Plasmodium infection. A comprehensive literature search of six electronic databases  was conducted. Of the 2158 initially identified records, 18 met the eligibility criteria. The majority of studies reported a significant decrease in GSH levels in malaria patients compared with uninfected controls, and this was confirmed by meta-analysis . Additionally, there was no significant difference in GSH levels between Plasmodium falciparum malaria and P. vivax malaria . Similarly, no significant variation was observed between symptomatic and asymptomatic malaria cases . In conclusion, although GSH levels appear to be generally lower in malaria patients, further detailed studies are necessary to fully elucidate this complex relationship.Reduced glutathione (GSH) is a crucial antioxidant with recognized roles in malaria pathogenesis and host response. Despite its importance, reports on the association of GSH with malaria are inconsistent. Therefore, this systematic review and meta-analysis investigated the differences in GSH levels in relation to  Plasmodium genus that are transmitted to humans through infecting bites of female Anopheles mosquitoes1. Among them, Plasmodium falciparum and P. vivax are the most prevalent and cause the most significant public health burden2. Malaria is characterized by cycles of fever, chills, and sweats. Severe cases can lead to complications, such as cerebral malaria, severe anemia, and multiorgan failure3. During the intraerythrocytic stage, Plasmodium parasites metabolize hemoglobin, producing heme as a by-product4. Oxidative stress, an imbalance between the production of reactive oxygen species (ROS) and the body’s ability to neutralize their harmful effects through antioxidants, has been implicated in malaria pathogenesis and progression5. To detoxify heme, the parasite polymerizes it into hemozoin. However, this process also generates free radicals, thereby inducing oxidative stress in the host4. Additionally, during Plasmodium infection, ROS are produced by activated host phagocytes, such as neutrophils6. Enzymatic antioxidants, such as superoxide dismutases, catalase, and glutathione peroxidases (GPxs), and nonenzymatic antioxidants, such as vitamins C and E, β-carotene, and reduced glutathione (GSH), are activated by the host’s antioxidant defense system in response to oxidant stress7.Malaria is a life-threatening disease caused by protozoan parasites of the 9. Besides its direct antioxidant activity, it plays several distinct roles. GSH acts as a cofactor for various enzymes, including GPx, glutathione S-transferases (GSTs), and glyoxalases8. Specifically, GPx uses GSH to detoxify peroxides, thereby converting GSH into glutathione disulfide (GSSG). Then, with the aid of the cofactor nicotinamide adenine dinucleotide phosphate hydrogen, glutathione reductase restores GSH from GSSG10. Additionally, GSH directly scavenges free radicals, such as superoxide anions, hydroxyl radicals, and nitric oxide, neutralizing their reactivity and preventing cellular damage11. It is also involved in regenerating other antioxidants, notably vitamins C and E12, and is a substrate for GPx, which reduces peroxides, including hydrogen peroxide and lipid peroxides. This function is crucial because it prevents the formation of more reactive species, like hydroxyl radicals13.GSH is a pivotal nonenzymatic antioxidant in mammalian cells16, while others reported increased GSH levels18 or no difference20. A comprehensive understanding of the role of GSH in Plasmodium infection, including the effects of specific species, clinical outcomes, and the relationship between GSH levels and parasite density, remains elusive. Our investigation addresses this knowledge gap, setting the groundwork for future research to translate our findings into tangible clinical and public health benefits. Elevated or diminished GSH levels may be critical markers of malaria’s clinical course, enabling earlier detection of severe cases and timely intervention. Furthermore, insights into the role of GSH may inform both clinical management and preventive strategies. Thus, this systematic review and meta-analysis investigated the differences in GSH levels in relation to Plasmodium infection, considering various Plasmodium species, clinical outcomes, and the correlation of GSH levels with parasite density.Despite the known roles of GSH, the results of studies examining its relationship with malaria are inconsistent and often limited by small sample sizes. Some studies have shown reduced GSH levels in malaria patients compared with uninfected controls21.The protocol for this systematic review and meta-analysis was registered with PROSPERO (CRD42023434937) and performed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines22 .The structure of this systematic review was based on the Population, Exposure, Comparator, Outcome framework23. Additionally, only the first 200 search records were screened for eligibility, as previously suggested23. The searches were restricted to articles written in English, but there was no restriction on the publication date. The searches began from database inception to June 12, 2023.A comprehensive literature search was conducted across six electronic databases . The search terms included “(“reduced glutathione” OR GSH OR “gamma-L-Glutamyl-L-Cysteinylglycine” OR “gamma L Glutamyl L Cysteinylglycine” OR “gamma-L-Glu-L-Cys-Gly” OR “gamma L Glu L Cys Gly”) AND ”, with slight variations for each database (Supplementary Table The study selection process was conducted in a stepwise manner by two independent authors (M.K. and A.M.). Any disagreement between the two authors was resolved via consensus.24. The exclusion criteria encompassed in vitro/in vivo studies, review articles, studies lacking GSH information, studies not specifying the GSH type, studies without malaria cases, conference abstracts, or studies analyzing post-treatment GSH levels.First, duplicate records from the various database searches were removed manually and using automated tools. Then, the remaining unique records underwent a screening process, where irrelevant studies and those without abstracts were excluded. Next, the full texts of the remaining articles were retrieved for a detailed eligibility assessment. Studies were included in the review if they were original research articles, reported GSH levels in malaria and uninfected controls, and provided sufficient quantitative data for meta-analysis, such as the mean (with standard deviation) or median (with interquartile range) GSH levelsPlasmodium species, clinical status, data on GSH levels, method of malaria detection, and method of GSH measurement. The quality of the included studies was assessed using Joanna Briggs Institute Critical Appraisal tools, depending on the study design 25. The cross-sectional studies were evaluated for clarity of inclusion criteria, validity of exposure and outcome measurements, and management of confounding factors. The cohort studies were appraised based on group similarity, validity of exposure measurement, strategies for handling confounding factors, adequacy of the follow-up period, and appropriateness of statistical analyses. The case–control studies were assessed for group comparability, appropriateness of case–control matching, validity of exposure measurement, management of confounding factors, and sufficiency of the exposure period. Responses were categorized as “Yes,” “No,” “Unclear,” or “Not applicable” based on the relevance and availability of information for each criterion. The quality rank of an individual study was determined by the percentage of “Yes” responses among all items as follows: > 75th percentile, high quality; 50th–75th percentile, moderate quality; and < 50th percentile, low quality26.The following data were extracted for each eligible study: first author’s name, year of publication, study design, year in which the study was conducted, geographic location, targeted I2 statistic27 as follows: 0–40%, low heterogeneity; 30–60%, moderate heterogeneity; 50–90%, substantial heterogeneity; and 75–100%, considerable heterogeneity27. To explore the potential sources of heterogeneity, a meta-regression analysis using various factors, like the publication year, study design, continent, participant groups, Plasmodium species, diagnostic method for malaria, and quality rank of included studies, was conducted28. Subgroup analysis was conducted based on publication year, study design, geographic location, participant group, Plasmodium species, diagnostic method for malaria, and quality rank of included studies. A sensitivity analysis was performed using the fixed-effect model and the leave-one-out meta-analysis29. The leave-one-out meta-analysis was used to determine the effect of each individual study on the pooled effect estimate of the remainder of the studies29. Publication bias was evaluated by funnel plot analysis and Egger regression test30. All statistical analyses were performed using Stata v17.0 software . P-values < 0.05 were considered statistically significant.The extracted data were used for qualitative synthesis. Additionally, a meta-analysis was conducted for quantitative synthesis. For the meta-analysis, the standardized mean difference (Hedges g) of GSH levels between groups of participants was calculated along with their 95% confidence intervals (CIs). Heterogeneity was quantified using the 41: 12 from the primary database search39, five from Google Scholar41, and one from a reference list40 . Initially, 663 duplicate records were eliminated, resulting in 1495 unique records for screening. Of them, 1164 were excluded due to their irrelevance to malaria or GSH or a lack of abstract, leaving 331 records for retrieval. Two records were irretrievable, so 329 reports were assessed for eligibility. This led to the exclusion of 317 records for various reasons, including being in vitro/in vivo studies, review articles, or conference abstracts, as well as lacking GSH information, the absence of malaria cases, not specifying GSH type, or analyzing post-treatment GSH levels. Eventually, 18 studies meeting the criteria were included in the reviewPlasmodium species targeted in these studies were mostly P. falciparum , indicating its global importance. Although participant demographics varied, suggesting the universal impact of malaria, it is notable that approximately a quarter of the studies (27.8%) focused on children. The common method of malaria detection was light microscopy (72.2%) (Tables The 18 included studies showed diverse characteristics. The majority were published during 2010–2023 (83.3%), indicating a greater interest in the investigation of the association between oxidative stress and malaria during this period. Of the studies included, only 50% specified the year in which they were conducted. The research designs were primarily cross-sectional studies (61.1%) and