Plasmodium falciparum, scarce data exist regarding the association of P. vivax and HIV. In this cohort study, we assessed the effect of HIV on the risk of vivax malaria infection and recurrence during a 4-year follow-up period in an endemic area of the Brazilian Amazon. For the purpose of this study, we obtained clinical information from January 2012 to December 2016 from two databases. HIV screening data were acquired from the clinical information system at the tropical hospital Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD). The National Malaria Surveillance database  was utilized to identify malaria infections during a 4-year follow-up period after diagnosis of HIV. Both datasets were combined via data linkage. Between 2012 and 2016, a total of 42,121 people were screened for HIV, with 1569 testing positive (3.7%). Out of all the patients diagnosed with HIV, 198 had at least one episode of P. vivax malaria in the follow-up. In the HIV-negative group, 711 participants had at least one P. vivax malaria episode. When comparing both groups, HIV patients had a 6.48  (adjusted relative risk) greater chance of acquiring P. vivax malaria. Moreover, being of the male gender , Amerindian ethnicity , and a resident in a municipality of the Metropolitan region of Manaus  were independent risk factors associated with an increased risk of clinical malaria. Education ≥ 8 years  and living in the urban area  were associated to a lower risk of P. vivax malaria. A total of 28 (14.1%) and 180 (25.3%) recurrences  were reported in the HIV-positive and HIV-negative groups, respectively. After adjusting for sex and education, HIV-positive status was associated with a tendency towards protection from P. vivax malaria recurrences . HIV status was not associated with hospitalizations due to P. vivax malaria. CD4 + counts and viral load were not associated with recurrences of P. vivax malaria. No significant differences were found in the distribution of parasitemia between HIV-negative and HIV-positive P. vivax malaria patients. Our results suggest that HIV-positive status is a risk factor for vivax malaria infection, which represents an additional challenge that should be addressed during elimination efforts.Globally, malaria and human immunodeficiency virus (HIV) are both independently associated with a massive burden of disease and death. While their co-infection has been well studied for  This overlap favors Plasmodium-HIV co-infections, which are responsible for deaths of millions of individuals each year4. Although the consequences of co-infection with HIV-Plasmodium are not fully understood, the available evidence suggests that infections act synergistically and together may result in worse clinical outcomes5. Recent studies show that people living with HIV have more episodes of severe and lethal P. falciparum malaria, and more malaria treatment failures9. HIV can increase parasitic loads in malaria patients and consequently increase malaria transmission rates12. Moreover, P. falciparum infection may increase HIV plasma viral load and reduce TCD4 + cells14; this, in turn, suggests that malaria can lead to a faster progression from HIV infection to AIDS and increased risk of therapeutic failure after antimalarial chemotherapy15. However, the evidence on HIV-Plasmodium co-infection is mostly restricted to P. falciparum endemic areas in sub-Saharan Africa where the two diseases have the highest burden16.Malaria and HIV/AIDS are major public health problems and globally have a great geographical overlap, with most affected people living in sub-Saharan Africa, the Indian subcontinent and Southeast Asia, Latin America, and the CaribbeanP. falciparum malaria is unknown, and further research should be conducted in other settings to assess the interaction of HIV infection with other Plasmodium species, such as P. vivax17. Globally, ~ 229 million malaria cases were reported in 2019, with at least 6.9 million cases due to P. vivax (3%)18. Despite its comparatively lower numbers, P. vivax is geographically the most widely distributed cause of malaria, with its highest endemicity found in Southeast Asia, Central Asia, and Latin America, and a much less frequent presence in Africa19. P. vivax is a challenging parasite to eliminate due to its peculiar biology, which causes the different origins of a P. vivax malaria episode that include acquired infection by mosquito bites, relapse from liver-stage hypnozoites, and recrudescence from blood-stage treatment failure20. Clinically, P. vivax infection ranges from asymptomatic individuals carrying low parasitemia to severe disease and death21. Reports of HIV-P. vivax co-infection are scarce in the literature, with only a few studies describing their clinical outcomes27. In one study in the Brazilian Amazon, of 21 patients with HIV-P. vivax co-infection hospitalized in a referral hospital, severe malaria was diagnosed in 5 (23.8%), and one patient died (4.8%)27.The impact of HIV on susceptibility to non-falciparum malaria among HIV-infected patients, research on co-infection with the most widely distributed vivax malaria has so far been neglected. In this study, we evaluated the impact of HIV infection on P. vivax malaria incidence and the frequency of recurrences in a malaria endemic area of the Brazilian Amazon. The hypothesis of this study was that HIV infection may increase P. vivax malaria susceptibility and recurrence rates.Although over the past three decades, there has been clear evidence of an association between the increased risk of Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD) between January 2012 and December 2016. After removing duplicates for individuals tested more than once, 42,121 were included in the study, with 1569 being classified as HIV- positive (3.7%) and 40,552 as HIV-negative (96.3%)  and a younger mean age (P < 0.001). Regarding municipality of residence, HIV-positive status was less prevalent in Manaus compared to the municipalities of the metropolitan region (P = 0.046) ; P < 0.0001], male gender , Amerindian ethnicity , and residence in a municipality in the Metropolitan region of Manaus  were independently associated with an increased risk of P. vivax malaria. Education ≥ 8 years  and living in the urban area  were independently associated to a decreased risk of P. vivax malaria.Table P. vivax malaria . After adjusting for sex and education, the association between HIV-positive status with protection from recurrences was not detected   and 180 25.3%) recurrences were reported in the HIV-positive and HIV-negative groups, respectively. In the univariate analysis, HIV-positive status was associated with protection from recurrences of % recurreP. vivax malaria were documented in the HIV-positive group (2.7%) and 40 in the HIV-negative group (4.5%) . No deaths from malaria were reported.Six hospitalizations due to P. vivax malaria. Individuals carrying a viral load that was < 50 copies/mL at baseline presented a higher risk of malaria  and P. vivax malaria  compared to those with a viral load > 100,000 copies/mL, after adjusting by gender, ethnicity, municipality of residence, years of education and zone of residence   of the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado . The ERB gave a waiver for informed consent. After database linkages, the final dataset was anonymized before statistical analysis.P. vivax infections28. The national HIV prevalence is estimated at 0.4%, with a noticeably higher HIV incidence rate of 34.8/100,000 population in the Brazilian Amazon, compared to the overall national rate of 17.8/100,00029.Out of all malaria cases in Brazil, more than 99% occur in the Brazilian Amazon region, of which about 89% are attributable to FMT-HVD is a tertiary, referral healthcare center for infectious diseases in Manaus, state of Amazonas, western Brazilian Amazon and, in its outpatient clinic and emergency department, it receives all patients seeking care for infectious and parasitic diseases or those referred from other health units in the city of Manaus and the surrounding areas. HIV testing is provided after careful health screening for possible epidemiological risk and exposure. Diagnosis and treatment are free of charge. For both diseases, any positive HIV or malaria diagnoses are compulsorily recorded in structured forms available on-line as part of the Brazilian Ministry of Health's notification system. FMT-FVD is responsible for the follow-up and management of ~ 80% of the HIV/AIDS cases in the state of Amazonas.30, as reported on the FMT-HVD Information System (iDoctor).This is a concurrent, cohort study, based on surveillance data from patients tested for HIV at FMT-HVD and malaria diagnosis data and case recurrences recorded in the National Malaria Epidemiological Surveillance Information System  between January 2012 and December 2016. In this study, exposure was defined as a confirmed HIV-positive status, according to the national HIV infection diagnosis guidelineBio-Manguinhos, Fiocruz) with 100% sensitivity and 99.8% specificity31 was used in mobile testing campaigns and at the FMT-HVD outpatient clinics. For each positive test, blood samples were collected at the FMT-HVD laboratory, and the western blot technique was used to confirm the diagnosis, and the patient was then referred to the HIV ward for consultations, psychological support and ART therapy. All the remaining individuals with HIV-negative test results were included as non-exposed controls. HIV-positive patients were stratified by CD4 cell counts (/mm3) and viral load (copies/mL), via data gathered from the FMT-HVD SISCEL (Laboratory Test Control System) database. The predictor variables sex, age, education (in years of schooling), self-reported ethnicity, place of residence , and municipality of residence were also used. All HIV-positive patients were treated according to the Brazilian Ministry of Health Guidelines32.In short, a rapid diagnostic test (RDT)  P. falciparum infection, (iv) P. vivax malaria recurrences up to 90, 180 days and 4-years, (v) malaria-associated hospitalizations, (vi) malaria-associated deaths, in a 4-year follow-up, and (vii) parasitemia. Recurrence was defined as the occurrence of at least one additional malaria infection during the follow-up period. Furthermore, the study aimed to determine any association between age, sex or parasitemia as a potential additional risk factor for malaria reinfection. In Brazil, thick blood smears (TBS) are routinely performed for the diagnosis of malaria, and are prepared according to the Walker technique33 and then evaluated by a local microscopist. The results of peripheral parasitemia are given using the following semi-quantitative system: < 1/2 + (< 200 parasites/mm3); 1/2 + (200–300 parasites/mm3); 1 + (301–500 parasites/mm3); 2 + ; 3 + ; and 4 + . All positive slides and 10% of negative slides are routinely reviewed at a referral unit with experienced microscopists. In case of divergence, the reviewed result is updated on the on-line system. Only participants with a positive TBS for Plasmodium spp. are treated according to the Brazilian Anti-Malarial Treatment Guidelines34. The malaria patients' data are entered into the SIVEP Malaria  database. Data on malaria-associated hospitalizations and deaths were extracted from HIS  and MIS  databases, respectively.The present study was designed to determine the risk of (i) malaria infection, (ii) 35 and was installed on a Linux Workstation, Intel Core i7-8700, with 32 GB of RAM and 12 physical processing cores. In the absence of a unique identifier, Tucuxi-BLAST used the patient's name, date of birth and mother's name. Malaria episodes reported before the participants' entries were then excluded from the generated database. All remaining matches were double-checked and false matches were also excluded. This inspection included visual confirmation of homonyms, possible siblings (twins), and duplicity. Finally, the list of malaria cases obtained was linked to the HIS  and MIS  databases to investigate these outcomes. We obtained a final selection of pairs identified as likely to be from the same patients by automatic verification, applying a probability threshold (probability > 0.7) for all linkages. A final identification (ID) was created for all included participants.Duplicated HIV tests were excluded from the iDoctor database  and, in the case of individuals that had had more than one HIV test in the period, the first positive result was considered. Patients exposed to disease (HIV-positive) and the controls (HIV-negative) were included in the study according to the day of confirmation of HIV-status. Patients entered and left the study at different points in time. The HIV-status database and the SIVEP-malaria database were both deduplicated and further the record linkage was performed  of Tucuxi-BLAST software, which uses the DNA-encoded approach2) test were used to compare proportions, as appropriate. Crude relative risk (RR) with its respective 95% confidence interval (95% CI) was determined in a univariate analysis. Logistic regression was used for the multivariate analyses and the adjusted RR (ARR) with 95% CI were also estimated. A log binomial multivariate generalized linear regression was performed using an automated backward and forward stepwise estimation. All variables that were associated with dependent variables at a significance level of P < 0.200 in the univariate analysis were included in the multivariate analysis. Statistical significance was considered when P < 0.05 in the Hosmer–Lemeshow goodness-of-fit test. Kaplan–Meier survival estimates were used to compare recurrences over follow-up time. Additionally, a survival analysis for recurrent events was performed36. A two-tailed P < 0.05 was considered significant. The statistical analyses were carried out using R software (version 4.1.0), R Studio (version 1.4.17), and Stata v.13.0 .Descriptive statistics were used for demographic variables. Student's t test was used to compare means while Fisher's exact or the Chi-squared (XSupplementary Information.


