Methodology. This was a cross-sectional study conducted among 413 primary school children from February to April 2020. Finger-prick blood samples were used to determine asymptomatic malaria and hemoglobin concentrations. Stool samples were collected and processed through formalin-ether concentration techniques to detect the presence of intestinal helminths. Data were double entered into Epi Data version 3.1 software and exported to SPSS version 20 for analysis. Pearson's chi-square and correlation analysis were performed as part of the statistical analyses.  Asymptomatic malaria and helminths coinfection occurs mainly in the tropics and subtropics where poverty and sanitary practice favor its high prevalence. In the tropics, where malaria is endemic, helminths also thrive resulting in coinfection. This study aimed to access the prevalence of asymptomatic malaria and helminths coinfection and its contribution for anemia in primary school children of Gedeo Zone, Southern Ethiopia. P < 0.05). 15.9% of helminths-infected school children were anemic, not statistically significant (P > 0.05). The prevalence of anemia was 12 (41.3%) among coinfected students, which is statistically significant (P < 0.005).  A total of 413 primary school children aged 6 to 16 years (mean age ± SD: 10.7 ± 2.64years) were enrolled in the study. 159 (38.5%) of school children were infected with at least one of the parasitic diseases. The overall prevalence of asymptomatic malaria and intestinal helminths was 46 (11.1%) and 113 (27.3%) respectively. Asymptomatic malaria and helminths coinfection was 29 (7%). Total of 39.1% of asymptomatic malaria-infected school children were anemic, which is statistically significant ( Asymptomatic malaria and helminths coinfection affects the health status of considerable number of primary school children in the study area. Therefore, simultaneous combat against the two parasitic infections is crucial to improve health of the school children. Plasmodium species [Asymptomatic malaria and helminths infection are among the most prevalent diseases in sub-Saharan Africa. It is estimated that over one-third of the world's population, mainly those individuals living in the tropics and subtropics, are infected by intestinal helminths or one or more of  species . Both in species .Plasmodium falciparum and P. vivax are the dominant parasites responsible for the majority of malaria cases in Ethiopia [Although there is no standard definition for “asymptomatic” malaria infections, it is generally accepted to be malarial parasitemia of any density, in the absence of fever or other acute symptoms in individuals who have not received recent antimalarial treatment . There aEthiopia –7, and mEthiopia .Intestinal helminths are also the cause of malnutrition, or aggravate existing malnutrition; through damage to the gastrointestinal mucosal epithelium and inflammation, reduced food intake, malabsorption, and increased nitrogen loss in faeces and diarrhea, malaria-helmiths coinfections further compromise the health condition of the patient –12.Ascaris lumbricoides, Trichuris trichiura, Hook worm, and Hymenolophis nana, are widely spread with variable prevalence [In Ethiopia, like other developing countries, infections with the major soil-transmitted helminths, including evalence –15.Asymptomatic malaria and helminths coinfection overlapping distribution contributes substantially to anemia . MalariaAscaris lumbricoides-associated vitamin A deficiency may further increase the risk of anemia in those coinfected with malaria [There is evidence that  malaria . Hookwor malaria .This parasitic disease affects the health status of the community in sub-Saharan Africa. Also, studies indicated that individuals coinfected with more than one parasite species are at risk of increased morbidity, as well as at a risk of developing frequent and more severe disease due to interactions among the infecting parasite species , 22. HowTherefore, this study aimed to assess the prevalence of intestinal helminths and asymptomatic malaria and its effects on anemia among primary school children. The results of this study could help concerned stakeholders to take action on the prevention of intestinal helminths and asymptomatic malaria.A cross-sectional study was conducted from February to April 2020 in selected Dilla town and Dilla Zuria woreda primary schools.Dilla is located 360 km to the south of Addis Ababa, the capital of Ethiopia, with an area of 1123.47 sq.kms. It is located 5°53′N to 6°27′N latitude and 38°8′ to 38°30′ east longitude. Dilla town and Dilla Zuria woreda are located in Gedeo zone . The altAll primary school children aged 5 to 16 years attending class in Dilla town and Dilla Zuria woreda and who did not take antimalarial and antihelminths drug two weeks prior to the study commencing were considered as the study population.There are 31 primary schools in Dilla and Dilla Zuria woreda. Six of them were selected by simple random sampling and by considering proportional allocation: three primary schools  and another three primary schools  were selected from Dilla and Dilla Zuria woreda, respectively. Finally, representative primary school children were selected by systematic random sampling techniques from the registration list of selected schools.Sample size was calculated using a single population proportion formula by taking a previous similar study conducted in Alaba kulito , 95% conSociodemographic data were collected using pretested structured questionnaires prepared in local languages (Gede'offa and Amharic). The study participants were also interviewed to obtain sociodemographic characteristics and other information . A total of 417 primary school children were enrolled in the study, and three of them were excluded from the study since they did not fulfill the inclusion criteria. Therefore, a total of 413 primary school children participated in the study.μl of blood for thick and 2 μl of blood for thin blood films were collected [The clean slides were labeled with patient identification number, date, and time of collection at the frosted end. The blood sample was collected from the middle or ring finger after cleaning with a 70% alcohol-moistened swab and dried with a piece of dry cotton after pricked using a disposable blood lancet. Through wiping off the first drop of blood, 6 ollected , 26; theollected , 28. SmeA stool sample was collected from each participant using a clean, leak-proof, and sterile stool cup and preserved using 10% formalin and then processed by the formalin-ether concentration procedure; 2 ml of preserved stool was added in a clean conical centrifuge tube containing 7 ml of 10% formol water. The suspension was filtered through a sieve into a 15 ml conical centrifuge tube. Then, 4 ml of diethyl ether was added to the formalin solution and the content was centrifuged at 300 rpm for 1 minute. The supernatant was discarded, and stool smears were prepared on sterile slides from the sediment and then three slides were prepared for each participant , 29. FinHemoglobin concentration was determined using a Hemocue Hb 201 analyser , 31. AneTo keep the quality of data, on-site training was given for data collectors on how to collect sociodemographic data and other associated factors. Data were checked for completeness before and after entry. Reagent and instrument quality was also checked for expiry dates and any functional problems.Mission® Hemoglobin Plus Hb .The slides were microscopically examined by two experts, while discrepancies were resolved by a third opinion for quality control. Similarly, quality control was performed on hemoglobin measures, Hemocue Hb 201 + analyser and P < 0.05.The data were double entered to EpiData software and exported to SPSS version 20 software for analysis. Descriptive statistics was performed for sociodemographic characteristics. Pearson's chi-square and multivariate logistic regression association were also performed to assess the association between malaria/helminths coinfection and anemia. Statistical significance were considered if Ethical clearance was received from the research and ethical review committee of Dilla University. Before data collection, the objectives of the study were explained to the study participants, parents, and the school community. Written informed consent was obtained from partners or the legal guardians. Children infected with any of the parasites were referred to the nearby health institution for treatment. All information obtained from the study participants was coded to keep confidentiality.A total number of 413 participants were enrolled in the study. About 322/413 (78%) of the study participants were between the age range of 10–15 years. Two hundred and twenty-seven study participants (55%) were males, 132 (32%) used bed net, 362 (87.5%) washed their hand before and after toilet use, and 305 (73.8%) and 108 (26.2%) of study participants wore shoe always and sometimes, respectively. About 374 (90.3%) used toilet, and the rest 40 (9.7%) used open space for defecation. The source of drinking water as pipe, well, and river was 322 (78%), 84 (20.3%), and 6 (1.5%), respectively. Two hundred and seventeen (51.8%) of them were living in urban areas .Plasmodium falciparum, 23 (5.6%) Plasmodium vivax, and 2 (0.4%) mixed infections. The majority, 29 (7%) of malaria-infected students were in the age group of 10–14 years. The prevalence of asymptomatic malaria among males and females was 21 (5.1%) and 25 (6%), respectively , 21 (5.1%) ectively , and higectively .Ascaris lumbricoides was the most prevalent with 57 (13.8)  were infected by at least one intestinal helminths. The prevalence of intestinal helminthic infection was higher, 85 (20.5%), among the age group of 10–14 years. 7 (13.8) .Ascaris lumbricoides, Hook worm, and Taenia species among malaria-infected students was 18 (4.3%), 10 (2.5%), and 1 (0.2%), respectively . The magnitude of ectively .Ascaris lumbricoides/plasmodium vivax and Ascaris lumbricoides/Plasmodium falciparum takes a major share. The prevalence of coinfection among males and females was 11 (38%) and 18 (62%), respectively . ectively .P value 0.004, and there was no significant association observed among sex groups, P value 0.37  were found to be anemic according to the WHO classification. The prevalence of anemia was significant among the age group of 5–9, lue 0.37 .P=0.037). However, among 113 (27.3%) students infected with helminths, 18 (15.9%) were anemic, not statistically significant (P=0.58), and among 29 (7%) malaria-helminths coinfected students, 12 (41.3%) were anemic, which is statistically significant (P=0.003) (Among 46 (11.1%) asymptomatic malaria-infected students, 18 (39.1%) were anemic, which is statistically significant (P=0.003) .The overlapping distribution of asymptomatic malaria and intestinal helminths coinfection is the main cause of anemia. In this study, we have assessed the magnitude of asymptomatic malaria and helminths coinfection, as well as its effect on the hemoglobin level, among primary school children of Gedeo Zone, Southern Ethiopia.Plasmodium vivax, Plasmodium falciparum, and mixed infection was 23 (5.6%), 21 (5.1%), and 2 (0.5%), respectively. This finding is higher than that of the study conducted in Southern Ethiopia, 3.6% [The prevalence of asymptomatic malaria among primary school children was 46 (11.1%). The magnitude of ia, 3.6% , and Demia, 3.6% , and lowia, 3.6% , and Côtia, 3.6% . The difThe prevalence of asymptomatic malaria decreased when the age of participants increased. The reason might be related with the development of protective immunity by repeated infections as well as acquisition of knowledge on malaria prevention and control mechanisms. A significant number of asymptomatic malaria cases were also observed in rural areas, which might be associated with suboptimal use of bed net. On the other hand, the prevalence of helminths was 113 (27.6%) and it was lower than that of studies conducted in Southern Ethiopia, 69% , and NorAscaris lumbricoides coinfection with Plasmodium falciparum and Plasmodium vivax was 8 (1.9%) and 10 (2.4%), respectively. The prevalence Hookworm coinfection with Plasmodium falciparum and Plasmodium vivax was 4 (1%) and 6 (1.45%), respectively. This finding is lower than that of the study conducted in Southwest Nigeria [The prevalence of asymptomatic malaria and helminths coinfection was 29 (7%). The prevalence of  Nigeria , Côte d' Nigeria , and Gha Nigeria  and larg Nigeria . This coThe prevalence of anemia among asymptomatic malaria and helminths coinfected students was 41.3%. This findings was higher than that of the study conducted in Northwest Ethiopia, 10.9% , SouthweAsymptomatic malaria and helminths coinfection was observed in the study area, and the prevalence of anemia was significantly associated with coinfection. Therefore, combined and integrated interventional measures should be taken including health education for school children about personal and environmental hygiene practices, use of bed net, encouraging footwear practice, and use of latrine.


