E malaria vaccine safety, effectiveness, and impact will be assessed in pre- and post-vaccine introduction studies, comparing the occurrence of malaria cases and adverse events in vaccinated versus unvaccinated children. Because those comparisons may be confounded by potential year-to-year fluctuations in malaria transmission intensity and malaria control intervention usage, the latter should be carefully monitored to adequately adjust the analyses. This observational cross-sectional study is assessing Plasmodium falciparum parasite prevalence (PfPR) and malaria control intervention usage over nine annual surveys performed at peak parasite transmission. Plasmodium falciparum parasite prevalence was measured by microscopy and nucleic acid amplification test (quantitative PCR) in parallel in all participants, and defined as the proportion of infected participants among participants tested. Results of surveys 1 (S1) and 2 (S2), conducted in five sub-Saharan African countries, including some participating in the Malaria Vaccine Implementation Programme (MVIP), are reported herein; 4,208 and 4,199 children were, respectively, included in the analyses. Plasmodium falciparum parasite prevalence estimated using microscopy varied between study sites in both surveys, with the lowest prevalence in Senegalese sites and the highest in Burkina Faso. In sites located in the MVIP areas (Kintampo and Kombewa), PfPR in children aged 6 months to 4 years ranged from 24.8% to 27.3%, depending on the study site and the survey. Overall, 89.5% and 86.4% of children used a bednet in S1 and S2, of whom 68.7% and 77.9% used impregnated bednets. No major difference was observed between the two surveys in terms of PfPR or use of malaria control interventions.RTS,S/AS01 Malaria remains a major cause of death worldwide, with approximately 93% of all malaria deaths in 2017 occurring in Africa.1 To reach the Global Technical Strategy for Malaria 2016–2030 target of reducing global malaria incidence and mortality rates by at least 90% by 2030,2 the need for safe and effective malaria vaccines that prevent disease and death and decrease transmission to enable malaria eradication was endorsed and documented in the WHO Malaria Vaccine Technology Roadmap.3Substantial investment to expand existing malaria interventions has resulted in a reduction in the global incidence rate of malaria between 2010 and 2017.Plasmodium falciparum malaria vaccine, RTS,S/AS01E , for routine immunization of infants and children living in sub-Saharan African (SSA) malaria-endemic countries. In 2015, the European Medicines Agency adopted a positive opinion for the use of the vaccine in children aged 6 weeks to 17 months at the first dose.4 In January 2016, the WHO recommended a pilot implementation of RTS,S/AS01E in children as of 5 months of age in three to five epidemiological SSA settings with moderate to high malaria transmission.5 In April 2017, the WHO announced the vaccine introduction based on a cluster-randomized design in pilot areas of Ghana, Kenya, and Malawi through the national expanded programs on immunization, in the framework of the Malaria Vaccine Implementation Programme (MVIP).6 Today, RTS,S/AS01E is the first vaccine implemented for the prevention of malaria.GlaxoSmithKline (GSK) has developed, in partnership with the PATH Malaria Vaccine Initiative, a pre-erythrocytic Trials.gov identifiers: NCT02374450 and NCT03855995, respectively), allowing comparison of the occurrence of malaria cases and adverse events in vaccinated versus unvaccinated children. In parallel with those studies, the present observational cross-sectional annual study (NCT02251704) is estimating P. falciparum parasite prevalence (PfPR) and the use of malaria control interventions during up to nine consecutive years, applying standardized methodologies and multiple diagnostic testing. More specifically, considering the WHO recommendation to operate the MVIP in moderate to high transmission areas of SSA, this study will allow 1) characterizing the malaria transmission intensity (MTI) before RTS,S/AS01E vaccine introduction in different countries/areas, including the ones participating in the MVIP; 2) monitoring overtime fluctuations of MTI and of the use of malaria control interventions before and after vaccine introduction in those areas to adjust the pre- and post-vaccine introduction comparison analyses for potential year-to-year and/or geographical variations.To assess vaccine safety, effectiveness, and impact, GSK designed a pre- and a post-vaccine introduction observational study , Ghana (GH), Kenya (KE), Senegal (SN), and Tanzania (TZ), and conducted in accordance with the provisions of the International Conference on Harmonisation and Good Clinical Practice guidelines.Supplemental Appendix Section 1). Children in care, or actively participating in any trial involving the administration of an investigational malaria vaccine and/or drug, were excluded.Individuals aged 6 months to < 10 years, whose parents or legally acceptable representative had provided informed consent for study participation, were randomly selected each year in each of the study sites (see Study design section) using population listings generated from local Health and Demographic Surveillance Systems (HDSS) and following a stratification by age-group , serological conversion rates (SCRs), and blood parasite prevalence. Although EIR is a standard method, the measure is challenging, and the interpretation and comparability of the setting may be difficult because of vector heterogeneity.8 The methodology and interpretation of SCRs to classify the intensity of malaria is still not commonly used. Plasmodium falciparum prevalence, despite requiring trained staff for slide reading, provides a standardized and relatively easy to implement method to assess MTI in study sites of varied transmission intensity, and has often been used in previous epidemiological studies.10 Therefore, PfPR was the selected method to assess potential variations in MTI in the present study.Malaria transmission intensity levels are consensus indicators developed by the Global Malaria Eradication Programme to measure malaria endemicityThe study is multicentric with study sites corresponding to geographically limited catchment areas located in low, moderate, or high MTI regions of SSA, and having an HDSS in place.E vaccination will be implemented through the national Expanded Programs on Immunization in the framework of the MVIP. It is important to note that this study is also conducted in SSA areas where the RTS,S/AS01E vaccine will not be introduced in the framework of the MVIP because GSK initiated the study before the WHO recommendation.11Up to nine annual cross-sectional surveys will be conducted during the malaria peak transmission period in each study site (from mid-September to mid-December in Western African sites and from mid-April to mid-August in Eastern African sites). To optimize the detection of the peak transmission, each study site has been equipped with a weather station to record meteorological data such as rainfall, temperature, and humidity. Surveys were conducted during the course of the rainy season preferably when rains decrease, which should correspond to the period of highest malaria transmission. In this article, results of the first two annual surveys are presented. Survey 1 (S1) and survey 2 (S2) were conducted in seven study sites in five SSA countries: BF , GH (Kintampo), KE (Kombewa), SN , and TZ (Korogwe). Sites in GH (Kintampo) and KE (Kombewa) are part of the areas where the RTS,S/AS01Demographic details (age and gender), medical history, and information on care-seeking behaviors ; malaria control intervention usage ; the usage of coils, repellents, and local herbs; and malaria potential risk factors  were recorded for all participants at the time of the survey. Axillary body temperature was measured and recorded during the survey visit.PfPR, study areas were mapped by villages using grid referencing and divided into 3–14 segments with a minimum of 10 enrolled individuals per segment. Segments will remain unchanged for the duration of the study.To assess within-site heterogeneity of 13 In brief, a blood sample was collected by finger prick for thin and thick blood films for the microscopy assessment and filter paper blood spots for NAATs. Blood smears were examined by two local independent microscopists, and any discrepancies were settled by a third reader. Parasitemia was measured by the examination of 100 high-powered fields on thick smear to determine the presence of parasites; in the case of a positive result, additional 100 fields were examined to assess the presence of multiple species. Plasmodium species and sexual forms were identified on thin blood film. Parasite density was computed as the geometric mean of two readings, counting parasites against 200 white blood cells on thin blood film, assuming 8,000 white blood cells/μL. Parasite density was categorized as low , medium , high , or very high . In the case of low density (< 10 parasites against 200 leukocytes), parasite count was conducted against 500 white blood cells. The parasite count technique was replicated to count gametocytes. In parallel with microscopy, asexual and sexual parasitemia was assessed by NAATs using both real-time quantitative PCR (QT-PCR) assay and real-time nucleic acid sequence-based amplification (QT-NASBA) assay. Quantitative PCR allowed detection of asexual and sexual parasites combined, the final output being qualitative (positive or negative) and semi-quantitative . QT-NASBA allowed detection of gametocytes, the final output being qualitative (positive or negative). Details for blood slide and NAAT assessment of parasitemia are available in the Supplemental Appendix .Both microscopy and Nucleic Acid Amplification Tests (NAATs) were used in parallel on all participants to assess parasitemia. The latter are expected to be more sensitive and specific, particularly in cases of low parasite density.If fever  was recorded at the time of the visit or reported to have occurred within 24 hours before the visit, a malaria rapid diagnostic test (RDT) was conducted using blood from the finger prick sampling. If the RDT was positive, then treatment was given according to national guidelines. Moreover, any participant identified as being parasite positive following microscopy was traced to receive treatment according to national guidelines.PfPR estimates .The planned sample size was 600 participants per study site and per survey distributed as 400 participants between the ages of 6 months to 4 years and 200 participants between the ages of 5 and 9 years. The sample size was calculated to ensure sufficiently narrow CIs around study site–specific Plasmodium falciparum parasite prevalence and prevalence of gametocytes were estimated as the proportion of participants infected, or carrying gametocytes, respectively, among participants tested. Prevalences were estimated by age and by site. The agreement between the two diagnostic methods used in the framework of this study (parasitemia measured by microscopy versus NAATs) was described using the Cohen’s kappa coefficient and assessed using the Landis and Koch scale.14 The prevalence of Plasmodium species other than P. falciparum was estimated as the proportion of infected participants among participants tested. The within-site heterogeneity between segments was tested using Cochran’s Q-test based on inverse variance weights.P. falciparum parasitemia as measured by microscopy) was conducted using a multivariable logistic regression with study site as cluster and using a backward strategy for the selection of significant explanatory variables, that is, predefined potential risk factors and/or the use of malaria control interventions . Age was computed as a continuous variable.Malaria control intervention coverage was estimated as the proportion of users among participants for which this information was available. The care-seeking behaviors  were described as the proportion of participants having sought for