Anti-Gametocyte Antigen Humoral Immunity and Gametocytemia During Treatment of Uncomplicated Falciparum Malaria: A Multi-National Study.

Introduction: Understanding the human immune response to Plasmodium falciparum gametocytes and its association with gametocytemia is essential for understanding the transmission of malaria as well as progressing transmission blocking vaccine candidates. Methods: In a multi-national clinical efficacy trial of artemisinin therapies (13 sites of varying transmission over South-East Asia and Africa), we measured Immunoglobulin G (IgG) responses to recombinant P. falciparum gametocyte antigens expressed on the gametocyte plasma membrane and leading transmission blocking vaccine candidates Pfs230 (Pfs230c and Pfs230D1M) and Pfs48/45 at enrolment in 1,114 participants with clinical falciparum malaria. Mixed effects linear and logistic regression were used to determine the association between gametocyte measures (gametocytemia and gametocyte density) and antibody outcomes at enrolment. Results: Microscopy detectable gametocytemia was observed in 11% (127/1,114) of participants at enrolment, and an additional 9% (95/1,114) over the follow-up period (up to day 42) (total 20% of participants [222/1,114]). IgG levels in response to Pfs230c, Pfs48/45 and Pfs230D1M varied across study sites at enrolment (p < 0.001), as did IgG seroprevalence for anti-Pfs230c and D1M IgG (p < 0.001), but not for anti-Pfs48/45 IgG (p = 0.159). In adjusted analyses, microscopy detectable gametocytemia at enrolment was associated with an increase in the odds of IgG seropositivity to the three gametocyte antigens (Pfs230c OR [95% CI], p: 1.70 [1.10, 2.62], 0.017; Pfs48/45: 1.45 [0.85, 2.46], 0.174; Pfs230D1M: 1.70 [1.03, 2.80], 0.037), as was higher gametocyte density at enrolment (per two-fold change in gametocyte density Pfs230c OR [95% CI], p: 1.09 [1.02, 1.17], 0.008; Pfs48/45: 1.05 [0.98, 1.13], 0.185; Pfs230D1M: 1.07 [0.99, 1.14], 0.071). Conclusion: Pfs230 and Pfs48/45 antibodies are naturally immunogenic targets associated with patent gametocytemia and increasing gametocyte density across multiple malaria endemic settings, including regions with emerging artemisinin-resistant P. falciparum.

Field evaluation of the diagnostic performance of EasyScan GO: a digital malaria microscopy device based on machine-learning.

BACKGROUND: Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue. METHODS: A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist. RESULTS: In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9-92.7), and specificity 75.6% (95% CI 73.1-78.0). With good quality slides sensitivity was similar (89.1%, 95%CI 86.2-91.5), but specificity increased to 85.1% (95%CI 82.6-87.4). Sensitivity increased with parasitaemia rising from 57% at < 200 parasite/µL, to ≥ 90% at > 200-200,000 parasite/µL. Species were identified accurately in 93% of Plasmodium falciparum samples (kappa = 0.76, 95% CI 0.69-0.83), and in 92% of Plasmodium vivax samples (kappa = 0.73, 95% CI 0.66-0.80). Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides. CONCLUSIONS: The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations. Trial registration ClinicalTrials.gov number NCT03512678.

Mapping the travel patterns of people with malaria in Bangladesh.

BACKGROUND: Spread of malaria and antimalarial resistance through human movement present major threats to current goals to eliminate the disease. Bordering the Greater Mekong Subregion, southeast Bangladesh is a potentially important route of spread to India and beyond, but information on travel patterns in this area are lacking. METHODS: Using a standardised short survey tool, 2090 patients with malaria were interviewed at 57 study sites in 2015-2016 about their demographics and travel patterns in the preceding 2 months. RESULTS: Most travel was in the south of the study region between Cox's Bazar district (coastal region) to forested areas in Bandarban (31% by days and 45% by nights), forming a source-sink route. Less than 1% of travel reported was between the north and south forested areas of the study area. Farmers (21%) and students (19%) were the top two occupations recorded, with 67 and 47% reporting travel to the forest respectively. Males aged 25-49 years accounted for 43% of cases visiting forests but only 24% of the study population. Children did not travel. Women, forest dwellers and farmers did not travel beyond union boundaries. Military personnel travelled the furthest especially to remote forested areas. CONCLUSIONS: The approach demonstrated here provides a framework for identifying key traveller groups and their origins and destinations of travel in combination with knowledge of local epidemiology to inform malaria control and elimination efforts. Working with the NMEP, the findings were used to derive a set of policy recommendations to guide targeting of interventions for elimination.