Plasmodium falciparum is often accompanied by thrombocytopenia. Treatment includes transfusion of whole blood, which contains erythrocytes, platelets, and other blood components. The objective of the study was to assess the effect of whole blood transfusion on survival in children with severe falciparum malaria and to examine the potential interaction of thrombocytopenia with malaria mortality and transfusion response. METHODS/STUDY POPULATION: We analyzed a retrospective cohort of 842 hospitalized children in Zambia with severe malarial anemia . Severe malarial anemia was defined as a positive rapid diagnostic test or blood smear in combination with an admission hemoglobin concentration ≤5 g/dL. RESULTS/ANTICIPATED RESULTS: Mortality was 13% (94/703) in the transfused group and 24% (34/139) in the non-transfused group. Kaplan-Meier survival estimates stratified by transfusion status and thrombocytopenia  showed increased mortality in children with thrombocytopenia who did not undergo transfusion, with no differences in mortality among the other transfused and non-transfused groups (log-rank test P = 0.0001). Effect modification analysis by Cox proportional hazards regression adjusted for age, sex, hemoglobin concentration, blood group type, and eosinophilia showed a significant interaction between platelet count and transfusion status (P = 0.028). Children with thrombocytopenia who were transfused and died had little or no post-transfusion increase in platelets, in contrast to those who survived. Freshness of transfused whole blood, construed from expiration dates, correlated with greater platelet recovery and improved survival. DISCUSSION/SIGNIFICANCE OF IMPACT: The role of platelets in malaria pathophysiology is complex and incompletely understood; prior studies describe preferential binding of platelets to parasitized erythrocytes and direct parasitocidal activity, whereas others detailed deleterious effects in malaria involving the central nervous system vasculature. These findings point to a potential clinical role for platelet-directed transfusion strategies to improve survival in children with severe falciparum malaria, which should be further assessed in randomized interventional studies.OBJECTIVES/GOALS: Severe malarial anemia due to


Malaria and hypertensive disorders of pregnancy (HDoP) affect millions of pregnancies worldwide, particularly those of young, first-time mothers. Small case-control studies suggest a positive association between falciparum malaria and risk of pre-eclampsia but large prospective analyses are lacking.We characterized the relationship between malaria in pregnancy and the development of HDoP in a large, prospectively followed cohort. Pregnant women living along the Thailand-Myanmar border, an area of low seasonal malaria transmission, were followed at antenatal clinics between 1986 and 2016. The relationships between falciparum and vivax malaria during pregnancy and the odds of gestational hypertension, pre-eclampsia, or eclampsia were examined using logistic regression amongst all women and then stratified by gravidity.There were 23,262 singleton pregnancies in women who presented during the first trimester and were followed fortnightly. Falciparum malaria was associated with gestational hypertension amongst multigravidae (adjusted odds ratio (AOR) 2.59, 95%CI 1.59–4.23), whereas amongst primigravidae, it was associated with the combined outcome of pre-eclampsia/eclampsia . In contrast, there was no association between vivax malaria and HDoP.Falciparum but not vivax malaria during pregnancy is associated with hypertensive disorders of pregnancy. Worldwide, hypertensive disorders of pregnancy (HDoP) are estimated to affect 3–10% of all pregnancies . The cliPlasmodium falciparum malaria in pregnancy with a peak of detection at 13–18 weeks’ gestation in higher transmission settings [Plasmodium vivax malaria does not display a placental tropism, but in the present study area, has also been associated with poor pregnancy outcomes similar to the effect of falciparum malaria [Primigravidae are also particularly susceptible to settings . Falcipasettings , and falsettings . Periphesettings ; pre-tersettings , small fsettings ; stillbisettings ; and neosettings . In cont malaria , 13, 16.In West Africa, an increase in pre-eclampsia  and mateWe therefore examined the association between falciparum and vivax malaria in pregnancy and HDoP along the Thailand-Myanmar border, an area of low seasonal malaria transmission, in a large cohort of women who attended antenatal clinics regularly from the first trimester of pregnancy.P. falciparum, malaria was detected actively by blood smears obtained at each ANC visit. Hematocrit was also assessed at each visit; moderate and severe anemia was defined as < 30% and < 20%, respectively. Blood pressure (BP) was measured by a manual sphygmomanometer with the woman recumbent. BP was monitored at least monthly, weekly in the last 4 weeks of pregnancy, and at every malaria episode. A high blood pressure reading was confirmed by repeating the reading after 30 min to confirm that the blood pressure remained elevated after the woman was allowed to relax. Proteinuria by urine dipstick was assessed if BP was elevated (systolic BP ≥ 140 and/or diastolic BP ≥ 90 mmHg).The Shoklo Malaria Research Unit (SMRU) has worked with displaced persons since 1986 and migrant populations since 1998. Clinics are situated along the Thailand-Myanmar border and offer free antenatal and birth care. Antenatal records of singleton births occurring at or after 28 weeks’ gestation between 1986 and 2016 were obtained. The analysis was restricted to women who began antenatal attendance in the first trimester (< 14 weeks’ gestation). Women were encouraged to attend an antenatal clinic (ANC) weekly when malaria transmission was high, and fortnightly as transmission decreased, and to attend at any time if ill. The woman’s age, parity, refugee status, number of consultations, weight at enrollment, weight gain during pregnancy, location and year of delivery, and birth outcome were recorded. Data on smoking in pregnancy were obtained between 1997 and 2016. Gestational age was assessed preferentially by ultrasound biometry (2001 to 2016) but if not available by Dubowitz Gestational Age Assessment (1992–2002) and finally by fundal height (1986–1994). As there are no safe and effective chemoprophylactics for pregnant women in settings of multidrug-resistant From the first ANC visit to delivery, vitamin supplements were provided: non-anemic women received iron (ferrous sulfate 200 mg daily), folic acid (5 mg weekly), and vitamin B1 (100 mg daily). Women with anemia received higher doses of ferrous sulfate (400 mg twice daily) and folic acid (5 mg daily) as well as vitamin C and vitamin B12 (both 100 mg twice daily).Throughout the many years of the study, a rigorous and consistent definition of the clinical syndromes was used.Hypertension was two confirmed episodes of high blood pressure > 6 h apart with systolic BP ≥ 140 or diastolic BP ≥ 90.Chronic hypertension was hypertension by medical history or documented before 20 weeks’ gestation.Gestational hypertension was new hypertension that occurred after 20 weeks’ gestation with no/trace of proteinuria.Pre-eclampsia was new onset hypertension that occurred after 20 weeks’ gestation with proteinuria (≥1+ on a dipstick), without alternative explanation. For women with a history of chronic hypertension, the onset of new proteinuria was after 20 weeks’ gestation.Eclampsia was observed generalized convulsion  in a pregnant woman without other clear explanation, with a record of proteinuria or hypertension.Hypertensive disorder of pregnancy was a combined outcome of gestational hypertension, pre-eclampsia, or eclampsia.Plasmodium species and parasitemia were determined by microscopy for all positive smears. All malaria infections were treated irrespective of symptoms; P. falciparum was treated with quinine or artemisinin-based combination therapy, and P. vivax was treated with chloroquine. The majority of treatments were supervised. Women with positive blood smears were followed with daily malaria smear until negative. Recurrence or reinfection after treatment completion and negative blood smear was considered possible from day 7 after the primary diagnosis.Malaria was defined as the presence of asexual parasites on a blood smear. P. falciparum detected during the pregnancy represented the exposed group, regardless of the number of infections, and women with no Plasmodium of any species detected served as the control group. The same approach was taken for the analysis of vivax malaria. Given the likely placental origin of HDoP, we restricted the analysis to women who experienced infection after 6 weeks of gestation. Logistic regression was used to estimate the association between falciparum and vivax malaria in pregnancy and each outcome separately. As eclampsia is considered to be a feature of severe pre-eclampsia and there were relatively few eclampsia outcomes, we additionally considered the composite outcome of pre-eclampsia or eclampsia. Models were adjusted for confounding variables: gravidity, maternal age, population (migrant versus refugee), first-trimester weight, pregnancy weight gain, chronic hypertension, year, and place of delivery. Women with pre-pregnancy hypertension were excluded from the model of gestational hypertension. First-trimester weight was used in place of body mass index (BMI) as height was available for a limited number of