Microscopy continues to be the mainstay for the evaluation of parasitaemia in malaria but requires laboratory support and microbiological experience. Other fast and simple methods are necessary.Plasmodium falciparum monoinfections diagnosed by peripheral blood smear and/or polymerase chain reaction (PCR),which also had a positive RDT result in the same blood sample, were included in the study.A retrospective observational study of imported malaria treated from July-2007 to December-2020 was carried out to evaluate the association between the degree of parasitaemia and both rapid diagnostic tests (RDT) reactivity patterns and haematological parameters. P. falciparum pattern) had ranges between 0 and 1%. Not a single case of P. falciparum pattern was found for parasitaemia ranges greater than 1%, showing a negative predictive value of 100% for high parasitaemia. All the correlations between haematological parameters and parasitaemia resulted to be weak, with a maximum rho coefficient of -0.35 for lymphocytes and platelets, and of 0.40 for neutrophils-to-lymphocytes count ratio. Multivariate predictive models were constructed reflecting a poor predictive capacity.A total of 273 patients were included. Most of them were male  and visiting friends and relatives (VFR) travellers . Patients with plasmodial lactate dehydrogenase (pLDH) or aldolase and histidine-rich protein 2 (HRP-2) co-reactivity (Pan/Pf pattern) had a parasitaemia range between 0 and 37% while those with just HRP-2 reactivity (P. falciparum parasitaemia in travellers with imported malaria, discriminating patients with lower parasite loads. Haematological parameters were not able to estimate parasitaemia with sufficient precision.The reactivity pattern of RDT allows a rapid semi-quantitative assessment of The online version contains supplementary material available at 10.1186/s12936-023-04781-2. Plasmodium falciparum is the species that most frequently produces severe symptoms and is responsible for most of the morbidity and mortality associated with the infection. Sub-Saharan Africa suffers the greatest burden of the disease, reporting 95% of all malaria cases and 96% of all malaria-related deaths globally occurred during the 2019–2020 period [Malaria is the most important parasitic disease in the world. Nearly half of the world's population lives in areas at risk for infection. In 2021, there were an estimated 247 million malaria new cases and about 619,000 related deaths [Microscopic examination remains the “gold standard” for laboratory confirmation of malaria, not only because of its high sensitivity and specificity, but also because it allows identifying the species and quantifying the degree of parasitaemia. However, it requires skilled microscopists often not available in many health facilities. This is especially concerning outside endemic zones where most laboratorians may not be used to tropical diseases diagnostic tests requiring the use of microscopy visualization techniques. Molecular techniques, on the other hand, are usually available only in reference centres.For all these reasons, rapid diagnostic tests (RDT) have become the preferred method for initial diagnosis since they are simple, fast, offer high sensitivity and specificity, are widely available, and do not require previous experience to interpret their results , 7. Yet,P. falciparum imported malaria.The present study aims to evaluate the association between the degree of parasitaemia and both RDT reactivity patterns and haematological parameters in a cohort of patients with P. falciparum monoinfections diagnosed by peripheral blood smear and/or PCR, who also had a positive RDT result in the same blood sample, were included in the study.A retrospective observational study of all malaria episodes treated at the Tropical Medicine Unit (TMU) of the Poniente Hospital  between July 2007 and December 2019 was conducted. All patients over 14 years of age with The Poniente hospital serves an area located in Southern Spain that holds a population close to 300,000 people, with a migrant share of 21%. Migrants from sub-Saharan Africa, mainly from Western Africa, are a very large group within this foreign population, and malaria is one of the most frequently imported diseases diagnosed among them , 17. MigPatients coming from endemic areas presenting with symptoms suggesting patent malaria were mostly diagnosed at the Emergency Department. In addition, malaria cases were also detected in the outpatient clinic for tropical diseases (TMU) as a result of the malaria screening tests that are offered to all migrants coming from endemic areas in the last 12 months, irrespective of the reason for consultation at the clinic.Malaria diagnosis was made by means of RDT, direct microscopic examination of thin smear and / or molecular tests. In all cases, the tests were performed using peripheral venous blood samples extracted in 5 mL EDTA tubes.P. falciparum, and aldolase, a pan-malarial antigen found in all Plasmodium species. The second one is the SD Bioline Malaria Ag P.f/Pan® that detects both HRP-2 and pLDH (Plasmodium lactate dehydrogenase), a plasmodial enzyme expressed by all Plasmodium species, and was used in the second part of the study, from 2016 on. Reactivity for both antigenic bands was defined as the Pan/Pf pattern, while reactivity for just the HRP-2 band was defined as the Pf pattern. The degree or level of parasitaemia in the thin smear was expressed as the percentage of parasitized erythrocytes. Blood smear examinations were performed by several microscopist, all experienced in imported tropical pathology. For Plasmodium DNA detection, a conventional Nested Multiplex Malaria PCR (NM-PCR), capable of identifying four human malaria species , was used [Two different RDT were used along the study period: the BinaxNOW® malaria test and the SD Bioline Malaria Ag P.f/Pan®. The first one, used from 2007 to 2015, is an immunochromatographic assay that detects both histidine-rich protein 2 (HRP-2), an antigen specific to was used . ParasitThe haematological parameters were obtained from the same blood sample used for the malaria diagnostic test of each case. The values of haemoglobin (g/dL), leukocytes (/µL), lymphocytes (/µL), neutrophils (/µL), monocytes (/µL), eosinophils (/µL), and platelets (/µL) were recorded. Additionally, the ratios of monocytes-to- lymphocytes count (MLCR), and of neutrophils-to-lymphocytes count (NLCR) were calculated.A descriptive analysis of all variables was carried out. Quantitative variables were assessed through the mean, median and range. To describe qualitative variables, absolute and relative frequencies (%) were used. The normality of all the variables was checked by the Shapiro–Wilk or Anderson–Darling tests.The predictive capability of parasitaemia to discriminate the rapid diagnostic test results was investigated through the ROC (receiver operating characteristic) curve and the Youden’s index. The best cut-off point was the one that maximized the value of the Youden’s index. In order to show the distribution of the RDT results regarding the level of parasitaemia, a first categorization of parasitaemia into four groups was made: < 0.01%, 0.01%-0.1%, > 0.1%-1%, and > 1%. These cutoff points were chosen to facilitate comparison with those other published studies that had used a similar categorization ..75) were deleted from the equation. The dataset was split into two subsets, the training set (75%) and the test or validation set (25%). The model was built applying tenfold cross validation (10-k CV) in the training set. This method requires to randomly divide the training dataset in 10 subsets. Each time, one subsample will be retained and used as a validation set, while the model will be set up using the rest of the subsamples. In 10-k CV, the cross-validation mechanism is repeated 10 times, and then, the 10 results are averaged to produce just one single estimation of the model. Afterwards, the model was verified in the test set. To assess the performance of the statistical learning process from the training data, the root mean square error (RMSE) was calculated in order to know how the predicted values fit the real data.Due to the lack of normality in most of the variable distributions, the correlation between haematological variables and parasitaemia was assessed by means of the Spearman’s rank correlation coefficient (rho). To predict parasitaemia, a generalized linear model was proposed. In this work, it was assumed that the response variable followed a quasibinomial distribution (proportion data). In fact, the dependent variable was divided by 100, so its range was restricted to the closed interval 0 to 1. For all the cases, the covariates which were highly correlated  severity criterion that considers parasitaemia > 2.5% as a mortality risk factor, and parasitaemia > 4% as hyperparasitaemia (6). The 1% limit was added to differentiate those patients with a very low parasite density among the rest of non-severe malaria cases (parasite density < 2.5%). The location parameters of all the haematological variables were compared between all these categories. Without the normality assumptions, Kruskal–Wallis rank sum test was used to analyse them, being followed by Nemenyi post-hoc test to establish between which two groups were the differences found. A predictive model was constructed to try to discriminate between parasitaemias < 1% and ≥ 1% following the same re-sampling strategy (tenfold CV). For this statistical model, selection of the variables was made following Akaike’s information criterion (AIC). The confusion matrix was obtained when the real observations were compared to the predicted ones in the validation dataset.Finally, the level of parasitaemia was categorized into another four different groups: < 1%, 1%-2Statistical analysis have been carried out with R software (R Core Team 2020). The significance level was set up at 0.05.P. falciparum and/or mixed infections were excluded from the analysis (n = 34). Those episodes in which the RDT was not performed with the same blood sample used for the blood smear and/or for the Plasmodium PCR test were also excluded (n = 85). Finally, 273 malaria episodes  met the study inclusion criteria.During the study period, 392 malaria episodes were treated. Infections by species other than The epidemiological, clinical and analytical characteristics of the patients included in the study are shown in Table Most cases (94.5%) were diagnosed in the Emergency Department. The rest were diagnosed as a result of the implementation of the imported diseases screening protocol used in our Tropical Medicine Unit. Mean parasitaemia was 1.8% (range: 0%-37.4%). A 17.9% of the cases required admission to the Intensive Care Unit (ICU), most of them (80%) just because of hyperparasitaemia, with no other signs or symptoms of severe malaria. There were no deaths.Plasmodium species were confirmed by the peripheral blood smear results. For the 25 patients with a positive RDT but no detectable parasitaemia