health care among all participants. In addition, a risk factor analysis for malaria infection . Across all sites, 51.3% of participants in S1 and 50.3% in S2 were males .Survey 1 and S2 data were collected between October 2014 and August 2015, and between September 2015 and July 2016, respectively. During S1 and S2, 4,215 and 4,204 children were enrolled and 4,208 and 4,199 were included in the analyses , with a Plasmodium falciparum parasite prevalence estimated using microscopy varied between study sites in both surveys, with the lowest prevalence figures in Senegalese sites and the highest in BF , BF Saponé , TZ Korogwe , KE Kombewa , and GH Kintampo (S2 only P = 0.0065).st in BF . In KintPfPR varying from 2.7% in SN Niakhar to 69.9% in BF Nouna (not measured in SN Keur Socé) in S1 and from 1.3% in SN Keur Socé to 76.3% in BF Nouna in S2 .Similar trends were observed when measured by QT-PCR, with Supplemental Table 3).The prevalence of gametocytes measured by microscopy ranged from 0.5% to 4.0% in S1 and from 0.0% to 5.3% in S2 in all sites, except in BF . Among participants tested positive for asexual parasites, 7.5–66.7% carried gametocytes in S1  and 0.0–83.3% in S2  . The proAcross surveys, approximately 21.6% of participants with a positive QT-PCR result had a negative microscopy reading  and around 8.8% of participants positive for microscopy had a negative result with QT-PCR  . The proAcross all sites, Cohen’s kappa coefficient between qualitative results of the two methods of measurement of parasitemia (microscopy versus QT-PCR) using the Landis and Koch scale showed a substantial agreement in both surveys . Cohen’s kappa coefficient between semi-quantitative results for parasitemia measured by microscopy versus QT-PCR also showed a substantial agreement in both S1  and S2 .Supplemental Table 4).Between 25.0% (192 of 767 in S2) and 33.8% (185 of 548 in S1) of participants tested positive for gametocytes by QT-NASBA were also detected positive by microscopy . Coinfection with both P. malariae and P. falciparum was more frequent than single infection with P. malariae alone in both surveys . Of the 62 participants in S1 and 128 participants in S2 infected with P. malariae, 55 (88.7%) and 91 (71.1%) were also infected with P. falciparum, respectively. Infection with Plasmodium ovale was low in both S1 and S2 (0.5% and 0.2% of participants). Plasmodium vivax was not observed in S1 and in only one participant in S2. Infections with species other than P. falciparum were mostly observed in sites of medium-to-high P. falciparum prevalence.Infection with Supplemental Table 6). The highest use of bednets was in KE Kombewa (97.5%) in S1 and TZ Korogwe (99.2%) in S2, and the lowest in GH Kintampo (70.2%) in S1 and SN Niakhar (69.6%) in S2  in S2 . A decreSupplemental Table 6). Details for the characterization of bednet usage  by study site are shown in Overall, 70–80% of the bednets were impregnated, 60–70% were new, and approximately 25% were torn .Participant’s recall of IRS in the past 12 months was recorded for a very low number of participants , mainly in SN .Overall, usage of coils and repellents was limited, around 10% of the population in both surveys with variations per site between 1% and 40% according to the survey . Across all sites, occurrence of fever was higher in P. falciparum–infected versus non-infected participants . In both surveys, fever was more frequently reported by participants with higher parasite densities .Fever in the last 24 hours was reported for approximately a quarter of the participants in both surveys, with differences across study sites ranging from 3.6% in BF Saponé to 63.8% in KE Kombewa in S1 and from 3.0% in SN Keur Socé to 71.6% in KE Kombewa in S2 . Plasmodium falciparum–infected children sought treatment against malaria or fever more often than non-infected children .In S1, 15.7% of participants had sought treatment against malaria or fever in the 14 days before the survey compared with 12.8% in S2, ranging from 0.0% to up to 33.3% depending on the study site and on the survey .The proportion of participants hospitalized for malaria was 2.6% in S1 and 2.8% in S2, with no marked difference between P. falciparum infection. Across both surveys, houses equipped with electricity , cement/plaster walls versus mud , and nets on all windows  were associated with a lower risk of infection with P. falciparum. In addition, older age was associated with a higher risk of infection .An exploratory multivariable model was used to assess the association between potential risk factors or malaria control interventions and PfPR as a proxy, this study aims at characterizing MTI15 in different SSA settings, including areas in GH and KE where the RTS,S/AS01E malaria vaccine is currently introduced in the framework of the MVIP. More specifically, considering the WHO recommendation to operate the MVIP in moderate to high transmission areas of SSA, PfPR in children aged 6 months to 4 years in Kintampo and Kombewa sites was high, ranging from 24.8% to 27.3% depending on the study site and the survey. Similar PfPR was estimated by Drakeley et al.,9 indicating a stable mesoendemic MTI level (PfPR = 10–50%) in those areas.16Plasmodium falciparum parasite prevalence varied largely between sites as it was expected from various preselected transmission intensity areas. In both surveys, the two sites in BF had the highest PfPR and the two sites in SN, the lowest. Plasmodium falciparum parasite prevalence rates recorded in this study are in line with previous findings.18Using PfPR was lower in the 6-month to < 5-year than in the 5-year to < 10-year age-group. In addition, a risk factor analysis highlighted an association between older age and higher risk of infection. Those results corroborate the findings of other studies, identifying increasing age as a risk factor for carrying malaria blood stage parasites.19 This may potentially be explained by the fact that younger children benefit from a more focused usage of control interventions (bednets).20 Another explanation would be increased immunity in older children due to repeated exposure to the parasite, leading to asymptomatic carriage and a lower probability to be treated than in symptomatic children.22Across most sites, the P. malariae was observed in a few participants, and infection with P. ovale was rare, which supports observations from other studies conducted across SSA.26 Across all sites and surveys, only one Plasmodium vivax infection was identified by microscopy in TZ, which differs from Twohig et al.’s27 recent findings of growing evidence of this species in SSA. A high proportion of coinfections with P. falciparum was observed in P. malariae–infected participants (between 70% and 90%) and P. ovale–infected participants (around 60%). Similarly, high percentages of P. falciparum coinfection in P. malariae–infected participants was previously reported in Guinea (97%),23 Uganda (91%),24 and in the Democratic Republic of the Congo (90%),25 with lower percentages reported in rural BF (67%)26 and Benin (34%).23Infection with P. falciparum by QT-PCR were undetected by microscopy. Classifying the study sites by MTI, this proportion was higher in low MTI sites. This might be explained by a relatively lower sensitivity of microscopy readings than nucleic acid related techniques, particularly in low parasite density infections, which are more frequently observed in low MTI areas.13 Nevertheless, the kappa statistic estimated a substantial agreement between microscopy and NAAT techniques using either qualitative or semi-quantitative real-time PCR.Approximately 20% of participants positive for PfPR and in the use of malaria control interventions, which are key variables to be monitored before and after vaccine introduction in the MVIP areas. Little variation in P. falciparum prevalence between the first two annual surveys was observed among most sites. Usage of bednets as a malaria control intervention was high in all sites and in both surveys (ranging 70–99%). Some variation in the usage of bednets was observed in all sites, except in BF.In addition, the present results are establishing a standardized baseline for further estimation of the year-to-year variations in P. falciparum ; however, no such association was observed in S1. Various entomological studies have questioned the efficacy of repellents and coils as effective malaria prevention measures and highlighted the false sense of protection perceived by the user.30 Our data do not allow to draw robust conclusions at this stage, and the trend observed in S2 should be closely monitored in the subsequent cross-sectional surveys.In S2, participants not using mosquito coils, insecticide sprays, or repellents against malaria vectors were significantly less likely to be infected with 31 Moreover, this should mainly impact low transmission settings due to the high proportion of low-density infections.As with all interview–questionnaire-based studies, this study could have been subject to information or recall bias as data related to bednet usage, control interventions usage, and care-seeking behaviors were collected from parents’ recollection as opposed to objective observation. The impact of these potential information biases on the study results is estimated to be limited because of the proximity in time between the occurrence of the event for which information is collected and the interview itself. Another study limitation may be related to both sensitivity and specificity of microscopy slide readings. Slide reading performances may indeed vary depending on the parasite density and species identification. However, the kappa statistic estimated a substantial agreement between microscopy and QT-PCR, the latter being able to detect a higher number of low density infections than microscopy and RDT.PfPR observed in study sites that are part of the MVIP is in line with the WHO recommendation to operate the program in moderate to high transmission areas. In addition, our results are key to inform on the potential occurrence of annual fluctuations in MTI that may influence the assessment of the RTS,S/AS01E vaccine safety, effectiveness, and impact. The observations based on the first two surveys of this study do not indicate major temporal changes in terms of Plasmodium prevalence or use of malaria control interventions, but more surveys are needed to confirm this trend. The data generated in this study will be used to create variables to adjust the temporal and concurrent comparison analyses of the RTS,S/AS01E vaccine safety, impact, and effectiveness study. More specifically, the year- and site-specific PfPR computed on unvaccinated study participants will be included as covariates in the regression models of the safety, impact, and effectiveness study to assess annual fluctuations and/or changes due to other malaria control interventions.The present article summarizes the results of the first two annual surveys of a larger malaria prevalence study aiming at characterizing MTI in light of the use of malaria control interventions and other environmental factors. Our results confirm that the high To a broader extent, the large sample size of this study  and the use of a standardized methodology across multiple sites in various countries in SSA using multiple testing will provide a unique perspective on malaria prevalence variations across Africa.Supplemental materials