women  according to the maximum signs and symptoms she experienced during the pregnancy. For example, if a woman first developed gestational hypertension but later developed proteinuria, she was considered as a final diagnosis of pre-eclampsia; if she went on to develop seizures, she was considered as a final diagnosis of eclampsia. For analyses of falciparum malaria, women with only To determine if the association between malaria and HDoP was modified by the gestational age at first detection, we added an interaction term between malaria and the gestational age of the first detection as a continuous variable to the adjusted models. The assumption that the change in the association between malaria and the outcome across levels of gestational age was linear was tested visually by plotting the estimated log odds ratios within 4-week gestational age windows and statistically by including an interaction with gestational age as a quadratic term. We also included an interaction between malaria and gravidity as in the primary analysis, but we did not let the interaction between malaria and gestational age be modified by gravidity as there was no evidence that this interaction was different between primigravidae and multigravidae.P. ovale, P. malariae, or infection with unknown species and were excluded from the analysis.Of 51,913 women who delivered at or after 28 weeks’ gestation between 1986 and 2016, 23,262 (45%) began antenatal care in their first trimester and had a singleton pregnancy  and delivered with a median gestational age at delivery of 39 weeks Table . The medWomen with malaria were more likely to be migrants, anemic, weigh less during the first trimester, gain less weight during the pregnancy, and deliver at home Table . These pp < 0.001) but not primigravidae  which was statistically significant when assessing the combined outcome of pre-eclampsia/eclampsia  .The overall risk of HDoP was low; most women, 21,168 (95.3%), did not become hypertensive, 620 (2.8%) had gestational hypertension, 346 (1.6%) had pre-eclampsia, and 43 (0.2%) had eclampsia. Falciparum malaria during pregnancy was associated with gestational hypertension in multigravidae  than at 28 weeks   suggesting a dose effect. The relationship between falciparum malaria and pre-eclampsia/eclampsia amongst primigravidae was preserved .Because of the inverse correlation between gestational age at the first episode and the number of falciparum episodes, we performed a sensitivity analysis in which we restricted evaluation to those women who had only a single detected episode compared to those who had no detected episodes. This attenuated the relationship between falciparum malaria and gestational hypertension amongst multigravidae .Additional file 2:Table S2. 1 P. falciparum to predict HDoP among women with known smoking status between 1997-2016 .Additional file 3:Figure S1. First detection of plasmodium on blood smear by gestational age.Additional file 4:Table S3. Association between falciparum malaria at 14 and 28 weeks’ gestation and HDoP.


P. vivax and 95 negatives) were included in the final analysis. Using nested-PCR as the gold standard, the sensitivity and specificity of Gazelle was 88.2% and 97.9%, respectively. Using a cutoff of 200 parasites/μl, Gazelle’s sensitivity for samples with more than 200 p/uL was 98.67% (95%CI: 92.79% to 99.97%) whereas the sensitivity for samples lower than 200 p/uL (n = 10) was 12.5% (95%CI: 0.32% to 52.65%). Gazelle’s sensitivity and specificity were statistically similar to microscopy  and higher than both SD Bioline  and BinaxNOW . The diagnostic accuracy of Gazelle for malaria detection in P. vivax infections was comparable to light microscopy and superior to both RDTs even in the presence of low parasitemia infections. The performance of Gazelle makes it a valuable tool for malaria diagnosis and active case detection that can be utilized in different malaria-endemic regions.A major challenge for malaria is the lack of tools for accurate and timely diagnosis in the field which are critical for case management and surveillance. Microscopy along with rapid diagnostic tests are the current mainstay for malaria diagnosis in most endemic regions. However, these methods present several limitations. This study assessed the accuracy of Gazelle, a novel rapid malaria diagnostic device, from samples collected from the Peruvian Amazon between 2019 and 2020. Diagnostic accuracy was compared against microscopy and two rapid diagnostic tests (SD Bioline and BinaxNOW) using 18ssr nested-PCR as reference test. In addition, a real-time PCR assay (PET-PCR) was used for parasite quantification. Out of 217 febrile patients enrolled and tested, 180 specimens (85  Plasmodium species  which puts more than 40% of the world’s population at risk [Malaria is an important public health threat that led to more than 228 million cases and 405 000 deaths in 2018 [P. falciparum [Currently, malaria treatment in all endemic regions, regardless of their transmission intensity, is prescribed after only cases are confirmed with a diagnostic test that is either an RDT or light microscopy. However, both methods suffer from several drawbacks that limit their use. Light microscopy requires continuous power, highly skilled personnel, a long processing time and its sensitivity is affected by low parasitemia . In the lciparum –6.Molecular approaches such as PCR and loop mediated isothermal amplification (LAMP) have been developed for highly sensitive and specific detection –9. HowevLAMP based methods have been widely explored and many variants have been tested showing good results in field and lab condition settings –13. The The photo- induced electron transfer (PET-PCR) is a real time assay that has been developed as a tool for malaria surveillance in endemic settings , 16. ThiHowever, there is still a need for high quality, point-of-care, cost-effective malaria diagnostic tools to enable detection of cases in the field in order to have prompt malaria detection that will not only improve patient health but will help to prevent transmission and reduce disease severity , 10.In order to address this gap, we evaluated a new rapid, point-of-care, battery-operated device that detects hemozoin particles in the blood of infected patients named Gazelle. Hemozoin crystals are formed by the polymerization of dimers ferriprotoporphyrin IX that is a byproduct from hemoglobin digestion generated by all malaria species . PreviouP. falciparum showed that this technology can potentially detect as low as 40 parasites per microliter of blood [P. vivax predominant elimination settings[However, there is promising evidence that magneto-optical detection can potentially allow sensitive identification of malaria. A previous in-vitro study on of blood . Furtherof blood . Further settings.P. falciparum detection in a high-transmission setting in India [P. vivax predominance in the Peruvian Amazon Basin.A previous study using Gazelle showed promising results for in India . Based oFig 1). This region accounts for 28.7% of Peru’s territory with an incidence of malaria of 1.8% in 2019  which represents more than 90% of all malaria cases reported by Peru during that year.Samples were collected from a passive surveillance study that enrolled febrile participants between 2019 and 2020 at nine health facilities located in the city of Iquitos and surrounding communities. Iquitos is the capital of the Loreto region which is located in the northwest side of the Peruvian Amazon basin  in compliance with all applicable federal regulations governing the protection of human subjects (protocol NMRCD.2007.0004). Adult participants provided written consent and minors provided written assent and parental/guardian consent.The sample size was estimated assuming a sensitivity of at least 70% for Gazelle with 10% relative precision at 95% confidence and 80% power. This resulted in a minimum sample size of 165 subjects.Trained phlebotomists collected four mL of whole blood by venipuncture from each participant into sterile EDTA treated vacutainer tubes. Samples were transported under cold chain within two hours of collection to NAMRU-6 facilities in Iquitos. Two thin and thick blood smears were prepared for each participant, stained with 10% Giemsa and read by two microscopists at the NAMRU-6 laboratory in Iquitos. Results were provided after reading 200 oil-immersion fields and positive slides were quantified using 6,000 WBC/μl of blood as reference. Collected blood was used for RDTs and Gazelle testing in Iquitos and an aliquot was shipped to the NAMRU-6 laboratory in Lima for DNA extraction and molecular assays.P. falciparum and lactate dehydrogenase (SD Bioline) or aldolase (BinaxNow) of Plasmodium species in human whole blood.Two RDTs were used to diagnose malaria infection at the NAMRU-6 Iquitos laboratory by one laboratory technician: i) SD BIOLINE (05FK80-40-0) and ii) BinaxNOW (665–025). These two RDTs are based on the detection of the histidine-rich protein II (HRP-II) antigen of Fig 2). A pipette was used to drop 15 μL of whole blood into the lower chamber of the cartridge along with 80 μL of Gazelle buffer (2 % Triton in water). Then the upper chamber was placed, and the sample was sonicated through the wall of the cartridge, using a sonicator incorporated in the Gazelle.The Gazelle device testing was performed in the Iquitos laboratory by the same laboratory technician with fresh samples that were processed within four hours from collection. The device consists of a table-top reader and