in the blood smear, confirmation was performed by PCR.In 90.8% of patients (n = 248), diagnosis of malaria and of the /aldolase and HRP-2 (Pan/Pf pattern) was observed in 195 patients (71.4%). The rest  showed reactivity just for HRP-2 (Pf pattern).Co-reactivity on the RDT for pLDHFigure The percentage of patients with a Pan/Pf pattern increased proportionally along with the increase in the parasitaemia range Fig. . The ROCP. falciparum result never have parasitaemias > 1% is again observed.The results of the RDT have been analysed separately in two temporal periods following the use of each of the test at the time: BinaxNOW® malaria test before 2016 and SD Bioline Malaria Ag P.f/Pan® from 2016 on). During the first period, 114 patients were included, 85 (74.6%) of them presenting a Pan/Pf pattern and 29 (25.4%) a Pf pattern. In the second period 159 patients were included, 110 (69.2%) presenting a Pan/Pf pattern and 49 30.8%) a Pf pattern. Figure 0.8% a PfThe correlation coefficients between each blood count variable and parasitaemia were calculated and are shown in Table A statistical model  to predict the degree of parasitaemia was constructed, initially including haemoglobin, leukocytes, monocytes, eosinophils, platelets, and NLC and MLC ratios. Haematocrit, lymphocytes and neutrophils were eliminated because they behaved as covariates with a high degree of correlation. The results of fitting the generalized linear model to the variable representing the degree of parasitaemia are shown in Additional file In Fig. The relationship between blood count parameters and parasitaemia was again analysed, this time after parasitaemia was categorized in ranges as < 1%, 1%-2.5%, > 2.5%—4%, and > 4%. Figure Given that the differences between the location parameters  were generally between the < 1% range and the rest , a predictive model was built to try to discriminate between parasitaemias < 1% and ≥ 1%. The coefficients of the statistical model are presented in Additional file Eosinophils, platelets, and the NLC and MLC ratios were the only covariates selected for the model. The model was able to predict correctly 70% of the observations; the achieved sensitivity was 58%, while the specificity was 79%. Positive predictive value and negative predictive value were 69% and 70%, respectively.P. falciparum imported malaria, reactivity solely for HRP-2 on the combined RDT (Pf pattern) allowed discriminating those patients with parasitaemia ≤ 1%. On the contrary, the haematological parameters during the acute malaria episode did not show a correlation strong enough to indirectly estimate the level of parasitaemia.In this cohort of patients with Plasmodium parasitaemia. The demonstration that the quantification of plasma levels of PfHRP2 allowed estimating the total body parasite biomass in acute falciparum malaria [Although microscopy continues to be the mainstay for malaria diagnosis, its widespread use is somewhat limited because of technical obstacles. The need for laboratory support in endemic regions and the scarce microbiological experience in non-endemic areas have promoted the search for alternative methods to assess  malaria , 20 was P. falciparum infection . On the contrary, such reactivity pattern was less frequent in patients with mixed infections  or with mono-infections due to P. falciparum with parasitaemia < 40,000/μl . They concluded that co-reaction of PfHRP-2 and aldolase indicates the presence of a monoparasitic infection due to P. falciparum with high parasitaemia. Later on, Van Gool et al. [P. falciparum imported malaria. As in our study, most patients acquired the infection in sub-Saharan Africa and belonged to the VFR traveller category. In this case, all patients with parasitaemia > 50,000/μl (> 1%) had PfHRP-2/pAldolase co-reactivity, while the absence of reactivity of pan-malarial antigen (HPR-2 reactivity pattern) allowed to exclude those patients with parasitaemia greater than 1% with a negative predictive value of 100%. This work reproduces the same results in a population of similar characteristics and also extends them to RDT that uses pLDH as pan-malarial antigen. Similarly, both studies contradict the hypothesis that PfHRP-2/pan-malarial antigen co-reactivity is associated with P. falciparum infections with high parasitaemia as this pattern also occurs in infections with very low parasitaemia. As shown in Fig. . [Richter et al.  had prevn 20/25; 0%. On thn 20/25; 0%. On thn Fig. . .P. falciparum band, concentrations 10 times higher (45 ng/mL) of pLDH are required for pan-malarial bands to be positive. This could be explained by the fact that PfHRP-2 possesses multiple epitopes capable of binding to immunoglobulins while pLDH possesses only one such epitope [It seems that the differences in antibody-binding avidity between PfHRP2 and pLDH may be reflected in the different sensitivity of the respective test bands. While blood concentrations ≥ 4 ng /mL of HRP-2 are needed to produce positive results in the specific  epitope . Based o epitope .Plasmodium parasite is a blood pathogen that causes different haematological manifestations due both to direct and indirect mechanisms. Target cells are not only red blood cells but white blood cells and platelets too. Anaemia, thrombocytopenia, lymphopenia, monocytosis, and eosinopenia are the most frequent haematological findings [The findings , 23, 24.findings , 26. Thefindings –31. In cSevere anaemia (Hb < 5 mg/dL) is the only haematological parameter that is part of the WHO criteria for severe malaria . MalariaDespite not being a criterion for severe malaria, thrombocytopenia is one of the most consistent features of malaria. Destruction of platelet by macrophages, bone marrow alterations, antibody-mediated platelet destruction, oxidative stress, platelet aggregation, coagulation disturbances, and splenomegaly are all related to its pathogenesis. In this study, platelet count is significantly reduced in patients with high parasitaemia compared to those in the low and moderate parasitaemia ranges, findings that are similar to those previously described in other studies , 12. AgaChanges in leukocyte counts are often less pronounced than in the other blood cell lineages. In general, total leukocyte counts have been found to be low to normal in malaria . Those sIn recent years, multiple studies have been published on the diagnostic and prognostic value of different haematological ratios and their ability to provide added information to the one given by standard blood count values. The ratio neutrophil-to-lymphocyte count (NLCR) has been proposed as marker of severe systemic inflammation in critical patients admitted to intensive care units , as a be. [P. falciparum infection, the presence of a high MLC ratio correlated with a greater susceptibility to clinical malaria and a lower clinical effectiveness of the RTS,S malaria vaccine. These results posited the MLCR as a possible marker of the individual's ability to mount an effective immune response against malaria. In their work, Antwi-Baffour et al. [P. falciparum infections in children up to 5 years compared to healthy controls. There was significant difference between case and controls and a significant positive correlation with parasitaemia, although very weak (rho 0.20). This work does not replicate these findings. In this study, no significant linear correlation (p 0.09) or differences between the different groups after categorizing parasitemia were found. Moreover, in the constructed predictive model, the MLC ratio behaves as a protective factor regarding parasitic load. These discordant findings could be explained by the differences in the populations included in both studies  and in their immune status with respect to Plasmodium infection. The findings of Berens-Riha et al. [Monocytes are essential components of the innate immune response and act as a link to the adaptive response by means of antigenic presentation to lymphocytes and the production of cytokines. However, they can also participate in the pathogenesis of severe conditions . The mon. , 46. Ther et al.  examineda et al.  are in lP. falciparum parasite clearance because the antigen lingers weeks after resolution of infection. This situation is particularly relevant in high-transmission setting were antigen presence does not necessarily imply active parasite infection. As a result, is not recommended to extrapolate findings from this work to other contexts than that of imported malaria [The current study presents the methodological limitations inherent to its retrospective nature, although in this case it has probably little influence on the results. The majority of patients included in the study are sub-Saharan VFR travellers and therefore, the findings may not be simply extrapolated to other population groups. On the other hand, the large number of patients included in the study strengthens the results. Despite numerous studies describing the behaviour of haemotologic parameters in patients with malaria, there are few studies describing their association with parasitaemia. Similarly, as far as we know, there are no other studies attempting to estimate such association using multivariate predictive models including different variables and haematological ratios. This research extends the semi-quantitative approach to parasitaemia based on the pattern of the results of RDT to those HPR2-pLDH tests since, to date, similar evidence had only been demonstrated with tests that use pAldolase as the pan-malarial band. HRP2-based diagnostic tests can remain positive for an extended period of time following successful  malaria . FinallyP. falciparum parasitaemia in travellers with imported malaria, particularly to point-out patients with lower parasite loads. The presence of HRP-2 reactivity in the absence of LDH or aldolase was a reliable predictive marker to detect parasitaemias < 1% with a 100% PPV. On the contrary, neither haematological parameters, nor NLC or MLC ratios, nor the multivariate predictive models developed, were able to estimate parasitaemia with sufficient precision.The results of this study show that the reactivity pattern of BinaxNOW® malaria test  and SD Bioline Malaria Ag P.f/Pan (HPR-2/pLDH) allows a rapid semi-quantitative assessment of This findings may be relevant and useful when treating patients suspected of having imported malaria in the acute care setting, especially in those centres where microscopic diagnosis is not readily available or where expertise in managing malaria is lacking. Further studies involving a larger number of patients and different populations are needed to confirm that these data are extrapolable to different scenarios.Additional file 1: Details of correlation between the haematological parameters and parasitaemia. Table S1. Parameter estimates of the definitive generalized linear model for the variable “parasitemia”. Table S2. Hematological parameters by parasitemia ranges. Table S3. Parameter estimates of the predictive model for the parasitaemia variable (< 1% and ≥ 1%).


