Plasmodium infection, remains a major global health problem. Monocytes are integral to the immune response, yet their transcriptional and functional responses in primary Plasmodium falciparum infection and in clinical malaria are poorly understood.Malaria, caused by P. falciparum blood stages and in children and adults with acute malaria. Monocyte gene expression and functional phenotypes were examined by RNA sequencing and flow cytometry at peak infection and compared to pre‐infection or at convalescence in acute malaria.The transcriptional and functional profiles of monocytes were examined in controlled human malaria infection with in vitro toll‐like receptor stimulation and increased IL‐10 production upon in vitro parasite restimulation. Longitudinal phenotypic analyses revealed sustained significant changes in the composition of monocytes following infection, with increased CD14+CD16− and decreased CD14−CD16+ subsets. In acute malaria, monocyte CD64/FcγRI expression was significantly increased in children and adults, while HLA‐DR remained stable. Although children and adults showed a similar pattern of differentially expressed genes, the number and magnitude of gene expression change were greater in children.In subpatent primary infection, the monocyte transcriptional profile was dominated by an interferon (IFN) molecular signature. Pathways enriched included type I IFN signalling, innate immune response and cytokine‐mediated signalling. Monocytes increased TNF and IL‐12 production upon Monocyte activation during subpatent malaria is driven by an IFN molecular signature with robust activation of genes enriched in pathogen detection, phagocytosis, antimicrobial activity and antigen presentation. The greater magnitude of transcriptional changes in children with acute malaria suggests monocyte phenotypes may change with age or exposure. P. falciparum infection in malaria naive healthy subjects and during an episode of clinical malaria in children and adults residing in a malaria‐endemic region. We specifically isolated monocytes for transcriptome analysis by RNA sequencing, to determine monocyte gene expression during subpatent and clinical malaria. Monocyte activation during subpatent malaria is driven by an inflammatory molecular signature and a greater magnitude of transcriptional changes in children with acute malaria suggest monocyte phenotypes may change with age or exposure.We investigate the transcriptional, functional and phenotypic changes in monocytes during an experimental blood‐stage  Plasmodium falciparum.Plasmodium parasites firstly infect hepatocytes to mature and multiply before release into the bloodstream and immediate infection of red blood cells (RBCs). Blood‐stage infection is characterised by cycles of asexual replication leading to RBC rupture and periodic malaria symptoms. Despite recent gains in reducing the burden of malaria, progress has stagnated in the last 3–5 years, with morbidity rising in several highly endemic countries.Malaria remains an important global disease, with an estimated 228 million cases and 405 000 deaths in 2018, the majority caused by Plasmodium infection may aid the development of strategies to enhance protective responses to improve the morbidity and mortality of malaria and assist in vaccine development.Innate and adaptive immune responses mediate both tolerogenic and antiparasitic protective mechanisms that can result in reduced clinical symptoms in future reinfections,Plasmodium infection,Monocytes express a broad range of pattern recognition receptors, including toll‐like receptors (TLR),+CD16−), intermediate (CD14+CD16+) and non‐classical (CD14−CD16+) monocytes.P. falciparum.Plasmodium infection can change the composition of circulating monocytes. In children with asymptomatic+ intermediate/non‐classical monocytes increase proportionally in the peripheral blood. However, there is a lack of information regarding prospective changes to monocyte composition during primary blood‐stage infection and acute malaria in non‐immune adults. Recent whole‐blood transcriptional analysis suggests that monocytes may have an anti‐inflammatory role in antimalarial immunity.P. falciparum infectionThree distinct subsets of monocytes are identified by differential expression of the LPS receptor (CD14) and FcγRIII (CD16), and are defined as classical , antigen presentation, neuroinflammation signalling , triggering receptor expressed on myeloid cells‐1 (TREM‐1) signalling, role of pattern recognition receptors in recognition of bacteria and viruses , activation of IRF by cytosolic pattern recognition receptors, phagosome formation [including FCGR1A (CD64)], FcγR‐mediated phagocytosis and production of reactive oxygen (RO) species  , cytokine‐mediated signalling, type I IFN signalling, regulation of immune system process, cell surface signalling , IFN‐γ‐mediated signalling, signal transduction, regulation of innate immune response [including CD40 and signal transduction (including chemokine CCL2 (MCP‐1) and its receptor CCR2)] , we identified DEG enrichment of canonical pathways, including IFN signalling, communication between innate and adaptive immune cells , and other inflammatory cytokines , pattern recognition receptors (TLR3 and TLR9), and TNF superfamily member CD40LG (CD40 ligand) , prostaglandin E2 receptor 4 (PTGER4), suppressor of cytokine signalling 1 (SOCS1) and interleukin 1 receptor antagonist (IL1RN), and tripartite‐motif protein 24 (TRIM24), the negative regulator of p53 stability.Using IPA, we further identified predicted upstream regulators of the DEGs Figure , with pr) Figure . UpstreaPlasmodium infection in adults.P. falciparum blood‐stage infection is dominated by an IFN‐driven signature accompanied by pathways relating to pathogen detection, phagocytosis, antimicrobial activity and antigen presentation.Across all identified pathways, the magnitude of response varied across participants Figure , consistP. falciparum infection  is rapidly lost during short‐term culture and stimulation.slamf7 gene expression , respectively; Supplementary figure P = 0.06), with a significant increase in TNF/IL‐10 co‐production in response to pRBC restimulation (P = 0.04)  Figure . There wP. falciparum blood‐stage infection, monocytes responded to TLR stimulation with increased inflammatory cytokine production, while they co‐produced regulatory cytokines in response to restimulation with P. falciparum parasites, that is increased IL‐10 producers, suggesting that during subpatent P. falciparum malaria monocytes develop a regulatory capacity, potentially to minimise tissue damage following chronic activation.Taken together, these data suggest that during a subpatent P. falciparum infection, classical (CD14+CD16−) monocyte frequency increased significantly on day 15 and remained significantly elevated at 45 days after infection  monocytes showed a corresponding decrease  gene expression was significantly increased in isolated monocytes at day 8 of blood‐stage infection. In accordance with the literature,CD40 gene expression following P. falciparum infection, we observed increased CD40 cell surface expression on all three monocyte subsets, with expression also remaining significantly elevated 45 days after infection  and non‐classical (CD14dimCD16+) monocytes. CD40 is a member of the TNF receptor family, and increased expression of CD40 on monocytes is reported to increase monocyte pro‐inflammatory cytokine production and expression of other co‐stimulatory receptors.We further measured cell surface marker expression relating to activated pathways. CC‐chemokine receptor 2 ; and mitogen‐activated protein kinase kinase kinase 1 (MAP3K1), a molecule involved in TNFR signalling] and NF‐κB signalling . OAT, the gene for ornithine aminotransferase, an enzyme involved in L‐proline metabolism from L‐ornithine, was also differentially expressed in adults, suggestive of arginine degradation  in both adults and children, and SLAMF7 and FCER1G, the Fc receptor gamma chain (FcRγ)FCGR1A, the gene for the high‐affinity Fc‐gamma receptor CD64, and FCGR1B (CD64b) in children]  monocytes was significantly higher in children during acute infection than convalescence  in children .in vitro TLR agonists such as LPS.In line with IFN‐γ having the highest activation score of the predicted upstream regulators, monocytes displayed a heightened responsiveness to TLR ligands during subpatent infection. This is in accordance with previous reports of PBMC hyper‐responsiveness to TLR ligation in febrile malaria.Plasmodium infection, with CD40LG being amongst the predicted activated upstream regulators. CD40 is considered a marker of monocyte activation, and CD40 ligation increases monocyte production of inflammatory cytokinesBesides IFNs, the CD40/CD40 ligand axis may contribute to monocyte activation following P. falciparum sporozoite infection.P. falciparum carriers during the malaria season in Mali.P. falciparum liver stages, and warrants further investigation into the control (and timing) of upstream regulators.Predicted inhibited upstream regulators of monocyte gene expression in subpatent primary infection included MAPK1, IL1RN and TRIM24, which overlapped with regulators predicted to be inhibited in malaria‐naive volunteers experiencing symptomatic malaria following P. falciparum infection with sporozoites,Plasmodium‐infected reticulocytesP. falciparum infection.+ DC,Multiple lines of evidence suggest that during malaria, monocytes also have important roles in initiating adaptive responses.Plasmodium falciparum infection resulted in changes in monocyte composition and phenotype which was sustained for up to 1 month after a first infection. These results suggest that P. falciparum may drive ‘innate immune training’,+CD16−) monocytes and reduced proportions of non‐classical (CD14−CD16+) monocytes in the periphery until at least day 45 following infection. In homeostasis, monocytes migrate from the bone marrow and can circulate in the periphery for approximately 1 day.P. falciparum infection may be a consequence of a sustained increase in bone marrow production of monocytes. Consistent with this, we show increased transcription and cell surface expression of CCR2, which mediates monocyte emigration from the bone marrow.Toxoplasma gondii infection.P. falciparum was suggested to be indicative of genomewide chromatin remodelling.During clinical malaria, the monocyte transcriptional profile had both similarities  and differences  to that seen during primary subpatent infection. Phagocytosis pathway was one pathway upregulated in both malaria cohorts and is a key function of monocytes in protective immunity.In acute clinical malaria, we show children have higher transcriptional activation of monocytes and more DEGs than adults. Despite greater transcriptional activation, the upstream regulators predicted for adults and children were similarly inflammatory, including IFN‐γ in children and IL‐6 and TREM1 (triggering receptor expressed on myeloid cells‐1) in adults. TREM1 is associated with amplifying the inflammatory response in sepsisIn summary, using transcriptional, functional and phenotypic analyses, we reveal monocyte function during primary subpatent infection as dominated by an IFN molecular signature, with increased proportion of activated classical monocytes. Alongside this inflammatory phenotype, monocytes increased IL‐10 production upon re‐exposure to parasite‐infected RBCs, suggesting a role in controlling inflammation upon multiple parasite exposure. Of note, malaria‐naive volunteers maintained phenotypic changes to monocytes up to 1 month post‐infection. Further studies are needed to understand whether these sustained changes are maintained upon repeat infection in previously malaria‐naive volunteers. Differences in transcriptional activation observed between adults and children with clinical