Plasmodium falciparum and Plasmodium vivax cause over 600,000 deaths each year, concentrated in Africa and in young children, but much of the world’s population remain at risk of infection. In this article, we review the latest developments in the immunogenicity and pathogenesis of malaria, with a particular focus on P. falciparum, the leading malaria killer. Pathogenic factors include parasite-derived toxins and variant surface antigens on infected erythrocytes that mediate sequestration in the deep vasculature. Host response to parasite toxins and to variant antigens is an important determinant of disease severity. Understanding how parasites sequester, and how antibody to variant antigens could prevent sequestration, may lead to new approaches to treat and prevent disease. Difficulties in malaria diagnosis, drug resistance, and specific challenges of treating P. vivax pose challenges to malaria elimination, but vaccines and other preventive strategies may offer improved disease control.Infections with  Plasmodium spp. that commonly infect humans – P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi – P. falciparum causes 95% of the cases and the great majority of the severe disease . Cir. Cir4]. sitaemia ,39. The sitaemia , althougsitaemia . A simild below) .The threshold at which parasitaemia is associated with fever increases with age and is higher in holoendemic settings than mesoendemic settings , suggestP. falciparum  compared to uninfected children [The clinical impact of low-density infections is unclear. In a study of 2801 febrile children, there were no differences in severe clinical outcomes or secondary hospitalizations for individuals with low-density children . In a 2-children . In contchildren . The conchildren .P. falciparum and 46% of P. vivax infections lasted for over 6 months (during periods of low transmission) [In the absence of mass drug administration regimes, low-density infections are untreated. Although individuals with submicroscopic infection are less likely to infect mosquitoes than microscopy detected infection , submicrSeveral RBC polymorphisms occur at higher frequency amongst populations that have historically experienced a high burden of malaria and therefore are likely to offer an evolutionary survival advantage . It is wThere are likely to be a number of host genetic traits that contribute to protection and are not yet characterized . Of recePIEZO1 E756del [Understanding the mechanisms by which naturally selected genetic traits contribute to protection enhances our understanding of malaria pathogenesis that may open doors to novel therapeutic strategies. For example, the Dantu mutation increases membrane tension and in turn inhibits merozoite invasion, suggesting that a membrane tension threshold may be a key factor for successful merozoite invasion . Several E756del  reduce s E756del . In the  E756del . Both roP. falciparum IEs sequester in the heart, lungs, brain, skin, gastrointestinal tract, placenta, and other organs [Asexual r organs . Sequestr organs . Some seP. falciparum IE cytoadhere to receptors on endothelial cells or syncytiotrophoblast lining the placenta via cell surface antigens, particularly PfEMP1. Several possible endothelial adhesion receptors have been identified by in vitro and post-mortem histological studies, including Cluster of Differentiation 36 (CD36), intercellular adhesion molecule-1 (ICAM-1), E selectin, P-Selectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial protein C receptor (EPCR), integrins and fibronectins [Sequestration occurs when onectins , while conectins . In addionectins  and in honectins ,68 and tonectins . The autonectins .P. berghei migrate through the sinusoidal barrier of the bone marrow, spleen, and liver and translocation into the bone marrow parenchyma was partially dependent on host P-selectin expression [in vitro and this activated the release of cytokines and growth factors, many of which are involved in angiogenesis [2O2 that ultimately kills gametocytes, and proposed that the colocalization of CYP2D6 and CPR in the liver and bone marrow explains the efficacy of primaquine to kill hypnozoites and P. falciparum and P. vivax gametocytes [In addition to asexual parasites, post-mortem histology has revealed sequestration of immature gametocyte IE in the extravascular bone marrow parenchyma , accountpression . Adhesiopression  but not pression . A recenogenesis . Adhesioogenesis . Gametocetocytes .P. vivax-infected reticulocytes do not display PfEMP1-like antigens or knobs; however, there is some evidence for their cytoadherence to lung and placental endothelial cells in vitro [ex vivo in the lung [P. falciparum, immature P. vivax gametocytes sequester in the bone marrow parenchyma as a potential site of development and reservoir for infection [Asexual in vitro . Low levthe lung , liver, the lung , and liknfection ,79.Plasmodium spp. infected, asymptomatic individuals undergoing splenectomy found massive sequestration of intact P. vivax IE of all stages in the spleen, at a rate 3590 times higher than the peripheral blood [P. falciparum IE also sequestered in the spleen but to a far lesser extent [Interestingly, a recent study of al blood . P. falcr extent . It remaP. falciparum IE [P. vivax [The spleen has multiple important functions in infection. Splenic macrophages and polymorphonuclear cells remove IE by phagocytosis  and thisparum IE ,85 and mP. vivax . This suP. falciparum can bind to two or more uninfected red blood cells to form “rosettes.” This phenomenon is observed with some parasite lines but not others, suggesting it is a variant specific mechanism. Rosetting is also not associated with parasite density [2 macroglobulin binding to IEs can also mediate adhesion to uninfected erythrocytes in laboratory adapted and field isolates [Trophozoite IE infected with  density , suggest density ,90. Rose density , in part density ,92. Rose density . Serum fisolates ,94.In several studies, rosetting was more common in isolates taken from individuals with cerebral malaria or with severe malaria anaemia  but not The mechanisms by which rosetting contributes to severe malaria are still unclear. Rosetting has been hypothesized to block or slow capillary blood flow, possibly enhancing cytoadherence . RosettiP. vivax were similar in strength to those of P. falciparum and may alter IE deformability to enhance sequestration [P. vivax rosetting has been associated with anaemia [in vivo requires further study.Rosetting has been occasionally described in other parasite species , althougstration . Rosettistration ,99. P. v anaemia ; howeverin vitro makes it difficult to discern the importance of clumping in severe malaria [Aside from adhesion to uninfected erythrocytes, parasite IEs are also able to bind to CD36 or gC1qR/HABP1 on the surface of platelets to mediate clumping of IEs ,105. Clu malaria , and it P. falciparum malaria. There are 4 main families of VSAs expressed during the blood stage of P. falciparum: PfEMP1, encoded by   60 var genes; RIFINs, encoded by 150–200 rif genes; Surface-associated interspersed gene family proteins (SURFINs), encoded by   10 surf genes; and STEVOR, encoded by 30–40 stevor genes [n vitro, cultures of IEs panned on a particular receptor will favour expression of PfEMP1 that can adhere to the receptor [in vivo is that var2csa becomes the dominant var gene to be expressed by parasites infecting pregnant women and encodes for PfEMP1 that binds to placental CSA receptors [var genes allows the parasite to adapt to different adhesion environments to avoid splenic clearance. Additionally, multiple var genes of the same binding phenotype exist, allowing the parasite to maintain adhesion to particular receptors despite antibody recognition [Variant surface antigens (VSAs) are central to the pathogenesis of or genes . The laror genes ,111. Bulor genes . PfEMP1 receptor . A well-eceptors . The widognition .P. vivax, including the vir gene family, but are less well characterized due to high antigenic variation and difficulty maintaining cultures in vitro. Different VIR protein subfamilies are trafficked to different cellular locations, including the reticulocyte surface (for VIR14 of subfamily C) where they have been speculated to be involved in cytoadherence [P. falciparum IE expressing VIR14 has been shown to bind to ICAM-1 and human spleen fibroblasts in vitro [P. vivax-infected pregnant women [P. falciparum and whether other VIR or non-VIR proteins are involved in cytoadherence of P. vivax.Multigene families have been identified in dherence . Transgein vitro ,116. A snt women . It remavar genes/PfEMP1 types. Var genes have been classified into groups A, B, C, and E and two intermediate groups B/A and B/C, based on their chromosomal location and type of upstream promotor sequence  [var genes compared to uncomplicated malaria patients [var genes, rather than group B or C [Several studies have found associations between expression of specific PfEMP1 subclasses, domains or combinations of domains (domain cassettes or DCs) and disease outcomes, supporting the idea that there are virulent C, or E) . Interespatients . Additiop B or C ,124. Expp B or C ,125 but p B or C , as has p B or C .var genes are composed of arrays of Duffy binding-like domains (DBLs) and cysteine-rich interdomain regions (CIDRs), in various combinations that can be subclassified into DBL α0.1–2, β1–10, γ1–18, δ1–9, ε1–14, ζ1–6 and CIDR α1–6, β1–7, γ1–12, γ1–2 [var genes [in vitro is independent of CD36 [Individual 12, γ1–2 ,127. Dom12, γ1–2 . Some do12, γ1–2 ,129 thatar genes . Adhesioar genes . CD36 isar genes  and IE b of CD36 , but pla of CD36 . Thus, tGroup A PfEMP1s with head structures containing a CIDRα1.1 or 1.4–1.8 domain bind to EPCR and this includes conserved domain cassette 13  and domain cassette 8  . EPCR isPost-mortem immunohistochemical studies of cerebral malaria patients first showed ICAM-1 mediated adhesion of IEs to cerebral blood vessels . ICAM-1 var genes containing the ICAM-1-binding motif, that are predicted to induce binding to ICAM-1 and EPCR, is thought to be involved in the pathogenesis of cerebral malaria [var genes containing the ICAM-1-binding motif are upregulated in parasites from patients with cerebral malaria compared to severe anaemia and uncomplicated malaria [var genes predicted to bind to EPCR but not ICAM-1 was associated with severe malaria but not with cerebral malaria or severe malaria anaemia alone, whereas transcription of var genes encoding both ICAM-1 and EPCR binding was associated specifically with cerebral malaria [Expression of  malaria . Transcr malaria ,140 and  malaria . Dual IC malaria . In a st malaria . In a re malaria .var gene is var2csa. Var2csa is highly conserved – the 6 commonly-identified DBL domains and interdomain regions have 58–90% sequence homology across 7 different parasite genomes – and has a unique promotor sequence, UPS E [Var2csa is the dominant var gene transcribed in parasites infecting the placenta [Perhaps the most well-characterized e, UPS E . Var2csaplacenta ,147,148.placenta , via theplacenta , to mediSterile immunity (i.e. absence of infection) is not acquired with natural exposure to malaria, however protective immunity against high-density parasitaemia and clinical disease is acquired with repeated exposure and is lost if exposure is interrupted (reviewed in ). ExposuA pioneering study in the 1960s showed that transfer of immunoglobulin G (IgG) from serum of immune adults to non-immune children with malaria could dramatically reduce blood-stage parasite burden and fever, whereas transfer of non-immune serum and immunoglobulin-depleted serum had a far lesser effect . This stin vitro, likely via CD36 recognition of trypsin-sensitive surface antigens [P. falciparum gametocytes grown in vitro [P. vivax [Little is known about the contribution of the immune system to reduced levels of circulating or sequestering gametocyte IEs. Stage 1 gametocyte IEs are phagocytosed by monocytes and macrophages