Plasmodium. Plasmodium spp. have a worldwide distribution and records of mortality extend across a range of bird orders and species. In wild bird populations, infection is maintained within reservoir hosts, which are able to tolerate a chronic, low level of parasitism generally without severe health consequences. The introduction of these adapted avian species and their malarial parasites to naïve bird populations has resulted in devastating impacts on some non-adapted bird species, with the demise of Hawaiian amakihi as a pertinent example. In New Zealand, avian malaria was first recorded in introduced blackbirds (Turdus merula) in the 1920s, and reports of mortality in endemic and native bird species have been sporadic over the last three decades. Of the five species of kiwi, four are considered at risk or threatened by conservation standards. Here, we aim to describe the pathology of avian malaria as a cause of mortality in kiwi (Apteryx spp.) and the species of Plasmodium involved to further our understanding of this disease in arguably the most iconic of New Zealand’s endemic birds.Avian malaria refers to the parasitic blood infection caused by species of Plasmodium species is a known cause of mortality in avifauna worldwide, however reports within New Zealand kiwi (Apteryx spp.) are scant. Postmortem reports from kiwi were obtained from the Massey University/Te Kunenga ki Pūrehuroa School of Veterinary Science Pathology Register from August 2010–August 2020. Gross lesions were described from postmortem reports, and archived H.E.-stained slides used for histological assessment. Nested PCR testing was performed on formalin-fixed paraffin-embedded tissue samples to assess the presence of Plasmodium spp. and Toxoplasma gondii DNA and cases with a PCR-positive result were sequenced to determine the lineage involved. Of 1005 postmortem reports, 23 cases of confirmed or suspected avian malaria were included in this study. The most consistent gross lesions included splenomegaly, hepatomegaly, and interstitial pneumonia with oedema. Histological lesions were characterised by severe interstitial pneumonia, pulmonary oedema, interstitial myocarditis, hepatic sinusoidal congestion and hypercellularity, and splenic macrophage hyperplasia and hyperaemia/congestion with numerous haemosiderophages. Cytoplasmic meronts were consistently found within endothelial cells of a variety of tissues, and within tissue macrophages of the liver, lung and spleen. A diagnosis of avian malaria was confirmed via PCR testing in 13 cases, with sequencing revealing P. matutinum (LINN1) and P. elongatum (GRW6) as the species involved. This is the largest case series describing the pathology of avian malaria as a cause of mortality in endemic New Zealand avifauna.Avian malaria caused by  Plasmodium and Haemoproteus, with other related genera of veterinary importance including Leucocytozoon. Collectively, these haemosporidian parasites are a significant cause of morbidity and mortality in a range of bird species worldwide. Avian malaria caused by Plasmodium spp. was first identified in blackbirds (Turdus merula) and song thrushes (Turdus philomelos) in New Zealand in the 1920s, with records of mortality events in several native and endemic bird species over the last few decades highlighting it as a potential emerging disease. In New Zealand, at least 17 Plasmodium spp. have been identified, infecting birds from a range of avian orders including native and introduced Psittacines, Passeriformes, Anseriformes and Coraciiformes [Culex mosquito vectors have been found in the North Island [Leucocytozoon in marine penguins [Haemoproteus in endemic avifauna.Avian malaria is a disease caused by apicomplexan haemosporidian parasites of the genera iiformes ,7,8,9,10h Island ,12,13. Oh Island . It shoupenguins ,16, and Plasmodium spp. has an indirect lifecycle, with sexual development (sporogony) occurring within a mosquito vector followed by two asexual stages within the avian host, including merogony (exoerythrocytic and erythrocytic) and gametogony. The outcome of infection is a complex and incompletely understood balance between the degree of parasitism, species and lineage of Plasmodium and the immune response of the avian host [Toxoplasma gondii [Plasmodium is now commonly done by with nested PCR and genetic sequencing [Haemoproteus and Leucocytozoon genera.ian host . In wildian host ,19; in tian host . In liveian host . In fataa gondii ,23, howea gondii  or in sia gondii  help to quencing , which dA. mantelli; NIBK) and little spotted kiwi (A. owenii), although the rowi (A. rowii), great spotted kiwi (A. haastii), and tokoeka  continue to be threatened and have either stable or declining populations [Plasmodium spp. were first reported in blood smears of two captive NIBK chicks at Auckland Zoo in 2000. One chick was treated with antimalarial medications, however both had organisms present in the blood three months later [Plasmodium spp. (LINN1) [Plasmodium (Huffia) elongatum (GRW6) infection [Plasmodium spp. organisms in the blood smears of 25/32 juveniles at the same facility, though testing of numerous other individuals across an additional five facilities failed to find any further cases and no other deaths were reported [Plasmodium spp. lineage GRW6 from Ponui Island [Plasmodium spp. lineage LINN1 from Waimarino Forest [Ongoing conservation efforts have led to improvements in the conservation status of the North Island brown kiwi  . A seconnfection . Subsequreported . Prevalegatum GRW infectioPlasmodium involved to further our understanding of this disease within arguably the most iconic of New Zealand’s endemic birds.The purpose of this study was to describe the lesions of avian malaria in kiwi and identify the species and lineage of Plasmodium spp. infection was made or suspected on the basis of gross and histopathological findings were included in the study. The species, age group, sex, captivity status and location were recorded for each kiwi, along with the year and month that the bird was submitted for postmortem examination. Age was classified as neonatal, juvenile, subadult or adult as recorded by the submitter. ‘Sanctuary’ was used to denote kiwi from outdoor locations with predator proof fencing. ‘Créche’ was used to denote juvenile kiwi being kept in a semi-managed outdoor facility.Postmortem reports from all five species of kiwi were obtained from the Massey University/Te Kunenga ki Pūrehuroa School of Veterinary Science Pathology Register database from between August 2010 to August 2020. Reports in which a diagnosis of avian malaria due to Gross lesions were characterised based on the details included in the postmortem report, which was available for 22/23 cases. The body condition score from each bird was classified as ‘poor’, ‘suboptimal’ or ‘good’, based on the postmortem assessment of visceral and subcutaneous fat stores, pectoral muscle mass and bodyweight, as determined by the reporting pathologist. Histological assessment was carried out on archived Haematoxylin & Eosin (H&E)-stained slides, or slides prepared from archived formalin-fixed paraffin embedded (FFPE) tissues. Archival material and FFPE tissues were available for 21/23 cases, and histology reports were available for the remaining two cases. Selected tissue sections were examined with Perls Prussian blue iron stain or Martius Scarlett Blue fibrin stain to assess the presence of haemosiderin and fibrin thrombi, respectively.Plasmodium spp. on fresh (5) or formalin-fixed (1) tissues, and five of these had FFPE tissues available for repeat testing. One case of suspected avian malaria had no FFPE tissues available or previous test results to confirm the diagnosis.FFPE tissues were selected for PCR analysis based on the presence of both organisms and lesions, where possible. Two kiwi with no gross or histologic evidence of apicomplexan infection were selected as additional negative controls. Six cases had previously tested PCR-positive for ® Blood and Tissue kit, Valencia, CA, USA). Nested PCR was performed for Plasmodium spp. as described by Hellgren et al. [Toxoplasma gondii was performed on the same DNA extracts, using the method described by Aspinall et al. [Pppk-dhps gene. The products from each nested PCR were electrophoresed on 1.5% agarose gel . Results were compared against known positive controls and negative controls. The DNA from each positive amplicon was purified using PureLink PCR purification kit  and sequenced using BigDye Terminator Version 3.1 Ready Analyser . The sequence was examined for presence of overlapping peaks that may indicate co-infection and aligned using Geneious Primer , and compared against published sequences in the National Centre of Biotechnology Information (NCDI) BLAST GenBank database. Representative isolates were submitted to the NCDI database.DNA extraction was performed on 10 um scrolls of FFPE tissues using the manufacturer guidelines for a commercially available kit  with the remainder being little spotted kiwi (5) and great spotted kiwi (2). All age groups were represented, with one neonate, 14 juvenile, four subadult and four adult kiwi. Sex was listed as male in eight kiwi, female in 13 kiwi and unknown in two kiwi. Most cases originated from the North Island (21). Eight were from the wild, six were from sanctuaries, six were from créches and four were from captive sites. All cases were clustered over the late summer to early autumn period with five in the month of February, nine in March, eight in April and one in May .The body condition score was noted as poor (6), suboptimal (7) or good (9) . The mosEleven kiwi were identified as having one or multiple comorbidities. Diagnoses included verminous ventriculitis (10/11), intestinal nematodiasis (4/11), intestinal coccidiosis (2/11), intestinal cestodiasis (3/11) and bacterial enteritis (2/11). Two of these also had lesions consistent with visceral (2) or neural (1) larval migrans. Eight of these kiwi were in suboptimal to poor body condition, four of which were noted to have heavy gastrointestinal parasitism. The three remaining kiwi were in good body condition and were all noted to have only mild gastrointestinal parasitism. The level of parasitism was not specified in the remaining cases.Expansion of the parenchymal blood vasculature was the most prominent feature and was noted in the lung (21/21), spleen (20/23), liver (18/22), kidney (11/19), heart (9/21), adrenal gland (7/20) and brain (3/8). Areas of parenchymal congestion frequently had increased numbers of peripheralized monocytes within the vessel lumina. This was noted in the liver (15/22), lung (11/21), kidney (2/19), adrenal gland (1/10) and brain (1/8). Globular pale tan to brown cytoplasmic pigment within tissue macrophages was a common feature in congested organs and was seen in the spleen (15/23), lung (13/21) and liver (12/22). Perls Prussian Blue iron stain was done on the