single-use disposable two-chamber cartridges . For the first reaction 5 μl of DNA template was used, while 5 μl of the amplified product were used in the second reaction. Both reactions were carried out in a 50 μl volume containing 1X buffer, 2mM MgCL2, 125μM dNTP’s, 250nM of each primer, and 1 unit of Taq Polymerase . Results were visualized in a 2% agarose gel stained with GelRed®. Technicians were blinded to microscopy, RDT’s and Gazelle results.Nested PCR was conducted by two laboratory technicians in NAMRU-6 Lima. First, DNA was isolated from 200 μL of whole blood using the DNeasy Blood & Tissue kit  and eluted in 70 μL of elution buffer and used for 18S rRNA-nested PCR . The firPlasmodium genus PET-PCR in order to quantify parasite density [P. falciparum and P. vivax.All samples were tested in duplicates using a  density , 30. AllThe genus reaction was carried out in a 20 μl volume that contained 5 μl of purified DNA, 2X TaqMan Environmental buffer 2.0 , and 250nM of genus forward and FAM-reverse primer. The singleplex species-specific reactions contained the same mix but with a concentration of 125nM of the HEX-labelled species-specific primer.PET-PCR reactions were run on an Mx3005P qPCR system  and results were visualized in the MXpro qPCR software. Thermal cycling conditions for the genus and species-specific PET-PCR consisted of an initial denaturation at 95°C for 10 minutes and 45 cycles of denaturation at 95°C for 10 sec followed by annealing at 60°C for 40 sec. A threshold cycle (Ct) below 41 was used to separate positive and negative samples. Technicians were blinded to microscopy, RDT’s and Gazelle results.We estimated turn-around time and cost per sample for expert microscopy, Nested PCR, SD Bioline, BinaxNOW and Gazelle. The cost estimate included sample preparation for microscopy; DNA extraction, amplification reaction and results visualization for PCR; RDTs cost per sample and cartridge and buffer costs for Gazelle. Labor nor equipment cost were included in the analysis.Sensitivity and specificity with 95% confidence intervals (95% CI) were calculated for microscopy, RDTs and Gazelle using 18S rRNA-nested PCR as reference test. The McNemar test was employed to detect statistical differences between microscopy, RDTs and Gazelle. The Kappa coefficient was used to estimate agreement between these diagnostic tests while the Mann-Whitney U test was used to assess differences in parasitemia between the Gazelle’s true positives and false negatives. These analyses were carried out in the “stats” package implemented in R . MicroscP. vivax, 3 P. falciparum and 18 negatives; BinaxNow resulted in 10 non-P. falciparum positives and 22 negatives whereas SDBioline resulted in 10 P. vivax and 22 negatives.During the period of the study, 217 participants were enrolled and screened for malaria. A total of 32 participants were excluded from the final analysis because of inadequate blood sample to perform all four tests. Out of these 32 samples, microscopy identified 11 S1 Table).Out of the 185 remaining specimens, the malaria positivity rate by microscopy was 44.9% (83/185), by SD BIOLINE 38.9% (72/185), by BinaxNOW 35.6% (66/185), by Nested PCR 48.6% (90/185) and by Gazelle 43.8% (81/185) .A total of four Table 1).The median age was 33 years (range 5–79) and 60.5% were male. Participants had a median parasite density of 606 parasites/μL (12–61 773 parasites/μL) and recent history of antimalarial treatment was reported in 6 participants (P. vivax of 91.8% (95%CI: 83.8%– 96.6%) and specificity of 100% (95%CI: 96.2%– 100%). SD BIOLINE had a sensitivity of 82.4% (95%CI: 72.6%– 89.8%) and specificity of 100% (95%CI: 96.2%– 100%) whereas BinaxNOW had a sensitivity of 71.8% (95%CI: 61%– 81%) and specificity of 97.9% (95%CI: 92.6%– 99.7%). Gazelle had a sensitivity of 88.2% (95%CI: 79.4%– 94.2%) and specificity of 97.9% (95%CI: 92.6%– 99.7%) .Using 18S rRNA-nested PCR as reference, microscopy had a sensitivity for Using a cutoff point of 200 parasites/μl as in the WHO RDTs quality assurance program , the senFig 4). The McNemar test did not show statistical differences between the results of Gazelle compared to microscopy (p = 0.983). However, statistical significant differences were found between Gazelle against both SD Bioline and BinaxNOW .Cohen’s kappa showed that Gazelle presented a kappa of 0.94 (95%CI: 0.89–0.99) compared with microscopy and a kappa of 0.87 (95%CI: 0.79–0.94) compared with nested PCR. The area under the ROC curve was 0.93 for Gazelle, 0.96 for microscopy, 0.91 for SD Bioline and 0.85 for BinaxNOW  were found for Gazelle. Out of those, all were FN by both RDTs and 7 by microscopy. The average parasitemia in the 3 microscopy positive, but Gazelle FN specimens was 131 p/μl and the average parasitemia for submicroscopic, Gazelle FN was 1.4 p/ul using PET–PCR to quantify the parasitemia. Gazelle had 2 false positives (FP) results using 18S rRNA-nested PCR as reference. However, one FP turned out positive by PET-PCR with a parasitemia of 0.63 p/μl.Fig 5).The Mann-Whitney U test showed significant differences in the median parasitemia of Gazelle true positives versus Gazelle FN (p<0.00001). In this regard, the optimal parasitemia limit for a Gazelle positive result was estimated between 207 and 323 parasites/μl using microscopy and PET-PCR quantification data, respectively .The magneto-optical method used in this study is a quantitative technique as it detects hemozoin levels in the sample. In this regard, our results show a positive correlation between parasite density and the hemozoin signal detected by Gazelle (r = 0.83) (Table 2). Gazelle cost per test was estimated at one US dollar per test which is lower than BinaxNOW and similar to microscopy and SD Bioline (Table 2).Our results show that Gazelle had a shorter turnaround time than SD Bioline and BinaxNOW with an estimated time of five minutes per test including sample preparation and reading  and assessed its performance against microscopy and two RDTs (SD Bioline and BinaxNOW) using Nested PCR as the gold standard. The results of the current study showed that Gazelle’s sensitivity and specificity were similar to microscopy and better than RDTs which are the primary diagnostic methods used in health centers in Peru and other endemic regions.P. falciparum [P. falciparum and P. vivax cases.Our results are in line with a recent study on the performance of Gazelle in a high-transmission region in India with predominance of lciparum .The prevP. falciparum than P. vivax or introduction of new strains as previously shown [This is an important finding since species predominance varies between endemic regions. Furthermore, shifts in predominant species can occur within the same region as a result of malaria control activities, which tend to be more effective against ly shown –36.Gazelle’s turnaround time is fast (5 minutes) compared to microscopy (2 hours), RDTs (20 minutes) and PCR (9 hours) and the cost per test is comparable to microscopy and RDTs. Furthermore, Gazelle’s device cost is approximately $700 which is comparable to the cost of a basic light microscope and the device can process more than 20,000 tests during its estimated lifecycle.In this regard, our data suggests that Gazelle could complement microscopy or could be used as an alternative for malaria diagnosis in remote or resource-limited settings. This potential for point of care diagnosis is further supported by the minimal training required for Gazelle operation and the lack of observer-dependent variability as in microscopy.Using the WHO RDTs quality assurance program as a reference, the sensitivity of Gazelle for samples with high parasite density (>200 parasites/μl) was 97.47% (95%CI: 91.15% to 99.69%). This is a relevant finding as a density of 200 parasites/μl is well below the mean parasite density reported in many endemic settings including our sites in Peru . FurtherThe limited sample size (n = 10) for extremely low parasitemia infections (<200 parasites/μl) did not allow us to calculate a robust sensitivity estimate for Gazelle. However, given that the magneto-optical method used here is a quantitative technique, it could be possible to increase the sensitivity for low parasitemia infections by modifying the input sample volume.It is important to highlight that the current Gazelle prototype does not have the capacity for species identification. This an important drawback in regions where multiple species circulate and where treatment schemes differ since incorrect case management can lead to poor response to treatment with worsening disease, relapse or recrudescence . In thisGazelle constitutes an important addition to the malaria diagnostic toolkit that is suitable for point-of-care malaria detection. Our results show the potential of Gazelle as a cost-effective malaria diagnosis device that can be used in resource-limited endemic settings. Knowledge gained from this study will inform further advances in translating the technology into an affordable, rugged, and operational ready device that can be utilized in all malaria-endemic regions.S1 FigThe figure shows microscopy determined parasitemia versus Gazelle’s hemozoin quantification. A positive correlation was found between both measures (r = 0.83).(TIF)Click here for additional data file.S1 TableThe table shows the results for each of the methods used in this study for each of the samples as well as basic data from each of the participants including age, sex and date of sample collection.(XLSX)Click here for additional data file.