Contribution of Functional Antimalarial Immunity to Measures of Parasite Clearance in Therapeutic Efficacy Studies of Artemisinin Derivatives.

BACKGROUND: Antibodies to the blood stages of malaria parasites enhance parasite clearance and antimalarial efficacy. The antibody subclass and functions that contribute to parasite clearance during antimalarial treatment and their relationship to malaria transmission intensity have not been characterized. METHODS: Levels of immunoglobulin G (IgG) subclasses and C1q fixation in response to Plasmodium falciparum merozoite antigens (erythrocyte-binding antigen [EBA] 175RIII-V, merozoite surface protein 2 [MSP-2], and MSP-142) and opsonic phagocytosis of merozoites were measured in a multinational trial assessing the efficacy of artesunate therapy across 11 Southeast Asian sites. Regression analyses assessed the effects of antibody seropositivity on the parasite clearance half-life (PC½), having a PC½ of ≥5 hours, and having parasitemia 3 days after treatment. RESULTS: IgG3, followed by IgG1, was the predominant IgG subclass detected (seroprevalence range, 5%-35% for IgG1 and 27%-41% for IgG3), varied across study sites, and was lowest in study sites with the lowest transmission intensity and slowest mean PC½. IgG3, C1q fixation, and opsonic-phagocytosis seropositivity were associated with a faster PC½ (range of the mean reduction in PC½, 0.47-1.16 hours; P range, .001-.03) and a reduced odds of having a PC½ of ≥5 hours and having parasitemia 3 days after treatment. CONCLUSIONS: The prevalence of IgG3, complement-fixing antibodies, and merozoite phagocytosis vary according to transmission intensity, are associated with faster parasite clearance, and may be sensitive surrogates of an augmented clearance capacity of infected erythrocytes. Determining the functional immune mechanisms associated with parasite clearance will improve characterization of artemisinin resistance.

Mapping imported malaria in Bangladesh using parasite genetic and human mobility data.

For countries aiming for malaria elimination, travel of infected individuals between endemic areas undermines local interventions. Quantifying parasite importation has therefore become a priority for national control programs. We analyzed epidemiological surveillance data, travel surveys, parasite genetic data, and anonymized mobile phone data to measure the spatial spread of malaria parasites in southeast Bangladesh. We developed a genetic mixing index to estimate the likelihood of samples being local or imported from parasite genetic data and inferred the direction and intensity of parasite flow between locations using an epidemiological model integrating the travel survey and mobile phone calling data. Our approach indicates that, contrary to dogma, frequent mixing occurs in low transmission regions in the southwest, and elimination will require interventions in addition to reducing imported infections from forested regions. Unlike risk maps generated from clinical case counts alone, therefore, our approach distinguishes areas of frequent importation as well as high transmission.

Association of the Quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) Score With Excess Hospital Mortality in Adults With Suspected Infection in Low- and Middle-Income Countries.

