Pan troglodytes) harbor rich assemblages of malaria parasites, including three species closely related to P. falciparum (sub-genus Laverania), the most malignant human malaria parasite. Here, we characterize the ecology and epidemiology of malaria infection in wild chimpanzee reservoirs. We used molecular assays to screen chimpanzee fecal samples, collected longitudinally and cross-sectionally from wild populations, for malaria parasite mitochondrial DNA. We found that chimpanzee malaria parasitism has an early age of onset and varies seasonally in prevalence. A subset of samples revealed Hepatocystis mitochondrial DNA, with phylogenetic analyses suggesting that Hepatocystis appears to cross species barriers more easily than Laverania. Longitudinal and cross-sectional sampling independently support the hypothesis that mean ambient temperature drives spatiotemporal variation in chimpanzee Laverania infection. Infection probability peaked at ~24.5 °C, consistent with the empirical transmission optimum of P. falciparum in humans. Forest cover was also positively correlated with spatial variation in Laverania prevalence, consistent with the observation that forest-dwelling Anophelines are the primary vectors. Extrapolating these relationships across equatorial Africa, we map spatiotemporal variation in the suitability of chimpanzee habitat for Laverania transmission, offering a hypothetical baseline indicator of human exposure risk.Chimpanzees ( Chimpanzees are known to suffer from malaria parasitism. Here, the prevalence and epidemiology of several malaria parasites is examined in a wild chimpanzee population. Plasmodium falciparum (sub-genus Laverania), the most prevalent and malignant malaria parasite of humans6. Although the emergence of P. falciparum in the human population has been traced to a gorilla parasite8, Laverania species appear to exhibit considerable host-specificity in wild ape populations11. Chimpanzees (Pan troglodytes)—the most abundant and widely distributed great ape species12—host three Laverania parasites 15, which are primarily transmitted by forest-dwelling Anopheles mosquitoes  that feed promiscuously upon both humans and wild apes17. Although habitats of humans and chimpanzees often interface and humans are therefore likely to be exposed to these zoonotic parasites, three surveys of rural human populations in West Central Africa have failed to document cross-species transmission18, suggesting that strong molecular  as well as other barriers govern the host-specificity of the Laverania22. Cross-species transmission of Laverania has, however, recently been documented among apes living in captive environments23, suggesting that a high magnitude of exposure can overcome molecular barriers to infection.African great apes harbor a wide diversity of malaria parasites, including seven species closely related to Plasmodium transmission among chimpanzee hosts remain largely unexplored. Previous studies employing non-invasive sampling and molecular diagnostics have shown that the prevalence of chimpanzee Laverania infection varies regionally within equatorial Africa 24. Infections are also temporally dynamic, manifesting seasonal and inter-annual trends in prevalence25. However, to our knowledge, no study has quantified the ecological drivers of this variation in infection probability. Doing so would offer a mechanistic explanation of these spatiotemporal dynamics and could pave the way to explicitly quantify the magnitude of human exposure in ecologically permissive settings.The capacity to identify potential exposure hotspots is currently limited because the ecological conditions that mediate Laverania infection remain largely unknown, both laboratory and field studies have revealed that ambient temperature is a strong determinant of Plasmodium transmission dynamics in human populations29. Ambient temperature influences both the population dynamics of the Anopheles mosquito vector and the rate of parasite development therein 31. While higher temperatures accelerate the rate of parasite development within the vector, mosquito survival tends to decline above a particular temperature threshold32. Taken together, empirical studies indicate that transmission of P. falciparum peaks at a mean temperature of ~25 °C in human populations33. In essence, this optimal temperature represents the value at which the maximal number of mosquito vectors survive long enough to harbor the development of infectious parasites, and deviation from this value is associated with a reduction in the magnitude of transmission. Thus, spatial variation in ambient temperature defines the suitability of a particular locale for transmission, and temporal variation in this parameter underlies the seasonality of infection36.Although the ecological drivers of chimpanzee 37. The effect of precipitation on malaria transmission is more complex. Rainfall is necessary to support breeding habitat for aquatic mosquito larvae 29, whereas high levels of precipitation may flush breeding sites, resulting in larval mortality38. Finally, given that chimpanzee malaria parasites are transmitted by forest-dwelling Anophelines17, the availability of forested habitat is also likely to be a necessary determinant of infection39, though the relationship between deforestation and human malaria parasitism has proven to be complex40.Additional ecological variables can influence transmission dynamics. Intra-day fluctuations in ambient temperature can amplify or dampen transmission relative to that which would be expected at a particular mean temperatureLaverania prevalence. We used molecular diagnostic assays to screen chimpanzee fecal specimens, collected via both longitudinal and cross-sectional sampling strategies  from a subset of samples. While Laverania parasites exhibit considerable host-specificity, phylogenetic analyses revealed that Hepatocystis appears to cross species barriers more easily. We also analyzed the relationship between environmental variables and fecal parasite rates to define the ecological niche of the chimpanzee Laverania. Longitudinal and cross-sectional sampling strategies each independently supported the hypothesis that mean ambient temperature drives spatiotemporal variation in Laverania infection probability among chimpanzees. Infection probability peaked at ~24.5 °C among chimpanzee populations, consistent with the empirical transmission optimum of P. falciparum. Forest cover was also positively correlated with spatial variation in Laverania prevalence, consistent with the observation that forest-dwelling Anophelines constitute their primary vectors. Finally, we extrapolated these relationships across equatorial Africa to define spatiotemporal variation in the suitability of chimpanzee habitat for Laverania transmission, offering a hypothetical baseline indicator of human zoonotic exposure risk.In this study, we characterize the ecology and epidemiology of malaria infection in wild chimpanzee reservoirs and test the hypothesis that ambient temperature constitutes a critical driver of spatiotemporal variation in chimpanzee ies Fig. , for mal25, but no analysis has elucidated the ecological underpinnings of these dynamics. To investigate the epidemiology of Laverania infection in wild chimpanzees, we first employed a high-density, longitudinal sampling strategy. Between June 2013 and August 2016, we collected 878 fecal samples from the Kanyawara chimpanzee community living in Kibale National Park, western Uganda  and 22 male (N = 382 samples) chimpanzees and stored in RNAlater at −20 °C until DNA extraction. At the time of sampling, subjects ranged in age between 0.3 and an estimated 55.8 years of age  was genotyped at 19 nuclear microsatellite loci, as described previously44. Microsatellite genotyping was successful in 97.7% of these samples (43/44), suggesting that fecal DNA was of sufficient quality for subsequent molecular analyses. Of the successfully genotyped samples, we observed a misidentification rate of 2.3% (1/43), indicating that identification fidelity was sufficient for analysis of demographic predictors of infection.Malaria prevalence varies both spatially and temporally among wild chimpanzee populationsPlasmodium infection within and among the Kanyawara chimpanzees, DNA was extracted from fecal samples and screened for malaria parasites using a single genome amplification (SGA) strategy via nested PCR, which targeted a 956-bp segment of the apicomplexan cytochrome B (cytB) mitochondrial gene as described previously7. To increase the sensitivity of this approach, we adopted an intensified PCR protocol, whereby all samples were screened in eight independent PCR reactions, as previously described4. All positive reactions were confirmed via direct sequencing without interim cloning. Of these 878 samples, 31.1% (N = 217) tested positive for one or more malaria parasite species in at least one replicate. Because the sensitivity of this assay to detect parasite DNA has been shown in humans to be greater in blood than in matched fecal samples, this value likely underestimates the frequency of blood-stage infection18. However, Loy et al.18 also revealed that human fecal samples that tested positive by this approach exhibited a 26-fold increase in parasite DNA copy number in the blood relative to fecal samples that tested negative. Thus, it is likely that the positive fecal samples identified in our study reflect chimpanzees that harbor elevated parasitemia, which is a correlate of both morbidity47 and transmission potential49, at least in humans.To quantify variation in 50  harbored parasite sequences attributable to Hepatocystis spp., a clade of mammalian malaria parasites primarily isolated from Old World monkeys and bats52. Despite its divergent nomenclature, the Hepatocystis clade is more closely related to the primate malaria clade  than either is to Laverania53. Previous studies have similarly identified Hepatocystis in chimpanzee fecal samples14. However, to our knowledge, no study has evaluated the evolutionary relationships of these parasites relative to the large diversity of previously described Hepatocystis lineages to evaluate whether these species constitute (A) a novel Hepatocystis clade endemic to chimpanzee hosts or (B) the product of multiple independent cross-species transmission events originating in other mammals.In addition to the Hepatocystis sequences  relative to 56 previously published Hepatocystis cytB sequences  and rainfall were collected from Kanyawara during the study period. We defined mean ambient temperature as the average of minimum and maximum air temperature estimates recorded during the 30 days prior to sample collection and defined intra-day temperature variation as the average difference between minimum and maximum temperature estimates during the same period. Mean ambient temperature during sample collection ranged from 20.0 to 23.0 °C (median: 20.7 °C) and intra-day temperature variation ranged from 7.9 to 15.6 °C (median: 11.0 °C). Mean daily rainfall during the 30 days prior to sample collection ranged from 0 to 14.5 mm per day (median: 3.0 mm).55 with binomial error structure and logit link function to evaluate the relationship between climatic variation and the probability of Plasmodium infection among the Kanyawara chimpanzees. For each sample, we specified malaria infection (binary) as the outcome variable, mean ambient temperature (quadratic) and