case–control studies (27.8%). Geographically, the studies were fairly evenly distributed between Africa (55.6%) and Asia (44.4%), with Nigeria and India being the predominant countries, respectively. ) Tables .Table 1C40 exhibited robust adherence to the predetermined criteria but failed to address confounding factors. However, three studies38 successfully met all quality parameters. Two studies41, although fulfilling most criteria and identifying confounding factors, neglected to proffer strategies to mitigate confounders. Regarding the cohort studies, only one34 met all quality parameters. However, the other cohort study39 displayed certain inadequacies and ambiguities, including exposure measurement and handling of confounding factors. For the case–control studies, one14 struggled with group comparability and case–control matching and did not address confounding factors. Another31 upheld all quality criteria. The remaining three case–control studies37, while fulfilling most criteria, were vague regarding case–control matching and the duration of the exposure period , whereas studies conducted before 2010 showed a significant difference . Cross-sectional studies showed significant differences in GSH levels between malaria cases and controls , while case–control studies did not . Geographically, studies from Africa demonstrated significant differences , but those conducted in Asia did not . Regarding participant groups, only studies involving children showed a significant difference in GSH levels , while others did not. Differences in Plasmodium species  did not show significant differences in GSH levels. In terms of diagnostic methods for malaria, studies using microscopy showed significant differences , but those using a combination of microscopy and rapid diagnostic tests did not . Concerning the studies’ quality, high-quality studies showed significant differences , but moderate-quality studies did not .The subgroup analysis results are shown in Table P. falciparum and P. vivax malaria cases, providing quantitative data applicable to the meta-analysis36. The results showed a significant difference in GSH levels between P. falciparum and P. vivax malaria , as confirmed by the meta-analysis of 17 studies . Therefore, both the asymmetrical funnel plot and the significant Egger test result collectively indicate the possible presence of publication bias due to the small-study effect in the meta-analysis.Two standard methodologies were implemented to evaluate the presence of publication bias: a funnel plot analysis and Egger regression test. The results depicted in the funnel plot were asymmetrical Fig. , suggest42. The observed decrease in GSH levels in malaria patients may be a consequence of the body utilizing its GSH reserves to counteract the oxidative stress caused by malaria. Reductions in host GSH levels during malaria can have dual implications for disease pathogenesis. On the one hand, diminished GSH renders host erythrocytes more vulnerable to oxidative damage, potentially exacerbating disease symptoms due to increased oxidative stress43. Conversely, the malaria parasite, particularly P. falciparum, relies on host GSH to detoxify and resist antimalarial drugs4. Therefore, while reduced GSH may weaken the host’s defense against oxidative damage, it can also hinder the parasite’s ability to survive drug treatments. Thus, the intricate balance between host and parasite GSH dynamics underscores the complex nature of malaria pathogenesis44. However, this generalized trend is subject to certain exceptions. For instance, Aqeel et al. found decreased GSH levels, specifically in patients with P. vivax malaria but not in patients with P. falciparum malaria31. This divergence may stem from the different pathophysiological mechanisms employed by P. falciparum and P. vivax. Since P. vivax and P. falciparum exhibit different levels of disease severity, this may influence the level of oxidative stress and, consequently, GSH consumption45. For example, infection with P. falciparum can lead to more severe complications, yet some P. vivax cases can also develop severe malaria47. Nevertheless, no difference in GSH levels between P. falciparum and P. vivax malaria was observed, suggesting that more studies are necessary to determine differences in the distinct antioxidant levels between these two species. Furthermore, the meta-analysis of the clinical status of patients revealed no significant difference in GSH levels between symptomatic and asymptomatic malaria cases. This suggests that alterations in GSH levels are consistent, irrespective of the presence of symptoms. Additionally, a persistent reduction of plasma GSH levels in the early stages of the disease has been observed, which was later compensated during the advanced phase31. Thus, reduced GSH levels may be common during the acute phase of Plasmodium infection, regardless of disease severity or whether the infection is caused by P. vivax or P. falciparum.Qualitatively, most studies concur that GSH levels significantly decrease in malaria patients compared with uninfected controls, corroborating the role of GSH in malaria pathogenesis. Furthermore, the meta-analysis confirmed that GSH levels significantly decreased in malaria patients compared with uninfected controls. GSH, a critical antioxidant in human cells, protects the body from damage caused by oxidative stress48. Additionally, children in Africa have lower antioxidant levels, which may lead to more severe malaria cases49.Other subgroup meta-analyses demonstrated differences in GSH levels between malaria patients and controls across specific parameters, such as geographic location and participant groups. Notably, African studies showed a significant difference in GSH levels between malaria patients and controls, whereas Asian studies did not. This discrepancy may be because the African studies mainly enrolled children. In the subgroup analysis of age groups of participants, only studies involving children showed significant differences in GSH levels, possibly indicating that malaria has a more pronounced effect on GSH levels in this age group. Furthermore, the less mature immune systems of children may impact the oxidative stress response19 and Fitri et al.20 reported no significant differences in GSH levels between different patient groups. This finding may be attributed to a range of factors, including the timing of sample collection, the disease stage, and individual variations in patients’ antioxidant response. In contrast, two studies reported increased GSH levels in malaria patients18. While this may initially seem counterintuitive given the body’s typical response to oxidative stress, it is important to note that the elevated GSH levels are likely attributed to the parasite itself. In their bid to survive and resist antimalarial drugs, Plasmodium species can upregulate GSH synthesis, leading to observed increases in overall GSH levels within the host51. Sohail et al. proposed that the observed increase in GSH levels in malaria patients could be due to transitional polymorphisms within GSTs, which might enhance the host’s GSH availability17. Tyagi et al. suggested that increased GSH levels among malaria patients might be due to decreased GSH utilization18.Interestingly, Babalola et al.38. These discrepancies may be attributed to factors such as variations in the host’s immune response, the parasite’s lifecycle stage when the sample was collected, or differences in Plasmodium species. Oxidative stress and sickle cell disease are related to each other52. One of the included studies demonstrated that patients with malaria and sickle cell disease experienced severe oxidative stress32. The authors reported that although the GSH levels were higher in patients without sickle cell disease compared with those with malaria and sickle cell disease, the difference was not statistically significant. Therefore, patients with both malaria and sickle cell disease may have a higher demand for GSH to detoxify the increased oxidative stress32.The relationship between GSH levels and parasite density varied across studies, with some reporting a negative relationship and others finding no significant associationI2: 99.12%) must not be ignored, suggesting substantial heterogeneity in the results. A limited number of studies investigated GSH levels in various contexts: (i) malaria patients with severe complications versus those without severe complications, (ii) asymptomatic versus symptomatic malaria, and (iii) P. falciparum versus non-P. falciparum malaria. Thus, the conclusions of our study were limited. Among the high-quality studies, there was a significant difference in GSH levels with a substantial effect size. Conversely, moderate-quality studies did not show a statistically significant difference. This may highlight the importance of study quality when interpreting results. Additionally, the level of heterogeneity remained high when the subgroup analyses were performed. Therefore, the true confounders of the relationship between GSH and malaria remain unidentified. Other potential confounders may include both infectious and noninfectious conditions that are co-endemic with malaria, such as nutritional deficiencies53, diabetes54, human immunodeficiency virus55, and coronavirus disease 201956. Based on the information from the included studies, the timing of sample collection was not explicitly stated in each study. Consequently, a meta-regression analysis to test whether the timing of sample collection influenced the effect estimate of the meta-analysis could not be performed. The final limitation is the evidence of significant publication bias, and the sensitivity analysis calls for a cautious interpretation of these findings. The considerable impact of a single study on the overall meta-analysis outcome underscores the importance of incorporating a diverse range of studies to mitigate potential biases.While the meta-analysis demonstrated a significant reduction in GSH levels in malaria patients compared with uninfected controls, indicating an association between malaria and lower levels of this critical antioxidant, the high variability among studies (Plasmodium species, malaria symptoms, and geographic location, revealed more nuanced findings. Further studies are necessary to corroborate these findings and delve deeper into the complex relationship between malaria and GSH levels.This comprehensive review and meta-analysis of the existing literature indicate a trend of decreased GSH levels in malaria patients compared with uninfected controls, which is consistent with the majority of the reviewed studies. Furthermore, the meta-analysis underlines the potential of GSH as a diagnostic biomarker for malaria. However, the relationship between GSH levels and specific characteristics, such as Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Figure 1.