Importance: The quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) score has not been well-evaluated in low- and middle-income countries (LMICs). Objective: To assess the association of qSOFA with excess hospital death among patients with suspected infection in LMICs and to compare qSOFA with the systemic inflammatory response syndrome (SIRS) criteria. Design, Settings, and Participants: Retrospective secondary analysis of 8 cohort studies and 1 randomized clinical trial from 2003 to 2017. This study included 6569 hospitalized adults with suspected infection in emergency departments, inpatient wards, and intensive care units of 17 hospitals in 10 LMICs across sub-Saharan Africa, Asia, and the Americas. Exposures: Low (0), moderate (1), or high (≥2) qSOFA score (range, 0 [best] to 3 [worst]) or SIRS criteria (range, 0 [best] to 4 [worst]) within 24 hours of presentation to study hospital. Main Outcomes and Measures: Predictive validity (measured as incremental hospital mortality beyond that predicted by baseline risk factors, as a marker of sepsis or analogous severe infectious course) of the qSOFA score (primary) and SIRS criteria (secondary). Results: The cohorts were diverse in enrollment criteria, demographics (median ages, 29-54 years; males range, 36%-76%), HIV prevalence (range, 2%-43%), cause of infection, and hospital mortality (range, 1%-39%). Among 6218 patients with nonmissing outcome status in the combined cohort, 643 (10%) died. Compared with a low or moderate score, a high qSOFA score was associated with increased risk of death overall (19% vs 6%; difference, 13% [95% CI, 11%-14%]; odds ratio, 3.6 [95% CI, 3.0-4.2]) and across cohorts (P < .05 for 8 of 9 cohorts). Compared with a low qSOFA score, a moderate qSOFA score was also associated with increased risk of death overall (8% vs 3%; difference, 5% [95% CI, 4%-6%]; odds ratio, 2.8 [95% CI, 2.0-3.9]), but not in every cohort (P < .05 in 2 of 7 cohorts). High, vs low or moderate, SIRS criteria were associated with a smaller increase in risk of death overall (13% vs 8%; difference, 5% [95% CI, 3%-6%]; odds ratio, 1.7 [95% CI, 1.4-2.0]) and across cohorts (P < .05 for 4 of 9 cohorts). qSOFA discrimination (area under the receiver operating characteristic curve [AUROC], 0.70 [95% CI, 0.68-0.72]) was superior to that of both the baseline model (AUROC, 0.56 [95% CI, 0.53-0.58; P < .001) and SIRS (AUROC, 0.59 [95% CI, 0.57-0.62]; P < .001). Conclusions and Relevance: When assessed among hospitalized adults with suspected infection in 9 LMIC cohorts, the qSOFA score identified infected patients at risk of death beyond that explained by baseline factors. However, the predictive validity varied among cohorts and settings, and further research is needed to better understand potential generalizability.

Activation of coagulation and endothelium with concurrent impairment of anticoagulant mechanisms in patients with typhoid fever.

OBJECTIVES: Typhoid fever caused by Salmonella Typhi remains a major burden worldwide. Gastrointestinal bleeding can be seen in up to 10 percent of patients and may be fatal. The coagulopathy, which may be the driver of this severe complication in patients with typhoid fever, however is ill defined. The aim of this study was to evaluate the activation of coagulation, anticoagulation, and fibrinolysis in patients with acute typhoid fever. METHODS: Parameters of coagulation and fibrinolysis were measured in 28 hospitalized patients with culture-confirmed or PCR-confirmed typhoid fever and compared to 38 age- and sex-matched healthy volunteers. RESULTS: Patients demonstrated activation of the coagulation system, as reflected by elevated in vitro thrombin generation and high plasma levels of fibrinogen, D-dimer and prothrombin fragment F1 + 2 in concert with consumption of coagulation factors resulting in a prolonged prothrombin-time and activated-partial-thromboplastin-time. Concurrently, the anticoagulant proteins, protein C and antithrombin, were significantly lower in comparison to healthy controls. Patients also demonstrated evidence of activation and inhibition of fibrinolysis and a marked activation of endothelial cells. The extent of coagulation activation was associated with the course of the disease, repeated testing during convalescence showed a return toward normal values. CONCLUSIONS: Activation of coagulation is an important clinical feature of typhoid fever and is associated with severity of disease.

Acidosis and acute kidney injury in severe malaria.