antigens . Naturalantigens  however antigens  and the antigens  and are in vitro  or in inin vitro . Early sP. vivax  althoughP. vivax . GametocP. vivax .Plasmodium in infected hepatocytes [CD8+ T cells are important in defence against pre-erythrocytic stage parasites . Individatocytes .P. falciparum CSP are naturally acquired with age [P. vivax CSP are acquired following controlled human malaria infection and are sustained in the absence of re-infection [P. falciparum or P. vivax CSP have been associated with protection from clinical malaria in some studies but not others [CSP is the major antigen coating sporozoites prior to invasion of hepatocytes. Antibodies to with age , albeit with age , inhibitwith age ,169. Antnfection . Sterilet others ,171–173.t others , and endt others . RTS,S/At others  but may t others . The R21t others . In an at others . The ongAdditionally, live-attenuated sporozoites or live sporozoites administered simultaneously with chemoprophylaxis can induce sterile protection from infection against homologous controlled human malaria infection (CHMI) challenge . The vacDuring the blood stage of infection, monocytes, neutrophils and dendritic cells recognize excess GPI on the merozoite via Toll like receptors , as well as haemozoin and parasite DNA released from the ruptured RBC. This initiates cell killing mechanisms such as phagocytosis and the production of proinflammatory cytokines that activate NK cells and T cells . The innP. falciparum merozoites function to block erythrocyte invasion ligands [P. falciparum glutamate-rich proteins (GLURPs), MSP1, MSP2, MSP3, MSP7, RH5, RIPR, and EBA175 have been associated with protection from experiencing an episode of symptomatic infection or protection from symptomatic compared to asymptomatic infection [Antibodies to  ligands ,188 as w ligands . Studies ligands , althoug ligands ,194. Antnfection . Severalnfection , Rh5 [20nfection , and comnfection . Of notenfection .P. vivax merozoite antigens can be used to predict recent infection [However, other studies have reported a lack of association between antibody responses to merozoite antigens and protective immunity, including in regards to MSP1 or AMA1 , or havenfection ,211. Hownfection .Innate immune cell scavenger receptors recognize surface markers on the IE membrane, such as lipid phosphatidylserines that are usually confined to the inner-erythrocyte membrane , increasPfEMP1 is the dominant IE VSA targeted by antibodies, since parasites that do not express PfEMP1 have markedly lower antibody recognition by immune sera . Severalin vitro and a lack of anti-GARP antibodies has been associated with risk of severe malaria in one study [Antibodies targeting trophozoite and schizont stage IEs have multiple functions, including inhibiting rosetting ; blockinne study . In the ne study . Antibodne study ,232 althne study . Unlike ne study , or the ne study . In a rene study .P. falciparum infection and the clear associated of particular var genes or PfEMP1 receptor binding phenotypes with severe cerebral and placental malaria, several studies have hypothesized that antibodies targeting virulent PfEMP1 types may protect from severe malaria. Antibodies to severe forms of malaria are acquired early in life in high transmission settings and severe infections are most common in children under 5 [var genes than other var genes or may reflect preferential expression of group A var genes in the naïve human host compared to hosts with pre-existing immunity, as shown in a CHMI study [Due to the key pathogenic role of PfEMP1 in mediating sequestration and rosetting during blood-stage  under 5 . In line under 5 ,237, alt under 5 . PrefereMI study .In high transmission settings, women in their first pregnancy and men have low levels of VAR2CSA-specific antibodies and women acquire VAR2CSA-specific antibodies in a gravidity-dependent manner . Althougin vitro [The relationship between antibodies to pregnancy-specific IE and low birthweight is complex. Antibodies to VAR2CSA-expressing IE have been associated with reduced risk of low birthweight in some studies , but antin vitro  and it win vitro . While tIE that adhere to CD36 have generally been associated with infections causing uncomplicated rather than severe malaria and antibodies targeting CD36-binding CIDRα among other domains, are predictive of a reduced prospective risk of clinical malaria and severe malaria in some studies ,251. MalIn line with a role of EPCR binding CIDRα1 domains in severe malaria, antibodies against these domains or downstream domains from EPCR binding PfEMP1, are significantly elevated after an episode of severe malaria ,252 and in vitro [Antibodies against the DBLβ3 domains with the Lennartz motif have been associated with reduced risk of uncomplicated malaria ,254 and in vitro , furtherin vitro . Interesin vitro . This suAntibodies raised against recombinant DBLβ1 domains of rosetting parasite lines are able to bind and inhibit rosette formation ,261 and Plasmodium-derived toxins that are released into circulation during cell lysis, including glycosylphosphatidylinositol (GPI), haemozoin, and cell-free DNA and RNA. Toxins are recognized by pattern recognition receptors, including TLR4 on the surface of innate immune cells, TLR7, TLR8, and TLR9 on endosomes and cytosolic cGAS and AIM2 to induce production of a type 1 interferon response and pro-inflammatory cytokines . Duringiewed in ,264. Maliewed in ,265. Cytiewed in . Interesnfection , suggestin vitro [in vitro [in vivo. In a study of children with cerebral malaria, there were no differences in peripheral cytokine levels of children with severe brain swelling compared to no severe brain swelling [Alongside the importance of type 1 interferons and pro-inflammatory cytokines/chemokines in mounting an effective immune response against liver and blood-stage malaria, several studies have associated a dysregulated pro-inflammatory cytokine response with severe malaria, cerebral malaria, and death ,267–269.in vitro  and circin vitro ,265. Endin vitro  that mayswelling . This maswelling .Malaria also disrupts the balance of pro-inflammatory and anti-inflammatory cytokines required for a healthy pregnancy and several maternal cytokines have been associated with poor pregnancy outcomes including preterm birth, small for gestational age babies, and pregnancy loss . Placentcerebral malaria. Histologically, it is characterized by the sequestration of IE in the brain, typically in the capillaries, as well as pre-capillary arterioles and post-capillary venules. In the majority of cases, sequestration is accompanied by microvasculature pathologies, particularly haemorrhages in the brain white matter, thrombosis and accumulation of monocytes [Unarousable coma from malaria is known as onocytes .Cerebral malaria in children with documented malaria infection is clinically diagnosed by the Blantyre coma score, based on motor, verbal and eye movement responses. This diagnostic method overestimates cerebral malaria cases by up to 30% in a high transmission settings and up to 54% in low or moderate transmission settings ,279. TheIncreased brain volume due to swelling in the brain white matter  is commoThe current understanding of the pathogenesis of cerebral malaria  centres in vitro, human brain endothelial cells can phagocytose dual ICAM-1-EPCR binding IE in an ICAM-1 dependent manner, and parasites engulfed in brain endothelial cells were observed in ex vivo samples from two cerebral malaria patients [Several factors have been proposed to contribute to activation and impaired integrity of brain blood vessel endothelial cells. Sequestration of IEs in the brain microvasculature is strongly associated with coma and it has been hypothesized that microvascular congestion due to sequestration of IEs, platelet mediated clumping or rosetting causes hypoperfusion and ischaemic damage to the endothelium . IE, parpatients , highligP. berghei ANKA mouse model of experimental cerebral malaria (ECM), CD3+CD8+ T cells in the brain vasculature are considered key drivers of intense inflammation and disruption of the blood–brain barrier [In addition to IE, several parasite factors are thought to contribute to endothelial cell activation, including haemozoin and products bound to haemozoin , HRP2 2, histone barrier . Recent  barrier , althoug barrier , therefoIn response to IEs, parasite factors and inflammatory mediators, the activated endothelium expresses local inflammatory cytokines to perpetuate inflammation and dysregulates factors that mediate endothelial cell growth and integrity, including angiopoietin 2 and vascular endothelial growth factor . The actIn summary, multiple factors contribute to endothelial cell activation, breakdown, and impaired blood–brain barrier integrity, and there is strong evidence for a role of sequestration and cytoadherence, particularly of dual EPCR-ICAM-1 binding IE. Further studies are needed to understand the relative importance of these factors and the mechanistic links to clinical outcomes of cerebral malaria, including coma, severe brain swelling that leads to death, and long-term neurological sequelae.Severe anaemia, defined as haemoglobin <7 g/dL in adults and <5 g/dL in children, is a common cause of death in Africa , particuSeveral studies have suggested a key role of systemic inflammation in the pathogenesis of severe malaria anaemia. The release of parasite factors during IE rupture can induce complement deposition on nearby non-IEs leading to the latter’s phagocytic clearance . AdditioIn vitro, IEs directly alter transcription of globin mRNA [IEs and secreted parasite factors may contribute to severe anaemia by the disruption of erythropoietic processes. bin mRNA . Secretibin mRNA  or indirbin mRNA . Additiobin mRNA ,318 and bin mRNA ,320 and bin mRNA .Delayed haemolytic anaemia following artemisinin treatment was first observed following infection in non-immune travellers  and is tP. falciparum malaria in both adults [P. vivax [P. knowlesi [P. malariae [The WHO defines Acute Kidney Injury  as impaired glomerular filtration, based on serum creatine of >3 mg/dL or blood urea >20 mM . Howeverh adults  and chilh adults , includih adults . AKI hasP. vivax ,333, P. malariae . AKI is malariae ,335 and P. falciparum, P. vivax and P. knowlesi) and haemozoin have been observed in the glomerular capillaries of some individuals with AKI [P. berghei ANKA) treatment with Angiopoietin 2 or Angiopoietin 2 receptor prevented pro-inflammatory cytokine production and mouse AKI [Multiple molecular mechanisms of AKI in malaria have been proposed. Renal ischaemia can directly cause tubular damage or necrosis, and it may be brought about by excessive fluid loss or impaired glomerular blood flow due to parasite obstruction. Histologically, IEs  due to foetal growth restriction or preterm delivery [P. vivax malaria in pregnancy is less well studied but has also been associated with preterm birth and low birthweight [P. falciparum and P. vivax malaria in the first trimester are associated with increased risk of miscarriage, and malaria during pregnancy is associated with increased risk of in utero still birth [Malaria in pregnancy causes adverse health outcomes for both mother and baby. It is strongly associated with an increased risk of maternal anaemia and delivery . Approxidelivery . P. vivathweight ,342. Botll birth ,344.P. falciparum placental malaria occurs when IE cytoadhere in the intervillous space of the placenta via VAR2CSA [P. vivax in pregnancy but seem to be uncommon [P. falciparum and P. vivax during pregnancy, including syncytial knotting, fibrin deposits, increased placental barrier thickness, and presence of mononuclear cells [ VAR2CSA . There a VAR2CSA . Placent VAR2CSA . Placentuncommon ,348. Howar cells ,349–352.P. falciparum infection can lead to placental intervillous infiltrates of monocytes [P. falciparum [P. vivax malaria in pregnancy, cytokines are also skewed towards a proinflammatory response although further studies are