Plasmodium spp. infection in malaria-endemic areas. Malaria control efforts since 2000 have aimed to reduce this burden of disease.Women are at risk of severe adverse pregnancy outcomes attributable to Plasmodium spp. infection. We categorised pregnancies as occurring in areas of stable and unstable P. falciparum and P. vivax transmission. We further stratified stable endemicity as hypo-endemic, meso-endemic, hyper-endemic, or holo-endemic, and estimated pregnancies at risk in 2000, 2005, 2010, 2015, 2017, and 2020.We used data from the Malaria Atlas Project and WorldPop to calculate global pregnancies at-risk of P. falciparum, two-thirds  were in areas of stable transmission; 85 2M pregnancies were at risk of P. vivax, 93.9% (80.0M) were in areas of stable transmission. An estimated 64.6M pregnancies were in areas with both P. falciparum and P. vivax transmission. The number of pregnancies at risk of each of P. falciparum and P. vivax worldwide decreased between 2000 and 2020, with the exception of sub-Saharan Africa, where the total number of pregnancies at risk of P. falciparum increased from 37 3M in 2000 to 52 4M in 2020.In 2020, globally 120.4M pregnancies were at risk of Historic investments in malaria control have reduced the number of women at risk of malaria in pregnancy in all endemic regions except sub-Saharan Africa. Population growth in Africa has outpaced reductions in malaria prevalence. Interventions that reduce the risk of malaria in pregnancy are needed as much today as ever. Plasmodium falciparum infection during pregnancy increases the risk of spontaneous abortion [P. falciparum transmission, pregnant women develop partial immunity to placental infection through repeated malaria exposure over successive pregnancies [P.falciparum are largely due to the sequestration of the parasite within the placenta and are particularly seen in women who have not been exposed to P. falciparum in any previous pregnancy. The density and frequency of infection and the risk of associated adverse pregnancy outcomes are greatest among primigravidae, decreasing with each subsequent pregnancy.abortion , stillbiabortion , pretermabortion , low birabortion , small fabortion , maternaabortion , and matabortion , 4, 6. Ignancies –10. The P. falciparum cases are in sub-Saharan African, whereas lower transmission persists across parts of South America and Asia [P. falciparum transmission is low or unstable, there are no specific WHO prevention recommendations.The vast majority of global and Asia , 12. To and Asia . Where PPlasmodium vivax is also associated with stillbirth [P. falciparum in part because P. vivax infects only reticulocytes which limits parasite densities [P. vivax infection can be challenging due to recrudescent and relapsing infections. Primaquine is used to treat P. vivax and is capable of eliminating liver-stage hypnozoites, but is contraindicated in pregnancy because of the potential to induce haemolysis and blood disorders in the fetus. Radical cure in pregnancy is not recommended until after delivery [P. vivax are equally vulnerable to having low birthweight newborns [P. vivax cases are in geographic areas of the WHO Regional Offices for South-East Asia and Eastern Mediterranean [illbirth , pretermillbirth , low birillbirth , 16, andillbirth , but infensities . Howeverdelivery , 18. Morerranean  where thP. falciparum in sub-Saharan Africa [P. falciparum [P. vivax [P. falciparum and P. vivax stratified by transmission intensity.Global estimates of pregnancies at risk of malaria have not been generated since 2007 [n Africa . Since 2lciparum  and P. vP. vivax , as wellP. vivax , 22, altP. falciparum infection among 2 to 10 year olds, as generated by the Malaria Atlas Project (MAP), has been used as a proxy for malaria among pregnant women [P. falciparum and P. vivax parasite rates  from MAP as raster datasets at 5km2 resolution. These estimates incorporate prevalence data from household surveys, case data, and socio-economic and environmental data to produce smoothed, global estimates of malaria parasite rate  [th and 97 5th percentile, respectively) prevalence estimate for every 5km2 globally from 100 realisations of the underlying joint posterior prevalence of P. falciparum and P. vivax. For years 2000, 2005, 2010, 2015, and 2017 we used mean prevalence estimates as generated by MAP.The rate of nt women , 24. We valence) , 25. For2 resolution from WorldPop. These data are based on age and sex structures of global population at 1km2 to estimate the number of children under one year of age. An extrapolation factor is then applied to correct for child mortality and to estimate the number of livebirths, which is then standardised to the United Nations total national number of livebirths [2 for all African, Asian, and Latin American and Caribbean countries.We obtained global estimates of total pregnancies at 1kmvebirths –28. Counvebirths . yieldin2) for all malaria-endemic countries and matched these to the corresponding year of malaria prevalence. Pregnancy-weighted malaria prevalence was averaged at the smallest administrative level of each country. For 2020, we classified pregnancies as being at risk of stable  or unstable (>0% and <0 01%) transmission based on the central, lower, and upper estimates of malaria prevalence [P. falciparum and P. vivax, stable or unstable, at risk of mixed infections. We further categorised stable malaria endemicity as hypo-endemic (0 01 to 10% inclusive), meso-endemic (11 to 50% inclusive), hyper-endemic (51 to 75 inclusive %) and holo-endemic (>75%) and summed pregnancy estimates by categorical endemicity for years 2000, 2005, 2010, 2015, 2017, and 2020 [P. falciparum endemicity using WorldPop pregnancy estimates for 2020 and MAP prevalence estimates for 2000 and assumed that the number of pregnancies in 2020 no longer at risk of infection, or at lower risk of infection, were attributable to antimalarial interventions since 2000.We aggregated pregnancy estimates to the same resolution as MAP estimates  making up 59.47% of the region’s at risk pregnancies. Eastern and South-Eastern Asia had the second highest number of pregnancies at risk of unstable P. falciparum transmission, 6.1M, accounting for 60 7% of at risk pregnancies in the region . In Central Africa, the Democratic Republic of Congo (DRC) had the most pregnancies at risk of P. falciparum infection, 5 3M, all in areas of stable transmission where the mean prevalence was 39 7% (IQR: 8 9 to 68 8)  .P. falciparum within sub-Saharan Africa, 49 3% (IQR: 31 8 to 62 9) in areas of stable transmission and 214,600 pregnancies at risk, followed by DRC, as described above, and Sierra Leone, mean prevalence 39 2% (IQR: 22 7 to 46.0) and 328,800 pregnancies at risk. The top five countries globally with most pregnancies at risk of stable P. falciparum transmission were, in descending order: India, Nigeria, DRC, Ethiopia, and Pakistan; mean prevalence was highest in DRC and Nigeria. The top five countries with the most pregnancies in areas of unstable P. falciparum transmission  were, in descending order: India, Indonesia, Brazil, Afghanistan, and Vietnam.Liberia was the country with the highest mean prevalence of P. falciparum [P. falciparum (37 5M), of which 38.0% were at risk of stable but low transmission   . Among tto 1.61)  Figs.P. vivax, of which 80 0M (93 9%) were in areas of stable transmission. The majority of pregnancies at risk of stable P. vivax transmission were in Central and Southern Asia (51 7M), accounting for 94 8% of all pregnancies at risk of P. vivax in the region. Globally, 5.2M pregnancies were in areas of unstable P. vivax transmission. A total of 7 0M pregnancies within sub-Saharan Africa were at risk of P. vivax (99.9% of which in areas of stable transmission).Worldwide, 85 2M pregnancies were at risk of P. vivax by prevalence is shown in P. vivax were in areas of stable transmission. India had the largest number of pregnancies at risk of P.vivax, 38 3M, 97.2% of these (37.2M) were in areas of stable transmission where mean prevalence was 0 4%, IQR: 0 06 to 0 97, followed by Pakistan with 10.3M pregnancies all at risk of stable transmission, mean prevalence 0.76, IQR: 0.07 to 1.65). Indonesia had the third largest number of pregnancies at risk of P.vivax; in total 6.7M pregnancies were at risk, 96.8% (6.5M) of these were in areas of stable transmission where mean prevalence was 0.55, IQR: 0.03 to 2.24).The proportion of pregnancies at risk of stable P. falciparum and P. vivax transmission, and therefore at risk of mixed infections. The majority of these  were within Central & Southern Asia  or meso-endemic (11 to 50%) P. falciparum in 2020 than in 2000  has decreased; 75.5% of all pregnancies in P. falciparum endemic countries in 2000 were in areas of malaria transmission, reducing to 62.8% in 2020   in countries with endemic  in 2020 . The totagascar) .P. falciparum transmission and 13 4M fewer pregnancies in areas of hyper-endemic (51 to 75%) transmission than there would have been had prevalence remained at 2000 levels but pregnancies continued to increase to 2020 levels , reflecting the reduction of 18 6M pregnancies in areas of hypo-endemic (0.1 to 10%) and meso-endemic (11 to 50%) transmission (In sub-Saharan Africa there were just over 2.4 million fewer pregnancies in 2020 in areas of holo-endemic (>75%) 0 levels . Thus, msmission .P. falciparum and P. vivax and examine changes in risk by categorical endemicity since 2000. Despite decreases in P. falciparum and P. vivax prevalence, and decreases in the global number of pregnancies at risk of malaria, the number of pregnancies at risk of moderate to high P. falciparum transmission in sub-Saharan Africa i.e. women requiring preventative intervention, has remained relatively unchanged, increasing between 2017 and 2020. The scale up of antimalarial interventions for pregnant women throughout the antenatal period is needed today as much as ever and, indeed, should precede conception given that an estimated 70% of infections among primigravidae are acquired before pregnancy [We present contemporary global estimates of pregnancies at risk of regnancy . RedoublOur analysis illustrates the challenges of reducing the number and proportion of pregnant women at risk of malaria infection in sub-Saharan Africa, where fertility rates are decreasing, but remain high , and popP. falciparum transmission within sub-Saharan Africa, and a corresponding increase in the numbers at risk of lower levels of transmission. Across other regions, pregnancies at risk of