malaria warrant further investigation to understand the biological basis driving these differences and the impact on immunity. Our data suggest that monocytes are active and integral immune cells assisting parasite elimination and controlling inflammation in malaria.ClinicalTrails.gov . Blood‐stage parasitaemia was initiated by inoculation of 1800 or 2800 parasitised RBCs (pRBC) as previously described.P. falciparum 3D7‐pRBCs, and peripheral parasitaemia was measured by qPCR as described previously.Nineteen volunteers aged 18–31 years  consented to participate in a phase Ib clinical trial testing the efficacy of antimalarial drugs. All studies were registered with US NIH P. falciparum malaria as part of artemisinin combination therapy efficacy studies conducted in southern Papua, Indonesia.Peripheral blood mononuclear cells (PBMCs) were collected from 15 adults aged 18–38 years  and eight children aged 5–13,  with acute uncomplicated IBSM was approved by the Human Research Ethics Committees of QIMR Berghofer Medical Research, NT Department of Health and Menzies School of Health Research. In the clinical malaria cohorts, written informed consent was obtained from participants, with the study approved by the Human Research Ethics Committees of the National Institute of Health Research and Development, Indonesian Ministry of Health , the NT Department of Health and Menzies School of Health Research.P. falciparum pRBC and on acute and convalescence samples from adults and children with acute clinical malaria. IBSM and clinical samples were selected based on the availability of PBMCs at paired time points. Isolation and RNA sequencing for IBSM and clinical samples were performed at separate times by different operators. Monocytes were isolated from PBMCs using the Pan Monocyte Isolation Kit . In brief, monocytes were enriched by indirect magnetic labelling for the isolation of untouched monocytes , from human PBMCs. Non‐monocytes, such as T cells, NK cells, B cells, dendritic cells and basophils, were indirectly magnetically labelled using a cocktail of biotin‐conjugated antibodies to anti‐biotin microbeads. Conjugated microbeads and untouched monocytes were washed over MAC isolation columns. Purity of isolated monocyte populations was checked using flow cytometry IBSM  and clinical cohorts . The distribution of subsets in isolated monocytes was comparable to pre‐sorted populations  using the Illumina TruSeq Stranded mRNA LT Sample Kit and the HiSeq 2500 instrument. Transcriptome data were analysed using a modified version of an existing variant detection pipelineRNA sequencing was performed on paired samples collected prior to and at peak infection from five subjects experimentally infected with % IQR = 8–98, conv+ and by differential expression of CD14 and CD16. In brief, 200 µL of whole blood or 1 million PBMCs were stained with surface antibodies, CD3 , CD14 , CD19 , CD56 (HCD56), HLA‐DR (L243), CD11c , CD16 (3G8), CD86 , CD319 , CD40 (5C3), CD32 (FUN‐2), CD64 (10.1), CCR2 (K036C2), CD206 (15‐2), CXCR4 (12G5) and CD163 (GHI/61), all antibodies were purchased from BD Biosciences  or BioLegend . For whole blood, RBCs were lysed with FACS lysing solution (BD Biosciences) and resuspended in 2% FCS/PBS to acquire.Monocytes were characterised as lineage ‐negative, HLA‐DR8 cells per mL, TLR4: Escherichia coli K12 LPS 200 ng mL−1 or TLR7: imiquimod 2.5 µg mL−1 , pRBC or unparasitised RBC (uRBC) prepared as previously described6/mL. Protein transport inhibitor  was added after 2 h at 37 °C, 5% CO2. At 6 h, cells were stained to identify monocytes: lineage − and CD14+ (M5E2), and RBCs were lysed with FACS lysing solution (BD Biosciences), washed with 2% FCS/PBS, cells permeabilised with 1× Perm/Wash™ (BD Biosciences) and stained with intracellular anti‐TNF‐α (MAB11), IL‐12/IL‐23p40 (C11.5), IL‐10 (JES3‐9D7) or IgG1 isotype controls (BioLegend). To determine the stimulant‐specific response, spontaneous cytokine production was subtracted from responses to pRBC, uRBC or TLR agonists.Monocyte cytokine production was assessed in 1 mL of fresh whole blood unstimulated or stimulated with TLR agonists; TLR1: Pam3CSK4 100 ng per mL/TLR2: HKLM 10® 1.3 (Beckman Coulter) or FlowJo version 10.6 (BD Biosciences).FACS data were acquired using a FACSCanto™ II (BD Biosciences), Gallios™  or Fortessa 5 laser (BD Biosciences) and data analysed using Kaluzapost hoc statistics, a pairwise sign test with the Bonferroni correction for multiple comparisons was performed. For clinical cohorts, the Wilcoxon matched‐pairs signed‐rank test was used as paired data. Tests were two‐tailed and considered significant if P‐values < 0.05. For pathway enrichment, the online tool STRING version 11Statistical analyses and graph generation performed using R studio and GraphPad Prism. The Friedman multiple comparisons test was used to compare longitudinal data in the IBSM cohort. For The authors declare no conflict of interest.Jessica R Loughland: Formal analysis; Investigation; Visualization; Writing‐original draft; Writing‐review & editing. Tonia Woodberry: Conceptualization; Funding acquisition; Investigation; Methodology; Supervision; Writing‐review & editing. Matt Field: Data curation; Formal analysis; Methodology; Software; Writing‐review & editing. Dean W Andrew: Methodology; Writing‐review & editing. Arya SheelaNair: Methodology; Writing‐review & editing. Nicholas L Dooley: Methodology; Writing‐review & editing. Kim A Piera: Methodology; Project administration; Writing‐review & editing. Fiona H Amante: Project administration; Writing‐review & editing. Enny Kenangalem: Project administration; Supervision; Writing‐review & editing. Ric N Price: Funding acquisition; Project administration; Resources; Writing‐review & editing. Christian R Engwerda: Funding acquisition; Project administration; Resources; Writing‐review & editing. Nicholas M Anstey: Conceptualization; Funding acquisition; Project administration; Resources; Writing‐review & editing. James S McCarthy: Funding acquisition; Project administration; Resources; Writing‐review & editing. Michelle J Boyle: Funding acquisition; Investigation; Resources; Supervision; Writing‐review & editing. Gabriela Minigo: Conceptualization; Investigation; Methodology; Supervision; Writing‐review & editing.Supplementary figures 1‐7Click here for additional data file.Supplementary tables 1‐3Click here for additional data file.


Theileria parva is TpUB05 (XP_763711.1), which has been tested and shown to be associated with protective immunity in East Coast fever. In a bid to identify potent markers of protective immunity to aid malaria vaccine development, TpUB05 was tested in malaria caused by Plasmodium falciparum. It was observed that TpUB05 was better at detecting antigen-specific antibodies in plasma compared to UB05 when tested by ELISA. The total IgG raised against TpUB05 was able to block parasitic growth in vitro more effectively than that raised against UB05. However, there was no significant difference between the two study antigens in recalling peripheral blood mononuclear cell (PBMC) memory through IFN-γ production. This study suggests, for the first time, that TpUB05 from T. parva cross-reacts with UB05 from P. falciparum and is a marker of protective immunity in malaria. Hence, TpUB05 should be considered for possible development as a potential subunit vaccine candidate against malaria.Despite the amount of resources deployed and the technological advancements in molecular biology, vaccinology, immunology, genetics, and biotechnology, there are still no effective vaccines against malaria. Immunity to malaria is usually seen to be species- and/or strain-specific. However, there is a growing body of evidence suggesting the possibility of the existence of cross-strain, cross-species, and cross-genus immune responses in apicomplexans. The principle of gene conservation indicates that homologues play a similar role in closely related organisms. The homologue of UB05 in  The malaria parasite has a complex life cycle, consisting of developmental stages in the liver (sporozoites) and blood  of vertebrates and Anopheles mosquitos , which serve as transmission vectors . AntigenClinical manifestations of malaria are attributed to the blood-stage of parasites that reside within red blood cells (RBCs); thus, vaccines developed against erythrocytic forms of the parasite may contribute considerably to the efficient control of the disease. There are now a number of blood-stage antigens that are being characterized for inclusion as vaccine components in clinical development . AmongstPlasmodium vivax, containing conserved CSP regions, generated antibodies that could also recognize CSP on the surface of P. berghei and P. falciparum. Antibodies provoked in mice by recombinant PfCelTOS induced cross-species protection against P. berghei challenge by cross-reactivity to heterologous P. berghei sporozoites [Theileria parva, TpUB05, has also been shown to be a marker of protective immunity in East Coast Fever (ECF) that affects cattle [One approach of identifying better markers of protective immunity would be to seek homologues of markers of protective immunity in closely related organisms. The idea of the conservation of gene products has been proven to suggest that orthologous gene products appear to perform similar functions in closely related species . For exarozoites . With ths cattle .P. falciparum antigens UB05, UB09, and chimeric UB05-09 and their respective polyclonal antiserums were analyzed using ELISA, ELISpot assays, and growth inhibition assays with samples from a malaria endemic region. These findings have been published in [P. falciparum as a control antigen to that of TpUB05 from T. parva in human malaria.In previous studies from our group, the ished in ,13,14. DTpUB05 was amplified from T. parva schizont RNA using reverse transcription to produce a 291 bp DNA fragment [UB05 was amplified from a P. falciparum cDNA library to produce a 243 bp DNA fragment [Escherichia coli cells, yielding recombinant fusion proteins migrating at 28 KDa and 26 KDa, respectively, in SDS-PAGE performed in 15% polyacrylamide. The recombinant fusion proteins contained a 6xHis tag, a S tag domain, and a 109 amino acid thioredoxin fusion protein partner. However, it was easier to overexpress and purify TpUB05 as compared to UB05.Using gene-specific primers, fragment , while Ufragment . Both frp = 0.0001) than children  semi-immune status (SIS), 34 frequently sick status (FSS), and 27 sick children (SC) samples were obtained; thus 101 samples were analyzed using ELISA. Plasma from the study subjects was tested with TpUB05 using ELISA and it was shown that SIS possessed greater amounts of antibodies compared to FSS. However, the difference was not observed to be statistically significant using analysis of variance (ANOVA) and Friedman tests. Adults (SIS + FSS) produced  value of 0.372) 3.62 times more antigen-specific antibodies to TpUB05 (t-test) and for the 74 adults . Both tests gave a p-value of 0.006  and FSS (0.34 for TpUB05 and 0.31 for UB05), the difference, which is statistically significant, does not appear to be biologically significant; however, the data suggest cross-reaction B.r = −0.393 and a significant p-value of 0.0001, there is a significant negative correlation between TpUB05-specific antibody levels and parasite load. What is driving this result is the one subject presenting 20 × 104 parasitemia and the four subjects presenting around 5–10 × 104 parasitemia. If those subjects are removed from the analyses, the correlation would disappear. Taken together, these results suggest that the responses to the two antigens are very similar.When the antibody levels to TpUB05 were plotted as a function of parasitemia, it was observed that that there was a negative correlation between parasitemia and antibody levels. This implies