In recent years there have been reports of viral haemorrhagic fever (VHF) epidemics in sub-Saharan Africa where malaria is endemic. VHF and malaria have overlapping clinical presentations making differential diagnosis a challenge. The objective of this study was to determine the prevalence of selected zoonotic VHFs and malaria co-infections among febrile patients seeking health care in Tanzania.This facility-based cross-sectional study was carried out between June and November 2018 in Buhigwe, Kalambo, Kyela, Kilindi, Kinondoni, Kondoa, Mvomero, and Ukerewe districts in Tanzania. The study involved febrile patients seeking health care from primary healthcare facilities. Blood samples were collected and tested for infections due to malaria, Crimean-Congo haemorrhagic fever (CCHF), Ebola virus disease (EVD), Marburg virus disease (MVD), Rift Valley fever (RVF) and yellow fever (YF). Malaria infections were tested using rapid diagnostics tests while exposure to VHFs was determined by screening for immunoglobulin M antibodies using commercial enzyme-linked immunosorbent assays. The Chi-square test was used to compare the proportions.P < 0.05). District was significantly associated with co-infection (P < 0.05) with the highest prevalence recorded in Buhigwe (1.2%) followed by Kinondoni (0.9%) districts. Headache (100%) and muscle, bone, back and joint pains (83.3%) were the most significant complaints among those infected with both VHFs and malaria (P = 0.001).A total of 308 participants (mean age = 35 ± 19 years) were involved in the study. Of these, 54 (17.5%) had malaria infection and 15 (4.8%) were positive for IgM antibodies against VHFs . Six (1.9%) individuals had both VHF  and malaria infections. The highest co-infection prevalence (0.6%) was observed among individuals aged 46‒60 years (Co-infections of VHF and malaria are prevalent in Tanzania and affect more the older than the younger population. Since the overlapping symptoms in co-infected individuals may challenge accurate diagnosis, adequate laboratory diagnosis should be emphasized in the management of febrile illnesses. Viral haemorrhagic fevers (VHF) have become more prevalent in sub-Saharan Africa during recent years, causing either sporadic or large-scale epidemics, thus posing significant risk to public health. The VHFs reported in sub-Saharan Africa in recent years include Crimean-Congo haemorrhagic fever (CCHF), Ebola virus disease (EVD), Lassa fever (LF), Marburg virus disease (MVD), Rift Valley fever (RVF), and yellow fever (YF) –4. On thOf the VHFs, RVF and CCHF outbreaks have been reported in Tanzania –8. AlthoIn sub-Saharan Africa, VHF and malaria co-infections have been reported in Gabon , SenegalMost VHFs and malaria initially present with similar non-specific symptoms and signs of fever, headache, joint pains and weaknesses, posing challenges in differential diagnosis , 25. CliThis study was carried out in eight districts in five distinct ecological zones of Tanzania. The zones are Western, Southern-highlands, North-eastern, Central and Lake Victoria. The western zone is characterised by tropical forest, unimodal rainfall pattern, and an altitude of 2300 m. The Southern-highlands zone covers regions with high precipitations, tropical forest, bimodal rainfall pattern, low temperatures and elevation > 2300 m. The North-eastern Tanzania is characterised by a bimodal rainfall pattern and seasons of high temperatures. The Central zone comprises of a semi-arid area at an elevation of about 1000 m characterized by moderate precipitation and unimodal rainfall pattern. The Lake Victoria area is characterized by a bimodal rainfall pattern and intermediate temperatures and an elevation of 1134 m. The study districts were Buhigwe, Kalambo (western), Kyela (southern-highlands), Kilindi, Kinondoni (north-eastern), Kondoa, Mvomero  and Ukerewe (Lake Victoria)  before being transported to the laboratory at Sokoine University of Agriculture in Morogoro, Tanzania, where they were stored at −80 ℃ until examination. Aliquots of the sera were tested for the presence of human IgM antibodies against CCHF, EVD, LF, MBV, RVF, and YF using commercial enzyme-linked immunosorbent assays (ELISA) kits , according to the manufacturer's instructions. To validate the ELISA, determination of the intra-assay coefficient of variation (CV), inter-assay CV, recovery, linearity and parallelism was performed. The intra-assay CV (%) and inter-assay CV (%) were less than 15% for reactive samples.P-value was below 0.05. Cut-off values for ELISA antibody analysis were determined following the manufacturer’s instructions. The assay performance was considered adequate when the intra- and inter-assay coefficient of variabilities were less than 15%.Data were analysed using Statistical Package for the Social Sciences (SPSS) Statistics version 23 . Frequencies, median, and associated interquartile ranges (IQRs) were calculated. The Chi-square test (or Fisher’s exact test where appropriate) was used to compare the proportions of positive malaria, VHF and co-infections frequency by sex and age. A statistically significant difference was considered when the A total of 308 participants were involved in the study. Of these, 185 (60.1%) were females, and 123 (39.9%) males. The mean age was 35 ± 19 years. Over half (54.2%) of participants were in the 16‒45 years age category. About two-thirds (62.3%) of participants had primary school education. Crop farmers accounted for the largest proportion (43.2%) of the participants (Table The most common symptoms among the study participants were body aches and pains which included headache (48.4%), muscle, bone, back, or joint pain (32.0%), and stomach pain 16.4%). The least observed signs were jaundice, bleeding/bruising, and red spots on the skin, eyes or mucosae (Table 6.4%. Then = 31) than males . Individuals aged 0‒15 years  were most affected by malaria. However, there was no significant difference between sex and the age groups (P > 0.05) (Table P < 0.05). Seroprevalence of RVF, CCHF, MVD and YF were similar in all age groups  were positive to the rapid diagnostic malaria test. The prevalence of malaria infection was higher among females . Location was significantly associated with co-infection (P < 0.05) as higher prevalence was recorded among those from Buhigwe (1.2%) followed by Kinondoni (0.9%) districts (Table Six (1.9%) individuals had both VHF  and malaria infections. Of those with VHF and malaria co-infections, four (66.7%) were males and two 33.3%) were females. In relation to age, the highest co-infection rate (0.6%), was observed among individuals aged 46–60 years (3.3% wereSevere headache was the most frequent (100%) complain among those with both VHF and malaria infections. Other aches, including muscle, bone, back, and joint pains were reported by 83.3% of those with co-infections of VHFs and malaria Table .Table 4MMalaria was the most important febrile associated infection in the study districts. Overall, about 5% of the study population were positive for IgM antibodies against VHF tested, with RVF accounting for the largest proportion of the infections. Co-infections of malaria and VHFs were detected in about 2% of the febrile individuals seeking care from health facilities. While RVF outbreaks have been reported to occur in low malaria endemic districts of Tanzania –32, CCHFThe VHF and malaria co-infection rate observed in this study was associated with older age. The higher prevalence of VHF and malaria co-infections among the adults is likely to be associated with higher exposure compared with children due to their livelihoods activities that bring them closer to sources of VHF infection . Many stVHFs  and malaria co-infections were most prevalent in Buhigwe district in Kigoma Region of western Tanzania, bordering the Democratic Republic of Congo. Available statistics indicate that Buhigwe is among the districts with high malaria prevalence 24%) in Tanzania , 38. Mor% in TanzAches and pains including severe headache were significantly noted in most of all the co-infected patients in this study compared to patients with VHF alone. However, this study could not determine whether malaria, VHF or both to be responsible for the observed symptoms. Our study agrees with others that patients with VHFs usually present with non-specific signs, including fever, vomiting, diarrhoea, fatigue, myalgia and headache . Most ofThe presence of antibodies against RVF, CCHF, EVD, MVD and malaria in this study was highly unexpected. The results are alarming because if sublethal VHF infections commonly occur, they are likely to be missed in co-infections with malaria.This study has limitations including use of rapid test to diagnose malaria, which could result to some cases been missed due to low parasitaemia. In addition, no information on the use of antimalarials prior to the test was obtained which could result to false positives. The study was conducted between June and November, a period associated with both high and low malaria season depending on the district, which could influence visits to health facilities and availability of febrile patients as treatment seeking is low during low malaria seasons, and levels of malaria risk obtained. The serological results are based on antibody detection which are likely to be a reflection of cumulative exposure and/or antibody persistence following single or multiple exposure. Moreover, point of care tests also have limitations associated with possibility that one or more as-yet-unknown pathogens are circulating among the human populations sampled in this study, producing antibodies cross-reactive. The results of this study should be interpreted with caution. Despite useful information gathered on the presence of VHF and malaria co-infections, the findings from this study are based on very small sample hence weaken the accuracy and may not be representative.The findings of this study show that the prevalence of co-infections of viral haemorrhagic fevers and malaria in Tanzania is generally low, though higher rates were observed among the older individuals. Since infections due to malaria and VHF are associated with similar symptoms, these findings suggest the need for improvement in case management of febrile illnesses based on adequate laboratory diagnosis. It is equally important that more studies are needed to establish how VHFs and malaria infections interact to improve case management and control in the future. The findings also highlight the need to conduct molecular studies to identify specific pathogens.