I2 statistics and Egger’s test were used to test heterogeneity and publication bias. The random-effects model was used to estimate the pooled prevalence at a 95% confidence interval (CI). The review protocol has been registered in PROSPERO, with the number CRD42019144812. In total, 24 studies  were identified in this study. The pooled analysis revealed that 20% of pregnant women were co-infected by malaria and helminths in sub-Saharan Africa. The pooled prevalence of malaria and helminths were 33% and 35%, respectively. The most prevalent helminths were Hookworm (48%), Ascaris lumbricoides (37%), and Trichuris trichiura (15%). Significantly higher malaria and helminthic co-infection during pregnancy were observed. Health systems in sub-Saharan Africa must implement home-grown innovative solutions to underpin context-specific policies for the early initiation of effective intermittent preventive therapy.Malaria and helminthic co-infection during pregnancy causes fetomaternal haemorrhage and foetal growth retardation. This study determined the pooled burden of pregnancy malaria and helminthic co-infection in sub-Saharan Africa. CINAHL, EMBASE, Google Scholar, Scopus, PubMed, and Web of Science databases were used to retrieve data from the literature, without restricting language and publication year. The Joanna Briggs Institute’s critical appraisal tool for prevalence studies was used for quality assessment. STATA Version 14.0 was used to conduct the meta-analysis. The  Globally, approximately 1.5 billion cases of infection from malaria and helminths pose a significant risk of mortality and morbidity to the population at risk including pregnant women and the foetus . RecentlTen countries in SSA—Burkina Faso, Cameroon, The Democratic Republic of the Congo, Ghana, Mali, Mozambique, Niger, Nigeria, Uganda, and The United Republic of Tanzania—that were hard hit by malaria endorsed the “High Burden to High Impact Approach (HBHI)” ; this seHookworm (32%) [Ascaris lumbricoides (52%) [Trichuris trichiura (2.9%) [Schistosomiasis (13%) [The burden of helminthic infection during pregnancy in SSA ranges from 11% to 31% . The mosrm (32%) , Ascarises (52%) , Trichura (2.9%) , and Schis (13%) . Concurris (13%) ,18.The World Health Organisation (WHO) 2030 road map aims to establish an efficient helminths control program specifically for women of reproductive age. Nevertheless, helminths continue to constitute major public health problems for pregnant women in SSA ,20,21. CAscaris lumbricoides [Hookworm [Schistosoma mansoni is also associated with anemia and undernutrition during pregnancy [A. lumbricoides is implicated with gallbladder perforation [A. lumbricoides and Hookworm has been associated with increased odds of P. falciparum infection [Plasmodium species and helminths revealed negative pregnancy outcomes such as anemia, fetomaternal haemorrhage, antepartum stillbirth syndrome, and low birth weight [Plasmodium falciparum and helminthiasis elucidates the incidence of cervical cancer among pregnant women [A. lumbricoides has been associated with an increased odds of P. falciparum infection [Plasmodium parasitaemia [Malaria during pregnancy increases the risk of miscarriage and stillbirth by 3 to 4 times, compared with pregnant women with no clinically confirmed malaria . Helmintricoides ,33, and Hookworm . In addiregnancy , while Aforation . Pregnannfection ,37, and h weight ,39,40. Mh weight ,49,50,51nt women ,53,54. Mnfection ,56, and sitaemia ,58.Plasmodium species infection [Currently, evidence on the burden of intestinal helminths and malaria co-infection, the nature of their interaction, and their impact on pregnancy is not well established in endemic countries ,60,61. Mnfection ,68,69,70nfection ,72. Malaria in pregnancy: This is an adverse clinical condition developed by pregnant women after being infected by Plasmodium species, which increases the risk of anemia, stillbirth, spontaneous abortion, low birth weight, and neonatal death [al death . Infantsal death ,75,76,77Co-Infection: This is a clinical condition of particular human health importance caused by the simultaneous infection of a host (human being) by multiple pathogen species, for instance, multiple parasite infections [fections ,80,81,82Helminths: These are worms that infect the gastrointestinal tract of humans upon accidental ingestion of their infective eggs [ive eggs .The Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) statement guidelines were used to fully record and report the search results and the reasons for exclusion of studies  Figure  Supplem. The revA robust search was performed on CINAHL, EMBASE, Google Scholar, Scopus, PubMed, and Web of Science databases to retrieve published and unpublished data from the literature. . No restAll the identified citations were exported into the EndNote version 15.0 reference manager. Two independent reviewers (M.T.B. and E.H.) rigorously screened the titles, abstracts, and the full text of selected literature against the inclusion criteria. The double-check of the included studies was performed by a third reviewer (H.T.A.). Discussions were made among the reviewers to resolve disagreements that arose at each stage of the study selection process.Inclusion Criteria: Observational studies published in SSA, which reported the co-infection of malaria in pregnancy with helminths as their main outcome were eligible for inclusion. Studies published in all languages of SSA until 20 January 2022 were included.Exclusion Criteria: Systematic reviews, studies with poor methodological quality after, and reports of studies conducted outside SSA were excluded. Studies that employed inappropriate sampling frames, inadequate sample sizes, and poor data analysis were excluded. Studies that reported malaria or helminthic infection alone during pregnancy were also excluded.The Joanna Briggs Institute’s (JBI) standardised critical appraisal instrument for prevalence studies was used to assess the methodological quality of included studies . The JBIPlasmodium falciparum, Hookworm, Ascaris lumbricoides, Trichuris trichiura, Schistosomiasis, the burden of helminths, prevalence of malaria, and malaria–helminthic co-infections, respectively.Data extraction was principally carried out by two reviewers (M.T.B. and E.H.). The validity and eligibility of the extracted data for the meta-analysis were cross-checked by a third reviewer (H.T.A.). Variables such as the name of the corresponding author and publication year, study design and data collection period, sample size and study setting, the test approaches for the diagnosis of malaria, and helminths were extracted . In addiMalaria and helminthic co-infection during the gestation period were considered to occur when a laboratory-confirmed case of at least one Plasmodium and helminth species identified from blood and faecal bio-specimens was obtained from pregnant women .A quantitative meta-analysis of eligible studies was performed to estimate the event rate  . Based oA total of 1525 publications  were obtA total of 14,087 pregnant women from 24 eligible studies from SSA participated in this systematic review. Studies with the highest  and lowest (n = 87) sample sizes were reported from Uganda and Nigeria, respectively . Only siThe prevalence of malaria ranges from 4.6% to 36.2% . The lowHookworm infection ranged from 2% to 69%     . The pre% to 69% . The poo6%, 61%) . The lowectively . The poo0%, 44%) . The preto 21.4% . The poo5%, 45%) . Only si5%, 45%) . The lowest and the highest prevalence rates of comorbidity with malaria and helminths were 3% and 69% . The pooThis study estimated the pooled prevalence of co-infection of malaria and helminths during pregnancy from a total of 24 eligible studies and 14,087 pregnant women in SSA. The pooled prevalence of comorbidity of malaria and helminths among pregnant women in SSA was 20%, ranging from 9% in Ethiopia to 40% in Ghana. The burden of simultaneous infection from Plasmodium and helminthic species among pregnant mothers living in Uganda and Kenya was similar (16%). This could be attributed to the poor implementation of the intermittent preventive treatment of malaria during pregnancy, barriers to access to clean water, and inadequate sanitation in these three countries ,122,123.The burden of malaria in the gestational period among women immune-compromised by helminthic infection in SSA was 33%. This finding was higher than those of studies in Colombia (3.4%)  and EthiThis implies a concerted need to intensify malaria vaccine coverage in SSA to save the lives of pregnant mothers, in addition to having preventive, therapeutic, and control strategies in place to end malaria during pregnancy ,164,165.Ascaris lumbricoides (37%), and Trichuris trichiura (15%), respectively, were the pooled estimates of the most prevalent helminths associated with unintended pregnancy complications in SSA. The findings of our study were higher than those reported as global burden of helminthic infection during pregnancy in terms of the aggregate (3.6%) and species-specific Hookworm (19%), Ascaris lumbricoides (17%), and Trichuris trichiura (11%) [Schistosoma mansoni and malaria was determined by narrative synthesis because only 6 studies from the eligible 24 articles were reported with (n = 692) pregnant women from five countries in SSA who were co-infected by malaria and Schistosoma mansoni. Only five countries in SSA have (n = 1159) and (n = 1803) pregnant women in primigravid and multigravida who were co-infected by malaria and helminths. Our review revealed that the pooled prevalence of helminthiasis among pregnant mothers in SSA whose immunity is weakened by malaria was 35%. Uganda had a burden of helminthic infection in pregnancy (70%), which was higher than Cameron and Malawi combined (22%). Hookworm (48%), ra (11%) . This cora (11%) ,155,156.Although there are universal malaria interventions such as bed nets and access to prompt diagnosis and treatment for pregnant women in malaria-endemic settings, universal access to sanitation and hygiene should be implemented to prevent malaria and helminths co-infection in women of reproductive age and schoolchildren in endemic settings . MoreovePlasmodium parasitaemia and helminthiasis requires a concerted global health leadership and commitment [The global effort to end the preventable death of the mother caused by the comorbidity of mmitment ,161. Susmmitment ,166,167.mmitment ,176,177.P. falciparum parasites with histidine-rich protein 2 (pfhrp2)and histidine-rich protein 3 (pfhrp3) gene deletions that can escape the current HRP2 based-RDTs detection [Ensuring adequate access and enforcing adherence to safety and hygiene practices among pregnant women and safeguarding gestational mothers from economically disadvantaged households by creating sustainable access to economic opportunities will be essential to meet the global effort to control, prevent, and eliminate helminthic infections in sub-Saharan Africa ,185,186.etection , the estSignificantly higher levels of malaria and helminthic co-infection during pregnancy were observed. Existing interventions, such as deworming, prioritisation, and distribution of insecticide-treated bed nets and other control measures addressing pregnant women need to be highly encouraged. In addition, health systems strengthening gatekeepers and health policy framers in sub-Saharan Africa must implement home-grown, innovative solutions to underpin context-specific policies and practice for early initiation of effective intermittent preventive therapy for the prevention of malaria in pregnancy. Investments in reverse vaccinology to augment cutting-edge research and innovations in the comorbidity of gestational malaria and helminths through public–private partnerships must be implemented by sub-Saharan African countries and their international development partners. Tailored advocacy on focused antenatal care must be in place to inform and raise awareness among pregnant women regarding the health benefits of universal sanitation and hygiene coverage, together with the effective establishment of integrated community-level early detection and treatment of malaria and helminthic co-infection in sub-Saharan Africa.