that the higher the specific antibody level, the lower the parasite density in the blood. In other experiments, antibody levels have been shown to be statistically higher in subjects without a fever than in subjects with a fever A,B. Withp = 0.0001 using t-test; In an attempt to investigate the possible ability of TpUB05 in T-cell mediated immunity we compared the T-cell responses of cohorts of semi-immune and susceptible malaria patients to these orthologous antigens. Sixty-three  peripheral blood mononuclear cell (PBMC) samples were used for a human ELISpot assay. More PBMCs from SIS produced IFN-γ than FSS when stimulated with the TpUB05 antigen (6 cells) was also used to assess the difference between TpUB05 and UB05 in recalling T-cell function by IFN-γ production. The magnitude of the response showed TpUB05 provokes more cells from SIS (average of 168 SFC/106 cells) to produce IFN-γ than FSS (average of 40 SFC/106 cells). The same trend was observed for UB05 .The determination of spot-forming cells per million (SFC/10p = 0.002) compared to those who had a fever (Using stimulatory index (SI), we also found significantly higher IFN-γ production by PBMCs from subjects with no fever , as described in the Materials and Methods section, and the data were compared with those obtained with the UB05-specific antiserum. Rabbit anti-TpUB05 antiserum was able to inhibit parasite growth in vitro significantly better than the performance of the anti-UB05 antiserum , indicating that these two are less cross-reactive than those directed against TpUB05 and UB05. The results presented in p = 0.0001 using ANOVA).In the positive control experiments, we tested in parallel two monoclonal antibodies available from BEI Resources, namely, anti-AMA1 and anti-EBAI1. Amongst the parasite strains tested, only the www.iedb.org, we also predicted the presence of human antibody epitopes in TpUB05 , amongst other cytokines and chemokines [p-values obtained when magnitudes of IFN-γ production were compared to fever status, as well as the significant negative correlation observed between magnitude of IFN-γ production and parasitemia, indicate that immune responses to TpUB05 are associated with protection against malaria. When these data were compared to those obtained with the UB05 antigen from P. falciparum [Studies to elucidate the mechanism(s) of the protective immune response to apicomplexan parasites implicate the role of the production of gamma interferon  has been shown to confound T-cell response assay results to malaria antigens. However, it is unlikely that there was a contribution of possible contaminating bacterial LPS to the observed responses in the present study, as the responses were specific to each of the groups and as the control antigen (UB05) was prepared under similar conditions and treated in the same way as the test antigen (TpUB05). Cytokines whose production appear to be influenced by LPS include IL-1beta and IL-6. However, IFN-gamma, whose production is not affected by LPS, was studied herein .T. parva have any effect on malaria parasite development by employing an in vitro growth-inhibition assay. The growth-inhibition assay, which involves impaired merozoite invasion and the subsequent development of parasites in erythrocytes, is currently being considered as one of most relevant assays to screen potential blood-stage vaccine candidates prior to moving to the stage of clinical development. Purified polyclonal total IgG induced in rabbits against TpUB05 from T. parva was able to significantly inhibit malaria parasite growth in vitro. This inhibition was statistically higher than that observed with the anti-UB05 polyclonal antibody as well as the anti-EBA175 monoclonal antibody, but not the anti-AMA1 monoclonal antibody. Hence, TpUB05 should be considered to be associated with protective immunity against malaria.The present study shows that TpUB05 possesses T-cell and B-cell epitopes that bind and recognize human MHC molecules and antibodies, respectively. This was confirmed using in silico algorithms. It was then necessary to determine if the polyclonal antibodies raised against TpUB05 from A bioinformatics comparison of TpUB05 and UB05 shows that they exhibit a degree of sequence homology, where 43.3% identity and 67% similarity was observed. The in silico analysis predicted the presence of human T-cell and antibody epitopes of TpUB05 and the cross-reactivity observed was expected, in view of the significant homology between the two antigens. However, TpUB05 may contain more potent B-cell epitopes compared to UB05 that are yet to be identified.Mycobacterium bovis (BCG vaccine) was used to vaccinate humans against M. tuberculosis [Onchocerca ochengi were protected against O. volvulus infection [Altogether, the results from this study imply that TpUB05 is associated with protection against malaria, and this confirms the notion that homologues could play similar roles in related organisms. This is in line with previous studies where rculosis  and whernfection . The potP. falciparum and T. parva. There is a strong cross-reactivity between TpUB05 and UB05 antigens, based on ELISA, ELISpot, and growth-inhibition data, strongly suggesting the use of TpUB05 for the development of a malaria vaccine.The importance of immune cross-reactivity may indicate the enhancement of protective immune responses amongst distantly related apicomplexans such as The study was carried out in Buea, which is an endemic center for malaria. Buea is a multi-ethnic town found along the flanks of Mount Cameroon in the southwestern region of the Republic of Cameroon. The prevalence of malaria in Buea varies between meso-endemic (dry season) and hyperendemic (rainy season) zones with perennial malaria transmission .T. parva: TpUB05  Subjects who were aged 18 years of age or older and who had been living in the study site for at least 3 years; (ii) subjects with no history of a malaria episode in the last 12 months, no fever, and parasitemia at sample collection, no use of a mosquito bed net, hence being exposed to mosquito bites, and no prophylaxis were designated to semi-immune subjects (SIS); (iii) subjects who had had at least one malaria episode in the last 12 months and had a fever an parasitemia at the time of sample collection were referred to as frequently sick subjects (FSS); and (iv) children aged five or younger who had a fever and parasitemia at the time of sample collection were referred to as sick children (SC). This cohort of individuals was highly selective and has been described previously . The ana2) = weight (kg)/height2 (m)It has been shown that being in good health reduces susceptibility to non-communicable and infectious diseases, including malaria . The assP. falciparum laboratory strains were used: FCR-1/FVO , 3D7 , and HB3  for the in vitro assay. Two field isolates, GH01 and SC01, were also used for the study, and were obtained from the Buea District Hospital and Solidarity Clinic, Buea, Cameroon, respectively. The overexpression of recombinant TpUB05 and its polyclonal antibody production in rabbits has been previously described [As previously mentioned , all labescribed .The procedure to isolate peripheral blood mononuclear cells (PBMCs) was performed according to manufacturer’s instructions. Briefly, the preparation of PBMCs for use in the ELISpot assay was done using the Percoll gradient method, as earlier described . Briefly6 cells were also used to determine the magnitude of response by PBMCs. The antigen-specific spots count was calculated as the mean of three wells minus the mean spot count from PBS negative control wells. Spot counts were multiplied by 3.33 to obtain spot-forming cells per million cultured PBMCs. All analyses were performed with these final counts, i.e., spots forming cells per million cultured PBMCs.The enzyme-linked immunosorbent spot assay was carried out as described . BrieflyPlasma was collected from the blood samples and stored until further analysis. At the time of performing the ELISA experiments, additional subjects were recruited into the study to obtain more plasma samples and increase the sample size for analysis. Plasma samples were also collected in EDTA tubes from children below the age of five whose parents gave their consent and tested positive for malaria (fever and parasitemia). The samples were then centrifuged and the plasma was aspirated and stored at −20 °C until use.E. coli extract or the r-UB05 antigen as a positive control for the antigen, while the tag-only (Fusion partner) antigen was used as a negative control antigen. The plates were washed thrice with 200 μL of a wash buffer  after overnight incubation, followed by blocking with 150 μL per well of 0.2% casein in PBS with 0.05% Tween-20. After the second washing, plasma was added at 1:150 dilutions in the wash buffer, containing 1% skimmed milk. The samples were then incubated at room temperature for 3 h. To obtain and record background signals (negative control for plasma), human plasma from malaria-naïve individuals, which was kindly given to us by Mrs. Philomena Gwanmesia of the Biotechnology Center, University of Yaounde I, Cameroon, was included in the assay. After the 3 h incubation at room temperature, the plates were washed with the wash buffer three times and 100 μL of the anti-rabbit IgG-HR.P. conjugate was diluted at 1:10,000 in a wash buffer containing 1% skimmed milk and then incubated for 1 h at room temperature, after which the plates were washed with the wash buffer and 100 μL of the substrate added. The optical densities (OD) of the wells were obtained at 405 nm using a microplate reader. A subject was considered positive if its OD value was equal to or greater than the mean control OD + 2 Standard Deviation (2SD). The experiment was run in duplicate.Antibody  measurement was carried out by ELISA, as earlier described , with soP. falciparum in vitro was tested by measuring parasite lactate dehydrogenase (pLDH) in the late trophozoite- /early schizont-stages in cultures, as described in Methods In Malaria Research 6th Edition [2 incubator for two cycles. Polyclonal antibodies raised against r-TpUB05 or r-UB05 were added at the start of the cultured at an optimized dilution of 1:10 . The monoclonal antibodies, anti-EBA-175 RII , and anti-AMA1 , were used as positive controls in the inhibition assays, while the negative control wells contained pre-immune sera  or no antiserum. The control wells were included in the assay that contained either only parasitized RBCs (pRBC only) or normal RBCs. Percentage growth inhibition was calculated by subtracting the average OD from the normal RBCs wells from all the other OD values obtained before proceeding to the calculation proper.The ability of purified rabbit IgGs induced against r-TpUB05 and r-UB05 to inhibit the replication of  Edition . Brieflywww.immuneepitope.org). The MHC I binding predictions were made on 7/30/2019 using the IEDB recommended method, which combines predictions from ANN [The in silico prediction of immune epitopes from TpUB05 and UB05 was done using algorithms from IEDB Analysis Resources . A value of Ethical clearance for this study (Ref.: 2013/144/UB/FHS/IRB) was obtained from the Institutional Review Board of the Faculty of Health Sciences, University of Buea. All participating adult subjects read, approved, and signed the consent form. A parent or guardian of any child participating in the study provided informed consent on the child’s behalf. The informed consent given was written. Ethical clearance for blood collection and polyclonal antibody production in rabbits was approved by the International Livestock Research Institute—Institutional Animal Care and Use Committee . ILRI-IACUC provided clearances based on “The Animal Research: Reporting In Vivo Experiments” (ARRIVE) guidelines on the care and use of animals in research.