Plasmodium falciparum malaria in Cambodia is a goal with both national and international significance. Transmission of malaria in Cambodia is limited to forest environments, and the main population at risk consists of forest-goers who rely on forest products for income or sustenance. The ideal interventions to eliminate malaria from this population are unknown.Rapid elimination of In two forested regions of Cambodia, forest-goers were trained to become forest malaria workers (FMWs). In one region, FMWs performed mass screening and treatment, focal screening and treatment, and passive case detection inside the forest. In the other region, FMWs played an observational role for the first year, to inform the choice of intervention for the second year. In both forests, FMWs collected blood samples and questionnaire data from all forest-goers they encountered. Mosquito collections were performed in each forest.P. falciparum and 14.6–25.0% positive for Plasmodium vivax among forest-goers in each study site. In vectors, malaria prevalence ranged from 2.1% to 9.6%, but no P. falciparum was observed. Results showed poor performance of mass screening and treatment, with sensitivity of rapid diagnostic tests equal to 9.1%  for P. falciparum and 4.4%  for P. vivax. Malaria infections were observed in all demographics and throughout the studied forests, with no clear risk factors emerging.Malaria prevalence by PCR was high in the forest, with 2.3–5.0% positive for Malaria prevalence remains high among Cambodian forest-goers, but performance of rapid diagnostic tests is poor. More adapted strategies to this population, such as intermittent preventive treatment of forest goers, should be considered. The development and spread of artemisinin resistant malaria in the Greater Mekong Subregion  has made elimination of malaria from this region a global priority . PreviouMalaria transmission in Cambodia and throughout the GMS is concentrated in forest environments , 12. HowThe proliferation of different approaches to elimination of forest malaria raises the question of which strategies could be most effective in quelling this source of transmission in the GMS . This stThis study reports preliminary findings of two separate in-forest malaria control studies Table . Both st2) in Mondulkiri and Ratanakiri provinces, near the Kratie province border  submitted a questionnaire and were asked to provide a dried blood spot (DBS). Forest goers who were continuously or repeatedly in the forest could be enrolled at most every 2 weeks. The data obtained from this first year was intended to be strictly observational and inform a choice of intervention for the following year, from March 2020–March 2021. Due to ethical concerns, however, FMWs were provided with small supplies of malaria RDTs  and artesunate-mefloquine (AS-MQ)  during the observation year in case they encountered any sick forest goers unable to return to the nearest health centre for treatment. The final outcomes of this study will be based on a comparison of the observation and intervention years, both within the forest and in neighboring health centers and village malaria workers. This study is referred to as the “observation-intervention” study.The first study is located inside a small forested area  and treatment , passive case detection of febrile cases, and reactive case detection among the colleagues of any identified cases. All forest goers of any age who provided informed consent were given a questionnaire, asked to provide a DBS, and tested for malaria by RDT. Parental consent was required for minors < 18 years old, as well as assent of the minor if at least 10 years old. Forest goers who were continuously or repeatedly in the forest could be enrolled every 2 weeks. Additionally, febrile cases could seek out the FMWs for testing and treatment. In case of a positive test, FMWs were encouraged to test any forest goers who had travelled or worked in the forest with the case and not yet been tested. Positive cases were provided with treatment (ASMQ) and, beginning in November 2019, with insecticide-treated hammock nets . Final outcomes of this study will be based on the comparison of malaria notifications from intervention health centers surrounding the study forest and control health centers in other parts of the country. This study is referred to as the “MSAT” study.The second study is located in a forested area of approximately 1000 kmP. falciparum malaria from the study forests by the end of the study period. The results presented here are preliminary results through to Dec 31, 2019.The overall goal of both studies is to fully or nearly eliminate The areas of the forests to be covered in these studies were chosen based on satellite imagery followed by repeated exploratory missions to the forest. Owing to the size and isolation of these forests, these exploratory missions involved spending multiple days in the forest by motorbike. Mobile phones were used to track the paths used by research staff inside the forest, and they recorded where they encountered forest goers, camps, and water sources.2. In the MSAT study, 36 sectors were initially selected, with a median (IQR) sector size of 25.9  km2. The number of FMWs was equal to the number of sectors. However, the sectors did not all remain fixed within the study. Data from the first few months of the observation-intervention study revealed that some sectors had few forest goers, while additional exploratory missions suggested other areas that should be included. As a result, four sectors  were removed and replaced with four new ones  the week of 8 July 2019. In the MSAT study, four of the initially selected sectors had no active FMW through the time of this analysis due to difficulties in obtaining permission from a company working in those areas.In the observation-intervention study, 30 sectors were initially selected, with a median (IQR) sector size of 15.2  kmFMWs were provided with Android smartphones for geolocation, monitoring, and electronic data entry. A 1 week training session was conducted for FMWs from each study site on malaria awareness, the study objectives and procedures, use of the smartphones, collection of dried blood spots, and malaria case management.Electronic data entry was done via Kobo toolbox. FMW smartphones were also equipped with the GPS Logger application to track their movements inside the forest and BackCountry Navigator to help them understand the boundaries of their sector. FMWs were provided with powerbanks to maintain smartphone power inside the forest. At the end of each work week, the FMWs returned the smartphones and powerbanks to study staff, who uploaded the data collected that week and charged the devices before returning them to the FMWs. Weekly feedback on each FMW was automatically produced based on their questionnaire results and GPS tracks, and was used to improve their performance.The questionnaires for the two studies were very similar and included questions on their activities and shelter inside the forest, their knowledge of malaria and malaria prevention methods, and their healthcare-seeking behaviour. Questions about identifying information such as name, village of residence, age, and phone number were also asked, to allow for better identification of repeat forest goers both for purposes of analysis and intervention . In the observation-intervention study, forest goers were asked if they thought they had a fever on the day of the interview; those who said no were assumed to be healthy, and axillary temperatures were measured among those who said yes. In the MSAT study, axillary temperatures were measured for all participants. Those participants with temperatures > 37.5C were classified as having a fever at the time of the interview. Testing and treatment was offered to any individuals in either study presenting with a fever. Temperatures measured outside a biologically plausible range were discarded and considered as missing.Dried blood spots were collected from forest goers for malaria real-time PCR screening and speciation. Parasite DNA was extracted using Instagene matrix resin .Screening and speciation of malaria infection was performed by targeting sequence coding for cytochrome b according to Canier et al. . Cycle tBeginning in January 2020, after the end of the results presented here, study staff have attempted to contact individuals in the MSAT study who tested positive by PCR to receive malaria treatment. Individuals testing positive by PCR in the observation-intervention study were not re-contacted for treatment.Anopheles sp. mosquitoes were morphologically identified using a taxonomic key [®, , according to the manufacturer’s instructions. A two-step semi quantitative real-time PCR was performed to detect malaria parasites, as previously described [Exposure to malaria vectors was determined by carrying out mosquito collection using odour-baited double net traps (BNTs as in ) baited omic key . Followiescribed .The interim analyses presented here were intended to assist in the choice of interventions for Year 2 of the “observation-intervention” study. All analyses were made in R. The main questions and analyses are as follows.Basic descriptions of forest goer demographics, activity patterns, and familiarity with malaria are reported as means and percentages. To describe forest goer movement patterns, it was necessary to identify repeat forest goers inside the database. The details of how this matching was done are reported in the Additional file P. falciparum infection and per P. vivax infection were calculated. The yield was calculated as number infected divided by number tested. The NNT was calculated as the inverse of the yield.To assess the risk-benefit profile of mass preventive treatment to forest goers, the yield and the number of people that would need to be treated (NNT) per P. falciparum or P. vivax) by PCR, or whose RDTs were considered positive by the FMW or any study staff member, were read by three different staff members and the majority opinion was used.To determine whether RDTs should be used to assign treatment in Year 2 of the observation-intervention phase, the sensitivity and specificity of RDTs (compared to PCR) were calculated from the MSAT study. RDT readings made by FMWs were compared to those made by study staff based on photos of the RDTs uploaded into Kobo Toolbox. All RDT photos were read by at least one study staff member. The RDT photos of all individuals who tested positive for malaria ; prevalence of the risk factor among individuals with P. falciparum infection (to ensure high intervention coverage); and consistency between the two studies . Associations between questionnaire variables (independent variables) and P. falciparum PCR positivity (dependent variable) were used to assess strength of the relationship. Odds ratios and p-values were obtained from sequential univariate logistic regression models.Additional analyses examined whether any risk factors from the questionnaire could be used instead of or in addition to RDTs to target individuals for treatment. Risk factors were evaluated according to the following criteria: strength of the relationship with P. falciparum malaria infection (odds ratio > 1.5) and that were present in a majority (> 50%) of P. falciparum infections in the output of both studies. Cut-offs based on odds ratios rather than p-values were used, as they provide measures of the magnitude of effect rather than statistical significance (which may be affected by other factors such as sample size); a high odds ratio implies high separation of risk between the different risk groups. An odds ratio of 1.5 implies only a 50% higher odds of infection inside the risk group compared to outside, and was considered the minimum acceptable value for this initial screen.These analyses emphasized primarily those variables that have been identified as risk factors in studies outside of the forest, namely: age, gender, logging, history of malaria treatment, staying overnight in the forest, and having fever in the last month , 27–29. P. falciparum infection in both study forests. Those individuals PCR positive for P. falciparum were considered as “cases” and those PCR negative for P. falciparum as “controls.” The R package sparr function “risk” was used to estimate the relative risk between the case and control 2D kernel density estimates, and considered as hotspots those regions identified as significantly higher risk (p < 0.05) [Finally, the analyses searched for geographic hotspots of  < 0.05) .Anopheles mosquitoes with daytime biting behaviour was considered, and the previous use of various vector control strategies among forest goers (incl. insecticide-treated hammocks) with and without