Inflammation and infections such as malaria affect estimates of micronutrient status. Medline, Embase, Web of Science, Scopus and the Cochrane library were searched to identify studies reporting mean concentrations of ferritin, hepcidin, retinol or retinol binding protein in individuals with asymptomatic or clinical malaria and healthy controls. Study quality was assessed using the US National Institute of Health tool. Random effects meta-analyses were used to generate summary mean differences. In total, forty-four studies were included. Mean ferritin concentrations were elevated by: 28·2 µg/l  in children with asymptomatic malaria; 28·5 µg/l  in adults with asymptomatic malaria; and 366 µg/l  in children with clinical malaria compared with individuals without malaria infection. Mean hepcidin concentrations were elevated by 1·52 nmol/l  in children with asymptomatic malaria. Mean retinol concentrations were reduced by: 0·11 µmol/l  in children with asymptomatic malaria; 0·43 µmol/l  in children with clinical malaria and 0·73 µmol/l  in adults with clinical malaria. Most of these results were stable in sensitivity analyses. In children with clinical malaria and pregnant women, difference in ferritin concentrations were greater in areas with higher transmission intensity. We conclude that biomarkers of iron and vitamin A status should be statistically adjusted for malaria and the severity of infection. Several studies analysing asymptomatic infections reported elevated ferritin concentrations without noticeable elevation of inflammation markers, indicating a need to adjust for malaria status in addition to inflammation adjustments. However, prevalence of micronutrient deficiencies can be incorrectly estimated because certain micronutrient biomarkers are affected by inflammation and infections such as malaria. Inflammation is characterised by the acute-phase response to infection, injury or environmental insults. Some acute-phase proteins are also micronutrient markers; for example, serum ferritin, the primary iron storage protein, is a positive acute-phase protein – that is, its concentration increases in response to inflammation – and retinol binding protein (RBP) is a negative acute-phase protein – that is, its concentration decreases in response to inflammation,3. Whilst in the absence of inflammation, the concentration of plasma or serum ferritin is positively correlated with the size of the total body iron stores, during inflammation plasma/serum ferritin is raised and does not represent iron stores. Infants and young children, as well as women of reproductive age, are at high risk of micronutrient deficiencies due to increased physiological needs. They are also at considerably greater risk of contracting malaria, and developing severe disease, than other demographic groups. According to the WHO, there were 229 million cases of malaria in 2019. More than 90 % of these cases were located in the WHO African region. The presence of parasites can produce a chronic or mild acute-phase response. In settings of higher and more holoendemic malaria transmission, more individuals in a population, especially non-pregnant adults, will have some degree of immunity to malaria. Asymptomatic malaria, that is, the presence of parasitaemia in the absence of fever or other malaria-related symptoms, is very common in malaria endemic areas, with some prevalence rates exceeding 50 %. There are five well-established malaria parasite species that infect humans, namely Plasmodium falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum accounts for 99·7 % of infections in sub-Saharan Africa, while P. vivax accounts for 75 % of infections in the Americas. Currently used diagnostic methods include microscopy which visualises parasites in stained blood smears, rapid diagnostic tests that detect parasite antigen/s in blood samples;and PCR which identifies the presence of specific malaria genes in a blood sample.Micronutrient deficiencies are a major public health burden, especially in low-income countries, and accurate prevalence estimates are important to guide planning and monitoring of nutritional interventions. Ferritin values may differ by malaria infection status after correcting for inflammation defined by raised CRP and/or AGP, and the updated WHO guidelines mention malaria as a possible factor for adjustment. Other biomarkers are also likely to be affected by malaria. Retinol, the predominant circulating form of vitamin A in the blood, is known to be affected by malaria infection. In children aged 6–59 months in Ghana, increasing malaria parasite density was significantly associated with decreasing serum retinol concentrations. These reductions have been attributed largely to the inflammatory response. As the measurement of serum retinol requires expensive laboratory equipment, some micronutrient surveys measure its carrier protein, RBP, instead of retinol itself. In young children in Liberia, Larson et al. found a significant added effect of malaria on RBP concentrations and vitamin A deficiency prevalence estimates even after adjusting for CRP and AGP using the regression approach. There is also a growing interest in the impact of malaria on hepcidin, the iron regulatory hormone,14. Hepcidin seems to be upregulated in malaria infection even in asymptomatic human infection. This results in a blockage of iron absorption from the diet and a redistribution of iron into the body, away from the serum.Recently, the WHO published an updated guide on the use of ferritin to assess iron status and the recommended adjustments for inflammation, measured on the basis of C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP) concentrations in blood serum/plasmaTaking into account the effect of malaria on micronutrient biomarkers has the potential to significantly modify the estimation of the prevalence of micronutrient deficiencies derived from large population-based surveys such as national micronutrient surveys. This research estimated the effect of malaria on several biomarker values  by performing a meta-analysis of studies comparing biomarker values in individuals infected with malaria and individuals without malaria infection.The protocol of this systematic review has been published on PROSPERO on the 24 September 2021: CRD42021279974. Ethical approval was not needed as the data used in the analysis are fully available in the public domain. We followed the Meta-analysis of Observational Studies in Epidemiology (MOOSE) reporting checklist for this systematic review and meta-analysis.Randomised controlled trials or quasi-randomised controlled trials, prospective observational studies with data collection at multiple time points and cross-sectional studies with a control group that measured selected biomarkers in malaria-infected individuals were eligible. In children and non-pregnant adults, studies that distinguished asymptomatic and clinical malaria cases were included; studies in these populations that combined asymptomatic and symptomatic infections in a single malaria group were excluded. Studies that provided an intervention believed to impact the iron or vitamin A status of the participants were only included if data from a control group, or baseline data, could be extracted. Studies of human participants of any age and sex were eligible. As individuals suffering from severe malaria are often not sampled in large population-based surveys that measure micronutrient status, we decided not to include reports that only recruited participants with severe malaria. However, if individuals with severe malaria were included in papers that meet other selection criteria, they were analysed separately. For similar reasons, studies that recruited individuals based on their being anaemic or having another disease that is likely to affect iron or vitamin A metabolism  were not included.1. There was no restriction on the date of publication. Reports written in English, French and Spanish were eligible. Abstracts and unpublished studies were not considered. Two reviewers, FS and LSR, screened each record independently, in a two-stage process: first the reviewers examined titles and abstracts. The full texts were then retrieved and the reviewers examined the full-text reports for compliance with the eligibility criteria. After retrieval of articles from the search, the reference lists of all selected articles were checked for other potentially relevant articles; two additional papers were identified. Disagreements were discussed between the two reviewers until an agreement could be reached. References were managed in EndNote 20 . The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) flow chart of identified studies is illustrated in Medline, Embase, Web of Science, Scopus and the Cochrane library were searched in April 2021. The search strategy included the use of Medical Subject Heading (MeSH) terms and text words, with the use of explosion technique. The complete search strategy, which was reviewed by a qualified librarian, is included in supplementary file Plasmodium falciparum prevalence rate (PfPR) among children aged 2–10 years, as described in the Malaria Atlas Project. The different categories were defined by the WHO: PfPR < 1 %: very low intensity, PfPR ≥ 1 % and < 10 %: low intensity, PfPR ≥ 10 and < 35 %: moderate intensity, PfPR ≥ 35 %: high intensity. The data from three clinical groups were included: healthy participants with negative malaria test results, asymptomatic participants with positive malaria test and without clinical sign of illness, and a clinical malaria group who had a positive malaria test and fever. For prospective studies, the biomarker measurement at admission was considered the measurement of the malaria group, and the measure at the final follow-up point was considered the measurement of the control group. We analysed separately three population groups: children, non-pregnant adults and pregnant women. When a study provided data for different malaria species, or for different age groups, the corresponding data were entered into different datasets to allow for subgroup analysis, which explains why there is a greater number of datasets than studies. If the data from the same group were used for two comparisons in the same meta-analysis, we halved the number of participants from this group in each comparison, following the method described in the Cochrane Handbook. Authors were contacted if the relevant information was not available in the report. The risk of bias of all included studies was assessed by FS, using the US National Institute of Health quality assessment tool for observational cohort and cross-sectional studies. This tool contains fourteen questions around key concepts for evaluating the internal