rainfall as fixed effects, chimpanzee individual as a random effect, and age (quadratic), sex, and intra-day temperature variation as covariates . As predicted, ambient temperature was positively correlated with the probability of infection, though the quadratic term had no effect and was omitted from the best-fit model es Table . A full del Fig. ; Table 213, fecal samples of younger chimpanzees  were more likely to test positive for malaria parasites than were older individuals . Although the father of this chimpanzee (OG) exhibited an unremarkable pattern of infection (positive in 6/15 samples), her mother (NP) tested positive for Plasmodium in 1/29 samples, and her maternal grandfather (LK) was positive in 0/25 samples  and unpublished (N = 500) samples  estimates from the MODerate Resolution Imaging Spectroradiometer (MODIS) thermal sensor on board the NASA-Terra satellite at 1  km2 (downsampled to 10 km2) spatial and 1-day temporal resolution 60. We derived minimum and maximum air temperature estimates by applying the transformation outlined in Weiss et al.36. As above, mean ambient temperature was defined as the average of minimum and maximum air temperature estimates recorded during the 30 days prior to sample collection and intra-day temperature variation as the average difference between minimum and maximum temperature estimates during the same period. Rainfall estimates were derived from the Global Precipitation Climatology Project 61 at 1-day temporal resolution and 1-degree spatial resolution. Forest cover estimates were derived from high-resolution global maps of tree canopy cover , published by Hansen et al.62. The resulting dataset of 2436 samples included only those data points for which all predictor variables were available. Mean ambient temperature during the 30 days prior to sample collection ranged from 9.8 to 27.3 °C (median: 23.8 °C). Mean daily rainfall during the 30 days prior to sample collection ranged from 0 to 14.8 mm per day (median: 4.0 mm). Intra-day temperature variation during the 30 days prior to sample collection ranged from 6.0 to 13.4 °C (median: 8.8 °C). Forest cover at sampling sites ranged from 16 to 100% (median: 90%).Although direct measurements of climate were not available from all sites, satellite remote sensing technology can estimate climatic variablesLaverania infection among wild chimpanzees across equatorial African sampling sites. We coded Plasmodium infection (binary) as the outcome variable, mean ambient temperature (quadratic), rainfall, and percent forest cover as fixed effects, sampling site as a random effect, and daily temperature variation and number of replicates  as covariates . As predicted, ambient temperature was positively correlated with infection probability at low-temperature values and negatively correlated with infection at high-temperature values  across the spatial extent of chimpanzee habitat . In addition to mean ambient temperature, intra-day temperature variation—which has also been shown to influence the epidemiological processes at the parasite-mosquito interface37—was correlated with infection probability in both datasets (Tables 28 or vector-specific effects37). Taken together, these results indicate that the temperature-sensitive epidemiology of the chimpanzee Laverania closely resembles that of P. falciparum, suggesting that conserved mechanisms govern the epidemiology of these taxa.Ambient temperature influences numerous elements of the on Figs. , Table 2An. vinckei, An. moucheti) constitute the primary vectors of these parasites17 and supports a recent analysis of malaria parasitism across non-human primates39. Although these signatures of forest-dependent transmission suggest that human encroachment into undisturbed habitat may pose a greater exposure risk than does habitat fragmentation, these relationships are likely complex40 and warrant further investigation at fine-grained spatial scales.Forest cover was also positively correlated with infection probability Fig.  which isLaverania transmission  high prevalence . Future studies should prioritize rural human populations living near forests containing Plasmodium-infected apes demonstrating active transmission in the Republic of Congo and the Democratic Republic of the Congo, as well as parts of southern Cameroon and eastern Gabon, between the months of January and May. Since gorillas are infected with the precursor of P. falciparum, this ape species in particular should be included in epidemiological studies. Future efforts that fine-tune and extend this risk map  are necessary to operationalize these predictions.In addition to highlighting variation in nce Fig. , (2) earnce Fig. , and (3)nce Fig. . These rLaverania transmission has never been documented. Strong molecular barriers to cross-species Laverania transmission have been identified22 and two surveys of rural human populations in Gabon and Cameroon11, as well as an extensive survey of indigenous hunter gatherers in Cameroon18, have failed to reveal evidence of zoonotic transmission. The strong host-specificity of the Laverania species contrasts with the lack of host-specificity of Hepatocystis parasites  may have been underestimated. Future studies that screen human and chimpanzee populations for this parasite using Hepatocystis-specific primers will be necessary to examine the frequency of productive cross-species transmission.Although it is likely that human exposure to chimpanzee malaria parasites occurs with regularity in parts of equatorial Africa, chimpanzee-to-human tes Fig. . Though ion Fig. . Althoug64, little is known about the pathogenicity of the Laverania in chimpanzees. While some case studies have documented several characteristic symptoms—such as fever, anemia, elevated parasitemia, and possibly mortality—in Plasmodium unexposed captive chimpanzees upon initial exposure to these parasites65, other studies have produced equivocal results66. If Laverania parasites are routinely pathogenic in wild chimpanzees, natural selection could plausibly have favored the evolution of mechanisms of resistance. Three closely related individuals in our sample had exceptionally high frequencies of testing negative for malaria parasites, raising the possibility that they shared a genetic polymorphism conferring resistance. It is also conceivable that chimpanzees have evolved a series of behavioral responses  to mitigate malaria pathogenesis. Future studies that synthesize molecular and behavior approaches at long-term study sites, such as Kanyawara, will facilitate investigation of these hypotheses.The analyses presented here may also catalyze future studies of host-parasite coevolution in wild chimpanzee hosts. Although malaria pathogenesis ranks amongst one of the strongest selective pressures to confront human populations during the past 50,000 yearsP. knowlesi69) and South America (P. simium70) have raised concerns that zoonoses could rollback these hard-fought gains. While the ape Laverania appear to exhibit considerable host-specificity on contemporary timescales, there is nevertheless a strong impetus to critically evaluate the zoonotic potential of these parasites. Chimpanzees and gorillas live in close proximity to humans in parts of equatorial Africa, two pandemic malaria parasites  have emerged from African great ape reservoirs in the past7, and evidence of contemporary human-to-ape transmission of P. falciparum has been documented72. In addition, chimpanzee and gorilla Laverania parasites have transcended primate species barriers in captivity and vectors of ape malaria parasites exhibit promiscuous biting preferences17. Accordingly, it is likely that human exposure occurs with regularity in parts of equatorial Africa. Given finite resources, it is critical that any effort to evaluate the zoonotic potential of the chimpanzee Laverania adopt a targeted approach. Our results offer an ecological framework that will facilitate the design of surveillance efforts by highlighting where and when human exposure is most likely to occur.Although efforts to control malaria have achieved considerable success during recent decades, the emergence of non-human primate malaria parasites in Southeast Asia (later (1:1 vol/vol) and stored at −20 °C until exportation and DNA extraction. For cross-sectional analyses, 2436 chimpanzee fecal samples were collected from 55 chimpanzee field sites, distributed across equatorial Africa , transported at ambient temperature, and stored at −80 °C. For both longitudinal and cross-sectional surveys, DNA was extracted using the QIAamp Stool DNA minikit . Confirmation of host species was obtained either by direct observation of defecation  or via amplification and sequencing of host mitochondrial DNA78 .In this study, we used a combination of longitudinal and cross-sectional sampling strategies to collect 3314 fecal samples from wild chimpanzees for molecular analyses of malaria parasitism. For longitudinal analyses, 878 fecal samples were collected from 54 chimpanzees inhabiting the Kanyawara chimpanzee community, located in Kibale National Park, western Uganda  when screened for Laverania infections using cross-reactive PCR primers  field site also yielded a fecal sample from an unknown human, which was identified by mtDNA analysis. This human fecal sample as well as a dried blood spot sample collected from a captive chimpanzee (50094) at the Limbe Wildlife Centre in Cameroon were both positive for 44 (with slight modifications), to genotype 44 fecal samples at 19 polymorphic microsatellite loci. Briefly, all microsatellite loci were first amplified in tandem via an initial multiplexing step. For each sample, 5 μL of extracted DNA was added to a 20 μL multiplex PCR reaction containing all 19 primer pairs at 0.15 mM concentration, 1X Expand Long Template Buffer without MgCl2, 1.75 mM MgCl2, 110 μM of each dNTP, 16 μg bovine serum albumin (BSA), and 0.5 U of Expand Long Template Taq DNA polymerase. Thermocycling was performed with an initial denaturation step for 5 min at 95 °C, 30 cycles of 30 s at 95 °C, 90 s at 58 °C, and 30 s at 72 °C, followed by a final extension at 72 °C for 30 min. Next, a singleplex PCR was performed for each locus in 10 μL volume by mixing 5 μL of the multiplex reaction (diluted 1:100), 0.25 mM of the corresponding fluorescently labeled  forward primer, 0.25 mM nested reverse primer, 1X Expand Long Template Buffer without MgCl2, 0.875 mM MgCl2, 110 μM of each dNTP, 8 μg BSA, and 0.25 U of Expand Long Template Taq DNA polymerase. Thermocycling was performed with an initial denaturation step for 5 min at 95 °C, 30 cycles of 30 s at 95 °C, 90 s at primer-specific annealing temperatures, and 30 s at 72 °C, followed by a final extension at 72 °C for 30 min. Amplification products were electrophoresed on an ABI PRISM 3100 Genetic Analyzer and sized relative to an HD400 (ROX) size standard using GeneMapper 5.0 (Applied Biosystems). This procedure was repeated up to six times for loci that failed to amplify. The microsatellite genotype derived from each of 44 samples was then compared to those of the Kanyawara chimpanzees, and incorrectly attributed samples were recorded as such.To evaluate the accuracy of Kanyawara chimpanzee identification, we used a semi-nested multiplex PCR protocol, as described previously7. To increase the sensitivity of this approach, a subset of fecal samples were subjected to an intensified PCR protocol, whereby samples