liver, lung and spleen of four cases showing that the pigment stained dark blue-black. The corresponding H&E sections were examined under polarised light and no birefringence of the pigment was seen. The pigment was interpreted as being consistent with haemosiderin C.In the lung, interstitial inflammation was characterised by marked hypercellularity of the interstitium with moderate numbers of foamy macrophages and smaller numbers of lymphocytes and granulocytes (15/21), along with occasional localised deposits of smooth hypereosinophilic material (fibrin) and severe generalised congestion as previously described. Pulmonary oedema was characterised by pooling of proteinaceous fluid within air spaces and expansion of the interstitium with clear space (5/21) A. In thePlasmodium species were positively identified in the spleen (22/23), lung (20/21), liver (20/22), heart (17/21), kidney (14/19), gastrointestinal tract (6/13) and adrenal gland (6/10). Organisms were not identified in any of the examined brain sections (0/8). Organisms were consistently found within capillary endothelial cells and within Kupffer cells of the liver and macrophages within the spleen  and Plasmodium elongatum (GRW6), including a previously submitted P. elongatum (GRW6) isolate from a North Island brown kiwi (GenBank HQ454000). Representative sequences from the isolates of P. matutinum (LINN1) from a great spotted kiwi and a little spotted kiwi were submitted to GenBank . Of the 21 tested cases, nine were PCR-positive for Plasmodium in this assay, three had previously tested PCR-positive using fresh tissues, including the single kiwi that had concurrently tested PCR-positive for T. gondii. This kiwi tested weakly PCR-positive for T. gondii with the current assay. Four kiwi tested PCR-positive for T. gondii only and sequencing confirmed the presence of T. gondii pppk-dhps gene DNA . Preliminary genotyping of the B1 gene revealed a Type I genotype at these loci. Five cases were PCR-negative for both agents. Of the 12 tested cases which were PCR-negative for Considering current and previous PCR-assay results, a diagnosis of avian malaria was confirmed in 13 cases, all of which had an original diagnosis of avian malaria based on gross and histological lesions. A summary of the tested tissues, sequencing and PCR results is presented in Most cases of avian malaria in kiwi were seen in the North Island of New Zealand, with a geographic distribution consistent with areas known to be inhabited by the affected species of kiwi  along wiPlasmodium spp., but there is still much about infection in wild avifauna that is not known. Owing to the protected status of kiwi, prospective infection studies are not possible. Human contact with non-captive kiwi is not allowed during breeding season between June to December, which significantly impedes epidemiological surveillance in some populations. There may also be interspecies differences in kiwi susceptibility, as NIBK are more numerous and probably more genetically diverse than smaller kiwi populations, including little spotted kiwi. This could partially explain previous reports of parasitaemia in wild-born captive juvenile NIBK [Plasmodium spp. Nevertheless, the cases presented here stand to highlight the potential for a fatal outcome, and it may at least be considered a sporadic cause of death in kiwi. In captive populations, netting of enclosures to reduce contact with mosquito vectors is warranted. Superficially, the isolated geography of New Zealand would suggest that kiwi are more likely to lie on the non-adapted end of the spectrum of susceptibility to ile NIBK ,31 or PCile NIBK ,9 withouChlorodrepanis virens) where infected females and males have been demonstrated to have higher serum prolactin and testosterone levels, respectively, compared to uninfected birds [Anthus campestris), nutmeg mannikins (Lonchura punctulata) and Iiwi (Vestiaria coccinea) [As part of ongoing conservation efforts, many neonatal kiwi are raised in captivity before being released back into the wild once they reach a bodyweight at which they are considered less likely to be predated . Increased birds . Nine ofoccinea) ,48, althPoor body condition has been associated with increased susceptibility to avian malarial infection and reduced survival time in some birds ,48, and Cell-mediated immunity plays a major role in elimination of organisms following acute infection and is primarily facilitated by monocyte-macrophage lineage cells in the haematopoietic organs. In adapted hosts, chronic infection may occur with development of acquired immunity and is associated with a decline in parasitaemia, antibody production and persistence of organisms in monocyte-macrophage lineage cells, often without significant ill effect ,50,51. DSpheniscus magellanicus) [Megadyptes antipodes) [Eudyptula minor) [Eudyptes pachyrhynchus) [Spheniscus demersus) [P. falciparum infection in humans, haematogenous spread of merozoites and the associated systemic inflammatory response lead to alterations in cell surface adhesion molecules on infected erythrocytes and capillary endothelial cells, resulting in increased adhesion, increased vascular permeability and leakage of plasma proteins into the alveolar spaces and interstitium [The most consistent lesions were severe interstitial pneumonia with pulmonary oedema, splenomegaly with macrophage hyperplasia and expansion of the red pulp by congestion or hyperaemia, hepatomegaly with mononuclear infiltration of portal areas and sinusoids, as well as increased numbers of circulating monocytes within the blood vasculature, which was noted in a variety of tissues. There is little information pertaining to the precise pathogenesis of interstitial pneumonia in avian malaria infections, although it is a common finding in reports of mortality in experimentally infected Hawaiian amakihi , blackbilanicus) , yellow-tipodes) , little a minor) , a Fiordhynchus)  and blacemersus) . Some reemersus) ,53. In Prstitium ; while mrstitium ,53,54,56Plasmodium spp. For some kiwi, interstitial myocarditis may have contributed to impaired cardiorespiratory function, although the degree of cardiac inflammation was relatively mild when compared to the lung. Severe anaemia and circulatory shock have been cited as a cause of death in acute infections, which typically peaks around one week post-infection with the onset of parasitaemia [Plasmodium spp. These may have resulted from organism-induced tissue necrosis, hypoxia or potentially ischaemia secondary to fibrin thrombosis, although the cause was not clear from histological examination.The cause of death was likely respiratory failure and hypoxaemia secondary to severe pulmonary pathology induced by the exoerythrocytic proliferation of sitaemia  and is psitaemia . While tsitaemia ,19. In tPlasmodium spp. lineage LINN1 was first reported in New Zealand in 2012 [Plasmodium (subgenus Haemamoeba) matutinum (LINN1) in 2017 [Plasmodium (subgenus Huffia) elongatum (GRW6) is a generalist parasite with low host specificity and following characterization in 2008 [Plasmodium lineages have a widespread worldwide distribution and have been implicated as a cause of pathology across a range of avian hosts [ in 2012 ,6 and su in 2017 . Plasmod in 2008 , has bee in 2008 ,5,6,9,14an hosts ,41,60,61Plasmodium spp. using fresh tissues tested negative in this study with FFPE tissues. There were five kiwi that tested negative in both Plasmodium spp. and T. gondii PCR assays. Four of these kiwi  had lesions and intracellular meronts consistent with Plasmodium spp. and are considered to be false negative results. The fifth (#7) had lesions suggestive of possible malaria but no organisms were seen in tissue section, hence it is probably unlikely that this kiwi died of avian malaria, although the possibility of prior resolving disease cannot be excluded. Formalin fixation compromises DNA integrity through formation of cross linkages, which is worsened with longer periods of fixation [One kiwi (#5) had no tissues or slides available for examination, hence the diagnosis of malaria could not be further investigated. Three cases  that had previously tested PCR-positive for fixation . Long pefixation ,63 and lfixation , but thiT. gondii. One kiwi was suspected to have succumbed to multifactorial disease including starvation and gastrointestinal parasitism (#11), and another had lesions suggestive of apicomplexan infection but was autolysed (#1), which may have impacted on assessment of some histological features. The significance of avian malaria infection to the death of these two birds is therefore uncertain. In the other two kiwi (#12 and #21), the lesions and morphology of organisms were indistinguishable from the confirmed cases of avian malaria. These kiwi did not have foci of parenchymal necrosis, the main lesion associated with toxoplasmosis, and organisms were visible in both endothelial cells and tissue macrophages, which is not a usual feature of natural T. gondii infections [T. gondii tissue cysts and zoites may be slightly larger than Plasmodium spp. [T. gondii exposure in kiwi are limited [T. gondii and Plasmodium spp. DNA amplification in the absence of visible organisms or lesions within the tissues [T. gondii positivity with compatible lesions was not made. The presentation of these kiwi (#12 and #21) remains suggestive of avian malaria; immunohistochemistry would be required to further ascertain the significance of these results but was unfortunately outside the scope of this study. The morphology and location of organisms was not consistent with other apicomplexan parasites including Atoxoplasma, (Isospora), Haemoproteus and Leucocytozoon hence these were not considered for molecular testing. In the authors’ experience, Haemoproteus has not been found in endemic avifauna samples submitted to our laboratory since surveillance for haemoparasites began in 2006.There were four kiwi which tested PCR-positive for only fections ,24,65. Tium spp. , and tenium spp. ,24. Neur limited , and a s tissues . In bothPlasmodium matutinum (LINN1) and Plasmodium elongatum (GRW6) as a cause of mortality in kiwi. The most consistent lesions include splenomegaly with macrophage hyperplasia, severe interstitial pneumonia with pulmonary oedema and interstitial myocarditis. Exoerythrocytic meronts were widespread and found within the cytoplasm of endothelial cells throughout the body, in tissue macrophages of the spleen and lung, and in Kupffer cells of the liver. Mortality is likely attributable to cardiorespiratory failure; whether kiwi which succumbed to infection survived long enough to develop anaemia as a comorbidity was uncertain. Several factors impair studies of natural disease in kiwi, however further investigation surrounding the host response could potentially be gleaned from monitoring captive birds in the North Island during peak transmission times.This is the largest case series linking molecular data with the lesions of avian malaria in an endemic bird of New Zealand and expands our knowledge of infection with