needed to determine the associations with poor pregnancy outcomes [There are multiple proposed mechanisms by which parasites in peripheral or placental blood may contribute to poor birth outcomes and inflammation is thought to be a major contributor. onocytes , T cellsonocytes , and polonocytes . If inteonocytes ,356, paronocytes . The rellciparum ,360, altlciparum . In P. voutcomes ,363. Plaoutcomes . A clearP. falciparum Kelch13 protein [in vitro [Treatment of malaria is continuously challenged by the emergence of drug resistant parasites. Delayed parasite clearance with artemisinins, the frontline anti-malarial drugs, has emerged in South East Asia  and has  protein . To curb protein . ACT par protein  and like protein  and Kelc protein . However protein  and high protein . Reduced protein ; therefo protein . In Afri protein  and subtin vitro .Cerebral malaria is treated with parenteral artesunate  and by mP. vivax can be treated with chloroquine or an ACT. Where chloroquine resistance occurs, an ACT is recommended [P. vivax and P. ovale hypnozoites and prevent relapse [P. falciparum to reduce gametocyte burden and transmission [In areas where it is chloroquine sensitive, ommended . In both relapse . Single smission . Individsmission . The recsmission , also insmission ,390 and smission .P. falciparum RDTs, and to improve detection of low-density infections. In pre-elimination settings, serology holds promise, especially for detection of recent exposures to P. vivax. Better, more widely available point of care G6PD testing is critical to the widespread implementation of primaquine or tafenoquine to eradicate P. vivax hypnozoites  could protect up to three times as many children from severe malaria and risk of dying each year. Increasing coverage with intermittent preventive treatment in pregnancy and determining the most effective drug combination will decrease the burden of placental malaria and associated low birthweight and neonatal and infant mortality.New drugs and smarter ways of administering existing drugs will be key to combating the rise of drug resistance. ACT with 3 drugs offers hope in the short term, while new classes of agents are developed. Controlled human malaria infections are a powerful tool to speed up the development of new drugs, vaccines and alternative therapies such as monoclonal antibodies, which could provide sustained protection against infection .Aotus nancymaae monkey model of placental malaria caused by P. falciparum, however, does recapitulate many of the features of human placental malaria [Models of sequestration using organoids  or engin malaria .P. vivax elimination has been the lack of robust experimental challenge and in vitro culture systems. The liver humanized mouse model shows significant promise as a tool to study liver stage infection and development of hypnozoites [P. vivax CHMI has been developed [P. vivax cultures [P. knowlesi in risk groups in South East Asia remains a significant concern [A major difficulty with nozoites , and a Peveloped . More recultures . Other ccultures . Meanwhi concern .The lack of progress in reducing global malaria deaths in recent years highlights the problems we face in controlling this disease. While better deployment of existing tools can make a significant impact we still lack the powerful tools, such as highly effective vaccines, that could significantly accelerate progress towards eradication.


Malaria has been identified as a significant public health burden, exhibiting a high risk of death and morbidity. In sub-Saharan Africa, most young children attending primary healthcare facilities are commonly diagnosed with malaria. Thus, introduction of malaria rapid diagnostic test (mRDT) kits and effective antimalarials has substantially improved the management of malaria cases. However, healthcare worker confidence and adherence to procedures dependent on malaria test results remain variable in high-burden settings due to lacking alternative point-of-care tests to diagnose other causes of fever. In this study, we compared the results of malaria screenings using mRDT and microscopy in febrile children presenting at a primary health facility.Plasmodium falciparum histidine-rich protein-2 mRDT kits. Blood samples were collected on slides for microscopy and in hematocrit tubes for hematocrit determination simultaneously, whereas the mRDT test was done by routine health staff. Children found positive for malaria via mRDT were diagnosed as uncomplicated malaria cases and treated as outpatients using artemether-lumefantrine. Blood slides were read independently by two trained microscopists blinded to the mRDT results. The parasite densities were defined as average counts by both microscopists. We then assessed the sensitivity, specificity, and predictive value of mRDT for the diagnosis of malaria.This study was conducted at a primary health center in Owo, Ondo State, Nigeria. Children with fever were assessed for malaria by health staff and, where indicated, screened using We compared the test results of 250 febrile children who are under 15 years old. The test positivity rates were 93.6% (234/250) and 97.2% (243/250) using microscopy and rapid RDTs, respectively. The sensitivity and specificity of mRDT compared to microscopy were 100.0% and 43.8%, respectively, with a positive predictive value of 96.3% (95% CI 93.1–98.3). The hematocrit value was <30% in 64% of the children.As per our findings, mRDTs have correctly detected infections in febrile children. Healthcare workers and caregivers should be encouraged to act in accordance with the test results by means of regular feedback on the quality of mRDTs in use in malaria case management. Plasmodium falciparum, remains a significant public health challenge worldwide, with an estimated 229 million cases in 2019 in 87 countries where it is endemic [Malaria, known to be caused by  endemic . The Wor endemic , 5.The differential impact of these interventions contributed to changes in the epidemiological profiles of malaria risk and transmission , 7. SomeThe current protocols for malaria case management require screening for infection before treatment in cases with associated symptoms such as a history of fever . MalariaThus, in this study, we compared the utility of malaria RDTs in the diagnosis of malaria in children under 15 years old presenting with fever at a primary healthcare facility in an area with perennial and high malaria transmission intensity.Plasmodium falciparum histidine-rich protein 2 (HRP-2)-based mRDT kit or microscopy, followed by treatment with artemisinin-based combination therapy if the test returns positive [This cross-sectional study was conducted from August to October 2020 at the Oke-Mapo Primary Health Center in Owo, Ondo State, Southwestern Nigeria. The town is located 305 m above sea level and has a population of about 222,000. The clinic is generally focused on the treatment of common childhood infections with referrals for more serious conditions to the district and federal hospitals in Owo. Malaria treatment guidelines in Nigeria recommend screening individuals presenting with a history of fever in the past 48 h or having a measured temperature of >37.5°C for malaria using a positive . The mRDInformed consent were provided by the caregivers for children aged less than 15 years where the decision to screen for malaria was made by the attending healthcare worker. Once permission was given, we documented the child’s age, gender, and complaints on presentation. In addition to the blood sample collected for the mRDT test, we collected three additional drops of blood on a slide for later malaria screening via microscopy. The slides were labeled with the participant’s initials and screening number.The blood smear was dried at room temperature and stained with 3% Giemsa stain for 30 min and was thereafter rinsed and dried. The stained slides were independently examined under the microscope by two laboratory scientists, and up to 100 high-power fields were viewed before the sample was declared negative. When a parasite was seen, quantification was done by counting parasites in relation to 200 white blood cells and assuming an average white cell count of 8000 cells/μL, following published protocols . For theTo determine the sample size for this study, we assumed a 5% discordance in results between microscopy  and mRDTData collected on paper forms were entered in an access database and were verified against the source document. We presented a description of the study population and the association between malaria covariates, including age and gender, assessed using a regression model. We also determined the sensitivity, specificity, and predictive values of mRDT compared to microscopy.P=0.84), with 63.4% (159/250) of children having PCV < 30%.We screened 250 children with a history of fever for malaria using both mRDTs and microscopy. Of those enrolled, 46.0% 115/250) were female (Table 15/250 wep = 0.349) than those younger than 5 years.From the mRDT results, 97.2% (243/250) were found to be positive for malaria and were treated with artemether-lumefantrine; 53.1% (129/243) were children younger than 5 years. Children over 5 years were twice more likely to have had a positive test , although mean parasite densities were not different between these age groups after adjusting for sex and PCV level .Microscopy screening showed that 93.6% (234/250) were positive, with a mean (SD) parasite density of 2446 (2634))/μL. Children over 5 years old were more likely to have a positive slide result . The sensitivity and specificity of mRDT relative to microscopy were 100.0% and 43.8%, respectively  initiative to scale up diagnostic testing, treatment, and surveillance for malaria in endemic countries. This program was developed to ensure that all malaria patients receive prompt treatment. Furthermore, T3 was developed to improve surveillance for malaria cases and deaths and help Ministries of Health to determine which areas or population groups are most affected and help target resources where they are most needed. However, the use of malaria diagnostics remains to be limited, and opinions are divided on its utility [Malaria diagnosis is available in most primary healthcare settings because of the ease of use and the stability of RDT cassettes, across a range of temperature and humidity. Results are also easy to interpret. In 2012, the WHO launched the  utility , 17.PfHPR2 mRDT and microscopy. Children with positive mRDT results were diagnosed as having uncomplicated malaria cases and were treated by routine healthcare workers with artemether-lumefantrine and discharged as outpatients following the national treatment guideline. The test positivity rate for malaria using microscopy was 93.6%, with slightly higher rates in children over 5 years old (95.7%. 111/116). Older children also had higher mean parasite densities than those who were under 5 years old. The sensitivity of the mRDT was 100%, with a positive predictive value of 96.3%.In this study, children presenting with fever in the past 48 h or having a measured temperature of >37.5°C were screened using routine Furthermore, approximately 64% of the children examined were moderately anemic with a hematocrit <30% . MalariaJegede et al. showed that an integrated intervention involving RDTs and Artemisinin-based combination therapy (ACTs) by community health workers (CHWs) in Burkina Faso, Nigeria, and Uganda was feasible and acceptable to their communities ; howeverThis study did not investigate other causes of fever in the participating children and there was no follow-up of treated participants to determine outcome after treatment.Malaria remains to be a significant cause of clinic presentations for febrile illness in children under 15 years old. Malaria RDTs are very sensitive and are determined to correctly predict infection in children. Healthcare workers and caregivers should be encouraged to adhere to test results in the case management of malaria in highly endemic settings.