validity of a study. They are not intended to create a final score but help to assess potential selection, information, measurement and confounding biases. The use of this tool for cross-sectional studies was recommended in a recent review by Ma et al.. For the outcomes specified earlier, we reported the mean value of different groups , as well as the 95 % CI or the standard deviation. We calculated the mean difference with 95 % CI between groups. We also attempted to calculate missing information from other reported measurements, if possible. When the geometric mean was provided, we transformed it into an arithmetic mean using the method explained by Higgins. We generated meta-analyses based on the severity of the disease, either asymptomatic or clinical malaria and specific outcomes, such as ferritin or retinol concentrations. We first calculated a summary statistic for each study to describe the observed malaria effect. As our data were continuous, the summary statistic was a difference between means and a 95 % CI. When the data came from prospective studies, we followed the method described in the Cochrane Handbook to impute a standard deviation change from baseline to endline. We then used a random effects meta-analysis for combining data, as we anticipated that there may be natural heterogeneity between studies attributable to the different populations and settings. The study weights were equal to the inverse of the variance of each study’s effect estimate according to the methodology developed by DerSimonian and Laird. We generated forest plots and we provided a CI, which communicates the precision (or uncertainty) of the summary estimate and a P-value.Data were extracted into MS Excel by FS using a tool which included author name, study design, sample size, population group, age group, year when the study took place, country, malaria endemicity profile, method of diagnosis of malaria, clinical definition of malaria, malaria species, and the summary statistics of ferritin, hepcidin, retinol and RBP. Although Hb and soluble transferrin receptors are sometimes used to describe iron status, we did not include them in our analysis, as Hb is not a specific indicator of iron deficiency and soluble transferrin receptors concentrations are not reported as often as ferritin concentrations. The malaria endemicity profile was defined by the v. vivax), malaria endemicity profile of the country , method of diagnosis of malaria (rapid diagnostic tests v. microscopy or both), age of children (under v. over 5 years old) and design of the study . Heterogeneity was assessed using the I2 statistic. Both a qualitative (funnel plot) and a quantitative (Egger’s regression test) approach were used to examine potential publication biases. An influence analysis was conducted to determine the effect of removing each included study on the overall effect and 95 % CI using the technique described by Viechtbauer and Cheung. The meta-analyses, funnel plot, Egger’s regression test and influence analysis were conducted using RStudio software version 1.3.959 with the dmetar (v. 0.0.9000) and meta-packages. The workbooks for meta-analysis (version 1.5) developed by Suurmond were used to perform the subgroup analyses. A P-value of < 0·05 for meta-analyses was considered statistically significant.When data were available, and when more than one study provided relevant data for meta-analysis, we conducted the following subgroup analyses: species of malaria  and one in the Americas  was larger than for adults (N 985) (1).Of 101 full-text reports screened, 43 papers describing 44 studies conducted in 27 countries met selection criteria13,26–65 . Twenty-Americas . Among t (N 985) . The ris,60,63,64 (twenty-three datasets) analysed the association between malaria and ferritin concentrations in asymptomatic children . Overall, ferritin concentrations were 28·2 µg/l  greater in children with asymptomatic malaria compared with control groups (2 = 99 %). Heterogeneity was not explained by descriptive study factors (1), and all studies had a low or unclear risk of bias (2).Fifteen studiesl groups . The subl groups . There w factors . The sen of bias . The funP = 0·02)  and the sensitivity analysis showed the stability of the pooled result  . This me,54,61,64 (nine datasets) analysed the association between clinical malaria and ferritin concentrations in children . These studies were conducted in Africa, in Oceania and in the Americas. Overall, ferritin concentrations were 366 µg/l  P < 0·003) greater in children with clinical malaria compared with control group (5) and after removing the studies with high risk of bias (6).Seven studiesol group . The subol group . The sen of bias . The funIn terms of clinical malaria in adults, 269 adults were included in the analyses of 5 datasets, including an experimental infection and a study of imported cases in Europe. All the studies reported a higher ferritin concentration in the clinical malaria group . In the meta-analysis, the CI was wide and included zero .The elevation of ferritin concentrations in clinical malaria patients increased with greater severity of malaria in all studies that included this analysis, in adults and children (data not shown).,49,51,53 (twelve datasets) were used to analyse the association between asymptomatic malaria parasitaemia and hepcidin concentrations in children. The studies were mainly conducted in Africa apart from one study (four datasets) that was conducted in Indonesia. Hepcidin concentrations were 1·52 nmol/l  greater in malaria-infected groups compared with controls (2 = 96 %). The leave-one-out analysis and the sensibility analysis by risk of bias showed the stability of the pooled result .Seven studiescontrols . No intecontrols . The het,51 reported on asymptomatic malaria infection in adults and hepcidin concentrations, including 242 adults in total. There was a non-significant elevation in hepcidin in the malaria group of 0·3 nmol/l . Two studies reported concentrations of hepcidin in clinical malaria infection in children,51 and reported a non-significant elevation in hepcidin of 10·8 nmol/l  in the malaria group. In adults,51, there was also a non-significant elevation of hepcidin of 6·3 nmol/l  in the malaria group.Two studies–31,33 analysed ferritin concentrations in pregnant women with or without malaria infection, as defined by a positive parasitaemia. The authors did not systematically report the presence of clinical symptoms. Pregnant women with malaria parasites had greater ferritin concentrations than control pregnant women without parasites  (2 = 100 %). The sensitivity analysis showed the stability of the pooled result . After excluding the study with high risk of bias, the difference in ferritin concentration was lower  (Seven studies = 0·02) . The sub = 0·02) . Heterog = 0·01) .,34,38 (four datasets) analysed the association between malaria and retinol concentrations in asymptomatic children . All studies were conducted in Africa and the species involved was always P. falciparum. Overall, retinol concentrations were lower, that is, −0·11 µmol/l  in children with asymptomatic malaria compared with control group (2 = 60 %) and the sensitivity analysis showed the stability of the pooled result after the leave-one-out analysis, and after removing the studies with high risk of bias . The analysis showed that retinol concentrations were reduced during an infection  (Six studies = 0·01) . When th = 0·01) . There w = 0·01) .,43,44 (five datasets) analysed the association between clinical malaria and retinol concentrations in adults . Two of the studies were conducted in Asia, and they were both conducted in settings with very low intensity of transmission. One was conducted in Europe with imported cases, and the author did not specify the endemicity profile of the country of origin. Retinol concentrations were lower in adults with clinical malaria compared with healthy control adults, by 0·73 µmol/l  (2 = 54 %). The leave-one-out analysis showed the stability of pooled results . Only one study in this analysis was considered at low risk of bias (Three studies= 0·005) . Due to  of bias . reported data on retinol concentrations in pregnant women with malaria. They found a significantly reduced concentration of retinol in pregnant women with malaria .Only one study conducted in Burkina Faso with 262 children found that children with asymptomatic malaria had lower RBP values than the control group, and the mean difference was −0·13 . One study conducted in India with 100 children presented data on RBP concentration in children with clinical malaria and found that children with clinical malaria had lower RBP values than the control group and the mean difference was −1·52 .One study, and the sample size was too small to report the data (seven patients).Only one study reported RBP data in adults during clinical malariaWe conducted several meta-analyses to estimate associations between malaria infection and nutrition biomarkers by using data from cross-sectional and prospective studies. Although mostly based on data from observational studies, our analyses indicate that malaria infection is associated with increased ferritin and reduced retinol concentrations even in asymptomatic infections, when individuals might not have elevated markers of inflammation.,66,67. In our analyses, the increase in ferritin concentration during an asymptomatic infection was similar in children and adults, and there were no differences noted in the subgroup analyses. The quality of the evidence for children seems strong as the sample size for analysis is large, the CI is relatively small and the sensitivity analysis did not reveal any significant influencer in the results or publication bias. Moreover, studies included in this analysis were considered of good quality based on the risk of bias analysis. The increase in ferritin concentration did not vary by age group. For residents in malaria-endemic areas, parasitaemia peaks in children younger than 5 years old and subsequently declines in an age-dependent manner. In populations living under heavy exposure to malaria and frequent infection, individuals tend to develop partial immunity against the disease earlier in childhood, which may explain why children under 5 years old did not have a greater increase in ferritin than older children. However, this interpretation is limited by the fact that age groups were not particularly well defined and therefore, there might have been differences between age groups that we were not able to observe.The results provide strong and consistent evidence that malaria infection, asymptomatic or symptomatic, is associated with increased ferritin concentrations in children. This result was expected, as ferritin synthesis is highly upregulated by inflammatory cytokines and by infections including malaria; both markers are elevated in large proportions of children in low- and middle-income countries. This definition is imperfect, as inflammation is a complex process than cannot be captured simply by the elevation of these two acute-phase proteins. However, in the absence of other widely available biomarkers of inflammation in population-based surveys, we continue to rely on a definition on inflammation based on these two markers. Namaste reported that ferritin concentration increases from 19·5 µg/l in the reference group to 50·8 µg/l during the early convalescence phase of an inflammatory episode, which in this analysis was defined