hypo-endemic (>0.01 to 10%) transmission have decreased while the numbers in areas of unstable (<0.01%) or no P. falciparum transmission have increased. Approximately a third of all pregnancies at risk of P. falciparum worldwide were in areas of very low, unstable transmission where prevention recommendation are missing, and if they were to be present, would be operationally challenging to deploy and may be difficult to justify on a cost-effectiveness basis unless the value of malaria elimination is also taken into consideration. Nonetheless, this burden of disease is not insignificant given the very large number of pregnancies in consideration, the lower acquired levels of immunity in these transmission settings and the higher likelihood of infections evolving to clinical disease [P. vivax are fewer in number and P. vivax commonly considered to be more benign than P. falciparum, research investment is urgently needed given the millions of pregnant women at risk of P. vivax infection for which there is no radical cure during pregnancy and increasingly strong evidence of its distribution across malaria-endemic Africa [We have shown that malaria control efforts have led to a reduction in the number of pregnancies at risk of higher levels of  disease . While pc Africa .P. falciparum during pregnancy and stillbirth is stronger in areas of low to intermediate transmission than in areas of high transmission [Women of all gravidities who reside in areas of unstable transmission are at risk of developing high-density placental infections with potential for adverse maternal and newborn health outcomes. The association between smission . Access P. falciparum transmission in Africa, an intervention that has potent anti-malarial and non-malarial effects on birthweight [P. falciparum transmission across the continent have been insufficient to translate into significant decreases in the number of pregnancies requiring IPTp, while IPTp coverage continues to lag [Within areas of stable transmission, malaria prevalence varied widely. The WHO recommends the provision of IPTp with sulfadoxine-pyrimethamine to pregnant women resident in areas of moderate to high thweight , 37 Decrs to lag .P. falciparum and 7.7M fewer pregnancies at risk of P. vivax in 2020 than were estimated by the previous global estimates for 2007 [km resolution enable us to estimate number of pregnancies, and the level of malaria transmission within which they reside, at a much finer geographic resolution than was applied to 2007 estimates [P. falciparum transmission in sub-Saharan Africa in 2020 [Our estimates suggest there were 35.1M more pregnancies at risk of  in 2020 , one milP. falciparum prevalence estimates generated by MAP for 2 to 10 year-old children. Previous estimates of pregnancies at risk of malaria have used the same metric [P. vivax, we used data available from MAP which represents the prevalence across all age groups, the only available global P. vivax estimates. Averaging prevalence at the smallest administrative region of each country is pragmatic, however, it means that all pregnancies within an administrative region that has only hot-spots of transmission are classified as being at risk of malaria. As transmission decreases and becomes more localised, a more granular approach to estimating risk of malaria may become more appropriate. Both MAP and WorldPop estimates come with their own limitations and uncertainties. The uncertainty in 2020 MAP estimates is captured in the range between 2.5th, mean, and 97.5th percentile prevalence. WorldPop estimates for pregnancies have been computed using the total number of children under one year of age to quantify the number of live births first and then pregnancies. This can result in ecological fallacies as demographic rates at national level are applied to sub-national levels, resulting in possible over- or under-estimation of pregnancies. Similar variability may occur when using defined administrative boundaries to summarise the pregnancies. This is known as the Modifiable Areal Unit Problem (MAUP), which describes how spatial summary measures are inherently influenced by the administrative boundaries that they are reported at [P. falciparum transmission, i.e. those at greatest risk of adverse outcomes.Our analyses have limitations. In the absence of malaria prevalence estimates specifically for pregnant women, we have used e metric , 38, ande metric . In the orted at . In addiThe Covid-19 pandemic has disrupted malaria control programmes and health systems in general and malaria prevalence and cases greatly increased in 2020 , 41. IndS1 Table(DOCX)Click here for additional data file.S2 TableNB: where Mean (2.5th to 97.5th percentile) prevalence (%) is noted as “<0.001” all three measures of prevalence <0.001%.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fignaturalearthdata.com.Note: Map made with Natural Earth. Free vector and raster map data @ (TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S6 Fig(TIFF)Click here for additional data file.S7 Fig(TIFF)Click here for additional data file.S8 Fig(TIFF)Click here for additional data file.S9 FigPlasmodium vivax transmission globally from 2000 to 2020 disaggregated by risk of malaria or no risk of malaria, and number (C.i) and proportion (C.ii) of pregnancies by risk of Plasmodium falciparum and Plasmodium vivax (D.i and D.ii) from 2000 to 2020 by geographic region.Number (A) and proportion (B) of pregnancies in countries of Plasmodium falciparum and (TIFF)Click here for additional data file.S10 Fig(TIFF)Click here for additional data file.S11 Fig(TIFF)Click here for additional data file.


The systematic review and meta-analysis highlighted inconsistencies in NO levels in malaria patients. Given the high heterogeneity of the results, further studies using standardized metrics for NO measurements and focusing on biochemical pathways dictating NO responses in malaria are imperative to understand the association between NO and malaria.Nitric oxide (NO) has been implicated in the pathology of malaria. This systematic review and meta-analysis describe the association between NO levels and malaria. Embase, Ovid, PubMed, Scopus, and Google Scholar were searched to identify studies evaluating NO levels in malaria patients and uninfected controls. Meta-regression and subgroup analyses were conducted to discern differences in NO levels between the groups. Of the 4517 records identified, 21 studies were included in the systematic review and meta-analysis. The findings illustrated significant disparities in NO levels based on geographic location and study time frames. Despite the fluctuations, such as higher NO levels in adults compared to children, no significant differences in mean NO levels between patients and uninfected controls ( Anopheles mosquitoes, is caused by Plasmodium parasites. P. falciparum and P. vivax are the most prominent species causing malaria infections globally [Malaria is one of the world’s most critical infectious diseases, posing considerable public health challenges, especially in tropical and subtropical regions where malaria is endemic . Malariaglobally ,2,3. Desglobally ,4. In adNitric oxide (NO) is a soluble gas that plays a crucial role in numerous physiological and pathological processes in humans ,6. NO rePlasmodium parasites and prevent propagation within the host [NO produced by host immune cells plays a dual role in malaria. Elevated NO levels restrict the growth of the host . NO elicthe host . Furtherthe host ,19. Howethe host ,20. ThisExploring the association between nitric oxide levels and malaria presents a nuanced yet vital avenue for understanding the pathology of malaria. Over the past decades, the complex relationship between NO and the pathology of malaria has been highlighted in many studies. On the one hand, high NO levels produced by the host immune system are associated with controlling parasite density and protecting against severe disease manifestations ,21,22. CThe research framework and strategy adhered to the PRISMA  guidelines to ensure a high standard of reporting . FurtherPlasmodium infection or severe disease, C was the uninfected controls or less severity, and O was the NO levels.The framework of this systematic review was constructed following the Population, Exposure, Comparator, Outcome (PECO) approach , encompaThe literature review was comprehensively performed to identify studies investigating NO levels in malaria patients compared to uninfected controls and across different malaria severity levels. Embase, Ovid, PubMed, Scopus, and Google Scholar were searched using the following specific keywords and phrases: “Nitric oxide” OR “Nitrogen Monoxide” OR “Endothelium-Derived Nitric Oxide” OR “Endogenous Nitrate Vasodilator” OR “Mononitrogen Monoxide” AND “malaria” OR “Plasmodium” OR “Plasmodium Infection” OR “Remittent Fever” OR “Marsh Fever” OR “Paludism”  critical appraisal tools for cross-sectional, cohort, and case–control studies were employed to assess each study across specific criteria . The cheA qualitative synthesis of the collected data was performed through a thematic analysis approach . The datI2 statistic; 0–40% indicated low heterogeneity, 30–60% indicated moderate heterogeneity, 50–90% indicated substantial heterogeneity, and 75–100% indicated considerable heterogeneity [Plasmodium species, diagnostic methods for malaria, methods for NO measurement, and types of blood samples. The influence of these factors on the pooled effect estimate was assessed. Based on these variables, subgroup analyses were conducted. The results of the meta-analysis are presented as forest plots. All statistical analyses were performed utilizing Stata v17.0 software , with p-values less than 0.05 deemed statistically significant.Studies were included in the meta-analysis if they reported NO levels as means or medians accompanied by standard deviations or ranges. The outcome of interest was the difference in NO levels between the two groups. Differences in NO levels between patients with severe and non-severe forms of malaria were also analyzed. Hedge’s g was used to determine the effect size, and the results were presented with a 95% confidence interval (CI). Heterogeneity between studies was assessed using the ogeneity . High hen = 1234), Ovid (n = 1429), PubMed (n = 658), and Scopus (n = 1196). Duplicate records (n = 1799) were removed, leaving 2718 records for screening. Records (n = 2237) were excluded for the following reasons: 593 records were not related to the participants of interest, and 