BACKGROUND: In severe falciparum malaria metabolic acidosis and acute kidney injury (AKI) are independent predictors of a fatal outcome in all age groups. The relationship between plasma acids, urine acids and renal function was investigated in adult patients with acute falciparum malaria. METHODS: Plasma and urinary acids which previously showed increased concentrations in proportion to disease severity in patients with severe falciparum malaria were quantified. Patients with uncomplicated malaria, sepsis and healthy volunteers served as comparator groups. Multiple regression and multivariate analysis were used to assess the relationship between organic acid concentrations and clinical syndromes, in particular AKI. RESULTS: Patients with severe malaria (n = 90), uncomplicated malaria (n = 94), non-malaria sepsis (n = 19), and healthy volunteers (n = 61) were included. Univariate analysis showed that both plasma and creatinine-adjusted urine concentrations of p-hydroxyphenyllactic acid (pHPLA) were higher in severe malaria patients with AKI (p < 0.001). Multiple regression analysis, including plasma or creatinine-adjusted urinary acids, and PfHRP2 as parasite biomass marker as independent variables, showed that pHPLA was independently associated with plasma creatinine (ß = 0.827) and urine creatinine (ß = 0.226). Principal component analysis, including four plasma acids and seven urinary acids separated a group of patients with AKI, which was mainly driven by pHPLA concentrations. CONCLUSIONS: Both plasma and urine concentrations of pHPLA closely correlate with AKI in patients with severe falciparum malaria. Further studies will need to assess the potential nephrotoxic properties of pHPLA.

Bans of WHO Class I Pesticides in Bangladesh-suicide prevention without hampering agricultural output.

Background: Pesticide self-poisoning is a major problem in Bangladesh. Over the past 20-years, the Bangladesh government has introduced pesticide legislation and banned highly hazardous pesticides (HHPs) from agricultural use. We aimed to assess the impacts of pesticide bans on suicide and on agricultural production. Methods: We obtained data on unnatural deaths from the Statistics Division of Bangladesh Police, and used negative binomial regression to quantify changes in pesticide suicides and unnatural deaths following removal of WHO Class I toxicity HHPs from agriculture in 2000. We assessed contemporaneous trends in other risk factors, pesticide usage and agricultural production in Bangladesh from 1996 to 2014. Results: Mortality in hospital from pesticide poisoning fell after the 2000 ban: 15.1% vs 9.5%, relative reduction 37.1% [95% confidence interval (CI) 35.4 to 38.8%]. The pesticide poisoning suicide rate fell from 6.3/100 000 in 1996 to 2.2/100 000 in 2014, a 65.1% (52.0 to 76.7%) decline. There was a modest simultaneous increase in hanging suicides [20.0% (8.4 to 36.9%) increase] but the overall incidence of unnatural deaths fell from 14.0/100 000 to 10.5/100 000 [25.0% (18.1 to 33.0%) decline]. There were 35 071 (95% CI 25 959 to 45 666) fewer pesticide suicides in 2001 to 2014 compared with the number predicted based on trends between 1996 to 2000. This reduction in rate of pesticide suicides occurred despite increased pesticide use and no change in admissions for pesticide poisoning, with no apparent influence on agricultural output. Conclusions: Strengthening pesticide regulation and banning WHO Class I toxicity HHPs in Bangladesh were associated with major reductions in deaths and hospital mortality, without any apparent effect on agricultural output. Our data indicate that removing HHPs from agriculture can rapidly reduce suicides without imposing substantial agricultural costs.

Host immunity to Plasmodium falciparum and the assessment of emerging artemisinin resistance in a multinational cohort.