Malaria and dengue are common mosquito-borne diseases around the world that cause high mortality and morbidity. The number of cases of both diseases is currently rising in Sudan and is associated with climate and environmental changes. Limited information is available on malaria and dengue co-infections and the severity of the two diseases among febrile patients in eastern Sudan. Thus, this study aimed to estimate the prevalence of malaria and dengue co-infections among febrile patients in Kassala, eastern Sudan.A cross-sectional hospital-based study was conducted among febrile patients from September to December 2019. A total of 395 patients were enrolled after consenting to participate in the study. Demographic and clinical data were collected by structured questionnaires. Blood samples were provided to diagnose malaria infections using microscopy and polymerase chain reaction (PCR) and for serology diagnosis of dengue using enzyme-linked immune sorbent assay (ELISA) IgM. Multiple logistic regression analysis was used to assess the association between demographic information, clinical symptoms and malaria and dengue co-infections.p-value = 0.018), while patients of co-infections were eight times more likely to have fatigue, and two times more likely to suffer from joint and muscle pain and this difference was statistically significant with  and , respectively.Out of 395 febrile patients examined 158 (40%) were malaria positive and 67 (17%) were dengue positive. The prevalence of malaria and dengue co-infections was 6.6% (26/395). Results of multiple logistic regression indicated that elder patients (41–60 years) had less rate of co-infections (OR = 0.3, 95% CI 0.11 to 0.81, This study confirmed the existence of malaria and dengue co-infections among febrile patients in Kassala, eastern Sudan for the first time. The severity of clinical symptoms of patients with malaria and dengue co-infections was observed, and the co-infections were found prevalent among young people. Malaria and dengue are common vector-borne diseases in eastern Sudan. However, co-infections with these two diseases are not reported among febrile patients. Malaria and dengue co-infections need a particular concern because this may increase morbidity and mortality if there are delays in diagnosis and appropriate treatment. So, this study has investigated the prevalence of malaria and dengue co-infections among febrile patients in Kassala, eastern Sudan. The findings of the study showed the prevalence of malaria and dengue co-infections in the study area according to the gender and age of enrolled patients. Results also showed the severity of clinical symptoms of patients with malaria and dengue co-infections were high compared to mono-infections, and the co-infections were found less among elder people. Plasmodium spp. which is usually transmitted by Anopheles spp. mosquitoes. Five parasite species cause malaria in humans; nevertheless, two of these species–P. falciparum and P. vivax–pose the greatest threat. Plasmodium falciparum is the deadliest malaria parasite and the most prevalent on the African continent [Aedes aegypti mosquitoes [Both malaria and dengue are known to be rapidly spreading mosquito-borne diseases and are of high importance in terms of morbidity and mortality, posing a worldwide public health problem due to the global expansion of their vectors . Malariaontinent . Dengue squitoes . Dengue squitoes .Malaria and dengue are endemic in similar tropical regions, and therefore, may result in the possibility of co-infection. Urban demographic expansion, deforestation, and agricultural settlements in peri-urban areas are known causes of the probability increasing of concurrent infections of these two diseases . ConcurrThere are many cases of malaria and dengue co-infections reported from various regions in the world following the first case that was reported in France in July 2005 . SeveralMalaria is endemic in Sudan, whereas dengue has established itself in an epidemic transmission cycle . The majEthical approval for the study was provided and issued by the Federal Ministry of Health, Sudan (No. 4-11-18). A signed informed consent was obtained from each participant that agreed to participate voluntarily in this study. All personally identifiable information was not disclosed to any person except the main investigators. The cost of ELISA and PCR tests for patients was paid by the authors and the participants had not any payment for these tests.2. Kassala city is located along the main Khartoum-Port Sudan highway which makes it the main trading center beside the connections with Eritrea and Ethiopia. The population of Kassala is estimated about 401,477 [Kassala state is located in eastern Sudan (10°12′N 34°19′E) . It is b 401,477 . In the A cross-sectional hospital-based survey for malaria and dengue co-infections was conducted in Kassala city. The study participants were enrolled during the dengue transmission season between September to December 2019 from eight hospitals and health centers namely Kassala Teaching Hospital, Rama Specialized Hospital, Alsika Hadid Health Center, Kassala Kuwaiti Hospital, Kassala Police Hospital, Doctors Health Center, Tarawa Health Center, and Alswagi Algnobia Health Center in Kassala city.In this survey, all febrile patients who were residents and admitted to the hospitals and health centers in Kassala city and agreed to participate in the study and signed the informed consent were enrolled in the study. Study participants were examined for malaria infection using microscopy and positive cases were confirmed using the polymerase chain reaction (PCR) method, patients were also examined for dengue infection using the serology method of enzyme-linked immune sorbent assay (ELISA IgM) .A well-constructed questionnaire was used to collect data from febrile patients. Demographic variables such as sex, age, and residence were included in the questionnaire. Also, clinical symptoms like fever, headache, fatigue, and nausea were collected from study participants. Data were collected through face-to-face interviews using a paper-based data collection tool.P. falciparum or P. vivax. The second outcome was the co-infections between dengue and P. falciparum. The third outcome variable was P. vivax and dengue co-infections. Each outcome variable had a binary outcome, either positive or negative.The first outcome variable was malaria and dengue co-infections whether it was A training workshop for laboratory technicians and health workers was conducted before the beginning of the study to review the standard procedures for malaria and dengue examinations and diagnostics. Venous blood (about 3 ml) was collected from eligible participants in the ethylenediaminetetraacetic acid (EDTA) containers. A few drops were used for the preparation of thin and thick smears that were stained with Giemsa stain and used for the detection of malaria parasites. All the positive microscopy samples were confirmed using the molecular technique. About 400 μl of blood from each positive microscopy sample was used for DNA extraction using the guanidine chloride protocol . Then, oThe PCR products were separated in 1.5% agarose gel stained with ethidium bromide and observed under UV using the BioDocAnalyze gel image documentation system .th day of dengue virus infection and the highest IgM mean titers were observed on day 7th of infection. The IgM antibodies were not detected after the 60th day of dengue infection [The IgM antibodies specific for dengue virus NS1 serotypes  were detected using an anti-dengue virus ELISA (IgM) kit   followinnfection .Chi-square analysis was used to test the significant associations between outcome variables and explanatory variables. Multiple logistic regression was used to estimate the odds ratio (OR) and 95% confidence interval (CI) for the outcome variables. For all the analyses, variables were considered statistically significant at p-value of ≤0.05.All demographic, clinical, and laboratory data were entered into the excel sheet and stored in a computer until the time of analysis. Each participant was assigned a unique identifier so that identifying information  was removed upon entering the questionnaire data into the database and confidentiality was maintained. Descriptive statistics were performed based on variables from the questionnaire using STATA version 14. Measures of central location such as mean, median, and measures of dispersion such as standard deviations, and interquartile range were calculated for continuous variables in the questionnaire. Proportions and ratios were calculated for categorical variables such as sex, age group, and others. Measure of statistical significance such as A total of 395 febrile patients were included in this study with a median age of 26 years (range: 5–84). Male patients counted for 229 (58%) and females for 166 (42%). The majority of patients  were found in the age group of 21–40 years .Out of 395 febrile patients examined 158 (40%) were malaria positive and 67 (17%) were dengue positive. The total of malaria and dengue co-infections was 26 (6.6%). Detailed results of malaria species infections with or without dengue co-infections were presented in . All theP. falciparum and dengue co-infections  and  respectively . In addition, patients infected with P. vivax and dengue co-infections are eight times more likely to have fatigue compared to those who did get the mono-infection . Also, patients with P. vivax and dengue co-infections are two times more likely to suffer from joint and muscle pain and this difference was statistically significant . For the P. falciparum and dengue co-infections, no statistical significant association was observed  had less rate of observed .Malaria and dengue co-infections are considered of great public health importance, especially in the tropics. This condition is usually underreported and could have fatal outcomes if left undiagnosed. However, malaria and dengue co-infections