Plasmodium falciparum malaria in 400 febrile patients from a low-transmission region (Jazan) in southwestern Saudi Arabia. Diagnostic performance of all three methods was compared using microscopy and nested PCR as reference methods. Overall, 42 (10.5%), 48 (12.0%), and 57 (14.3%) samples were found positive by microscopy, RDT, and PCR, respectively. With PCR as reference method, the RDT showed higher sensitivity (79% vs. 71.9%), similar specificity (99.1% vs. 99.7%), and better NLR (0.20 vs. 0.27) and area under the curve (89.0% vs. 85.8%) than microscopy. The sensitivity of RDT and microscopy decreased as age increased, and false negatives were associated with low parasite density. In addition, the sensitivity of RDT and microscopy was higher in non-Saudi than in Saudi participants. Against microscopy, both RDT and PCR showed high sensitivity (83.3% vs. 97.6%), specificity (96.4% vs. 95.5%), and NPVs (98.0% vs. 99.7%), but reduced PPVs (72.9% vs. 71.9%), respectively. The results showed that the performance of the AllTest Malaria p.f./p.v RDT was better than that of microscopy in diagnosing P. falciparum malaria among febrile patients in the Jazan region when nested PCR was used as the reference. However, further studies are required to assess malaria diagnostic methods among asymptomatic individuals in the region.This cross-sectional study aimed to assess the performances of a rapid diagnostic test (RDT)—the AllTest Malaria p.f./p.v., microscopy, and nested polymerase chain reaction (PCR) for diagnosing  Plasmodium species transmitted by female Anopheles mosquitoes, namely Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. In addition, P. knowlesi—a zoonotic Plasmodium species that naturally infects macaques, causing simian malaria—has been identified as a fifth species that causes malaria in humans. Naturally acquired human P. knowlesi cases have been reported in many parts of southeast Asia, particularly Malaysia [Malaria is a leading cause of death in developing countries, particularly in tropical and subtropical regions . In 2020Malaysia ,5.Plasmodium parasite and estimating parasite density. However, the sensitivity and specificity of microscopy depend on the quality of the stained slide and the competency of the microscopists [A prompt, accurate diagnosis followed quickly by effective treatment is critical for effective management and surveillance of malaria; these are considered the main pillars of the global technical strategy for malaria 2016–2030 . Microscscopists . Moreovescopists ,10.P. falciparum histidine-rich protein 2 (Pf-HRP2), lactate dehydrogenase (LDH), which can be either pan-specific  or species-specific (P.f.-specific or P.v.-specific), and genus-specific aldolase which detects all Plasmodium species [Rapid diagnostic tests are easy to use, fast, cost-effective, and field-deployable tools for malaria diagnosis . Three a species . The fir species ,14. Howe species ,16,17,18In addition, PCR is highly sensitive (2–5 parasites/μL) compared with RDTs (>100 parasites/μL) and microscopy (50–500 parasites/μL) ,20. TherIn Saudi Arabia, the national malaria control programme, established in 1948, has achieved remarkable success in reducing the annual number of malaria cases and controlling autochthonous and indigenous malaria transmission. This has been achieved through some control and elimination strategies that include annual indoor residual spraying, long-lasting insecticide-treated nets, and proper management of infection, with individual case follow-up and reactive surveillance . CurrentA cross-sectional study was used to compare three techniques used in malaria parasite detection in the Jazan region. The malaria cases included in this study were detected through passive case detection among clinically suspected cases (febrile patients) who presented at healthcare facilities in selected governorates of the Jazan region.2 and a population of approximately 1.8 million [Anopheles arabiensis the main vector [P. falciparum malaria. This was replaced by artemisinin-based combination therapy, with artesunate plus sulfadoxine-pyrimethamine and a combination of artemether-lumefantrine used as first- and second-line treatments [P. falciparum malaria [The Jazan region is in the southwest of the Kingdom of Saudi Arabia, 1100 km from the capital, Riyadh. It is the smallest region in the country, with a total land area of approximately 11,671 km million . The reg million . The regn vector . Until 2eatments . In addi malaria .Blood samples were collected from febrile patients  presenting at selected hospitals in the study area. All patients were enrolled regardless of their nationality, age, or sex. The participants’ demographic data were collected from their medical records.® filter papers . The prepared blood spots were kept in individual zipped plastic bags at 4–6 °C and used for molecular examination.Approximately 2–3 mL of venous blood was collected from each patient into an EDTA tube clearly labelled with the patient’s reference number, name, sex, and age. Directly after collection, blood samples were examined using a malaria RDT. Thin and thick blood films were prepared on clean, labelled glass slides and stained with diluted Giemsa stain. In addition, blood spots were prepared on clean, labelled 3MM WhatmanPlasmodium parasites under a 100 × objective. The thick smears were used to assess the presence of malaria infection and parasite density, while the thin smears were examined to identify the parasite species and stages. At least 200 high power fields of the thick smear had to be examined before a sample was considered negative [The thin and thick blood smears were stained for 45 min with 3% buffer-diluted Giemsa stain solution (pH 7.2) and then examined for negative .For positive smears, parasite density was estimated by counting the asexual stages against 300 WBCs and then multiplying by 25, assuming the mean total WBC count of any human being is 8000 cells per 1 μL of blood . About 2P. falciparum-specific HRP2 on the P.f. test line and P. vivax-specific pLDH on the P.v. test line. A third line is used for positive control. A sample was considered negative if only one band appeared on the control, whereas the presence of another band either in the P.f. or P.v. area together with the control band was considered a P. falciparum- or a P. vivax-positive result, respectively. Furthermore, when all three bands (a control band and two test area bands) appeared simultaneously, a P. falciparum/P. vivax mixed infection was recorded. If no control band appeared, the test was considered invalid.This study used AllTest Rapid Test—Malaria p.f./p.v. kits, product code IMPV-402 . This RDT detects ® Blood & Tissue Kit, Cat. no. 69506, Hilden, Germany). Using a sterile puncher (6 mm diameter), one or two discs of the dried blood spots were cut, placed into a 1.5 mL microcentrifuge tube, and processed for DNA extraction; DNA was eluted using 50 µL AE  elution buffer and kept at −20 °C until used. The samples were examined using a conventional nested PCR assay using different oligonucleotide primers based on Plasmodium 18s rRNA genes following an established protocol [P. falciparum, P. vivax, P. malariae, and P. ovale).Following the manufacturer’s instructions, DNA was extracted using a Qiagen blood and tissue kit  and presented with corresponding 95% confidence intervals (CI). In addition, kappa statistics were used to assess agreement between the tests. The area under the curve (AUC)—a powerful way to summarize a diagnostic test’s overall accuracy—was evaluated using the receiver operating characteristic (ROC) curve analysis. A p-value of < 0.05 was considered significant.Data were analysed using IBM SPSS version 20 . The performances of microscopy, AllTest Malaria RDT and PCR in diagnosing malaria were evaluated using the following indicators: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), negative likelihood ratio (NLR), positive likelihood ratio (PLR), and accuracy. These indicators were calculated using the MedcalcP. falciparum, with 34 of these found positive by all three methods. Overall, 42 (10.5%), 48 (12.0%), and 57 (14.3%) samples were found positive by microscopy, RDT, and PCR, respectively.This study tested 400 blood samples using light microscopy of Giemsa-stained blood films, RDT  and nested PCR. The results are summarised in Most of the 45 samples found positive by both PCR and RDT were among male (77.8%), rural (64.4%), and non-Saudi (82.2%) participants; 44.4% were among participants aged 18–30, and 58.5% were in samples with low parasite density. Similar results were found in the 41 samples found positive by both microscopy and PCR. Two RDT-positive samples were considered false positives based on both microscopy and PCR; 16 (4.5%) were considered false negatives among the microscopy-negative samples. Interestingly, 12 PCR-positive samples were found negative by RDT, one of them with moderate parasite density.P. falciparum positive cases were 89.0%  and 85.8% , respectively, indicating outstanding and excellent discriminating ability (The comparative performances of the methods are presented in  ability A.p < 0.001) and 96.6% , respectively . Compared with microscopy, ROC curves showed outstanding discriminating ability for both RDT and PCR, with AUC of 89.9%  and 28.1% (16/57) of cases that had been missed by RDT and microscopy, respectively; 8.3% (5/60) were identified only by PCR. Data analysis was performed based on two scenarios considering PCR and microscopy as reference methods.An accurate, prompt diagnosis is crucial for managing, controlling, and eliminating malaria. This study evaluated the performance of an RDT, microscopy, and PCR in diagnosing Plasmodium species. It also accurately detects mixed-species infections; thus, it has been widely used for diagnosis, epidemiological surveys, and drug efficacy trials [P. falciparum malaria. The results revealed that the diagnostic performance of the AllTest Malaria p.f./p.v. RDT  was better than that of light microscopy . The results also showed that the RDT and microscopy had high PPVs and NPVs compared with nested PCR, minimising false positives and negatives. These findings agree with the only previous study in the Jazan region among patients attending King Fahd Central Hospital, which also evaluated the three methods [PCR is highly sensitive when detecting malaria parasites and accurately identifies y trials ,20. Furty trials ,34,35,36 methods . Hawash  methods  reportedP. falciparum malaria using PCR as a reference [The accuracy of malaria diagnostics depends on several factors, including the level of malaria endemicity, parasite density, mutation