Plasmodium knowlesi causes zoonotic malaria across Southeast Asia. First-line diagnostic microscopy cannot reliably differentiate P. knowlesi from other human malaria species. Rapid diagnostic tests (RDTs) designed for P. falciparum and P. vivax are used routinely in P. knowlesi co-endemic areas despite potential cross-reactivity for species-specific antibody targets.P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH.Ten RDTs were evaluated: nine to detect clinical P. knowlesi mono-infections. Median parasitaemia was 788/µL . Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6–61.5) (SD BIOLINE) to 87.0% (95% CI 75.1–94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of six highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.Samples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. No false-positive results were observed in either P. falciparum-pLDH or histidine-rich-protein-2 channels.For cultured P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria.Selected RDTs demonstrate sufficient performance for detection of major human malaria species including  Plasmodium knowlesi, known to infect humans across most of Southeast Asia, poses significant challenges to regional malaria control programs and World Health Organization (WHO) human malaria elimination progress  for malaria includes seven tests that can detect the genus-conserved parasite lactate dehydrogenase (pLDH) antigen  as an alternative to microscopy . Althoug antigen . Howeverf 200/µL . McCutch species . Howeverfections .P. falciparum-specific histidine-rich protein 2 (HRP2) commercially sourced between endemic countries. For example, Malaysia uses microscopy as the first-line detection tool, whereas a variety of different malaria RDTs are primarily used within certain areas of other countries such as Indonesia  was tested only on clinical samples, and another (Meriscreen OneStep™) on only laboratory samples.The study included all seven WHO-prequalified malaria RDTs that target pan-pLDH . Based oselected Table 1.2), accepting referrals from 16 primary health clinics, including all cases of malaria for in-patient management. Patient blood samples were collected and demographic and clinical data were recorded on standardized case record forms as part of an ongoing prospective observational malaria study. Patients presenting to the hospital study site were included if they were positive for P. knowlesi by microscopy, were more than 1 year of age, and provided appropriate written informed consent. Malaria-negative individuals presenting with or without a fever at the same healthcare facility were also prospectively enrolled as controls. The study was approved by the Medical Research and Ethics Committee of the Ministry of Health, Malaysia (NMRR-10-584-6684) and Menzies School of Health Research, Australia (HREC 10-1431).Patients were enrolled from Ranau District Hospital in Sabah, Malaysia, a secondary referral center servicing the administrative district (area of 3609 kmP. knowlesi parasitaemia (parasites per microlitre) was conducted by an experienced research microscopist equivalent to WHO Level 1 competency; parasitaemia was calculated from the number of parasites per 200 white blood cells on thick blood film, multiplied by the individual patient’s total white cell count  according to the manufacturer’s manual, with a final elution volume of 200 μL. PCR detection of Plasmodium species was performed by laboratory research members blinded to the microscopy results. A real-time PCR assay, QuantiFast™ targeting the 18S SSU rRNA gene was conducted once for each sample in accordance with the manufacturers’ and Sabah Public Health Laboratory protocols, using the Bio-Rad CFX96 Touch™ PCR machine . QuantiFast™ real-time PCR was carried out over two separate reactions, with the first reaction for Plasmodium genus screening, followed by subsequent P. knowlesi and other human Plasmodium species-specific detection if positive  and P. falciparum (Pf3D7) parasites were maintained in vitro as described previously (Plasmodium parasites) and positive controls  were supplied by FIND, the global alliance for diagnostics  in this cohort. A total of 202 microscopically determined P. knowlesi clinical cases and 26 malaria-negative controls who were able to be approached by the research team and gave appropriate consent were prospectively enrolled for RDT testing.From July 2020 to August 2021, 1,698 febrile patients presented to the hospital site and were screened by microscopy, with 508 identified as P. knowlesi-positive samples with RDTs tested, 127 (63%) were collected from patients prior to anti-malarial treatment and were included in the primary analysis. The median age of patients with P. knowlesi malaria was 34 years (range 4 – 87 years). PCR-confirmed negative controls had a median age of 34 years (range 12 – 78 years) and included 21 (81%) febrile patients. Most malaria patients and controls were male . There were no statistically significant differences in age or sex between malaria cases and negative controls across the RDTs conducted.Of the P. knowlesi infected patients was 734 parasites/µL (95% CI 563 – 956), with a range of 18 – 41,883 parasites/µL. Of 202 P. knowlesi enrolled patients, 55 (27%) had a parasite count of less than 200/µL, which included 26 (47%) with samples collected prior to treatment. Three patients were classified with severe P. knowlesi malaria using WHO research criteria  and lowest detected parasitaemia of 50/µL for both , the highest for detecting P. knowlesi among all RDTs tested. The Biocredit™ RDT also had the lowest recorded parasite counts of 32 parasites/µL and 40 parasites/µL for clinical samples tested either before or after antimalarial treatment, respectively  and lowest detected clinical parasitaemia of 50 parasites/µL. The median parasitaemia of patients with a false-negative Pv-pLDH result was 83 parasites/µL (IQR 41 – 203), with the highest false-negative result for Biocredit™ recorded at 251 parasites/µL and for FalciVax at 422 parasites/µL.The ectively Table 3.Pf-pLDH component for which no cross-reactivity was observed for any of the P. knowlesi clinical samples tested. Of the seven RDTs which contain a Pf-HRP2 component, a single false-positive result was recorded for the FalciVax RDT at a P. knowlesi count of 31 parasites/µL , First Response  and FalciVax .Amongst the RDTs tested, only the Biocredit™ RDT included a sites/µL Table 2.P. knowlesi-infected patients whose samples were collected prior to treatment, 26 (20%) had a parasite count less than 200/µL, with a median parasitaemia of 88 parasites/µL (IQR 50 – 133) (Pv-pLDH from the Biocredit™ RDT with a sensitivity of 73.7% (95% CI 48.8 to 90.9%), followed by the FalciVax Pv-pLDH test at 50%. The pan-pLDH tests performed poorly at these low parasite counts, with the SD Bioline unable to detect a single P. knowlesi-positive sample and both the CareStart™ PAN and First Response® tests demonstrating a low sensitivity of 42.9% (95% CI 9.9 - 81.6%); Of P. knowlesi-infected participants, with a median parasitaemia of 1,245/µL . Both pan-pLDH and Pv-pLDH targets demonstrated excellent utility for P. knowlesi detection, with three RDTs exceeding the predefined sensitivity threshold of 95%  and 95.8% (95% CI 88.3 – 99.1%) respectively, in addition to the pan-pLDH of Standard Q at 95.7% sensitivity (95% CI 87.8 – 99.1%). Comparable performance was seen for the pan-pLDH of the CareStart™ PAN and First Response® RDTs with a sensitivity of 93.6% for both. Performance further improved for the 74 (58.3%) P. knowlesi malaria cases with parasitaemia of more than 500 parasites/µL, with the same five highest performing RDTs all recording 100% sensitivity. The SD Bioline pan-pLDH test also performed poorest in this parasite count group with a sensitivity of 62.0%.The parasite count group of 200 parasites/µL and above comprised of 101 (80%) d of 95%  at whichd of 95% Figure 1P. knowlesi infections collected prior to antimalarial treatment. The mean proportions of early trophozoite (rings), late trophozoite and schizont asexual life-stages in a single infection were 1.9% (95% CI 0.8 – 2.9), 92.8% (95% CI 90.0 – 95.6) and 5.3% (95% CI 2.7 – 8.0) respectively. Asynchronous life-stages were found in 52% (66/127) of P. knowlesi infections, with synchronous late trophozoites and schizonts present in 46% and 2% respectively. The proportion of ring stages inversely correlate with test positivity for CareStart™ combo, CareStart™ PAN and Biocredit™ RDTs ; Pv-pLDH positive test outcome decreases by 0.9% (95% CI 0.8 – 1.0) for CareStart™ combo (p=0.044) and 0.8% (95% CI 0.7 – 1.0) for Biocredit™ (p=0.016) with each percentage increase in mean ring stage proportion. In comparison, in vitro cultures were all asynchronous with geometric mean proportions of rings and late trophozoites of 59.1% (95%CI 49.7 - 70.4) and 37.6% (95%CI 28.8 - 49.0) respectively.Microscopic evaluation of parasite life-stages were carried out on the 127 clinical samples with Pv-pLDH RDT tests exhibited 100% specificity for malaria diagnosis, as demonstrated by the lack of response for all malaria-negative controls in individually tested RDTs. Two false-positives were recorded for Pf-HRP2 antibody, one each by First Response® and Parascreen® respectively  and the Pv-pLDH based RDTs . The other 4 submicroscopic P. knowlesi infections were negative on all RDTs. Inclusion of these submicroscopic P. knowlesi infections in the primary analysis would not have resulted in statistically significant differences in reported RDT sensitivities.Five patients excluded from the primary analysis were initially enrolled as microscopy-negative febrile controls; however, subsequent reference PCR detected . knowlesi-infected patients after administration of weight-based antimalarial treatment with oral artemether-lumefantrine, including 6.5% of whom initially received at least 1 dose of intravenous artesunate. RDTs were conducted a median of 3.3 hours (IQR 1.7 - 7.1 hours) after antimalarial treatment, with no statistically significant difference in time among the evaluated RDTs. Of the seven RDTs with pan-pLDH as the target antigen, two had significantly higher sensitivities when tested on pre- versus post-treatment samples: SD Bioline  and Parascreen®  . Diagnostic sensitivity for all primary target antigens when categorized to parasite count groups of below and above 200/µL was not otherwise affected by whether samples were taken before or after administration of anti-malarial treatment   to moderate  agreement, whereas the Pv-pLDH lines gave weak kappa values of 0.50 (Biocredit™) and 0.57 .All control channels for all RDTs were graded 4 and there were no invalid results recorded. Line intensity gradings positively correlated with increasing p<0.001) Figure 3P. knowlesi and P. falciparum parasites cultured in vitro were detected by all pan-pLDH channels of the RDTs tested. For the detection of PkA1-H.1 via the pan-pLDH channel, the most sensitive RDT was the CareStart™ PAN with a LoD of 25 parasites/µL, followed by CareStart™ combo and First Response® with a LoD of 98 parasites/µL for both , whereas SD Bioline was the least sensitive . All RDTs with HRP2 channels were able to detect Pf3D7 with LoD values of >25 parasites/µL.PkA1-H.1 were also detected by the Pv-pLDH channels of Biocredit™ and FalciVax®. No cross-reactivity occurred in the corresponding P. falciparum-pLDH