cause febrile illness with similar symptoms that are not easy to differentiate clinically. Therefore, understanding the distribution of malaria and dengue co-infections is vital for improving accurate diagnosis and proper therapeutic interventions. Information provided by this study can be used as guidance for clinicians, policymakers, and public health workers for choosing appropriate diagnostics and treatment in Sudan and similar endemic areas.Many areas in Sudan reported high prevalence of both malaria and dengue infections such as in the eastern region, but there were no previous reports about co-infections from this region. In the current study, the prevalence of malaria and dengue co-infections was reported  among febrile patients for the first time in Kassala, eastern Sudan. This finding is lower than a study conducted in western Sudan where the prevalence of malaria and dengue co-infections was 15% .vivax malaria showing a prevalence of 8.3% [The finding of previous studies conducted in endemic areas of dengue fever and malaria found a high prevalence of co-infections such as a study conducted in Brazil in 2009, where 11 co-infections were recorded among 132 patients with  of 8.3% . In anot of 8.3% . Additio of 8.3% . The dif of 8.3% . This mi of 8.3% .Based on our findings, it was observed that malaria and dengue co-infections were found not to be associated with age groups, but less associated among elder age group (41–60) years. Also, malaria and dengue co-infections were not associated with gender. This finding is in agreement with a study conducted in Cameroon where gender and age were not associated with malaria and dengue co-infections . ContrarIt was observed that malaria and dengue co-infections increase the likelihood of suffering from fatigue, joint, and muscle pain. This finding was supported by a finding of study conducted in Nepal, where malaria and dengue co-infections were associated with fatigue, joint pain, and chills . This fiIn this study, the prevalence of malaria and dengue co-infections among febrile patients was estimated for the first time in Kassala, eastern Sudan. Malaria and dengue co-infections are considered more severe than mono- infection, such as in a study conducted in French Guiana . MalariaOne of the limitations of the current study is that although the study is based on health facilities the clinical information is incomplete, because of deficiencies in the resources in the participating facilities. Some basic laboratory data could have been collected to characterize this condition and enable comparison with published reports on this condition, like malaria parasite count, haemoglobin concentration, platelet count and white blood cell count. Additionally, in the cross-sectional study design, causal link between the outcome and the exposure cannot be established.The prevalence of malaria and dengue co-infections was estimated in Kassala, eastern Sudan. The severity of clinical symptoms of patients with malaria and dengue co-infections was observed, and young ages were more affected by the co-infections. A clear understanding of the epidemiology of malaria and dengue co-infections is essential to inform decision-makers to institute an appropriate control strategy for both diseases. More research on malaria and dengue co-infections is needed in other endemic areas of Sudan. In addition, more vector studies are required for a better understanding of their role in transmission of malaria and dengue in Kassala, and in the country as a whole.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


The current study aimed to estimate prevalence of malaria infection, especially sub-patent infection, in pregnant women residing in high malaria-endemic, hard-to-reach pockets of the Indian state of Odisha; and also measure its impact on birth-weight of their new-borns.n = 308) adjusted for residence (block), gravida, caste, age and gestational age at testing.A time-to-event analysis of prospective longitudinal follow-up study nested within a cross-sectional survey of people residing in high malaria-endemic six districts of Odisha was conducted during July–November 2019. Malaria status in pregnant mothers was categorized as malaria free; sub-patent, and patent. Hazards Ratio (HR) of low birth-weight  was estimated in these three categories (p = 0.032) in pregnant women with patent infection and 1.82  in women with sub-patent infection when compared to no malaria group.50.3% pregnant women had sub-patent malaria infection, 3.9% had patent infection. In fully adjusted model, hazards ratio of LBW was 3.76 (95% CI 1.12, 12.64, The study showed that half of the pregnant women in high-endemic pockets had sub-patent infection which posed deleterious influence on birth-weight of their new-borns. The study thereby flags the prevalence of sub-patent infection as a public health concern, because sub-patent infection in pregnant mothers may persist as a “silent” reservoir, with the potential to derail the malaria control program, especially when the country plans malaria elimination by 2030. Plasmodium falciparum , has devastating consequences for both the mother and baby, especially in terms of birth-weight and infant mortality [Malaria continues to be an international public health problem. Most of the 241 million malaria cases and 62,700 malaria deaths, reported worldwide during 2020, are from Africa and Southeast Asia region. India alone accounted for 83% of estimated malaria cases and about 82% of all malaria deaths in the World Health Organization (WHO) South-East Asia Region –7. In Inortality , 11.P. falciparum infection with adverse pregnancy outcomes [P. falciparum infection and LBW, preterm delivery. From India, there is hardly any evidence regarding prevalence of sub-patent parasitemia and its health impacts. Realizing the scarcity of evidence in this domain, we set out to examine the burden and effect of malaria among pregnant women in the Indian state of Odisha, so that the Indian malaria program can be informed appropriately. Odisha with 4% of the country’s population contributes to 40% of the malaria cases of the country [Malaria can be detected by either microscopic examination of a blood smear or rapid diagnostic test (RDT) or molecular detection assays like polymerase chain reaction (PCR). The sub-patent infection is that infection which is negative for parasites by light microscopy  or RDT  but positive for parasites only by PCR-based assay , the test with highest sensitivity , 13. Suboutcomes . But theoutcomes  and Benioutcomes  have sho country . Therefo country . We alsoSince 2017, in Odisha, a colossal state-run public health program, namely Durgama Anchalare Malaria Nirakaran , in short DAMaN, is being implemented to complement the ongoing malaria control activities of the National Vector-borne Disease Control Programme of India, to accelerate the decline of malaria in the state. DAMaN is being implemented in hard-to-reach pockets of 23 malaria-endemic districts of Odisha. The components of DAMaN mainly included biannual active case detection, enhancement of Long-lasting Insecticidal Net (LLIN) use and strengthening of Indoor Residual Spray (IRS). An assessment of the activities and impact of DAMaN was undertaken in 2019, the details of which have been described elsewhere .n = 338). Only 12 pregnant women refused to provide specimen. The current study, nested within the DAMaN assessment, was a time-to-event analysis of prospective longitudinal follow-up of pregnant women encountered during the survey [As a part of this assessment exercise, a multi-phase cross-sectional survey was conducted in six DAMaN-implementing districts of Odisha, representing three distinct geophysical regions of the state : Rayagada and Kalahandi). Two DAMaN-implementing districts from each region, one representing higher and the other lower than median Annual Parasite Index (API), the number of confirmed cases of malaria per 1000 population (2015), were selected. Consequently, 1823 households were surveyed between June and November 2019, which is considered as high malaria transmission season in India. These households were selected randomly using a multi-stage clustered sampling technique . ApproxiAll enrolled women were longitudinally followed-up until they underwent abortion , experienced a still-birth or delivered a live fetus. Out of 338 enrolled women, 308 delivered live fetuses with validated records of birth-weights of their neonates. Birth-weights were recorded by Auxiliary Nurse and Midwives (ANMs) or Accredited Social Health Activists (ASHAs) immediately after birth using digital weigh scales, reporting weight to the nearest 10 g. LBW was defined as a birth-weight of < 2500 g. The gestational age when blood specimen was collected and when a live fetus was born was estimated using the date of last menstrual period before pregnancy, which was recorded during the survey.