or deletion of the gene encoding the HRP2/HRP3, format and type of the RDT product, and storage conditions ,18,38,39eference ,45,46,47Plasmodium infectious agents, such as African Trypanosoma, and immunological factors such as rheumatism have also been reported [In this study, the AllTest Malaria p.f./p.v. RDT failed to detect 12 (21.1%) positive cases that PCR detected, 7 of which were confirmed by microscopy. This may lead to patients going untreated and becoming parasite carriers and malaria reservoirs in their communities . Such HRreported .This study showed that microscopy missed 16 (27.6%) positive cases; however, 11 were detected by RDT and treated; microscopy performance is associated with parasite density . A meta-P. falciparum parasites. The sensitivity of the AllTest Malaria p.f./p.v. RDT and PCR were 83.3% and 97.6%, respectively, and the specificity was 96.4% and 95.5%, respectively. However, the PPVs of the RDT and PCR were 72.9% and 71.9%, respectively; this indicates that these methods overestimated the positivity rate by approximately 27% and 28%, respectively. Nonetheless, both methods showed PLRs over 10 and NLRs below 0.2, indicating that they are useful diagnostic methods for P. falciparum malaria [P. falciparum malaria using microscopy as a reference [Plasmodium species circulating in the targeted area [Using microscopy as a reference method, the present study revealed that both RDT and PCR methods performed well in detecting  malaria . PPVs an malaria ,61. Alth malaria . Previou malaria  and elseeference ,49,50,51ted area .This study showed that the performance of RDT and microscopy decreased as age increased, agreeing with previous studies ,36,47; tP. falciparum and P. vivax, its performance in detecting the latter or mixed infections was not assessed. Fourth, according to the literature, the AllTest RDT has not been assessed by the WHO’s Malaria RDT Product Testing Programme; however, another product  by the same manufacturer was tested and failed to meet the performance criteria [This study had some limitations. First, the data relied on suspected cases in patients presenting at the participating hospitals. This limited the ability to detect asymptomatic, sub-microscopic malaria infections; sub-microscopic infections are predominant in low-transmission settings . Second,criteria . The percriteria ; therefoP. falciparum malaria in symptomatic patients in the Jazan region, a low-transmission area. Using PCR as a reference method, the AllTest RDT showed better performance than light microscopy and had higher sensitivity and approximately equivalent specificity, PPVs, NPVs, and PLRs, but desirably lower NLRs. However, further studies to evaluate malaria diagnostics among asymptomatic individuals in the region are required.This study showed that the AllTest Malaria p.f./p.v. Rapid Test Cassette is a useful tool for diagnosing


Severe malaria (SM) is a leading cause of global morbidity and mortality, particularly in children in sub-Saharan Africa. However, existing malaria diagnostic tests do not reliably identify children at risk of severe and fatal outcomes. Dysregulated host immune and endothelial activation contributes to the pathogenesis of SM. Current research suggests that measuring markers of these pathways at presentation may have clinical utility as prognostic indicators of disease progression and risk of death. In this review, we focus on the available evidence implicating soluble urokinase-type plasminogen activator receptor (suPAR) as a novel and early predictor of severe and fatal malaria and discuss its potential utility for malaria triage and management. Plasmodium falciparum and Plasmodium vivax infections. Regardless of the method used to detect malaria parasites, no current diagnostic test can reliably predict which children will progress to severe or fatal disease. Dysregulated host immune and endothelial activation contributes to the pathogenesis of severe malaria (SM)  . Host-baria (SM) –6.Soluble urokinase-type plasminogen activator receptor (suPAR) is an indicator of immune and endothelial activation and plays an important role in processes including cell migration, adhesion, and chemotaxis. Elevated suPAR levels have been linked to poor prognosis in various infections including HIV-1, tuberculosis, and sepsis. Emerging evidence also suggests that increased circulating levels of suPAR are associated with disease severity and mortality in children with malaria. Here, we report an overview of urokinase/urokinase-type plasminogen activator receptor (uPA/uPAR) biology, review what is known about suPAR in pediatric malaria, and discuss the ability of suPAR to predict disease outcome and its potential as a druggable target. We summarize putative mechanisms through which suPAR may contribute to the pathogenesis of malaria. Lastly, we outline the need for research investigating the causal role of suPAR in malaria and the evaluation of a currently available suPAR point-of-care test as a potential triage tool for severe malaria in prospective trials.uPAR is a glycosylphosphatydilinositol (GPI)-anchored protein that contains three homologous domains  and is expressed on the surface of various cell types including immune, endothelial, epithelial, and smooth muscle cells , 8. suPAuPA, a serine protease, and its cellular receptor uPAR exhibit pleiotropic functions during both physiological and pathological processes. The uPA/uPAR system mediates numerous critical cellular functions including extracellular proteolysis, chemotaxis, cell adhesion, vascular homeostasis, and tissue remodeling and repair , 15–17.The uPA/uPAR system has a central role in fibrinolysis and the modulation of host inflammatory and immune responses , 19. BinThe uPA molecule is directly involved in mechanisms of migration, adhesion, and chemotaxis . Importavia integrated mechanisms including proteolysis, adhesion, migration, and mitogenesis  , interferon gamma (IFN-γ), and interleukin 2 (IL-2)] . For exaammation . These fukocytes , 47. In igration . uPA-kno(in vivo . Further(in vivo . In respnfection . Collectrophils) . HoweverIn vivo, suPAR may stimulate the recruitment and activation of leukocytes, which promotes inflammation and immune activation  including tuberculosis, bacteremia and/or sepsis, meningitis, hepatitis B and C, HIV-1, hemorrhagic fever, hantavirus, malaria, and most recently coronavirus disease 2019 (COVID-19) –72. AcroIn patients with HIV-1 infection, suPAR is associated with disease severity, mortality risk, and ineffective immune recovery , 68. EleIn a study of patients with symptoms of COVID-19, suPAR levels were significantly higher in patients who developed severe and critical illness compared with those who were moderately ill, and it was shown that suPAR cutoffs could be used to risk-stratify patients , 75. In Another set of studies used suPAR POC testing to quantify suPAR levels, risk-stratify patients to identify those at higher risk of respiratory failure or death, and provide early targeted treatment for these individuals with a drug intervention (anakinra) , 80.P. falciparum malaria, Perch et al. found that serum suPAR levels at study inclusion were associated with parasite density and children with the highest parasitemia had significantly higher suPAR levels than children who had lower parasitemia or had a negative blood film  might be beneficial. In cell culture experiments, uPA binds to human erythrocytes infected with mature forms of P. falciparum and is required for the rupture of erythrocytes and the release of merozoites, which suggests that uPA may be an additional adhesion molecule for PEs  Foundation grant (FDN-148439 to KK) and the Canada Research Chair Program (KK). This work was also supported by a Collaborative Research Agreement Grant from Intellectual Ventures/Global Good (KK), the Bill and Melinda Gates Foundation Trust through Intellectual Ventures/Global Good, and donations from Kim Kertland and the Tesari Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In comparison to all other groups, school-age children (5–15 years) had the highest prevalence of malaria. Our results show that mRDT may miss up to 6% of cases of malaria mainly due to low-density parasitemia when compared to LM and PCR. Routinely used mRDT will likely miss the sub-microscopic parasitemia which will ultimately contribute to the spread of malaria and hinder efforts of elimination.Global malaria epidemiology has changed in the last decade with a substantial increase in cases and deaths being recorded. Tanzania accounts for about 4% of all cases and deaths reported in recent years. Several factors contribute to the resurgence of malaria, parasite resistance to antimalarials and mosquito resistance to insecticides being at the top of the list. The presence of sub-microscopic infections poses a significant challenge to malaria rapid diagnostic tests (mRDT). Our cross-sectional surveys in Handeni and Moshi, Tanzania assessed the effect of low parasite density on mRDT. Handeni had higher malaria prevalence by mRDT (39.6%), light microscopy (LM) (16.9%) and polymerase chain reaction (PCR) (18.5%), compared to Moshi with prevalence of 0.2%, 1.3% and 2.3%, respectively. A significant difference ( Anopheles mosquito densities [In recent years, there has been little progress in the fight against malaria globally and malaria cases and deaths are on the rise . Accordiensities ,4. Studiensities . There iensities ,7.Individuals with asymptomatic and sub-microscopic infections rarely seek medical treatment and they can act as potential reservoirs for malaria transmission ,9,10,11.The low-density parasitemia will likely contribute to the new transmission circles when mosquito numbers increase in the rainy seasons . Sub-micThe introduction of mRDT in Tanzania more than ten years ago led to the replacement of LM in most health facilities . AlthougThis study aimed at investigating the impact of sub-microscopic malaria infections on mRDT in comparison with LM and PCR across areas with diverse malaria transmission endemicity.The study was conducted in Handeni, Tanga region and Moshi in Kilimanjaro region . HandeniAnopheles arabiensis, which are the secondary vectors in malaria transmission. Lower Moshi is a low malaria-endemic area with a prevalence of about 1% in the last 10 years [The second study site, Moshi, is located 10 km from Moshi municipality, and 800 m above sea level, south of Mount Kilimanjaro. Most of the population in the area is engaged in agriculture with irrigated rice and sugarcane cultivation as the main crops. Irrigation activities provide an important breeding site for 10 years .Participants of the study were residents from the two areas with an age of 6 months and above.A community sensitization campaign was carried out in the study areas, where village residents were invited to designated dispensaries to participate in the study. Potential participants were informed in detail of the study and invited to voluntarily participate in the study by signing an informed consent form. About 1003 participants gave consent to participate, and their demographic information were collected following a questionnaire.