Malaria related mortality is associated with significant deregulation of host inflammatory factors such as interferon-inducible protein 10, a member of the CXC or α-subfamily (CXCL10), and host angiogenic factors such as angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2). However, detection of these factors in malaria patients requires the drawing of blood, which is invasive and increases the risk of accidental blood-borne infections. There has been an increased interest in the use of saliva as the body fluid of choice for the diagnosis of many infectious diseases including malaria. Here, saliva levels of CXCL10, Ang-1, and Ang-2 previously shown to be predictive of severe malaria in malaria patients in Ghana were assessed in malaria patients.This study was conducted in the Shai-Osudoku District Hospital in Dodowa, Accra, Ghana and the study population comprised 119 malaria patients and 94 non-malaria subjects. The non-malaria subjects are healthy community participants with no malaria infection. Plasma and saliva levels of CXCL10, Ang-1 and Ang-2 of the study participants were measured using an enzyme-linked immunoassay. Complete blood counts of each participant were measured with a haematology autoanalyzer. Pearson correlation was used to evaluate the correlation between plasma and saliva levels of each biomarker in malaria patients. A p-value of < 0.05 was considered significant. Box plots of median biomarker concentrations were plotted. SPSS version 14.2 software was used for statistical analysis.2 = 0.7, p < 0.0001) and Ang-1 . Malaria patients had lower saliva levels of Ang-1 (p = 0.009) and higher saliva levels of CXCL10 (p = 0.004) and Ang-2 (p = 0.001) compared to non-malaria subjects.The non-malaria subjects had a median age of 29 years compared to 23 years for malaria patients (p = 0.001). Among the malaria patients, there was a strong significant relationship between CXCL10 (RThis study provides the first evidence of elevated levels of CXCL10 and Ang-2 in the saliva of malaria patients. Detection of CXCL10, Ang-1 and Ang-2 in saliva may have a potential application for non-invasive malaria diagnosis. Malaria remains a major public health problem with an estimated 229 million cases and 409,000 related deaths globally in 2019, with 64% of the deaths occurring among children aged under 5 years .Plasmodium antigen in blood was promoted [Microscopic examination has been the mainstay of malaria diagnosis. However, depending on the microscopist the sensitivity and specificity under field conditions may be as low as 10% and 71%, respectively . In addipromoted . Howeverpromoted . Thus, tRecently, saliva has become a viable alternative body fluid for analytical purposes and experimentation due to its ease of use and access by healthcare staff with limited training and minimal need for specialized equipment use . BiomarkPlasmodium falciparum-mediated severe malaria [Angiopoietins are distinct members of a family of vascular growth factors which play important roles in mediating inflammation and quiescence within the vascular endothelium . Plasma  malaria –24. To dIn this study, the levels of CXCL10, Ang-1, and Ang-2 in saliva and plasma were evaluated and compared to the levels in malaria patients with non-malaria subjects  in Ghana with the goal of assessing their use for non-invasive diagnosis of the disease.The study was a case–control study performed between March and June 2017 in the Shai-Osudoku District (SOD) Hospital in Dodowa, Accra Ghana. Malaria patients and non-malaria subjects were recruited from SOD Hospital. The study recruited 213 participants including 119 malaria patients and 94 non-malaria subjects. A structured questionnaire was used to collect relevant socio-demographic and clinical data. Participation in the study was voluntary, with an informed consent form signed by the patient, or in the case of children, by the parents/ guardians.Plasmodium infection. Malaria was confirmed as positive thick and thin blood film, RDT positive, plus parasitaemia ≥ 2500/μl in the blood. The controls were healthy community participants with negative RDT for pLDH/HRP-2 and have the absence of P. falciparum parasitaemia on a thick and thin peripheral blood-stained smear. These healthy community participants were classified as non-malaria subjects in this study. The non-malaria subjects also had no history of fever, temperature, known infectious diseases or meningitis and showed no signs of impaired consciousness. The non-malaria subjects were recruited from the communities in which the patients with malaria reside. Patients with conditions which affect plasma Ang-1, Ang-2, and CXCL10 levels, such as diabetes mellitus, hypertension, coronary artery disease, chronic inflammatory, and autoimmune diseases as well as cancer [The study protocol was approved by the Ethical and Protocol Review Committee of the College of Health Sciences, University of Ghana. In this case–control study, the presence of Ang-1, Ang-2 and CXCL10 biomarkers levels in saliva of malaria patients were evaluated. Malaria case was defined as participants having acute febrile illness as defined by an axillary temperature ≥ 37.5 °C at presentation or history of fever within the last 72 h of s cancer  were excg using the Forma 3L Gp 4500R centrifuge  to separate plasma, red cells and peripheral blood mononuclear cells (PBMCs) and the processed plasma was stored at − 80 °C. A complete blood count (CBC) was done with an Automated Hematological Analyzer . Thick and thin blood film slides were prepared with the EDTA blood sample within 2 h of collection, Giemsa-stained and examined for parasitaemia. About 1.5 ml of unstimulated saliva was collected from each participant into an OMNIgene-Oral tube  containing preservatives. The sample was stored at 28–30 °C prior to analysis.Four millilitres (4 ml) of venous blood from each study participant was collected into ethylene diamine tetraacetic acid (EDTA) tubes. An aliquot of 2.0 ml of the blood sample was put into CPT and centrifuged for 15 min at 1000×The plasma and saliva levels of CXCL10, Angiopoietin-1, and Angiopoietin-2 of each participant were measured using commercial Sandwich enzyme-linked-immunosorbent assays  according to the manufacturer’s instructions. The optical density was determined within 30 min using Spectra Max 190 fluorescence microplate reader  with the wavelength set to 540 nm.The data was entered in Statistical Package for the Social Sciences (SPSS) version 14.2 software . Frequency tables generated for categorical demographic characteristics and anaemia status are summarized as percentages. The results for continuous variables are expressed as median with the interquartile range (IQR). Chi-square tests were used to examine the differences in categorical variables. Fisher’s exact test was used when cell count was < 5. The Kruskal Wallis test or Mann–Whitney U test was used to compare differences in median values continuous variables among malaria patients and non-malaria subjects. Pearson correlation was used to evaluate the correlation between plasma and saliva levels of each biomarker in malaria patients. A p-value of < 0.05 was considered significant. Box plots of median biomarker concentrations with 25th and 75th percentiles were plotted. SPSS version 14.2 software  was used for statistical analysis. GraphPad Prism version 6  for windows was used to generate all the graphs.A total of 213 participants were enrolled in the study which included 94 non-malaria subjects and 119 malaria patients   and Ang-1  levels between saliva and plasma  (Fig. To determine the relationship of Ang-1, Ang-2 and CXCL10 between saliva and plasma, we performed a Pearson correlation to determine the association of these markers in saliva and plasma samples. There was a strong significant relationship in CXCL10 (RSaliva Ang-1 levels were significantly lower in malaria patients than in non-malaria subjects, p = 0.009 Fig. A. SimilaDifferences in several haematological factors among malaria and non-malaria study participants were observed in this study. Consistent with findings from other studies, there was a significant difference in haemoglobin levels among malaria patients compared to the non-malaria group Table . This coPlasmodium infections [P. falciparum infection, the production of angiogenic factors has been found to be associated with an increase in the cytoadherence of infected erythrocytes to the vascular endothelium [Previous studies have demonstrated low plasma levels of Ang-1 and high values of Ang-2 to be associated with severe fections . During othelium . Ang-1 aothelium , 28. Othothelium . This stothelium , 29.P. falciparum malaria infection than other markers of disease severity. Meanwhile, a similar study also reported an increase in plasma Ang-2 levels in adults and children with severe malaria and found Ang-2 to be a better predictor of death than other markers of disease severity [In line with this study, Sahu et al.  reportedseverity .Elevated Ang-2 levels have also been associated with endothelial damage , 31. AltThis study provides the first evidence of Ang-1, Ang-2, and CXCL10 in saliva discriminating between malaria patients and non-malaria subjects. With the future goal of incorporating these factors in point-of-care diagnostic kits for malaria detection, the discovery of these markers in the saliva of malaria patients could allow for accurate and early detection of malaria for immediate intervention. Furthermore, this will provide a non-invasive approach that will facilitate and make for easy screening of large populations in epidemiological surveys in endemic countries.The small number of patients examined restricted the possibilities of extrapolating the results to other populations and will require further studies to enable firm conclusions to be drawn. Additionally, the subjects in the study were not tested for other infections that have been associated with these biomarkers to determine if these biomarkers will also differentiate malaria parasite infection from other infections associated with these biomarkers. Therefore, further studies need to be conducted to test for the differentiation of these biomarkers between malaria and other infections.This study indicate that key biomarkers of malaria, Ang-1, Ang-2, and CXCL10, could be detected in saliva and may have potential application for clinical diagnosis of malaria. After further optimization of the assay protocol to improve sensitivity, it could serve as a supplement or alternative to blood sampling for testing malaria in malaria-endemic areas and large-scale epidemiology surveys. Further studies in different settings or populations as well as the sensitivity and specificity of these biomarkers in saliva are however needed to validate our current findings.