® Ethylene Diamine Tetraacetic Acid (EDTA) tubes from these women for parasite detection by RDT and PCR-based assay. RDTs were performed at the point of care using the Pf/PAN RDT  as per the manufacturer’s recommendation. A positive test result was defined as the presence of the control band and P. falciparum and/or other Plasmodium infection. Those who tested positive by RDT were referred to the local malaria program for administration of standard anti-malaria treatment. PCR detection of parasites was performed using the species-specific primers  targeting 18S ribosomal Ribonucleic Acid (rRNA) and cycling parameters as described by Snounou and others [® DNA Blood Mini Kit  and eluted in 50 μl of distilled water. The primary PCR was performed in a 25 μL reaction mixture that contained 0.2 Units of Taq DNA polymerase , 0.2 mM each Deoxynucleoside triphosphate (dNTP) , 0.4 mM each primer  and 2.0 mM MgCl2 . The reaction was allowed to proceed for 35 cycles after an initial denaturation at 94 ℃ for 1 min (min), annealing at 50 ℃ for 1 min, and extension at 72 ℃ for 1 min; final extension was at 72 ℃ for 10 min in a Thermal Cycler . The nested PCR reaction condition was the same as primary PCR except for the annealing temperature, 55 ℃. The PCR products were visualized under ultraviolet light following the electrophoresis on 1.2% (w/v) ethidium bromide-stained agarose gel, and images were captured using a gel documentation system .During the survey, 1 ml of venous blood specimens was collected in BD Vacutainerd others . BrieflyThe demographic characteristics of the sampled respondents were summarized using descriptive statistics. Survival (time-to-event) analysis was carried out employing Cox proportional-hazards regression model  to estimThe Cox models were only adjusted for maternal age initially. We a priori hypothesized that remote location, tribal membership , gestational age at malaria testing and parity of the pregnant women can confound the malaria–LBW relationship. Therefore, we controlled for these variables (full-adjusted models) to estimate the independent relationship between MiP and LBW. All the statistical analyses were performed in R version 4.1.2.The ethical approval has been obtained by both the Institute Human Ethical Committee (ICMR-RMRC/IHEC-2019/012 dated 27/02/2019) and State Research & Ethics Committee of Department of Health and Family Welfare, Govt. Of Odisha (453/SHRMU/187/17 Dated 22/8/2017). A signed informed consent was obtained from all the respondents. Respondents consenting to provide their demographic and study related information were only interviewed. Further, 1 ml of their venous blood specimens was collected from the consenting respondents. The details of all the respondents were anonymised at the analysis stage. Moreover, positive malaria test results done at the point of care as well as laboratory were conveyed to the respondents and concerned health officials for necessary action as per program guidelines.The study included 308 pregnant mothers who delivered live fetuses and whose birth-weights were available Fig. . Sixty pp value for unadjusted trend < 0.05).The proportion of LBW was found to be 10.6% in malaria-free controls followed by 17.4% in sub-patent malaria infection group and 33.3% in pregnant women with patent infection (the p = 0.0132) times higher in pregnant women with patent infection followed by 1.74 times  in women with sub-patent infection as compared to the malaria-free control group. The Hazard Ratios (HRs) attenuated to 3.76  and 1.82 , respectively, in the fully adjusted models .The age-adjusted hazards of delivering a new-born with LBW was 4.06  1.34, 12.32, The current study reveals that more than half of the pregnant women residing in hard-to-reach high-endemic malaria pockets of the Indian state of Odisha were infected with malaria during high-transmission season. Of them, only a small fraction could be detected by RDT, which is routinely used by the malaria control program. However, 50% of all the pregnant women, who returned a negative rapid test, tested positive for malaria by PCR-based assay. According to Rogerson and others 2018), RDT have been poor at detecting malaria in pregnant women, while PCR-based assay was highly efficient in detecting the low-density infection prevalent among pregnant women, , RDT hav. The higP. falciparum, are commonplace in high-endemic areas [The low-density parasitemia, attributable to immune tolerance due to multiple and often untreated infection with ic areas . Recent ic areas . Sub-patic areas . Many stic areas –34.Apart from its public health importance as a ‘silent’ reservoir, another likely yet unclear impact of sub-patent infection during pregnancy is its effect on the fetus, evidence regarding which is scanty and nebulous. Some studies reported no clinical impact of sub-patent infection during pregnancy , 35, whePlasmodium infection. P. falciparum-infected erythrocytes express a unique variant surface antigen, Variant Chondroitin Sulfate A (VAR2CSA), which mediates sequestration in the placenta leading to placental infection and inflammation. Sequestration leads to dysregulated expression of cytokines and chemokines, abnormal placental development and reduced placental nutrient transport to fetus, contributing to higher rates of LBW in malaria-infected mothers [MiP threatens the well-being of the mother and her developing fetus. An infected mother is likely to be an important reservoir of  mothers , 30. ButOur study has a few limitations, the primary among them was the lack of statistical significance of the association between sub-patent infection and LBW, although the effect size of the relationship measured using hazard ratio was clinically significant. We calculated post hoc the power of our sample and found it to be only 40% for identifying an effect size of 1.9, the hazard ratio of LBW we found in the sub-patent group. Therefore, the lack of statistical significance probably does not undermine our all-important conclusion that sub-patent infection leads to LBW, because it is most likely due to lack of power  and not due to absence of actual deleterious relationship between sub-patent malaria and birth-weight. Moreover, the dose–response relationship that we observe between malaria and LBW further strengthens the posited causal association between MiP and birth-weight. The second limitation was the ascertainment of malaria infection only once during pregnancy and that too at different gestational sages of the participants. However, it does not undermine our inferences, because malaria-infected women overall showed greater propensity for delivering LBW neonates even after adjustment for the timing of the test. The third limitation of the study is non-use of microscopy for initial parasite detection. The study instead used the RDT method, which is the routine test used in national malaria programs. The strengths of our study include laboratory-based PCR tests of more than 300 pregnant women in a hard-to-reach Indian setting, adjustment with the area (block) variable to control for ecological heterogeneity between regions—a crucial determinant of malaria infection—and lastly adjusting for differences in social determinants, such as education, caste and parity which also have important bearing on birth-weight.To conclude, our findings show high prevalence of sub-patent malaria in pregnant women, residing in high-endemic pockets of the Indian state of Odisha. Additionally the study also demonstrates that sub-patent infection has substantial deleterious impact on the birth-weight of their new-borns. The study thereby underlines that sub-patent infection during pregnancy is a major public health concern which requires immediate attention as the country heads towards malaria elimination by 2030. It underscores the need to further (1) evaluate the importance of sub-patent infection as a reservoir for transmission, (2) determine the importance of sub-patent infection during pregnancy in terms of various health outcomes of mother and child, and (3) intensify the research for development of point-of-care molecular diagnostic tools to identify individuals harboring sub-patent infection in the community.


Coinfection with malaria and typhoid fever is a major public health issue in developing countries. In endemic areas, including Ethiopia, people are at risk of acquiring both malaria and typhoid fever at the same time. Therefore, this study aimed to determine the magnitude of malaria-typhoid fever coinfection in febrile patients attending hospital at Southern Ethiopia. st October to 30th December 2021. The data was collected using a pretested structured questionnaire. Capillary and Venus blood samples were collected for assessing malaria and typhoid fever, respectively. Blood smear, culture, and biochemical tests were performed based on standard parasitological and microbiological methods. The P-value ≤ 0.05 was considered statistically significant.  A hospital-based cross-sectional study was carried out on 416 febrile patients attending Arba Minch General Hospital from 1Plasmodium falciparum. The malaria-typhoid fever coinfection showed a statistically significant association with a clinical presentation of a continuous pattern of fever  and chills . About 29.6% of Salmonella isolates were multidrug-resistant (MDR).  The magnitude of malaria, typhoid fever, and their coinfections was 26.2% (109/416), 6.5% (27/416), and 3.1% (13/416), respectively. Among the confirmed malaria cases, about 66% of infections were Salmonella spp. and higher prevalence of malaria‐typhoid fever coinfection, proper diagnostic procedure should be implemented for proper use of drugs. The total rate of coinfection with malaria and typhoid fever was comparable to that of previous studies. With the consideration of higher prevalence of drug resistance of  Malaria and typhoid fever are the most frequent acute febrile infections with no discernible symptoms and leading causes of morbidity and mortality in sub-Saharan Africa –5. AcutePlasmodium species are known single-celled protozoan parasites that cause malaria in humans . This finding is more or less similar to previous studies done in Shashemene, Ethiopia (5.0%) , West, GP. falciparum, 4 (30.8%) were with P. vivax, and 2 (15.4%) were with mixed infections . This finding agrees with the results reported for Ghana (1.91%)  were positive for malaria-typhoid fever coinfection. Of these, 7 (53.8%) were coinfected with  (1.91%) , India ( (1.91%) ,36, and parum, 4 .8% were  (1.91%) , Cameroox, and 2 .4% were  (1.91%) , Calabar (1.91%) , Southea (1.91%) , and Aku (1.91%) . The low (1.91%) ,37 involparum, 4 .8% were  (1.91%) , North-W (1.91%) , New Del (1.91%) .P < 0.05), which is in contrast to a study done in Northwest Ethiopia [Clinical features, such as continuous patterns of fever and chills, were found to be statistically significant with malaria-typhoid fever coinfection  of febrile patients as well as the prognosis of the patient were not considered in this study due to budget and resource constraints.