P. falciparum infection was compared.To determine the potential impact of vector control kits, the proportion of The observational year of the “observation-intervention” study was approved by the Cambodia National Ethics Committee for Health Research on 19 October 2018 with study number 281 . It was renewed on 6 December 2019 under study number 306. The MSAT study was approved by the Cambodia National Ethics Committee for Health Research on 28 June 2019 with study number 148 .In the “observation-intervention” study, 1696 forest goer encounters, of which 1612 (95.0%) provided informed consent and completed the questionnaire, were recorded from 11 March to 31 December 2019. In the MSAT study, 1145 (99.6%) of 1150 forest goers in Stung Treng from 27 July to 31 December 2019 provided informed consent and completed the questionnaire and 467 (88.8%) of 526 forest goers in Kratie from 2 September to 31 December 2019.The matching algorithm detected 5.7% of FGs in the “observation-intervention” and 3.6% of forest goers in the MSAT study as repeat interviews with participants already included. However, the applied algorithm cut-off ensured a high probability (99%) that identified pairs were true matches, at the cost of allowing many (> 50%) of matches to be missed  in the observation-intervention study and 0.9 km  in the MSAT study; 12% of such repeated interviews in the observation-intervention study and 0% in the MSAT study were > 15 km apart. Only 33% of forest goers from the observation-intervention study and 15% of forest goers from the MSAT study provided contact phone numbers. Most forest goers (52% in the observation-intervention study and 57% in the MSAT study) reported not having a mobile phone.The main activities of forest goers differed by study site and time. Logging (45%) and gathering forest products (43%) were the most common activities reported in the observation-intervention study, whereas in the MSAT study gathering forest products was by far the most commonly reported activity (75%). The proportion of forest goers in the observation-intervention study who reported logging declined between June and September, corresponding to a period of crackdowns on illegal logging throughout Cambodia  were positive for P. vivax infection, and 2 (0.1%) were positive for Plasmodium malariae infection. Of these, 10/73 (13.7%) positive for P. falciparum and 36/363 (9.9%) positive for P. vivax had out-of-range temperature values recorded. Of those remaining, 2/63 (3.2%) positive for P. falciparum had a measured fever at the time of the interview ; among those positive for P. vivax, including mixed, this result was 3/327 (0.9%). If all forest goers were to receive preventive treatment for malaria, these findings would correspond to a number needed to treat (NNT) of 19.9  individuals per P. falciparum infection and 4.0  individuals per P. vivax infection  were positive for P. falciparum infection, 234 (14.6%) were positive for P. vivax infection, and 10 (0.6%) were positive for P. malariae infection. Among those positive for P. falciparum, 3/37 (8.1%) had a measured fever at the time of the interview ; among those positive for P. vivax, including mixed, this result was 6/234 (2.6%). If all forest goers were to receive preventive treatment, the NNT would be 43.2  per P. falciparum infection and 6.8  per P. vivax infection  were positive for Results of rapid diagnostic tests are based on the MSAT study. Of 1598 interviews with PCR results available, 297 (19%) had no RDT photo available or the photo was unreadable; 306 (19%) had photos taken too early, while the RDT was still bloody; and 995 (62%) had acceptable RDT photos.P. falciparum, there was 99.6% agreement between RDT readings by research staff and FMWs, and Cohen’s kappa was 0.60 . For P. vivax, there was 99.8% agreement between RDT readings, and Cohen’s kappa was 0.89 .Based on the acceptable photos only, for P. falciparum and P. vivax. The sensitivity of RDTs to detect P. falciparum infections in the forest was estimated at 9.1%  and the specificity was 99.8% . The sensitivity of RDTs to detect P. vivax infections was estimated at 4.4%  and the specificity was 99.8% .Table P. vivax cases that were RDT-positive; for P. vivax cases that were RDT-negative, the median (IQR) cycle threshold was 36.6 . Among P. falciparum infections that were negative on RDT but positive for PCR, the median (IQR) cycle threshold was 37.3 .The number of cycles required for PCR to reach positivity was lower for individuals who were positive also by RDT, suggesting higher parasite density. The screening cycle thresholds ranged from 22.7 to 30.5 for P. falciparum cases detected by both RDT and PCR.This suggests that poor RDT sensitivity to diagnose malaria in forest goers is the consequence of low parasite density. The screening cycle thresholds were 26.7 and 31.2 for the two P falciparum parasitaemia measured in both studies. These six risk factors were selected based on previous studies outside of the forest. None of these risk factors met the criteria of being both strong (odds ratio > 1.5) and common  across both studies. In addition, no other risk factors were identified that met these criteria in both studies. In the observation-intervention study, residence in Kratie as opposed to Mondulkiri or other provinces was a highly significant risk factor for P. falciparum infection . In the MSAT study, residence in Kratie as opposed to Stung Treng or other provinces was not significantly associated with higher risk of P. falciparum infection .Table P. falciparum infections and 17% of all interviews  was detected in the observation-interventions study area containing 33% of all iews Fig . HoweverAnopheles dirus represented 24% (56/234) of the collected Anopheles and 3 individuals were infected with malaria parasites . Three species represented more than half of the collected anopheles: An. dirus (23.77%), Anopheles maculatus sensu lato (s.l.) (23.78%) and Anopheles philippinensis s.l. (13.99%). The day biting behaviour (mosquitoes collected between 6am and 6pm) frequency was high with 22.4% of the mosquitoes biting during daytime in the human BNTs and 25% in the cow BNTS. In addition, day biting behaviour in human BNTs was usually above 20% (20.5 % to 25%) in the forest sites whereas it was below 15% in the villages . At the time of the interim analyses, the overall mosquito malaria parasite prevalence was 5.1% with 68 P. vivax infected mosquitoes out of the 1,329 screened individuals. Infected mosquitoes were collected equally in cow BNTS (35/727) and in human BNTs (33/602). Furthermore, a high diversity of infected mosquitoes was observed with at least 12 different Anopheles species carrying human malaria parasites  infections, P. falciparum (including mixed) infections, and no infections who said they had ever heard of or ever used individual vector control measures in each study. Although the proportions of people familiar with each vector control measure differed between study sites, they were generally similar among people with and without prevalent malaria infections.Additional file inside the forests in Cambodia. Though the two studies are still on-going, these interim analyses were planned to assist in the determination of an intervention for the second year of the observation-intervention study.The epidemiology of malaria in forest-going populations in the GMS including Cambodia is poorly characterized. This paper reports results from the first two malaria studies based P. falciparum and 4.4%  for P. vivax. Similarly poor RDT sensitivity has previously been seen at the village level in Cambodia, in a study in which 91 participants were PCR-positive for any Plasmodium infection, but only 2 were positive by RDT [Malaria infections were detected in both humans and mosquitoes in the study forest areas, as had been expected based on the presence of cases at nearby health centres. One of the primary conclusions is that, despite a high prevalence of malaria infection as measured by PCR, very few of these infections are detected by RDTs. The sensitivity of the RDTs in this context was very low, at 9.1%  for e by RDT . Other se by RDT , 32. RDTThe results suggest that a low parasite density explains the poor RDT sensitivity. Considering their occupational profile, forest goers are likely to be subjected to a chronic exposure to malaria parasites and therefore may have developed a relative immunity enabling the host to maintain a low parasite density. Previous studies have shown that submicroscopic malaria is common in the Greater Mekong Subregion, that it can last for months, and that infection density can change over time , 34. AltAlthough a PCR-based MSAT (in place of RDTs) is theoretically appealing given its much higher sensitvity, this would be operationally irrelevant and is not a scalable option in Cambodia. Even in these smaller pilot projects it often took one month or longer for the dried blood spots to be transferred from the forest to the field site to the study central laboratory for testing and the results to be available. Furthermore, re-contacting forest goers after such results are available is not trivial given the small percentage who provide mobile phone numbers. Forest-goers participating in illegal logging may be particularly unwilling to provide identifying information that could allow them to be re-contacted.P. falciparum infection and present in a majority of people with P. falciparum infection in both studies. Other studies have identified presence in the forest as the main risk factor for malaria infection in Cambodia [P. falciparum infections were found outside of identified hotspots; however, it was not possible to identify where these individuals were initially infected.A risk factor based treatment strategy could be appealing as a replacement for RDTs if an appropriate risk factor could be found. However, no risk factors were identified that were highly associated with Cambodia , 28, 37.Anopheles mosquitoes in the forest commonly bite during daytime hours, making traditional insecticide-treated nets or forest-adapted insecticide-treated hammock nets less appealing tools for this environment. Similar numbers of people infected and not infected with malaria reported ever using various vector control measures including wearing protective clothing and using repellent.In terms of vector control, this study shows that Given these identified issues, and the continued high prevalence of malaria infection inside the forest , the observation-intervention study will provide preventive malaria treatment to everyone in the forest during its second year. This appears likely to be the most efficient strategy to rapidly eliminate malaria in the forest. Artemisinin-based combination therapy (ACT), using AS-MQ or dihydroartemisinin-piperaquine (DP) are appealing for this purpose as they would both eliminate existing infections and provide temporary protection from infection given the long half-life of the partner drugs. Similar to chemoprophylaxis, such “intermittent preventive treatment of forest goers” (IPTfg) would probably need to be repeated multiple times at intervals determined by the partner drug half-life, and thus has similar requirements in terms of drug safety. The two drugs with the most appealing safety and acceptability profile in Cambodia are AS-MQ and DP .The problem of using AS-MQ is that it is the current first-line treatment in Cambodia, and use as IPTfg might accelerate development of resistance. However, data suggest resistance levels are currently low, and theoretical models have shown that chemoprophylaxis could in some circumstances reduce resistance in the population by reducing treatment of symptomatic cases , 39; thiP. falciparum elimination, motivated in part by widespread resistance to artemisinin in the region. However, an effect on P. vivax may also be observed as repeated ACT administration could offer temporary protection from P. vivax relapse. Nevertheless, any observed effect on P. vivax will likely be short-lived, and more adapted strategies such as targeted use of