® protein saver cards  followed by drying at room temperature overnight. Dried filter papers were stored in a bag containing silica gel at room temperature.A finger prick blood was collected for mRDT, microscopy and dried blood spots. Blood spots were prepared from 50 µL of blood on WhatmanSampling in both the study sites was done at the end of the long rainy season between April and June 2018.Malaria detection on site was done using SD BIOLINE pf/pan Ag test kit (Standard Diagnostic INC-Korea) following testing procedures as per manufacturer’s instructions. Participants found to be positive were treated using Artemether-lumefantrine (ALu), the recommended first-line antimalarial treatment for uncomplicated malaria in Tanzania.Thick and thin blood smears were prepared using freshly collected blood. The thin smear was fixed using absolute methanol. Both smears were stained for 10 min with 10% Giemsa solution, washed with distilled water and dried on a slide warmer . Slides were packed in slide boxes and stored at room temperature and the reading took place within 7 days after preparation. Slide reading and recording were done by qualified microscopists who are examined for competence 3 times a year with control slides from College of American Pathologists (CAP). Parasite/µL was estimated by the function of parasite counted against 200 white blood cells in thick smear and 8000 divided by the number of leucocytes as described in the WHO protocols . ParasitDNA for the Polymerase Chain Reaction was extracted following a modified-chelex method , where fPlasmodium species were detected using a nested-PCR (PCR) technique targeting 18S ribosomal RNA, specifically for P. falciparum, P. malariae, P. vivax and P. ovale identification as previously described by Snounou et al. [u et al. .Statistical analysis was conducted using Stata 16 software .p < 0.05. A two-sample test of proportions (prtest) was used to determine if there were statistically significant differences in prevalence (proportions) between groupsParasitemia was log-transformed and presented in a scatter plot. Proportions and frequencies were presented in tables and figures. Wilcoxon rank-sum (Mann-Whitney) test was used to test statistically significant differences between age groups if Cohen’s kappa statistic was used to measure reliability of PCR, RDT and LM results with 95% confidence intervals. Positive likelihood and negative likelihood ratio tests were calculated as follows :PositiveTotal agreement percent was calculated as the sum of true positives and true negatives divided by total tests. The study enrolled 1003 participants, but we only included 985 participants in this analysis, who had complete data collected, of which 513 (52.1%) were from Handeni and 472 (47.9%) from Moshi. The majority of the participants were over 15 years of age, constituting 54.1%. Children aged 5–15 years and ˂5 years comprised 23.5% and 22.4%, respectively. p ˂ 0.001) when compared to Moshi. Furthermore, when comparing the individual tests within sites, mRDT detected a significantly higher proportion of malaria cases in the Handeni site than LM and PCR (p ˂ 0.001) different from the Moshi site, where PCR detected more malaria cases than RDT and LM (p ˂ 0.001). There were also significant differences in malaria prevalence across age groups in Handeni. School-age children (5–15 years) had a higher proportion of malaria cases than the rest of the age groups (p ˂ 0.001) in the Handeni site. Malaria prevalence in Moshi was too low to perform an age-wise analysis , with the lowest parasitemia of 159 p/µL and 100 p/µL, respectively. There was no significant difference in parasitemia between school-age children and those below five years of age (Children under five years of age had the highest median parasitemia (3162 p/µL) when compared to other age groups. There was a significant difference in parasitemia between participants aged below 5 years and those aged >15 years (s of age .Submicroscopic parasitemia refers to the samples negative by LM and mRDT (assuming detection thresholds are the same) but positive by PCR. Of the 204 total positive samples in all study sites, (26) 13% were positive by PCR alone, and almost equally distributed in Handeni and Moshi (12) 46% and (14) 54%, respectively. Most of the samples had low parasitemia, 22 of the 26 (85%) had parasite density below 200 p/µL and only two samples had high parasitemia. More than 58% (15) of sub-microscopic cases were from participants 15 years and above followed by school-age children (39%) and children below 5 years of age (3%) .The performance of the mRDT to detect malaria was different when compared to LM and PCR in Handeni and Moshi sites. The sensitivity and specificity of mRDT were 75.3% and 85%, respectively, when compared to PCR results. Compared to PCR, LM had a slightly higher sensitivity and specificity than mRDT. The positive predictive value for mRDT (34.31%) was almost half that of LM when compared to PCR (62.22%). RDT and LM showed similar negative predictive values. LM had a higher relative agreement value of around 94% with PCR at κ of 0.7 .Anopheles gambiae ss and Anopheles funestus mosquitoes [Anopheles arabiensis, which are zoophilic in nature, are abundant in Moshi, with a low transmission rate [In this community-based study, we report a considerable difference in malaria prevalence between the study sites. Handeni had a significantly higher prevalence when compared to Moshi, and generally there were no statistically significant difference in malaria prevalence between female and male participants. Based on the historical malaria transmission patterns in Handeni, a lowland inhabited by anthropophilic squitoes ,27, Anopion rate . These rion rate ,21,29.Rapid test (mRDT) detected more cases in Handeni compared to LM and PCR, suggesting the presence of mRDT false positivity. This was different from the Moshi site, where PCR detected more cases than mRDT suggesting the presence of submicroscopic parasitemia. Observation from several studies in high malaria transmission areas estimates up to 5% false positivity due to circulating HRP2 antigen up to five weeks after malaria treatment ; this coFalse positivity ramifications are far less compared to false negativity. False-positive individuals will still receive malaria treatment instead of for the actual disease they are suffering from. Treating false-positive malaria cases wastes resources and delays the treatment of non-malaria conditions .As compared to PCR, which is a more sensitive diagnostic tool and a comparator in this analysis, mRDT is relatively less sensitive, although low agreement was observed when compared to LM and PCR. In this study, mRDT gave positive results to 75 out of every 100 true positive individuals who lived within the study area. The low sensitivity of mRDT may be due to submicroscopic parasitemia, which cannot be detected by mRDT ,34,35. AAn age-dependent prevalence analysis showed that school-age children had higher malaria prevalence than the other groups, even though they represented only a quarter of all study participants. Children under the age of five and pregnant women are the main targets of malaria interventions. There is a significant disparity in the delivery of bed nets whereby school-age children are prioritized the least, despite being equally at risk, and this could be the reason for their high malaria prevalence ,39. ThisFindings from this study showed a high proportion of sub-microscopic parasitemia in adults (>15 years) and children of age between 5 and 15 years. This explains the possibility that adults and children of age 5 to 15 years might be experiencing repeated malaria exposure and can sustain sub-microscopic parasitemia . AsymptoA sub-microscopic infection results from immunological inhibition of parasite multiplication ,46,47,48pfhrp2 deletion, we ruled out the possibility of false negatives due to pfhrp2 deletion. The high detection threshold of mRDT compared to LM and PCR could also support this finding [Results from this study showed that mRDT failed to detect six cases and four cases in every 100 participants in Handeni and Moshi, respectively, when compared to LM and PCR combined. Since we have already submitted data on  finding . This ra finding ,55,56.This cross-sectional study reported a substantial proportion of cases of low parasitemia (˂200 p/µL) from Handeni and Moshi. There were also two cases with high parasitemia  and negative mRDT results. There have been studies showing that these submicroscopic cases tend to be asymptomatic ,58. It iFalse-negative mRDT is common in low-endemic areas and is believed to have implications for malaria elimination. The evidence of sub-microscopic infections in low malaria-endemic areas suggests that up to 50% of seasonal transmission is attributed to these infections . StudiesRDT performance was compared only for LM and PCR since these are the most commonly used diagnosis methodologies for malaria detection, especially in research. We did not test other techniques such as Loop-mediated Isothermal Amplification (LAMP) which is a nucleic acid amplification assay, which is sensitive, rapid and cheaper compared to PCR. In this study we did not test for immunological factors that might give mRDT false-positive results.There is evidence that sub-microscopic parasitemia infections occur in both high and low malaria transmission settings. Although mRDT is easy to use and convenient in low resource settings, it has a higher detection threshold than LM and PCR, making it less viable in low-endemic settings with high sub-microscopic malaria prevalence. The PCR technique is sophisticated and expensive to operate making it inappropriate for large scale deployment in low income countries. Although LM is usually referred to as a gold standard for malaria diagnosis, lack of expertise has been the greatest challenge. Sub-microscopic malaria will ultimately facilitate human-mosquito transmission and the effect is more pronounced in low-endemic areas which hinders elimination campaigns. A comparative evaluation of the relative benefits of mRDT should be conducted in areas where malaria prevalence is less than 5%.