Little information is available about malaria and scrub typhus co-infection. This study aimed to investigate the pooled prevalence of malaria and scrub typhus co-infection in febrile patients. Further, it aimed to estimate the prevalence of scrub typhus infection among patients with malaria and the odds of co-infection. This will aid the diagnosis and management of co-infected patients in endemic areas.I2 statistics. We performed subgroup analyses of countries and scrub typhus diagnostic tests to explore the sources of heterogeneity of the included studies. We assessed publication bias if more than 10 studies were used to estimate the outcome. All data analyses were conducted using Stata version 14 .We searched for relevant studies in three databases: PubMed, Scopus, and Web of Science. We assessed the quality of the included studies using the Joanna Briggs Institute checklist for analytical cross-sectional studies. We estimated (1) the pooled prevalence of malaria and scrub typhus co-infection, (2) the pooled prevalence of scrub typhus infection in malaria-positive patients, and (3) the pooled odds of co-infection using the DerSimonian–Laird method for random-effects models. The study results and summary estimates were visualized on a forest plot as point estimates  and 95% confidence intervals (CI). We assessed the heterogeneity of the studies by Cochrane Q or I2: 78.28%), while the pooled prevalence of scrub typhus infection (321 cases) in patients with malaria (1418 cases) was 21% . Subgroup analysis showed that the pooled prevalence of scrub typhus infection among patients with malaria in India was 8% , while the pooled prevalence of scrub typhus infection among patients with malaria in Thailand was 35% . The co-infections did not occur by chance . In the sensitivity analysis, the pooled prevalence of malaria and scrub typhus co-infection among febrile patients was 0% .Of the 542 studies retrieved from three databases, we included 14 meeting the inclusion criteria in the systematic review and meta-analysis. The pooled prevalence of malaria and scrub typhus co-infection (56 cases) among febrile patients (7920 cases) was 1% (95% CI: 0–1%, The present study showed the pooled prevalence and a significant association between malaria and scrub typhus. The results show the status of co-infection. Further research into co-infection in endemic areas is needed, in particular, to determine whether co-infection can accelerate disease progression or protect against severe disease.The online version contains supplementary material available at 10.1186/s13071-021-04969-y. Anopheles mosquitoes transmit malaria by one of the six Plasmodium species, P. falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri, and P. knowlesi; however, some zoonotic Plasmodium species, such as P. cynomolgi and P. simium, have been associated with human cases of malaria [Blood-feeding  malaria –5. In a  malaria . In a lo malaria , 8. In 2 malaria . Within  malaria .Orientia tsutsugamushi. It is transmitted via trombiculid mite chigger (larva) bites [Orientia species are related to scrub typhus, including “Candidatus Orientia chiloensis” [Candidatus Orientia chuto” [Scrub typhus, which causes an acute febrile illness, is caused by the Gram-negative obligate intracellular bacillus a) bites  and is aa) bites , 11. Recloensis”  and “Cana chuto” , 14. Scra chuto” . Scrub ta chuto”  and the a chuto” , with a a chuto” , 14, 18.Clinical signs and symptoms of scrub typhus are fever, headache, myalgia, cough, and gastrointestinal symptoms, with or without escharation . A recenLittle information is available about malaria and scrub typhus co-infection. We aimed to investigate the pooled prevalence of malaria and scrub typhus co-infection among febrile patients, the prevalence of scrub typhus infection among patients with malaria, and the odds of co-infection. The data will help diagnose and manage co-infected patients in endemic areas.The protocol of this systematic review was registered at PROSPERO with ID CRD42021255893. We followed the preferred reporting criteria for systematic reviews and meta-analyses (PRISMA) [We used three databases to search for relevant studies, PubMed, Scopus, and Web of Science. We used the keyword combination “ AND scrub typhus.” The date range was from inception to May 12, 2021. The searches were unrestricted for language or year of publication. We also searched reference lists and Google Scholar to maximize studies and prevent relevant studies from being missed. The search strategy is described fully in Additional file Plasmodium infections by microscopy (gold standard), rapid diagnostic test (RDT), or a molecular method. We detected scrub typhus infections using indirect fluorescence assay (IFA) (gold standard), IgM enzyme-linked immunosorbent assay (ELISA), or RDT. All studies that reported co-infections with malaria and scrub typhus were considered. Case reports and case series, reviews, correspondence, animal studies, experimental studies, and test evaluation studies were excluded.We used the PICo  or PICO  approach to select eligible studies. For the primary outcome, we used (i) P: febrile patients suspected of malaria or scrub typhus infection, (ii) I: patients with co-infection with malaria and scrub typhus, (iii) Co: none. For the secondary outcome, we used (i) P: patients with malaria infection, (ii) I: patients with co-infection of malaria and scrub typhus, (iii) Co: none. For the tertiary outcome, we used (i) P: febrile patients suspected of malaria or scrub typhus infection, (ii) I: patients with co-infection of malaria and scrub typhus, (iii) C: febrile patients without malaria and scrub typhus, and (iv) O: chance of co-infection. We detected We reviewed all studies reporting malaria and scrub typhus co-infection. First, we excluded duplicates. Second, we screened the titles and abstracts, excluding non-related studies. Third, we read the full text and excluded irrelevant studies for various reasons. The study selection process was performed using EndNote X8 for reference management. Two authors  selected the studies independently and resolved any disagreements by a discussion with the third author (PW).We extracted the following information: first author’s name, year of publication, study site, year conducted, study design, age group, gender, number of participants, number of co-infections, number of malaria cases, number of scrub typhus cases, diagnostic test for malaria, and diagnostic test for scrub typhus. We recorded the data in a spreadsheet for further analysis. Two authors (MK and SK) independently extracted the data and resolved any inconsistency or disagreement by consensus.We assessed the quality of the selected studies using the checklist for analytical cross-sectional studies developed by the Joanna Briggs Institute. The checklist was based on study design, study conduct, and analysis of the outcome of interest. High-quality studies received seven to eight points, while moderate-quality studies scored four to six points. Studies scoring fewer than four points were excluded. Two authors (MK and SK) assessed the included studies’ quality.P > 0.05 indicated significant heterogeneity) or I2 statistics, with I2 values < 25%, 25–75%, and > 75% interpreted as low, moderate, and high heterogeneity, respectively [The pooled prevalence of malaria and scrub typhus co-infection among febrile patients was estimated using the DerSimonian–Laird method for the random-effects model, based on the inverse variance approach for measuring weight as described previously , 23. Theectively . We analAmong the 542 studies retrieved from the three databases, 10 studies –35 met tFourteen studies that reported malaria and scrub typhus co-infections were included in the study Table . All theThe quality of the included studies was assessed using the Joanna Briggs Institute checklist for analytical cross-sectional studies. Four studies , 35, 38 I2: 78.28%)  among febrile patients 7920 cases) was estimated from nine studies , 38, 39.0 cases wI2: 85.87%, nine studies with 59/794 cases).The pooled prevalence of scrub typhus infection, 321 out of 1418 patients with malaria (cases), was estimated from 13 studies , 31–39. I2: 98.9%, four studies with 262/624 cases). Combined with the results of the estimated prevalence in India and Thailand, the pooled prevalence of scrub typhus infection among patients with malaria was 21%  , in which the studies by Kotepui et al.  and Chan%, in whiI2: 90.15%) according to IgM ELISA alone, 31% with IgM ELISA/polymerase chain reaction (PCR) (95% CI: 13–58%), 31% with IFA alone , 3% with IgM ELISA/IFA (95% CI: 2–6%), 5% with PCR (95% CI: 1–10%), 63% with RDT (95% CI: 55–69%), and 100% with the Weil Felix test    in India and 1%  in Thailand  Fig. . The fixand Fig. .Fig. 6SeP = 0.023, coefficient: 6.55, standard error: 2.43, t: 2.69). Further contour-enhanced funnel plot analysis showed that most of the studies missing from non-significant areas (P > 0.01) indicated that the cause of funnel plot asymmetry was likely to be due to publication bias  and standard error of the ES (seES) from the 12 studies , 31–38 tMalaria and scrub typhus are vector-borne diseases transmitted by different arthropod vectors , 42. TheOur meta-analysis showed a high prevalence of scrub typhus infections among patients with malaria in Thailand (35%). This was associated with high heterogeneity (98.9%), possibly caused by differences in the study participants, scrub typhus diagnostic tests, study area, or the year of the studies. In the study by Kotepui et al. , RDT wasIn India, a meta-analysis demonstrated a low prevalence of malaria and scrub typhus co-infection in febrile patients and a low prevalence of scrub typhus infection in malaria patients. There was heterogeneity in the prevalence of malaria and scrub typhus co-infection in febrile patients among the studies conducted in India (0–1%). However, the prevalence of co-infection tended toward homogeneity when the meta-analysis excluded the results of Ahmad et al. . The lowThe present meta-analysis showed significantly decreased odds of co-infection, which indicates that malaria and scrub typhus co-infections occurred by chance rather than infection by one pathogen leading to an increased risk of contracting the other infection. However, co-infection risk was demonstrated in only two studies , 31, whiThe present study had limitations. First, only a small number of studies in Thailand and India reported malaria and scrub typhus co-infections. Therefore, the low prevalence of co-infection among febrile patients might be due to the underreporting of scrub typhus or misdiagnosis. Second, the high heterogeneity of measurements among the studies might have influenced the interpretation of the meta-analysis. Therefore, the pooled prevalence must be interpreted in consideration of the results of individual studies and sensitivity tests.The findings of this study emphasize the importance of further research on malaria and scrub typhus co-infection in endemic areas. The outcomes also highlight the importance of interpreting diagnostic tests together with clinical signs and symptoms to facilitate the precise management of febrile patients who live in areas endemic for both diseases.Additional file 1: Table S1. Search terms.Additional file 2: Table S2. Quality of the included studies.