radical cure, should be considered secondarily for this species.The IPTfg intervention is targeted primarily at These studies were based inside forests and conducted by FMWs with strong forest knowledge but limited medical education, which led to some unique challenges. Full coverage of the population was not achieved in these studies and it is not possible to say with certainty how many forest goers were missed, as missing forest goers were not recorded systematically and may have avoided any contact with the FMWs altogether. The length of the consent process and questionnaire  likely lowered coverage, as forest goers often work in groups and could not postpone their work to participate. Furthermore, reports of the number of people in the forest are assumed to be an underestimate, as FMWs reported that sometimes forest goers would run away when they saw them. Forest goers are often participating in illegal logging activities, which could make them suspicious of FMWs or study staff, though this risk was lowered by recruiting FMWs from the forest going community. If nonparticipation was systematic, this could affect the description of the forest goer population, but the results are still accurate for the subset of the population interviewed. The accuracy of the questionnaire responses could have been reduced if FMWs or forest goers did not accurately understand the questions. Furthermore, all risk factors were measured retrospectively, and the questionnaire did not attempt to determine the timing of past exposures such as when insecticide-treated nets were last used. However, this should be less important as the main goal was to determine associated factors that could be used for allocation of treatment, rather than elucidate causal relationships.P. falciparum and P. vivax. RDT sensitivity is low inside the forest, and the risk appears fairly homogeneous across all categories of individuals in these high-risk locations. Forest-going populations are likely to maintain a strong antimalarial immunity leading to a majority of low density infections, limiting the utility of MSAT in this environment. Given the tight deadlines to eliminate malaria in this region, intermittent preventive treatment of forest goers will be implemented inside the observation-intervention study for its intervention year. If this strategy proves feasible and effective, it could be expanded to other locations in Cambodia and in other countries attempting to eliminate forest malaria. The results suggest that the forest goer chemoprophylaxis strategies being explored by other stakeholders in Cambodia may be appropriate given the dynamics seen within these forests, but that the efficacy of hammock nets or mass testing and treatment by mobile malaria workers may be more limited, given the low sensitivity of RDTs among forest goers and the daytime biting behaviour of mosquitoes inside the forest.Individuals within the forest in Cambodia have high PCR prevalence of Additional file 1: Table S1. Specificity and sensitivity of the matching algorithm at various cutoff values. . Table S2. Infected Anopheles mosquitoes collected in the two study. Fig S3. Proportion of individuals with P. falciparum infection, P. vivax infection, and no malaria infection (as measured by PCR) who have ever heard of or used the listed vector control measures, in the observation-intervention study. Fig S4. Proportion of individuals with P. falciparum infection, P. vivax infection, and no malaria infection (as measured by PCR) who have ever heard of or used the listed vector control measures, in the MSAT study.Additional file 2. De-identified study database.


The filter removes the leukocytes and platelets from the blood samples, which interfere with the accurate detection of malaria by the blue-ray devices. We confirmed that the filter, which can be operated automatically by centrifugal force due to the rotation of the disc, achieved a high removal rate of leukocytes (99.7%) and platelets (90.2%) in just 30 s. The automated system exhibited a higher sensitivity (100%) and specificity (92.8%) for detecting Plasmodium falciparum from the blood of 274 asymptomatic individuals in Kenya when compared to the common rapid diagnosis test (sensitivity = 98.1% and specificity = 54.8%). This indicated that this system can be a potential alternative to conventional methods used at local health facilities, which lack basic infrastructure.There is an urgent need to develop an automated malaria diagnostic system that can easily and rapidly detect malaria parasites and determine the proportion of malaria-infected erythrocytes in the clinical blood samples. In this study, we developed a quantitative, mobile, and fully automated malaria diagnostic system equipped with an on-disc SiO Malaria is a vector-borne disease, which is caused by infection from Plasmodium spp. and is transmitted by Anopheles mosquitoes. The United Nations Sustainable Development Goals had proposed to end the epidemic of malaria by 20302. Although the annual fatality rate has decreased since 2016, about 405,000 malaria-related deaths were reported in 2018. Similarly, new malaria cases have increased slightly since 2014 with 231 million recorded cases in 2017 and 228 million recorded cases in 20183. Several factors have stagnated global progress in eradicating malaria4. Particularly, most tools to tackle the current malaria infection were developed before 2000. Thus, there is a need to develop new tools using novel technologies to accelerate the efforts toward malaria elimination4.Globally, malaria is one of the “big three” infectious diseases with an incidence rate of 57 cases per 1000 individuals5. Furthermore, the deletion of the HRP2 gene results in a false-negative result6. The microscopic examination of Giemsa-stained blood smear, which is another common diagnostic method, is a low-cost method for the determination of parasitaemia. However, microscopic examination involves labour-intensive steps and requires technical expertise for an accurate diagnosis.Accurate diagnosis is essential to ensure effective patient management and to prevent unnecessary treatment. Rapid diagnostic tests (RDTs), which detect malaria-specific antigens, such as histidine-rich protein 2 (HRP2), are easy to use, rapid, and affordable. Hence, RDTs are widely used even in remote areas. However, RDTs are unable to determine the quantitative content of parasites in the blood (parasitaemia) that is generally associated with the severity of malaria infection. Additionally, as the clearance time of HRP2 antigen in the patient’s blood is very long, the presence of residual antigen potentially produces a persistent false-positive result even after several weeks post-parasite clearance7. The system comprises a scan disc and a fluorescence image reader. The erythrocytes are dispersed onto the disc surface where malaria parasites are fluorescently stained. The fluorescence-positive erythrocytes are then detected by the fluorescence image reader. There was a linear correlation between our malaria diagnostic system and microscopic examination for the detection of parasitaemia. However, further improvements are needed to render the current system a fully automated malaria diagnosis equipment that can be used in malaria-endemic area. Previously, the samples that contained only erythrocytes  were used. However, the patient blood samples also contain leukocytes and platelets, which potentially produce fluorescent noises. These noises interfere with the accurate detection of malaria. In some cases, these noises are incorrectly recognised as malaria parasites. Additionally, P. falciparum strains in the patient blood in malaria-endemic regions are highly diverse. The previous study was performed in a well-equipped laboratory. The laboratory conditions are different from the conditions in the malaria-endemic regions, which have limited infrastructure. The current system needs a centrifugation step to remove the leukocytes and platelets, which usually requires huge electric power and several complicated manual steps before applying blood samples into the system.Recently, we had developed a highly sensitive and rapid malaria diagnosis system using fluorescent blue-ray optical devices2 nanofiber filter device and incorporated it into the scan disc, which can be operated automatically without the need for centrifugation. This modification enabled the conversion of the current system into a fully automated malaria diagnosis equipment. We then validated the applicability of the newly developed equipment for the diagnosis of P. falciparum in individuals from the malaria-endemic region of Kenya.In this study, we first developed a SiOPlasmodium staining unit. The function of the scan disc is to isolate the erythrocytes and deploy them in a monolayer formation onto the staining unit. In the staining unit, the malaria parasites are fluorescently stained with a nuclear-specific fluorescence stain, Hoechst 34580 . The fluorescence image reader detects the fluorescently stained nuclei of the malaria parasites. The identification of P. falciparum and the quantitative measurement of the proportion of infected erythrocytes among all counted erythrocytes are performed using custom-made software.The automated malaria diagnostic system is primarily composed of the following two devices Fig. : scan di8. Based on this, we developed a scan disc SiO2 nanofiber filter unit. We strictly controlled the pore size and the time required for the formation of the SiO2 nanofiber  in 274 Kenyan blood samples   underwent a diagnosis test for malaria parasites.Among the 288 school children enrolled in this study, 11 were excluded as we did not obtain an adequate amount of blood samples n = 3) or because the blood coagulated (n = 8) . The staining yielded fluorescent-positive images of the malaria parasites in the infected cells Fig. . Erythrotes Fig. . As haemtes Fig. .Figure 3P. falciparum-positive samples (n = 53) and 912,556  in the P. falciparum-negative samples (n = 221) : 737,174–936,552) in the 13. The CV was determined as 0.0048% based on the mean (0.0018%) and standard deviation (SD) (0.0018%) of the percentage of parasites determined by our diagnostic system in parasite true negative samples (n = 221)  as the cut-off value for detecting parasitaemia using our diagnostic system.We first determined the critical value (CV) of our diagnostic system, which is defined as the value that produces an error probability of 0.05 when parasite true negative samples are measured. We used the Currie method that is recommended by the International Union of Pure and Applied Chemistry and commonly used for this purpose–16, McNemar’s test). To assess the variability in diagnostic accuracy across different groups of participants, we analysed the blood samples obtained from Japanese volunteers (n = 40)  to the blood samples and the samples were analysed using our diagnostic system. The sensitivity and specificity of the malaria diagnostic system were 93.3%  and 92.0% , respectively. This indicated that the diagnostic values obtained in the blood samples of Japanese individuals were similar to those obtained in the blood samples of Kenyan individuals.The sensitivity and specificity of the malaria diagnostic system were 100%  and 92.8% , respectively Table . The pos13. The calculated LOD was 0.0077% (347 parasites/µL) . A strong correlation was also obtained in Spearman’s rank-correlation test . These results indicated a linear correlation between the percentage parasitaemia value obtained by the automated malaria diagnostic system and that obtained by microscopy.We evaluated the degree of correlation between the percentage parasitaemia obtained by our diagnostic system and that obtained by microscopy in 53 malaria parasite-positive samples . As mentioned previously, the CV and LOD for our diagnostic system were 0.0048% and 0.0077%, respectively . The automated malaria diagnostic system could detect malaria parasites with a high sensitivity value (100%). Furthermore, the specificity (92.8%) of our diagnostic system was markedly