The Malaria Evolution in South Asia (MESA) International Center for Excellence in Malaria Research (ICEMR) was established by the US National Institutes of Health (US NIH) as one of 10 malaria research centers in endemic countries. In 10 years of hospital-based and field-based work in India, the MESA-ICEMR has documented the changing epidemiology and transmission of malaria in four different parts of India. Malaria Evolution in South Asia-ICEMR activities, in collaboration with Indian partners, are carried out in the broad thematic areas of malaria case surveillance, vector biology and transmission, antimalarial resistance, pathogenesis, and host response. The program integrates insights from surveillance and field studies with novel basic science studies. This is a two-pronged approach determining the biology behind the disease patterns seen in the field, and generating new relevant biological questions about malaria to be tested in the field. Malaria Evolution in South Asia-ICEMR activities inform local and international stakeholders on the current status of malaria transmission in select parts of South Asia including updates on regional vectors of transmission of local parasites. The community surveys and new laboratory tools help monitor ongoing efforts to control and eliminate malaria in key regions of South Asia including the state of evolving antimalarial resistance in different parts of India, new host biomarkers of recent infection, and molecular markers of pathogenesis from uncomplicated and severe malaria. Plasmodium vivax. Shalini Hospital, associated with Krishi Gram Vikas Kendra (KGVK), is the site in Ranchi district of Jharkhand. The most remote MESA-ICEMR site is at the Northeast corner of Assam in Dibrugarh district, within 160 km from the India–Myanmar border. It is hosted by the Regional Medical Research Center-Northeast (RMRC-NE) and has associations with Assam Medical College (AMC). The two Eastern sites KGVK and RMRC-NE see a higher proportion of Plasmodium falciparum infections compared with GMC and AVBRH  International Center of Excellence in Malaria Research (ICEMR) program sponsored by the US National Institutes of Health (NIH) has worked actively in India for a decade.H Figure . Within H Figure . Of thesTogether, the MESA-ICEMR sites are uniquely positioned to capture variables, which contribute to heterogeneity and complexity of malaria transmission in different healthcare accessibility settings in India. The sites regularly report on parasite species distribution, pathogenesis of severe and uncomplicated malaria, changes in malaria transmission, and monitor risk of importing Artemisinin Combination Therapy (ACT) resistance from Southeast Asia. The present perspective reveals the clinical and translational value of MESA-ICEMR conducted research in India, and how such research can contribute to improved malaria control strategies and its implementation. The relative impact of this research on malaria policy-making in India is discussed in an accompanying article titled “International Center of Excellence for Malaria Research for South Asia and Broader Malaria Research in India.”All four sites under MESA-ICEMR have been conducting health facility-based surveillance. The GMC has been conducting it from 2012 to current date. The AVBRH, KGVK, and RMRC-NE conducted surveillance between 2014 and 2017. In addition, RMRC-NE has also conducted longitudinal household-based surveys in Karbi Anglong district in Assam  at 8.4% for GMC, which went up to 10% at the peak of transmission season during the monsoon.a Figure . Having A common questionnaire used at all the sites collects information about age, gender, occupation, and travel history. We noted a higher proportion of cases in the above 5 years age group compared with 5 years and below age group at all the sites. This is characteristic of a low-transmission setting like India. The median age of infection at GMC was 27 years.P. falciparum malaria treatment regimen in India, which involves different ACTs such as artemether-lumefantrine (AL), artesunate-mefloquine (AS-MQ), and artesunate-sulfadoxine-pyrimethamine (AS-SP) along with primaquine (PQ). Northeast states  and Ranchi used AL + PQ, Wardha used AL + PQ, and AS-SP + PQ, Goa used AS-MQ + PQ (before December 2015) and AS-SP + PQ (post December 2015) for P. falciparum malaria treatment. All sites used chloroquine (CQ) and PQ for P. vivax malaria treatment. These treatment regimens follow the national guidelines for first line of antimalarial use in India,,Treatment data collected by the study sites reflect the heterogeneity of The data collected in the MESA-ICEMR sites complement the data collected by National Vector Borne Disease Control Program (NVBDCP) in real time monitoring of the malaria situation and outbreaks in the selected study sites. The MESA-ICEMR data also provide a useful historical record, which can be used to analyze the impact of malaria control measures as well as to improve malaria case management (diagnosis and treatment) in these regions.Vector-related research in MESA-ICEMR involves monitoring the extent and impact of vector-control methods and entomological surveillance Table . Vector-Anopheles stephensi, An. Culicifacies, An. Fluviatilis, An. Dirus (An. Baimai), and An. minimus are responsible for malaria transmission at the Indian study sites.,An. stephensi, An. culicifacies and An. fluviatilis have been shown to be responsible for transmission in Goa. We identified An. subpictus as the fourth vector for transmission in urban construction sites of Panjim, Candolim, Porvorim, and Margao.An. subpictus as the second species along with An. stephensi to be responsible for malaria in urban regions of Goa. Seasonal distribution of these two species indicates that numbers of both the species peaks during rainy months (May–October). However, An. subpictus might be the dominant species responsible for transmission in the dry season (November–April), when An. stephensi numbers are low.An. stephensi in Goan survey sites to be resistant to the commonly used insecticides; deltamethrin (8%), Malathion (18%), and DDT (48%)An. subpictus in Goa still needs to be determined.An. stephensi larvae from construction sites and later grew them under laboratory conditions in an insectary. These larvae were then propagated to establish a pure colony. We have assessed the ability of these laboratory-raised colonized mosquitoes to support P. vivax infection from malaria-infected clinical samples collected at GMC and compared with similar ability of wild mosquitoes. The mosquito infection rates and sporozoite load were higher in wild mosquitoes compared with the colonized mosquitoes. The results may reflect the effect of genetic differentiation associated with long-time mosquito colonization on levels of vector susceptibility to P. vivax.P. vivax parasitemia/gametocytemia with mosquito infection rates but weak correlation was seen with parasite development in the mosquitoes. We also found that a higher mosquito infection intensity in wild An. stephensi in this region corresponds to a female to male gametocyte sex ratio close to 1.P. vivax sporozoites,P. vivax hypnozoites, but also aid in the design of future point-of-care (POC) hypnozoite diagnosis. We found that optimizing the mosquito rearing method and replacing patient plasma with naïve serum led to more than 2-fold increase in mosquito infection and sporozoite levels.In a MESA-ICEMR study, we collected P. falciparum prevalence remains steady.Overall, malaria cases have been declining in India since 1996, however, N = 22) from Northeast India  and Goa had parasites with elevated tolerances to dihydroartemisinin.Pfkelch13, which previously had been declared by the World Health Organization (WHO) as a candidate marker for artemisinin resistance.We demonstrated using RSAs that a relatively small patient set  from Southeast Asia have not been found in India yet. It is unclear which mutations originate from founder populations within India and which are imported from its neighbors. We evaluated the intrinsic capacity of Indian parasites to acquire in vitro resistance to novel inhibitors and their rates of mutagenesis are comparable to those from Southeast Asia (manuscript in progress). Although the capacity to mutate genomes may be similar, South Asia may still be in a good position to minimize the development and spread of artemisinin resistance by vigilant molecular and clinical surveillance. The MESA-ICEMR collection sites spread throughout India have the capacity to identify founder and/or imported resistant populations.The four confirmed (in vitro and in vivo) artemisinin resistance Pfdhfr (59R + 108N) and a single Pfdhps mutation (437G), or none, were phenotypically as resistant as triple mutants for both genes (unpublished). We suspect that folate salvage was the primary modulator of antifolate resistance levels in the Goan parasites. Although rapid parasite clearance is dependent on artemisinins quickly reducing biomass, the day 3 positivity and late-treatment failures point to possible preexisting antifolate resistance genotypes.,–Choice of partner drugs included in ACTs is critical to preserving artemisinin’s ability to serve as a frontline antimalarial. Although Northeast India relies on the artemether–lumefantrine combination, most of India is using artesunate-sulfadoxine-pyrimethamine to care for uncomplicated malaria cases.The MESA-ICEMR team has collected data on around 600 patients hospitalized with severe malaria (SM) and about 2,000 uncomplicated malaria patients, so far (unpublished data). Clinical assessment of the degree of malaria severity was done in all sites. Apart from estimating the burden of SM, we use laboratory-based data from severe and uncomplicated patient samples to explore different facets of the complex pathogenesis involving several intricate processes in the malaria parasite and host Table .P. vivax patients at GMC has allowed the development of tools to overcome experimental shortcomings with the study of P. vivax. First, the parasitemia of P. vivax infections are usually low within patients and we have improved counting methodologies for low parasitemia infections by light microscopy.P. vivax biology are severely limited by the lack of a continuous in vitro culture system. Access to a steady collection of P. vivax patient isolates has facilitated significant advances in our ability to short-term culture P. vivax. We have discovered that short-term culturing P. vivax in a hematopoietic stem cell medium permits highly efficient maturation of parasites from the ring to schizont stage of the intraerythrocytic developmental cycle (IDC) of the parasite.P. vivax drug and invasion assays using both frozen parasites as well as fresh clinical isolates from GMC. This, in turn, has set the stage for exploring the molecular