were screened in 8–10 independent PCR reactions, as previously described4. All Kanyawara fecal samples and a subset of pan-African samples  and DW4 (5′-TGTTTGC TTGGGAGCTGTAATCATAATGTG-3′) in the first-round PCR, and internal primers Pfcytb1 (5′-CTCTATTAATTTAGTTAAAGCACA-3′) and PLAS2a (5′-GTGGTAATTGACATCCWATCC-3′) in the second round, as described previously79 to identify the best-fit nucleotide substitution model and MrBayes (version 3.2.6)50 to generate Bayesian posterior probabilities, using a chain length of 5.5 million and 10% burn-in. Convergence was achieved when the average deviation of split frequencies was <0.01. Parasite species identity was inferred via NCBI nucleotide BLAST and confirmed using the phylogenetic relationships generated above. To analyze the evolutionary relationships of chimpanzee Hepatocystis isolates, we used the methodology outlined above, except sequences were trimmed to 815 bps to allow for comparison to a greater diversity of Hepatocystis reference sequences. In this case, chimpanzee and human Hepatocystis isolates were aligned to a representative diversity of previously published Hepatocystis isolates, spanning parasites of African Old World monkeys, Asian Old World Monkeys, and other mammals.For analysis of malaria parasitism within the Kanyawara chimpanzee community, we trimmed sequences to 863 bps and used Geneious aligner (version 10) to align sequences to a set of phylogenetically informative reference sequences, corresponding to a representative diversity of simian and human malaria parasites. Sequences shorter than 863 bps were omitted from phylogenetic analyses. Alignments were then visually inspected and sequences containing ambiguities were removed from subsequent analyses. We used jModelTest (version 2.0)2 spatial resolution and 1-day temporal resolution 60 at the geospatial point of sample collection. We subsequently performed quality control to remove low quality or missing data  and downsampled spatial resolution to 10 km2 to increase applicability of variables across the sampling site. We then converted these daytime and nighttime LST estimates to minimum and maximum ambient temperature using the equations presented in Weiss et al.36 :Tmin is minimum ambient temperature, Tmax is maximum ambient temperature, LSTnight is the nighttime LST estimate, LSTday is the daytime LST estimate, and Daylength is the number of daylight hours on the day of sampling. Given these values, we calculated mean ambient temperature as the average of minimum and maximum ambient air temperature measurements and intra-day temperature variation was calculated as the difference between these values, as above. Rainfall estimates were derived from the Global Precipitation Climatology Project (GPCP V2.3)61 at 1-day temporal resolution and 1-degree spatial resolution (https://www.esrl.noaa.gov/psd/). Again, for each sample, we defined mean ambient temperature, intra-day temperature variation, and rainfall as the average of measurements of the corresponding variable during the 30 days prior to sample collection. Forest cover estimates were derived from high-resolution global maps of tree canopy cover , published by Hansen et al.62. The resulting dataset included only those data points for which all predictor variables were available (N = 2436 samples).We estimated ecological variables using two alternative methodologies: direct measurement and remote sensing. For longitudinal analyses of the Kanyawara chimpanzee community, CAC recorded measurements of ambient air temperature  and rainfall twice daily during the study period via local weather monitoring stations installed at the field site. Mean ambient temperature was calculated as the average of minimum and maximum ambient air temperature measurements and intra-day temperature variation was calculated as the difference between these values. For each sample, we defined mean ambient temperature, intra-day temperature variation, and rainfall as the average of measurements of the corresponding variable during the 30 days prior to sample collection. For cross-sectional analyses, ecological variables were estimated using remote sensing datasets. For each sample, we downloaded daytime and nighttime Land Surface Temperature (LST) measurements from the MODerate Resolution Imaging Spectroradiometer (MODIS) thermal sensor on board the NASA-Terra satellite at 1 kmLaverania prevalence in wild chimpanzee hosts. For longitudinal analyses , we specified a generalized linear mixed model (GLMM)55 with binomial error structure and logit link function to evaluate the relationship between climatic variation and the probability of Plasmodium infection among the Kanyawara chimpanzees. For each sample, we specified malaria infection (binary) as the outcome variable, mean ambient temperature (quadratic) and rainfall as fixed effects, chimpanzee individual as a random effect, and age (quadratic), sex, and intra-day temperature variation as covariates  as the outcome variable, mean ambient temperature (quadratic), rainfall, and percent forest cover as fixed effects, sampling site as a random effect, and daily temperature variation and number of replicates  as covariates  across the IUCN-designated spatial extent of chimpanzee habitat, according to the following equation:AT, TV, and FC are each corrected by subtracting the means of the chimpanzee dataset . All geospatial analyses were performed in ArcMap .Given the ecological relationships identified in the Pan-African GLMM, outlined above, we extrapolated infection probabilities across wild chimpanzee habitat in equatorial Africa. To characterize ecological variation across equatorial Africa, we derived composite rasters from mean ambient temperature and intra-day temperature variation measurements recorded between March 2000 and February 2017 across the African continent at 1-degree spatial and 1-month temporal resolution. Using these composite temperature rasters and the Hansen et al.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1-4Reporting Summary


Plasmodium) species. In this study, clinical testing of the XN-31p was performed using blood samples from patients with imported malaria in Japan.The automated haematology analyzer XN-31 prototype (XN-31p) is a new flow cytometry-based device developed to measure the number and the ratio of malaria-infected red blood cells (MI-RBC) with a complete blood count (CBC). The XN-31p can provide results in about one minute and also can simultaneously provide information on the malaria parasite , and the other 47 were negative. The XN-31p detected 32 patients as “MI-RBC positive”, which almost matched the results by the nested PCR and, in fact, completely matched with the microscopic observations. The ratio of RBCs infected with malaria parasites as determined by the XN-31p showed a high correlation coefficient of more than 0.99 with the parasitaemia counted under microscopic observation. The XN-31p can analyse the size and nucleic acid contents of each cell, and the results were visualized on a two-dimensional cytogram termed the “M scattergram”. Information on species and developmental stages of the parasites could also be predicted from the patterns visualized in the M scattergrams. The XN-31p showed a positive coincidence rate of 0.848 with the nested PCR in discriminating P. falciparum from the other species.Thirty-three patients were diagnosed by the nested PCR as being malaria positive (28 Plasmodium species. It was regarded to be of great help for the clinical diagnosis of malaria.The XN-31p could rapidly provide instructive information on the ratio of MI-RBC and the infecting The online version contains supplementary material available at 10.1186/s12936-022-04247-x. Plasmodium that cause human malaria include Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. In addition, Plasmodium knowlesi, one of the causative pathogens of malaria in monkeys, sometimes infects human and causes severe malaria symptoms in Southeast Asian regions [Plasmodium species in a patient’s blood can lead to adequate treatment of malaria. In particular, discrimination of P. falciparum from other species is very important, since patients with P. falciparum can rapidly develop high parasitaemia leading to unexpected lethal outcomes.The species of  regions –3. RapidPlasmodium infected red blood cells (RBCs) can be calculated, and indeed, Plasmodium species can be identified according to their morphological characteristics. However, the sensitivity and specificity of the diagnosis depend on the skill of the microscopists.The gold standard in the diagnosis of malaria is detection of parasites by microscopic observation of Giemsa-stained blood smears . The ratPlasmodium species using the different genetic sequences of parasite DNAs [Additional differential diagnostic techniques are currently available. Rapid diagnostic tests (RDTs) are immunochromatographic test kits designed to detect malaria parasite-specific antigens in the patient’s blood qualitatively . The RDTite DNAs –8. HowevTo solve those challenges, the automated haematology analyzers XN-31p were developed based on the principle of flow cytometry by the Sysmex Corporation  to detect malaria parasites in patient’s blood. Information on MI-RBC is suggested by the XN-31p in about 1 min after placing a whole blood sample in the system without any special pre-treatment of the sample.P. falciparum in blood samples were well confirmed at some clinical sites in malaria-endemic areas [As a matter of fact, the XN-30 was first released for research use only, and the accuracy and reliability of its counting parasite numbers of ic areas , 10. Theic areas –13.In this study, clinical testing of the XN-31p for the detection of malaria parasites in patient blood was performed to evaluate the confidence of the data in comparison with the results provided by the standard diagnostic methods of the RDT, microscopic observation, and PCR in a clinical setting using patients with suspected imported malaria in Japan.Eighty-six suspected patients who visited the Center Hospital of the National Center for Global Health and Medicine (NCGM) from January 2017 to January 2019 were asked to be subjects in the clinical testing of the XN-31p. Of them, 82 were enrolled, and the other 4 patients did not give their consent to this study.Of the 82 registered cases, 1 case that was found to be incompatible with the selection criteria  was dropped. In addition, another case whose measurement results could not be obtained within 24 h of blood collection was excluded from the analysis. Finally, 80 cases were included and monitored through their treatment procedure by collecting daily EDTA-preserved venous blood samples.Plasmodium infected RBCs are effectively analysed according to the intensity of the FSC and SFL of each cell. The intensity of the FSC signal mainly reflects the size of the cells, whereas that of the SFL reflects the amount of each cell’s nucleic acids. These signals are consequently used to detect malaria-infected RBCs (MI-RBC) with the help of unique algorithms.The automated haematology analyzer XN-31p (Sysmex Corporation) can detect malaria parasite-infected RBCs, CBC based on the principle of flow cytometry. The XN-31p automatically counts and classifies cells by irradiating cells with a 405-nm laser beam and analysing the resultant forward-scattered light (FSC) and side fluorescent light (SFL). In the XN-31p, the blood cells are