P. falciparum and/or P. vivax malaria are at risk of severe anaemia and death. We hypothesized that in an area of high malaria transmission, intermittent screening and treatment of infants with malaria (ISTi) will reduce morbidity compared to passive case detection (PCDi).In Papua (Indonesia), infants with We conducted a cluster randomised, open label, superiority trial. A total of 21 clusters of village health posts (VHP) were randomised 1:1 to either IST for infants coinciding with 4 routine immunisation visits or PCDi. Healthy term infants born to consenting mothers enrolled into a maternal malaria cluster randomised trial were included in the study and followed for 12 months. Point of care malaria rapid diagnostic tests were used to detect peripheral parasitaemia at 2, 3, 4 and 9 months old in all infants in ISTi clusters and when symptomatic in PCDi clusters. Infants with detected peripheral parasitaemia were treated with dihydroartemisinin-piperaquine. The co-primary outcomes were the incidence rate of clinical malaria in the first year of life and the prevalence of parasitaemia at age 12 months. The incidence rate ratio and prevalence ratio between ISTi and PCDi were estimated using mixed-effects Poisson and log-binomial regression modelling (accounting for clustering at VHP level).p = 0.280). The prevalence of parasitaemia at 12 months was 13% (33/254) in the IST clusters and 15% (57/379) in the PCD clusters  = 0.92 , p = 0.55). There was no difference in the risk of anaemia between treatment arms.Between May 2014 and February 2017, 757 infants were enrolled into the study, 313 into 10 ISTi clusters, and 444 into 11 PCDi clusters. Overall, 132 episodes of parasitaemia were detected, of whom 17 (12.9%) were in symptomatic infants. Over 12 months, the incidence rate (IR) of clinical malaria was 24  per 1000 children-years at risk in the ISTi arm and 19  per 1000 children-years in the PCDi arm (adjusted incidence rate ratio [aIRR] 1.77 ; In high malaria transmission area outside of Africa, our study suggests that compared to PCDi, ISTi offers no significant benefit in reducing the risk of clinical malaria in infants born to women receiving effective protection from malaria during pregnancy.NCT 02001428, registered on 20 Nov 2013.ClinicalTrials.gov The online version contains supplementary material available at 10.1186/s12916-022-02394-1. Plasmodium falciparum and P. vivax are highly prevalent and a third of hospitalised infants are diagnosed with malaria, often associated with severe anaemia and respiratory distress in the first year of life [P. vivax malaria than older children and adults. If undetected and inadequately treated, recurrent parasitaemia leads to a cumulative risk of severe anaemia and death [In malaria endemic areas, the susceptibility of infants to malaria increases as maternal antibodies wane after birth, rendering infants at risk of severe disease and dying –4. In Ti of life . Infantsnd death –7.In infancy, the signs and symptoms of malaria are not specific, leading to a delay in diagnosis and treatment . Early dP. falciparum in Africa, the implementation of intermittent preventive treatment in infancy (IPTi) with curative dose of sulfadoxine-pyrimethamine (SP) has been shown to be an effective malaria control strategy [In areas of high transmission of strategy , 10. Howstrategy . In areastrategy .DHP has been the first-line treatment for uncomplicated malaria in Indonesia since 2006, and its use as monthly IPT during pregnancy has been shown to be effective in reducing the risk of maternal malaria . HoweverBetween May 2014 to March 2017, we conducted a cluster randomised, open-label superiority trial to compare ISTi (intervention group) with PCDi (control group). Intermittent screening coincided with the infant’s local immunisation schedule. The unit of randomisation was the village health post (VHP), a community-based maternal and child health care facility, providing monthly antenatal care for pregnant women, as well as an immunisation and nutritional program for infants and children under 5 years old. A total of 21 VHPs were randomised to implement either ISTi or PCDi.Healthy term infants born from mothers participating in a maternal cluster randomised trial were eligible to be enrolled into the infant study. The maternal study compared three arms: intermittent screening and treatment (ISTp), intermittent preventive treatment (IPTp) and single screening and treatment (SSTp), as described previously . InfantsP. falciparum and P. vivax parasitaemia are similarly prevalent. Previous clinical trials in this region have shown high rates of treatment failure for patients with either P. falciparum or P. vivax malaria following chloroquine monotherapy, CQ plus SP or unsupervised quinine [P. falciparum and P. vivax infections [The study was conducted in Timika, Papua (Indonesia), an area where  quinine . In 2006fections .The 21 clusters were stratified by the three maternal intervention groups (7 per group) as described elsewhere . Within Study participants, field team, investigators and study statistician were aware of the intervention allocation, however laboratory technicians responsible for deriving the clinical outcomes were blinded to intervention allocation.At birth, information on maternal and newborn’s demographic, socioeconomic, clinical and laboratory data were collected from mother and child records reported in the maternal study. After 6 weeks of age, clinical and laboratory data were collected prospectively by the investigators of the infant study.All infants were followed up until 12 months of age. Infants in all clusters underwent a routine clinical questionnaire and examination (including anthropometric data) at 2, 3, 4, 6, 9 and 12 months old. The diagnosis of malaria and asymptomatic parasitaemia was made at the VHP using rapid diagnostic tests , with microscopic peripheral blood examination conducted later the same day at the supporting study laboratory. Slides were read by two experienced microscopists. In cases where readings were discordant, the slides were reread by a third microscopist and a consensus reached. Haemoglobin (Hb) concentration was measured using a portable haematocrit analyser (Hemocue®).In the ISTi clusters, infants were screened routinely for peripheral parasitaemia and had a Hb measurement at 2, 3, 4 and 9 months of age coinciding with diphtheria, pertussis, tetanus (DPT) 1-2-3 and measles vaccination, respectively. In the PCDi group, infants were only assessed for peripheral parasitaemia and Hb if they presented with fever or history of fever in the preceding 24 h. Additional capillary blood samples were collected during home visits from a heel prick from all infants in ISTi and PCDi clusters at age 6 and 12 months of age. Submicroscopic infections and speciation were assessed at the 12-month visit using qPCR methods to blood samples collected, as described previously , 21.Infants with clinical malaria in either group and those with asymptomatic peripheral parasitaemia in the ISTi arm were treated with DHP once daily for 3 days  without primaquine according to the national treatment guidelines . Individ0C) or history of fever in the preceding 24 h plus detection of peripheral asexual parasitaemia by either RDT or microscopy. The co-primary study outcomes were the incidence rate of clinical malaria in the first 12 months of life and the prevalence of peripheral parasitaemia  at 12 months of age regardless of symptoms. Secondary outcomes included the prevalence of anaemia (Hb < 10 g/dl) at 6 and 12 months.Clinical malaria was defined as the presence of fever . With 757 infants, the trial was powered to detect a 20% reduction in the incidence rate of clinical malaria from 3.0 to 2.4 case per child in the first year of life .Data were double entered and validated using EpiData 3.02 software  and analysis performed using Stata version 17.0. Demographic data were presented by intervention arm with categorical variables presented as frequency (%) and continuous variables as mean (SD) or median (25th-75th percentiles). The adjusted incidence rate ratio ) of clinical malaria in the first year of life comparing IST to PCD intervention group was estimated using mixed-effects Poisson regression analysis, with the exposure period calculated using the age at final visit, adjusted for maternal intervention group, maternal parasitaemia at enrolment or during pregnancy, maternal socioeconomic status (low/medium/high) and infant sex. Random effects at the VHP level were used to account for clustering. Clinical assessments of the 12-month visit that occurred after 14 months (425 days) were censored in the analysis.The adjusted prevalence ratio  of parasitaemia at age 12 months was estimated using log binomial regression analysis with a robust estimator of standard error adjusted for maternal intervention group, maternal parasitaemia at enrolment or during pregnancy, maternal socioeconomic status (low/medium/high) and infant sex. The adjusted prevalence ratio  of anaemia at age 6 and 12 months was estimated using a log binomial regression analysis with a robust estimator of standard error adjusted for maternal intervention group, maternal parasitaemia at enrolment or during pregnancy, maternal socioeconomic status, maternal anaemia (Hb < 10 g/dl), maternal malnutrition (mid upper arm circumference < 23.5 cm) and infant’s sex. The modified intention to treat population included all randomised participants with outcome data available.Ethical approval was obtained from the Human Research Ethics Committee of the Northern Territory Department of Health, Australia (HREC 2013-2108), and the Health Research Ethics Committees of the Faculty of Medicine Universitas Gadjah Mada, Indonesia (KE/FK/1023/EC/2013).The study was conducted over 33 months with the first recruitment on 7 May 2014, the last patient enrolled on 6 April 2016 and the last follow-up visit on 23 February 2017. A total of 757 infants (755 singletons and 1 set of twins) were enrolled into the study  were in symptomatic infants. At 6 months, 1.0% (7/668) of infants had peripheral patent parasitaemia detected by RDT or microscopy  at 12 months was 13% (33/254) in the IST clusters and 15% (57/379) in the PCD clusters; adjusted prevalence ratio (aPR) = 0.92 , p = 0.55), Table At the end of follow-up the incidence rate (IR) of clinical malaria in the ISTi arm was 7 per 287 children years at risk  compared to 8 per 417 