Plasmodium falciparum born to nonimmune women, who typically present at birth or soon thereafter. Semi-immune women are less likely to transmit malaria, and their children often become ill weeks after delivery with less severe symptoms. Cases in the USA usually trace to semi-immune immigrant mothers whose last exposure to malaria may have preceded the pregnancy, leading to misdiagnoses. Congenital malaria may be under-recognized in malaria-endemic areas since parasitemia occurring after the first week of life is usually attributed to mosquito transmission. Malaria prophylaxis and the absence of fever during pregnancy do not preclude congenital malaria in a newborn. Quinine plus clindamycin is commonly used to treat P. falciparum congenital malaria, and chloroquine is used to treat other malaria parasites, such as Plasmodium vivax. Severe cases should be managed with intravenous quinine (available as its enantiomer quinidine in the USA) or with intravenous artesunate, which was recently approved for investigational use by the US FDA. Primaquine is not required for infants with congenital P. vivax or Plasmodium ovale, but should be offered to their mothers after excluding G6PD deficiency.Congenital malaria is rare and usually indolent but can be fatal. Mortality risk is high in newborns with  Since the deadliest malaria parasite, Plasmodium falciparum, sequesters in the maternal vascular spaces of the placenta, sometimes at extraordinarily high densities, the infrequent occurrence of congenital malaria cases has puzzled observers for more than a century. Despite the general observation that congenital malaria is uncommon and often indolent in its appearance, its true incidence is controversial and its outcome may be rapidly fatal when the mother lacks immunity. Clinicians should be alert to the possibility of congenital malaria in febrile neonates when the mother is from or has recently traveled to an endemic area, even if she reports no illness during pregnancy Malaria will cause half a billion clinical episodes and one million deaths this year, with most deaths occurring in young African children. In areas of stable transmission, malaria has a distinct epidemiologic signature at the mother–newborn interface: pregnant women are relatively susceptible and newborns are relatively resistant P. falciparum and most Plasmodium vivax as well. According to the few available studies, susceptibility to the less common malaria species Plasmodium malariae and Plasmodium ovale remains unchanged during pregnancy Pregnancy increases susceptibility to P. falciparum assumes a novel phenotype during pregnancy that may explain maternal susceptibility. Mature parasite stages sequester in the intervillous  vascular spaces of the placenta by binding to the receptor chondroitin sulfate A (CSA) The gravidity-specific pattern of susceptibility is not observed among nonimmune pregnant women. These women are at high risk of severe syndromes and death if they become infected, regardless of gravidity, and abortion or fetal death can occur during acute febrile or severe malaria episodes P. falciparum, P. vivax has been reported to be more common during first rather than later pregnancies P. vivax do not sequester in the placenta or other vascular beds, nor do P. malariae or P. ovale have this property. Mature stages of P. vivax, P. malariae and P. ovale circulate freely in peripheral blood, and are therefore frequently detected in peripheral blood smears, unlike P. falciparum in which only the young parasite stages appear in peripheral blood smears.Similar to Congenital malaria is caused by blood-stage malaria parasites that are transmitted from mother to child. Pre-erythrocytic stages of the parasite – including the sporozoite form inoculated by the mosquito, and the liver stage forms that develop in hepatocytes during the first week of infection – are not known to cross the placenta nor to cause congenital malaria. Therefore, treatment of congenital malaria is intended to eradicate blood-stage and not liver-stage parasites in the newborn.P. falciparum can persist for months to years P. malariae can persist for decades as blood-stage infections (the stage that multiplies in red blood cells). P. vivax and P. ovale form hypnozoites in the liver that can relapse after months or sometimes years to cause new blood-stage infections.The mother’s history can be misleading for clinicians who are assessing an infant with congenital malaria, because malaria parasites can persist in asymptomatic individuals. According to a review of all US cases between 1966 and 2005, many mothers who deliver a child with congenital malaria report no exposure and no symptoms during the pregnancy Many US mothers who transmit malaria to their children deny having fevers or other illnesses during pregnancy in utero. Transmission might also occur across the placental barrier during labor, or as a result of newborn contact with infected maternal blood during parturition.The mode of parasite transmission from mother to newborn is usually uncertain. Older literature documents abortuses and stillbirths with parasites or parasite pigment in the spleen or liver Maternal blood is sometimes transferred to the fetal circulation during healthy pregnancy, but this is less frequent than transfers of blood from the fetus to the mother. By typing blood group antigens (ABO or Rh) in discordant mother–newborn pairs, fetal red cells have been detected in 41.5% of maternal samples Placental malaria may or may not disrupt the placental barrier and alter the rate of mother-to-child transmission. One study reported that cord-blood parasitemia was associated with features of chronic placental malaria such as intervillous inflammation Reports on congenital malaria have suggested a wide variation in prevalence among populations residing in areas of stable transmission P. falciparum at delivery However, large studies conducted at established research centers have uniformly found that congenital malaria defined by Giemsa-stained blood smear is rare in areas of high malaria transmission. Garnham, who authored the standard illustrated guide to malaria parasites, did not observe parasitemia during the first week of life in any of 146 neonates born to Kenyan women infected with Among offspring of semi-immune women who deliver in nonendemic areas, congenital malaria typically appears several weeks after birth. Presumably, offspring in malaria endemic areas would similarly manifest congenital malaria several weeks after birth, but in these cases, congenital malaria cannot easily be differentiated from mosquito-borne infection. For this reason, researchers in endemic areas have attempted to use more sensitive approaches than Giemsa-stained blood smear to detect parasites in cord or newborn blood, such as PCR detection of parasite DNA or RNA Researchers using PCR assays report detection of parasites in a considerable proportion of cord blood samples, ranging from 10.6% in utero and not at the time of delivery, since transmission at the time of delivery would be expected to yield genetically identical parasites in placental and cord blood samples. Nevertheless, the relevance of cord blood parasites detected by PCR to symptomatic congenital malaria or neonatal outcomes is unclear. Just as maternal erythrocytes are cleared from the neonatal circulation during the first days of life, so too have parasites been observed to clear from neonatal blood in endemic areas Genetic discordance has supported the idea that parasites detected by PCR in cord blood do not reflect contamination of samples by placental blood. Discordance in parasite species Congenital malaria in nonimmune populations has been studied less frequently, but the available data consistently suggest that mother-to-child transmission is more likely in such cases, compared with semi-immune populations. On the Thai–Burmese border, where malaria transmission is low and women therefore have low levels of immunity, three congenital malaria episodes were documented among 213 deliveries Similar to observations elsewhere, the reported incidence of pregnancy malaria in the USA far exceeds the incidence of congenital malaria. Congenital malaria in the US is a rare phenomenon In recent years (1966–2005), congenital malaria in the USA has primarily affected immigrant families. In many of these cases, the last exposure to malaria occurred before pregnancy P. malariae, which can persist for decades in asymptomatic individuals P. falciparum congenital malaria reported her last exposure in Haiti 7 years before delivery, which would be an unusually long period for this parasite species to persist, if true The mother’s time-to-last-exposure also reflects the persistence of malaria parasites in the human host. Between 1966 and 2005, 85% of mothers were exposed within 1 year prior to delivery P. falciparum, but not other species concentrate in placental blood, one might expect that falciparum malaria would disproportionately cause congenital malaria, but this is not the case. In cases reported in the USA between 1997 and 2004, the distribution of parasite species differs substantially between congenital malaria, pregnancy malaria and nonpregnancy malaria cases P. falciparum infection compared with infants with congenital malaria  or to nonpregnant women (p = 0.0001). Conversely, P. vivax disproportionately appears as a cause of congenital malaria in US cases reported to the CDC (82%), followed by P. falciparum (9%) and P. malariae (9%). Only one case of P. ovale congenital malaria has been reported since 1966 P. vivax and 18.5% were due to P. falciparumSince • P. vivax has greater transmission potential, for example since it causes less placental inflammation;• P. falciparum have greater systemic immunity, which is transplacentally transferred and protects the fetus;Women with • P. vivax are less likely to receive treatment that eradicates infection, due to persistent liver stages or to submicroscopic parasitemia;Women with • P. falciparum is more likely to become symptomatic and, therefore, prompt treatment during pregnancy • Antimalarial drugs such as quinine and mefloquine that may be used to treat malaria during pregnancy can cross the placenta and may contribute to clearing parasites from fetal blood This may have any of several hypothetical explanations, including:The geographic origins of cases in the USA generally parallel immigration patterns. An increase in congenital malaria cases starting in 1979 reflected the influx of immigrants and refugees from South-East Asia The severity of disease and the age of onset in the newborn are highly dependent on maternal exposure history and, therefore, immunity. Unlike congenital malaria cases reported in US immigrant families P. falciparumP. falciparum died of severe malaria within 6 days of life P. falciparum and two with P. vivax, and the average time to first symptom was 1.3 days of life P. falciparum. In the series by Wickramasuriya from Sri Lanka, five stillbirths were also documented to have been infected in uteroInfants of nonimmune women develop symptoms at a younger age, and are at significant risk of serious morbidity and mortality from congenital malaria, particularly when infected by In the USA, congenital malaria cases have been limited to immigrant families over recent years. These women acquired their infection before immigration or during subsequent travel, but in most cases experienced few or no symptoms during pregnancy, suggesting that they had immunity sufficient to suppress clinical signs of malaria P. malariae, and at 25 days of age for infants with P. vivax or P. falciparumAccording to a recent review of cases between 1966 and 2005 P. malariae has been described in a baby born to a heroin addict who had parenterally acquired malaria Among US cases occurring between 1991 and 2004, the most common finding was fever, followed by pallor or anemia, thrombocytopenia and poor oral intake Preterm newborns with congenital malaria may have an altered presentation. Symptoms may appear