higher than that of other commercial RDTs (55%). The high specificity of diagnosis may prevent unnecessary administration of antimalarial drugs to non-malaria parasite-infected individuals. Consequently, this may alleviate the risks associated with the emergence and spread of drug-resistant malaria parasites15.Previously, we had performed an 16. Further improvement of imaging resolution in the detection area can also improve the ability of the system to distinguish the malaria parasites from the accidentally stained platelets. Although a resolution of 500 nm has been already achieved, a higher resolution can be achieved using super-resolution techniques, which include fine measurements with a smaller spot size and an objective lens having a higher numerical aperture and/or digital reconstruction of fine images by image convolution18. When the blood samples that were pre-treated for erythrocyte transfusion were analysed, the platelets were almost completely removed and the LOD for detecting the parasite was 0.0002% (9 parasites/μL)7. Therefore, we believe that a similar LOD level can be achieved using the above-mentioned improvements.The estimated CV and LOD of our diagnostic system in Kenyan individuals were 0.0031% (140 parasites/µL) and 0.0061% (275 parasites/μL), respectively, which are comparable to the LOD of the microscopic examination with Giemsa staining by a skilled microscopist and RDT. The factor that negatively affects the CV and LOD is platelets, which could not be removed in the system designed in our previous study. Further improvement of the filtration performance can potentially decrease the number of platelets in the detection area. Platelet aggregation using collagen activation is a potential strategy because it is inexpensive and simple19, flow cytometry20, automatic counting from digitally captured images of Giemsa-stained blood smears22, and the evaluation of acoustic signals of vapour-generated nanobubbles from hemozoin23. Although some technologies are highly sensitive for the detection of malaria parasites with minimal blood volume, they might not be ideal for POCT because of the size of systems and/or requirement of huge power supply, which are generally limited in the remote areas with poor infrastructure.The automated diagnostic system can determine parasitaemia, which provides a comparative advantage over RDT that has been used as a point of care testing (POCT). The determination of parasitaemia enables the evaluation of therapeutic efficacy by monitoring the patient’s parasitaemia status after treatment and the early detection of antimalarial-resistant cases. It also provides clinical benefit for patients with severe malaria because high parasitaemia is one of the important manifestations of suspected severe malaria. Therefore, it is reasonable to develop a novel malaria diagnostic system that can determine parasitaemia. Various new technologies have been used for this purpose, including magnetic resonance relaxation7. However, we further improved the packaging using an aluminium bag. Additionally, the inert N2 is cycle purged, which prevents humidity and oxygen permeation that may degrade the fluorescent dyes. Furthermore, the automated malaria diagnostic system is easy to operate and enables the analysis of stable parasitaemia independent of technical expertise. The scan discs can be manufactured at low-cost because a nanofiber filter chip inside the scan disc is manufactured by mass-production using a wafer. The cost of 1 $/sample can be realised even if necessary components, such as pipette tips and phosphate-buffered saline (PBS) are included. The image reader can be used on any tablet or laptop PC by installing the software, and the main body is expected to be priced at similar levels to commercial blue-ray players.Contrastingly, the automated malaria diagnostic system is portable, battery-driven, and robust. These characteristics are important as malaria diagnosis is commonly performed in tropical regions with severe conditions, such as high temperature, humidity, and dusty environment. The system is designed for protection against particles and water based on the criteria stipulated by the International Electrotechnical Commission IP52 ,” 2013). We have not observed the errors caused by heat, humidity, or dust after repeated experiments with the automated diagnostic system in the field. The scan discs were also stable for several months at a wide temperature range (10–35 °C)In conclusion, we have developed an automated and quantitative malaria diagnostic system. Field testing of the system in Kenya revealed that the diagnostic system has high diagnostic accuracy. Compared to the Giemsa staining method, the automated system is associated with shorter diagnosis time, minimal human errors, higher specificity, and the ability to quantitatively measure parasitaemia. These results indicate the potential of this diagnostic system as a valid alternative to conventional methods used at local health facilities, which lack basic infrastructure. However, further studies using a large sample size are needed to verify the applicability of the automated malaria diagnostic system.2 nanofiber chips were made from silicon wafer using the micro-electromechanical system (MEMS) processing technology, which involves lithography and etching  method25. The VLS method is a vapour phase growth method in which SiO2 vapour is melted into liquid metal (platinum) particles and the nano solid phase (SiO2) precipitates when SiO2 reaches supersaturation26. SiO2 nanofiber is produced only in the presence of Si and platinum catalyst26. Hence, it is possible to form SiO2 nanofiber only at an arbitrary position by patterning platinum catalyst or patterning SiO2  to be placed inside the scan disc  is then manually diluted using PBS as a buffer solution (198 µL); Step 2, the diluted blood sample is injected into the scan disc; Step 3, the scan disc is set up in the fluorescence image reader; Step 4, the diluted sample is automatically passed through the SiOrce Fig. . The purrce Fig. ; Step 5,rce Fig. ; Step 6,2 and included 14 villages27. In this site, the annual rainfall ranges from 700 to 1200 mm with two rainy seasons (from March to June and from November to December). All Plasmodium species that cause malaria in humans, except Plasmodium vivax, were reported in this region. Additionally, P. falciparum (>90%) was reported to be the most prevalent species in this region28. The prevalence rate of malaria parasites in the study region evaluated by microscopic diagnosis and PCR was approximately 15–24% and 30–44%, respectively28. Anopheles gambiae sensu stricto, Anopheles arabiensis, and Anopheles funestus are the main malaria vectors in the study region30.A field test was conducted between 14 and 23 February 2018 to evaluate the performance and field application of our diagnosis system. The area of the study region, which is in the Gembe East Sub-location in Homa Bay County , was approximately 12 kmet al.31. According to their study, 50 infected individuals are sufficient to detect P. falciparum with an expected sensitivity of 95% and a lower bound 95% CI value of 80%. We assumed that the prevalence of P. falciparum infection in the study area was 20%, which estimated that 250 individuals must be recruited for the study. This estimate is well matched to the sample size used in our study.The minimum number of subjects to be enrolled for the study was determined based on the table of power estimates reported by Flahault 2EDTA . We analysed a maximum of 40 samples in one day at the central laboratories of the International Centre of Insect Physiology and Ecology . The haemoglobin level was measured using a HemoCue Hb201+ system . P. falciparum infection was screened using a commercial RDT kit . We prepared thin and thick blood smears on site. All smears were stained with 10% Giemsa solution for 10 min and examined under oil immersion objected of a light microscope  at 1000X magnification. For molecular analysis, whole-blood samples (20 µL) were transferred onto Whatman FTA® microcards . The samples were allowed to dry at room temperature and stored separately in plastic bags at −20 °C. DNA was extracted from the 5.5-mm-diameter blood spots using the QIAamp DNA Micro Kit . The final elution volume was 20 µL.The study was conducted using a community-based cross-sectional survey in all villages and 14 primary/secondary schools. The blood samples were collected from school children aged 1-16 years using a finger-prick technique and stored in BD Microtainer Tubes containing KThe purpose and procedure of the study were informed to the participants through local interpreters. Written informed consent was obtained from their parents or legal guardians. Individuals who tested positive for malaria were treated with Artemether–lumefantrine. We obtained ethical approval to conduct the human study from the Kenya Medical Research Institute Ethical Review Committee , and the National Institute of Advanced Industrial Science and Technology (AIST) ethics committee (No. 2017-156). All experiments were performed in accordance with Ethical Guidelines for Medical and Health Research Involving Human Subjects stipulated by the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare .P. falciparum infection as described previously33. This method targets the 18S rRNA gene of P. falciparum33. The genome of P. falciparum has 5–8 copies of 18S rRNA. The 18S rRNA is commonly used for DNA-based malaria detection methods34. The reported LOD of this method varies from 1 to 50 parasites/μL37. In this study, we determined the LOD of parasite density using a laboratory-adapted 3D7 clone at a density range of 0.0375–4 parasites/μL (using 2-fold dilutions in 12 different rows). Probit analysis was performed to determine the minimal density at which the parasite would be detected with 95% confidence. Parasitaemia in P. falciparum-positive cases was determined by counting 10,000 erythrocyte using thin blood smears or counting 500 leukocytes using thick blood smears.Species-specific nPCR was used to detect the 13. The CV, which is defined as the value that produces an error probability of 0.05 when true negative samples are measured, was used as the cut-off level to distinguish the parasite-positive cases from the parasite-negative cases by this system. The CV was determined based on the following formula: CV = mean + 1.645 SD. The LOD, which is defined as the value that produces an error probability of 0.05 when samples having a LOD level are measured, was calculated using the following formula: LOD = mean + 3.29 SD13. In this study, the samples that tested negative for the parasites in nPCR and were verified by microscopic evaluation were used as true negatives for determining the CV and LOD. When the parasitaemia percentage determined by our automated system was higher than the CV, the sample was considered as parasite-positive. Conversely, when the parasitaemia percentage determined by the system was lower than the CV, the sample was considered as parasite-negative.To estimate the CV and LOD, we used the Currie method that is recommended by the International Union of Pure and Applied Chemistry and commonly used for this purpose38. Pearson’s correlation test and Spearman’s rank-correlation test were used to evaluate the degree of correlation between the percentage parasitaemia obtained by our diagnostic system and that obtained by microscopy. A linear regression analysis was also performed to correlate the parasitaemia values obtained by these two detection methods39. The difference was considered statistically significant when the P-value was less than 0.05.The significance of discordance was measured using the Welch’s t-test, Chi-squared test or McNemar’s test. All statistical analyses were performed using R version 3.6.0. Exact 95% CI were computed for sensitivity, specificity, positive and negative predictive values, and accuracy using binomial distributions with the Clopper-Pearson methodSupplemental manuscript.Dataset 1.Dataset 2.Dataset 3.Dataset 4.Dataset 5.