initially treated automatically with Lysercell M and Fluorocell M (Sysmex Corporation). Lysercell M shrinks the RBCs, and the size of these shrunken cells well reflects the parasite cell size within the RBCs . Then, tPlasmodium infected RBCs. The PD mode uses blood samples diluted 7 times with CELLPACK DCL (Sysmex Corporation). Sample of 60 µL was used for measurement for the LM mode and 70 µL was used for the PD mode. The blood samples were measured by both modes of the XN-31p within 24 h of blood collection from patients. For the PD mode, the samples were measured immediately after the blood dilution.This study measured patient blood samples by two modes of the XN-31p: the LM  mode and the PD (pre-dilution) mode. The LM mode uses whole blood samples and was optimized to count low numbers of P. falciparum, and “Malaria? (Others)” is the flagging suggesting Plasmodium species other than P. falciparum. When the malaria parasite is unable to be classified, flagging of “Malaria? (UNC)”, meaning unclassified parasites, is suggested. Moreover, in rare instances, the detection of unknown particles in the blood sample sometimes causes indeterminant results, and then, flagging of “MI-RBC Abn Scattergram” is suggested, which means abnormal scattergram.Information on the species of malaria parasites can be suggested by flagging performed by the haematology analyzer algorithm. Namely, “Malaria? (P.f)” is the flagging suggesting Plasmodium infected RBCs were calculated based on visual counting under microscopic observation. Gametocytes were also included in the parasite density if any. The counting could be stopped if 50 or more infected RBCs were found when 5000 RBCs were observed (parasitaemia > 1%). In the case that not less than 10 infected RBCs were found when 10,000 RBCs were observed, the counting could also be stopped (parasitaemia > 0.1%). Then, no more than 100,000 RBCs were observed until at least 10 infected RBCs were found (parasitaemia: 0.1–0.01%). The counting was to be terminated when 100,000 red blood cells were observed regardless of the number of the infected RBCs detected (parasitaemia < 0.01%). Thus, samples were declared negative after having observed 0 parasite per 100,000 RBCs. The counting manner is illustrated as a flowchart in Additional file Thin blood smears were prepared with the patient’s blood. After fixation with methanol, the blood smears were stained with Giemsa solution. Ratios of P. falciparum-specific histidine-rich protein 2 antigen is detected. The T2 line appears when aldolase, a pan-Plasmodium antigen, is captured. Namely, the T1(+)T2(+) result can be interpreted as a single infection with P. falciparum or combined infection with P. falciparum and other Plasmodium species, T1(+)T2(−) as a single infection with P. falciparum, T1(−)T2(+) as an infection with non-falciparum Plasmodium species, and T1(−)T2(−) as negative for malaria infection.For the RDT, BinaxNOW® Malaria  was used according to the manufacturer’s procedure. The T1 line is formed when P. knowlesi was performed to verify the Plasmodium species [The DNA samples were extracted from fresh or frozen patient blood, and 200 µL of blood was used for the extraction. DNA extraction was accomplished with a Maxwell DNA RSC kit  or Qia DNA mini kit . The nested PCR targeting the 18SrRNA gene of 4 human malaria parasite species and A flow diagram of all patients recruited to this study is shown in Fig. P. falciparum positive by the flagging message of “Malaria? (P.f)” and 3 received the message of “Malaria? (Others)” ” Fig. a. One saP. falciparum positive by the LM mode of the XN-31p, 26 samples were also diagnosed as P. falciparum infection by microscopic observation. The other 2 samples were detected by microscopic observation to be mixed infection of P. falciparum and P. ovale and P. knowlesi, infection, respectively. Of the 3 samples flagged with “Malaria? (Others)” by the LM mode, 2 samples were confirmed to be P. vivax infections, and the other 1 sample was proved to be P. malariae infection by microscopy.Of the 28 samples detected as P. falciparum positive by the PCR, and 1 P. malariae positive by microscopy that was revealed to be a P. falciparum and P. malariae mixed infection by the PCR. In this P. falciparum and P. malariae mixed infection, the specific band for the PCR product made with the P. falciparum -specific primers on gel electrophoresis was very pale (data not shown), and the RDT result was T1(−)T2(+), suggesting that the density of P. falciparum parasites in the peripheral blood of the patient was low enough to be at submicroscopic level.The results of microscopic observations were well concordant with the nested PCR, except for 1 negative by microscopy that was, in fact, The results with the PD mode of the XN-31p were almost the same as those with the LM mode except for 3 samples  infection, and P. falciparum and other mixed infections was evaluated. The coincidence rates were calculated using the results of 33 PCR-positive samples. The LM and PD modes of the XN-31p showed a coincidence rate of 0.848 and 0.906 respectively, which were lower than that of 0.938 by microscopy ” by both modes of the XN-31p, and P. falciparum and P. ovale mixed infection detected by microscopy was detected as “Malaria? (P.f)” by the LM and PD modes of the XN-31p  were chosen and tested in 10 repetitive measurements ”.Figure P. vivax cases, RBCs infected with malaria parasites of various growth stages such as ring form, trophozoite, schizont, and gametocyte were found by microscopy. Those parasites were considered to correspond to each coloured cluster shown in the M scattergrams ”.In the slide smears of 2 ams Fig. d, e. TheP. malariae case was detected microscopically  appeared at a position where the FSC intensity was low , and the PCR result indicated P. falciparum infection. A pale but obvious cluster was detected in the M scattergram ” by the LM mode of the XN-31p. In this case, a quite low number of Two cases were flagged as “MI-RBC Abn Scattergram” by the LM mode of the XN-31p. The M scattergram of one case is shown ” flagging message.The M scattergram of a case of P. falciparum infection, only ring forms can be found in the patient’s peripheral blood because trophozoites and schizonts are usually sequestrated in vascular endothelium [Plasmodium falciparum gametocytes can also be found in patients’ peripheral blood, but this is not always the case. This study detected only ring stages in most of the P. falciparum-positive cases both by microscopy and the XN-31p, but in several cases, P. falciparum gametocytes or schizonts were found under microscopic observation. These non-ring stage P. falciparum observations were also detected by the XN-31p and showed specific clusters in the M scattergram , 2 cases of P. vivax (case nos. 10 and 42), 1 case of P. knowlesi (case no. 47), and 1 case of P. malariae-P. falciparum mixed infection (case no. 14). In all cases, according to the progress of recovery from malaria, daily M scattergrams visually showed the density of the clusters of Plasmodium infected RBCs to become thinner day by day , a small number of parasites were still detected by the LM and PD modes of the XN-31p on day 4 when parasite clearance was declared based on microscopic observation, and the patient was discharged. Further monitoring with the XN-31p was not feasible. For the P. malariae–P. falciparum mixed infection (case no. 14), the day of parasite clearance confirmed by microscopy was 2 days after the day when parasites were no longer detected by the LM and PD modes of the XN-31p. For 1 P. vivax case (case no. 42), monitoring was terminated before confirmation of parasite clearance both by microscopy and the XN-31p because the patient was discharged. In this case, on day 2, only gametocytes were detected by the microscopic observation at 0.014% parasitaemia, and the M scattergram showed a green-coloured cluster of parasites only, which corresponded to the gametocytes.Changes of parasitaemia until parasite clearance as determined by microscopic observation and the XN-31p were well concordant ”, but negative by the PD mode. Thus, the LoD (limit of detection) of the LM mode of the XN-31p could be much lower than the LoQ value defined by the company. This result is consistent with a recent report [P. falciparum. Recently, asymptomatic malaria cases with low-density parasitaemia have been discussed as potential reservoirs for transmission in endemic areas [The general detection limit for malaria parasites by microscopic observation was reported to be 50 parasites/µL of blood . Howevert report  that estic areas . Nucleicic areas  or LAMP ic areas , which aIn the present study, 2 samples were suggested to show abnormal patterns of M scattergrams by the flagging message of “MI-RBC Abn Scattergram”. In previous studies using the XN-30, it was pointed out that such abnormal patterns were related to the health conditions of the participants. For example, infants or malnutrition of the participants was related to the appearance of such abnormal patterns . The exiP. falciparum and non- P. falciparum cases. In the case of the P. falciparum-P. ovale mixed infection, the flagging message given by the XN-31p was “Malaria?(P.f)”, but the existence of non- P. falciparum parasite infected RBCs could be predicted by the M scattergram as a small sub-cluster appearing beside the main P. falciparum clusters.In most of the present cases, the flagging messages suggested by the XN-31p well differentiated the P. knowlesi infection, the flagging message was also “Malaria?(P.f)”, but the location of the cluster of parasite infected RBCs in the M scattergram was slightly different from the typical P. falciparum patterns, suggesting the possibility of infection by some other Plasmodium species. In this case, the negative RDT result strongly supported the prediction, and microscopic observation and the nested PCR verified this case to be a P. knowlesi infection. Imported cases of P. knowlesi are quite rare in Japan, and this case was the 2nd reported case [In the case of ted case .P. malariae infection, the shape of the cluster shown in the M scattergram was different from that of P. falciparum or P. vivax. Although it is known that the trophozoites of P. malariae and P. knowlesi found in the microscopic observations of thin-blood smears are similar by showing a “band form” [P. malariae and P. knowlesi were absolutely different in the M scattergram of the XN-31p. Such characteristics are expected to be better reflected in the algorithm of the XN-31p, and detailed differentiation of Plasmodium species may be possible in the near future.In the case of nd form” , 3, 21, P. falciparum, such as schizonts and gametocytes. The appearance of schizonts in the peripheral blood of patients with P. falciparum infections is an important sign of severe malaria [P. vivax cases, both included the red-coloured cluster indicative of ring stages  during the treatment of malaria. a The M scattergrams obtained by the PD mode from daily measurements. b Changes in parasitaemia in each case during the monitoring period. The vertical axes show parasitaemia detected by microscopy (dashed lines) and PD (solid lines) mode. The black arrows indicate the days when parasite clearance in the patients’ blood was declared by microscopy, and the gray arrows indicate those detected by PD mode. Pf: P. falciparum, Pv: P. vivax, Pm: P. malariae, Pk: P. knowlesi.