or HRP2 channels, similar to test results for clinical samples. The LoD of cultured parasites in the Pv-pLDH channels were determined to be 49 parasites/µL for both RDTs, also consistent with the lowest parasitaemia detected from clinical samples  and FalciVax (LoD 12 parasites/µL) RDTs, with no cross-reactivity occurring in the Pv-pLDH channels.Culture-derived  samples Table 3.P. knowlesi. Given the lack of routine microscopy available in many areas of Southeast Asia, the need to develop and validate RDTs for P. knowlesi diagnosis was identified as a key research priority in 2017 by the P. knowlesi WHO Evidence Review Group  and one pan-pLDH based RDT (Standard Q) demonstrating sensitivity above 95%, increasing further to 100% for parasite counts more than 500/μL. These RDTs may have reasonable utility in areas where microscopy is not routinely available, although in endemic regions such as Sabah, Malaysia, symptomatic P. knowlesi infections commonly occur at low parasite densities, with previous large studies reporting around 12% of patients having a parasite count less than 200/μL  and VOM -pLDH (CareStart™) RDTs demonstrated poor sensitivities of less than 42%  and pan-Plasmodium aldolase (ParaHIT) RDTs also demonstrated insufficient sensitivities of 74% and 23%, respectively (®)  . Test reformance  and are formance . This reP. knowlesi LDH antigens with Pv-pLDH antibodies used in two commercially available RDTs tested . P. knowlesi LDH has previously been shown to have high sequence identity (>96%) with the P. vivax ortholog  evaluated in this study did not demonstrate any cross-reactivity with P. knowlesi, despite a Pf -pLDH containing RDT in a previous study demonstrating low sensitivities of up to 29%  of these extremely low-level infections. The small number of patients with submicroscopic infections included in this subgroup analysis limits meaningful interpretation, although the true sensitivity is likely to remain low. These findings are consistent with the lowest reported limit of detection of 25 parasites/µL demonstrated with the CareStart™ PAN RDT, marginally below the microscopic detection threshold of approximately 40 parasites/µL  and Australian Government Research Training Program (RTP) Scholarship at Charles Darwin University. MJG is supported by the Australian Centre for International Agricultural Research, Australian Government (LS-2019-116). The analytical performance analysis part of this work was conducted with funds from the Bill & Melinda Gates Foundation (grant no. OPP1172683).This work was supported by the Australian Centre of Research Excellence in Malaria Elimination, the National Health and Medical Research Council, Australia , the National Institutes of Health, USA (R01AI160457-01 and R01AI160457-02) and Malaysian Ministry of Health (grant number BP00500/117/1002) awarded to GSR. AFT is supported We thank the study participants, the IDSKKS malaria research team , Ranau District Hospital director and clinical/laboratory staff, Dr. Fiona Binzini of Ranau Public Health District (PKD Ranau), Dr. Dyang Ruksuna Md Ruhul, Dr. Wan Mohamad Haziman Bin Wan Taib and Dr. Mohd Fakhrurizal Bin Matdiris as well as the Sabah State Public Health Laboratory. We would like to thank the Director General of Health Malaysia for the permission to publish this article.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.The reviewer MN declared a past co-authorship with the author TW to the handling editor.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malaria has been associated with bacterial co-infections, but the importance of bacterial co-infections in uncomplicated malaria is poorly described. We report a unique case of a 27-year-old female with concomitant Plasmodium falciparum and Bacillus cereus bacteremia who acquired those infections while traveling in Tanzania but became ill only after returning to the United States. Blood parasites screen revealed Plasmodium falciparum and blood cultures obtained at presentation showed Bacillus cereus. Even after completing treatment for malaria, she continued to have abdominal pain and watery diarrhea, which improved only after IV vancomycin. Bacillus cereus bacteremia cases are reported in travelers and immigrants returning from countries where malaria transmission occurs, mainly from sub-Saharan Africa but co-infection with Plasmodium falciparum and Bacillus cereus has not been described in the literature yet. In this case, malaria symptoms resolved after targeted treatment was initiated but persistent diarrhea improved only after appropriate therapy against Bacillus cereus. Persistent watery diarrhea and dehydration in patients with malaria should raise concerns about Bacillus cereus co-infection. Malaria has been associated with bacterial co-infections and in particular with bacteremia . The mosIn individuals with malaria, intestinal translocation of bacteria and increased erythrophagocytosis have been suggested as possible mechanisms for increased susceptibility to bacteremia . The malDue to limited awareness in non-endemic regions, imported malaria in high-income settings can have high mortality and risk of delayed diagnosis . We repoA 27-year-old female presented to the emergency department (ED) in Ohio because of fever, nausea, vomiting and watery diarrhea for three days. Thirteen days before ED presentation she had returned from a 30-day trip to Tanzania. While in Tanzania, she was asymptomatic and stayed in an urban environment. On the day of her trip back to the US, she developed watery diarrhea and nausea. Three days later, she complained of fevers that were occurring multiple times per day, worsening diarrhea, and dehydration. She then sought medical care. She denied having any headache, skin rash, respiratory symptoms or sore throat. While in Tanzania, she reported mosquito bites. She endorsed eating home-cooked meals and drinking bottled water, but she drank tap water a few times while in Tanzania. She denied any sick contact. She was advised to take a prescription medication for malaria prevention, but could not get it before traveling.The patient had no relevant past medical history and was not taking medications. She had received vaccines against yellow fever, tetanus, influenza, COVID-19, hepatitis A and meningococcus. She was born and raised in Tanzania and moved to the US when she was a teenager and mentioned no other recent international travel. This trip was her first trip to Africa since she moved to the US.In the ED, her temperature was 100.9° F, pulse was regular and 120/minute, blood pressure was 149/75, respiration rate was 17/minute and unlabored, and O2 saturation was 99% while breathing ambient air. On physical examination, she appeared uncomfortable. She did not have any jaundice or neck lymphadenopathy. On auscultation, lung auscultation and heart sounds were normal. Her abdomen was tender in the epigastrium, and there was no hepatosplenomegaly. Her neurologic exam was unremarkable.Laboratory test revealed thrombocytopenia (129 k/uL), her hemoglobin was 12.5 g/dL with signs of hemolysis with a lactate dehydrogenase of 314 U/L. Her aspartate aminotransferase (AST) was 31 U/L and alanine aminotransferase (ALT) was 54 U/L. Her sodium level was 132 mEq/L, potassium 3.1 mEq/L, blood urea nitrogen (BUN) was 12 and creatinine 0.76 mg/dL (estimated glomerular filtration rate 110 ml/min). Initial lactate was 2.4 mmol/L and improved to normal after fluid resuscitation. A blood parasite screen was done with a Binax NOW Enzyme Immunoassay (EIA) for Plasmodium spp. and Giemsa stained thick and thin smears were obtained. The tests were confirmatory for P. falciparum with a parasitemia level of 1.9%. Enteric Bacterial Panel by polymerase chain reaction (PCR) of stool specimen was negative for Salmonella, Shiga-like toxin-producing E. coli, Campylobacter, Shigella, enteropathogenic E. coli, enterotoxigenic E. coli, and enteroinvasive E. coli. Ova and parasite examination were negative for amoeba, helminths, or protozoa. Clostridium difficile PCR was negative. On day two of hospital stay blood cultures showed Gram-positive bacilli, confirmed on the next day as a Bacillus cereus species (MALDI-TOF mass spectrometry). Further testing for syphilis, HIV, dengue, Chikungunya and Zika virus were negative.She was treated for uncomplicated malaria with artemether/lumefantrine for three days. Fever resolved by day three at the hospital but diarrhea and dehydration persisted. Repeat blood smear on day three showed parasitemia < 0.1%. Regarding the B. cereus bacteremia, a decision was made to consider this a pathogen and not a contaminant due to the persistent abdominal tenderness and watery diarrhea requiring IV fluid resuscitation daily. After completing the treatment for malaria on day three, she was started on IV vancomycin 1g every 12 hours on the same day. On the second day of vancomycin her diarrhea improved and on day three of vancomycin diarrhea was resolved. She was discharged on the fifth day of hospital stay, with instructions to take levofloxacin 750 mg daily for additional seven days.Bacillus cereus is a facultatively anaerobic, spore-forming Gram-positive bacilli. The ingestion of food contaminated with enterotoxigenic B. cereus or the emetic toxin causes infection . The diaThere is limited information available on the incubation period for Bacillus cereus bacteremia in the literature. According to a review article on Bacillus cereus infections, the incubation period for B. cereus bacteremia can range from a few hours to several days, with a median incubation period of 18-24 hours . HoweverB. cereus bacteremia is rare and reported in the setting of intravenous drug use, central venous lines or mucosal injuries, especially in patients with immunosuppression. The clinical presentation in B. cereus bacteremia can be dramatic, starting with gastrointestinal symptoms and progressing to alteration of consciousness or even infective endocarditis. A 2007 study testing susceptibility of Bacillus species to several antibiotics reported universal resistance to trimethoprim/sulfamethoxazole and beta-lactams . In thatAbout 2,000 cases of malaria are diagnosed in the US annually, commonly in travelers and immigrants returning from countries where malaria transmission occurs . Co-infeIt is unclear why bacteremia is more common in patients with malaria. Theories have been suggested to investigate the impact of malaria on the immune system. The infection alters the production of pro-inflammatory cytokines by dendritic cells and suppresses the oxidative burst capacity of neutrophils, allowing for sustained bacteria replication . DiffereThis case report highlights the importance of considering Bacillus cereus bacteremia co-infection in patients with malaria, particularly in those who present with persistent watery diarrhea and dehydration. A thorough medical history and laboratory workup, including blood cultures and parasitemia levels, can aid in the accurate diagnosis of such cases. It is crucial to routinely obtain these tests from febrile patients returning from malaria-endemic areas, as bacterial co-infections may be contributing to unresolved symptoms. This report contributes to a greater understanding of the relationship between malaria and Bacillus cereus bacteremia, and underscores the need for increased awareness and vigilance in the diagnosis and management of these infections.