Artemisinin-resistant falciparum malaria, defined by a slow-clearance phenotype and the presence of kelch13 mutants, has emerged in the Greater Mekong Subregion. Naturally acquired immunity to malaria clears parasites independent of antimalarial drugs. We hypothesized that between- and within-population variations in host immunity influence parasite clearance after artemisinin treatment and the interpretation of emerging artemisinin resistance. Antibodies specific to 12 Plasmodium falciparum sporozoite and blood-stage antigens were determined in 959 patients (from 11 sites in Southeast Asia) participating in a multinational cohort study assessing parasite clearance half-life (PCt1/2) after artesunate treatment and kelch13 mutations. Linear mixed-effects modeling of pooled individual patient data assessed the association between antibody responses and PCt1/2.P. falciparum antibodies were lowest in areas where the prevalence of kelch13 mutations and slow PCt1/2 were highest [Spearman ρ = -0.90 (95% confidence interval, -0.97, -0.65), and Spearman ρ = -0.94 (95% confidence interval, -0.98, -0.77), respectively]. P. falciparum antibodies were associated with faster PCt1/2 (mean difference in PCt1/2 according to seropositivity, -0.16 to -0.65 h, depending on antigen); antibodies have a greater effect on the clearance of kelch13 mutant compared with wild-type parasites (mean difference in PCt1/2 according to seropositivity, -0.22 to -0.61 h faster in kelch13 mutants compared with wild-type parasites). Naturally acquired immunity accelerates the clearance of artemisinin-resistant parasites in patients with falciparum malaria and may confound the current working definition of artemisinin resistance. Immunity may also play an important role in the emergence and transmission potential of artemisinin-resistant parasites.

Optimal health and disease management using spatial uncertainty: a geographic characterization of emergent artemisinin-resistant Plasmodium falciparum distributions in Southeast Asia.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum malaria parasites are now present across much of mainland Southeast Asia, where ongoing surveys are measuring and mapping their spatial distribution. These efforts require substantial resources. Here we propose a generic 'smart surveillance' methodology to identify optimal candidate sites for future sampling and thus map the distribution of artemisinin resistance most efficiently. METHODS: The approach uses the 'uncertainty' map generated iteratively by a geostatistical model to determine optimal locations for subsequent sampling. RESULTS: The methodology is illustrated using recent data on the prevalence of the K13-propeller polymorphism (a genetic marker of artemisinin resistance) in the Greater Mekong Subregion. CONCLUSION: This methodology, which has broader application to geostatistical mapping in general, could improve the quality and efficiency of drug resistance mapping and thereby guide practical operations to eliminate malaria in affected areas.

Effectiveness Study of Paromomycin IM Injection (PMIM) for the Treatment of Visceral Leishmaniasis (VL) in Bangladesh.

BACKGROUND: This study was conducted in Bangladeshi patients in an outpatient setting to support registration of Paromomycin Intramuscular Injection (PMIM) as a low-cost treatment option in Bangladesh. METHODOLOGY: This Phase IIIb, open-label, multi-center, single-arm trial assessed the efficacy and safety of PMIM administered at 11 mg/kg (paromomycin base) intramuscularly once daily for 21 consecutive days to children and adults with VL in a rural outpatient setting in Bangladesh. Patients ≥5 and ≤55 years were eligible if they had signs and symptoms of VL (intermittent fever, weight loss/decreased appetite, and enlarged spleen), positive rK39 test, and were living in VL-endemic areas. Compliance was the percentage of enrolled patients who received 21 daily injections over no more than 22 days. Efficacy was evaluated by initial clinical response, defined as resolution of fever and reduction of splenomegaly at end of treatment, and final clinical response, defined as the absence of new clinical signs and symptoms of VL 6 months after end of treatment. Safety was assessed by evaluation of adverse events. PRINCIPAL FINDINGS: A total of 120 subjects (49% pediatric) were enrolled. Treatment compliance was 98.3%. Initial clinical response in the Intent-to-Treat population was 98.3%, and final clinical response 6 months after end of treatment was 94.2%. Of the 119 subjects who received ≥1 dose of PMIM, 28.6% reported at least one adverse event. Injection site pain was the most commonly reported adverse event. Reversible renal impairment and/or hearing loss were reported in 2 subjects. CONCLUSIONS/SIGNIFICANCE: PMIM was an effective and safe treatment for VL in Bangladesh. The short treatment duration and lower cost of PMIM compared with other treatment options may make this drug a preferred treatment to be investigated as part of a combination therapy regimen. This study supports the registration of PMIM for use in government health facilities in Bangladesh. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01328457.