as when CRP and AGP concentrations are at their highest. This represents an increase of about 30 µg/l, which is in the same range as the increase we see during a malaria asymptomatic infection. For women of reproductive age, they report an increase in the same range (c. 30 µg/l).Overall, in the case of an asymptomatic malaria infection, the average increase in ferritin concentration is estimated at c. 28·5 µg/l, across all settings and all groups of population. Considering that the mean value of ferritin concentration in control groups was c. 25 µg/l, this indicates that the ferritin concentration in asymptomatic malaria infection was approximately doubled compared with a control group. As a comparison, based on sixteen datasets included as part of the BRINDA collaboration, Namaste and colleagues have assessed the increase in ferritin concentrations during an inflammation process, based on elevated CRP and/or AGP. Righetti found similar results in Cote d’Ivoire. Among children 6–8 years old with asymptomatic malaria, 55 % had neither CRP concentration > 5 mg/l nor AGP concentration > 1g/l. In non-pregnant women, this proportion was even higher (65 %). Similarly, de Mast found low circulating concentrations of CRP in Indonesian schoolchildren with asymptomatic parasitaemia, and 84 % of them had CRP concentrations < 5 mg/l, the threshold to define inflammation. These findings could be attributed to the use of thresholds for CRP and AGP and might mask a mild elevation in inflammatory markers. Several studies found that even after adjusting for raised CRP and/or AGP with the regression method, ferritin concentrations were higher in children suffering from asymptomatic malaria than in the control group,70. In children 6–23 months old in Burkina Faso, Wessells found that after adjusting for acute-phase proteins, children with asymptomatic malaria had greater plasma ferritin concentrations than the control group . Muriuki found that children with malaria had greater ferritin concentrations at every decile of CRP, compared with those without malaria. Even in the lowest decile of CRP, the difference in ferritin between children with malaria and without malaria was of about 20 µg/l. They found that malaria parasitaemia also increased ferritin levels independently of increased CRP and/or AGP in multivariable analyses. In longitudinal studies looking at ferritin concentrations and inflammatory markers concentrations after a malaria infection, it is notable that, even if CRP and AGP concentrations are slightly elevated during an asymptomatic infection, their concentrations go back to normal rapidly once the malaria infection is cleared, while ferritin concentrations remain elevated for about 1 month after the infection,49. Considering this, we can assume that individuals with an asymptomatic malaria infection are either not suffering from inflammation or have elevated CRP and AGP for a period of time that is shorter than the time needed for their ferritin concentration to return to normal.The recent WHO guidelines on the use of ferritin concentrations to define iron status recommend adjusting for inflammation and indicate that it is possible to adjust for malaria. There are, however, currently no details on why this adjustment should be made or whether some specificities, such as the severity of the infection, the population group or the endemic profile should be considered. An important question for micronutrient surveys is whether elevated acute-phase proteins fully capture the effects of malaria on micronutrient markers, as might be assumed from the similar differences in ferritin due to malaria or CRP plus AGP, or whether we should account for both inflammation and malaria. Several studies included in this review report that not all children with asymptomatic malaria have elevated CRP or AGP. In the study in Burkina Faso in children with asymptomatic malaria, Barffour reported that only half of the children with malaria also had elevated AGP during low malaria season, based on AGP concentrations >1 g/lThe highest increase in ferritin is unsurprisingly seen in clinical malaria, even though we could not reach a conclusion on clinical infection and ferritin in adults, due to the high heterogeneity and the small number of adults included. In children, the highest increase in ferritin was observed in the countries with a moderate parasite rate. In pregnancy, there was also a significant increase in ferritin concentrations in pregnant women with malaria, of c. 27 µg/l. The difference in mean concentration was greater in settings with high transmission intensity. and a non-inflammatory pathway, as suggested previously,53. Hepcidin reduces iron absorption from the gut and increases iron sequestration, resulting in a decrease of iron in the blood and a decrease in erythropoiesis. Considering the high proportion of the population suffering from asymptomatic malaria infections in sub-Saharan Africa, this increase in hepcidin concentration could help to explain why iron deficiency prevalence remains high in population surveys and why iron supplementation and iron fortification programmes have been less effective than expected.Hepcidin concentrations were also increased during malaria infections. As for the ferritin data, these datasets included children with and without raised CRP or AGP, and we can assume that the increase in hepcidin concentration might be occurring through both an inflammatory. However, the populations are different as BRINDA is more likely to include datasets coming from healthy participants while in these specific analyses, patients were ill and hospitalised due to malaria. Also, in these analyses, not all endemic profiles were represented. These results need to be interpreted with caution as the sample size for these analyses were small. The acute-phase response to either infection or inflammation affects retinol homoeostasis, and substantial vitamin A can be lost in the urine during illness accompanied by high fever. There may also be increased tissue retinol uptake due to increased needs for retinol in infection which could decrease plasma retinol concentrations. An alternative explanation is that sick children tend to eat less, and malaria could lead to vitamin A deficiency in children who already had low reserves of vitamin A.Children with an asymptomatic infection had lower values of serum retinol than the control group. In case of a clinical infection, the reduction was greater. In adults, lower values of serum retinol were also observed in case of a clinical infection. The reductions we observed in children and adults with a clinical infection (respectively 0·43 and 0·73 µmol/l) were greater than the reductions in retinol due to inflammation defined by elevated CRP and/or AGP, reported by the BRINDA collaboration. The BRINDA collaboration reported that inflammation reduces retinol by 0·27 µmol/l in preschool children and by 0·24 µmol/l in women of reproductive ageP. vivax, and it requires further research. In our analyses, the use of different malaria diagnostic methods did not seem to impact the magnitude of the effects.The magnitude of change in ferritin and retinol concentrations in the case of an asymptomatic infection is likely to affect the estimations of iron and vitamin A deficiency in population-based surveys. Asymptomatic individuals are often present in these surveys. They are likely to experience an increase in ferritin without a significant elevation of CRP and AGP values, and therefore their ferritin values would not be adjusted by the BRINDA method as it is recommended by WHO. With regard to retinol, even if BRINDA has recommended to adjust for inflammation in children, WHO does not currently recommend any adjustment. Children and adults with asymptomatic infection could be wrongly considered as iron replete or vitamin A-deficient, and the validity of the deficiency prevalence estimates could be affected. Adjusting for malaria is only referred to as a ‘possible adjustment’ in the WHO guidelines on ferritin, and there is no mention of asymptomatic infections. However, we observed that ferritin concentrations were elevated by 28·2 µg/l in asymptomatic children and by 366 µg/l in children with clinical malaria compared with healthy children. Retinol concentrations were reduced by 0·11 µmol/l in asymptomatic children and by 0·43 µmol/l in children with clinical malaria. These data suggest an important difference according to the severity of infection, and this could have important repercussions in the assessment of iron and vitamin A status in populations where different forms of malaria infections co-exist. Even if individuals with clinical malaria are not likely to be included in surveys, people recovering from clinical malaria might be. Applying a single correction factor to all forms of malaria, as is currently recommended in the WHO guidelines, would certainly affect estimates coming from micronutrient surveys and other surveys that assess the iron status of a sample of a population in a malaria endemic setting. More research should be done to confirm whether the study setting should be considered when applying an adjustment, as our data seem to indicate that a clinical infection could have different repercussions on ferritin concentrations depending on the endemic profile. We did not have enough datasets to analyse infections with The primary limitation to these analyses is the variability between studies, including the large age range among children in some of the datasets. Another limitation is the population used as a control group, as most studies had different strategies to recruit their control group. We did not investigate whether the children were suffering from hookworm or Schistosoma infections, which could have affected further their iron status. Another limitation is the generalisability of the results related to clinical infections. Most of the studies including clinical infections were conducted at the hospital, whereas in population-based surveys conducted to measure the micronutrient status of a population, participants would be sampled in the community. We also acknowledge that our search strategy, despite being large and inclusive of three languages of publication, did not include grey literature or regional databases, which might have introduced a selection bias in the systematic review. Finally, although there were many studies on the association of ferritin and malaria in children, fewer studies were included in other meta-analyses, and these included studies had higher risk of bias, suggesting caution is required in interpreting these results.The findings of this systematic review and meta-analysis suggest that malaria infection should be measured and adjusted for in nutritional surveys of populations living in malaria endemic areas, particularly for assessments of iron status. Malaria test results should be reported in population-based surveys, as well as a measure of clinical symptoms in the participant. This will allow more accurate adjustment of serum ferritin concentrations to define individual iron status. Preliminary analyses indicate that retinol concentration also is affected by malaria, but not enough data are currently available to support firm conclusions for children and adults. Further research is needed to develop individualised adjustment methods that can take into account the concentrations of acute-phase proteins and the presence, and severity, of a malaria infection.