Plasmodium falciparum malaria infections in a cohort study of ∼25,000 people living in a rural hypoendemic area of about 179 km2 in a small area of the Chittagong Hill Districts of Bangladesh. Asymptomatic infections were identified by active surveillance; symptomatic clinical cases presented for care. Infections were identified by a positive rapid diagnostic test and/or microscopy. Fifty-three subjects with asymptomatic P. falciparum infection were compared with 572 subjects with symptomatic P. falciparum between mid-October 2009 and mid-October 2012 with regard to seasonality, household location, and extent of spatial clustering. We found increased spatial clustering of symptomatic compared with asymptomatic infections, and the areas of high intensity were only sometimes overlapping. Symptomatic cases had a distinct seasonality, unlike asymptomatic infections, which were detected year-round. In a comparison of 42 symptomatic Plasmodium vivax and 777 symptomatic P. falciparum cases from mid-October 2009 through mid-March 2015, we found substantial spatial overlap in areas with high infection rates, but the areas with the greatest concentration of infection differed. Detection of both symptomatic P. falciparum and symptomatic P. vivax infections was greater during the May-to-October high season, although a greater proportion of P. falciparum cases occurred during the high season compared with P. vivax. These findings reinforce that passive malaria surveillance and treatment of symptomatic cases will not eliminate the asymptomatic reservoirs that occur distinctly in time and space.Mapping asymptomatic malaria infections, which contribute to the transmission reservoir, is important for elimination programs. This analysis compared the spatiotemporal patterns of symptomatic and asymptomatic  Plasmodium falciparum is the predominant species and Plasmodium vivax occurs to a lesser extent.,,–,,,In Bangladesh, malaria is endemic in 13 of 64 districts, with more than 14 million people at risk for infection. The highest rates are in the Chittagong Hill Districts, where In 2009, a cohort study—supported by The Johns Hopkins Malaria Institute, in collaboration with International Center for Diarrheal Disease Research, Bangladesh—began in the Chittagong Hill Districts to understand more fully the epidemiology of malaria in the region.As more countries shift from highly endemic to hypoendemic settings, finding the remaining hotspots for malaria and responding appropriately is imperative to the success of control and elimination programs. Most of the current strategies are based on locating hotspots of symptomatic, clinically presenting cases. In this article, we explore whether this strategy will likely adequately capture asymptomatic or oligosymptomatic infections that had mild enough symptoms not to present to care by understanding the extent to which symptomatic and asymptomatic infections overlap in space and time. If asymptomatic infections are not occurring in the same geographic pattern or at different times of the year, more active surveillance is then necessary to locate and treat them.2 and has approximately 24,000 residents. The methods and structure of the cohort study are explained elsewhere in more detail.This study was conducted in two geographic unions  located north of Bandarban city in the Bandarban District . The areActive surveillance began in mid-October 2009 in Kuhalong Union, and in May 2010 was expanded to include Rajbila Union, and continued through mid-October 2012. Each union was divided into 12 geographic clusters, and one person from each cluster was sampled weekly, rotating between three age groups . In a year, 624 persons in each union were selected randomly for this active surveillance, or 1,248 each year including both unions. During the 3-year period approximately one sixth of the entire population was actively sampled. Each week, 2 of 12 people were also selected for longitudinal follow-up, with three more visits at 3-month intervals. All pregnant women were also sampled for longitudinal malaria detection during pregnancy. All people sampled provided blood for rapid diagnostic testing and microscopy, and were deemed an asymptomatic infection if positive by either method. All those tested were surveyed for symptoms at time of testing, before results were known. Although we labeled those identified in the passive sample as asymptomatic, a proportion of them were oligosymptomatic, or had mild symptoms that were not enough to result in seeking care. For simplicity, all those testing positive in the active survey are termed asymptomatic infections. For this analysis, we combined all actively sampled persons, including the randomly selected (those selected in the weekly random sampling) and the additional women sampled because they were pregnant. The location of survey participant households was mapped  and straP. falciparum infections was conducted for just those randomly selected. This included spatial intensity, seasonality, and clustering analysis.Because of potential biases introduced from oversampling pregnant women, a sensitivity analysis in which all the comparisons between symptomatic and asymptomatic P. falciparum and P. vivax infections, analysis was extended through mid-March 2015.The passive surveillance system also began in mid-October 2009 in Kuhalong Union, and in May 2010 was expanded to include Rajbila Union and still continues. Infections or cases included were diagnosed with malaria based on a positive rapid diagnostic test (RDT) and/or microscopy when patients sought care for suspected malaria, either through the local Bangladesh Rural Advancement Committee clinic or directly through the study doctor or field workers, which visited each community regularly and were available by cell phone. All cases identified by passive surveillance were symptomatic by nature of presenting with symptoms. For comparison with asymptomatic infections, we limited the follow-up to match the period when active surveillance was conducted (mid-October 2009 to mid-October 2012). For comparing symptomatic P. falciparum infections, and a sensitivity of 76.9% and specificity of 100% for P. vivax infections. When compared with microscopy as the gold standard, FalciVax had a sensitivity of 98.2% and a specificity of 97% for P. falciparum infections, and a sensitivity of 90.5% and a specificity of 99.7% for P. vivax infections.During visits for both active and passive surveillance, information related to symptoms of malaria was obtained, and a blood sample was collected for a malaria smear for microscopic examination and an RDT . In a prior study in the Chittagong Hill Tracts of Bangladesh, FalciVax RDT, when compared with real-time polymerase chain reaction (PCR) as the gold standard was found to have a sensitivity of 97.6% and a specificity of 95.8% for Household locations were captured using a global positioning satellite device. The latitude and longitude coordinates were converted to Universal Transverse Mercator zone 46N for analysis using ARCGIS 10.1 .P. falciparum infections, and 2) comparing symptomatic P. falciparum and P. vivax infections.The main aspects of the analysis focused on the spatial intensity, spatial clustering, and potential effects of seasonality 1) comparing asymptomatic and symptomatic ,Spatial intensity (the expected number of infections per square kilometer) was estimated for symptomatic and asymptomatic malaria infections using a non-parametric kernel density approach and was mapped to highlight spatial variation in the concentration of infections by household location.P. falciparum infections and was then stratified by season. Significant differences in spatial clustering were assessed using the Monte Carlo random-labeling approach.Spatial clustering describes how dispersed or tightly compact a set of mapped events are, complementary to spatial intensity. It characterizes spatial variation in the location and concentration of events. The K-function was used to estimate the expected number of infections within various distances of other infections of the same type  to assess spatial clustering.The cross K-function is similar to the K-function, but it assesses the spatial interaction between two sets of event locations by estimating for a range of distances the expected number of one type of event around the second type. The cross K-function assessed the extent of clustering of asymptomatic infections around symptomatic cases. The results were compared under the null hypothesis that asymptomatic and symptomatic infections are spatially independent.P. falciparum and P. vivax infections captured through the passive surveillance system from October 2009 through March 2015 were compared. Because both were incident cases, the spatial odds was used to estimate and visualize the spatial variation in risk of each type of infection. Seasonality was assessed by comparing proportions of infection during the high and low seasons, and a Z-test compared proportions.Using spatial intensity and K-function methodology, the spatial intensity and clustering of symptomatic ,–Data were analyzed using R 3.1.1  and ArcGIS 10.1.P. falciparum infections and one (1.9%) was positive for P. vivax infection by microscopy with no co-infections. Thirty-five of the asymptomatic P. falciparum infections were identified by random sampling and 18 were detected during pregnancy surveillance. The geometric mean parasite density of the 44 microscopy-positive infections was 902 parasites/μL . Of those participants sampled randomly as positive for asymptomatic P. falciparum malaria infection, 68.6% were noted to have fever, headache, fatigue, and/or muscle aches in the prior 2 weeks compared with 17.5% of those who tested negative . Of those who were pregnant, 85.0% of those who tested positive for P. falciparum malaria infection had fever, headache, fatigue, and/or muscle aches in the prior 2 weeks compared with 48.8% in those who tested negative .Of the 3,978 people surveyed by active screening, 3,971 (99.8%) had laboratory information available. Demographics of this group and the cohort study population are shown in P. falciparum mono infections, 35 (4.3%) with P. vivax mono infections, and seven (0.9%) with P. falciparum and P. vivax co-infections. Of the 812 positive cases, 808 (99.5%) were positive by RDT and 655 (80.7%) were positive by microscopy, with 95 (11.7%) not having complete blood films and one (0.1%) not having a documented RDT.In the passive survey between mid-October 2009 and mid-March 2015, there were 812 people with documented malaria by RDT and/or microscopy, 770 (94.8%) with P. falciparum cases, 572 (73.6%) were documented during the same period the asymptomatic infections were detected by active survey (mid-October 2009 to mid-October 2012) and were used as the comparison group for assessing differences between symptomatic and asymptomatic infections. A total of 570 (99.7%) were positive by RDT, whereas 456 (79.7%) were positive by microscopy, and 63 (13.8%) did not have completed blood films. The geometric mean parasite density of the 456 symptomatic microscopy-positive infections was 4,740 parasites/μL .Among passively detected symptomatic P. falciparum infections. For the passive survey, spatial information was available for 39 of the 42 P. vivax infections and 763 of the 777 P. falciparum infections (565 of the 572 from mid-October 2009 to mid-October 2012). Infections without spatial information were dropped from the mapping and spatial intensity aspects of this analysis but were kept for seasonality statistics.Household location data were available for 52 of the 53 asymptomatic P. falciparum infections to symptomatic cases was structured, with areas in darker blue in The spatial odds of asymptomatic There was pronounced seasonality in the symptomatic cases when compared with asymptomatic infections. Symptomatic cases had an extensive peak in June and July, whereas the asymptomatic infections were detected year-round . For pasSupplemental Figure 1).The regions of greater risk intensity differed between asymptomatic and symptomatic infections  and 4B. Supplemental Figure 2) at distances up to between 1 and 2 km. These differences were more evident during the high season  than during the low season .The patterns between symptomatic and asymptomatic infections presented in the maps  and 4B aThe cross K-function  demonstrSupplemental Figure 3). The difference in K-functions in the sensitivity analysis showed greater spatial clustering at borderline significance out to about 2.5 to 4 km compared with 1.5 km seen in the primary analysis year-round and during the high season, and borderline significance during the low season at around 5 to 6 km not seen in the primary analysis . However, the overall conclusions, as earlier, were unchanged.The sensitivity analysis (excluding samples selected due to pregnancy) showed few differences from the primary analysis. There was one area of greater intensity of pregnant woman with asymptomatic malaria infections in the southeastern region of the map that, when excluded, favored high intensity of symptomatic cases , indicate a substantial overlap in the areas of greater intensity of infection, although the areas with the greatest rates differed, with the northwestern corner of the map in northern Rajbila Union having the greatest intensity of P. vivax infection whereas the center of the map in northern central Kuhalong Union had the greatest intensity of P. falciparum infection.Comparisons for nfection . Howeverax cases  and 4D, P. falciparum and P. vivax  showed no significant difference in P. vivax compared with P. falciparum cases overall and during the high season, and mostly no difference but borderline increased clustering of P. vivax compared with P. falciparum cases during the low season up to 1 km.The difference in K-function comparing symptomatic P. falciparum compared with symptomatic P. vivax infections occurred during the high season . However, both increased during the high season, with 661 P. falciparum cases (85%) compared with 26 P. vivax cases (62%) occurring during this time.A greater proportion of symptomatic This study using only rapid diagnostic testing and microscopy demonstrated that asymptomatic infections may not occur at the same time and place as symptomatic infections, differing both in seasonality as well as geographic locations. This possibility is absolutely critical to consider