earlier, either at birth or within the first week of life. The earlier presentation has been speculatively attributed to a diminished level of passively transferred antibody Offspring of semi-immune women residing in endemic settings are usually protected from clinically significant malaria for the first 2 to 3 months of life Congenital malaria should be entertained as a diagnosis in an infant with suggestive symptoms including fever or thrombocytopenia More than one blood smear may be needed to diagnose congenital malaria, as parasite density tends to be low and the chance of failing to detect parasites on a single smear is high P. falciparumIn addition to blood smear, highly sensitive and specific PCR-based assays have been developed to make species-specific determinations Separately, because HIV increases the risk of malaria during pregnancy Unfortunately, very little is known about the pharmacokinetics and pharmacodynamics of antimalarials in neonates. To date, no randomized trials of treatments for congenital malaria have been reported, and therefore treatment guidelines are largely the product of case reports, field experience and extrapolation from older children. Early treatment is essential. In many cases, pediatric preparations may not be available, and thus adult preparations, dissolved in liquid, may need to be substituted.Treatment regimens are based on parasite species, region where the infection was acquired (parasite resistance profile), and the severity of disease P. vivax or chloroquine-sensitive strains of P. falciparumP. falciparum in the USA Chloroquine is commonly used for congenital malaria due to For severe cases of congenital malaria, such as those complicated by seizures, respiratory distress, hyperparasitemia or severe anemia, therapy should be given parenterally until a fall in parasitemia is noted Intravenous (iv.) or intramuscular (im.) artesunate has been recommended as first-line therapy outside the USA, where alternatives include im. artemether and iv. or im. quinine P. vivax and P. ovale is not necessary in the infant P. vivax or P. ovale congenital malaria should receive treatment with primaquine following delivery, after confirming normal G6PD status. For pregnant women with P. vivax, primaquine is contraindicated; weekly chloroquine prophylaxis may suppress parasitemia Since congenital malaria is acquired through mother-to-child transfer of blood-stage parasites, treatment with primaquine to eradicate liver-stage P. falciparum infections with severe symptoms Transfusion may be needed to treat severe anemia due to congenital malaria P. falciparum is transmitted by nonimmune women to their offspring. Clinical suspicion of malaria is often low as the mothers’ exposure to malaria may be distant and many mothers do not report fever or illness during pregnancy. Fever is the most common presenting symptom in the infant. Treatment should be tailored to the drug-susceptibility profile of the parasite, according to the species and country of origin. P. vivax congenital malaria does not require primaquine treatment of the newborn, as congenital malaria only involves blood-stage parasites.Congenital malaria is a rare condition. In the USA, congenital malaria is a malady that mainly affects immigrant families. Outcomes are good where passively transferred maternal immunity protects the child, but can be fatal when P. vivaxTreatment recommendations may change as drug susceptibility profiles of parasites around the world evolve, including the possible spread of chloroquine-resistant The frequency, timing and mode of mother-to-child transmission remain poorly defined, and merit further study. Similarly, the long-term consequences of congenital malaria, particularly in endemic areas where the condition is rarely recognized and could modify malaria outcomes in the child, are unknown but are the focus of ongoing research.Box 1. in utero or during the peripartum period. Parasites transmitted from mother to child may be detected in the cord blood at birth, or in the infant’s peripheral blood. In areas without mosquito transmission, malaria arising in an infant is presumed to be congenital malaria. In endemic countries, parasites must be detected within the first week of life to confidently diagnose congenital infection, as the incubation period of mosquito-transmitted infection is approximately 1 week. Clinical diagnosis of congenital malaria has relied on Giemsa-stained blood smear analysis. Detection of cord-blood parasitemia may be confounded by contamination of the sample with infected maternal blood. Soluble malaria antigen and genetic material can be transferred transplacentally, which may confound antigen- and DNA-detection assays.Congenital malaria refers to the transmission of parasites from the mother to the fetus, either Box 2. Plasmodium falciparum malaria. She delivered a boy while still undergoing treatment. At the time of delivery, P. falciparum parasites were detected by Giemsa-stained smear in placental blood and in the newborn’s peripheral blood, but not in the cord blood. The infant was symptomatic at birth. Despite treatment, the newborn deteriorated and died of severe malaria on the sixth day of life.A pregnant Karen woman was diagnosed with Taken from Box 3. Plasmodium falciparum. The baby experienced fever shortly after birth that resolved, and blood smears at birth, 24 h and 48 h were all negative. At age 5 and a half weeks the baby presented with 3 days of fever and irritability. Laboratory studies showed anemia, thrombocytopenia and elevated lactate dehydrogenase. Cultures were taken and the baby was started on ceftriaxone for presumed sepsis. A blood film demonstrated 3% P. falciparum parasitemia. The baby was treated with oral quinine sulfate and clindamycin and made a full recovery.In 2004, a baby boy was born in the USA to a woman from Nigeria who was asymptomatic during pregnancy, but who experienced fever during delivery and was diagnosed with Taken from Box 4. Plasmodium vivax (<1% parasitemia) were seen on blood smear. The infant was admitted and received chloroquine therapy and a blood transfusion. The patient recovered fully, and all subsequent blood smears were negative. At the time of malaria diagnosis of the child, the mother had a negative blood smear, but a positive PCR test for P. vivax. She was treated with chloroquine and primaquine after being screened for G6PD deficiency.A 4-week old male infant was hospitalized for fever and decreased oral intake. The infant was the product of spontaneous vaginal delivery and had no malaria exposure history. The infant’s mother had immigrated from Guatemala 1 year prior to the infant’s birth and had experienced a febrile episode that resolved without therapy when she was 6 months pregnant. The infant was was started on ampicillin and gentamicin for presumed sepsis. At admission, the infant was anemic with a hemoglobin of 9.2 g/dl on day 1 and 8.0 g/dl on day 2. Bacterial cultures were negative, and the infant was discharged on day 4. 3 days later, the patient was found to have a hemoglobin level of 6.0 g/dl. Trophozoites and gametocytes of Taken from • Congenital malaria in the USA primarily affects immigrant families.• It is frequently misdiagnosed, because symptoms overlap with other infections and the mother often reports no travel and no illness during pregnancy.• Congenital malaria is more likely when the infected mother has low immunity than when she has high immunity.• Plasmodium falciparum to her child.It can be rapidly fatal when a nonimmune woman transmits • P. falciparum is the causative agent.Congenital malaria tends to appear earlier when the mother lacks immunity and when • It can occur in offspring of mothers who report adherence to malaria prophylaxis during travel or during residence in endemic areas.• Plasmodium vivax is the most common cause of congenital malaria in the USA, and P. falciparum is the most common cause of pregnancy malaria.• Congenital malaria should be considered as a cause of fever in an infant whose mother has recent or distant travel to an endemic area.• The diagnosis of congenital malaria is made by Giemsa-stained blood smears, and a single negative blood smear is not sufficient to exclude the diagnosis.• P. vivax is not indicated for the infant, but should be given to the mother after G6PD testing.Treatment should be based on parasite species, region where the infection was acquired (parasite resistance profile) and the severity of disease. Treatment with primaquine to eradicate liver stage


Background: There were about of 124 to 283 million cases of malaria with 367,000 to 755,000 deathsannually. This study aimed to assess the prevalence of malaria cases and associated risk factorsamong attendants at Chichu and Wonago health centers, South Ethiopia.Methods: In this health institution based cross sectional study, 324 subjects, attendants fromoutpatient department who came for any kind of medical services, were included during May to June2016. A blood film examination format and structured questionnaire were used for data collection.Peripheral blood samples were collected and the presence of malaria cases was observedmicroscopically. The collected data were analyzed by SPSS version 20.0.Results: Malaria cases were detected in 91 (28.1%) of the participants with higher infection rateamongst (56.04%). The predominant Plasmodium species detected was P. vivax (52.75%) followed byP. falciparum (35.16%) and mixed malaria infection by both of the species (12.09%). Housingconstruction and not using of insecticide treated bed nets for the last 6 months were significantlyassociated with the risk of getting malaria. Individuals who had stagnant water in their compound weremore likely to get malaria than those who did not . Houses that hadbeen sprayed with insecticide in the past 6 months were protected against malaria infection . Moreover, bed net utilization was associated with a significantly lower risk ofinfection .Conclusions: Type of housing construction, not using bed net, insecticide spraying and residing nearstagnant water were associated risk factors with malaria positivity in the study area. Plasmodium; P. falciparum, P. vivax, P. ovale and P. malariae and transmitted via the bites of female anopheles mosquito vectors2. Of these, P. falciparum and P. vivax are the most common2. From the estimated 1 million malaria deaths worldwide, 90% occur in Africa, killing mostly under 5 years of age children3, who account for 78% of all deaths4.Malaria is endemic throughout most of the tropics and is caused by the protozoan parasites of one of the four species of 5. The control of the disease and its vectors in Africa is less successful because of the occurrence of antimalarial resistant parasites and insecticide resistant vectors, the shifting behavior of mosquitoes (from indoor to outdoor) as a result of frequent indoor insecticide sprays, lack of efficient infrastructure, shortage of trained manpower, lack of appropriate management, shortfalls in funding and inability to integrate different methods of control6.Malaria affects non-immune individuals in many parts of the African continent7. It is one of the leading causes of morbidity and mortality in Ethiopia3. Nearly 3/4 of the land is malarious, with malaria primarily associated with altitudes as high as 2500 meters and high risk with rainfall patterns above 100 mm (but peak during and just after the rainy season),9. According to the report of The Carter Centre3, approximately 55.7 million people in Ethiopia faced the risk of getting malaria, and approximately 80% of the 736 districts in the country are considered malaria endemic. Malaria transmission peaks bi-annually from September to December and from April to May in most areas of the country10.Ethiopia is one of the seriously affected countries in sub-Saharan African countries, as malaria is the top ranking in the list of common communicable diseases in the country11. Major contributing factors such as housing type, house proximity to mosquito breeding sites or stagnant