P. vivax. However, in Malaria patients and co-infected there was a positive correlation between the IL-6 and IL-10 levels (P < 0.0001). This is the first report of sTREM-1 levels in P. vivax infected. Moreover, the results revealing a divergent effect of co-infection with the increased balance between pro-and anti-inflammatory cytokines and reduced IL-6 levels but increases the anemia occurrence. The results also highlight the potential use of IL-6 as a biomarker for P. vivax and enteroparasites coinfection.Malaria is a major health issue with more than 200 million cases occurring annually. Moreover, in Malaria endemic area are frequently observed Malaria-enteroparasite co-infections associated with the modulation of inflammatory response. In this aspect, biomarkers play an important role in the disease prognosis. This study aimed to evaluate inflammatory mediators in malaria during coinfection with enteroparasites. A subset of serum samples already collected was analyzed and divided into four groups: Malaria (n = 34), Co-infected (n = 116), Enteroparasite (n = 120) and Control (n = 95). The serum levels of sTREM-1 and IL-6 were measured by ELISA. TNF-α, and IL-10 levels were previously carried out by flow cytometry. Higher serum levels of sTREM-1 and IL-6 were showed in malaria patients compared to healthy controls. In co-infected malarial patients sTREM-1 serum levels were similar to control group. Interestingly, co-infected malaria patients showed IL-6 serum levels decreased compared to individuals only infected with  Plasmodium [P. vivax is the species with greatest epidemiological interest as it is the most widely distributed species in regions outside of the African continent. P. vivax is responsible for high morbidity caused by the infection, and brings social and economic losses to the countries where this disease is present [Malaria is a major parasitic disease with high morbidity and mortality worldwide, with 228 million cases occurring only in the year 2018 [ present , 4. Case present –8.Plasmodium vivax, may alter the immunopathological course. A previous study conducted by our group [Plasmodium-enteroparasite in the municipality of Oiapoque, located in a border area between Brazil and French Guiana. The results showed an increase in the levels of the cytokines TNF-α, and IL-10 in individuals co-infected with P. vivax and enteroparasitoses. However, this data raises new questions about the impact of other inflammatory immunological markers since it was not possible to verify the influence of co-infection on the cytokines profile that characterizes the Th1 and Th2 pattern.In 2018 more than 194,000 cases of the disease occurred in Brazil, of which more than 99% are from the Amazon region –12 whichur group  analyzedP. falciparum [Receptors present on the surface of innate immune cells can recognize components of microorganisms and initiate the inflammatory response. Among these, the Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) is a receptor expressed in macrophages, monocytes, and neutrophils that is known to increase inflammatory responses of myeloid cells in innate and adaptive responses that were initiated by the Toll-like receptors , 15. Thelciparum –19.P. vivax in Brazil’s extra-amazon [In addition to sTREM-1, several cytokines have been described that play a role in controlling infection and symptom development . Betweena-amazon  and Amaza-amazon  however This study therefore aims to evaluate the levels of inflammatory mediators in malarial patients and to compare with individuals co-infected with enteroparasites residents of the municipality of Oiapoque, Amapá state, North Brazil.A subset of samples already collected was analyzed out of individuals previously evaluated by Menezes et al. . The samP. vivax who were negative for intestinal parasites (n = 34); (2) Co-infected: individuals with malaria and intestinal parasite co-infection (n = 116); (3) Enteroparasite: individuals without malaria and positive for an intestinal parasite (n = 120); (4) Control: individuals who were negative for malaria and intestinal parasite (n = 95). Blood collection was performed during diagnosis for malaria so none of the participants were using anti-malarial drugs to the time of blood collection. Ten millilitres of venous blood was collected from each patient by venipuncture. Four millilitres was dispensed into a tube with EDTA to perform haematological analysis and to prepare thick blood smear slides, and six millilitres was dispensed into a tube with no anticoagulant for immunological analysis. The serum samples were stored at -20°C in the Center for Microorganisms’ Investigation of the Fluminense Federal University until use. The diagnosis and pro and anti-inflammatory cytokine profiles was conducted according to the methodology described below [Participants were divided into four groups: (1) Malaria: individuals with malaria by ed below .P. vivax and enteroparasite infection.Estimation of the hematological and parasitological parameters against The diagnosis for malaria, the quantification of parasitemia, the quantification of gametocytes, and hemoglobin dosage have been previously performed, as well as the enteroparasites diagnosis . The diaIn addition, hemoglobin dosage was performed according to the protocol recommended by the World Health Organization through hematocrit index. The hematological parameters evaluated were the total number of erythrocytes  and hemoglobin levels . Individuals were considered anemic when their haemoglobin blood levels were 13 g/dL for males, 12 g/dL for females, and 11 g/dL for the children.Cytokine levels were detected by flow cytometry according to previously published guidelines in the plasma samples . QuantifIn addition to the existing cytokine panel, IL-6 and sTREM-1 levels were also dosed. The levels of sTREM-1 in the serum were measured by using Human TREM-1 DuoSet ELISA kit  and IL-6 levels were measured by IL-6 Human Uncoated ELISA Kit . The absorbance was measured using a microplate reader Epoch  at 450 nm and a wavelength correction at 570 nm to subtract the background. A standard curve was generated for each plate using the manufacturer’s recommended protocol.Differences between the two groups were calculated using the Mann–Whitney U-test, or the Kruskal-Wallis test with the Dunn showing mean and standard error. Differences with p < 0.05 were considered statistically significant. Analyses were performed using Prism 8.4.3 software (GraphPad).The samples included in this study were collected with the approval of the Research Ethics Committee of the Federal University of Amapá. The participants who agreed to participate signed a written informed consent form. For minor participants, their parents or guardians signed the consent forms.This study was also approved by the Ethics Committee of the Fluminense Federal University (CAAE number 40790120.7.0000.0003) to reuse the samples and waiving the consent of the participants and parents or guardians of the minors for this new analysis.Serum levels of sTREM-1 and IL-6 were measured in patients with malaria and controls. Considering the high prevalence of enteroparasitosis circulating in the region and that this infection can alter the immune course of malaria this was also measured the levels in two other comparison groups: co-infected and enteroparasites. Results showed a higher level of sTREM-1 and IL-6 in individuals with malaria compared with individuals without the infection (P < 0.05). The level of sTREM-1 was similar between the groups, although a decreasing trend was seen for the co-infected group . InteresGiven the importance of the pro-and anti-inflammatory balance during malaria, a comparison was performed between the ratio of the main pro-inflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines identified in the malaria and coinfected groups. In this analysis, the coinfected group had a higher level of IL-10 than the inflammatory cytokines, compared to the profile seen in the malaria group .Considering previous findings that these samples showed an increase in anemia in the coinfected group , an evalP. vivax and has identified significantly increased levels in infected individuals (P < 0.05).The inflammatory response generated during malaria is essential for parasite clearance however enhanced inflammation can trigger immunopathological damage to the host , 25. ThiP. falciparum identified sTREM-1 as a new biomarker of severe and fatal malaria in children with this malaria profile [The sTREM-1, the soluble portion of TREM-1, has been extensively investigated as a potential biomarker of severity , 26–28. P. vivax which is the most widely distributed species of Plasmodium worldwide [Our study is thus the first to include only non-severe patients infected by orldwide , 3 and oorldwide , 20, 26.Furthermore, experimental studies of sTREM-1 induction with LPS in animal models demonstrated that when low-dose LPS (2 ng/kg) was used, the increase in sTREM-1 was transient and decreased progressively within the first 24 hours . If the P. falciparum infection was observed to be associated with increased clinical episodes [P. vivax, since no variations were observed in the production of anti-PvMSP-119 IgG antibodies in our previous study with these same samples [Our results also demonstrated that IL-6 serum levels increased 6.9 times in malaria patients compared to healthy individuals. In addition, IL-6 was associated positively with IL-10, which is compatible with the mild forms in the patients included in this study. Athough, higher IL-6 production during episodes , the IL- samples .P. vivax infection. Interestingly, the difference observed between malaria and the coinfected group seems to indicate a modulation of inflammation in individuals coinfected with enteroparasites. As expected, the IL-10/IL-6 ratio was lower in malaria group when compared to the coinfected, showing that in the coinfected the response was leaning more towards a suppressive profile. This may indicate a potential protective effect of co-infection on malaria by P. vivax. In malaria by P. falciparum, this protective effect has already been seen in acute renal failure and jaundice [Ascaris lumbricoides was protective against cerebral malaria [P. falciparum and soil borne helminth, coinfection led to increased IL-10 and IL-6 levels and a down-regulation of TGF-β [P. vivax, the coinfected had increased IL-10/IL-6 and IL-10/TNF-alpha ratios and decreased IL-6 levels when compared to patients who were infected with malaria infection only.In contrast to what was found for sTREM, IL-6 