when designing interventions. In prior analyses, we noted that the majority of infections in the area were asymptomatic when comparing passive to active incident data.The study also saw a general decrease in the number of symptomatic infections seen in the later years of the study compared with earlier years. This may very well relate to the large push of national control programs in the area.The factors that contribute to an infection being symptomatic rather than asymptomatic are complex. Environmental, behavioral, and genetic factors may influence exposure, immunity, and access to clinical care for the populations at risk. However, we suspect, in our study, the presumptive ease of access and availability of the study clinician to make house calls meant that these barriers likely did not decrease substantially the number of people who sought care, and the development of symptoms was likely a more important predictor of a clinical disease. It is possible there are different isolates of malaria parasites that have varying degrees of virulence, possibly impacting the development of symptoms. In our study, there was an increase in geometric mean parasite density among the symptomatic compared with asymptomatic infections. More research is needed to characterize the role of virulence, with a focus on those strains that do have the potential to cause clinical disease.P. falciparum and P. vivax infections, there were some areas of overlap in the hotspots. However, the areas with the greatest number of P. vivax infections were not the most common areas for P. falciparum. These differences may be based on the preferred habitats of vector species that carry these infections, although more research is necessary.,With respect to P. falciparum and 76.9% for P. vivax compared with real-time PCR. We added microscopy secondarily to improve sensitivity. We thus are likely to catch the large majority of P. falciparum infections, but may be missing a slightly greater fraction of P. vivax infections.There are several limitations to this analysis. First, the lack of PCR means that a number of sub-microscopic infections could have been missed in the active survey. However, the sensitivity of the FalciVax RDT was estimated at 97.6% for Second, in comparing just the asymptomatic infections and symptomatic cases, we assumed that the sampling in the active study reflects the population in general. Their recruitment does appear similar to the distribution of the general population . AlthougAnother limitation relates to the nature of the active survey data collection. Only one person a week was surveyed in each of the 24 geographic clusters for our prevalence survey. As a result, clusters in the same space and time may have been missed. This issue is mitigated in part by the long period of infection likely for most of those in the active survey.Furthermore, the asymptomatic infections, rather than the proportion testing positive, were compared during this analysis. The infection intensity is reasonably reflective of the intensity of the proportion that was positive, because the population was sampled randomly in the active survey and all pregnant women, regardless of location, were included. Nevertheless, it is possible the sampling design introduced some bias.P. falciparum/P. vivax infections equally, so spatial odds ratios should not be affected.For both passive and active surveillance, the coverage from October 2009 to April 2010 was for only one  of the two unions. Thus, the number of infections picked up was less than during the same period in following years and may have been slightly weighted spatially to Kuhalong. However, this would impact both passive and active studies equally, and impact detection of P. falciparum infections in the area were found to be asymptomatic and persisted year-round. Although the symptomatic and asymptomatic hotspots overlapped, symptomatic hotspots did not predict consistently areas of ongoing asymptomatic transmission, particularly during the low season. It is thus necessary to develop active surveillance for elimination programs.In conclusion, the majority of Supplemental materials


Aedes aegypti or A. albopictus, and malaria, a parasitic disease transmitted by Anopheles gambiae, are prevalent in Nigeria and neighbouring countries, but their burden and possible coinfections are poorly understood. In this study, we investigated the seroprevalence, hidden burden and endemicity of chikungunya and malaria in three regions in Nigeria. Methods: A cross-sectional sero-survey was conducted on 871 participants in three regions of Nigeria. The samples were collected from outpatients employing simple random sampling. All serum sample analyses were performed using CHIKV virus-like particle recomLine Tropical Fever for the presence of arboviral antibody serological marker IgG immunoblot for chikungunya and malaria RDT (Rapid Diagnostic Test) for malaria parasites. Results: The seroprevalences of chikungunya and malaria mono-infection were 64.9% and 27.7%, respectively, while the coinfection seroprevalence was 71.9%. The central (69.5%) and northern (67.0%) regions showed more significant seroprevalences than the southern region (48.0%). The seroprevalence and the hidden burden of chikungunya and malaria infections varied across the three geographical regions. Conclusions: This study highlighted an unexpectedly high seroprevalence and hidden endemicity of chikungunya and a less surprising high malaria endemicity in three regions of Nigeria.Background: Mosquito-borne infections are of global health concern because of their rapid spread and upsurge, which creates a risk for coinfections. Chikungunya, an arbovirus disease transmitted by  Aedes. Aedes aegypti and Aedes albopictus are the primary vectors of the chikungunya virus (CHIKV), but it is also maintained in sylvatic cycles involving primates and forest dwelling Aedes species. Chikungunya is one of the most common vector-borne diseases  to identify the sociodemographic and behavioral characteristics of the study population. The results are presented in tables and figures. A Chi-square test of independence and correlations were performed to examine the relationship between the variables and chikungunya–malaria seroprevalence. The results were deemed statistically significant at a The study protocol was reviewed and approved by the local ethics committee on human research at the Universitatsklinikum, Freiburg, [No. 21-1233] and the local ethics committee on human research at the Tertiary Hospitals and national ethics committee on human research of Nigeria [No KF/REC/02/21].p = 0.01). There was a low positive correlation and a statistically significant association between Abia (ABIA), Nasarawa (NAS), and Kaduna (KAD) with respect to chikungunya infection (p = 0.03).A total of 871 participants  were investigated for the seroprevalence of chikungunya and malaria parasites in three Nigeria regions. The mean age of the study population was 36.7 years (range 0 month to 80 years). In the three profile regions, the seroprevalence of chikungunya was 64.9% (95% CI (0.63–0.67)) and that of malaria was 35.1% (95% CI (0.32–0.38)). A significant seroprevalence of chikungunya was observed among participants from the central (Nasarawa) region (69.5% (95% CI (0.68–0.70))) and northern (Kaduna) region (67.0% (95% CI (0.65–0.69))), while a slightly lower seroprevalence of 48.0% (95% CI (0.46–0.50)) was revealed among the participants from the southern (Abia) region . The difp = 0.65). However, there was a negative correlation and a significant association between the regions and malaria infection (p = 0.01).The seroprevalence of malaria was slightly higher in the southern region ) than in the northern ) and central regions ). There was no statistically significant difference between malaria infection and the regions ; however, the difference between the sexes  and chikungunya was not statistically significant (p = 0.82).In the present study, a lower chikungunya seroprevalence of 64.3% (95% CI (0.61–0.67)) was observed among males than among females (65.1% (95% CI (0.62–0.68))). Similarly, malaria seroprevalence was also higher among females ) than males ) . There wp = 0.01).The seroprevalence of chikungunya was highest (70.8% (95% CI (0.69–0.73))) among slum dwellers, compared to 68.8% (95% CI (0.69–0.72)) rural and 61.1% (95% CI (0.59–0.63)) urban dwellers. There was a statistically significant association between place of domicile and chikungunya infection (p = 0.01), with a negative correlation  than in rural  and urban participants . The results showed a significant association between place of residence and malaria (relation .p = 0.14). In contrast, a significant malaria seroprevalence of 33.0% (95% CI (0.22–0.44)) was recorded among pregnant women compared with nonpregnant women ). There was a statistically significant association between pregnancy status and malaria (p = 0.04), with a negative correlation ) was obtained in nonpregnant women than in their pregnant counterparts ). The correlation was negative, and the difference was not statistically significant ) chikungunya–malaria coinfection was observed among the study participants in the three regions. A subgroup analysis of the demographic groupings revealed an overall coinfection seroprevalence of 68.4% among all age groups, which was closely followed by the domicile group (66.9%) and the male and female groups (65.1%). The lowest prevalence was observed among the pregnant and nonpregnant women groups (62.6%). The relationship between these demographic variables and chikungunya–malaria coinfection was statistically significant .Chikungunya and malaria coinfections could result in a significant public health impact and fatal outcomes through aggregates of various factors and inaccurate and misdiagnosis. In the present study, we investigated the seroprevalence and burden of chikungunya hidden endemicity and malaria mono- and coinfection among subpopulation groups in three regions of Nigeria. Our findings showed that the study population has a high seroprevalence and a hidden burden of chikungunya 64.9%) and malaria mono- and coinfection 71.9%) across various sociodemographic groups. The seroprevalence and heterogeneity of infections across the various geographical regions may be explained by differences in vegetation, human population, climate change , vector adaptations, variations in temperatures and humidity, changes in habitats and microclimates, and unplanned urbanisation in the various regions that favour transmission dynamics of the mosquito-borne vectors of these diseases ,16. Thes.9% acros.9% and mChikungunya infection was more prevalent in older age groups than malaria, which was more common in younger age groups . It is aAedes mosquito bites (day feeding activity of Aedes aegypti), while children are more vulnerable to malaria than chikungunya. This age bias has been previously reported [The high seroprevalences among the population could also be attributed to past infection, reinfection or ongoing transmission or increased vector exposure in relation to socioeconomic activities close to mosquito breeding habitats. Furthermore, older people maintain sedentary lifestyles, and because they sit for long periods in unscreened places, they are increasingly exposed to reported  and may Anopheles and Aedes mosquitoes [The seroprevalences of chikungunya and malaria mono- and coinfections in female participants were higher and more discrete than those in their male counterparts. These differences could possibly be due to the number of females recruited in this study compared to males . Additionally, females in Nigeria engage in outdoor activities such as trading, fetching water from ponds and streams, and gathering wood for fuel and farming, just as much as their male counterparts, thereby exposing them to the breeding habitats of squitoes ,10,14.There was a higher seroprevalence of chikungunya and malaria in slums than in rural and urban settlements in the present study. This could be explained by the fact that slums are informal settlements located in urban areas or cities across the three profiled regions ,2,3. As The prevalence of malaria was higher among pregnant women, whereas chikungunya was more prevalent among nonpregnant women. These results could be explained by the differences in immune-physiological state during pregnancy, thus resulting in reduced or weakened immunological responses to mosquito-borne infections ,24, malaIt was evident in the present study that there was a high and widespread seroprevalence of chikungunya–malaria coinfection. These findings could be attributed to the spread of chikungunya in various regions in Nigeria where malaria is endemic and could possibly result in an increase in concurrent infection ,15,16,29The study population is hospital-based; therefore, the seroprevalence could not be a true reflection of what is obtainable in the general Nigerian population as such, and it could be underestimated. Although we employed a novel molecular diagnostic test that is highly specific but not a seroneutralisation test, it still remains a challenge to diagnose chikungunya and other alpha viruses using serological tests due to cross-reactivity, which complicates the interpretation of the results. The interpretation of IgG antibodies may also be complicated  by previous exposure to mosquito-borne arbovirus vaccinations. Furthermore, the obvious clinical presentations of arboviral infection, apart from being subclinical, can mimic other infections, such as bacterial and fungal infections, which are also endemic in Nigeria. Among the participants in the present study, there were more females than males, which may lead to bias and confounding other variables as well as age.We were able to show that there is a high seroprevalence and hidden endemicity and burden of chikungunya and malaria mono- and coinfection in Nigeria. Several intrinsic and extrinsic factors were responsible for these high seroprevalences. It is evident that these two infections could go unnoticed, especially when causing fever, and will end up being treated as other common febrile illnesses or bacterial and fungal infections because of their subclinical presentation. According to our findings, molecular laboratory diagnostics and serosurveillance epidemiological facilities are essential for the accurate and early diagnosis of arboviral mono- and coinfections in Nigeria.