water, toilet facilities, and malaria preventive measures have been identified in different studies14. Other factors such as bed nets availability per household, individual’s age and residence altitude, as well as household wealth, temperature, peak monthly rainfall can affect malaria prevalence15.Documenting the current epidemiologic status of malaria and identification of its risk factors can urge the decision makers to act timely to develop targeted interventionsThe overriding objective of this study was therefore to determine the current prevalence of malaria cases and associated risk factors among the study subjects.The study was conducted in Chichu and Wonago health centres Gedeo Zone, Southern Nations, Nationalities and Peoples’ Region, among outpatient department attendants who came for medical services. Gedeo zone is 365 km away from Addis Ababa, the capital of Ethiopia. The health centres provide different medical services including malaria microscopic diagnosing services for inpatient and outpatient attendants. The two major malaria transmission periods in the area are from September to December and from April to June. The study was conducted from May to June 2016.A health institution based cross-sectional study design was conducted among subjects attending the Chichu and Wonago health centers. Patients, attended for any medical services, were included in the study at their follow-up visits during the study period at outpatient department of the health centres. All individual members who have been living for more than six months, having history of fever during the past 24 h, and volunteer to participate in the study were enrolled. In addition, all individual members with anti-malarial treatment for the last 8 days were excluded from the study.16, 0.05 precision with 95% confidence interval (CI), and 10% of non-response rate. Finally, 324 individuals were enrolled. For sampling method, the 324 sampled participants were distributed to the two health centres proportionate to the number of study subjects. These health centres were selected purposely classified as the catchment health facilities for malaria endemic districts. Estimated patients in both health centres (Chichu 162 participants and 162 study subjects from Wonago) from outpatient department were selected. Study subjects were randomly selected and all eligible attendants were invited to participate.Sample size was calculated using the single population proportion formula, and prevalence was assumed to be 25.8% of the study conducted in Hadiya Zone health facilities, Ethiopia17. Slides were labeled and air-dried horizontally in a slide tray in the field, and thin films were fixed with methanol after drying. Slides were stained with 3% Giemsa for 30-45 min at laboratory unit18. Blood slides were read and crosschecked at the laboratory unit in the health center for the positivity and negativity of the slides. All procedures were conducted by following the standard protocol of WHO. Then, the positivity of parasite was determined from thick smear preparations and species identification was carried out from thin smear. Forms were checked by the supervisors and inconsistencies were verified with the respondents. To ensure the validity of the slide test, all samples were re-examined by another independent experienced medical laboratory technologist. Finally, kappa statistics was performed to check the reliability between the two results (kappa=0.926).Before blood sample collection, the finger was cleaned with alcohol-moistened cotton. Then, a drop of blood, approximately 50 μL (capillary blood from fingertip) was collected. Two blood slides each composed of thick and thin films, were taken from each study subject according to the standard operating procedure19. The questionnaire was administered to volunteers from whom the blood film was drawn according to the time schedule of each subject.For associated risk factors, the survey questionnaires were used, based on the malaria indicator survey household questionnaires modified for the local conditions, filled by the participantsThe collected data were entered and cleaned using Epi INFO vision 3.1. After cleaning, the data were transported to SPSS version 20  for analysis. Both descriptive and inferential statistics were performed. Frequencies, proportion and summary statistics were used to describe the study population in relation to relevant variables. The degree of associations between malaria cases and selected risk factors were computed using logistic regression analysis together with their corresponding 95% confidence interval.The study obtained ethical approval from the Institutional Review Board of Dilla University. Permission to conduct the study was also obtained from the health bureau director office. Only children who assented and whose parents or guardians consented to involve in the study.Overall, 324 individuals were included with 100% response rate. One hundred two (31.4%) of the participants were in the age group of 15-24 yr while 173 (53.4%) of individuals were male. The majority of the respondents (25%) were in the grade level of 9-12. One hundred seventy one (52.8%) of the participants had a family size of 4 and below persons per head and most of them (68.2%) had monthly income of <45 US$ .P. vivax was the predominant species, accounting for 48 (52.7%), followed by P. falciparum 32(35.2%) and mixed infection with both P. vivax and P. falciparum 11(12.1%) . The species wise distribution showed 1(12.1%) .Regarding malaria cases with respect to the housing condition of the subjects, , gettingLogistic regression analysis findings showed that participants whose house had been sprayed with insecticide in the past 6 months were three times less likely to get malaria infection . Approximately two fold increased prevalence of malaria was observed in individuals’ who had been living in the nearby stagnant water than those who were living far away (by at least 1 km away from their residence) from these risky areas . Individuals having and using insecticide treated nets for the last six months were 0.2 times less likely to get malaria parasites than those of not having adequate bed nets . Similarly, participants accessing health facilities were 0.3 times less likely to be infected by malaria parasites than those who had no access  .P. vivax infection. The logistic regression analysis showed that the infection was associated with different factors.Malaria infection is high in the area among the selected population. A large proportion of the infected individuals were male, and 52.75% of the cases were 20. Similarly, the result from Hadhramout, Yemen visited to health facilities 18.8%21 and from Hadiya, Ethiopia16 with 25.8%. The current report was higher than reports from Arsi Negele Ethiopia22, Butajira Ethiopia23, and Bangladesh24 with 11.45%, 0.93%, 3.97% and 2.2% respectively. In contrary, it is less than that reported in Kersa Woreda Jimma 43.8%25, Hallaba, Ethiopia26 with 82.8% and Nigeria by 39.5%27. The observed difference might be due to the seasonality of malaria and the different control measures in the current study area for the previous years.The finding of the current study revealed that malaria infection in the study participants was 91(28.1%). This report is comparable with the study conducted among patients attending public health facilities of Maputo City, Mozambique with 15.7% of prevalenceP. vivax was the predominant species, accounting for 48(52.7%), followed by P. falciparum 32(35.2%) and mixed infection with both P. vivax and P. falciparum 11(12.1%). This is in line with the studies conducted in Arsi Negelle Health Center, Ethiopia22 and Hallaba, Southern Ethiopia26. However, this finding is disagreeing with the national prevalence indicating of P. falciparum and P. vivax, which is 65%-75% and 25%-35% respectively3. In addition, studies conducted in hospitals, Islamabad, Pakistan28 and Hadhramout, Yemen21 are not supporting the present finding, reporting a higher prevalence of P. falciparum infection. The deviation between the finding of this study and the national figure of epidemic regarding P. vivax along with P. falciparum might be due to relapsing by P. vivax. A more than two-fold increase in the rate of drug resistance has been reported in south-central Ethiopia, close to the study area29. Thus, chloroquine resistant vivax malaria may have accounted for the dominance of vivax observed in the current study.The species wise distribution shows 14, Ferede et al.30 and Alemu et al. 31 who reported infection rate is higher among males in Northwest Ethiopia but differs from the findings of Tefera26 in similar study, in Ethiopia, where female subjects were more infected.Concerning gender-wise prevalence of malaria, the cases were high among males with 51(56.04%), but did not show significant association. This is consistent with the findings of Ayele ET AL.14. This is because it is most suited for porous surfaces such as brick, thatch, dung and mud walls for residence of mosquito vectors.It is observed that rate of infection with the disease was significantly higher when the wall of the house was mud blocks/bricks; house floor was local dung and when living in stick and mud roofs. This is similar with some of the findings from previous reports32 and Maputo City, Mozambique20. Approximately twofold increased prevalence of malaria was observed in individuals’ who have been living in the nearby stagnant water than those who were far away (by at least 1 km away from their residence places) from these risky areas . Living in the nearby stagnant water was also identified as a risk factor. The significantly higher parasite rate was found among the individuals having stagnant water in their compound. It can be explained from the fact that they are more exposed to mosquito bites, because these areas are suitable for breeding of mosquitoes around their homes. Individuals having and using insecticide treated nets for the last six months were around 0.2 times less likely to get malaria parasites than those of not having adequate bed nets . Similarly, participants accessing health facilities were 0.3 times less likely to be infected by malaria parasites than those who have no access . This finding is in agreement with the reports of Nahum et al.33 with .Moreover, participants whose house has been sprayed with insecticide with the currently supplied insecticides by the government (Proxus and Bifenthrin) in the past 6 months are three times less likely to get malaria infection . This is similar with the studies done in East Shoa Zone, EthiopiaThere were some limitations in our study. The study was conducted at one point in time and did not redirect the yearly seasonal pattern of the disease. Since, it is possible that seasonal variation will have affected the study findings. Malaria cases in the area could vary with the seasons, rainfall patterns and years. Next, the finding is not representative of the whole community since data was collected from only those individuals who came to health centres for health services.P. vivax species. There was a significant association of malaria cases with the type of housing construction, bed net ownership, presence of mosquito breeding places and insecticide spray of the respondents’ houses. Further studies needed to be conducted for the status and contributing factors of epidemiological shifting of malaria parasites in the study area in large scale.Most of the infected participants were male with high prevalence of We would like to forward our appreciation to Dilla University, Research and Dissemination Office for financial and material support of the research.The authors declared that no competing interests exist regarding the publication of this paper.Prevalence of malaria is high in the study area .Plasmodium vivax cases are more prevalent than Plasmodium falciparum.Type of housing construction and bed net use showed association with malaria infection. Malaria infection did not show significant association with gender and monthly income.