Malaria rapid diagnostic tests (RDTs) are alternative diagnostic methods that have enabled reliable biological diagnostic testing in all situations where previously only clinical diagnosis was available. Varying diagnostic accuracy of malaria RDTs makes policymakers confused while choosing malaria test kits for their country.  The aim of this study was to evaluate the diagnostic performance of currently being used malaria RDT in Southern Ethiopia.  A cross-sectional study design was conducted from October 1 to December 15, 2016. A total of 160 patients were included in the study. Finger-prick blood sample was obtained from study subjects for the RDT test and microscopic examination. Collected data were entered and analyzed using SPSS version 20.0. P. falciparum detection were 63.27% and 94.3% and for P. vivax detection were 86.96% and 95.62%, respectively. The agreement between microscopy and RDT for specific identification of malaria species was moderate with a kappa value of 0.568.  The test kit evaluated had an overall sensitivity, specificity, PPV, and NPV of 97.44%, 93.67%, 93.83%, and 97.37%, respectively, to detect the presence or absence of malaria. Sensitivity and specificity of the kit for  The overall performance of the kit was below the WHO standard. Further study on a large sample size is recommended to be carried out in the study area to use the test kit instead of microscopy for malaria diagnosis. Providing training on quality malaria laboratory diagnosis and availing necessary supplies for malaria diagnosis shall also be considered. Anopheles [In Ethiopia, malaria is the leading cause of morbidity and mortality. Almost 75% of the country is malarious, and an estimated 51 million people (68% of the population) live in areas at risk of malaria [nopheles . It is onopheles .The nonspecific nature of signs and symptoms of malaria made a clinical diagnosis of the disease unreliable . MicroscRDTs are alternate diagnostic methods which enabled reliable biological diagnostic testing in situations where previously only clinical diagnoses were available, although it has varying diagnostic accuracy across different geographical regions , 7. TheiPlasmodium malaria was very good, with 99.8% sensitivity and 97.7% specificity, according to the study conducted on acute febrile patients visiting the Felegeselam Health Center, North Ethiopia [P. falciparum and P. vivax infections according to a study performed in Afar region, Northeast Ethiopia [The ability of Care Start™ Malaria Pf/Pv Combo test to diagnose Ethiopia . SimilarEthiopia .Even though Arba Minch and adjacent hot rural areas are one of the most highly malarious places in Ethiopia and using malaria RDTs as an alternative diagnostic tool, its performance was not evaluated so far. This study aimed at evaluating the diagnostic performance of malaria RDT by determining its sensitivity and specificity compared with microscopy to help local malaria eradication program presenting the actual performance of currently used malaria RDT.The study was conducted in Arba Minch General Hospital (AGH) and Arba Minch Health Center. Arba Minch town is located in Gamo Gofa Zone of the Southern Nations, Nationalities, and People's Region (SNNPR) about 500 kilometers south of Addis Ababa. It is found at an altitude of 1200–1300 meters above sea level with an average annual temperature of 29.7°C and rainfall of 700 mm [A cross-sectional study design was employed from October 1 to December 15, 2016.The study participants were all individuals who came to AGH and Arba Minch Health Center being suspected of malaria and have a request paper for malaria diagnosis at the respective laboratory unit.All age group patients with clinical signs and symptoms of malaria who presented to AGH and health center laboratory department for confirmation during the study periods were eligible for enrollment into the study.Clients who do not fulfill the inclusion criteria and refused participation were excluded from the study.Assuming a confidence level of 95%, an error risk of 1.96, an expected minimum sensitivity and specificity of all RDTs of 95%, compared with microscopy according to the World Health Organization , and a mA convenient sampling technique was employed to obtain those study subjects.n=((z2)p(1 − p)/d2)=80 , where n is the sample size, z is the 95% confidence interval (1.96), d is the margin of error (5%), and p is the sensitivity or specificity (95%).Questionnaires were developed for the purpose of simple sociodemographic data. Formats were used for reporting parasite density and the laboratory result of malaria status by microscopy and RDT. They were prepared originally in English after reviewing relevant literature and then translated to Amharic. Pretest of the questionnaire for the clarity and consistency of questions was performed, and the necessary correction was made based on the findings of the pretest.A blood sample was obtained from eligible study subjects visiting Arba Minch General Hospital and Arba Minch Health Center in the specified study period, having signs and symptoms of malaria, and sent to laboratory diagnosis. From each study participant, a finger-prick blood sample was collected for malaria testing with both Care Start™ RDT (Cat No.G0161 T) and microscope. Two blood films were prepared; the first blood film was processed at a hospital or health center laboratory setup as a routine way, and the result at such facility setup was used as primary reading. Such primary result was recorded separately for the research purpose according to the prelabeled identification number. The second blood film was dried and fixed (the thin film) and examined at Arba Minch College of Health Science teaching laboratory by the principal investigator for quality assurance purposes. Any discrepancy between primary reading and secondary reading was cleared by third-person final reading. The Care Start™ RDT test was performed at the site of sample collection by different laboratory personnel so that the one who did the microscopy do not know the result of the RDT.P. falciparum and P. vivax. These antigens are the histidine-rich protein -2 (PfHRP-2) and lactate dehydrogenase (LDH).The available RDT kit was used in this study according to the manufacturer's instructions. It is used for the qualitative detection of antigens produced by Blood smears were stained with 10% Giemsa and examined using 100X oil immersion objective lens of a light microscope by two independent laboratory technologists ) who are blinded to each other's results.Parasite density estimation was performed by measuring the level of infection in red blood cells by examination of a thin blood film which is confirmed to be more accurate . To quanData collected from this study were entered and analyzed using SPSS version 20. The data include participant demographic information, clinical signs and symptoms, parasite type, previous treatment history, and RDT result. Sensitivity, specificity, positive predictive values (PPVs), and negative predictive values (NPVs) of the Care Start™ Malaria RDT were calculated. Kappa value was calculated to determine the agreement between the results of microscopy and the diagnostic test kit.Fresh blood samples were transferred directly to the sample pad by the provided sample applicator. All Care Start™ malaria test kits were labeled with the patient ID number, and the procedure and the result recording period were according to the manufacturer's instruction. To eliminate observer bias, quality control was performed by the principal investigator at Arba Minch College of Health Science teaching laboratory blindly repeating all test results at the facility level.In this study, a total of 160 study participants suspected of malaria were examined for malaria parasites by thick/thin blood smear microscopy and Care Start™ combo RDT test kit. Of the total participants, 89 (55.6%) were males and 71 (44.4%) were females with ages ranging from 1 year to 70 years with a mean age of 24 and a median of 21 .P. falciparum test result for 31 study participants, P. vivax test result for 20 study participants, and mixed infection with P. falciparum and P. vivax for only 5 study participants. As presented in P. falciparum by microscopy.In this study, microscopy and Care Start™ malaria RDT gave similar P. falciparum, 23 (28.75%) were infected with P. vivax, and the remaining six (7.50%) had mixed infection with P. falciparum and P. vivax  blood smear samples. Among microscopically examined blood smear samples of study participants, 51 (63.75%) were infected with P. vivax . No studTaking a thick blood smear as a gold standard test for malaria, the overall sensitivity and specificity of Care Start™ RDT were found to be 97.44% (95% CI = 91.04%–99.69%) and 93.67% (95% CI = 85.84%–97.91%), respectively. The PPV and the NPV of the device were found to be 93.83% (95% CI = 86.18%–97.97%) and 97.37% (95% CI = 90.82%–99.68%), respectively. The agreement between the light microscopy and Care Start™ RDT to detect the presence or absence of the malaria parasite has a kappa value of 0.877 .P. falciparum malaria. The PPV and NPV of Care Start™ RDT test kit to diagnose P. falciparum were 83.78% and 85.12%, respectively. The corresponding sensitivity and specificity of Care Start™ for the diagnosis P. vivax malaria were 86.96% (95% CI = 66.41%–97.22%) and 95.62% (95% CI = 90.71%–98.38%), respectively, with 76.92% PPV and 97.76% NPV. The sensitivity, specificity, PPV, and NPV of Care Start™ for the diagnosis of mixed infection  were 83.33%, 91.56%, 27.78%, and 99.30%, respectively. Accordingly, the overall agreement between light microscopy and Care Start™ malaria RDT test kit for specific identification of malaria species has a kappa value of 0.568 (The Care Start™ RDT test kit was 63.27% (95% CI = 48.29%–76.58%) sensitive and 94.50% (95% CI = 88.40%–97.95%) specific to detect of 0.568 .On analysis of the diagnostic performance of the kit used by percent parasitemia, generally, the sensitivity of the kit increased when the percent parasitemia increased in the individual study subject. Assessment of sensitivities at percent parasitemia threshold below 0.049% was not displayed because there is no positive test result by microscopy within that percent parasitemia range. Starting from 1.01% parasitemia up to the maximum percent parasitemia (2.85%), the Care Start™ RDT showed 100% sensitivity. Association of percent parasite load with RDT and microscopy result is presented in P. falciparum had a sensitivity of 100% for study participants with percent parasitemia >1.51%. As indicated in P. falciparum) and 90.91% (for P. vivax) to 46.67% and 75.00%, respectively, even though the percent parasitemia increased  study participants who had P. falciparum