Although China has entered the post-malaria-elimination era, imported cases remain a public health concern in China.We retrospectively analyzed data from cases of imported malaria from January 2017 to December 2020 in Chengdu Public Health Clinical Center. We assessed potential clinical, epidemiological, geographical, and seasonal effects on duration of hospital stay. Cox proportional hazards model was used to identify predictive factors for prolonged hospital stay. Multivariate logistic regression was used to assess the potential risk factors associated with severe cases.Plasmodium falciparum. The Edwards test indicated no significant seasonality in imported cases of malaria . Bacterial infection  and thrombocytopenia  were risk factors for prolonged hospital stay. The C-reactive protein  and procalcitonin  were risk factors for severe cases.The highest number of imported cases of malaria were from the Democratic Republic of the Congo  and most patients  were infected by Bacterial infection and thrombocytopenia are risk factors for prolonged hospital stay among imported malaria cases. The C-reactive protein and procalcitonin level were risk factors for severe cases. Following implementation of the National Malaria Elimination Program (NMEP) in 2010 , China fChengdu City is the provincial capital of Sichuan province, which occupies much of Western China including the eastern Qinghai-Tibet plateau. Chengdu Public Health Clinical Center (CPHCC) is the only tertiary care center for infectious diseases in Chengdu City and provides services for much of Sichuan province. Most imported malaria patients in Sichuan province are hospitalized in CPHCC. In 2019, CPHCC passed the national examinations for the NMEP; requirements include standards for malaria patient management, disease reporting, treatment, diagnosis, laboratory equipment, and staff training. Thus, data from patients with imported malaria in CPHCC may be considered representative of imported malaria in China.Treatments for malaria are effective only if administered in a timely fashion , 8. HoweWe retrospectively analyzed data for imported malaria patients admitted in CPHCC from Jan. 2017 to Dec. 2020. All the patients firstly went to emergency department or fever clinic of CPHCC, many of them referred by other hospitals. A total of 150 cases were enrolled. Demographic and clinical data were collected, including medical history, main complaints, and source country. Laboratory parameter results included routine blood tests, liver function tests, and medical imaging records.Plasmodium parasites. All confirmed cases were included in this study. Patients with suspected malaria who were subsequently diagnosed with other conditions by additional medical investigations were excluded from the study.Malaria was confirmed in all patients by pathologic evidence of Ethical approval for this study was obtained from the Chengdu Public Health Clinical Center Ethics Committee. The requirement for informed consent was waived because of the retrospective nature of the study and the data were anonymous.Malaria diagnosis was based on national recommendations for malaria diagnosis WS 259-2015) [-2015 [9]Plasmodium parasites in the blood by microscopy of blood smears; (2) significant improvement in clinical signs, manifestations, and laboratory examinations; and (3) normal body temperature for more than 3 days.The criteria for discharge were based on the rules of CPHCC as follows: (1) no evidence of Statistical analyses were performed using STATA/SE 14.1 software  and R version 4.0.2 . The SEAp < 0.05 were considered statistically significant.Normally distributed continuous data were presented as means and standard deviations and non-normally distributed continuous data were presented as medians and ranges. Multivariable Cox proportional hazard regression and Multivariate logistic regression with stepwise variable selection were used to find the potential factors associated with prolonged hospital stay and severe cases. The Edwards test for seasonality was used for time series case number data . The logAll patients were Chinese nationals and acquired malaria infection outside China. All patients were providing labor services in Africa or two Asian countries (Myanmar and Pakistan). All were male with a median age of 42 years (range: 19–62 years). All were in good health and had undergone physical examinations before going abroad  reported fever with varying duration . Most patients  reported shivering associated with fever, while roughly half  suffered from headaches. Other signs and symptoms included cough , productive cough , fatigue , anorexia , and muscular soreness .Plasmodium species parasites twice or more, and 68% of patients had been infected at least once. Of note, 17% of patients (25/150) had been misdiagnosed at other medical facilities. Because the clinical signs of malaria are non-specific, physicians in non-endemic areas have limited experience with this diagnosis. The DOF ranged from 0.2 to 30 days with a median of 3 days.The Hospital Information System (HIS) recorded details of each patient’s medical history as well as onset of malaria. Half of all patients  had been infected by P. vivax infection and three patients who returned from Pakistan with P. vivax infection, all cases originated in Africa. Most patients were infected  or co-infected  by P. falciparum. The second most common Plasmodium species was P. vivax, infecting and co-infecting 16% (24/150) and 3% (4/150) of patients, respectively.Except for one patient who returned from Myanmar with P. falciparum. The second-highest number of imported cases of malaria originated in Equatorial Guinea ; 81% (17/21) of these patients were infected by P. falciparum ; approximately three-quarters of these patients  were infected by %, 34/150Cases of imported malaria over the four years of our study were plotted Fig. . No sign2 = 2.51, p = 0.28), there appeared to be no significant seasonality in imported malaria cases at CPHCC   and 0.66 , respectively). The results are shown in Table Using stepwise variable selection, bacterial infection and thrombocytopenia were statistically significant  and thus protective against prolonged LOS, because the antimalarial agent was ACTs normally. The ACTs have shorter course of treatments.2 values 8.0 and 8.16, respectively; both p < 0.001)  Figs.  and 4.p = 0.00). To identify the potential factors associated with severe cases, the multivariate logistic regression with stepwise variable selection was used. The independent variables were same with Cox proportional hazard model (previous section). The multivariate logistic regression revealed that concentration of CRP was a risk factor for severe cases . The PCT was identified as a risk factor for severe cases as well . Five patients in the nine severe cases had complication of cerebral malaria.Nine cases  were severe cases in this study. The median LOS was 10 days (ranged 6–14 days) which was significantly longer than other cases  . The antMalaria prevention efforts should target laborers working abroad given the increasing number of workers returning from overseas . A majorHowever, misdiagnosis and long DOF (> 5 days) were not associated with prolonged LOS as shown by Cox proportional hazard modeling.Through Cox proportional hazard modeling, we found the low PLT counts and bacterial infection were predictive factors of prolonged hospital stay. Prolonged hospital stays imply a more serious clinical situation, with longer time required to fulfill the discharge criteria. Patients with longer LOS also consume more medical resources and introduce higher local transmission risks. A previous study also found that PLT count was predictive of the severity of imported malaria . VaradarP. falciparum infection also had bacterial infections [Bacterial infections are common in malaria patients . A systefections . In our fections .P. falciparum disease [The previous studies indicated the CRP was a prognostic factor and a biomarker for malaria infection and monitoring of malaria severity , 22. PCT disease . In our We found no evidence of seasonality in the 150 imported malaria cases admitted to our hospital. However, previous studies revealed a seasonal pattern of malaria incidence in Burundi . AnotherPlasmodium species. However, P. falciparum was the most common pathogen .Understanding the geographical distribution of pathogens is important for clinical diagnosis and treatment. If clinicians can deduce the causative pathogens of infection based on the regions patients have visited without waiting hours for laboratory testing results, treatment may be initiated more rapidly. A review confirmed that rapid treatment initiation reduced the risk of severe disease , 28. In We studied the epidemiological and clinical features of patients with imported malaria, which represent the bulk of malaria infections in the post-elimination era in China. Misdiagnosis at other medical facilities was common, and efforts to improve this situation must be taken. Thrombocytopenia and bacterial infection were predictive factors for prolonged hospital stay. No seasonality was observed for imported malaria cases. The higher PCT and CRP associated with higher risk of severe cases.