The early detection of oral cancer and oral potentially malignant disorders can facilitate minimum intervention and subsequent improvements in prognosis and quality of life after treatment. Recently, several non-invasive adjunctive fluorescence-based detection systems have improved the accuracy of detection and diagnosis of oral cancer and oral potentially malignant disorders. This article summarizes current knowledge about fluorescence-based diagnostic methods and discusses their benefits and drawbacks from a clinical viewpoint.Oral health promotion and examinations have contributed to the early detection of oral cancer and oral potentially malignant disorders, leading to the adaptation of minimally invasive therapies and subsequent improvements in the prognosis/maintenance of the quality of life after treatments. However, the accurate detection of early-stage oral cancer and oral epithelial dysplasia is particularly difficult for conventional oral examinations because these lesions sometimes resemble benign lesions or healthy oral mucosa tissues. Although oral biopsy has been considered the gold standard for accurate diagnosis, it is deemed invasive for patients. For this reason, most clinicians are looking forward to the development of non-invasive diagnostic technologies to detect and distinguish between cancerous and benign lesions. To date, several non-invasive adjunctive fluorescence-based detection systems have improved the accuracy of the detection and diagnosis of oral mucosal lesions. Autofluorescence-based systems can detect lesions as a loss of autofluorescence through irradiation with blue-violet lights. Photodynamic diagnosis using 5-aminolevulinic acid (ALA-PDD) shows the presence of very early oral cancers and oral epithelial dysplasia as a red fluorescent area. In this article, currently used fluorescence-based diagnostic methods are introduced and discussed from a clinical point of view. Oral cancer originating from the lip and oral cavity has been ranked as the 16th most common cancer worldwide, but the global incidence rate has seen a decline. In South and Southeast Asia in particular, alcohol intake and the use of tobacco including betel quid, which are risk factors for oral cancer, are high. Thus, the incidence rate remains elevated. In addition, human papillomavirus (HPV) is a high risk factor, and the incidence of HPV-related oral cancer has increased in specific areas . When orIn 2017, the World Health Organization (WHO) stated that oral precancerous lesions and oral precancerous conditions are called oral potentially malignant disorders (OPMDs) . OPMDs oThe conventional oral examination (COE) has been identified as a standard diagnostic procedure for clinicians in detecting oral mucosal changes by inspection and palpation ,13. EpstConsidering this point, it can be concluded that the COE alone cannot accurately detect and discriminate between OEDs or OSCC and other lesions or classify high-risk and low-risk OPMDs, even though oral cancer specialists perform the COE ,14,15. OIn this article, some light-based diagnostic technologies for early-stage OSCC and high-risk OPMDs are introduced, with a special emphasis on photodynamic diagnosis using 5-aminolevulinic acid (ALA-PDD), and discussed from a clinical viewpoint.®  system consists of a disposable chemiluminescent light source and a 1% acetic acid solution . Th. Th53]. After administration, exogenous ALA is transported to the cytoplasm by membrane transporters such as PEPT 1/2 and converted to PpIX through the heme biosynthetic pathway ,54, and PpIX has the Soret-band around 405 nm. Thus, PpIX absorbs blue light (405 nm) and excites red fluorescence (635 nm) . BrieflyThe routes of administration of ALA are primarily oral and topical administration for ALA-PDD . The oraMost clinicians in the head and neck cancer field have applied the topical administration of 0.4–1.0% ALA solutions and incubated for 1–2.5 h to accumulate PpIX in malignant cells. They suggested that ALA-PDD with topical application of 0.4% ALA could facilitate the visualization of OSCC and OEDs. Therefore, the use of low ALA concentrations must be sufficient for visualization of the superficial mucosa lesion. Furthermore, the usage of as low concentration of ALA as possible would be beneficial to avoid potential side effects, such as skin phototoxicity.The side effects of ALA depend on the route of administration. Oral administration is reported to cause mild gastrointestinal disorders such as nausea and vomiting ,58. AlthIn the field of oral cancers ,41,44, aUsing the ALA-PDD method, 0.4–1% ALA solution is topically administered in the oral cavity via continuous oral rinsing for 15 min, and then PDD using blue-violet light at a wavelength of 375–440 nm is performed after incubation for 1–2.5 h . This inLeunig et al. reported the clinical application of ALA-PDD for detecting OSCC for the first time . They usp < 0.053 and p < 0.073, respectively). Therefore, the combination of ALA-PDD and fluorescence spectroscopy can discriminate between OEDs and normal tissues or benign lesions; moreover, this strategy can potentially be used to divide OEDs into high- and low-risk dysplasia. However, large-scale case studies  will be required in the near future to verify that ALA-PDD can discriminate between high- and low-risk dysplasia.Sharwani et al. focused on diagnosing oral premalignancy lesions and benign lesions, including inflammatory and hyperplastic tissue changes, using a combination of ALA-PDD and the spectroscopic assessment of fluorescence intensity . SpecifiALA-PDD along with the qualification of fluorescence can discriminate OSCC and high-risk OPMDs from low-risk OPMDs or benign lesions with high sensitivity. In terms of dysplasia, Speight et al. indicated that the ultimate goal of grading OEDs is to provide patients with the most effective management and care . HoweverThe histopathological diagnosis of a biopsy depends on the selection of the optimal biopsy area in the lesions. For example, if a biopsy specimen is not obtained from an optimal area of the lesion, especially in a heterogeneous, multifocal, or wide lesion, which likely contains various grades of OED and CIS, the lesions may subsequently undergo malignant transformation into advanced cancer. However, ALA-PDD could help prevent underdiagnosis and/or misdiagnosis associated with a sampling error by identifying the worst area of a lesion as red fluorescence.The drawback of ALA-PDD is that it produces a few false positives, which in turn results in lower specificity. Hyperplastic or chronic inflammation lesions and radiated mucosa may be observed as red fluorescence ,43. ThisOn the other hand, false-negative lesions can be high-risk dysplasia or cancerous lesions covered with hyperkeratosis or necrotic tissue . This is® and an auto-fluorescence-based system would be highly convenient. Contrarily, spectroscopic analysis is useful for the objective judgment of ALA-PDD results; however, this analysis is particularly complicated because the optical splitter, spectrometer, and repeated calibration are required to measure fluorescence spectra [Currently, the ALA-PDD system that is used is an endoscope type. However, all clinics and hospitals do not own this type of device due to relatively poor cost effectiveness. Therefore, a more compact PDD system such as ViziLite spectra . MoreoveThe improvement of diagnostic techniques for oral mucosa lesions could decrease patient suffering associated with oral cancer. Each light-based diagnostic device can assist in the diagnosis of oral mucosa lesions. Examination under chemiluminescent light could delineate oral lesions because of the improved brightness and sharpness of margins in the identified lesions. Autofluorescence-based systems can discriminate oral cancer and dysplasia from benign lesions in combination with the quantitative analysis of autofluorescence. ALA-PDD may be suitable for detecting small changes or malignant transformation in cells within superficial mucosa lesions that cannot be detected by COEs. In fact, it enables the earliest detection of malignant lesions. Moreover, the combination of the ALA-PDD system and simple imaging processing has been proven to have higher sensitivity and specificity for identifying high-risk dysplasia and cancer and objectively discriminating between high-risk and low-risk OPMDs. Hence, this system might contribute to the application of minimally invasive therapies and improvements in patient prognosis and the longitudinal surveillance of patients with OPMDs.


Pneumonia and malaria are the leading causes of global childhood mortality. We describe the clinical presentation of children diagnosed with pneumonia and/or malaria, and identify possible missed cases and diagnostic predictors.Prospective cohort study involving children (aged 28 days to 15 years) admitted to 12 secondary‐level hospitals in south‐west Nigeria, from November 2015 to October 2017. We described children diagnosed with malaria and/or pneumonia on admission and identified potential missed cases using WHO criteria. We used logistic regression models to identify associations between clinical features and severe pneumonia and malaria diagnoses.P < .001), and less likely to die . Of 2299 (14.2%) children who met WHO severe malaria criteria, 365 (15.9%) lacked a malaria diagnosis. Compared with “potential missed” diagnoses of severe malaria, children with “detected” severe malaria were less likely to die , with no observed difference in antimalarial administration . We identified predictors of severe pneumonia and malaria diagnosis.Of 16 432 admitted children, 16 184 (98.5%) had adequate data for analysis. Two‐thirds  of children were diagnosed with malaria and/or pneumonia by the admitting doctor; 31.5% (567/1799) of those with pneumonia were also diagnosed with malaria. Of 1345 (8.3%) children who met WHO severe pneumonia criteria, 557 (41.4%) lacked a pneumonia diagnosis. Compared with “potential missed” diagnoses of severe pneumonia, children with “detected” severe pneumonia were more likely to receive antibiotics (odds ratio [OR], 4.03; 2.63‐6.16, Pneumonia should be considered in all severely unwell children with respiratory signs, regardless of treatment for malaria or other conditions. Our exploratory analysis identified potential missed cases of severe pneumonia and severe malaria, explored predictors of “correct” pneumonia and malaria admission diagnoses, and considered reasons for clinical overlap and alternative diagnoses in malaria and pneumonia syndromes.22.1We conducted a prospective cohort study nested within a stepped‐wedge trial evaluating pulse oximetry and oxygen systems in 12 Nigerian hospitals.2.2We conducted this study in southwest Nigeria, recruiting 12 small‐ to medium‐sized hospitals that were representative of secondary healthcare facilities that admit children.We included all children aged 28 days to 15 years who were admitted to participating hospitals during the study period  and generalist nurses (nursing observations and nursing admission note). Admitting doctors were typically junior doctors who did not have pediatric specialty training, who performed their pediatric clinical duties while also covering other wards, and who would complete admission documentation at the point of admission (outpatient or emergency department) or on the pediatric ward. Admitting nurses were typically generalist nurses or nursing assistants who were based on the pediatric ward and who would complete admission documentation when the child arrived on the ward.As part of our broader pulse oximetry and oxygen intervention,2.4Trained data entry clerks used EpiData 3.1χ2 test to evaluate differences between categorical dependent and independent variables, t test for dichotomous independent variables on a continuous outcome, and analysis of variance for multiple categorical independent variables on a continuous outcome.We identified children diagnosed with pneumonia or malaria on admission, permitting multiple diagnoses. We accepted all diagnostic terms relating to lower respiratory tract infection for pneumonia (including bronchiolitis) and similarly for malaria. We categorized participant diagnoses as “malaria,” “malaria + pneumonia,” “pneumonia,” or “neither malaria nor pneumonia.” For each category, we summarized demographic and clinical symptoms and signs using descriptive statistics and tests of difference. We summarized categorical variables by number and percentages. We summarized continuous variables by mean and standard deviation (SD) if normally distributed, and by median and interquartile range (25th and 75th centiles) if non‐normally distributed. We used WHO Pocketbook of Hospital Care for Children, these WHO clinical case definitions are intended for use by generalist doctors and nurses working at first‐referral level health hospitals with limited diagnostic capacity. WHO Pocketbook diagnostic and treatment advice is consistent with Nigeria's Standard Treatment Guidelines Using WHO case definitions Box , we idenBox 1Severe pneumoniaCough or Difficult breathing AND at least one of the following:severe respiratory distress;2<90%);hypoxemia  AND positive malaria test (blood film or rapid