levels were significanty lower in the coinfected group compared to the malaria group. The malaria group showed higher values of this cytokine compared with all other groups, demonstrating that IL-6 levels are highly stimulated during jaundice  and co-i malaria . A studyof TGF-β . In our However, another possible scenario must be considered. In the acute phase of infection, a predominantly pro-inflammatory response is important for parasitemia control and parasite clearance . Given tAdditionally, increased anemia was seen in the coinfected compared to the malaria group  which maPlasmodium replication that leads to red blood cells lysis.It is worth of noting that despite the decrease in IL-6, this coinfected group showed higher values of TNF-α than malaria . TNF-α iAs expected, in malaria patients, parasitemia was positively correlated with the anti and pro-inflammatory cytokines IL-10 and TNF-α. In a study conducted in a municipality in Amazonas, Brazil increased IL-10 and IL-6 were associated with parasite load . In our We also demonstrated a potential modulation of IL-6 during coinfection with intestinal parasites. It is important to mention that most malaria-endemic regions have a high prevalence of enteroparasites , 32, 39.TREM-1 gene on the susceptibility, inflammatory role and prognosis of malaria and other diseases [TREM-1 in populations of endemic areas and their impact on the course of malaria by P. vivax. Finally, is the coinfection with enteroparasites beneficial during P. vivax infection in non-severe cases, and is the anemia developed during coinfection caused by immune damage or due to parasite persistence? are two key questions that need further clarification.Therefore, studies are needed to determine the impact of these immune molecules on each of these groups, especially sTREM-1 which has been scarcely studied in these populations. Moreover, studies have demonstrated the impact of polymorphisms in the diseases , 40–44. S1 Data(XLSX)Click here for additional data file.


In areas where malaria has been eliminated, delayed care-seeking and diagnosis of imported malaria are constant threats. This study aimed to describe the profile and determinants of delayed care-seeking and diagnosis among patients with imported malaria in China.This retrospective study assessed surveillance data obtained from 2014 to 2021 in the Chinese provincial-level administrative divisions (PLADs) of Anhui, Henan, Hubei, and Zhejiang, and Guangxi. Epidemiological characteristics were analyzed using descriptive statistics. Furthermore, factors associated with delayed care-seeking and diagnosis among imported malaria cases were identified using multivariate logistic regression.χ2 = 36.099, P < 0.001) and diagnosis . In multivariate analysis, independent risk factors associated with delayed care-seeking include PLADs (Guangxi as reference), consultations in high-level facilities for the first medical visit, infections with non-Plasmodium falciparum species, and older age. However, PLADs (Guangxi as reference), the purpose of traveling (labour as reference), and infections with non-P. falciparum species increased the risk of delayed diagnosis. Delayed care-seeking  and diagnosis  were risk factors for severe disease development.Overall, 11.81% and 30.08% of imported malaria cases had delays in seeking care and diagnosis, respectively. During the study period, there was a decreasing trend in the proportion of imported malaria cases with delayed care-seeking (P. falciparum species should be highlighted, and more sensitive and specific point-of-care detection methods for non-P. falciparum species should be developed and implemented. In addition, education programs should be enhanced to reach target populations at risk of malaria infection. All these factors may reduce delayed care-seeking and diagnosis of imported malaria.Based on this study’s findings, we strongly advocate for improved access to quality healthcare to reduce the rate of misdiagnosis at the first visit. Infections caused by non-The online version contains supplementary material available at 10.1186/s40249-022-01050-3. Remarkable progress has been made in malaria elimination; however, malaria remains a serious public health problem worldwide, with populations in 85 endemic countries at risk . The numIn China, the National Malaria Elimination Action Plan was launched in 2010 to eliminate malaria nationally by 2020 . SignifiChina has been under tremendous pressure from ‘imported’ malaria, that is, the infections acquired outside the country over the years . SimilarThis retrospective study used surveillance data from 2014 to 2021 in five Chinese provincial-level administrative divisions (PLADs) to describe profiles of delays and to identify predictors of delayed care-seeking (DC) and delayed diagnosis (DD) of imported malaria cases. These findings might contribute to developing innovative interventions for the prevention of re-establishment of malaria, which may improve early healthcare-seeking behavior, prompt diagnosis, and treatment of imported malaria cases.The provinces of Anhui, Henan, Hubei, and Zhejiang, and Guangxi Zhuang Autonomous Region, China, were selected for this study due to their burden of imported cases and risk of re-establishment  where it was diagnosed . Severe According to WHO guidelines, malaria should be promptly diagnosed and treated within 24 h of symptom onset to reduce the risk of severe disease and transmission . In pracThis study included imported malaria cases reported in five PLADs from 2014 to 2021. Indigenous malaria cases were excluded. This study included 5765 imported malaria cases.This retrospective study evaluated surveillance data from January 2014 to December 2021 in five Chinese PLADs to identify the factors contributing to DC and DD. Malaria is a nationally notifiable disease in China, and each case must be reported to the Centers for Disease Control and Prevention through two reporting systems: the China Information System for Disease Control and Prevention (CISDCP) and the Information System for Parasitic Disease Control and Prevention (ISPDCP), a subsystem of the CISDCP. Moreover, staff from county-level Centers for Disease Control and Prevention must complete an epidemiological report to identify the origin of the infection and record other valuable information. Demographic, epidemiological, and clinical data of patients with malaria, including age, sex, occupation, travel history, date of symptom onset, diagnosis, treatment, symptoms, complications, and prognosis, were extracted from the CISDCP, ISPDCP, and epidemiological survey reports.χ2 or Fisher’s exact test, as appropriate. The Bonferroni-corrected post-hoc test was conducted to adjust the observed significant level for multiple comparisons. The chi-square test for trend was used to describe the statistical significance of the differences between years. Factors associated with DC and DD were determined using hierarchical logistic regression analysis. The association between delays and exposure variables was estimated using odds ratios (ORs) and 95% confidence intervals (CIs). In the first layer, only one variable (PLAD) was included. In the second layer, variables considered significant (P < 0.1) in the univariate analysis were included in the multivariable model, and non-significant variables were removed using a forward stepwise approach. Only significant variables (P < 0.05) were included in the final model. Forest plots were used to display the results of multivariate logistic regression. Imputation for missing data was addressed using the dummy variable method. Statistical significance was set at P < 0.05. ROC analysis  was used to determine the optimal cut-off value for this study. All statistical tests were performed using SPSS version 26.0 . ROC curve analysis was performed using MedCalc statistical software version 20.115 .Normally distributed quantitative variables were presented as means and standard deviations, whereas non-normally distributed variables were reported as medians and interquartile ranges (IQR). Categorical variables were expressed as percentages. Differences in proportions were assessed using Pearson’s chi-squared Plasmodium falciparum , followed by P. ovale , P. vivax , P. malariae , and P. knowlesi . There were 82 mixed-species infections (1.42%). During the study period, 297 patients (5.15%) developed severe malaria, leading to 20 deaths , with a fatality rate of 0.35%. Table This study included 5765 imported malaria cases , and the annual number of cases from 2014 to 2021 ranged from 139 to 1035. The number of infections peaked in 2016 at 1035 (17.95%). Only 139 cases were reported in 2021 because of travel restrictions due to the COVID-19 pandemic. The mean age of all patients was 41.11 ± 9.92 years, and 5532 (95.96%) patients were males. Migrant laborers comprised 81.53% (4700/5765) of the patients. Most infections  originated in Africa, especially Ghana , Nigeria , Cameroon , Angola , and the Democratic Republic of the Congo . The predominant species was At the first consultation, the number of patients who received care in PLAD, city, county, township, and private/village health centers was 627 (10.88%), 1314 (22.79%), 2724 (47.25%), 278 (4.82%), and 697 (12.09%), respectively. One hundred and twenty-five patients (2.17%) sought other health services . In addition, 1478 patients (25.64%) were misdiagnosed during their first visit. Misdiagnosis rates in private/village and township health centers were 91.68% (639/697) and 49.64% (138/278), respectively  and DD  decreased . Among 4795 cases with information on the date of arrival in China, 3648 (76.08%) experienced symptoms within 30 d. The median interval between symptom onset and the first visit was 1 d (IQR: 0–3 d), and 5084 (88.19%) patients sought medical care within 3 d. The median time between the first visit and diagnosis was 1 d (IQR: 0–2 d), and 4031 (69.92%) cases were correctly diagnosed within 1 d of the first visit. The median time between symptom onset and diagnosis was 2 d (IQR: 1–5 d), and 4228 cases were diagnosed within 4 d after symptom onset. During the study period, the proportions of DC  and 30.08% (1734 cases) of the patients, respectively. Univariate analysis was performed to explore the potential contributors to these delays. The factors associated with DC included age, travel region, malaria species, level of healthcare facilities for the initial medical visit, and time between arrival in China and symptom onset. The purpose of travel, travel region, malaria species, level of healthcare