Plasmodium falciparum and Plasmodium vivax infections, clinical presentations, treatment effectiveness, vector transmission, and reinfections. With Government of India partners, Indian and U.S. academics, and trained researchers on the ground, the MESA-ICEMR team contributes information on malaria in selected parts of India.The Malaria Evolution in South Asia (MESA) International Center of Excellence for Malaria Research (ICEMR) conducted research studies at multiple sites in India to record blood-slide positivity over time, but also to study broader aspects of the disease. From the Southwest of India (Goa) to the Northeast (Assam), the MESA-ICEMR invested in research equipment, operational capacity, and trained personnel to observe frequencies of  NIH, and in collaboration with the Indian Council of Medical Research, the Malaria Evolution in South Asia (MESA) International Center of Excellence for Malaria Research (ICEMR) was organized to perform basic science and clinical research in India. The goal was to assess our present understanding of malaria pathogenesis, virulence, and transmission at multiple locations across India. In this framework, the MESA-ICEMR has witnessed new and evolving Government of India initiatives and policies that have been advanced to check transmission and treatments . Given lA single platform—called the Malaria Elimination Research Alliance (MERA)–India, led by scientists and clinicians within the Indian Ministry of Health, was established by the Government of India in 2019 to identify research priorities.For sampling diverse malaria cases across the country, the MESA-ICEMR team established several malaria study sites in India during the past decade. These include research laboratories at Goa Medical College (GMC), Goa; NIMR-Goa, Goa; the Regional Medical Research Center–Dibrugarh (RMRC-Dibrugarh), Dibrugarh, Assam; and field sites at Shalini Hospital–Krishi Gram Vikas Kendra, Ranchi, Jharkhand; and Acharya Vinoba Bhave Rural Hospital, Wardha. At the field sites, patients with malaria are recruited into the study, blood samples are collected, and data are recorded. The MESA-ICEMR laboratory at GMC-Goa also serves as a central site where new scientists and staff undergo training in patient recruitment and laboratory techniques, and where visiting scientists and collaborators gather for ICEMR-related scientific meetings. Moreover, research collaborations with government organizations such as NIMR-Delhi and public institutions such as the Indian Institute of Technology–Bombay, Mumbai, have also been established.Plasmodium falciparum and Plasmodium vivax malaria endemic in the state, GMC treats both the local and migrant populations in large enough numbers to allow for adequate sampling of both mild and severe malaria infections of both Plasmodium species. In Goa, the MESA-ICEMR program was able to ascertain a demographic profile of both P. falciparum and P. vivax malaria, with diagnostic and clinical indicators, over a 4-year period.P. falciparum– and P. vivax–infected red blood cells, particularly P. falciparum erythrocyte membrane protein 1 on the surface of infected erythrocytes. These proteins varied in their ability to bind to endothelial protein C receptors and to inhibit activated protein C– endothelial protein C receptor interactions.GMC is the only government tertiary health-care facility in a peri-urban setting in the small state of Goa. With both P. vivax cases seen annually at the GMC, the experimental approaches used in the MESA-ICEMR research laboratory at the GMC have led to insightful observations and results. MESA scientists initially focused on comparing cryopreservation efficacy between Indian P. vivax isolates and previously used Brazilian isolates.P. vivax parasitemia and reticulocytemia, which are frequently low.P. vivax prefers to infect reticulocytes ex vivo.P. vivax isolates that was greater than that of reticulocytemia, suggesting that Indian isolates differ in their reticulocyte preference.Plasmodium vivax has proved to be more challenging to culture through in vivo or even ex vivo assays compared with P. falciparum, mainly because P. vivax infects reticulocytes predominantly, which are difficult to isolate.P. vivax–infected reticulocytes and also found that P. vivax infection greatly decreases the osmotic stability of the infected reticulocyte.,P. vivax continue to be a high priority for the MESA-ICEMR, which has a strong interest in understanding variations in disease severity in malaria.With the relatively high volume of non-severe P. falciparum and P. vivax. In the initial work, the MESA-ICEMR team discovered a new vector, Anopheles subpictus, to be a contributor to perennial transmission.Anopheles stephensi mosquitoes hatched directly from field larvaeAn. stephensi laboratory colonies.,Plasmodium infections induced in both field-derived and colonized An. stephensi mosquitoes, and subsequent sporozoite production.P. vivax sporozoite production in An. stephensi at an Indian field site, and the techniques learned will be beneficial for other vector biologists both inside and outside India.P. vivax liver stage assays and transmission-blocking experiments are currently underway.The MESA-ICEMR laboratory at NIMR-Goa, built with a fully functional, state-of-the-art insectary, was crucial for the controlled vector infection studies conducted in India using both P. falciparum or P. vivax, or both. Using protein arrays, seroreactivity was measured against recombinant P. falciparum and P. vivax antigens. This studyP. falciparum antigen was pronounced and is comparable to the seroreactivity as seen from endemic areas such as Africa. Of 248 seropositive P. falciparum antigens, MSP10, heat shock protein 70, PTP5, AP2, AMA1, and SYN6 Merozoite Surface Protein 10 (MSP10), Heat Shock Protein 70 (HSP70), PfEMP1-trafficking protein (PTP5), Apetala 2 (AP2), Apical Membrane Antigen 1(AMA1), and SNARE protein (SYN6) showed strong reactivity to patient serum IgG. For P. vivax patient sera, however, ETRAMPs, MSPs, and ApiAP2, sexual stage antigen s16, RON3 Early Transcribed Membrane Proteins (ETRAMPs), Merozoite Surface Proteins(MSPs), Apicomplexan Apetela 2(ApiAP2), sexual stage antigen Pfs16, and Rhoptry Neck Protein 3 (RON3) were the key antigens that showed strong seroreactivity. This studyAs a part of the ongoing coordinated effort by all ICEMRs to identify shared antibody responses to a number of parasite antigens, the MESA-ICEMR group initially teamed up with Indian Institute of Technology–Bombay, Mumbai, to determine the serological profiles for 200 Indian patients infected with either P. falciparum malaria. The hilly terrain, vast forests, tribal inhabitants, poor infrastructure, and frequent floods present challenges in terms of diagnosis and treatment of malaria infections. These environmental conditions not only hamper malaria research, they also interfere with effective malaria interventions, such as the distribution of treated bed nets and effective indoor spraying. To gain a deeper perspective on the complexity of malaria transmission in these areas, longitudinal population studies with extensive follow-up would be most informative. The MESA-ICEMR team, with partners in Assam, set up a large cohort study in the high-transmission region of the Karbi Anglong District (manuscript in preparation). The goal was to assess the impact of epidemiological and socioeconomic factors on progression of disease in individuals and households over a 2-year period. The MESA-ICEMR study observed a greater malaria prevalence in the community, a high burden of asymptomatic cases, and some delayed parasite clearance (manuscript in preparation) compared with earlier reports.,In addition to MESA-ICEMR studies in Goa, a second highly active partnership is with a Government of India research laboratory in RMRC , Dibrugarh, in the northeastern state of Assam. Assam and other neighboring states of India, share ∼4,000 km of international borders with surrounding countries and provide a potential route for entry of novel forms of malaria drug resistance into India.Dissemination of relevant evidence from northeastern India in publications, and presentations at Indian scientific meetings and symposia, have enabled the MESA-ICEMR team to bridge the gap regularly between field practices and laboratory malaria research in India. Inter-site communication within MESA-ICEMR has ensured that work is not duplicated, and continual contact between collaborators ensures that imaginative new ideas and techniques are transferred quickly from the laboratory to field sites. Continued close interactions among practicing clinicians, university medical departments, and state-level health committees remain a priority as the MESA-ICEMR team monitors malaria trends across India.P. falciparum-to-P. vivax ratios, vector species distribution, and varying sensitivity to artemisinin combination therapy (ACT). This heterogeneity requires that subnational malaria control policies must be tailored to particular regions. For example, India has adopted two different first-line ACT regimens, one for Northeast India and another for the rest of India.,India is a big, populous country, and malaria in India is complex and heterogeneous. The heterogeneity includes region-specific variations in malaria transmission intensity, ,,P. vivax, but the MESA-ICEMR team has used a collection of live patient isolates to develop invasion assays. This will aid in mapping parasite invasion ligand expression and their interactions with red blood cell receptors. Furthermore, the invasion assay platform will allow MESA-ICEMR to test directly whether potential vaccine candidates such as recombinant invasion ligands,P. vivax, for which very little is known about invasion, the MESA-ICEMR team will continue to establish much needed methods essential to blocking invasion. Such fundamental initiatives are unachievable in other settings.Study outcomes from both our current research and planned research can provide complementary data for evidence-based policymaking at the regional level . The MESEfforts to monitor ACT sensitivity at the MESA-ICEMR study sites in India are still in preliminary form. Future expansion of these research efforts will provide a more detailed view of the effectiveness of current antimalarial regimens in India, and whether the current treatment policies need to be updated.Future research activities of the MESA-ICEMR will have an emphasis on malaria core interventions . The cohP. falciparum and P. vivax in surveys conducted in their natural settings. This could build on earlier work pointing to poorly appreciated vectors possibly contributing to regional transmission, as we have reported previously.The MESA-ICEMR team will expand its entomological surveillance capacity, and laboratory vector studies, to include membrane feeding and parasite development studies. A high priority will be placed on poorly characterized Indian vectors that tested positive for P. vivax, which has historically been much less explored compared with P. falciparum. In particular, a full understanding of the molecular basis of P. vivax pathogenesis is needed, and the MESA-ICEMR team aims to pursue investigations on P. vivax pathogenesis, red blood cell invasion, and reticulocyte tropism. Plasmodium vivax has been shown to cause severe malaria and deaths in South Asia, and it is important to determine how much of this is a result of particular pathogenic strains of P. vivax and/or a region-specific host vulnerability.