1644 records were not related to the outcome. Of the remaining 481 reports, 2 could not be retrieved. The remaining 479 reports were assessed for eligibility. Studies were excluded (n = 463) for the following reasons: 181 were in vivo studies, 87 were reviews, 75 were in vitro studies, 40 were mosquito studies, and the rest were excluded for other reasons such as no information on NO (n = 22), inhaled NO (n = 10), and genetic/polymorphism studies (n = 10). Finally, 21 studies were included in the review [n = 16), Google Scholar (n = 1), and reference lists (n = 4)  .P. falciparum was the most common Plasmodium species studied (76.19%). The participant ages were evenly split between children and adults (42.86% each); only 9.52% of the studies included both children and adults. Most studies focused on symptomatic malaria (66.67%). The microscopic method was used for Plasmodium detection in most studies (52.38%). The Griess assay was the most frequently used method for NO measurement (57.14%), and plasma was the predominant choice for blood sampling for NO measurement (57.14%).Nine studies were evaluated using the JBI critical appraisal checklist for analytical cross-sectional studies ,42,44,45The NO levels were compared between patients with malaria and uninfected controls . NO leveP. falciparum malaria were detected in a study by Agbenyega et al. conducted in children in Ghana [P. falciparum malaria were detected in a study by Dondorp et al. in Thailand [The NO levels were compared between patients with different malaria severity levels. No significant differences in the NO levels between severe and non-severe malaria cases, or between survivors and fatal cases were detected in several studies. For example, no differences in NO concentrations between severe and non-severe cases of in Ghana . Similarin Ghana . No signThailand . In a stThailand . In contThailand .p = 0.25, Hedge’s g: 0.35, 95% CI: −0.25–0.96, I2: 97.39%, The NO levels in patients with malaria and uninfected controls were compared using a meta-analysis of 12 studies that reported NO levels as means or medians with standard deviations or ranges ,44,46,47I2: 97.39%) was observed in the studies included in the meta-analysis. Consequently, meta-regression and subgroup analyses were conducted to explore the sources of heterogeneity. The meta-regression analysis incorporated factors such as publication years, study design, continent, age group, Plasmodium species, diagnostic methods for malaria, methods for NO measurement, and types of blood samples. Publication years, continent, age group, methods for NO measurement, and types of blood samples were identified as factors that significantly influenced the pooled effect estimate . In contrast, studies published before 2000 showed that NO levels significantly decreased in patients with malaria compared with uninfected controls . In terms of study design, case–control studies did not demonstrate significant differences in NO levels (p = 0.44), whereas cohort studies presented a high Hedges’ g value of 1.09, although the number of studies was limited. Geographically, studies from Africa demonstrated a significant increase in NO levels (p = 0.02), while studies from Asia did not demonstrate any differences (p = 0.39). A significant increase in NO levels was noted in adults (p = 0.01), but not in children (p = 0.45). A significant increase in NO levels was detected in studies involving multiple Plasmodium species, such as P. falciparum and P. vivax (p < 0.01). No significant differences in NO levels were detected between symptomatic versus asymptomatic or severe versus non-severe cases. Specifically, symptom severity did not yield notable differences . A significant increase in NO levels was noted in studies that employed the colorimetric method for detecting NO (though the specific assay was not stated) (p < 0.01). Studies using serum samples demonstrated a significant increase in NO levels (p < 0.01), while those using plasma did not show a significant difference (p = 0.10).The subgroup analyses are shown in p = 0.09, Hedge’s g: 0.71, 95% CI: −0.11–1.54, I2: 96.07%, In the meta-analysis, the NO levels in patients with severe and non-severe malaria were compared. The analysis incorporated data from five studies that reported NO levels as means or medians with standard deviations or ranges ,36,37,38p < 0.05), suggesting publication bias due to the absence of smaller studies. After trimming and filling to adjust for this bias, the results showed that NO levels increased in patients with malaria compared to uninfected controls . The assessment of publication bias in the meta-analysis to determine differences in NO levels between patients with severe and non-severe malaria was not performed due to the few studies included in the meta-analysis (less than 10).Publication bias was assessed by visually inspecting the funnel plot symmetry and using Egger’s test. The funnel plot displays an asymmetrical distribution of effect estimates across the middle line . Egger’sLeave-one-out analyses were performed for the meta-analyses to determine the differences in NO levels between patients with malaria and uninfected controls, and between patients with severe and non-severe malaria  were detected in the meta-analysis, which was based on a considerable number of studies. The meta-analysis exhibited a high level of heterogeneity (I2: 97.39%). This heterogeneity prompted a more detailed analysis using meta-regression and subgroup analyses to determine the factors influencing the results, including publication years, geographic location, age group, NO measurement methods, and blood sample types. Recent studies (from 2020 to 2023) and studies conducted in Africa showed significant increases in the NO levels in malaria patients compared with uninfected patients. This trend was not observed in studies conducted before the year 2000 and studies conducted in Asia. A pressing question arises regarding the reasons behind the elevated NO levels observed in the more recent studies. However, only two recent studies were included in the subgroup meta-analysis; thus, the reasons for these discrepancies are unclear [Although the thematic analysis revealed a variety of observations, no significant differences in mean NO levels between malaria patients and uninfected controls . Although a high degree of heterogeneity (I2: 96.07%) was noted in this subgroup analysis, severe Plasmodium infection did not markedly alter the NO levels compared to non-severe forms.A noteworthy demographic finding from the meta-analysis was the significant elevation of NO levels in adults compared to children. This discrepancy might stem from physiological differences, immune responses, or environmental influences ,49. SeveP. vivax parasites in the laboratory setting [P. yoelii-infected mice [P. falciparum malaria by inhibiting cytoadherence [The reasons for the lack of differences in NO levels in the meta-analyses between malaria patients and uninfected individuals or between severe and non-severe malaria cases are unclear. In vitro studies demonstrated that the combination of NO and chloroquine reduces parasitemia in chloroquine-resistant malarial infections . Exogeno setting . Iron ch setting . NO is ated mice . As for ted mice . NO may dherence .Variations in NO levels and NOS expression occur worldwide due to a combination of genetic, dietary, environmental, and disease-related factors. Specific polymorphisms in NOS genes can influence their expression and activity, leading to different NO production levels among global populations ,57,58. DSevere malaria is associated with reduced NO production and lower plasma L-arginine levels . L-arginI2 values exceeding 96%, was encountered in both meta-analyses, highlighting the considerable variability in the outcomes of the incorporated studies. This variability may undermine the reliability of the pooled results. The range of methodologies employed across the studies, encompassing different diagnostic approaches, NO measurement techniques, and blood sample types, introduces another layer of complexity and potential source of bias, hindering the derivation of unified conclusions. Moreover, the scarce representation of recent studies, particularly those published between 2020 and 2023, raises questions regarding the current relevance of the findings, given the evolving dynamics of malaria epidemiology and advances in diagnostic techniques. The subgroup analyses, vital for a nuanced understanding of the data, were constrained by the limited data, which was especially apparent in the evaluation of NO levels between severe and non-severe malaria cases. Additionally, the review noted significant geographical and demographic disparities in the NO levels, without a thorough exploration of the underlying causes, leaving a crucial gap in the interpretation of results. This systematic review and meta-analysis present several limitations. A high degree of heterogeneity, as demonstrated by This systematic review and meta-analysis underscore the need for further research into the relationship between NO and malaria. Inconsistencies and heterogeneity were observed across the studies included in the analysis. While no significant differences in NO levels were detected between various groups, the role of NO in malaria remains a topic of interest. Future studies should adopt a standardized approach for measuring NO to ensure consistency across studies. Moreover, delving deeper into the biochemical pathways that govern NO responses to malaria may offer valuable insights into therapeutic advances. Determining NO levels in patients, when combined with other metrics and harmonized conditions, may be instrumental in understanding the association between NO and malaria.This systematic review and meta-analysis revealed inconsistent NO levels among patients with malaria. Overall, the results indicate no significant differences in the NO levels between patients with malaria and uninfected controls, or between patients with severe and non-severe malaria. As evidenced by a marked increase in recent studies showing elevated NO levels in malaria patients, especially in Africa and in studies utilizing specific NO measurement techniques and blood sample types, a directional shift in understanding the role of NO in malaria physiology necessitates further exploration with refined methodology. Further research exploring the biochemical pathways governing NO responses in malaria and how these responses can be leveraged for therapeutic advancements is warranted.


