC  or lowerHBA gene . These oTherefore, age, sex, iron parameters, inflammation and nutritional status were evaluated further in this large well-defined cohort to identify predictors of malaria parasitaemia in asymptomatic individuals.Interestingly, despite parasitaemia peaking over 80% in the age group between 10 and 14 years, the parameter parasite density decreased with age linearly from one until 20 years old individuals. While the literature agrees that most clinical malaria cases occur during early life and in pregnant women , the linAs previously outlined, haemoglobin concentration as a robust marker for general anaemia classification was significantly associated with parasitaemia only in the univariate analysis and not in the multivariable model. Since all-cause anaemia is very common in individuals of the investigated area, haemoglobin alone might not serve as surrogate predictor of parasitaemia.Associations of IL-6 and IL-10 with parasitaemia may indicate an ongoing inflammatory response in infected individuals. Both were significantly higher in parasitaemic compared to aparasitaemic individuals. IL-6 is known to play a pro-inflammatory role, and IL-10 plays an anti-inflammatory role during inflammation. In contrast, IL-10 was proposed to influence the susceptibility to coinfections in asymptomatic malaria parasitaemia , which mIn contrast, the iron biomarkers ferritin (storage iron) and soluble transferrin receptor  were not associated with parasitaemia in the current study. This relationship of parasitaemia and iron homeostasis is discussed controversially in the literature –45. The 95: 1.00–1.03, p = 0.043). Control of peripheral hepcidin expression in macrophages is influenced by IL-10 according to in vitro studies [A central regulator of iron homeostasis, hepcidin, was marginally associated with parasitaemia . Table S2. Frequency of all 21 haemoglobin genotypes covered by the strip assays , two individuals showed co-inheritance of more than one mutation. Table S3. Frequency of all 22 haemoglobin genotypes covered by the strip assay , 16 individuals showed co-inheritance of more than one mutation. Table S4. Univariate logistic regression results of parasitaemia as response variable. Table S5. Univariate linear regression results of parasite density as response variable. Table S6. Multivariable logistic regression of parasitaemia as response variable . The overall probability of parasitaemia was 56%. Figure S1. Relationship of anaemia and asymptomatic malaria parasitaemia (log10 of parasite density). (A) A lower parasite density in the non-anaemic group is documented . (B) Lower haemoglobin concentrations are documented for the aparasitaemic group . Figure S2. Parasitaemia positive frequency for the basic anaemia categories  is plotted in bars and lines describing 95% CI from binomial distribution. A similar difference as in Fig. S3 (A) is observed as the parasitaemia and parasite density both appear lower in the non-anaemic group. Figure S3. Inflammation marker concentrations (log10 transformed) are illustrated and grouped by qualitative parasitaemia categories (n = 688). Highly significant differences are observed for all the parameters. Figure S4. Positive parasitaemia frequency for the gametocytaemia categories . Individuals were categorised depending on their age, and upper-arm circumference or body mass index. For the group < 19 years malnutrition status was determined by MUAC-for-age z-score and for the adult group of 20 years and older body mass index classification was used. Both variables were calculated and previously published cut-offs applied.erature, ) is plotAdditional file 2. A CSV with the analysed dataset that can be used to reproduce the results of this study.