basis of P. vivax invasion, host cell tropism, and pathogenesis. The MESA-ICEMR has shown that gametocytes from cryopreserved patient P. vivax isolates are stable for use in mosquito infection.P. vivax and the high frequency of gametocytes that we observe in clinical isolates at GMC, will be important resources for future transmission studies.Steady, year-round access to infected blood from P. vivax invasion of red blood cells is an obligatory step in the parasite life-cycle but there is a marked deficiency in our understanding of the invasion process in P. vivax, again primarily due to the lack of a continuous in vitro culture system for this parasite. Plasmodium vivax is unique due to its strong preference for invading reticulocytes, the youngest red blood cells.,P. vivax infection.P. vivax invasion ligands, the existence of discrete invasion pathways, and their association with disease severity are poorly understood. The PvDBP/DARC interaction is thought to be essential for P. vivax invasionP. vivax.,P. vivax infection in DARC-negative individuals–P. vivax isolates from India, suggestive of greater geographical variation in P. vivax parasites.P. vivax parasite burdens in malaria patients at the GMC.The variation in the usage of P. vivax invasion assay has also allowed us to investigate P. vivax invasion ligand gene expression and variations in invasion. In addition to the PvDBP/DARC invasion pathway, the MESA-ICEMR team has also investigated the newly identified reticulocyte-tropic invasion pathway between the P. vivax RBP2b invasion ligand and host transferrin receptor (TfR1).P. vivax isolates using host-targeted small molecules/antibodies (anti-DARC and anti-TfR1) and we observed that while invasion could be inhibited in all strains, there was significant variation in usage of each invasion pathway.P. vivax vaccine development.Our P. falciparum pathogenesis has involved var gene (PfEMP1) profiling in complicated and uncomplicated malaria patients from Goa.,var genes in pathogenesis of SM through infected RBC (iRBC) sequestration and the role of parasite biomass.var transcripts were overrepresented in SM than the type B and C var transcripts. Group A var variants, specifically DC8 and DC6 PfEMP-1 were previously found to be associated with pediatric SM.–var phenotypes in SM patients. Our data suggests that specific types of PfEMP-1 may promote higher parasite biomass, ultimately contributing to severe pathophysiological outcomes. Machine learning analysis indicated the combination of high parasite biomass (assessed through PfHRP-2 levels) along with DC6 and DC8 encoding var transcripts was the strongest indicator of severity and hospitalization in the study subjects from Goa.Our work in var gene expression profiles play independent and complementary roles in development of SM, not only in African children, but also in Indian adults.var gene expression profiles show variation within host and among sites, the var gene profile signatures associated with SM were highly similar across three cohorts included in the study. There are differences in disease presentation in African children and Indian adults, which is to be expected owing to differences in transmission intensity in African and Indian regions. Despite these differences, SM in African children and Indian adults is definitively linked to increased transcription of var variants predicted to bind EPCR.–var transcripts were implicated in SM.,Our subsequent meta-analysis study involving three different cohorts with pediatric malaria in Tanzania and Malawi, and adult malaria in Goa, indicated that parasite biomass and ,Plasmodium species.Plasmodium species and their vectors, and diverse epidemiological profiles that can directly contribute to the development of NAI. Seroreactivity and antibody response against parasite proteins are useful biomarkers of population-wide NAI, and significantly different levels of these markers had previously been observed at different Indian sites.falciparum and 515 P. vivax protein respectively.Plasmodium falciparum patient Sera showed a broader immune response than P. vivax patient Sera. This observation in India is consistent with previous ICEMR protein array data from Kenya.P. falciparum and two P. vivax antigens with differential seroreactivity among inpatients and outpatients. Severe falciparum and vivax malaria patients showed significantly higher antibody response than uncomplicated patients. Interestingly, several conserved proteins like MSP, PHIST, and NOT family proteins were found to be seroreactive only in nonsevere patients in both falciparum as well as vivax infections. Altogether, 248 P. falciparum and 73 P. vivax seroreactive proteins were identified in the MESA-ICEMR study, which is consistent with similar studies published earlier.–P. falciparum antigens in severe versus nonsevere cohorts,The interaction/s of malaria parasites with the host that are responsible for clinical outcome vary with parasite biology and host physiology. Naturally acquired immunity (NAI) of the human host to parasites play an important role in malaria pathogenesis. Development of NAI depends on duration and degree of exposure to the parasites,Pfhrp-2 deletions. We also monitor the current status of ACT sensitivity in the Indian parasites from these sites. Together, these efforts put the MESA-ICEMR team in a competent position for complementing and informing diagnosis and treatment regimens in this region. Moreover, the MESA-ICEMR has built strong research facilities at the study sites, particularly Assam and Goa. This makes us uniquely poised to help deliver on the malaria research and development needs outlined by the WHOMalaria accounted for 5 million cases in India in 2020.


Axillary body temperature was measured using a digital thermometer. The prevalence of anemia was found to be 55.3% , of which 45.1% had mild , 52.1% moderate  and 2.9% had severe anemia . Among anemic, 70.8%  resided in LMED and the rest in HMED. The median age of the anemic population was 12 years (IQR: 7–30). Overall, malaria positivity was 8.9% , of which HMED shared 79.6% (584/734) and LMED 20.4% (150/734) malaria burden. The village-wise malaria frequency was concordant to asymptomatic malaria (10–20%), which showed that apparently all of the malaria cases were asymptomatic in HMED. LMED population had significantly lower Hb than HMED  and low-density Plasmodium infections had higher Hb levels than high-density infections . Women of reproductive age had higher odds for malaria . Females  and febrile individuals  have shown lower Hb levels, but malaria positivity did not show any effect on Hb. Young children and women of reproductive age are prone to anemia and malaria. Although there was no relation between malaria with the occurrence of anemia, we found low-density Plasmodium infections, female gender, and LMED were potential determinants of Hb.Anemia and malaria are the two major public health problems that lead to substantial morbidity and mortality. Malaria infection destroys erythrocytes, resulting in low hemoglobin (Hb) levels known as anemia. Here we report the determinants of anemia in high and low malaria-endemic areas that would help understand which parasite densities, age, and gender-associated low Hb levels. Therefore, a cross-sectional mass survey ( In malant women .P. falciparum was the predominant species that caused the majority of malaria infections (>80), and the rest of the malaria cases were due to P. vivax   from 14.5 to 2.6 cases per 1,000 population. The transmission of malaria in this district starts in April, and the peak is observed in June/ July. In 2018, P. vivax . Contrar< 0.001) . Our pren = 7,754), and polymerase chain reaction (PCR) . Out of 8,233, 5,525 participants were from the district Udalguri  and 2,708 were recruited from East Garo Hills district . There were 57%  female participants in the study and the rest were male. The RDT kits used in the study were SD Bioline malaria Ag Pf/Pv, Haryana, India. Giemsa staining was used for microscopic examination of slides to detect the presence of Plasmodium species. Dried blood spots (DBS) were prepared on Whatman filter paper, grade 1  for identification of Plasmodium parasite species using PCR. Axillary body temperature was recorded using a digital thermometer and a body temperature of ≥ 37.5°C at the time of examination was defined as febrile. The proportion of afebrile individuals in the study population was 92.4%  and febrile illness was found in 7.6%  participants. Afebrile participants detected with Plasmodium parasite through RDT were defined as asymptomatic malaria cases. Low-density Plasmodium infections were defined as those malaria cases which were detected by PCR but undetected by microscopy and/or RDT. An individual was considered “malaria positive” only when detected with Plasmodium parasite either by light microscopy, RDT or by PCR. Anemia was defined according to the age-group as classified by WHO  and anemia  was calculated as percentages for the selected villages, blocks and districts . Two PHCs from each district were randomly selected  recognized as a high-endemic PHC, and another with low API as a low-endemic PHC) based on the past 5 years of malaria epidemiological data of PHCs under the study districts. Further, 10 villages from each PHC were selected using a probability proportional to size (PPS) sampling technique. In a situation where the required sample from the selected village was not available, the shortfall was covered from adjacent villages. The map in d by WHO . The outistricts . The vilPlasmodium parasites were found after examining 100 microscopy fields. For all positive slides, identification of Plasmodium species was performed. All the microscopists were kept blinded to RDT results and later the discordant results (RDT+ve Microscopyve− and RDTve− Microscopy+ve) were re-examined by another experienced microscopist and/ or a WHO certified microscopist who was not aware of previous microscopy/ RDT results. The final microscopy results were considered positive or negative depending upon the quality check results of the microscopy.Thick and thin blood smears were prepared at the study site and transported daily to the local laboratory, where fixation of thin blood smears was performed with methanol followed by staining of both thick and thin smears with 3% Giemsa stain solution for 30 min. All the slides were examined under oil emersion (10 × 100 magnification) by experienced microscopists. A blood slide was considered negative if no Plasmodium species identification using PCR was done on DBS prepared at ambient temperature and sealed in zipped plastic bags containing desiccant and stored at−20°C until analysis. To perform the diagnostic PCR, DNA was isolated from DBS using the QIAamp Blood Mini Kit  according to the manufacturer's protocol. The protocol