Plasmodium infection was detected using microscopy (and nested-PCR for submicroscopic infection). The genes coding for merozoite proteins-1 and -2 were genotyped by allele-specific nested PCR. Totals of 397 (72.4%) and 151 (27.6%) P. falciparum isolates were collected in rural and urban areas, respectively. The K1/msp-1 and FC27/msp-2 allelic families were predominant both in rural  and urban (45.4% and 54.5% respectively) areas. The multiplicity of infection (MOI) was higher (p = 0.0006) in rural areas (2.9) compared to urban settings (2.4). The rainy season and the positive microscopic infection were associated with an increase in MOI. These findings reveal a higher P. falciparum genetic diversity and MOI in the rural setting of the Republic of Congo, which is influenced by the season and the participant clinical status. Polymorphisms in the genes encoding the merozoite surface proteins msp-1 and msp-2 are widely used markers for characterizing the genetic diversity of Plasmodium falciparum. This study aimed to compare the genetic diversity of circulating parasite strains in rural and urban settings in the Republic of Congo after the introduction of artemisinin-based combination therapy (ACT) in 2006. A cross-sectional survey was conducted from March to September 2021 in rural and urban areas close to Brazzaville, during which  Plasmodium falciparum  infection. In 2021, the number of new cases of malaria reached 247 million, causing 619,000 deaths. More than 90% are from sub-Saharan African countries [P. falciparum to develop resistance to antimalarial drugs, and the unavailability of effective malaria vaccines due to the high genetic diversity within the parasite population are major obstacles to malaria control and elimination [Globally, almost half of the population is at risk of mination .P. falciparum species being the leading cause of infection and mortality [P. falciparum infection is characterized by high genetic diversity associated with high multiplicity of infection implying the evolutionary fitness of the parasite and therefore a higher survival advantage in overcoming malaria control and elimination efforts [In the Republic of Congo, although efforts are devoted to reducing the burden of malaria, it remains one of the major public health problems. Malaria is the leading cause of hospital consultations 63%), hospitalizations (20%), and deaths (9%) , with P.ortality . In high3%, hospiP. falciparum strains circulating in an endemic area.Demographic and entomological data showed that malaria transmission can be reduced via urbanization, including sanitation and education of the population. This increases the number of non-immune individuals, leading to differences in epidemiological characteristics of malaria in rural and urban settings, as many interventions are conducted in urban zones . InterveP. falciparum isolates in a malaria endemic area is based on the amplification of the genes encoding for the msp-1 and msp-2 [P. falciparum and its multiplicity of infection (MOI) describes the intensity of malaria transmission and is a good indicator of the effectiveness of the malaria control and intervention strategy used in a country [P. falciparum genotypes, as well as on the factors determining gene flow between sites, is crucial for successful malaria elimination. However, after the revision of the treatment policy in 2006 in the Republic of Congo, genetic diversity studies have been mainly conducted in the hospital setting and urban areas in order to assess the impact of the implementation of malaria control measures on the circulating parasite strains [P. falciparum parasite population in the country. The msp-1 and msp-2 genetic polymorphism and the multiplicity of infection in isolates collected from rural and urban setting have been investigated in the Republic of Congo. This work will provide evidence of the effectiveness or not of current malaria control measures in both settings.The method commonly used to characterize the genetic diversity of nd msp-2 . These gnd msp-2 ,9. In ad country . A high  strains . This woThe study was conducted in rural areas of the Goma Tsé-Tsé district (Ntoula and Djoumouna villages) in the Department of Pool and in the urban area of Brazzaville Department (Mayanga in the west quarter of Madibou District) of the Republic of Congo. The two departments have a tropical humid climate divided into two seasons: a short period of dry season (June to September) and a long period of rainy season (October to May). These zones are geo-localized at 4.36 S, 15.15 E and an average altitude of 217 m. Djoumouna is a village located 25 km from Brazzaville. It is characterized by the presence of a gallery forest bordering the Djoumouna River. The locality is surrounded by four rivers—the Lomba, Kinkoue, Loumbangala, and Djoumouna—which supply a series of fish-farming ponds  that canA community-based cross-sectional survey was conducted from March to September 2021 covering both the rainy (March to May) and dry seasons (June to September). Individuals at least 1 year old residing in the study areas since at least 3 months prior were included in the study. A well-structured questionnaire was used to record sociodemographic and clinical data, including age, gender, weight, fever or fever history in the last 48 h, headache, vomiting, nausea, etc. Fever was defined based on axillary temperature ≥ 37.5 °C [Plasmodium infection was screened via microscopy using the Lambaréné method [é method . BrieflyAll participants with symptomatic or asymptomatic parasitemia were treated by medical staff according to World Health Organization (WHO) and National Malaria Control Program treatment protocols.Plasmodium species characterization was performed as we have previously described [P. falciparum were further characterized for msp-1 and msp-2 with specific oligonucleotide primer pairs.Parasite genomic DNA was extracted from whole blood using the QIamp DNA kit  following the manufacturer’s instructions. DNA extract samples were then checked for purity and quantity using a nanodrop spectrophotometer. The escribed . Amplifimsp-1 and msp-2 (block 2 and 3) was performed as previously described [msp-1 and msp-2 genes, respectively. The amplicons of the first reaction were used as templates in six separated secondary PCR reactions using different pairs of primers  and msp-2 (3D7 and FC27). The mix of primary and secondary reactions contained 500 nM of each primer, 2.5 mM MgCl2, 100 µM of each dNTP and 0.05 units of Taq DNA polymerase (Qiagen). DNA from P. falciparum reference strains  was used in each run as positive control. The amplified products were then analyzed via 2.5% ultrapure agarose gel electrophoresis  for msp-1 and 2% for msp-2. The SYBER-Green-stained DNA fragments were visualized using UV transillumination  and analyzed with Image Lab Software Version 6.0. Allele sizes were determined with two molecular weight standards . Alleles were considered identical if the fragment sizes were within the range of 10 bp [msp-1 and msp-2 respectively.Nested PCR of the polymorphic regions escribed  using th primers  that tarof 10 bp  and 20 bPlasmodium positive participants were classified in this study:Uncomplicated malaria, also referred as symptomatic malaria—those with positive microscopy and having less than 24 h of fever history and/or symptoms).Asymptomatic malaria was defined as the presence of positive microscopy and no clinical symptoms.Participants presenting no clinical malaria symptoms with a negative microscopy and positive in PCR were classified as sub-microscopic infection.Three groups of Polyclonal infection is defined by the presence of more than one allele for a given gene in a single isolate, whereas the presence of a single allele was considered monoclonal infection. The multiplicity of infection (MOI) is defined as the mean number of distinct parasite clones per infection .msp-1 and msp-2 were calculated as the proportion of alleles detected for each allelic family out of the total alleles detected. The Mann–Whitney  test was used for comparing non-parametric variables, while Student’s t or analysis of variance (ANOVA) tests were used for parametric variables. χ2 tests (or Fisher’s exact test) was used to compare proportions. Statistical significance was defined as p-value < 0.05.Data were recorded in Microsoft Excel  version 2016 and analyzed using GraphPad Prism (version 6.0.1). Continuous variables were expressed as means ± standard deviations (SD) or as medians with interquartile range (IQR). The allele frequencies of This study was approved by the Institutional Ethics Committee of the Congolese Foundation for Medical Research (N°013/CIE/FCRM/2018); administrative authorization was obtained from the Marien Ngouabi University (N°247/UMNG.FST.DFD.FD-SBIO) was obtained, and approval from the head of each study area was also obtained. Written informed consent or assent was obtained from all participants or parents/guardians for those under 18 years of age.Plasmodium falciparum positive samples were analyzed, including 397 (72.4%) samples from rural and 151 (27.6%) from urban areas. The age of the infected subjects ranged from 1 to 88 years with an average of 14 (1–88) years in rural areas and 16 (3–81) years in urban areas. Participants aged more than 20 years were the primary population both in rural and urban areas. Women were also more represented than men in both study areas. Individuals with positive microscopic infection represented 33.7% (185/548) of the total population, among whom 39.1% (155/397) were from rural areas and 19.9% (30/151) were from urban areas, with geometric means of parasite density of 753 (575–985) parasites/μL and 592 (290–1208) parasites/μL, respectively ) and 3D7-type alleles (62/151 (41.1%)) were the most detected in isolates from urban areas. The prevalence of the allelic families in general was statistically (p < 0.05) different between the two study areas, with the exception of the K1-type family  association of allelic frequency was observed with the study site (msp-2 gene (p = 0.0098) association of allelic frequency was observed with the study site  but not in urban areas (p = 0.0839). The K1 allelic family was predominant in all age groups both in rural and urban areas, and a significant association of the allelic frequency was observed with the age groups in rural areas (p < 0.0001) but not in urban area (0.5568). No significant association of the allelic frequency was found with the type of infection (microscopic and sub-microscopic) or with malaria clinical status (symptomatic and asymptomatic) in the two study sites. Among the sociodemographic parameters used for analyzing the msp-2 allelic frequencies (p = 0.0113) associated with allelic frequency in rural areas. The 3D7-type alleles were predominant in children less than 5 years (52.2%) of age, while FC27-type alleles were found to be more abundant in participants aged 5 to 14 years (67%) and >15 years (64.4%).The msp-1 and msp-2 allelic frequencies were also compared in this study. In rural areas, the K1 allelic family was predominant in the rainy season (43.2%), while the RO33 allelic family was the most abundant in the dry season (41.3%) . In urbaquencies , only thmsp-1 and msp-2 genes showed totals of 1743 (879 for msp-1 and 864 for msp-2) and 473 (262 for msp-1 and 211 for msp-2) individual fragments in rural and urban areas, respectively. Overall, the P. falciparum multiplicity of infection (msp-1+msp-2) was significantly (p = 0.0006) higher in rural (2.9) areas compared to the urban areas (2.4). However, there was no significant (p = 0.1006) difference in polyclonal infection (PI) rate between the two areas  vs. urban PI: 75.6% (102/135)). The proportion of polyclonal infection was significantly (p < 0.0001) higher in rainy season compared to the dry season, while this parameter was similar between the two seasons in the urban zone (p < 0.0001) higher P falciparum MOI compared to the dry season both in rural and urban areas. The grouping of participants according to the type of infection (p < 0.05) lower rate of polyclonal infections in sub-microscopic infected  participants compared to their counterparts presenting a microscopic infection . The same trend was true when comparing P. falciparum MOI between participants presenting microscopic and sub-microscopic infection in the two areas. No association was observed when the proportions of polyclonal infections and the MOI were compared according to the age groups and the clinical status of the participants.The characterizations of ban zone . Howevernfection  in each P. falciparum in different geographical contexts is of crucial importance for developing new and effective control strategies [P. falciparum infection in rural areas in comparison with urban areas using both msp-1 and msp-2 gene markers has been conducted in the Republic of Congo since the implementation of malaria control in 2006.Understanding the genetic diversity of rategies . To the msp-1 and msp-2 in the study sites, but this was significantly higher in rural areas compared to urban areas. Site-specific alleles were common in rural areas in comparison with the urban zone, as reported in Burkina Faso [P. falciparum infection in rural areas compared to urban areas [msp-1 and FC27/msp-2 allelic families both in rural and urban areas and was in line with previous reports in urban areas in the Republic of Congo [The present work showed a high genetic diversity of ina Faso  and Gaboina Faso . The disan areas . The levan areas ,20,21. Iof Congo ,20,21.msp-1 allelic frequencies was observed with season and age groups in rural areas in this study but not in urban areas. In rural areas, the K1 allelic family and the RO33 allelic family were predominant in the rainy and dry seasons, respectively, while the urban areas were dominated by the K1 allelic family during the two seasons. The K1 allelic family was predominant in all age groups both in rural and urban areas. The msp-2 allelic frequency was significantly associated with age group in rural areas, with 3D7-type