RTS,S/AS01 has been recommended by WHO for widespread implementation in medium to high malaria transmission settings. Previous analyses have noted lower vaccine efficacies in higher transmission settings, possibly due to the more rapid development of naturally acquired immunity in the control group.To investigate a reduced immune response to vaccination as a potential mechanism behind lower efficacy in high transmission areas, we examine initial vaccine antibody (anti-CSP IgG) response and vaccine efficacy against the first case of malaria  using data from three study areas  from the 2009–2014 phase III trial (NCT00866619). Our key exposures are parasitemia during the vaccination series and background malaria incidence. We calculate vaccine efficacy (one minus hazard ratio) using a cox-proportional hazards model and allowing for the time-varying effect of RTS,S/AS01.We find that antibody responses to the primary three-dose vaccination series were higher in Ghana than in Malawi and Gabon, but that neither antibody levels nor vaccine efficacy against the first case of malaria varied by background incidence or parasitemia during the primary vaccination series.We find that vaccine efficacy is unrelated to infections during vaccination. Contributing to a conflicting literature, our results suggest that vaccine efficacy is also unrelated to infections before vaccination, meaning that control-group immunity is likely a major reason for lower efficacy in high transmission settings, not reduced immune responses to RTS,S/AS01. This may be reassuring for implementation in high transmission settings, though further studies are needed.The online version contains supplementary material available at 10.1186/s12879-023-08699-7. Plasmodium falciparum. Overall efficacy of the vaccine in phase III trials was modest, resulting in a 36.3% reduction in the incidence of clinical malaria over a median of 4 years in children aged 5–17 months across 11 sites in sub-Saharan Africa [Malaria remains a public health challenge, with 241 million cases in 2020 despite the increasing implementation of control measures such as artemisinin combination therapies, insecticide-treated bed nets, and indoor residual spraying [Various theories attempt to explain the geospatial heterogeneity in RTS,S/AS01 vaccine efficacy . “DelayeP. falciparum can modulate the immune response upon infection, resulting in the depletion of T cells and altering the functional characteristics of B cells, potentially reducing the immune response to vaccination [However, lower efficacies in higher transmission settings have elicited hypotheses that children in endemic regions experience immune modulation mediated through infections before or during vaccination (three doses over three months) resulting in lower immune responses. Two analyses of phase II data and one of phase III data found support for this hypothesis –9. Potencination –14. Infecination , 16 so icination . Converscination  resultincination , 20. In cination , 22.Here, we evaluated the impact of malaria infections, before or during vaccination, on RTS,S/AS01 antibody response and efficacy. From three phase III trial sites in Gabon, Ghana, and Malawi, we obtained longitudinal malaria infection data and baseline and post-vaccination anti-circumsporozoite antibody levels. Further, we obtained parasitemia counts during the three-dose vaccine schedule. We used ecological data at the individual, household, and neighborhood levels to estimate the background malaria transmission intensity as a proxy for pre-vaccination infections.It is vital that we understand the specific mechanisms through which malaria transmission intensity does, or does not, alter the impact of RTS,S/AS01. With this understanding, we can improve implementation and design interventions to supplement RTS,S/AS01 vaccination where necessary.Study participants included in this analysis were children (5–17 months) enrolled in the 2009–2014 phase III trial of RTS,S/AS01 in Kintampo, Ghana; Lilongwe, Malawi; and Lambaréné, Gabon. The details of the parent study can be found elsewhere [The development of naturally acquired immunity in the control group was a cause of reduced vaccine efficacy in high-incidence areas in the phase III trial of RTS,S/AS01 , 6. WhenBackground malaria incidence was estimated using our previously described method  which prParasitemia during the vaccination series was measured directly by microscopy during passive surveillance in between the first and third dose of RTS,S: any individual which had a blood film microscopy with visible parasites (> 0 parasites per microliter) was considered to be infected.Anti-CSP antibody (IgG against the NANP repeat region) levels, which were associated with protection at the time of the trial, were previously measured using standardized enzyme-linked immunosorbent assays (ELISA) on the day of the first dose and one month after the third dose of RTS,S/AS01 . AntibodTo calculate the efficacy of RTS,S/AS01 against the first case of malaria depending on background malaria incidence and parasitemia during the vaccination series, we fit a Cox proportional hazards (CPH) model. The time period was bounded by the date of the third vaccine and the earliest date among the first case of malaria, loss to follow-up, receipt of the fourth dose of RTS,S/AS01, or study end. Those with non-malaria outcomes were considered to be censored. Main covariates included vaccination status , predicted background malaria incidence, and whether parasitemia during the initial 3-dose vaccination series period was observed. Interaction terms between treatment and background incidence as well as treatment and parasitemia were included. We used repeated measures  to allow for the effect of time on vaccine protection: an interaction term between time and treatment was included and we accounted for the repeated measures in standard error calculations. We also adjusted for the child’s age at the third vaccination; this age was set to the median when estimating from model outputs. Efficacy was calculated as one minus the hazard ratio for vaccination. Functional forms of background incidence were assessed using AIC.Finally, we fit an accelerated failure time (AFT) model to estimate the impact of vaccination on the time to the first case of malaria. Censoring was conducted in the same manner as in the CPH model and we assumed the outcome followed a log-logistic distribution. Our covariates in the AFT model were the same as the CPH model. The functional form of background incidence was assessed using AIC.We conducted a sensitivity analysis detailed in the supplemental materials by replacing our predicted background incidence values with average transmission intensity values from the Malaria Atlas Project over 2009–2014 . We repeIn total, 2,427 children were included in this analysis, 992 from Ghana, 771 from Malawi, and 664 from Gabon  in Ghanaian children, compared to 15,377  in Malawian children and 6,706  in Gabonese children.Antibody data were available for 641 children . On the day of vaccination with the first dose of RTS,S/AS01 or a control vaccine, participant anti-CSP levels were below 1.0 ELISA units/ml, regardless of background malaria incidence . This difference remained statistically insignificant after stratification by study area (p = 0.68)  being largely responsible for lower efficacy in high transmission in areas is strong, , 29, 30 Our results suggest that the lower RTS,S/AS01 efficacy in higher malaria transmission areas may be largely due to delayed malaria and the development of naturally acquired immunity in the control group, and not due to infections before or during vaccination which led to reduced immune responses. However, the literature remains divided over the influence of infections before vaccination on RTS,S/AS01 efficacy, with each published study (including ours) suffering from various important limitations. A future study that directly measures infections before vaccination is needed to elucidate the influence of such infections on RTS,S/AS01 efficacy. The roles of delayed malaria and the development of immunity in control group, however, have a strong evidence base in the current literature. During the widespread rollout of RTS,S/AS01, it may be sensible to concentrate our energy on combating delayed malaria later in follow-up in order to maximize vaccine impact in high transmission areas.Below is the link to the electronic supplementary material.Supplemental Figure 1: Distribution of MTI values within and across study sites using a) our original predicted values b) Plasmodium falciparum incidence rate and c) Plasmodium falciparum infection prevalence among 2-10 year olds. Supplemental Figure 2: Anti-CSP Antibody Response by Study Area (1 Month Post-Vaccination). Supplemental Figure 3: Anti-CSP Antibody Response by Transmission Intensity, All Study Areas and Study Area Adjusted  using Plasmodium falciparum incidence rate per person per year. Supplemental Figure 4: Anti-CSP Antibody Response by Transmission Intensity, All Study Areas and Study Area Adjusted  using Plasmodium falciparum infection prevalence among 2-10 year olds. Supplemental Figure 5: Efficacy Against the First Case of Malaria Over Time, Transmission Intensity, and Cases During Vaccination using Plasmodium falciparum incidence rate. Supplemental Figure 6: Multiplier Effect of RTS,S/AS01 Vaccination on the Time to First Case of Malaria A higher multiplier implies a greater delay until the first case of malaria . The null value is 1. Supplemental Figure 7: Efficacy Against the First Case of Malaria Over Time, Transmission Intensity, and Cases During Vaccination, Stratified by Study Area.