followed in this study to identify Plasmodium species using PCR is described elsewhere, which was based on targeting coding sequences of the small subunit of ribosomal RNA specific to the parasite species. The limit of detection ranged from 1 to 10 parasites/μl of blood. The details of the primer sequences used in this study have been described previously (+ve Microscopic+ve PCRve− and RDTve− Microscopicve− PCR+ve) were re-confirmed using light microscopy and PCR.The eviously . BrieflyThe data were entered into Microsoft Excel 2007 worksheet and cross-checked for typographical errors. Further, lists of quality checks have been applied in order to ensure the quality of data. Non–numerical categorical variables were coded numerically and frequency with percentage distribution was tabulated and Pearson's Chi square or Fisher's exact test was applied for statistical comparison of 2 × 2 contingency tables as appropriate. Standardized beta coefficient (unadjusted and adjusted) was calculated using univariate and multivariate regression model to find out determinants of Hb concentration in study population. A univariate linear regression was performed to calculate unadjusted standardized beta coefficient; whereas, adjusted standardized beta coefficient was calculated using multivariate regression model in which the effect of one variable on another was studied in the presence of other covariates. Logistic regression analysis was applied to calculate the Odds ratio (OR) with 95% confidence intervals (CI) to understand the likelihood of anemia severity among females with reference to males. Critical value for statistical significance was considered at alpha 0.05. Data were analyzed using statistical software R 3.4.3 for Windows .2 = 11.29; p = 0.001), whereas malaria positivity was more or less similar in this age-group. This difference was more pronounced among women of reproductive age, where anemia prevalence was 66.2%  in females and 38.2% (335/876) in males . Women of reproductive age-group 7.5%  were also found to have higher odds for malaria positivity (Odds ratio (OR) 1.42 at 95% confidence interval (CI): 1.00–2.05; p = 0.04) than men 5.4% (47/876). Malaria positivity in this population declined with the advancement of age, i.e., young children (<5 years) had the highest malaria positivity (13.2%) compared to adults (Plasmodium infections were found in 38.2% (139/364) cases, of which 49.6% (69/139) were anemic. Among low-density infections, 56.8% (79/139) cases were from high malaria-endemic district and 43.2% (60/139) from the low malaria-endemic district. Overall asymptomatic Plasmodium infections were 8.3% , of which the proportion of anemic cases was 51.8% (329/635). Among asymptomatic cases, 77.5% (492/635) cases were from high and, 22.5% (143/635) were from the low malaria-endemic district.In this study, overall malaria positivity irrespective of any diagnostic method used was 8.9% , of which high endemic district shared 79.6% (584/734) and low endemic 20.4% (150/734) of malaria burden. o adults . In thisp < 0.0001). Females also had higher odds for moderate anemia  and severe anemia  in comparison to males. Among males, the frequency of mild anemia was highest (25.1%), whereas the majority of females had moderate anemia (32.7%), and 2.1% were severe anemic . Among anemic individuals, 45.1% were mild , 52.1% were moderate , and 2.9% were severe anemic . Of anemic population, 70.8%  belong to low malaria-endemic district, and the rest are from high endemic district. The study map shows the prevalence of anemia and malaria RDT positivity in the investigated villages . Anemia e anemic . The ovee anemic .Plasmodium infections between 10-20% or above (Plasmodium infections were similar (<5%) in villages under high endemic PHC, and apparently, no malaria (only 1 RDT positive) was found in low endemic PHC. However, mild anemia was nearly 20–40% in most villages, frequency of moderate anemia was 40–60% in villages under high endemic PHC and 20–40% in villages under low endemic PHC. In high endemic PHC, the frequency of severe anemia cases was above 10% in five, and between 1–10% in 11 out of 30 villages; whereas out of 31 villages under low endemic PHC, two villages had severe anemia >10% and nine villages had severe anemia ranged between 1–10%  and asymptomatic malaria cases. In high endemic district severe anemia prevalence was below 5% in most villages except in two where prevalence was 10–20%, and moderate and mild anemia was between 20–40% in most villages. In the high endemic district, villages under high endemic PHC (dark brown region) showed high malaria positivity. Most villages, except a few, had asymptomatic or above . Nearly or above . RDT posen 1–10% .p < 0.0001]. Low-density Plasmodium infections had significantly higher Hb concentration in comparison to high density infections . Gender female in comparison to male  and febrile in comparison to afebrile subjects  had a lower concentration of Hb, but there was no effect of malaria positivity on mean Hb concentration of study participants (Hb concentration (mean ± sd) in high and low malaria-endemic districts was estimated at 11.7 ± 1.7 and 11.5 ± 1.6 g/dl, respectively. The analysis revealed that mean Hb concentration was significantly lower in low as compared to the high malaria-endemic districts [standardized beta (β) = −0.067; icipants . After aicipants .This study presents the prevalence of anemia and malaria in two  north-eastern districts of India. We found an 11.5 g/dl mean Hb concentration in the study population, and more than one-half of the study population was anemic, of which the majority was moderate anemic. Previous studies have shown nearly 60% anemia prevalence in the tribal people of Assam  and arouPlasmodium infections in the high malaria-endemic district (East Garo Hills), anemia prevalence was low compared with the low malaria-endemic district . Our findings were contrary to anemia estimates reported in NFHS-4 (2015-2016) but similar to the recent NFHS-5 (2019-2020) data that reported higher anemia prevalence in district Udalguri, Assam than East Garo Hills, Meghalaya  district had higher malaria positivity and low-density infections, there was a better Hb profile of residents from this district. This might be due to their dietary habits, mainly consuming meat products , a direcOur study suggests no relation between malaria positivity with anemia prevalence in the study districts. The low malaria-endemic district had higher anemia but lower malaria positivity than the high endemic district. We observed that younger children and females from reproductive age groups were most susceptible to anemia and malaria. Low-parasite density, low malaria endemicity, and female residents were the potential determinants influencing Hb concentration in this study population.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Institutional Ethics Committee of National Institute of Malaria Research. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.HS contributed substantially in data collection, data analysis, interpretation, and drafting of manuscript. MS: data cleaning, statistical analysis, and interpretation. SH: mapping village wise data and compilation of results. SP: study design, data collection, and study management. KS: data collection and logistic arrangements at the study sites. NM: conception and design of the study, study management, and editing of the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by Indian Council of Medical Research, New Delhi, India vide grant no. NER/55/2015-ECD-I.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pain was assessed using a numeric rating scale (NRS) 7 days before and immediately before surgery. The AUC0–24 h in the synovium were 4401.24 and 4862.70 ng·h/g in the SFPP and FPT groups, respectively. Maximum esflurbiprofen concentrations were observed in the synovium, synovial fluids, and plasma after SFPP application for 12 h. The NRS results indicated a long‐lasting effect of SFPP. The AUC of the synovial esflurbiprofen concentration of SFPP indicated that the SFPP is transferred to the synovium and synovial fluid in high concentration. The efficient deep‐tissue transfer of esflurbiprofen suggests that its pharmacokinetic characteristics differ from those of conventional topical NSAIDs. This study was prospectively registered in the Japan Registry of Clinical Trials (registration number: jRCTs031180228).This open‐label, multicenter, prospective, randomized controlled trial aimed to determine the effectiveness of esflurbiprofen plaster (SFPP) and flurbiprofen tablets (FPTs) on knee osteoarthritis in patients scheduled for total knee arthroplasty by comparing the transfer of esflurbiprofen and flurbiprofen to tissues and fluids. Thirty‐eight patients were randomly assigned in a 1:1 ratio to receive SFPP or FPT. Both groups were then divided into four subgroups, according to whether they received the final dose of SFPP or FPT at 2, 7, 12, or 24 h before planned surgery. The primary endpoints were the esflurbiprofen concentrations in synovium, synovial fluid, and plasma. Areas under concentration–time curves  . Maximum esflurbiprofen concentrations were observed in the synovium, synovial fluids, and plasma after SFPP application for 12 h. The area under the curve (AUC) of the synovial esflurbiprofen concentration of SFPP indicated that the SFPP is transferred to the synovium and synovial fluid in high concentration. The efficient deep‐tissue transfer of esflurbiprofen suggests that its pharmacokinetic characteristics are different from those of conventional topical NSAIDs. Moreover, articular cartilage degeneration results in subsequent changes in proliferative subchondral bone. According to the large‐scale cohort study called Research on Osteoarthritis/Osteoporosis Against Disability (ROAD) conducted in Japan, ∼8 million people are affected by painful knee OA, and this is likely to increase as the population ages  was launched in 2016 to treat knee OA and it has better transdermal drug absorption  in patients with knee OA who were scheduled for total knee arthroplasty (TKA). We postulated that SFPP would provide comparable deep tissue drug transfer to that of the oral form, with less transfer to blood and fewer adverse events.22.1The study complied with the principles of the Declaration of Helsinki and the Clinical Research Act of Japan. The Clinical Research Review Committee of Tokyo Medical and Dental University approved the protocols . The study was registered in the Japan Registry of Clinical Trials before it started (registration number: jRCTs031180228).2.2This open‐label, multicenter, prospective, randomized controlled trial initially enrolled 52 patients who met all the inclusion criteria and none of the exclusion criteria. Data were finally analyzed from 38 patients who were randomly