–Plasmodium vivax–specific mechanisms of pathogenesis remain largely unknown. The MESA-ICEMR study site in Goa at GMC has a high proportion of P. vivax infections (∼70% mono infections), of which ∼5% lead to severe disease manifestation. GMC thus provides an important opportunity to study questions relating to P. vivax pathogenesis. The major clinical complications of patients with severe P. vivax at GMC are anemia, jaundice, and respiratory distress.P. vivax case data with severe disease, including parasite biomass, reticulocyte preference, endothelial activation, plasma biomarkers of coagulation, and inflammation.The MESA-ICEMR, operating in South Asia, has a strong interest in P. vivax, particularly the parasite’s capacity to produce hypnozoites. Such research, in patient samples and in laboratory models, has the potential to develop diagnostic tools for identifying latent hypnozoites and to improve our understanding of relapsing infections. Improving technical approaches to understand P. vivax liver-stage biology is a high priority to MESA-ICEMR.The MESA-ICEMR will also explore liver-stage infections of The MESA-ICEMR has extensive technical resources available through its collaborators, both within India and abroad.Plasmodium falciparum culturing, genomics, and serology, which are well established at the central MESA-ICEMR GMC site. In addition, our research scientists also have the opportunity to visit parent laboratories in Seattle  or Boston . MESA-ICEMR scientists in India have also attended international workshops and conferences on malaria, and regularly upgrade their technical knowledge and skills.Even as MESA-ICEMR-mediated India–US collaborations have added fresh perspectives and innovations in malaria research for both sides, the MESA-ICEMR teams have also jointly helped build secondary expertise in statistical analysis, in novel molecular probes such as bead-based antibody detection, and in state-of-the-science insectaries. Last, the MESA-ICEMR team worked early and aggressively with the rapidly emerging biotechnology companies in India to gain high-quality, cost-effective services for primer design and DNA sequencing. This included local Indian startups and Indian branches of established companies such as Eurofins, Illumina, nanochip technology, and more. An internal, deep understanding of our questions and scientific needs allowed us to assess the quality of work outsourced in India, and set an example for others on what molecular research is possible in India, without large movements of patient samples abroad. MESA-ICEMR staff have varied educational backgrounds, with both basic and advanced science degrees from national and international educational institutions. Most newly hired staff train in laboratory techniques, including New protocols, which include community surveys at various sites, were under the ethical board review process at most sites, but progress has been hindered as a result of the COVID-19 pandemic. We have planned community surveys in conjunction with local community members and relevant government health workers to engage cooperation among local inhabitants. We have found local involvement to be invaluable for achieving community survey objectives (unpublished data). These surveys will be region specific and will focus on assessing health-seeking behaviors, disease surveillance, presence of asymptomatic infections, and more, in the local communities.The MESA-ICEMR team and its Indian partners have developed infrastructure to survey, assess, and conduct malaria research in select parts of India. Although India has numerous resources to monitor national malaria control campaigns, there are components of the MESA-ICEMR activities that can add value with fresh public health questions and unique technical approaches, including deeper scientific queries into asymptomatic infections and mechanisms that initiate antimalarial resistance.The team will also introduce administrative structures for sound and safe research. MESA-ICEMR can provide Indian authorities with an independent assessment of the malaria burden at each MESA-ICEMR study site,


Malaria is the most deadly parasitic disease and continues to claim more than a half million of deaths across the world each year, mainly those of under-fives children in sub-Saharan Africa. The aim of this study was to determine the epidemiological, clinical and laboratory features of patients with severe malaria at the Centre Hospitalier Régional Amissa Bongo (CHRAB), a referral hospital in Franceville.It was an observational descriptive study conducted at CHRAB over 10 months. All admitted patients at the emergency ward of all ages presenting with positive test to falciparum malaria diagnosed by microscopy and rapid test with clinical signs of severe illness describe by World Health Organization were enrolled.During this study, 1065 patients were tested positive for malaria, of them 220 had severe malaria. Three quarters (75.0%) were less than 5 years of age. The mean time to consultation was 3.5 ± 1 days. The most frequent signs of severity on admission were dominated by neurological disorders 92.27% (prostration 58.6% and convulsion 24.1%), followed by severe anemia 72.7%, hyperlactatemia 54.6%, jaundice 25% and respiratory distress 21.82%.The other forms such as hypoglycemia, haemoglobinuria, renal failure were found in low proportions < 10%. Twenty-one patients died, coma , hypoglycemia , respiratory distress  and abnormal bleeding  were identified as independent predictors of a fatal outcome. Anemia was associated with decreased mortality.Severe malaria remains a public health problem affecting mostly children under 5 years. Classification of malaria helps identify the most severely ill patients and aids early and appropriate management of the severe malaria cases. Plasmodium infections result in a spectrum of clinical effects, including asymptomatic parasitemia, uncomplicated malaria, severe malaria, and death [Plasmodium which can infect humans, Plasmodium falciparum is the most pathogenic species and accounts for the majority of severe and fatal malaria in Africa and particularly in Gabon [In 2020, malaria remains a devastating health problem and the deadliest parasitic disease for humans. Nearly 241 million cases of malaria occurred worldwide in 2020, most of which were in the WHO African Region. The same year, 627,000 malaria-related deaths were recorded [in Gabon , 4, prinin Gabon , 6. In eP. falciparum by microscopy or a rapid diagnostic test and at least one criterion for severe disease  [Severe falciparum malaria definition (SFM) has evolved these last two decades. In 1990, the World Health Organization (WHO) established criteria for the diagnosis of severe malaria . In 2000itaemia) .Malaria transmission in Gabon is stable and perennial . There iThis study was conducted at the emergency ward of Centre Hospitalier Régional Amissa Bongo (CHRAB). The CHRAB is located in the town of Franceville and it is the most important public healthcare facility in the Haut-Ogooué province. Franceville is an urban region of south-east Gabon , the capital city of Haut-Ogooué, it is the third main town of Gabon in terms of population  [The study was carried out from June 2019 to April 2020 (03 June 2019–09 April 2020).All febrile (or those with history of fever in the last 48 h) participants admitted at the emergency ward and pediatric emergency ward of all ages were referred to the study team and seen on admission by a clinical staff of the study. Summary data were recorded on a pro-forma sheet. Only patients presenting with positive falciparum malaria test with clinical signs of severe illness  from EDTA blood samples. Capillary blood glucose was measured with an ACCU-CHEK® Active blood glucose meter . Biochemical analyses were carried out by an ABX PENTRA device  on plasma samples. Macroscopic haemoglobinuria was measured using SD UroColor® Urine Test Strip .Laboratory analysis were performed at the CHRAB’s laboratory and the Centre International de Recherches Médicales de Franceville (CIRMF). Malaria was confirmed by thick blood film under the light microscope. The investigations were performed within 30 min of blood collection at the CHRAB’s laboratory. Thick blood films were stained with 3% Giemsa and parasite load was determined using the Lambarene method . All thiructions . A previructions . MeasureThe chest X-ray was performed when clinical examination is suggestive of lung damage at the CHRAB radiography department.P. falciparum by microscopy or a rapid diagnostic test and at least one WHO criterion for severe disease (see above). Fever was defined as axillary temperature  ≥ 37.5 °C or reported history of fever in the past 48 h.Severe malaria was defined by the detection of Data of patients were recorded in Excel 2013 spreadsheets. Statistical analysis was performed using the Epi-Info 6 and R version 4.0.5 (2021-03-31) software. Simple proportion were calculated for qualitative variables and quantitative variables by mean, standard deviation (SD), median with inter-quartile range (IQR). The qualitative variables were compared using the Chi-square test or Fisher’s exact test for numbers below 5. Binary logistic regression model was used to compute the adjusted odds ratios (aOR) of independent risk factors. The confidence interval was set at 95% (95% CI). Statistical significance was set at α = 5%.From June 2019 to March 2020, a total of 5642 patients were admitted to the CHRAB emergency department. One thousand sixty-five (1065) of them were tested positive for malaria and the 4575 other patients were admitted for another pathology. Of 1065 patients with malaria, 847 (79.4%) had simple or moderate malaria and 220 (20.6%) had severe malaria  as show in Table No patient with severe malaria went to the hospital at the start of symptoms. The mean time to consultation was 3.5 ± 1 days (extremes 1–7 days). Patients with severe malaria usually had a delay in consultation of more than 48 h (53.64%). Time to consultation from symptoms onset was not associated with death (p = 0.13). A total of 21 (9.54%) patients died during study. Sixteen (76.19%) patients died less than 24 h after their hospitalization . The highest number of deaths was observed in the age group of 0–5 years.Table Table Malaria is one of the world’s leading parasitic diseases and affects a considerably large number of people especially children under 5 years old. This study was designed to describe the epidemiology, clinical and laboratory presentations of severe falciparum malaria according to the last WHO updates  in patieMalaria prevalence was 18.9%, this prevalence has remained stable in Franceville since 2011 , 10, 11.P. falciparum malaria with further progression to severe malaria. In this study the mean time between the onset of the disease and admission was 3.5 ± 1 days (extremes 1–7 days). Indeed, several studies in other African settings have reported an increased risk of severe malaria with delay [In this study, the most represented age group was 0–5 years i.e. three quarters (75.0%) of population. This finding is in accordance with other studies, which states that children less than 5 years are most vulnerable to malaria , 21, 22.th delay –25 in prAnopheles gambiae species complex.) which develops during the rainy season. Rains create a moist climate, resulting in a rapid parasitic cycle in the mosquito, thus increasing malaria transmission during this period.The frequency of severe falciparum malaria cases was associated with pluviometry, with most cases seen in the months of September to November (rainy season in Gabon). This result is in accordance with another study performed by Maghendji-Nzondo et al. . This obThe recent definition of severe malaria requires additional investigations that are difficult to carry out sub-Saharan countries, especially in an emergency situation. In this context, the study was unable to diagnose metabolic acidosis. This difficulty is reported by Camara et al.  in SenegThe most frequent clinical feature of severe malaria on admission was neurological disorders, particularly prostration and convulsion, followed by severe anaemia, hyperlactataemia and respiratory distress. Similar results were also observed by other authors , 29, 30.P. falciparum infections, and the mortality rate is approximately 70% [The independent prognostic factors associated with mortality in this study were coma, respiratory distress, hypoglycaemia and abnormal bleeding. These observations are entirely consistent with other studies . The sevtely 70%  that contely 70% , 19, 35 tely 70% , 36. Hyptely 70% .This study highlight only 5 cases of abnormal bleeding but this feature was associated with mortality. It is also rare in other studies. Severe anaemia was the second most frequent clinical feature of severe malaria in this study but was associated with decreased mortality. A similar observation in other studies showed a better outcome in children with severe anaemia , 30. ThiAt the CHRAB hospital, severe malaria is one of the main cause of hospitalization and its related mortality is high The most common clinical features of severe malaria in this study were neurological disorders, severe anaemia and respiratory distress and coma, respiratory distress, hypoglycaemia and abnormal bleeding were independent prognostic factors associated with mortality in this study.