children-years at risk in the PCDi group ; adjusted incidence rate ratio (aIRR) = 1.77 , p = 0.012. The prevalence of anaemia (Hb < 10 g/dl) was 24% (66/278) in the ISTi arm and 29% (112/392) in the PCD arm at 6 months , p = 0.280) 28% (70/254) and 30% (114/378) respectively at 12 months , p = 0.810); Table p = 0.820.Anaemia (Hb < 10 g/dl) was present in 26.7% (178/670) of infants at 6 months and 29.1% 184/632) at 12 months , one infant with falciparum malaria and hyperpyrexia (PCDi arm) and one with malaria due to mixed species infection (PCDi arm). All infants were treated with intravenous artesunate and DHP and made a full recovery  was detected in 11% (74/678) of infants at 6 months and 15% (98/634) at 12 months with similar prevalence between intervention arms.By 12 months of age, 39 infants were admitted to the hospital without malaria, with a similar risk of admission in both treatment arms: 6.4% (20/313) in the ISTi arm and 4.3% (19/444) in the PCDi arm. One infant in each arm required 2 admissions . Although there were a higher number of clinical malaria episodes in the ISTi clusters, the overall benefit of ISTi compared to PCDi arms in reducing clinical malaria was inconclusive. Overall 12.9% of the 132 detected episodes of parasitaemia were associated with symptomatic illness. By 12 months, 14% of infants had detectable peripheral parasitaemia, but almost 80% of these infections were submicroscopic.The annual parasite incidence in the study area is approximately 250–400 cases per 1000 population, and thus the incidence of malaria in the infants enrolled was significantly lower than expected. Malaria in early life can result from vertical transmission (that may manifest months after birth), development of immune tolerance to malaria antigen in utero  or sporozoite inoculation by infected mosquitos in early life –27. The p = 0.280) and at 12 months  suggesting minimal effect of maternal anaemia status in these infants. As well as malaria, nutritional deficiencies including iron are major causes of anaemia in early life [Despite the low risk of infant malaria, almost 30% of participants were anaemic (Hb < 10 g/dl) by the time they were 12 months old. Although greater number of infants in PCDi arms had mothers with anaemia and severe anaemia, the risk of anaemia was similar between infants in ISTi and PCDi arms at 6 months .Maternal anaemia status was not considered a priori to be a confounding factor for the primary outcome. In a sensitivity analysis in which maternal anaemia was included as a covariate in the analysis; the estimated adjusted incidence rate ratio was similar to the findings of the primary analysis  reduced the risk of clinical malaria by 29% and the risk of anaemia by 25% . HoweverIn areas of high malaria endemicity in Africa, maternal and infant malaria intervention strategies are often combined to optimise impact. However, our study in a coendemic high transmission area suggests that the benefits of effective malaria control activities provided during antenatal care may have protected the babies in the first year of life , 32–34, Our study has a number of limitations. First, the study population may have been biased to individuals with a more positive attitude towards malaria, since mothers were attending antenatal care in village health posts, bringing their babies for immunisation and contact with the clinic staff. Additionally, children in the ISTi intervention arm were tested for malaria at 2, 3, 4, 6 and 9 months, increasing the likelihood of detecting a clinical malaria episode compared with the passive detection in the PCDi arm. These potential biases may have reduced the observed effect impact of ISTi. Second, infants in the ISTi arm were more likely to have come from the maternal IST group whereas infants in the PCDi arm were more likely to have been in the maternal IPTp-DHP group. This may have contributed to the reduction in the observed benefit of ISTi since maternal IPTp-DHP was associated with a significant reduction in the risk of malaria during pregnancy compared to maternal IST . Third, P. falciparum and P. vivax are coendemic, there was no clear benefit to infants receiving ISTi compared to current passive case detection, although this may have reflected reduced power of the study associated with concomitant interventions to reduce maternal malaria. Robust malaria control strategies during pregnancy may provide protection of infants from malaria but not from anaemia. In early life the clinical consequences of submicroscopic asymptomatic infections need to be explored, to inform complementary interventions for mothers and infants at high risk of malaria.In an area of high malaria transmission, where Additional file 1.


Vector-transfected SH-SY5Y-MOCK cells served as controls. Using these cells, we investigated mitochondrial respiration (OXPHOS), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, and citrate synthase activity (CS) in cells treated with ALA. Cells were treated for 24 h with different concentrations of ALA and with or without the complex I inhibitor rotenone. Results: Incubation with ALA showed a significant increase in ATP levels in both SH-SY5Y-APP695 and SH-SY5Y-MOCK cells. MMP levels were elevated in SH-SY5Y-MOCK cells, treatment with rotenone showed a reduction in MMP, which could be partly alleviated after incubation with ALA in SH-SY5Y-MOCK cells. ALA treatment showed significant differences in respiration chain complex activities in SH-SY5Y-MOCK cells. Citrate synthase activity was unaffected. ROS levels were significantly lower in both cell lines treated with ALA. Conclusions: ALA increased the activity of the different complexes of the respiratory chain, and consequently enhanced the MMP, leading to increased ATP levels indicating improved mitochondrial function. ALA only marginally protects from additional rotenone-induced mitochondrial stress.Introduction: Alpha lipoic acid (ALA) is a sulphur-containing organic compound, derived from octanoic acid, and an important cofactor for mitochondrial respiratory enzymes. It has strong antioxidant properties that improve mitochondrial function. We investigated if ALA improves mitochondrial dysfunction in a cellular model of Alzheimer’s disease (AD). Methods: SH-SY5Y-APP Alzheimer’s disease (AD) is a slowly progressing neurodegenerative disorder that starts with mild memory loss and, as it progresses, culminates in severe impairment of executive and cognitive functions ,2. The mA promising molecule is alpha lipoic acid (ALA). ALA is widely used as a pharmaceutical and nutraceutical due to its strong antioxidant and anti-inflammatory properties ,12,13.+/multivitamin transporter (hSMVT) is responsible for the transport of ALA. However, in vitro evidence suggests the existence of more than one cellular transport mechanism involving other fatty acid transporters, such as the monocarboxylic acid transporter (MCT)  and malate [2 mM] were added and the leak respiration was measured (CI(L)). The addition of ADP [2 mM] was used to determine coupled complex I respiration CI(P). CI and CII respiration in the coupled state (CI&CII(P)) was measured after the addition of succinate [10 mM]. Subsequent titration of carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), of up to 2.5 µM, led to uncoupling of the ETC, and the maximal uncoupled activity of CI&CII(u) was measured. After the addition of 0.5 µM rotenone uncoupled CII(u) respiration was monitored. By the addition of oligomycin [2 µg/mL], the leak respiration of CII(L) was determined. To exclude the oxygen consumption from enzymes that are not part of the oxidative phosphorylation, antimycin A was added and subtracted from all readings obtained in the experiment. By adding the electron donor N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) [0.5 mM], an artificial substrate for CIV, and ascorbate, which serves to keep TMPD in a reduced state, the maximal uncoupled respiration of CIV(u) was measured. At the end of the experiment, NaN3 was added to inhibit CIV. The remaining oxygen consumption was due to the autooxidation of TMPD and was additionally subtracted from the raw CIV readings.To evaluate the oxygen consumption of the ETC an Oxygraph-2k respirometer  and the software DatLab v. 4.3.2.7 was used . The protocol used to test mitochondrial respiration was developed by Greiner . For thi (MIR05) . At diffTo assess the citrate synthase activity, cell samples from the respiration measurements were previously frozen in liquid nitrogen and stored at −80 °C. The following steps were taken to determine the CS activity. (1) Cells were mixed with a reaction medium containing 5,5′-dithio-bis-(2-nitrobezoic acid) (DTNB) [0.1 mM], acetyl coenzyme A [0.31 mM], EDTA [50 µM], oxalacetate [0.5 mM], triethanolamine hydrochloride [5 mM], and Tris HCl [0.1 M]. (2) The reaction medium was warmed to 30 °C for 5 min. (3) Cells were mixed with this medium. On the basis of conversion from DTNB into TNB, the citrate synthase activity was spectrophotometrically determined at a wavelength of 412 nm .To determine protein content, Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. The measurements were carried out on a CLARIOstar plate reader (BMG Labtech). Each sample was measured in triplicates.Cellular ROS production was determined using a DCFDA/H2DCFDA Cellular ROS Assay Kit . Cells were incubated with ALA for 24 h. Afterwards, the manufactures instructions were followed.t-test . Statistical significance was defined for p values as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.Unless stated otherwise, data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using either one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post-hoc test or student’s In neuronal cells serving as a model for early AD and its controls, low ALA concentrations increased the activity of respiratory chain complexes and consequently ATP levels. Thus, ALA can compensate for mitochondrial dysfunction in cells that is not disease related. The increased activity of the mitochondrial respiratory complexes led to an increased formation of ROS, which can be intercepted by ALA and, thus, protect the mitochondria from oxidative stress. To better understand the observed positive results of ALA, the morphology and structure of mitochondria should be examined. Moreover, mitochondrial-associated factors should be further explored. In the future, in vivo validation of the ALA results should be performed.