Plasmodium falciparum erythrocyte membrane protein-1s (PfEMP1s), diverse malaria proteins expressed on the infected erythrocyte surface, play an important role in pathogenesis, mediating adhesion to host vascular endothelium. Antibodies to particular non-CD36-binding PfEMP1s are associated with protection against severe disease. We hypothesized that given lifelong P. falciparum exposure, Malian adults would have broad PfEMP1 serorecognition and high seroreactivity levels during follow-up, particularly to non-CD36-binding PfEMP1s such as those that attach to endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1). Using a protein microarray, we determined serologic responses to 166 reference PfEMP1 fragments during a dry and subsequent malaria transmission season in Malian adults. Malian adult sera had PfEMP1 serologic responses throughout the year, with decreased reactivity to a small subset of PfEMP1 fragments during the dry season and increases in reactivity to a different subset of PfEMP1 fragments during the subsequent peak malaria transmission season, especially for intracellular PfEMP1 domains. For some individuals, PfEMP1 serologic responses increased after the dry season, suggesting antigenic switching during asymptomatic infection. Adults were more likely to experience variable serorecognition of CD36-binding PfEMP1s than non-CD36-binding PfEMP1s that bind EPCR or ICAM-1, which remained serorecognized throughout the year. Sustained seroreactivity to non-CD36-binding PfEMP1s throughout adulthood amid seasonal fluctuation patterns may reflect underlying protective severe malaria immunity and merits further investigation. Plasmodium falciparum disease1. Older children in these areas acquire immunity to clinical malaria1, leading to predominantly asymptomatic malaria infections in adulthood. Although non-sterile natural immunity to malaria is incompletely understood, acquiring antibodies to variant surface antigens (VSA)—proteins expressed on the infected red blood cell surface—plays an important role. VSAs mediate microvascular sequestration of infected red blood cells via host receptor binding2, permitting immune system evasion and parasite propagation.In regions with high malaria transmission, children under five years of age are most vulnerable to severe Plasmodium falciparum erythrocyte membrane protein-1s (PfEMP1s) comprise the major VSA family implicated in severe malaria pathogenesis and are believed to be the main targets of protective immunity against clinical malaria5. PfEMP1s are encoded by ~ 60 var genes per genome, each containing an upstream promoter sequence and two exons that code for a highly variable extracellular human receptor-binding domain and a relatively conserved intracellular cytoplasmic tail or acidic terminal segment (ATS) residing within the erythrocyte. The extracellular region extrudes into the plasma from the erythrocyte surface and is composed of cysteine-rich interdomain regions (CIDRs) and Duffy binding-like (DBL) domains. The DBLα domain includes a 300–400 amino acid sequence unique to individual var genes that has been used as a fingerprint “tag” in clinical studies9. PfEMP1s have previously been divided into five subgroups according to chromosomal location, upstream promoter sequence, and direction of transcription: A, B/A, B, B/C, and C11. var genes have extreme sequence diversity and PfEMP1s have different antigenic and adhesion properties enabling binding to a range of host endothelium receptors. The CIDRα domains of some PfEMP1s bind CD36, the most common target receptor of PfEMP1s14. CD36 is a leukocyte differentiation antigen and scavenger receptor involved in fatty acid metabolism, phagocytosis, and angiogenesis17. PfEMP1s binding CD36 via CIDRα2 domains have been associated with uncomplicated or asymptomatic malaria19, whereas PfEMP1s binding the endothelial protein C receptor (EPCR)20 via CIDRα1 domains and PfEMP1s binding the intercellular adhesion molecule-1 (ICAM-1) via DBL27. Mathematical models suggest that immunity to severe malaria is acquired relatively rapidly in early childhood in malaria-endemic regions, while immunity to uncomplicated malaria is acquired much more slowly28. Studies in Tanzanian and Malian children have shown that antibodies to domains of EPCR-binding PfEMP1s are acquired prior to antibodies to other PfEMP1 domain types, supporting the notion that parasites expressing EPCR-binding PfEMP1s are linked to severe malaria in naïve individuals30. Conversely, parasites that express less pathogenic PfEMP1s may dominate future infections30.31. We used a custom protein microarray to analyze seroreactivity changes to a diverse group of PfEMP1 proteins longitudinally in Malian adults. We hypothesized that adults with lifelong exposure to P. falciparum acquire serorecognition of most PfEMP1 antigens and maintain significant PfEMP1 antibody responses throughout the year, particularly to non-CD36-binding PfEMP1s, reflecting sustained protection against severe disease. We also predicted that PfEMP1 serologic responses decrease during the dry season and increase during the malaria transmission season due to the intense, seasonal nature of parasite exposure in Mali.Protein microarrays are a powerful tool to measure seroreactivity to a large number of PfEMP1s simultaneously32, microarray construction34 included (1) polymerase chain reaction amplification of complete or partial P. falciparum open reading frames, (2) recombination cloning in Escherichia coli, (3) in vitro transcription and translation, and (4) chip printing. Each microarray contained standard controls, as described elsewhere7. PfEMP1 fragments were selected for inclusion on the microarray based on their successful amplification and cloning.As described previouslyPlasmodium falciparum reference strain 3D7 were included on the microarray 35. We have previously published a detailed description of this microarray and the locations of each of these fragments on 3D7 PfEMP1s35. This microarray was probed with sera from 18 adults aged 18–52 years old from the control arm of a malaria vaccine trial in Bandiagara, Mali36. Participants were predominantly from the Dogon ethnic group and included 14 males and four females. Malaria transmission in Bandiagara is strictly seasonal with no transmission in the dry season (January–May) and up to 60 infected mosquito bites per person per month during peak malaria transmission (June–December)38 . Sera from the 18 adults were collected at three time points: December 2004, the start of the dry season (pre-dry season); June 2005, the start of the malaria transmission season ; and December 2005, the end of the malaria transmission season . Serum samples from 11 North American, malaria-naïve adult blood donors were used as controls.A representative set of 166 PfEMP1 protein fragments from the 39. A PfEMP1 fragment that contained a domain known to bind EPCR or ICAM-1 or a domain that is a component of a domain cassette known to bind EPCR or ICAM-1 was included in the EPCR/ICAM-1-binding subgroup. Similarly, a PfEMP1 fragment that included a domain known to bind CD36 or a domain that is a component of a domain cassette with a CD36-binding member was included in the CD36-binding subgroup. A PfEMP1 fragment containing domains that typically follow the CD36-binding domain cassette was included in the “CD36-Associated” subgroup. PfEMP1 fragments that did not fall into any of these categories were included in the Miscellaneous subgroup. The remaining protein fragments included DBLFor analyses, we grouped PfEMP1 fragments based on the two or three domain composition linked to a known human receptor binding target36. Clinical trial protocols were approved by institutional review boards of the University of Bamako Faculty of Medicine, the University of Maryland, Baltimore, and the US Army Surgeon General. Written informed consent was obtained for screening and enrollment in the clinical trial. Verbal consent of illiterate subjects was verified by independent witnesses and documented using thumbprints.Serum samples were obtained from consenting adults in compliance with the International Conference on Harmonization Good Clinical Practices, the Declaration of Helsinki, and Malian regulatory requirements α tag for each serum sample at each time point. DBLα tags were treated as separate from extracellular PfEMP1 fragments in statistical analyses. Seroprevalence refers to the proportion of serum samples that recognized an individual PfEMP1 fragment or group of fragments at a given time point32. “Highly seroprevalent” refers to PfEMP1 fragments serorecognized by at least 90 percent of adult serum samples. Seroreactivity trends across PfEMP1 fragments are reported as the mean percentage of fragments ± standard deviation.Seroreactivity, measured by microarray fluorescence intensity, and serorecognition, defined as a fluorescence intensity greater than or equal to two standard deviations above the arithmetic mean of North American control sera, were determined for each extracellular and intracellular PfEMP1 fragment and for each DBLComparisons of seroreactivity and serorecognition were made for both fragments and for serum samples between time points. Unmatched comparisons between PfEMP1 seroreactivities of Malian adult sera and North American malaria-naïve controls were carried out by the Kolmogorov–Smirnov Test to determine group serorecognition at a given time point. Paired comparisons of PfEMP1 seroreactivity between time points were carried out by the Wilcoxon matched-pairs signed-rank test. For paired comparisons of the extent of seasonal change in CD36-binding and non-CD36-binding PfEMP1 seroreactivity seen in Fig. P < 0.05 for all statistical tests, without adjustment for multiple comparisons, consistent with previous microarray analyses42.Paired comparisons of PfEMP1 serorecognition between time points were carried out by the two-tailed McNemar test or the two-tailed binomial test for low sample size comparisons. Unmatched comparisons of serorecognition between time points were carried out by the This information has been previously presented at the Student Research Forum in July 2018 in Baltimore, Maryland as part of the University of Maryland School of Medicine Scholar Program, at Bioscience Research Day 2018 in November 2018 in College Park, Maryland as an undergraduate, and at the American Society of Tropical Medicine and Hygiene Annual Meeting in November 2019 in National Arbor, Maryland (abstract number: 361).