Characterization of the genetic diversity would provide essential information about the ongoing control efforts. This study aimed to explore allelic polymorphism of merozoite surface protein 1  and 27.6% (62/225) of the samples were collected in high and low transmission areas, respectively. Polymorphic msp1 and msp2 genes were used to explore the genetic diversity and multiplicity of falciparum malaria infections. Genotyping of msp1 was successful in 86.5% (141/163) and 88.7% (55/62) samples collected from high and low transmission areas, respectively. Genotyping of msp2 was carried out among 85.3% (139/163) and 96.8% (60/62) of the samples collected in high and low transmission sites, respectively. Plasmodium falciparum msp1 and msp2 genes were amplified by nested PCR and the PCR products were analysed by QIAxcel ScreenGel Software. A P-value of less or equal to 0.05 was considered significant.Parasite genomic DNA was extracted from a total of 225 dried blood spots collected from confirmed uncomplicated msp1  and the two allelic families of msp2 (FC27 and 3D7), were observed in samples collected in high and low transmission areas. However, MAD 20 and FC 27 alleles were the predominant allelic families in both settings. Plasmodium falciparum isolates circulating in western Ethiopia had low genetic diversity and mean MOI. No difference in mean MOI between high transmission sites (mean MOI 1.104) compared with low transmission area (mean MOI 1.08) (p > 0.05). The expected heterozygosity of msp1 was slightly higher in isolates collected from high transmission sites (He = 0.17) than in those isolates from low transmission (He = 0.12). However, the heterozygosity of msp2 was not different in both settings .High prevalence of falciparum malaria was identified in children less than 15 years as compared with those  ≥ 15 years old . The three allelic families of Plasmodium falciparum from clinical malaria cases in western Ethiopia has low genetic diversity and multiplicity of infection irrespective of the intensity of transmission at the site of sampling. These may be signaling the effectiveness of malaria control strategies in Ethiopia; although further studies are required to determine how specific intervention strategies and other parameters that drive the pattern. Plasmodium falciparum remains the most dangerous malaria parasite species to humans with important public health consequences mostly in Africa [(1 − Σpi2)], where n is the number of isolates sampled and pi is the allele frequency at a given locus. A p-value of less or equal to 0.05 was considered suggestive of a statistically significant difference between sites.Ethical clearance was obtained from the Ethiopian National Ethics Review Committee and Addis Ababa University, Aklilu Lemma Institute of Pathobiology, IRB. Permission to conduct the study at the health facilities was sought from the relevant regional and district health authorities. Written informed consent was obtained from adult study participants and a parent or guardian of a child. Written informed assent was also taken from children.P. falciparum cases enrolled in the study, 225 of them tested positive by PCR. 72.4% (163/225) and 27.6% (62/225) PCR positive samples were collected from high and low malaria transmission sites, respectively. The mean age of study participants was 17.8 ± 12.7 years and with an age range of 7 months–75 years old. The ratio of males to females was 1.27: 1. The prevalence of falciparum malaria was higher in children less than 15 years as compared with those  ≥ 15 years old . No significant difference in P. falciparum infections in gender was observed (p > 0.05),  and 88.7% (55/62) of samples collected from high and low transmission areas, respectively. Similarly, for msp2 genotyping, 85.3% (139/163) and 96.8% (60/62) samples were successfully genotyped from high and low transmission sites, respectively. The three allelic families of msp1  and FC27 and 3D7 of msp2 allelic families were identified in all study sites. There was no difference in the frequency of msp1 or msp2 allelic families by study sites (P > 0.05) , 25.5% (36/141), and 5% (7/141) of the samples from high transmission sites had MAD20, K1, and RO33 allelic families of msp1, respectively. Similarly, 92.7% (51/55), 14.5% (8/55), and 5.45% (3/55) samples collected from low malaria transmission settings had MAD20, K1, and RO33 msp1 allelic families, respectively. Multiclonal infections with K1 + MAD20, and MAD20 + R033 combinations were also observed. In the high transmission setting, K1 + MAD20 accounted for 14.9% (21/141) while the frequency of this allelic combination was 10.9%(6/55) in the low transmission setting . A similar pattern was also observed with the K1 alleles, where sizes in high malaria transmission ranged from 167 to 282 bp while those from low malaria transmission sites ranged from 167 to 269 bp. In addition, a similar pattern was observed in RO33 where P. falciparum isolates in high and low malaria transmission areas had fragment sizes of (160–162 bp) and (164–165 bp), respectively.The fragment size of MAD 20 alleles observed in high malaria transmission area (103–308 bp) was similar to that seen in msp2 allelic types identified among the samples collected from both study sites. However; FC27 was the frequent allelic type in western Ethiopia. There was no difference in the frequency of this allele in high transmission 97.8% (136/139) and low transmission 98.3% (159/160) areas. Few multi-allelic infections combining 3D7 + FC27 families were detected  and 87.3% (48/55) of the samples genotyped for msp1 in high and low transmission sites, respectively. Similarly, 97.2% (135/139) and 96.7% (58/60) of isolates from high and low transmission areas were monoclonal msp2, respectively. No significant difference was observed in the proportion of msp1 or msp2 multiplicity of infection with sex, and age group of study participants and intensity of transmission (P > 0.05)  and low transmission (mean MOI = 1.08) (p > 0.05). The expected heterozygosity of the msp1 gene was slightly higher in high malaria intensity sites (He = 0.17) than isolates from low malaria intensity (He = 0.12). On the other hand, heterozygosity of the msp2 gene was not different in both settings , (Table ) p > 0.0. The expP. falciparum population circulating in western Ethiopia, these didn’t represent populations with relatively high and low transmission sites as presented here. The prevalence of uncomplicated falciparum malaria in the populations studied was higher in children  ≤ 15 years than those  ≥ 15 years old  and this was in agreement with previous reports from Ethiopia and Kenya .Measuring the complexity of infection and genetic diversity of parasite populations across different endemicities may be used as an indicator to evaluate the efficacy of ongoing control and elimination strategies. While a few studies have investigated MOI and genetic diversity of the nd Kenya , 26. Immnd Kenya , and thend Kenya , 29. In msp1  and FC27 and 3D7 of msp2 allelic families were identified in the study areas. However, MAD 20 of msp1 and FC27 of msp2 allelic families were the predominant alleles in both areas with variable malaria endemicity. There was no difference in the frequency of msp1 or msp2 allelic families by the intensity of transmission. Therefore, the population composition of P. falciparum isolates in western Ethiopia shows monoclonality infections regardless of the difference in intensity of transmission intensity and geographic separation of the parasites. This finding suggests the likely presence of a high level of inbreeding among the circulating clones between the study sites. Previous reports had also indicated that malaria parasites in Ethiopia have moderate levels of genetic diversity and a similar population structure of the parasite . However, the lack of differences with the relative variance in transmission intensities might be an indication that malaria control measures that reduce entomological inoculation rate [Anopheles mosquito vector might be shaping P. falciparum population structure in malaria- endemic sites [P. falciparum may also determine the diversity of the parasite [Anopheles arabiensis, while Anopheles pharoensis, Anopheles funestus and Anopheles nili are secondary vectors [Anopheles gambiae is mostly seen.Most of the participants had monoclonal infections at both e groups , 44. Thie groups , though e groups . Overalle common , 48, 49.ion rate , 53, andion rate  have beeic sites –57. With vectors , 59, unlPlasmodium falciparum isolates were mainly monoclonal with low MOI and lesser genetic diversity. Mean MOI and diversity of the parasite were not associated with the intensity of malaria transmission. Hence, MOI and the genetic diversity of P. falciparum might not be a good predictor of transmission intensity in all malaria- endemic areas. There is a need to find out an inclusive measurement of the intensity of transmission in all malaria-endemic areas. The observation of low genetic diversity and MOI in the study area may signal the effectiveness of the recent deployment of massive malaria control strategies in Ethiopia; however, this needs further studies to map the different intervention strategies and other parameters that could affect parasite genetic diversity.


Plasmodium falciparum infection amongst patients presenting with suspected malaria were investigated at a major urban hospital in Douala, Cameroon with a particular focus on anaemia.Urban malaria has received insufficient attention in the literature. The prevalence and clinical characteristics of P. falciparum using rapid diagnostic tests and PCR, and anaemia was defined by haemoglobin level according to WHO definitions. Likelihood ratios (LR), odds ratios (OR), and population attributable risk percent (PARP) were calculated.A cross-sectional, 18-week demographic and clinical survey was conducted of patients presenting to the Emergency Department of Douala Military Hospital with suspected malaria, largely defined by the presence or recent history of fever. Venous samples were tested for Plasmodium falciparum was detected in 37.2% by nested PCR. Among all participants, several factors were associated with clinically significant LR for P. falciparum infection, including age 10–14 years (positive LR: 3.73), living in the island district of Douala VI (positive LR: 3.41), travel to any of three northern regions (positive LR: 5.11), and high fever > 40 °C at presentation (positive LR: 4.83). Among all participants, 8.7% of anaemia was associated with P. falciparum infection, while the PARP was 33.2% among those < 15 years of age and 81.0% among 10–14-year-olds.Participants were ages 8 months to 86 years, 51% were women (257/503), and all districts of Douala were represented. Overall, 38.0% (n = 189/497) were anaemic, including 5.2% (n = 26/497) with severe anaemia. Anaemia prevalence was significantly higher  among children < 15 years  compared to adults . P. falciparum infection in the urban hospital was high. Mirroring trends in many rural African settings, older children had the highest positivity rate for P. falciparum infection. Anaemia was also common in all age groups, and for those 10–14 years of age, 80% of the risk for anaemia was associated with P. falciparum infection. Malaria rates in major urban population centres can be high, and more research into the multifactorial causes of anaemia across the age spectrum are needed.The prevalence of The online version contains supplementary material available at 10.1186/s12936-022-04315-2. Anopheles stephensi vector [Malaria remains one of the most important global infectious diseases, with an estimated 241 million incident cases and 627,000 estimated malaria deaths in 2020 [i vector –7.Plasmodium falciparum is estimated to account for > 95% of malaria cases in Cameroon, though Plasmodium malariae, Plasmodium ovale, and even Plasmodium vivax have been detected [Located in the WHO Central Africa region, Cameroon is one of the WHO’s designated high burden to high impact malaria countries with > 6.9 million malaria cases and 14,841 malaria deaths estimated for 2020 . While tdetected –19.The economic hub and most populous city of Douala has a smaller representation in the malaria literature than the capital, Yaoundé. Lying within the Littoral Region of the Forest zone, Douala is home to a population of approximately 3 million people . This zoUncomplicated malaria often presents with nonspecific symptoms including malaise, headache, fatigue, abdominal discomfort, and myalgias/arthralgias followed by fever, chills, diaphoresis, anorexia, nausea, and emesis . In infaP. falciparum infection and the clinical characteristics of these infections, with a specific emphasis on anaemia.Urban studies looking at rates of malaria and anaemia in those presenting to high level care centers with fever are also less prevalent in the literature. In Cameroon, the few existing studies in Douala over the past 20+ years have been limited by reliance on microscopy for diagnosis, recruitment of a narrow age range, inconsistent haemoglobin thresholds used to define anaemia, and minimal assessment of additional symptomatology. To characterize the burden and presentation of malaria and anaemia in the most populated urban centre in Cameroon, a descriptive survey of symptomatic patients presenting to the Emergency Department (ED) of Douala Military Hospital (DMH) was conducted. This manuscript reports on the prevalence of A cross-sectional epidemiological and clinical survey of symptomatic patients presenting to the ED of DMH was conducted. The hospital sits in the Douala I district and treats both military and civilian patients. Its ED consists of 2–3 physicians, a triage station staffed by 5–7 nurses, and approximately 18 beds. The study planned to recruit a convenience sample of 450 patients presenting to the ED for whom there was a suspicion of malaria. Recruitment was planned for consecutive, weekday enrollment due to the availability of the laboratory staff and equipment. ED physicians and nurses were asked to refer patients meeting the following eligibility criteria to the study team:1) Age of 6 months and older.2) At least one of the following: (a) temperature ≥ 37.5 °C on presentation to the triage nurse station; (b) subjective history of fever in the previous three days; or (c) suspicion of malaria infection by the ED physician.The recruitment team administered a questionnaire including demographics, travel history, prior medications and traditional remedies used, and symptomology. The team also assessed for clinical signs of anaemia . Symptoms and signs were rated on a four-point scale from 0 to 3 with 0 indicating the absence of the symptom/sign, 1 indicating borderline or mild (or once per day in the case of emesis and diarrhoea), 2 indicating moderate (or twice per day for emesis and diarrhoea), and 3 indicating severe symptoms (or ≥ 3 times per day for emesis and diarrhoea). Temperature from the triage station was recorded from the patient’s medical card if available, and an oral