Z‐score (WHZ) < −3 are used for screening for severe acute malnutrition (SAM). Underweight and concurrent wasting and stunting may better target those at the highest risk of mortality. We compared anthropometric outcomes in children enrolled in a trial of antibiotics for SAM based on categories of baseline anthropometry, including indicators for programme admission  and alternative indicators . Participants were followed weekly until nutritional recovery and at 8 weeks. We evaluated changes in weight gain (g/kg/day), MUAC, and WHZ in children admitted by admissions criteria  and by underweight or concurrent wasting and stunting. Of 301 admitted children, 100 (33%) were admitted based on MUAC only, 41 (14%) WHZ only, and 160 (53%) both MUAC and WHZ, 210 (68%) were underweight and 67 (22%) were concurrently wasted/stunted. Low MUAC and low WHZ children had the lowest probability of nutritional recovery (17% vs. 50% for MUAC‐only and 34% for WHZ‐only). There was no difference in weight gain velocity or WHZ by admissions criteria (WHZ and/or MUAC). Underweight and concurrently wasted/stunted children had lower MUAC and WHZ at 8 weeks compared with those who were not underweight or concurrently wasted and stunted. Children with both low MUAC and low WHZ had the worst outcomes. Relying on MUAC alone may miss children who have poor outcomes. Other indicators, such as WAZ, may be useful for identifying vulnerable children.Mid‐upper arm circumference (MUAC) < 11.5 cm and weight‐for‐height  Z‐score (WHZ) and mid‐upper arm circumference (MUAC) are used for screening for severe acute malnutrition, but alternative indicators such as weight‐for‐age Z‐score (WAZ) may identify children at high risk of mortality. We evaluated anthropometric outcomes in children with severe acute malnutrition by baseline anthropometric status. Children with both low WHZ and MUAC had the worst outcomes, and low WAZ additionally identified children with poor outcomesWeight‐for‐height  Mid‐upper arm circumference (MUAC) and weight‐for‐height Z‐score (WHZ) are currently used for severe acute malnutrition (SAM) screening, but alternative indicators such as weight‐for‐age Z‐score (WAZ) may be additional options.Children admitted to the nutritional program based on both low MUAC and low WHZ had the worst outcomes, including the lowest probability of nutritional recovery and the worst anthropometric outcomes.While MUAC identified most children with SAM, WHZ may provide additional information about poor outcomes beyond MUAC alone.WAZ may be a useful alternative indicator to identify children who would benefit from a therapeutic feeding program without requiring height measurement. Other clinical or nutritional factors, such as breastfeeding, may also significantly modulate the risk of poor outcome regardless of admission criterion.Currently, there is no gold standard for the diagnosis of SAM. Both MUAC and WHZ are imperfect measures of SAM and do not provide a definitive diagnosis  at enrolment, as these markers may be useful in identifying the highest‐risk children who may benefit from admission to a nutritional programme.Response to treatment for outpatient SAM may be different for children admitted based on different criteria given that these criteria identify different subpopulations of children. We utilized data from a randomized controlled trial of azithromycin compared with amoxicillin for outpatient management of SAM  and had height  and MUAC  in triplicate and the median was used for analysis. Weight gain was calculated in g/kg/day since baseline (primary outcome) and also as period‐specific weight gain, for example, baseline to Week 1, Weeks 1–2, Weeks 2–4 and Weeks 4–8. Weight and height measurements were used to calculate WHZ, WAZ and HAZ based on 2006 World Health Organization standards  for malaria was performed on all children at 8 weeks after enrolment  at baseline and Weeks 1, 2, 4, and 8. To assess if anthropometric outcomes were significantly different at 8 weeks, we used unadjusted and adjusted linear regression models for the admission criterion for the nutritional programme predicting each outcome . Models were adjusted for the child's age, sex and the baseline measure for the outcome, with the exception of g/kg/day, which includes baseline weight in its calculation. To assess if there was a differential change in each anthropometric outcome over time, we used repeated‐measures models with an interaction term for the admissions criterion by study week adjusting for the child's age and sex. We then used unadjusted and adjusted logistic regression models to assess odds of achieving nutritional recovery by admissions criterion, with models adjusted for the child's age, sex and baseline MUAC and WHZ. Analyses pooled data from both trial arms and we did not adjust for the randomized treatment assignment because randomization and treatment happened after admission to the nutritional programme and adjustment for a post‐exposure variable may induce selection bias. There was no evidence of a difference in anthropometric outcomes by randomization arm in the trial's original analysis  at baseline and those who were concurrently severely wasted and stunted (defined as WHZ < −3 and HAZ < −3) at baseline. Models were identical to those described using the admission criteria to the trial. Models for underweight and concurrent wasting and stunting were run separately, as their categories are not mutually exclusive. All analyses were conducted in Stata 15.1 (StataCorp).3Of 301 children enrolled in the trial, 100 were enrolled based on MUAC < 11.5 cm, 41 WHZ < −3, and 160 MUAC < 11.5 and WHZ < −3 Figure . Of the Among all enrolled children, 210 (70%) were underweight (WAZ < −3) and 67 (22%) were concurrently stunted (HAZ < −3) and wasted (WHZ < −3). There was substantial overlap in low WAZ and HAZ by admissions criteria based on WHZ and MUAC Figure . Childrep for interaction = 0.15) or at 8 weeks . MUAC at 8 weeks was significantly lower in models adjusting for baseline MUAC in the WHZ only and MUAC and WHZ cohorts compared with MUAC only, reflecting that the children admitted based on MUAC only had more rapid changes in MUAC over time compared with the other cohorts  and WHZ stayed consistently lower for children admitted based on WHZ only and MUAC and WHZ. Similarly, WAZ increased over time for all three groups at a similar rate . HAZ did not change over time in any of the three admissions groups . Children who were admitted based on both MUAC and WHZ had consistently lower WAZ and HAZ throughout the study period.Although all three admissions criteria groups saw improvements in WHZ, there was no difference in change in WHZ over time by admission criteria  and WHZ and HAZ (concurrent wasting and stunting). WAZ has been shown to be a strong predictor of mortality in community‐based settings, and studies are increasingly recognizing the double burden of concurrent wasting and stunting that may place children at additional risk of poor outcomes (Gao et al., This study must be considered in the context of several limitations. Data arose from a randomized controlled trial of azithromycin versus amoxicillin for SAM. All measurements were standardized and taken by trained anthropometrists as part of the trial, but the sample size was limited by the trial's overall objective (O'Brien, Sié, Dah, Ourohire, Arzika, et al., In this secondary analysis of data arising from a randomized controlled trial of antibiotics for SAM, we found that children admitted based on both low MUAC and WHZ had worse anthropometric and clinical outcomes compared with children admitted based on a single criterion. MUAC‐only screening would have identified 86% of children ultimately enrolled in the trial. This analysis suggests that children admitted based on both MUAC and WHZ do not catch up to those meeting only one admission criterion in terms of anthropometric outcomes. Although MUAC is a much simpler screening tool than WHZ, those screening positive for SAM based on WHZ only had persistent anthropometric deficits and likely benefit from admission to the nutritional programme. Other indicators such as WAZ, which is easier to measure than WHZ as it does not require height measurements, may be considered to identify children at particularly high risk of poor outcomes.None of the authors have any conflicts of interest to report.Clarisse Dah, Millogo Ourohire, Moussa Ouédraogo, Ali Sié, Moussa Ouédraogo, Mamadou Bountogo, Elodie Lebas, Benjamin F. Arnold, Kieran S. O'Brien and Catherine E. Oldenburg designed the study. Millogo Ourohire, Moussa Ouédraogo, Valentin Boudo, Elodie Lebas and Fanice Nyatigo supervised data collection. Valentin Boudo and Fanice Nyatigo developed the database and managed data cleaning. Fanice Nyatigo, Benjamin F. Arnold, Kieran S. O'Brien and Catherine E. Oldenburg analysed the data. Clarisse Dah and Catherine E. Oldenburg wrote the paper. All authors have read and approved the final manuscript.Supporting information.Click here for additional data file.


Plasmodium infections complicate the conduct of malaria clinical trials. These infections confound infection detection endpoints, and their immunological effects may detract from intended vaccine-induced immune responses. Historically, these infections were often unrecognized since infrequent and often analytically insensitive parasitological testing was performed before and during trials. Molecular diagnostics now permits their detection, but investigators must weigh the cost, complexity, and personnel demands on the study and the laboratory when scheduling such tests. This paper discusses the effect of pre-existing and intervening, low-density Plasmodium infections on malaria vaccine trial endpoints and the current methods employed for their infection detection. We review detection techniques, that until recently, provided a dearth of cost-effective strategies for detecting low density infections. A recently deployed, field-tested, simple, and cost-effective molecular diagnostic strategy for detecting pre-existing and intervening Plasmodium infections from dried blood spots (DBS) in malaria-endemic settings is discussed to inform new clinical trial designs. Strategies that combine sensitive molecular diagnostic techniques with convenient DBS collections and cost-effective pooling strategies may enable more thorough and informative infection monitoring in upcoming malaria clinical trials and epidemiological studies.Pre-existing and intervening low-density  Plasmodium infection status of the participants is established at the time of trial enrollment. In malaria-endemic regions, participants may have been recently exposed to Plasmodium parasites, so it is possible that participants may be actively infected at the time of trial eligibility and enrollment assessments. Consequently, many studies are designed to start with anti-malarial drug treatment of some or all participants to eliminate any pre-existing Plasmodium parasites at the outset of the trial , which highlights the need for pre-enrollment testing worldwide. Pre-enrollment testing is a requirement for any non-endemic CHMI study intending to use a recently qualified Plasmodium 18S rRNA biomarker in lieu of thick blood smears (TBS) for detecting infections in such studies  completely eliminated the otherwise high efficacy achieved when blood stage parasites were absent during vaccination with a two-fold higher dose of the same vaccine given at 5-day intervals  at some point during the 28-day collection period. This study is notable because DBS samples were self-collected using daily finger prick sampling to observe the dynamic behavior of these asymptomatic low-density infections. This study is discussed in a later section as well to provide a roadmap for improving infection detection in future endemic site clinical trials. Prevalent, dynamic asymptomatic low-density Plasmodium infections have also been reported by other investigators in different regions of malaria endemicity  blood or peripheral whole blood, which can be collected and stored as liquid blood or preserved as DBS. In clinical trials, Phase 1-2 studies typically schedule more frequent testing and use more analytically sensitive tests compared to Phase 3 studies. Test selection considerations also include the study population , the clinical and laboratory capabilities of the site, and assay costs. It should be noted that some malaria clinical trials have used clinical signs and symptoms of malaria as an eligibility criterion for enrollment  for P. falciparum and lactate dehydrogenase (LDH) for all species). They have the advantages of ease of use, rapid turnaround time suitable for point-of-care or near point-of-care use, and therefore, deployability. However, the LoD for most marketed RDTs is ~200 parasites/µL  have been developed for many infectious diseases including malaria. In simplest terms, NAATs generally involve a nucleic acid extraction step followed by an amplification/detection step using oligonucleotide-specific reagents. Methods include PCR, qRT-PCR, and nucleic acid-based sequence amplification, which have been reviewed previously . MethodsPlasmodium parasites across a variety of settings   as well as the University of Washington Institutional Review Board (STUDY00009434). No potentially identifiable human data is presented in this article.TO, SM, JK, and TE conceived the idea. TO drafted the paper. DH, AS, WS, CC, and MC reviewed the first draft. All authors contributed to the article and approved the submitted version.OPP1212966/INV-009313 (SM), NIH R21AI146763 (SM).