Plasmodium infections and disease. Malaria in pregnancy is a major public health problem in endemic areas. Assessment of the burden and risk factors of malaria in pregnancy across different malaria transmission settings is required to guide control strategies and for malaria elimination. Thus, the current study is generating such evidence from parturient women in northwest Ethiopia.Pregnant women have an increased risk of A cross-sectional study was conducted among 526 pregnant women admitted to the delivery rooms of selected health facilities in Jawi district, northwest Ethiopia, between November 2021 and July 2022. Data on the socio-demographic, clinical, obstetric, and malaria prevention practices of pregnant women were collected using interviewer-administered questionnaires and from women’s treatment cards. Malaria was diagnosed by light microscopy, rapid diagnostic test, and multiplex real-time polymerase chain reaction. Risk factors for malaria were evaluated using bivariable and multivariable logistic regression models. A P-value of < 0.05 was considered statistically significant.Plasmodium infections. The prevalence of peripheral, placental, and congenital malaria was 12.2% (95% CI 9.5–15.3%), 10.9% (95% CI 8.2–14.1%), and 3.7% (95% CI 2.3–6.1%), respectively. About 90.6% of peripheral and 92% of placental Plasmodium infections were asymptomatic. Plasmodium infection at parturiency was independently predicted by maternal illiteracy , primigravidity , lack of antenatal care follow-up , and history of symptomatic malaria during pregnancy . Moreover, the blood group O phenotype was significantly associated with placental malaria among the primiparae.Among the examined parturient women, 14.3% (95% CI 11.4–17.5%) had Plasmodium infections were prevalent among parturients in northwest Ethiopia. Maternal illiteracy, primigravidity, lack of antenatal care follow-up, and history of symptomatic malaria during pregnancy were the risk factors for malaria during parturiency. Thus, promotion of a healthy pregnancy through ANC follow-up, strengthening malaria prevention and control practices, and screening of malaria in asymptomatic pregnant women are suggested to reduce its burden in pregnancy.Overall, asymptomatic  Plasmodium falciparum and/or Plasmodium vivax occurs in about 68% of the landmass and threatens over 60% of the population [Malaria remains a major public health and socioeconomic challenge in Africa, where it causes the highest proportions of global infections and deaths every year . In Ethipulation . The coupulation .Pregnant women are more susceptible to malaria infection than non-pregnant women due to pregnancy-associated hormonal, nutritional, and immunological changes . MalariaIn addition to the increased susceptibility, pregnant women and their newborns endure severe adverse outcomes associated with malaria, such as severe maternal morbidity, a high risk of maternal mortality, low birth weight, infant mortality, maternal anaemia, and congenital malaria , 10, 11.To reduce the burden of malaria, Ethiopia implements insecticide-treated bed nets (ITNs), indoor residual spray (IRS), and effective case management as core malaria prevention and control strategies , 16. TheOn the other hand, malaria in pregnancy remains poorly studied in Ethiopia, particularly among parturient women. Previous studies on malaria among Ethiopian parturients reported prevalences ranging from 2.3% to 15.2% and identified the association of primigravidity with malaria , 20. HowA health facility-based cross-sectional study was conducted among pregnant women admitted to the delivery rooms of Jawi Primary Hospital, Jawi Health Centre, and Bambluk Health Centre in Jawi district, Awi Zone, Amhara Regional State, northwest Ethiopia, for delivery or spontaneous abortion between November 2021 and July 2022  , \documeEthiopia , at a 95Probability proportionate to size sampling was used to determine the number of participants required from each health facility. The number of pregnant women from each health facility was determined based on the proportion of pregnant women who attended delivery rooms at each health facility in the last 6 months prior to the study. Accordingly, 350 women were included from the delivery rooms of Jawi Primary Hospital, 113 from Jawi Health Centre, and 63 from Bambluk Health Centre Fig. .Fig. 2SaPregnant women who were admitted to the health facilities’ delivery rooms during the study period and were willing to participate in the study were eligible. Pregnant women who were HIV positive, hypertensive, diabetic, had multiple pregnancies, and had serious delivery complications were excluded. Those who met the eligibility criteria and consented to participate were included in the study.Pretested structured questionnaires and checklists were used to collect socio-demographic, clinical, obstetric, and risk factor data from women who provided consent to participate in the study at the labour ward or postpartum. The questionnaire was initially prepared in English and later translated to the local languages (Amharic and Awungi) by native speakers of the languages. To ensure consistency, data collectors read and completed the Amharic or Awugni questionnaire for the participants, regardless of the participants’ literacy level.The questionnaire included the women’s socio-demographic characteristics, malaria prevention practices, the presence of stagnant water in the household vicinity, and their estimated monthly household income. Similarly, information on obstetric and clinical profiles, including antenatal care follow-up, gravidity, parity, malaria symptoms, history of malaria during pregnancy, and usage of anti-malarial drugs were collected by interview and from medical follow-up cards. Gestational age was determined by the last menstrual period and the fundal height method in health centres, whereas the Mindray DP-50 digital ultrasound machine was used in the hospital. The temperature was recorded from the maternal armpit using a digital thermometer.Mid-upper arm circumference (MUAC) was measured halfway between the tip of the shoulder (olecranon process) and the tip of the elbow (acromion process) to the nearest 0.1 cm (cm). An insertion-type MUAC tape that is non-elastic and non-stretchable was used to take the measurement. The measurement was taken at the mid-point on the relaxed left arm, without any clothing, and with optimal tape tension (not too loose or too tight) following the standard instructions and steps. Undernutrition was defined as MUAC less than 23 cm . MaternaMaternal capillary blood was collected by finger pricking. Immediately after delivery, the maternal side of the placenta was cleaned with 0.9% normal saline, incised with a surgical blade, and blood was collected from the intervillous space with a syringe and transferred to a 0.5 millilitre Ethylene diamine tetraacetic acid (EDTA) tube. Similarly, the umbilical cord was clamped, cleaned with 0.9% saline , sectioned with a lancet and blood was collected with a syringe and transferred to an EDTA tube.P.f/P.vtest kit  as per the manufacturer’s instructions. Thick and thin blood smears were prepared on a single microscope slide and allowed to air-dry at room temperature. Thin smears were fixed using absolute methanol and both the thick and thin smears were stained with a 10% Giemsa solution for 10 min, rinsed with tap water, and air dried [Malaria was diagnosed using light microscopy, rapid diagnostic tests (RDTs), and multiplex quantitative polymerase chain reaction (qPCR). The RDTs diagnosis of malaria was performed using the Abbott SD Bioline Malaria Ag ir dried . Stainedir dried . An expePlasmodium falciparum, Plasmodium vivax, and P. falciparum and P. vivax mixed infections was estimated on thick film against 200 leucocytes, assuming a total white blood cell count of 8000/µl. The parasite density was then classified as low (below 1000 parasites per microlitre of blood), intermediate (1000–4999 parasites per microlitre of blood), and high (≥ 5000 parasites per microlitre of blood) [The density of both sexual and asexual stages of f blood) .Finger-prick, placental and umbilical cord blood samples were spotted on Whatman filter papers, air-dried, packed in a ziplock containing desiccants, and transported by cold chain to the Aklilu Lemma Institute of Pathobiology (ALIPB) and stored at − 20 °C until analysed. A total of 1159 dry blood spots (DBSs) were collected from 372 delivering women and 43 abortion women. Accordingly, 12 peripheral blood DBSs were collected from 12 microscopic and/or RDT-positive aborting women, and 141 DBSs were collected from 47 delivering women who had microscopic and/or RDT-positive results in one or more of the compartments . The other 1006 DBSs were collected from 31 microscopic and/or RDT-negative aborting women  and 325 delivering women . The real-time polymerase chain reaction assay was done at the Ethiopian Public Health Institute.Genomic DNA (gDNA) extraction was performed using the Geneius™ Micro gDNA Extraction Kit . Briefly, 3 mm-diameter circles of DBS were punched out and processed following the manufacturer’s instructions in 1.5 ml Eppendorf tubes. The resulted DNA was eluted with 100 µl volume of elution buffer and stored at − 20 °C until assayed.. [All microscopy and/or RDT-positive samples were extracted and analysed individually. For microscopy and RDT-negative samples, malaria prevalence was estimated using pooled DBS sample extraction and analysis with slight modifications as described by Zhou et al.  Fig. 3). [3).FigPlasmodium genus using multiplex real-time PCR (qPCR). Samples in genus qPCR-negative pools were taken as negative. For any pool with a positive genus qPCR, individual DBSs were extracted following the protocol, tested for genus-specific qPCR and then species-specific qPCR assays were performed.In short, ten-punched out 3 mm-diameter circles were pooled together in 2 ml Eppendorf tubes and incubated overnight with lyse buffer and proteinase K solution to ensure proper lysis, and then DNA extraction was performed following the normal procedures. The extracted DNA was tested for the Plasmodium-specific and P. falciparum-specific primers; whereas P. falciparum and P. vivax-specific primers were multiplexed during the second run, as described in Belachew et al. [The DNA amplification and detection were performed by the QuantStudio 5 Real Time PCR system (Applied Biosystems) using the TaqMan fluorescence assay in a final volume of 10 μl in two rounds. In the first run, all samples were tested by multiplexing pan-w et al.  .Plasmodium infection in umbilical cord blood at delivery.Congenital malaria: : history of malaria infection during pregnancy recorded in the maternal treatment card.Malaria history in pregnancyPlasmodium infection:Plasmodium infection in the absence of malaria symptoms, a history of fever within the past 48 h, and an axillary temperature < 37.5 °C.Asymptomatic Plasmodium infection:Plasmodium infection from women who exhibited at least one of the signs and/or symptoms of malaria like axillary temperature ≥ 37.5 °C, fever, joint pain, malaise, vomiting, chills, and headache during delivery room admission or within the past 48 h.Symptomatic Plasmodium infection: Having positive RDT and/or detection of malaria parasites by real-time qPCR but not by microscopy.Submicroscopic : Malaria parasites not detected by microscopy and negative by malarial RDT but detected by real time qPCR.Subpatent infectionMultigravida: A woman who is pregnant for the second time and above.Multipara: A woman who delivered for the second time and more.Plasmodium infection was defined as having a positive RDT and/or detection of malaria parasites using light microscopy and/or real-time qPCR. The prevalence of Plasmodium species infections in peripheral, placental, and cord blood samples was estimated by dividing the number of women diagnosed positive for Plasmodium infection by the total number of pregnant women examined for peripheral, placental, and umbilical cord infections, respectively.Data was double entered into Epi data 3.1 software to reduce clerical errors and inconsistencies. The entered data was exported to Statistical Package for Social Sciences (SPSS) version 25 statistical software , cleaned, and analysed. Bivariable and multivariable logistic regression models were used to identify independent predictors of malaria. Only variables that showed statistical significance at p < 0.05 in the bivariable logistic regression analysis and those known to be associated with malaria based on previous studies were included in the multivariable logistic regression model. Multicollinearity among predictors was tested by using the variance inflation factor (VIF < 10). For the multivariable logistic regression analysis, the standard (Enter) method was used for variable selection. The model’s fitness was checked using the Hosmer and Lemeshow test (p > 0.05). The Odds ratio (OR) with a 95% confidence interval (CI) was used to measure the strength of the statistical association. A p-value < 0.05 was used to indicate statistical significance.Plasmodium infections, among which 87.5% (n = 460) were admitted for delivery and 12.5% (n = 66) for spontaneous abortion. The median (interquartile range) age of the women was 25 (8) years, and the majority (80%) were young adults aged 20–34 years. About 48.1% of the women resided in rural areas, 47.7% were illiterate, and 51.9% were engaged in farming. More than 55% of them were primigravidae or secundigravidae, and 63.7% had at least one ANC follow-up during the current pregnancy. Moreover, only 29.8% of them reported that they slept under ITNs on the previous night of the delivery room admission, and 46.4% of the households did not sprayed with IRS within the last 12 months. Among the women, 40.9% had the O blood group phenotype, and 2.9% had symptoms of malaria during or within 48 h of admission to the delivery rooms  had peripheral malaria and 10.9%  had placental malaria. About 3.7%  of the newborns had congenital malaria. All congenitally infected newborns were from mothers who had both peripheral and placental malaria. However, twenty women had discordant results between peripheral and placental blood specimens . Plasmodium falciparum accounted for 76.6% of peripheral, 82% of placental, and 88.2% of congenital malaria infections. Asymptomatic malaria was 11.4% (58/511) in peripheral blood and 10.3% (46/448) in placental blood specimens, which accounted for 90.6% of the peripheral and 92% of the placental Plasmodium infections. On the other hand, 40% of the symptomatic parturient women had peripheral malaria and 33.3% had placental malaria, which accounted for 9.4% of the peripheral and 8% of the placental malaria cases, respectively  of the admitted parturient women had Plasmodium infection than their multigravid counterparts . Similarly, parturient women who did not have ANC follow-up during pregnancy had more than a double increased risk of malaria at the end of pregnancy compared to women who had ANC follow-up . Moreover, women who had a history of symptomatic malaria during pregnancy had a more than fourfold increased risk of malaria at delivery  . The primigravidae were 1.88 times more likely to acquire Plasmodium infections, and the majority of the infections were due to P. falciparum. About 12.2% of the parturient women had peripheral malaria, 10.9% of the delivered women had placental malaria, and 3.7% of the newborns had congenital malaria. Maternal illiteracy, primigravidity, lack of ANC follow-up, and history of malaria during pregnancy were independent predictors of malaria at the end of pregnancy. Unlike the multiparae, blood group O primiparae had a significantly increased risk of placental malaria compared to the non-O ABO blood primiparae. Such findings of malaria in pregnancy could have a substantial impact, as malaria in pregnancy is associated with poor maternal, fetal, and childhood outcomes [In this study, 14.3% of the parturients admitted to the delivery rooms in three health facilities in Jawi district, northwest Ethiopia, had outcomes .Plasmodium infection in pregnancy in the current study was comparable with a study in southern Ethiopia [P. falciparum [P. vivax in pregnancy [Plasmodium species distribution in the areas, the socio-demographic characteristics and conditions of pregnant women , and the diagnostic efficiency of the malaria detection methods used could be possible explanations for the variation in malaria prevalence and Plasmodium species distribution between the current study and previous studies.The prevalence of Ethiopia , but relEthiopia , 21. On Ethiopia , Sudan [Ethiopia , and ColEthiopia . In linelciparum . On the regnancy . DifferePlasmodium infections were from women who had both peripheral and placental Plasmodium infections. However, nine women had a peripheral Plasmodium infection only, and eleven women had a placental Plasmodium infection only. This finding was consistent with previous studies, which reported different levels of discordance [Plasmodium infection might be due to early infections (particularly in low parasitaemia cases), a lack of VAR2CSA protein expression on infected red blood cells [In this study, all congenital cordance , 33. Perod cells , and theod cells .On the oPlasmodium infections were asymptomatic and more than 30% were submicroscopic. Plasmodium infections were significantly higher among adolescent women (< 20 years) and independently predicted by primigravidity. In line with the present study, studies in Papua New Guinea and Uganda reported that primigravid women had an increased risk of placental and peripheral malaria compared to multigravid women [Studies have shown that susceptibility to malaria and the severity of infections during pregnancy differ with the intensity of malaria transmission in the area, the level of exposure, and the anti-malarial immunity acquired by the pregnant woman , 36. Thuid women . Since oid women , this stid women . MoreovePregnant women in Ethiopia are targeted during malaria prevention and control activities. Malaria preventive and curative interventions are also provided on the ANC platform . In the Plasmodium infections at delivery, despite treatment of women with anti-malarial drugs upon diagnosis. Since only symptomatic cases are screened for malaria and light microscopy is the standard of malaria diagnosis in health centres and hospitals in Ethiopia [In the current study, there was a significant association between a history of malaria during pregnancy and Ethiopia , this daEthiopia . SimilarEthiopia .Plasmodium infection among blood group O primiparae is consistent with studies in Gambia [Anopheles gambiae mosquitoes to blood group O [In this study, a significantly increased risk of placental malaria was observed among blood group O-women than the non-O phenotypes. In the subgroup analysis, blood group O primiparae had about a fivefold higher risk of placental malaria than the non-O blood groups, which was not observed among multigravid women. The higher risk of placental n Gambia , Sudan [n Gambia , and Maln Gambia . The susn Gambia , 47. Morn Gambia  and a pr group O  could bePlasmodium infections.Taken together, this study provided important information that was limited in Ethiopia and the first in the study area, from a relatively representative sample size, using different diagnostic methods , and considering diverse factors compared to previous studies in Ethiopia, such as the ABO blood group. However, the findings of the current study should be considered with their limitations. This study did not use histopathological techniques for the diagnosis of placental malaria which could underestimate the prevalence. The qPCR diagnosis of malaria was not done for all study participants. Moreover, it would be better if malaria was screened from newborn peripheral blood in addition to umbilical cord blood for screening of congenital Plasmodium infections, the majority of which were asymptomatic cases, were prevalent among parturients in northwest Ethiopia. Maternal illiteracy, lack of ANC follow-up, primigravidity, and malaria infection during pregnancy were significantly associated with malaria at the end of pregnancy. Moreover, blood group O was associated with a higher risk of placental malaria, particularly in primiparae. Thus, promotion of a healthy pregnancy through ANC follow-up, strengthening malaria prevention and control practices, and routine screening of malaria in asymptomatic pregnant women are suggested to reduce the burden of malaria in pregnancy.Overall, this study showed that