temperature  was taken by study personnel for all participants.Plasmodium lactate dehydrogenase (pLDH) malaria rapid diagnostic test (RDT) commonly used throughout Cameroon  [In the ED, a 2 mL venous blood sample was collected, used to measure haemoglobin level using the HemoCue® 201 + system , and tested with a WHO pre-qualified histidine-rich protein 2 (HRP-2)/h Korea) .P. falciparum, 144 bp for P. malariae, 120 bp for P. vivax, and approximately 800 bp for P. ovale [Whole blood samples were centrifuged at 3000 rpm for 5–10 min and then pipetted into plasma and red blood cell (RBC) pellet samples. All samples were stored at -20° C in the DMH lab and later transported on ice to the Malaria Unit of the Centre Pasteur of Cameroon (CPC) in Yaoundé, Cameroon. DNA was extracted from the RBC pellets at the CPC using DNeasy® Blood and Tissue DNA extraction kits  then shipped to the Yale School of Public Health in New Haven, USA on dry ice. Extracted DNA was amplified by nested polymerase chain reaction (PCR)  with 95% confidence intervals (CI) were calculated using binomial logistic regression in which a single predictor variable (e.g. sex) with two categories was entered [All recruitment data were recorded onto paper standardized case record forms by study staff and then double entered into the database. Participants were grouped into ages < 15 years-old (children) and ≥ 15 years old (adults). Children were further divided into those < 5 years old, 5–9 years old, and 10–14 years old. Detection by PCR constituted the reference standard for the presence of malaria infection. Anaemia was defined by WHO criteria  had a history of fever Table , as expeAmong all participants, the prevalence of anaemia was 38.0% n = 189/497), including 16.3% (n = 81/497) with moderate and 5.2% (n = 26/497) with severe anemia. Clinical findings associated with anaemia can be found in Additional file 89/497, iP. falciparum infection only), 12.6% (n = 63/500) positive for both HRP-2 and pLDH , and only 0.4% (n = 2/500) positive for pLDH alone .One quarter  of participants were positive by RDT, with 12.2% (n = 61/500) positive for HRP-2 only  P. falciparum mono-infection, 1.8% (n = 9/503) P. falciparum/P. malariae mixed infection, and 1.0% (n = 5/503) P. falciparum/P. ovale mixed infection. P. falciparum infection status varied by age group  of < 5 year-olds, 48.5% (n = 16/33) of 5–9 year-olds, 69.0% (n = 20/29) of 10–14 year-olds, and 34.0% (n = 136/400) of ≥ 15 years old. There was a significant association between age group and either positive RDT (χ2(3) = 31.31, p < 0.001) or detection of P. falciparum by PCR (χ2(3) = 16.09, p < 0.001) with the highest standardized residuals for the 10–14-year-old age group. The prevalence of P. falciparum as diagnosed by PCR was significantly higher among participants aged 10–14 compared to participants < 5 years old  and ≥ 15 years old .Malaria rates were higher by nested PCR; among all participants, 37.2% (n = 187/503) were positive for Regarding measurement of haemoglobin level among children 6–59 months old, haemoglobin was < 11.0 g/dL in 58.3% (compared to 56.5% in the DHS), between 7.0–9.9 g/dL in 19.4% (compared to 25.1%), < 8.0 g/dL in 13.9% (compared to 5.8%), and < 7.0 g/dL in 11.1% (compared to 1.7%). Regarding prevalence of malaria infection as diagnosed by RDT, 25.0% of participants 6–59 months were RDT positive, compared to 7.8% in the DHS.P. falciparum PCR-positive participants were younger  and more likely to have reported already trying an antipyretic (χ2(1) = 4.31, p = 0.038) . Among epidemiological factors, clinically significant positive LRs for P. falciparum infection by PCR were associated with age 10–14 years (positive LR: 3.73), living in the island district of Douala VI (positive LR: 3.41), and travel to any of three northern regions (positive LR: 5.11). Clinically, high fever > 40 °C at the time of presentation (positive LR: 4.83) and frequent emesis ≥ 2 times per day (19.85) were associated with clinically significant positive LRs. Several other gastrointestinal symptoms weakly increased the probability of P. falciparum infection with positive LR >2.0 . Considering PCR as the gold standard, RDTs demonstrated high positive and negative predictive value in this urban setting. A positive RDT (any band) increased the probability of malaria dramatically (positive LR: 28.70), and an HRP-2/pLDH positive result had a positive predictive value close to 1.0 . The absence of a positive RDT  was associated with a clinically significant negative LR (0.37). Table P. falciparum infection among children (positive LR: 3.24–4.32), but not adults (positive LR: 0.70–0.91).PCR results were used as the reference standard for malaria diagnosis. Compared to PCR-negative participants, retic  among P. falciparum-positive  versus P. falciparum-negative participants , and the association between overall rates of anaemia and P. falciparum infection trended toward statistical significance   was significantly higher  among participants with P. falciparum  compared to those negative for P. falciparum . For those ≥ 15 years old, anaemia was not associated with malaria (PARP -0.8%) and anaemia rates did not differ statistically by P. falciparum infection status . The percent of anaemia associated with P. falciparum infection varied by age, as the PARP was 14.3% among children < 5 years old, 30.7% among children 5–9-year-olds, and 81.0% among 10–14-year-olds.Among all participants, 8.7% (the PARP) of anaemia (any severity) was associated with P. falciparum infection and anaemia. As DMH is a fee-for-service hospital in a major metropolitan area, a lower prevalence of malaria infection may have been expected, as such centres receive febrile patients with a variety of aetiologies; however, over one third of individuals presenting with fever, history of fever, or other suspicion for malaria were PCR-positive for P. falciparum. This study echoes findings from an urban hospital in Sierra Leone during the same year [P. falciparum, using a highly sensitive multiplex real-time PCR assay. Recent research shows that new vectors may emerge in large, urban environments [P. falciparum infection may remain high even within the largest population centres traditionally felt to be at lower risk.An 18-week cross-sectional study of symptomatic patients across the age spectrum presenting to an urban hospital in the most populous city in Cameroon was conducted and found a high incidence of both ame year . In thatronments , 36 and ronments . Within P. falciparum infection among these participants. Residence on the island district of Manoka  or travel to any of the three northern regions  increased the probability of P. falciparum detection. Travel as an important risk factor among an urban population has been previously described in Malawi [P. falciparum positive, which may reflect broader trends in higher prevalence among older children across sub-Saharan Africa [P. falciparum infection. Among children, more severe symptoms generally  increased the probability of P. falciparum. Importantly, while more severe degrees of anaemia increased the probability of P. falciparum infection among children < 15 years old, this was not seen among participants ≥ 15 years old.Several epidemiologic and clinical characteristics were associated with a higher probability of n Malawi . Children Africa , 37. PosP. falciparum infection was 51%, and a child presenting with fever > 40 °C and severe anaemia would have over 95% probability of P. falciparum infection. Among adults ≥ 15 years old, the prevalence (pre-test probability) of P. falciparum infection was lower at 34%. However, an individual in this age group presenting with frequent emesis and recent travel to the North would have a near 100% probability of P. falciparum infection. While not a replacement for malaria diagnostic tests, a better understanding of characteristic epidemiological and clinical trends can help health care providers risk stratify patients to streamline testing and treatment. Criteria and calculators to stratify risk for other infections are already employed in urgent care and emergency settings, for example the Centor score for Streptococcal pharyngitis [Applying these results to children < 15 years old, the prevalence (pre-test probability) of ryngitis  or the Aryngitis . CreatinP. falciparum infection by PCR testing. Self-medication prior to presentation to the hospital is quite common. Private pharmacies are proliferating in urban centres, where most drugs are available without a need for clinical consultation. A recent survey of customers at pharmacies in Douala IV found 47% of customers buying antibiotics did so without a prescription [In this cohort, over 17% had already taken an antimalarial, and among these, fewer than 50% were found to have cription . DetermiAlmost 40% of participants had anemia, with > 5% meeting criteria for severe anemia. While the burden of anaemia was not confined to younger age groups, the prevalence of anaemia was higher among children and women compared to adult men. In comparison to the community sample of children < 5 years old in the 2018 DHS, there was a higher percentage of more severe anaemia . Such a P. falciparum infection than young children or adults, and over 80% of anaemia was associated with malaria infection. The role of malaria in the burden of anaemia in Cameroon may extend even to asymptomatic malaria infection as a recent longitudinal study across multiple villages in Cameroon’s Central Region found asymptomatic malaria infection (diagnosed by PCR) was associated with 69.2% of anaemia in the study population [This study further demonstrates that a considerable risk for anaemia among symptomatic children is associated with malaria infection. Considering 10–14-year-olds for example, this group had a higher prevalence of pulation . Despitepulation , 43. Cerpulation .P. falciparum and anaemia were detected has shown that over 50% may remain with low haemoglobin even weeks after treatment for malaria [These findings have implications for clinical practice. First, effectively detecting and treating malaria infection may reduce the overall burden of anaemia among children. Indeed, chemoprophylaxis against malaria was recently found to reduce death and readmissions among children admitted for severe anaemia . Second, malaria . While i malaria .P. falciparum infections were either incidental findings or one or multiple comorbid infections responsible for the patient’s febrile illness. This study can therefore only comment on prevalence of P. falciparum infection rather than the clinical entity of P. falciparum malaria, including severe malaria. Second, recruitment took place at a single urban site over less than half the year, specifically during and after the summer rainy season when malaria transmission would be expected to be highest, and only during weekdays when laboratory resources were available. The DMH itself is a private, fee-for-service secondary care facility located proximal to major government facilities, and it serves as the regional medical centre for the Cameroonian Army, therefore serving a unique client base. Thus, participants in this study likely do not reflect the population of Douala as a whole. Third, the study was not powered to test specific hypotheses, and the small number of participants in younger age groups greatly limited the power of the study to detect differences among these age groups. Fourth, additional laboratory data to classify the type of anaemia was not obtained, nor were laboratory tests for haemoglobinopathies or helminth infections performed. As mentioned above, there are a myriad of possible etiologies for anaemia among these participants. Thus, this study cannot identify the etiology of anaemia among the participants and can only comment on possible association, rather than causation, between malaria and anemia. Finally, the assessment of presenting symptoms was subjective.There are important limitations to this study. First, parasitaemia levels by microscopy or quantitative PCR were not available, and the patient’s final diagnosis was not known. Thus, some percentage of P. falciparum infection in Douala, Cameroon. A high burden of both P. falciparum infection and anaemia was found in this populous urban setting. Older children tended to be at higher risk for P. falciparum infection, and several clinical and epidemiologic characteristics significantly increased the likelihood of malaria infection. Anaemia was higher among children and woman compared to adult men, and the presence of anaemia was associated with P. falciparum infection only among children. With recent slowing or even reversal of gains in malaria control efforts, additional epidemiologic studies in urban centres as well as across the full age spectrum in sub-Saharan Africa are recommended, so that appropriate interventions can be developed and deployed for these unique settings and demographics.The current study describes clinical characteristics of Additional file 1:Table S1. PCR Mastermix reagents and concentrations for PCR speciation assays. Table S2. PCR amplification parameters for PCR speciation assays. Table S3. WHO haemoglobin classification of anaemia. Table S4. Interpreting likelihood ratios. Table S5. Diagnostic characteristics of history and physical findings for anaemia.