assigned in a 1:1 ratio to receive SFPP or FPT. These groups were further divided into four subgroups of four or five patients each according to whether they received the final dose at 2, 7, 12, or 24 h before the planned surgery.Plasma concentrations of esflurbiprofen peaked at 12–22 and at ∼6.7 ± 2.1 h in a study of SFPP administered for 24 h and 7 days, respectively , which could influence knee evaluation, complicated with rheumatoid arthritis, scheduled for analgesics, intra‐articular injections, systemic adrenal corticosteroids, target knee puncture, knee joint aspiration, or arthroscopy within 7 days of exposure to the study medication, requiring preoperative target knee shaving, under medication or planning medication with drugs that interact with esflurbiprofen or flurbiprofen, a history of concurrent atopic dermatitis, skin disorders such as eczema on the target knee(s), a history of skin disorders caused by plaster preparations, infectious diseases, contraindicated for or requiring careful administration of study drugs, pregnancy, breastfeeding, difficulty providing consent and others, including patients who were considered unsuitable by an investigator or other medical personnel. The attending physician provided a written explanation of the study plan to eligible patients who were free to provide written informed consent after reading the explanation.2.4All patients discontinued analgesics 7 days before being assigned either of the test medications. The SFPP group applied 40 mg/day of esflurbiprofen  once daily for 7 days before surgery to the medial or lateral side of the target knee, so that it would not cover the surgical skin incision. The final plaster was removed immediately before surgery. The FPT group was orally administered with three doses of FPT per day   for 7 days before surgery. The last tablet on day 7 was the final dose before surgery.2.5Patient demographic characteristics were obtained at the start of the study, and NRS scores were assessed for self‐reported pain levels while walking, resting, starting a movement, and climbing stairs 7 days before and immediately before surgery. Esflurbiprofen concentrations were measured by liquid chromatography‐mass spectrometry  in synovium and synovial fluid samples collected from target knees during surgery, and in blood plasma collected immediately before surgery. All patients were monitored from 7 days before, until the day of surgery. Adverse events that developed during these 7 days were recorded.2.6max), and the area under the concentration–time curve (AUC0–24 h) of esflurbiprofen for each group was calculated. Either SFPP or FPT was administered for 7 days before surgery, and we considered that esflurbiprofen concentrations in tissues and fluids had reached the steady state. We assumed that values at 0 and 24 h were equivalent.The primary endpoint was the concentration of flurbiprofen in tissues or fluids. Esflurbiprofen concentrations in the synovium, synovial fluid, and plasma were determined in the SFPP and FPT subgroups when the final dose was administered. The highest tissue/fluid concentration  was calculated as follows:0–2 h = (mean concentration at 24 h + mean concentration at 2 h) × 2/2.AUC2–7 h = (mean concentration at 2 h + mean concentration at 7 h) × 5/2.AUC7–12 h = (mean concentration at 7 h + mean concentration at 12 h) × 5/2.AUC12–24 h = (mean concentration at 12 h + mean concentration at 24 h) × 12/2.AUC0–24 h = AUC0–2 h + AUC2–7 h + AUC7–12 h + AUC12–24 h.AUCThe AUC was calculated using mean esflurbiprofen concentrations in tissues and fluids and the formula for measuring the area of a trapezium. The AUCThe secondary endpoints were changes in NRS scores 7 days before and immediately before surgery. The safety endpoints included adverse events and side effects.2.7U‐tests. Within‐group comparisons of tissues or fluids at the time of final dose were analyze using Kruskal–Wallis tests. Within‐group and between‐group comparisons of parameters and changes in NRS scores at the time of final dose were respectively assessed using Wilcoxon signed‐rank tests and Mann–Whitney U‐tests. All data were statistically analyzed using R version 3.4.0 (R Core Team), and the two‐sided level of significance was set at 5%.The concentrations of esflurbiprofen in tissues and fluids were compared between the groups at the time of the final dose using Mann–Whitney 3Initially, 52 patients met the inclusion criteria, and 27 were assigned to the SFPP group. Among them, 5 withdrew from the study before SFPP exposure, and data from 19 patients were statistically analyzed. Five of 25 patients who were initially assigned to the FPT group were excluded from the study before FPT administration, and 1 withdrew before surgery. Finally, data from 19 of the patients who had received FPT, were statistically analyzed .Table max was 245.20 ± 234.67 and 367.25 ± 50.18 ng/g after 12 and 2 h in the SFPP and FPT groups, respectively  before and after administration (1.39–1.65)  of the 22 patients in the SFPP group developed dermatitis after SFPP exposure and 1 (5.0%) of the 20 patients in the FPT group developed two adverse reactions to FPT, one of which was an episode of nausea.4Suppressing inflammation is key to knee OA pharmacotherapy. Hence, drug pharmacokinetics around an affected area are as important as those in the blood. NSAIDs decrease prostaglandin E production in synovial fluid, where drug concentrations are low according to binding protein concentrations . However, all NRS scores of the SFPP group decreased more than the MCSD before and after administration. These results indicated that SFPP should be effective for long‐term pain relief.This study has some limitations. First, the study groups contained few patients. Most patients with knee OA were older, and many had comorbidities, such as chronic renal dysfunction and gastrointestinal problems. These patients were contraindicated for oral NSAIDs and could not be included in this study. Second, only esflurbiprofen, which is the active form of flurbiprofen, was measured in the present study. The R‐flurbiprofen included in the FPT was not considered. Third, the concentration of esflurbiprofen was not measured at 0 h immediately before surgery. We assumed that the values at 0 and 24 h were equivalent. Fourth, we administered FTP three times daily according to the package insert, but we used the same design as a previous study (Sekiya et al., 5We investigated the pharmacokinetics of esflurbiprofen in the synovium, synovial fluid, and plasma after administering SFPP and FPT. Drug concentrations in tissues and fluids were maximal at 12 h after SFPP application, and the NRS results indicated that the SFPP had long‐lasting effects. The AUC of the synovial esflurbiprofen concentration of SFPP indicated that the SFPP is transferred to the synovium and synovial fluid in high concentration. This high transfer efficiency of the drug into deep tissues suggested that the pharmacokinetic characteristics of esflurbiprofen differ from those of conventional topical NSAIDs.The authors declare no conflicts of interest associated with this manuscript.


Malaria is caused by Plasmodium species and is called a “Rainy season disease”. Scrub typhus is a zoonotic disease caused by Orientia tsutsugamushi. Both diseases occurring as co-infection are very rare. If a patient presents with a fever complaint, all the possible causes should be ruled out even if the patient shows infectivity for one of the vector-borne diseases. This case report shows the rare occurrence of the co-infection and its presentation. Early detection and timely management can prevent the patient from various fatal complications. P. falciparum is responsible for nearly half of these cases. Malaria can be cured if therapy is begun early enough. Treatment delays might have catastrophic effects, including death. Controlling disease transmission also necessitates prompt and efficient treatment. Patients who live in or have recently visited endemic regions should be suspected of having malaria. The incubation period of P. falciparum malaria is 12-14 days, and plasmodium vivax is 12-17 days [orientia tsutsugamushi, which is spread by trombiculid mite larvae. Eschar may or may not be visible in every situation [Malaria is a severe parasite illness that causes significant morbidity and mortality in India. Early diagnosis and comprehensive therapy are crucial to controlling the disease . The Indituation ,4. ThougA 24-year-old male came to the hospital with complaints of low-grade fever for eight days, irritability, and disoriented for one day. A blackish rash on the back of the chest, as shown in Figure Investigations were done as shown in Table The patient was initially started with injectable Artesunate 120 mg at 0-12hour-24hour followed by 120 mg for three days, injectable doxycycline 100 mg twice a day, tablet primaquine 15 mg twice a day and other supportive medications. The patient's condition improved during the hospital stay and symptomatically better. After completing an intravenous anti-parasitic drugs for seven days, the patient was discharged. On discharge, patient was adviced for screening of the relatives. On follow-up, the patient was doing well. Plasmodium vivax (P. vivax) and Plasmodium falciparum  malaria are prevalent. Despite the fact that malaria has symptoms that are similar to those of many other infectious illnesses, all clinically suspected malaria cases should be explored using microscopy and/or a Rapid Diagnostic Test (RDT) as soon as possible [P. vivax and a 95.83 percent malaria-negative specificity. To confirm the co-infection by P. vivax and P. falciparum, a thick and thin film analysis of the peripheral smear was performed [P. vivax and P. ovale) capable of generating latent liver stages and eventual malarial relapses [India is a tropical nation where possible . The RDTerformed . The patrelapses . PCR, dePlasmodium vivax can remain latent in liver cells as hypnozoites, which can evolve into merozoites months or years later. Scrub typhus has a nine-day incubation period. As a result, the patient who was already suffering from Plasmodium vivax fever might have been infected with scrub typhus [Scrub typhus should be suspected in every instance with protracted fever, and a thorough search for eschar should be conducted, as eschar is the most helpful clinical diagnostic clue . Plasmodb typhus . Scrub tb typhus . Scrub tb typhus . Empiricb typhus .Malarial and scrub typhus infection can be controlled with different measures taken at different levels. Early diagnosis and prompt treatment can help in preventing life-threatening complications. Along with treatment, and prevention at different levels vector control measures to public health education for awareness of transmission, symptoms, and prevention of diseases is important, especially in endemic areas.