alleles predominating in children less than 5 years of age and FC27 type alleles predominating in participants more than 5 years of age. Taken together, these findings suggest the capacity of P. falciparum to adapt itself to the changing environment and seasonality and to the level of immune response of the host. Overall, the genetic diversity of the three allelic families in these study areas was higher compared to what has been reported in several localities in Cameroon [A significant association of the Cameroon  and GaboCameroon .The MOI is an important parameter for the evaluation of level of malaria transmission and human immunity in a given locality . In thisP. falciparum MOI were significantly higher during the rainy season compared to the dry season. This result suggests an optimal malaria transmission likely coordinated by a high biodiversity of Anopheles mosquitoes during the rainy season, which is a conducive factor to malaria vector development [P. falciparum harbored a higher number of parasite genotypes during the rainy season compared to the dry season [P. falciparum in children with a microscopic infection compared to those with a sub-microscopic infection [In addition to the type of transmission area , the season and the type of infection (microscopic or sub-microscopic) were also factors associated with the variation of the number of parasite genotypes in this study. The rate of polyclonal infection and elopment . A receny season . Severalnfection ,21. The nfection .P. falciparum MOI was similar between the symptomatic and the asymptomatic patients, the findings of a longitudinal study reported a decrease of P. falciparum multiplicity infection when the clinical status of children moved from the asymptomatic to the symptomatic stage [In contrast to our study in which the ic stage . Howeveric stage ,18, it wic stage ,30.P. falciparum isolates in rural and urban settings south of Brazzaville after the deployment of numerous malaria control measures in 2006. The findings revealed high allelic diversity and MOI of P. falciparum, reflecting high intensity of malaria transmission in rural areas. The rainy season and the presence of microscopic infection were associated with increases in diversity of P. falciparum isolates. Thus, these findings will contribute to strengthening malaria control measures, taking into account factors such as the seasonality and the level of malaria transmission in the areas.This study provides new data on the genetic diversity of


Plasmodium parasites, mainly in tropical and subtropical countries. Plasmodium falciparum  is the most prevalent cause on the African continent and responsible for most malaria-related deaths globally. Important medical needs are biomarkers for disease severity or disease outcome. A potential source of easily accessible biomarkers are blood-borne small extracellular vesicles (sEVs).Malaria is a life-threatening mosquito-borne disease caused by We performed an EV Array to find proteins on plasma sEVs that are differentially expressed in malaria patients. Plasma samples from 21 healthy subjects and 15 malaria patients were analyzed. The EV array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies.We detected significant differences in the protein decoration of sEVs between healthy subjects and malaria patients. We found CD106 to be the best discrimination marker based on receiver operating characteristic (ROC) analysis with an area under the curve of > 0.974. Additional ensemble feature selection revealed CD106, Osteopontin, CD81, major histocompatibility complex class II DR (HLA-DR), and heparin binding EGF like growth factor (HBEGF) together with thrombocytes to be a feature panel for discrimination between healthy and malaria. TNF-R-II correlated with HLA-A/B/C as well as CD9 with CD81, whereas Osteopontin negatively correlated with CD81 and CD9. Pathway analysis linked the herein identified proteins to IFN-γ signaling.sEV-associated proteins can discriminate between healthy individuals and malaria patients and are candidates for future predictive biomarkers.The trial was registered in the Deutsches Register Klinischer Studien (DRKS-ID: DRKS00012518).The online version contains supplementary material available at 10.1007/s15010-023-02022-x. Plasmodium parasites are transferred by mosquitos. Plasmodium falciparum  is the most prevalent causative agent on the African continent and is responsible for 99.7% of malaria-related deaths globally [Malaria is a life-threatening disease and a major health issue in tropical and subtropical countries with 247 million cases of malaria in 2021, mainly in children under 5 years of age. Plasmodium falciparum infects red blood cells (RBCs) and causes their adherence to the lining of small blood vessels, hampering tissue perfusion [erfusion . Infecteerfusion . Severe erfusion . One imperfusion . It is aerfusion .Plasmodium-infected RBCs (iRBCs) are described to release small extracellular vesicles (sEVs) containing a variety of molecules mediating pathogenesis and intercellular communication between host cells and between host and parasite [Plasmodium itself, there might be a release of EVs from other cell types into the bloodstream, making them easily-accessible biomarkers for malaria, organ dysfunction, and even disease progression, as their acquisition only requires minimally invasive procedures [parasite . sEVs caparasite . The relparasite . EVs canparasite . It is kparasite . Apart focedures .Here, we analyzed the surface proteome of sEVs in the blood plasma of malaria patients in comparison to healthy controls by EV array and analyzed whether these sEV surface proteins might be suitable biomarkers for disease severity.P. falciparum infection (Giemsa stain). The patients included in this study did not use antimalarial prophylaxis. Patients with specific immunosuppressive therapy, as well as pregnant or HIV-positive patients, were excluded from the study. Additionally, healthy subjects were recruited. Blood plasma was isolated by centrifugation  of one collected Vacutainer® EDTA-tube. After centrifugation, the plasma phase was transferred and stored at -80 °C. Patients underwent routine laboratory testing, healthy volunteers only underwent a blood count.Patients suffering from malaria were recruited within 24 h after hospitalization and before the beginning of treatment. Inclusion criteria of patients in this analysis comprised acute illness after traveling in areas endemic for malaria and positive microscopy for ® Extreme Protein Edition Microarray Printer with a 946MP4 pin , as described [The microarray was produced and performed as previously described . In shorescribed . As a poet al. 2013 [®20)) was added. Samples were incubated for 2 h at ambient temperature followed by an overnight incubation at 4 °C. The slides were then washed and incubated with a cocktail of biotinylated detection antibodies . Detection antibodies were diluted 1:1500 in wash buffer. Cy5-labelled streptavidin  was used for spot visualization. After 30 min incubation, slides were washed in wash buffer and then in ultrapure water. The dried slides were then scanned as described [The EV Array analysis was performed as described by Jørgensen al. 2013 . In shorescribed .t test with Welch’s correction was performed to assess statistical significance. p values < 0.05 were considered significant. Pathway and protein–protein interaction network analyses were performed with STRING version 11.0.GraphPad Prism version 6  and R version 3.5.1 were used for statistical analysis. Background correction of EV Array data was performed on the mean signal of triplicates and before subsequent analysis, antibody signal values were log2 transformed. An unpaired http://efs.heiderlab.de) [Importance analysis of the sEV surface proteins and ranking of features was performed using the web-interface for ensemble feature selection  , 17. EFSAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The BioInflame study was approved by the ethics committee of the Charité-Universitätsmedizin Berlin (EA2/030/09) and the University Medical Center Marburg (55/17). All blood donors were at least 18 years of age and provided written informed consent for use of their blood samples for scientific purposes.P. falciparum-infected malaria patients (n = 15) and healthy donors (n = 21). Patient characteristics are given in Table p < 0.0001; Figure S1b). The parasite density ranged from 0.1–5%. In the group suffering from malaria, creatinine averaged 0.98 mg/dL, bilirubin 0.78 mg/dL, LDH 246 U/L and CRP 8.4 mg/L . Malaria patients were returning from travelling to Ghana (four patients), Cameroon (three patients), Gambia, Kenya, Sierra Leone, Nigeria or Cambodia (one patient each).We analyzed the plasma sEV surface protein composition and compared ents n = 5 and heaents n = 5 and heaThe individual statistical analysis of all 40 analyzed sEV surface proteins revealed 16 of them to be differentially expressed between malaria and healthy controls , which were also significantly regulated between healthy and malaria. As the male group contained many diseased individuals and showed a high standard deviation in this analysis, we additionally separated healthy and malaria samples and compared female healthy to malaria and male healthy to malaria. This resulted in no significant difference for HBEGF and Osteopontin based on gender, indicating that HBEGF and Osteopontin are indeed regulated based on malaria and not on gender.p < 0.0001), CD81 , Osteopontin  and HLA-DR  was performed and revealed that the differentially expressed plasma sEV surface proteins found in Fig. As the diagnostic potential of a sum of sEV surface proteins might be even higher than single proteins or parameters alone, we applied the machine learning tool Ensemble Feature Selection (EFS) that combines eight feature selection algorithms and thereby improves the prediction performance compared to every single algorithm . This mur = 0.941) and CD9 with CD81  or CD9 , though trends of subtle up- (CD81) or down-regulation (HLA-DR) could be observed. When performing EFS analysis for the discrimination between malaria and the severely ill malaria patients, CD81 (0.59) and CD106 (0.36) were the best markers for discrimination according to their normalized ensemble importance .As it is critical in clinical routine to distinguish between different severities of malaria, we asked whether the selected feature proteins on sEVs might help for this discrimination. According to World Health Organization (WHO) criteria, severe malaria has been defined as the occurrence of at least one of the subsequent features: impaired consciousness, prostration, multiple convulsions, acidosis, hypoglycemia, severe anemia (hemoglobin < 5 g/dL), acute renal failure (serum creatinine > 3 mg/dL), jaundice, pulmonary edema, significant bleeding, high parasitemia (> 10%) or death [p < 1.0e−16 compared to a random set of proteins of similar size. The generated PPI network was then tested for possible functions and the top five results for biological processes (GO-terms) and Reactome pathways (HSA) are presented  guidelines , they shesponses . As malaal cells  and thatal cells , which mal cells , and theactivity . In malaactivity . Moreove tissues . The mic tissues . Plasma  tissues . TNF-α l tissues . Endothe tissues  and ther tissues . The comBased on ROC analyses, CD106, CD81, osteopontin, and HLA-DR are best-suited for discrimination between malaria and healthy controls based on their high AUC values (> 0.92). To improve the discrimination, we additionally performed multivariate EFS analysis, which combines eight feature selection algorithms and thereby improves prediction performance compared to each individual analysis . The sel+ sEVs, along with reduced HLA-DR levels, the amount of monocyte-derived EVs might increase during malaria. When monocytes and macrophages receive pro-inflammatory stimuli, they release increased amounts of sEVs [Plasmodium in malaria infection, as they phagocytose iRBCs [+ DCs are the only DC subset activated during primary blood-stage human malaria. As they can produce TNF-α as well as IL-10, they can contribute to inflammatory as well as regulatory innate immune processes [CD81 is highly expressed on monocyte-derived EVs  and was  of sEVs . In partse iRBCs  and intese iRBCs  or with se iRBCs , leadingse iRBCs . DC-derise iRBCs , which ise iRBCs . The posse iRBCs . Besidesse iRBCs . CD16, arocesses . CD16 carocesses . Its exprocesses  and as wrocesses .Plasmodium infection, as it can control parasitemia on the one hand, but on the other hand it can exacerbate its severity by targeting sequestered iRBCs in the brain or the lungs leading to life-threatening complications in severe malaria [The observed protein pattern on plasma sEVs in malaria patients suggests that immune cells need their receptors for their own activation and do not release them via sEVs. To better understand the potential biological implication of the observed protein composition, pathway analysis by STRING was performed. It revealed a complex PPI network of the differentially expressed proteins and their link to interferon-γ (IFN-γ) signaling. IFN-γ is crucial during  malaria . This meIn future studies, a group with other febrile diseases could be included to assess the difference in sEV surface proteome between different febrile diseases. However, our previous study conducted with plasma samples from community-acquired pneumonia (CAP) patients showed a comparable surface proteome compared to malaria patients for some markers , while others were unique for malaria  . The shaTaken together, we characterized the surface proteome of plasma sEVs that allows discrimination between malaria and healthy controls. We cannot conclude yet which effect the herein studied sEVs will have in vivo, as the ultimate function of EVs also depends on the protein and RNA cargo and the recipient cell. For clarification of these questions, future studies are needed.Supplementary file1 (DOCX 1905 KB)Below is the link to the electronic supplementary material.