α tags, was probed with Malian sera from 14 males and four females aged 18–52 years. No participants were hospitalized for severe malaria during the study. We detected a range of antibody responses to all fragments, including intracellular and extracellular PfEMP1 fragments  and intracellular PfEMP1 fragments (n = 12 adults). About a third of adults experienced overall increases in seroreactivity to extracellular or intracellular PfEMP1 fragments (n = 7 and 6) Fig. C. A simiα tag) in the dry season. In the following malaria transmission season, adults had increased seroreactivity to three of these PfEMP1 fragments and maintained seroreactivity to an additional 11 fragments. The group’s average seroreactivity to intracellular PfEMP1 fragments and CD36-binding and associated PfEMP1 fragments declined in the dry season and increased in the malaria transmission season but remained at similar levels across the entire year  and increased in the malaria transmission season, resulting in a significant increase across the year as a whole , malaria transmission season (16 extracellular and one intracellular PfEMP1 fragments), and entire year (18 extracellular and two intracellular PfEMP1 fragments) . Indeed, a study that compared asymptomatic Malian children in the dry season to symptomatic children in the malaria transmission season found distinct var genes. It may not be surprising that adults as a group had shared serologic responses to intracellular PfEMP1 fragments, which they are more likely to have been exposed to previously, given sequence conservation. In contrast, adults as a group had less consistent responses to extracellular PfEMP1 fragments, which are more diverse and thus less likely to be elicit consistent serologic responses from adults. Likewise, individuals had seasonal fluctuations in serorecognition of these diverse extracellular PfEMP1 fragments.We also noted seasonal oscillation patterns in PfEMP1 seroreactivity and serorecognition. Individuals that experienced decreased or increased PfEMP1 serologic responses in the dry season tended to have changes in the opposite direction in the malaria transmission season. These serologic fluctuations occurred for extracellular PfEMP1 fragment subgroups and intracellular PfEMP1 fragments. While the group’s average seroreactivity to the extracellular CD36-binding and CD36-associated PfEMP1 subgroups remained constant across the year overall after decreasing in the dry season and increasing in the malaria transmission season, the group experienced increased seroreactivity to the EPCR/ICAM-1-binding PfEMP1s across the entire year. Additionally, seroreactivity changes were more pronounced for CD36-binding and CD36-associated PfEMP1 fragments across the dry and malaria transmission seasons than they were for EPCR/ICAM-1-binding PfEMP1s, but this was not the case across the entire year. We also noted pronounced seasonal seroreactivity changes to intracellular PfEMP1 fragments, which are encoded by a relatively conserved exon in 47. Sustained seroreactivity to EPCR/ICAM-1-binding PfEMP1 fragments included on our microarray, which included domain cassette 8 PfEMP1 fragments, into adulthood may reflect sustained protective clinical immunity to severe malaria. PfEMP1s that bind EPCR and ICAM-1 may lead to the development of severe malaria48.Individuals maintained serorecognition of EPCR/ICAM-1-binding PfEMP1 fragments more often than they did of the CD36-binding and CD36-associated PfEMP1 fragments. Among highly seroprevalent PfEMP1 fragments, EPCR/ICAM-1-binding PfEMP1s were overrepresented. A previous study of pediatric cerebral malaria cases found increased transcript abundance of PfEMP1 domain cassette eight (DC8) and group A EPCR-binding domains in children with brain swelling, a main cause of mortality in pediatric cerebral malariaThese results suggest a potential model for explaining sustained protection against severe malaria in adults in highly seasonal malaria settings. We found that most adults maintained serorecognition of the majority of PfEMP1s, especially EPCR/ICAM-1-binding PfEMP1s, throughout the study period. This occurred amid some seasonal serologic response fluctuations, during which decreased antibody responses in the dry season were met with maintained or increased antibody responses in the malaria transmission season. Despite not having severe malaria during the study period, these adults maintained serologic responses to PfEMP1s associated with severe disease. Blood stage parasite exposure most likely sustains PfEMP1 serologic responses, including in the dry season, when adults may have asymptomatic parasite carriage. We hypothesize that any malaria exposure—even with asymptomatic infection—sustains serologic responses to non-CD36-binding PfEMP1s in adults. Declines in PfEMP1 serologic responses likely reflect a lack of parasite exposure and could signal increased vulnerability to infection.P. falciparum proteins (including 19 PfEMP1 fragments) showed only minor increases in serorecognition for adults across a malaria transmission season in Mali31. We previously found that for 21 PfEMP1 fragments from eight PfEMP1s, the proportions of adult sera that recognized intracellular and extracellular PfEMP1 fragments across the malaria transmission season did not change32. Another study with microarrays featuring 123 recombinant PfEMP1-DBL-α “tags” (a ~ unique 100 amino acid PfEMP1 extracellular region) found similar antibody responses between infected and uninfected adults in Papua New Guinea7, a region with limited PfEMP1 genetic diversity9. Interestingly, in this Papua New Guinea population, antibody responses against group A PfEMP1s appear to reduce the risk of severe disease in children, supporting the idea that sustained responses to these PfEMP1s may protect adults from severe disease as well49. The fluctuation in serologic responses across seasons and between adults observed in the present study demonstrates the importance of including a diverse and broadly representative set of large PfEMP1 fragments when analyzing changes in serologic responses across time periods and between infected and uninfected adults.A previous study of antibody responses to multiple immunogenic 50. This higher seroreactivity to intracellular domains reflects increased sequence conservation of intracellular PfEMP1 fragments versus extracellular PfEMP1 fragments. Thus, while some extracellular antigens tend to be more seroreactive than intracellular antigens51, conserved intracellular portions of a PfEMP1 have been more seroreactive than diverse extracellular portions of the same PfEMP132. These unusually low serologic responses to intracellular PfEMP1 fragments that we have observed in a minority of Malian adults suggests an altered immune response due to an unknown host factor that was not ascertained in this study.Additionally, several adults consistently lacked serorecognition of intracellular PfEMP1 fragments despite high levels of seroreactivity to extracellular PfEMP1 fragments. Seroreactivity to intracellular domains has previously been regarded as a measure of malaria exposure and has been observed to be higher in adults versus children in Mali and Kenya and higher versus extracellular PfEMP1 domains36, so we do not know whether any of the 18 adults experienced such malaria episodes. Our limited sample size of 18 adults also limited our ability to detect subtle trends in serologic responses. Additionally, 20 percent of PfEMP1 domains in the reference strain 3D7 were not successfully expressed and so were not represented on the microarray. Another potential limitation is that there may be seasonality to the predominant PfEMP1s in a community. If this is the case, then seroepidemiological trends may reflect this seasonality, but such PfEMP1 seasonality has not yet been demonstrated.This study had several limitations. The vaccine trial that was the source of sera for this study did not include systematic monitoring for uncomplicated or asymptomatic malaria episodesFuture analysis should study PfEMP1 serologic responses over longer time periods and in a large, closely followed cohort of adults in malaria-endemic regions to identify specific PfEMP1 domains that are crucial for conferring protection against clinical malaria.Supplementary Dataset 1.Supplementary Dataset 2.Supplementary Information


Plasmodium gametocytes that are responsible for furthering malaria transmission. Assessing the relationship between total parasitaemia and gametocytaemia in field surveys can provide insight as to whether detection of low-density, asymptomatic Plasmodium falciparum infections with sensitive molecular methods can adequately detect the majority of infected individuals who are potentially capable of onward transmission.Further reductions in malaria incidence as more countries approach malaria elimination require the identification and treatment of asymptomatic individuals who carry mosquito-infective P. falciparum infections were screened by rapid diagnostic tests, blood smear, and quantitative PCR of dried blood spots targeting the varATS gene in genomic DNA. A multiplex quantitative reverse-transcriptase PCR assay targeting female and male gametocyte genes , a gene with a transcriptional pattern restricted to asexual blood stages (piesp2), and human GAPDH was also developed to determine total parasite and gametocyte densities among parasitaemic individuals.In a cross-sectional survey of 1354 healthy children and adults in three communities in western Kenya across a gradient of malaria transmission , asymptomatic varATS-detectable asymptomatic infections was greatest in Ajigo (42%), followed by Webuye (10%). Only two infections were detected in Kapsisywa. No infections were detected in Kipsamoite. Across all communities, children aged 11–15 years account for the greatest proportion total and sub-microscopic asymptomatic infections. In younger age groups, the majority of infections were detectable by microscopy, while 68% of asymptomatically infected adults (> 21 years old) had sub-microscopic parasitaemia. Piesp2-derived parasite densities correlated poorly with microscopy-determined parasite densities in patent infections relative to varATS-based detection. In general, both male and female gametocytaemia increased with increasing varATS-derived total parasitaemia. A substantial proportion (41.7%) of individuals with potential for onward transmission had qPCR-estimated parasite densities below the limit of microscopic detection, but above the detectable limit of varATS qPCR.The