diagnostic test) AND at least one of the following:severe anemia;decreased conscious state;seizures.Both the admission diagnosis and the clinical case definition classifications were derived from the admitting doctor and nurse's documentation at the time of admission. If a clinical sign was not recorded, we assumed that it was not present.By focusing on admission diagnoses and clinical case definitions, we were able to explore real‐life diagnostic and management practices in the critical first hours of a severely ill child presenting to hospital. We categorized those meeting WHO criteria for severe pneumonia as “detected” or “potential missed” depending on whether they had a corresponding admission diagnosis of pneumonia. We categorized those meeting WHO criteria for malaria similarly.To understand diagnostic decision making, we compared “detected” vs “potential missed” severe pneumonia subgroups. We built univariate logistic regression models to look for associations between presenting demographic characteristics, clinical signs, investigations, treatment, and clinical outcomes, with a “detected” pneumonia and malaria diagnosis. We then used a mixed effects logistic regression model to evaluate the relative contribution of demographic characteristics, clinical signs, and investigations to pneumonia and malaria diagnostic status . We selected variables for inclusion in the multivariate model using backward and forward stepwise regression. We used random effects to adjust for clustering at the hospital level.We report odds ratios (ORs), adjusted odds ratios (aORs), and 95% confidence intervals (95% CI) using complete case analysis approach .3In the 24‐month study period (November 2015 to October 2017), 16 432 children were admitted to participating hospitals. Case notes were available for 16 184 participants (98.5%)  of admitted children had an admission diagnosis of malaria and/or pneumonia  (Table P < .001). Children diagnosed with pneumonia were more likely to have cough or difficult breathing  and severe respiratory distress  than those diagnosed with malaria. They also tended to have a higher respiratory rate  and were more likely to have hypoxemia . Children diagnosed with malaria were more likely to have seizures , altered conscious state , and signs of severe dehydration  than those diagnosed with pneumonia.Children diagnosed with pneumonia were younger than those with malaria or other diagnoses . HIV prevalence was low (<1% of admissions). Chest X‐ray radiography was performed infrequently , even among those diagnosed with pneumonia .Most children were tested for anemia  and malaria  (Table P < .001) or be transferred to another facility  compared with those diagnosed with malaria  met the WHO case definition for severe pneumonia Figure . Of thesP < .001), more likely to have severe respiratory distress  and hypoxemia , less likely to have diarrhea , feeding difficulty , seizures , confusion/lethargy , severe dehydration , positive malaria test , or severe anemia , and had lower admission temperature   and lower odds of receiving intravenous fluids  compared with those with “potential missed” severe pneumonia (Table P = .067).Results from univariate logistic regression show that those with “detected” severe pneumonia had greater odds of receiving parenteral antibiotics on day 1  met the case definition for severe malaria. Of these, 365 (15.9%) were not diagnosed with malaria on admission, representing potential missed severe malaria diagnoses.Compared with those with potential “missed” severe malaria diagnoses, those with “detected” severe malaria were more likely to have a history of fever  and anemia , and less likely to have a history of cough or difficult breathing , severe respiratory distress , diarrhea , and severe dehydration  (Table P = .209) (Table P = .017).Antimalarial and intravenous fluid therapy were used frequently (90.6% and 99.6%), and univariate logistic regression found no association between children with missed versus detected severe malaria with regard to antimalarial therapy  Table . There w3.5In total, 177 (1.2%) of children met WHO criteria for both severe malaria and severe pneumonia, representing 7.7% (177/2299) of those with severe malaria and 13.2% (177/1345) of those with severe pneumonia , and showed a statistically nonsignificant but clinically significant lower odds of death .We found that current diagnostic practices rely on clinical signs, pulse oximetry, and malaria testing , with little use of chest radiology. Almost half  of children presenting with documented evidence of severe pneumonia (WHO case definition) were not given a pneumonia diagnosis. Compared with these “potential missed” diagnoses, children with “detected” severe pneumonia were four times more likely to receive parenteral antibiotics on day 1 of admission , survival was better in those with a malaria diagnosis . As a post hoc exploratory study, our findings should be interpreted with caution; however, they raise some interesting hypotheses.In contrast, only 16% (365/2299) of children presenting with evidence of severe malaria (WHO case definition) were not given a malaria diagnosis. While there was no difference in the likelihood of parenteral antimalarial administration between children with “potential missed” and “detected” severe malaria .We found that the majority  of children with “missed” severe pneumonia had signs of severe respiratory distress  or hypoxemia , suggesting substantial respiratory compromise. These results are consistent with other recent studies from large hospitals in Africa reporting similar hypoxemia prevalence in hospitalized children (aged <5 years) with pneumonia (28‐49%).P < .001 | aOR 0.20; 0.09‐0.45, P < .001). These findings suggest that doctors had a lower index of suspicion for pneumonia in children that did not exhibit obvious respiratory signs, or who tested positive for other conditions. While this may be appropriate, it does raise the question of whether bedside testing for malaria and anemia contributed to missed pneumonia diagnoses. Importantly, the routine use of pulse oximetry in participating hospitals is atypical for many hospitals in low‐ and middle‐income countries, and diagnostic practices may be different where pulse oximetry is not used routinely.Conversely, positive malaria testing or detection of severe anemia were associated with lower likelihood of a pneumonia diagnosis , it is possible that some children included in our study had other primary diagnoses and were therefore not diagnosed with pneumonia. We found that two‐thirds  of children with WHO‐defined severe pneumonia were diagnosed with a different respiratory or systemic condition that may mimic severe pneumonia: sepsis ; meningitis/encephalitis ; or asthma .P < .001). This suggests there is room for improvement in prompt antibiotic administration to all children meeting criteria for severe pneumonia, and raises questions about whether other standards of care for severe pneumonia may also have been lacking .Even if some of these cases could be explained by alternate diagnoses, in the absence of definitive testing, existing guidelines suggest that all children meeting WHO clinical criteria for severe pneumonia should receive treatment, including parenteral antibiotics.P = .067), we cannot necessarily attribute this to diagnostic or treatment practices. The “missed” severe pneumonia subgroup were more likely to have neurological signs  and be given a diagnosis of seizures, meningitis, or severe acute malnutrition—all features that may be associated with poorer clinical outcomes. We face similar challenges interpreting the lower mortality in those with “detected” vs “missed” severe malaria.While we observed a statistically nonsignificant but clinically relevant association with lower mortality in those with “detected” vs “missed” severe pneumonia , overlap was not so obvious among those with WHO case definition severe pneumonia and severe malaria . Previous studies have suggested that while clinical presentation of malaria and pneumonia may overlap, true severe disease overlap is uncommon, particularly in areas and times of lower malaria transmission.The shared clinical features of severe pneumonia and severe malaria include: fever; tachypnoea and respiratory distress; altered conscious state. In severe pneumonia, respiratory distress is related to the degree of lung injury, the age of the child , and the presence of comorbid conditions . Altered conscious state typically reflects impaired cerebral oxygenation, related to hypoxemia and/or impaired perfusion.In severe malaria, respiratory distress is a relatively common finding and is a well‐established predictor of mortality among children with severe malaria, increasing their risk of death approximately fourfold.In malaria, compensatory hyperventilation related to metabolic acidosis is the most common driver of respiratory signs—causing tachypnoea, chest indrawing, nasal flaring, and deep breathing—and is probably the primary mechanism for excess mortality.Adult studies suggest that acute respiratory distress syndrome (ARDS) is relatively common in severe malaria, noting that respiratory symptoms may develop following treatment when malaria parasite blood levels have dropped.4.4Previous studies in similar contexts have failed to find clinical or laboratory signs that reliably distinguish between severe malaria and pneumonia.Our findings suggest that doctors were using signs of severe respiratory distress and hypoxemia to diagnose pneumonia appropriately. However, doctors may have been overly reliant on these insensitive respiratory signs and unduly influenced away from a pneumonia diagnosis when other signs were present that could still be consistent with severe pneumonia —or when bedside testing suggested an additional diagnosis . While the proportion of “missed” severe malaria diagnoses was relatively low, our results suggest that doctors appropriately used severe anemia as a predictor of malaria diagnosis but may have been unduly swayed away from a malaria diagnosis by other signs that could still be consistent with severe malaria .4.5This study involved post hoc exploratory data analysis and is therefore primarily hypothesis generating. We relied on documented clinical information and had minimal missing data, thanks to the close attention of our research nurses at each site. A pediatrician assessed the level of detail in case note documentation before commencing data collection, but we did not independently verify the documented clinical signs or diagnostic results. Where clinical signs were not documented, we assumed that they were not present; however, this may have resulted in an underestimate of signs. We focussed comparative analysis on those meeting WHO definitions of severe pneumonia and malaria to exclude those with mild respiratory disease or incidental parasitaemia. We were limited by the lack of independent confirmatory testings, such as routine chest X‐ray radiography or blood cultures, which could have more accurately defined severe pneumonia and invasive bacterial disease.While we did not exclude children with HIV from analysis, this study was conducted in an area with low HIV‐prevalence and results may not be generalizable to contexts with higher HIV prevalence. Southwest Nigeria is relatively wealthier and better served with health services and staff than other regions of Nigeria (and sub‐Saharan Africa more broadly), and participating hospitals had received particular assistance with pulse oximetry and oxygen therapy. Diagnostic practices may be different in other areas of Nigeria and Africa that have lower socioeconomic indices, fewer healthcare workers, and less available hospital services. Routine use of pulse oximetry by nurses in our study is relatively unusual for hospitals in sub‐Saharan Africa,5Clinical overlap between children presenting with malaria and pneumonia continues to present diagnostic challenge to healthcare workers in Nigerian hospitals. Current diagnostic practices miss up to half of severe pneumonia cases and may result in delayed or undertreatment and poorer clinical outcomes. Severe malaria diagnoses are less frequently missed, and antimalarial treatment coverage is high. Healthcare workers should be encouraged to consider pneumonia in all severely unwell children with respiratory signs, even if they are also treated for malaria or another condition.HG, AAB, AIA, EN, TD, and AGF received payment for services on this project from the funder . The other authors declare that there are no conflict of interests.HG, AIA, AGF, and TD conceived and designed the study. HG, AAB, AIA, and AGF participated in project implementation. AAB and OBO coordinated data collection and database entry. HG did the data analysis. HG, AAB, AG, EN, SQ, TD, and AGF contributed to the interpretation of results. HG drafted the manuscript. AAB, AG, EN, SQ, TD, and AGF provided substantial comments to the writing of the manuscript. All the authors read and approved the final manuscript.This study obtained ethics approval from the University of Melbourne (1543797.1) and the University of Ibadan/University College Hospital Ethics Committee, Ibadan, Nigeria (UI/EC/16/0413).