Post-artemisinin delayed hemolysis (PADH) is a serious complication in patients who recover from severe malaria after receiving artemisinin-based combined therapy (ACT), including artemether-lumefantrine. In Japan, among the antimalarial drugs recommended by the World Health Organization (WHO) guideline for severe malaria, intravenous quinine gluconate is available only in 29 designated hospitals, and intravenous artesunate is unavailable. Therefore, oral artemether-lumefantrine is occasionally administered as an alternative, even though it may be a suboptimal treatment. In non-endemic settings like Japan, a lack of knowledge of malaria and the side effects, such as post-artemisinin delayed hemolysis caused by the ACT, can have critical consequences. Like our patient, being a primigravida in the early stages of pregnancy is a serious risk factor for severe malaria and must be carefully monitored.Plasmodium falciparum malaria complicated by fetal loss and prolonged anemia, requiring frequent blood transfusions. The patient was a previously healthy pregnant Japanese female in her 30 s. She developed a high fever 2 days after returning from Nigeria. The patient fulfilled the severe malaria criteria by WHO. On arrival, an abdominal ultrasound incidentally revealed a fetus of 5 week gestational age with a heartbeat in the uterus. Given her pregnancy and the severity of the disease, she was administered intravenous quinine 16 mg/kg as a loading dose. However, the second dose of quinine was not administered due to frequent vomiting and QTc prolongation. We initiated treatment with oral artemether-lumefantrine, and clearance of parasitemia was confirmed by microscopic observation on day 4. Miscarriage was noted on day 6 after admission. Moreover, the patient became feverish again up to 39 °C, and from days 14 to 22, the patient required multiple blood transfusions due to PADH. On day 40, follow-up was discontinued as the hemoglobin level exceeded 10 g/dL.This report describes a severe case of imported In patients who recover from severe malaria after ACT treatment, monitoring the hemoglobin level for at least a month is strongly recommended for prompt identification of PADH. Travelers to malaria-endemic countries, especially primigravida women, should be provided with adequate information on the risk and prevention of infection. Plasmodium falciparum malaria [In Japan, more than 50 imported malaria cases were reported annually before the COVID-19 pandemic, and of these, more than 60% were due to Intravenous Artesunate is strongly recommended as the first line of treatment for severe malaria , 3. GuidPost-artemisinin delayed hemolysis (PADH) is a severe complication that may occur following recovery from malaria after artemisinin-based combined therapy (ACT) , 6. AlthA pregnant Japanese woman in her late 30 s was referred to our infectious disease clinic in Tokyo for further management. Her chief complaint was fever for 4 days. Two days before the presentation, the patient sought medical attention at a local clinic near her home but could not obtain an appropriate diagnosis. She reported having returned to Japan 2 days before the onset of her fever after a 2-month stay in Nigeria. She also reported having experienced multiple mosquito bites during 2 weeks she spent there. She denied using malaria chemoprophylaxis, insect repellent, or mosquito nets. At the time of presentation at our clinic, her body temperature was 40.2 ℃, and her heart rate was 112/min; other vital signs were normal. She was alert, though mildly agitated and unable to sit due to weakness. Generalized jaundice was noted. On auscultation, both lungs were clear, and the chest X-ray was normal. Slight vaginal bleeding was also noted.Screening tests for the human immunodeficiency virus and blood cultures were negative. Blood examination revealed hyperbilirubinemia, severe thrombocytopenia, elevated liver enzymes, and elevated d-dimer Table . The artUrine human chorionic gonadotropin test was positive, and transvaginal ultrasound (TVU) revealed a gestational sac (GS) with a fetal heartbeat (FHB) equivalent to 5 week gestation. She had no history of pregnancy.P. falciparum-infected red blood cells at 7.3% parasitemia  of red blood cell concentrate on days 14 and 17, respectively. On day 17, she left the hospital at her request. After discharge, she continued monitoring her hemoglobin level at our outpatient clinic, and blood transfusions were still needed on days 19 and 21. Direct and indirect Coombs tests were negative on day 19, and haptoglobin was below the detection limit (< 10 mg/dL.) LDH reached the second peak (1226 IU/L) on day 21. On day 40, hemoglobin follow-up was discontinued as the hemoglobin level exceeded 10 g/dL.TVU on days 6 and 11 revealed a deformed GS without FHB, and subsequently, a diagnosis of miscarriage was made. On day 46, manual intrauterine aspiration was performed to evacuate uterine contents Fig. .Fig. 2ClWe have reported a case of imported falciparum malaria complicated with fetal loss and persistent transfusion-dependent anemia. PADH is a life-threatening condition seen 2–6 weeks after the ACT initiation in 20–30% of non-immune malaria patients, around 60% of whom require blood transfusions. PADH is defined as follows; a 10% decrease in hemoglobin associated with haptoglobin < 0.1 g/L and either an increase in LDH to > 390 IU/L or a > 10% rise 7 days after treatment initiation with the ACT , 6. Like. suggested the involvement of direct anti-globulin, but the literature review did not show a clinical difference between direct anti-globulin test-positive and negative patients [The possible pathophysiology for artemisinin-induced hemolysis is the shorter lifespan of erythrocytes infected with falciparum malaria compared with uninfected erythrocytes, known as the "pitting phenomenon" . Howeverpatients . A Frencpatients .To assess the risk of PADH following AL therapy, we searched articles in the English literature in PubMed describing oral AL-associated PADH following severe malaria. We excluded the cases relating to black water fever or uncomplicated malaria Table  and founSome literature also suggests using corticosteroids if hemolysis is associated with an autoimmune mechanism, but evidence for this is lacking. In our case, corticosteroids were not used, because both direct and indirect Coombs tests were negative. We conducted a literature review on corticosteroid use in such cases. This review revealed five patients had positive direct Coombs test results –19. AmonP. falciparum. A possible explanation is that the genome sequence of P. falciparum found no receptor homologous to that of humans [P. falciparum, causing severe disease [P. falciparum proliferates in the placenta [P. falciparum-infected red blood cells interact with placental tissue and induce activation of inflammatory cytokines, such as tissue necrosis factor-alpha (TNF-α), causing intervilliositis. Intervilliositis reduces megalin, which transports essential proteins to the placenta, and an amino acid transporter supplies amino acids to the trophic membrane [The patient in this report also happened to be a primigravida in the first trimester. Pregnancy is a well-known risk factor for severe malaria, and being a primigravida may predispose an individual to more severe disease , 3, whicf humans . However disease . The patplacenta . P. falcmembrane . These mUntil recently, ACT was regarded as responsible for miscarriage, because animal experiments showed potential teratogenic effects. The previous versions of the WHO guideline  suggesteThis case emphasizes the importance of ascertaining obstetric status while assessing the risk status of female malaria patients. Non-immune women of reproductive age should be more carefully advised of the dangers before travel. Moreover, it may be justified to imbue them with the importance of contraception in risky areas or check pregnancy status before traveling.In conclusion, physicians must monitor hemoglobin levels daily until parasitemia is cleared and weekly for at least 4 weeks after the clearance, especially in cases of severe malaria with high parasitemia. In addition, as being a primigravida and early stage of pregnancy are risk factors for severe malaria, non-immune women should be well-educated about risks and precautions to be taken before traveling to endemic areas.