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The proportion of severe pneumonia (546 cases) among patients with malaria  was 20% . The proportion of deaths among patients with co-infection was 13% . In conclusion, nearly one-fifth of patients with severe pneumonia have malaria, one-fifth of patients with malaria have severe pneumonia, and about 13% of co-infections lead to deaths. This information raised the clinical importance of diagnosis and management of concurrent infections. Patients with severe pneumonia should be investigated for malaria, and vice versa. Detection of co-infections might provide the information to inform the physician to manage and cure co-infected patients who live in areas where both diseases were endemic.Malaria and pneumonia are the leading causes of childhood mortality in children under 5 years of age. Nevertheless, the proportions and deaths of malaria co-infection among patients with severe pneumonia, particularly in children under 5 years of age, and characteristics of co-infection remain poorly explored. Hence, the present study aimed to collate the evidence of malaria among patients with severe pneumonia, severe pneumonia among patients with malaria, and the proportion of deaths among patients with co-infections. Potentially relevant studies were searched in six databases including PubMed, Scopus, Web of Science, Embase, Ovid, and MEDLINE to identify studies on malaria and severe pneumonia co-infections that were published until 21 July 2022 with a restriction for the non-English language but no restriction for the publication year. The quality of the included studies was determined using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). The pooled estimates, including the pooled proportion of malaria among patients with severe pneumonia, and the proportion of deaths among patients with co-infections, were estimated by the random-effects model. Of the 4094 studies examined, 11 studies that met the eligibility criteria were included in the review. Meta-analysis results showed that the proportion of malaria (2162 cases) among patients with severe pneumonia (9738 cases) was 19% (95% CI 12–26%, I For malaria, 229 million cases and 409,000 deaths were estimated worldwide in 20195. More than half of malaria cases globally were reported from Nigeria (27%), the Democratic Republic of the Congo (12%), Uganda (5%), Mozambique (4%), and Nigeria (3%)5. Meanwhile, more than half of malaria deaths globally were reported from Nigeria (23%), the Democratic Republic of the Congo (11%), the United Republic of Tanzania (5%), Mozambique (4%), Nigeria (4%) and Burkina Faso (4%)5. For pneumonia, it was reported as 14% of all deaths of children under 5 years old with a total number of 740,180 deaths in 20196. The high burden of cases and deaths from malaria and pneumonia were found in malaria‐endemic regions, particularly in low-income countries1.Malaria, pneumonia, and malnutrition are the leading causes of childhood mortality in children under 5 years of age10. In resource-limited settings where diagnostic tools for pneumonia and malaria may not be accessible or available, distinguishing between pneumonia and malaria in children is challenging as these two diseases present with some overlapping clinical signs and symptoms11. A study suggested that overlapping clinical features of severe malaria and severe pneumonia included fever, respiratory distress, and impaired consciousness12. Dehydration, lower packed cell volume (PCV), and hyperbilirubinemia were suggested to be the predictive markers of severe malaria. Meanwhile, hypoxemia, preadmission antibiotic treatment, chest signs, and higher leukocyte counts were indicated to be the predictive markers of severe pneumonia11. The risks of underestimating the overlapping clinical symptoms of malaria and severe pneumonia could lead to missed treatment for both diseases14.In areas where malaria is endemic, the clinical manifestations of malaria might overlap with those of bacteremia, meningitis, or pneumonia15. Another study suggested that although the actual overlap of severe pneumonia and malaria was uncommon, no reliable tools could distinguish between these two diseases11. Previous studies also indicated that children were positive for malaria in endemic areas, and children with respiratory signs, both pneumonia and malaria should be treated14. Although the differentiation of malaria from severe pneumonia had been attempted in literature16, the prevalence and probability of co-infection between the two diseases remain poorly explored. Malaria and severe pneumonia comorbidity are of wide interest to clinicians, public health professionals, and policymakers in considering the appropriate time to add antibiotics for the treatment of pneumonia in children with severe malaria. Hence, the present study aimed to collate the evidence of malaria among patients with severe pneumonia, severe pneumonia among patients with malaria, and the proportion of deaths among patients with co-infections.The previous study showed that among children admitted to the hospital with severe pneumonia, 19% of them had malaria17. Potentially relevant studies were searched in six databases including PubMed, Scopus, Web of Science, Embase, Ovid, and MEDLINE to identify malaria and severe pneumonia co-infection in children admitted to hospitals published until 21 July 2022. There was a restriction for non-English language but no restriction for the publication year. The combination of search terms “” AND (Pneumonias OR Pneumonia OR “Lung Inflammation” OR “Lung Inflammations” OR “Pneumonitis” OR “Pneumonitides” OR “Pulmonary Inflammation” OR “Pulmonary Inflammations”) AND (severe OR complicated)” were used to identify relevant studies (Table The systematic review was registered at PROSPERO (ID: CRD42021290750). The systematic review and meta-analyses adhered to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelineses Table .18.Malaria: The occurrence of malaria infection in a person in whom the presence of malaria parasites in the blood has been confirmed by a diagnostic test such as microscopy, serology, or rapid diagnostic testing (RDT)19.Severe pneumonia: Children who present with any general danger sign or chest drawing or stridor in calm child according to the Integrated Management of Childhood Illness (IMCI) definition which is developed by the World Health Organization (WHO)Malaria and severe pneumonia co-infections: Children who present with both malaria and severe pneumonia.PICo was applied for eligibility criteria; P: participants hospitalized for severe pneumonia or malaria, I: outcome of interest was co-infections, Co: context was endemic regions for malaria. Studies that reported the co-infection of malaria and severe pneumonia in participants were examined for inclusion. Study designs could be cross-sectional studies, cohort studies, case–control studies, retrospective or prospective observational studies, or clinical trials. Short reports, case series, case reports, letters to the editor, review, systematic review, comments, and opinions were excluded.Plasmodium spp., number of malaria, number of severe pneumonia, clinical outcomes of co-infection, number of deaths among patients with co-infection, diagnostic methods for malaria, and diagnostic methods for severe pneumonia. Similar to study selection, two authors  participated in data extraction, and the third author (PW) adjudicated if the first two authors did not agree.Two authors  used the predetermined eligibility criteria to screen titles, abstracts, and full texts of relevant studies. Meanwhile, a third author (PW) adjudicated if the first two authors did not agree. After relevant studies were selected, the following data were extracted from each study to the pilot excel datasheet; first author, publication year, study sites, characteristics of participants, age, sex , number of malaria and severe pneumonia co-infection, 20. A total of 22 items  were used to assess the overall quality of the included studies. We also assessed the methodological quality of the included studies based on nine items including study design, setting, participants, variables, data sources/measurement, bias, study size, quantitative variables, and statistical methods . Any study rated more than 75% indicated a high-quality study. Meanwhile, any study rated less than 75% showed moderate/low quality.The quality of the included studies was determined using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) which provided the checklist items for observational studies21. Results of the meta-analysis were shown as effect estimates, 95% confidence interval (CI), and weights in forest plots. The meta-regression analysis based on the methodological quality of the studies, publication year, country, study design, age groups, and diagnostic methods for malaria had been performed to test whether these parameters were the source (s) of heterogeneity of the pool proportion of the effect estimates or not. Subgroups analysis of covariates that were significant in the meta-regression analysis were performed to inspect the difference in the individual subgroup. Between-study heterogeneity was estimated using the I2 statistic to determine between-study heterogeneity of effect estimates. I2 statistic with < 50%, and 50–75%, and > 75% indicating moderate, substantial, and high heterogeneity22. Sensitivity analysis using the different diagnostic criteria for severe pneumonia was performed to determine the robustness of the meta-analysis results if some studies using non-standardized criteria for severe pneumonia confounded the effect size calculated by the random-effects model. Publication bias among the included studies was not performed due the meta-analyses were based on the prevalence data. All statistical analyses were carried out using Stata version 14 .Outcomes of interest were (1) the proportion of malaria among patients with severe pneumonia, (2) the proportion of severe pneumonia among patients with malaria, and (3) the proportion of deaths among patients with co-infection. The pooled estimates, including the pooled proportion of malaria among patients with severe pneumonia, the pooled proportion of severe pneumonia among patients with malaria, and the pooled proportion of deaths among patients with co-infection, were estimated by the random-effects model29 met the eligibility criteria and were included in qualitative and quantitative syntheses , Embase (997 studies), PubMed (678 studies), Scopus (641 studies), Web of Science (333 studies), and MEDLINE (308 studies). After 1429 duplicates were excluded, the remaining 2665 studies were screened for titles and abstracts, and 2548 non-relevant studies were excluded. Then the remaining 117 studies were assessed for eligibility, and 106 full-text articles were excluded, for specific reasons; 28 none severe pneumonia cases, 18 no records of articles, 10 malaria and bacteremia co-infection (no severe pneumonia), 10 full-texts