prevalence of P. falciparum. Experimental studies are needed to definitively determine whether the low-density infections in communities such as Ajigo and Webuye contribute significantly to malaria transmission.This assessment of parasitaemia and gametocytaemia in three communities with different transmission intensities revealed evidence of a substantial sub-patent infectious reservoir among asymptomatic carriers of The online version contains supplementary material available at 10.1186/s12936-021-03905-w. Plasmodium parasites, gametocytes, present in the blood of infected humans be ingested by female Anopheles mosquitoes during feeding. Strategies that combine effective control of the mosquito vector through use of insecticide-treated nets (ITNs) and indoor residual spraying alongside rapid diagnosis and effective treatment of malaria with artemisinin-based combination therapy (ACT) have reduced the prevalence of Plasmodium falciparum infection and the incidence of clinical malaria in endemic areas of Africa since 2000, albeit at a slower rate in recent years  were cultured in vitro using standard techniques [P. falciparum parasites were tenfold serially diluted in whole blood of an uninfected North American donor to obtain a final density of 440,000 down to 0.44 parasites/µl and spotted on 903 Protein Saver cards. To produce parasite RNA for use in standard curves for gametocyte density estimates, P. falciparum NF54 (MR4) in vitro cultures were enriched for gametocytes by decreasing asexual parasitaemia [To produce parasite genomic deoxyribonucleic acid (gDNA) for use in standard curves for parasite density determination, chniques  with twositaemia . Total gTotal DNA was extracted from three 0.32 cm diameter circles punched from each DBS using the QIAamp 96 Blood Kit  per the manufacturer’s instructions and eluted in 50 µl EDTA buffer. RNA was extracted from whole-blood RNA in Tempus using Norgen RNA extraction kit  and treated with RNase-Free DNase I Kit (Norgen Biotek) to a final elution volume of 50 µl, per manufacturer’s instructions. Extracted RNA samples were assessed for quality and quantity using automated parallel capillary electrophoresis .P. falciparum genomic DNA isolated from the DBS, primers targeting P. falciparum varATS that were originally designed for use in a Taqman-based qPCR assay [varATS amplicons and wells with Tm1 < 71.14 °C contained primer dimers. Subsequently, the criteria for a P. falciparum positive sample were set as having ≥ 2 of 3 replicate wells with a Ct < 39 AND a Tm1 > 71.14 °C. Using these criteria, genomic DNA samples isolated from the blood of 20 of 20 (100%) healthy North American controls with no malaria exposure history were confirmed to be P. falciparum negative, and 86 of 87 (98.9%) samples positive by conventional P. falciparum 18s rRNA PCR [varATS-based assay. The one discordant sample had only 1 of 3 replicate wells meeting the Ct and Tm1 criteria. A standard curve of gDNA extracted from serially diluted P. falciparum-spiked DBS samples (described above) and no-template negative controls were run on every plate, which allowed for estimation of parasite densities using Ct values.To detect the presence of CR assay  were adarRNA PCR , 23 werepfs25 [pfs230p were used as the female- and male-specific gametocyte targets, respectively. The gene encoding for parasite-infected erythrocyte surface protein  was chosen based on a transcriptional pattern restricted to asexual blood-stages, particularly trophozoites, in three P. falciparum gene expression datasets available on PlasmoDB (http://plasmodb.org) [pfs25 were adapted from Wampfler et al. [pfs230p and piesp2 were developed de novo using Primer 3 software [An initial aim of the study was to develop a four-plex real-time quantitative reverse transcription PCR (RT-qPCR) that would detect female gametocytes, male gametocytes, and asexual parasites, as well as a human housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which served as a control for RNA extraction and relative quantification. The genes pfs25  and pfs2odb.org) . Primersr et al. . Primerssoftware  followinGAPDH master mix (Applied Biosystems), Taqman multiplex master mix (Applied Biosystems), and cDNA to a final reaction volume of 10 μl. Field samples identified as positive for P. falciparum by varATS qPCR, no reverse transcriptase controls, amplification controls, and tenfold parasite RNA dilution standards were assayed in triplicate in 384-well MicroAMP Optical PCR plates (Applied Biosystems). The targets pfs25, pfs230p, piesp2, and human GAPDH were run in a QuantStudio6 Flex qPCR system (Applied Biosystems) with NFQ-MGB Quencher and VIC, FAM, ABY, and JUN reporter dyes, respectively  from 50 ng RNA for each sample replicate using LunaScript RT Supermix (New England Biolabs) under standard cycling conditions, 20× triplex master mix was prepared from appropriate final concentrations of primers and probes for the three parasite targets and combined with 20× human https://www.r-project.org). Sample size estimates were performed using the epiR package. Multiple logistic regression was performed with PCR-confirmed gametocytaemia as the dependent variable and gender, age (in years), recent bed net use, recent travel, log10 transformed parasite density, and community as independent variables. Plots were rendered using the ggplot2 package. Statistical tests used to determine significance are indicated in tables and figure legends, and p values < 0.05 were considered significant.All statistical analyses were performed using R version 4.0.1  and total PCR-detectable  asymptomatic infections  as having gametocytaemia based on the quantifiable expression of either gene. Among individuals with varATS-detectable parasitaemia (n = 122), there were no significant differences in gender distribution, age, use of ITNs, history of recent travel, or site distribution between those with and without gametocytes by univariate analysis  had > 5 gametocytes/μl estimated by pfs25  of individuals with potential for onward transmission, defined in our study as having at least 1.25 female and four male gametocytes per 2.5 µl of blood . High malaria transmission was observed in Ajigo, where 42% of individuals had asymptomatic parasitaemia. Webuye demonstrated moderate transmission with 10% prevalence of asymptomatic parasitaemia, which is lower than what has been previously described at this site [The current descriptive study provides a cross-sectional assessment of asymptomatic er 2016. . Similarer 2016. , 32, whehis site , 34, posP. falciparum infections were more prevalent in younger children age 5–14 years (~ 34%) relative to older children > 14 years (~ 9%) [The substantial reservoir observed among 6–15 year old children in Ajigo and Webuye is consistent with a prior study in the Kakamega district of western Kenya that showed PCR-confirmed asymptomatic s (~ 9%) . Howevers (~ 9%) . The difP. falciparum infections who are also potentially capable of onward transmission, the relationship between asexual parasitaemia and gametocytaemia was assessed. The initial goal was to correlate gametocyte densities with asexual parasite densities using a multiplex RT-qPCR that would contain targets specific to female gametocytes, male gametocytes, and asexual blood-stage parasites in a single assay, which would facilitate comparisons as this strategy eliminates both within subject differences in template preparation and assay variability. However, parasite densities determined using the chosen asexual-specific target piesp2, which encodes for parasite-infected erythrocyte surface protein and previously shown to be maximally transcribed in the trophozoite stage in laboratory isolates [varATS qPCR using gDNA (Additional file piesp2 could be due to lower piesp2 expression in ring stages, which had previously been thought to be the predominant asexual form of P. falciparum found in peripheral circulation. However, a recent study in Mali revealed that more developed trophozoite stages were commonly found in asymptomatic P. falciparum infections [piesp2 could also vary among the field isolates, perhaps due to differential transcriptional regulation related to precise stage at the time of collection or host immune pressure. Although speculative, these potential explanations are intriguing given that antibodies against PIESP2 associate with protection from malaria [piesp2 is a poor target for quantifying asexual parasite densities.To determine whether sensitive molecular assays can sufficiently detect the majority of individuals carrying low-density isolates –27, 37, fections . Express malaria , and PIE malaria . The curvarATS-derived parasite densities, asexual parasitaemia and residence in a high-transmission setting independently predicted gametocytaemia, consistent with a recent longitudinal analysis of gametocyte carriage in Kilifi, Kenya [varATS qPCR, which had a limit of detection of ~ 0.4 parasites/μl using dried blood spots, the proportion of individuals with potential for onward transmission drops off below 10 parasite/μl. This finding is in line with recent studies suggesting that mosquito infectivity occurs primarily when parasitaemia is > 1 parasite/μl [Nevertheless, using i, Kenya . On the i, Kenya , would ci, Kenya , 43, 44.asite/μl , 45, whiThere are several limitations to the current study. Although the study excluded individuals who endorsed symptoms of acute illness at the time of enrollment and sample collection, clinical or laboratory examinations that may have uncovered a more subacute or indolent disease process beyond self-reported symptoms were not performed. No short-term follow-up was conducted to assess whether asymptomatic individuals progressed to symptomatic malaria as other studies have done , 47. By P. falciparum infections among asymptomatic individuals in western Kenya provides an assessment of the relationship between parasitaemia and gametocytaemia in three communities with different transmission intensities. These data provide evidence of a substantial sub-patent infectious reservoir among asymptomatic carriers in these communities and supports prior findings that conventional molecular diagnostics may be capable of detecting the vast majority of infections capable of onward transmission [P. falciparum could accelerate progress towards malaria elimination by reducing the infectious reservoir.In summary, this cross-sectional survey of the prevalence and densities of smission . DevelopAdditional file 1: Table S1. Supplementary tables and figures.