only by the standard microscopy were found to have mixed infection by the kit used. A similar finding was observed in a study performed in Madagascar [P. falciparum. This may result in unnecessary drug and resource expenditure in areas where only RDT kit is being utilized as a means of malaria diagnosis.The performance of Care Start™ RDT was noted to be significantly influenced by the level of percent parasitemia. In this study, the percent parasitemia ranged from 0.05% to 2.85%. The sensitivity and specificity of the RDT increased when the percent parasitemia increased in the individual study subjects. A similar scenario was observed in other studies , 21, 22.dagascar  where thThe main limitation of the study was not using PCR as a reference method, since microscopy has lower sensitivity even when compared with loop-mediated isothermal amplification (LAMP) , and obvMalaria RDT kits may be used in areas where there are no trained personnel, electricity, necessary equipment, and reagents which are necessary for malaria microscopy. Since the performance of RDT may vary with various factors such as loss of integrity of kits during transportation, different climate conditions, and/or inappropriate storage condition, frequent local sensitivity testing shall be carried out.P. falciparum and P. vivax. So, further studies should be carried out to use the Care Start™ Malaria Pf/Pv Combo test kit instead of microscopy for the diagnosis of malaria. Providing training on quality malaria laboratory diagnosis for the staff and availing necessary supplies for malaria diagnosis shall also be considered. Gamo Gofa Zone Health Department should select and purchase the right type of malaria RDT which displays good performance by conducting area-specific performance evaluation tests.In this study, the performance of the currently used malaria RDT kit was poor for accurate diagnosis of malaria. It has also a drawback in correctly identifying malaria species of


Impaired microvascular perfusion is central to the development of coma and lactic acidosis in severe falciparum malaria. Refractory hypotension is rare on admission but develops frequently in fatal cases. We assessed cardiac function and volume status in severe falciparum malaria and its prognostic significance.Patients with severe (N = 101) or acute uncomplicated falciparum malaria (N = 83) were recruited from 2 hospitals in India and Bangladesh, and healthy participants (N = 44) underwent echocardiography.Patients with severe malaria had 38% shorter left ventricular (LV) filling times and 25% shorter LV ejection times than healthy participants because of tachycardia; however, stroke volume, LV internal diameter in diastole (LVIDd), and LV internal diameter in systole (LVIDs) indices were similar. A low endocardial fraction shortening (eFS) was present in 17% (9 of 52) of severe malaria patients. Adjusting for preload and afterload, eFS was similar in health and severe malaria. Fatal cases had smaller baseline LVIDd and LVIDs indices, more collapsible inferior vena cavae (IVC), and higher heart rates than survivors. The LVIDs and IVC collapsibility were independent predictors for mortality, together with base excess and Glasgow Coma Scale.Patients with severe malaria have rapid ejection of a normal stroke volume. Fatal cases had features of relative hypovolemia and reduced cardiac index reserve. Echocardiography in a cohort of patients with severe malaria showed that systolic and diastolic dysfunction was infrequent but that patients who died had evidence of hypovolemia and reduced cardiac index reserve. Severe falciparum malaria caused an estimated 435 000 deaths globally in 2017 , large vThe causal chain of events resulting in death from severe malaria has multiple links. The factors that cause severe disease to develop (including sequestration of a high parasite biomass infection) are not necessarily equivalent to the factors affecting subsequent survival. Although hypovolemia does not cause coma or acidosis, macrovascular stability could determine survival after the start of treatment because refractory hypotension is a common terminal event in patients with severe malaria . The carWe studied the relationships between disease severity, cardiac function, and volume status in malaria. We hypothesized that reduced cardiac index reserve resulting from hypovolemia and impaired systolic function are associated with increased mortality.Consecutive consenting patients admitted to the adult medical wards during the malaria seasons at Chittagong Medical College Hospital (CMCH), Chittagong, Bangladesh in 2011–2014 and ISPAT General Hospital (IGH), Rourkela, India in 2011 with severe or uncomplicated falciparum malaria as defined previously  were enrU tests (2 groups) or Kruskal-Wallis tests (more than 2 groups). Correlations were assessed using Spearman’s rank. Linear regression was used to adjust indices of ventricular function for preload, afterload, and ejection time  a measure of the rate at which the ventricle relaxes)  and mitrP = .007), MVET , but not early mitral peak E-wave velocity (E), IVRT, or IVC diameter or collapsibility , suggesting that in the severe malaria group, filling time and possibly relaxation are the main determinants of LVIDd. The IVRT and average E’ were inversely associated . The LVIDs was not associated with E, average E’, or IVRT .In severe malaria, LVIDd was correlated positively with average mitral peak early diastolic tissue velocity (E’)  but was negatively associated with average E’  and E  and was positively associated with IVSd , relative LV wall thickness (RWT)  and IVRT , consistent with the known age-related transition to concentric geometry and reduced relaxation.In patients with severe malaria, age was not associated with LVIDd or LVIDs (both P = .094), but it was positively correlated with temperature . Tachycardia was associated with smaller LVIDd , LVIDs , and stroke index values  and larger medial S’  and eFS  values but not E or average E’ (both P > .05).Tachycardia was not associated with hematocrit , stroke index , and eFS . In a univariate analysis, markers of volume status or filling pressure were not associated with hematocrit; there was no correlation with LVIDd or IVC dimensions or E/E’ (P > .05). Hematocrit correlated inversely with the systolic function parameters average S’  and LVOTVmax . Hematocrit was also associated positively with the diastolic parameter IVRT  and inversely with E  and E’ . Examining the associations with afterload indices, hematocrit correlated with SVRI  but not MRESS or mean arterial pressure (P > .05). Linear regression was used to explore whether the larger eFS in anemia was related to an increase in contractility or loading conditions. Consistent with hematocrit increasing eFS through changes in loading as opposed to contractility, in a linear regression model for eFS, after adjusting for sex, MRESS and LVIDd hematocrit was not a significant predictor . In a linear regression model for LVIDd, adjusted for gender and MVET, hematocrit was a positive predictor, suggesting that preload is higher in anemia.Hematocrit was inversely correlated with cardiac index  and eFS  and negatively associated with LVIDd  and LVIDs  but not stroke index, mean S’, IVRT, or IVC dimensions, fever, or hematocrit (P > .05).Venous plasma lactate concentrations were positively associated with heart rate , whereast heart rate, IVC collapsibility, IVC inspiratory diameter, and LVIDs index were significantly associated with a fatal outcome independent of base excess and GCS , but more substantial changes separated severe malaria survivors from fatal cases. Patients who died had higher heart rates, with smaller LV and RA diameters, increased IVC collapsibility, and high LV fractional shortening. Patients presenting with these cardiovascular profiles are expected to have lower cardiac index reserve and hence might be less able to compensate further peripheral vasodilation or other hemodynamic stresses. There was no evidence that systolic LV or RV dysfunction predicted outcome.Previous studies of hemodynamics in severe malaria have lacked power to assess the association between macrovascular hemodynamic parameters and mortality. In severe malaria, no clinically significant acute change in LVIDd was noted in 2 studies in children , 19 and In this study, malaria was associated with an increase in heart rate, peak ejection velocity, and S’, and a decrease in LVET and MVET, but preservation of LV end diastolic dimension, stroke volume, and eFS. There was no decrease in LV end systolic dimension, suggesting that although the rate of peak LV contraction is increased, this is counterbalanced by shorter ejection times, and therefore it does not increase stroke volumes. This more rapid ejection of a normal stroke volume could be caused by an increase in contractility due to sympathetic stimulation, circulating catecholamines, or the Treppe phenomenon (finding of contractility increasing with heart rate) . Alternavmax and S’) and filling (larger E). These findings are in keeping with studies in children with malaria and anemia, where cardiac index has been found to increase [Within the severe malaria group, tachycardia, which was associated with fever but not with anemia, inversely correlated with LV dimensions and stroke volume. Anemia was associated with a larger fractional shortening and stroke volume, likely maintained by a more rapid ventricular ejection , and moIn bacterial sepsis, as in severe malaria, vascular resistance is typically reduced and the circulation is typically hyperdynamic  . Left veUsing a noninvasive approach maximized recruitment into the study. However, this meant that chamber pressures and central venous oxygen saturations were not measured. In particular, in assessment of contractility and diastolic function, the assessment of pressure-volume loops would be informative. We have not assessed ventriculo-arterial coupling, a factor that can affect cardiac performance . AdolescIn conclusion, our study shows that impaired systolic function is typically absent in malaria and does not predict mortality. Diastolic LV function in malaria patients is usually not impaired and varies with age. Stroke volume index is maintained in adult patients with malaria despite a reduction in LV filling and ejection time. Patients who died from severe malaria had evidence of hypovolemia at baseline and evidence of reduced cardiac index reserve. Future studies should investigate whether tailoring fluid therapy to optimize volume status in these patients is beneficial.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiz568_suppl_Supplementary_MethodsClick here for additional data file.jiz568_suppl_Supplementary_TablesClick here for additional data file.