unavailable, 9 severe pneumonia only , 8 no malaria and severe pneumonia co-infection, 6 malaria only (no severe pneumonia), 6 data on severe pneumonia and malaria were unable to extract, 4 review/systematic reviews, 3 diagnostic test comparison, 3 conference abstracts, and 1 with knowledge assessment about severe pneumonia/malaria. Finally, 11 studies30, Tanzania 29, Kenya 24, Nigeria 12, Uganda 26, and Malawi 25. Most of the studies were prospective observational studies , prospective and retrospective observational studies , and retrospective observational studies . Most of the included studies did not report species of Plasmodium , meanwhile, two studies (18.2%)27 reported P. falciparum. Five studies (45.5%)27 reported deaths among co-infected patients, while others did not. The microscopic method was the most frequently used for malaria parasite detection . In contrast, other studies used RDT 25, microscopy/RDT 12 for malaria parasite detection, and two studies 29 did not report a method for parasite detection. Five studies (45.5%)27 used the IMCI definition for pneumonia (WHO defined severe pneumonia) and four studies (36.4%)23 used chest X-rays for diagnosing severe pneumonia  were published between 2011 and 2022. Most of the included studies were conducted in Mozambique 29; meanwhile, one study was moderate quality7. Based on the methodological quality with overall nine scores, 10 studies (90.9%) were high-quality studies29; meanwhile, one study was of moderate quality26. The details of quality assessments are provided in Table Based on overall 22 scores, 10 studies (90.9%) were high-quality studies29. The highest proportion of malaria among patients with severe pneumonia was demonstrated in the study conducted in Kenya during 1999–2000 7. Meanwhile, the lowest proportion of malaria among patients with severe pneumonia was demonstrated in the study conducted in Tanzania during 2007–2008 16. Overall, meta-analysis results showed that the pooled proportion of malaria (2162 cases) among patients with severe pneumonia (9738 cases) was 19%  of heterogeneity of the pool proportion of the effect estimates or not. The meta-regression results showed that publication years (P = 0.049) and study design (P = 0.017) were the sources of the heterogeneity of the effect estimates. Meanwhile no statistical difference in the methodological quality of the studies (P = 0.096), diagnostic methods for malaria (P = 0.98), diagnostic methods for severe pneumonia P = 0.162), country (P = 0.43), and age groups (P = 0.924) in the meta-regression analysis were found . Meanwhile, the pooled proportion among patients with severe pneumonia in the year 2000–2010 was 27% , and 1% , respectively. Meanwhile, the pooled proportion of malaria among patients with severe pneumonia among studies that performed both prospective and retrospective observational studies was 38% . Meanwhile, the lowest prevalence of severe pneumonia among patients with malaria was 2% (95%CI 2–3%). Overall, the pooled proportion of severe pneumonia (546 cases) among patients with malaria 10,325 cases was 20% , and study design (P = 0.378), the methodological quality of the studies (P = 0.93), diagnostic methods for malaria P = 0.721), diagnostic methods for severe pneumonia (P = 0.916), country , and age groups (P = 0.603) that affect the pooled estimates  than those with severe pneumonia without malaria . Källander et al.26 reported that malaria contributed to 9% (4/44) of pneumonia deaths. O’Callaghan-Gordo et al., reported that patients with co-infection had a comparable proportion of deaths  compared to those without malaria . Sigaúque et al.28 reported that 27% (16/59) of those with malaria and severe pneumonia co-infection led to deaths.Four studies28. The highest proportion of deaths among patients with co-infection was 27% (95% CI 17–40%). Meanwhile, the lowest proportion of deaths among patients with co-infection was 5% (95% CI 3–9%). Overall, meta-analysis results showed that the proportion of deaths among patients with co-infection was 13%  and were not included in the pooled proportion of deaths. The meta-regression analysis was not performed due to only three studies included in the meta-analysis.The proportion of deaths among patients with co-infection was estimated using the available data from three studies2: 99.06%, 4 studies, Fig. 2: 98.23%, 5 studies, Fig. The sensitivity analysis was performed to demonstrate the pooled proportion of malaria and severe pneumonia among studies that used chest X-ray and those that used WHO criteria for severe pneumonia (IMCI definition). Results showed that the pooled proportion of malaria and severe pneumonia among studies that used chest X-rays to confirm severe pneumonia was 11% . In addition, subgroup analysis showed the pooled proportion of malaria among patients with severe pneumonia was highest among both prospective and retrospective observational studies (38%). Therefore, the difference in the year of study or the study design might demonstrate the difference in the proportion of malaria among patients with severe pneumonia. In addition, the possible reason for the low proportion of malaria among severe pneumonia might be explained by the fact that patients with co-infection reported by the included studies were not representative of all malaria cases in a malaria-endemic area. For example, in the study by Rubach et al.7 that conducted the study in Kenya. Meanwhile, only 2% of patients with malaria have severe pneumonia in Graham et al.12 that conducted the study in Nigeria. The possible explanations were the difference in the study sites as all four studies were conducted in different countries of Africa. Nevertheless, based on the limited number of studies, the information was not sufficient for meta-regression analysis using country as a covariate. Furthermore, no probable candidate factor that might explain the difference in the proportion of severe pneumonia among patients with malaria in the meta-regression analysis exists.The present study showed that nearly one-fifth of patients with malaria have severe pneumonia. There was a difference in the proportion of severe pneumonia among patients with malaria between studies included in the meta-analysis. The highest proportion of severe pneumonia among patients with malaria (53%) was found in English et al.12. However, there are some local hospitals where blood cultures and chest X-rays are not performed. Moreover, the only combination of any clinical signs might not be able to reliably distinguish between malaria and severe pneumonia, especially in the absence of chest X‐ray facilities12. The laboratory tests for severe pneumonia including blood culture are also exploited as biomarkers to diagnose the type of real etiology in patients such as bacterial or viral severe pneumonia. There are limitations of blood cultures in the diagnosis of childhood pneumonia that the result was only positive in a small number of patients32. Therefore, half of the severe pneumonia cases were not given a diagnosis30, turning results in a high proportion of malaria among severe pneumonia or a low proportion of malaria among patients with severe pneumonia. A previous study showed that positive malaria testing was associated with a lower likelihood of a pneumonia diagnosis, indicating that physicians had a lower underestimation of pneumonia in children without respiratory signs12. In addition, patients with malaria admitted to the hospital might not have respiratory symptoms, and malaria incidence was dropped in many African countries33, affecting the number of severe pneumonia diagnosed in the hospitals in malaria-endemic countries. There were other factors that affect the proportion of malaria among patients with severe pneumonia or severe pneumonia among patients with malaria. For example, malaria infection caused respiratory complications that lead to pneumonia34. In addition, malaria and severe pneumonia co-infections might increase the odds of HIV infection as some included studies enrolled patients with HIV27. The impact of HIV on clinical malaria had been exclusively described in the previous systematic review37. The co-infection of malaria and HIV could lead the patients to severe malaria35 and might impact the clinical presentation of severe pneumonia11. In addition, HIV-related pneumonia had been reported as a cause of hospital admission38.The current diagnostic practices of both diseases are based on the clinical findings, chest radiology, culture, and malaria testing, including microscopy/RDT28 demonstrated 27% deaths among patients with co-infection; meanwhile, one study25 demonstrated 5% deaths among patients with co-infection. Sigaúque et al.28 which reported the highest proportion of deaths among patients with co-infection at 27% showed that fever and severe anemia were more frequent when clinical malaria and severe pneumonia were present together. In addition, the case fatality rate (CFR) was higher (14%) among co-infection compared with clinical malaria alone (9%)28. Eckerle et al.25 reported the lowest proportion of deaths among patients with co-infection at 5% showing that children who died were more likely to have malaria and lower hemoglobin than those who survived. The difference in the proportion of deaths between studies might be explained by a difference in the methodology quality among those two studies. Those two studies used different criteria for diagnosing both malaria and severe pneumonia. While Sigaúque et al.28 used chest X-rays as a criteria for diagnosing severe pneumonia; Eckerle et al.25 used IMCI definitions that lack specificity. Eckerle et al.25 used RDT for diagnosing the malaria; meanwhile, Sigaúque et al.28 used microscopy which is the gold standard for diagnosing the malaria. Malaria cases might be missed in studies using RDT for diagnosing malaria, and severe pneumonia might be misdiagnosed causing the lower proportion of co-infection and deaths. There has been reported that the poor and missed diagnosis of pneumonia and severe pneumonia, especially in malaria-endemic areas that did not recognize severe pneumonia, has caused respiratory signs in children and is misdiagnosed as severe malaria13. This leads to inappropriate treatment of two diseases and can increase the mortality rate. Therefore, a high level of awareness among physician who manage the overlapping of these two infections is required if the diagnostic criteria for both infections is not standardized. The previous study revealed that most children who died from pneumonia were first treated as malaria, which caused delays in seeking healthcare facilities40.Results of the meta-analysis showed the difference in the proportion of deaths among patients with malaria and severe pneumonia co-infection. One studyThe present systematic review had limitations. First, as there were a limited number of studies reporting malaria and severe pneumonia co-infection, some of the subgroup analyses contained two or three studies which might affect the robustness of the data. Second, there was high heterogeneity in the pooled prevalence of co-infection between studies. Therefore, a careful interpretation of the pooled estimates was suggested. Third, there was some publication bias caused by the small-study effect. Fourth, some forest plots that present a combined measure with only two studies might reduce the statistical rigor of the present study. Fifth, there was an uncertainty in the diagnosis of severe pneumonia. Some studies used chest X-rays as a diagnostic test for severe pneumonia and some studies did not. Therefore, there were uncertainty in the diagnosis of severe pneumonia.In conclusion, nearly one-fifth of patients with severe pneumonia have malaria, one-fifth of patients with malaria have severe pneumonia, and about 13% of co-infections led to deaths. This information raised the clinical importance of diagnosis and management of concurrent infections. Patients with severe pneumonia should be investigated for malaria, and vice versa. Detection of co-infections might provide the information to inform the physician to manage and cure co-infected patients who live in areas where both diseases are endemic.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Information 4.Supplementary Information 5.