facilities for the initial medical visit, diagnostic result in the first visit, and history of malaria infection were statistically associated with DD , the purpose of travel , and infections with non-P. falciparum species increased the risk of DD. In contrast, consultations in high-level health facilities for the initial medical visit, correct diagnosis at the first visit, and history of infection were conducive to timely diagnosis. Figures Considering the differences between PLADs, multivariate hierarchical logistic regression was used to identify the factors influencing DC and DD. The results indicated that the independent risk factors associated with DC were PLADs , older age, infections with non-ORs for the DC, DD, and overall delay associated with severe malaria were 1.59, 3.44, and 2.68, respectively. After adjusting for covariates, DC , DD , and overall delay  increased the risk of severe malaria  and DD  significantly contributed to severe disease development. This is consistent with previous findings that a 4–12 d delay in diagnosis increases the risk of developing severe malaria [In China, local malaria transmission was interrupted since 2017 , and pre malaria . TherefoEarly healthcare-seeking behavior, prompt diagnosis, and treatment were consistent pursuits; however, there were differences in managing imported malaria across the PLADs studied. Guangxi had the best performance in terms of care seeking and diagnosis among imported malaria cases, followed by Zhejiang. Subsequently, Anhui, Hubei, and Henan came close in performance. One possible explanation is the epidemiological profile differences between the PLADs. For example, 85.43% of malaria cases were reported in Nanning City , with high spatial aggregation . In 2013P. falciparum species were risk factors for DC and DD. During the research, 80.92% (4665/5765) of patients sought care in county health facilities or higher at the first medical visit, in line with previous studies [In this study, multivariate logistic regression models were used to identify factors influencing DC and DD to guide prevention of re-establishment of malaria practice better. Obtaining care in higher-level health facilities on the first medical visit and non- studies , 28. The studies , 30, whe studies , 31. ThiP. falciparum species contributed to DC and DD. The following are possible explanations. First, these infections are more benign than P. falciparum infections, which can progress rapidly to severe forms of the disease [P. falciparum spp. [P. falciparum species, can develop 1 to 2 years after infection [P. falciparum infections difficult. Non-P. falciparum infections can cause severe malaria and, occasionally, even death [P. vivax, whose vector is widespread in China [P. vivax cases increases the risk of local transmission [P. vivax emerge at an early stage of infection, before onset of illness [P. falciparum infections were reported [P. falciparum species; therefore, more sensitive and specific point-of-care detection methods for non-P. falciparum species must be developed and implemented.In contrast, infections with non- disease . Second,rum spp. . Third, nfection –36. All en death , 38. Anoin China . The DD  illness . Additioreported . The aboPrompt diagnosis and treatment of malaria are fundamental to adequate disease management. This study revealed that a correct diagnosis at the first visit was beneficial for timely diagnosis. This underlines the importance of the first diagnosis responsibility, requiring an improvement in clinicians’ awareness of malaria epidemiology, diagnosis, and treatment. A history of infection was another protective factor against DD. This was because patients previously infected with malaria were more likely to report a travel history, possibly due to an increased awareness of symptoms. In contrast, patients without a previous infection tend to self-treat their symptoms . InteresThe retrospective nature of this study may have led to recall bias. In addition, healthcare-seeking behavior among patients and malaria prevention and control programs may have affected measures during the COVID-19 pandemic. Finally, the practice of prevention of re-establishment of malaria in border regions is more complex. The findings of this study may not be directly applicable to these regions.P. falciparum species should be highlighted, and more sensitive and specific point-of-care detection methods for non-P. falciparum species must be developed and implemented. In addition, educational programs should be enhanced to reach target populations at risk of malaria infection. All these factors may improve DC and DD among imported malaria cases.Based on this study’s findings, we strongly recommend improving access to quality healthcare to reduce misdiagnosis rate during the first visit. Infections caused by non-Additional file 1: Appendix S1. Location of the study area in China.Additional file 2: Appendix S2. The ROC analysis between severe malaria and the time between symptom onset and diagnosis.Additional file 3: Appendix S3. Diagnostic results of imported malaria patients at their first medical visits by healthcare facility levels.Additional file 4: Appendix S4. Diagnostic accuracy at the first medical visit among imported malaria cases in China from 2014 to 2021.Additional file 5: Appendix S5. Delayed care-seeking and diagnosis among imported malaria in China, 2014–2021.


Malaria is an infection caused by the Plasmodium malaria (PM) parasite. There are still cases of malaria that are reported in the United States on an annual basis. All these cases were a result of travelers who did not receive or follow their prescribed chemoprophylaxis, recommendations for avoiding mosquito bites while traveling, or relapsed dormant plasmodium. The malaria parasite can be transmitted by the bite of an infected female mosquito, through contact with infected blood products, or from mother to child during pregnancy through the placenta. It can take anywhere from 12 to 20 days for symptoms to appear, but there are cases of delayed development and/or relapse that can occur up to 13 years after the infection. We report a 31-year-old female with a history of malarial infection in Liberia, which had been treated ten years prior to her arrival in the United States. She presented to the hospital with abdominal pain, fever, and headache. She was eventually diagnosed with plasmodium malaria infection relapse and treated with a 14-day course of primaquine 300 mg daily, with the symptoms resolving a few days after. We believe her malarial infection was caused by a dormant malarial parasite that evaded the immune system and relapsed without having a risk factor for relapse or re-infection 10 years after her original infection. Plasmodium malaria (PM) is a parasite that has been recognized as the cause of malaria since around 2000 years ago. It has previously been observed that there is a relationship between the parasite's 72-hour lifecycle and the development of paroxysms of chills and fever when compared to the parasite's 48-hour lifecycle, which later led to the separation of findings between different parasites of malaria . More thA 31-year-old African American woman with a history of latent tuberculosis treated with three-month therapy and a malaria infection presented to our hospital in August 2021 with complaints of abdominal pain, headache, and fever. Her fever began three to four days prior to her presentation and was associated with two episodes of nausea and vomiting at home.She lived at home with her husband and two children, ages five and seven years old, all in good health. She worked as a caretaker and claimed to have no other sick people in her life. She did not have any pets at home and did not interact with any other animals. She had not been camping, hiking, or participating in any other outdoor activities. She denied visiting other states or leaving the country since her arrival in 2014 from Liberia, where she has not returned since. She had malaria in Liberia about ten years ago and was treated with medications that she does not remember.9/L , hyperbilirubinemia with indirect bilirubin at 2 mg/dl , hemoglobin of 9.4 g/dL , MCV was 85 fl , and platelet count was 43×109/L . A blood smear revealed intraerythrocytic parasitic elements as in  count was 3.2× 10P. falciparum can survive in humans for long periods of time causing chronic infection. The longest reported is 13 years ,12. An iImmigrants and refugees who have left malaria-endemic areas were observed to have persistent falciparum infections and a sub-microscopic parasite prevalence. One of the studies showed that 195 African migrants living in Italy in 2007 had a parasite prevalence by the findings of 62 individuals with malaria who tested positive utilizing molecular detection methods, 13 of whom were gametocytic; 14 of the 62 cases were verified to have microscopic findings, 13 of which were P. falciparum [For blood products, the presence of subclinical parasitemia in migrants from areas where parasites are prevalent is an issue. Blood transfusion-transmitted Plasmodium falciparum malaria from migrants who have been away from their country for years has been documented ,15.It is critical to distinguish between relapse and reinfection to establish the best treatment strategy. This is because antimalarial medicine resistance is determined by factors such as location, type, and prior treatments. Although this patient had previously been treated for malaria, the treatment and duration were unknown. She has not been in a situation that would put her at substantial risk of a new malarial infection, such as an organ transplant, blood transfusion, recent travel history, or living near an airport. Furthermore, she did not have any of the factors that typically lead to relapse, such as being pregnant, taking drugs, undergoing an organ transplant, or having a history of splenectomy, by altering her innate immunity -9. Also,The importance of reporting this case is to have a high index of suspicion when treating patients with prior history of malaria infection. Being outside of the infectious zone for a long period of time does not rule out the possibility of malarial infection, and the index of suspicion should remain high. In our patient's case, her history of malaria led to the acquisition of a peripheral smear, and rapid PCR testing confirmed her diagnosis. Furthermore, it was reported that relapses can occur in a condition that alters the host immune system, however, our patient did not have any of those conditions and she was a healthy young female.