Baseline data of parasite clearance in patients with falciparum malaria treated with an artemisinin derivative: an individual patient data meta-analysis.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum manifests as slow parasite clearance but this measure is also influenced by host immunity, initial parasite biomass and partner drug efficacy. This study collated data from clinical trials of artemisinin derivatives in falciparum malaria with frequent parasite counts to provide reference parasite clearance estimates stratified by location, treatment and time, to examine host factors affecting parasite clearance, and to assess the relationships between parasite clearance and risk of recrudescence during follow-up. METHODS: Data from 24 studies, conducted from 1996 to 2013, with frequent parasite counts were pooled. Parasite clearance half-life (PC1/2) was estimated using the WWARN Parasite Clearance Estimator. Random effects regression models accounting for study and site heterogeneity were used to explore factors affecting PC1/2 and risk of recrudescence within areas with reported delayed parasite clearance (western Cambodia, western Thailand after 2000, southern Vietnam, southern Myanmar) and in all other areas where parasite populations are artemisinin sensitive. RESULTS: PC1/2 was estimated in 6975 patients, 3288 of whom also had treatment outcomes evaluate d during 28-63 days follow-up, with 93 (2.8 %) PCR-confirmed recrudescences. In areas with artemisinin-sensitive parasites, the median PC1/2 following three-day artesunate treatment (4 mg/kg/day) ranged from 1.8 to 3.0 h and the proportion of patients with PC1/2 >5 h from 0 to 10 %. Artesunate doses of 4 mg/kg/day decreased PC1/2 by 8.1 % (95 % CI 3.2-12.6) compared to 2 mg/kg/day, except in populations with delayed parasite clearance. PC1/2 was longer in children and in patients with fever or anaemia at enrolment. Long PC1/2 (HR = 2.91, 95 % CI 1.95-4.34 for twofold increase, p < 0.001) and high initial parasitaemia (HR = 2.23, 95 % CI 1.44-3.45 for tenfold increase, p < 0.001) were associated independently with an increased risk of recrudescence. In western Cambodia, the region with the highest prevalence of artemisinin resistance, there was no evidence for increasing PC1/2 since 2007. CONCLUSIONS: Several factors affect PC1/2. As substantial heterogeneity in parasite clearance exists between locations, early detection of artemisinin resistance requires reference PC1/2 data. Studies with frequent parasite count measurements to characterize PC1/2 should be encouraged. In western Cambodia, where PC1/2 values are longest, there is no evidence for recent emergence of higher levels of artemisinin resistance.

The role of previously unmeasured organic acids in the pathogenesis of severe malaria.

INTRODUCTION: Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria. METHODS: In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death. RESULTS: Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean ± SD) was elevated in severe malaria (8.2 mEq/L ± 4.5) and severe sepsis (8.6 mEq/L ± 7.7) compared with uncomplicated malaria (6.0 mEq/L ± 5.1) and encephalopathy (6.6 mEq/L ± 4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and ß-hydroxybutyric acid (all P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death (odds ratio [OR] 3.5, 95 % confidence interval [CI] 1.6-7.5, P=0.001), comparable to LA (OR 3.5, 95 % CI 1.5-7.8, P=0.003) (combined area under the receiver operating characteristic curve 0.81). CONCLUSIONS: Newly identified acids, in addition to LA, are elevated in patients with severe malaria and are highly predictive of fatal outcome. Further characterisation of their sources and metabolic pathways is now needed.

Microvascular obstruction and endothelial activation are independently associated with the clinical manifestations of severe falciparum malaria in adults: an observational study.

BACKGROUND: Microvascular obstruction and endothelial dysfunction have both been linked to tissue hypoperfusion in falciparum malaria, but their relative contributions to the disease's pathogenesis and outcome are unknown. METHODS: Microvascular blood flow was quantified in adults with severe falciparum malaria on their admission to hospital; plasma biomarkers of endothelial function were measured simultaneously. The relationship between these indices and the patients' clinical findings and in-hospital course was examined. RESULTS: Microvascular obstruction was observed in 119/142 (84 %) patients; a median (interquartile range (IQR)) of 14.9 % (6.6-34.9 %) of capillaries were obstructed in patients that died versus 8.3 % (1.7-26.6 %) in survivors (P = 0.039). The proportion of obstructed capillaries correlated with the estimated parasite biomass (rs = 0.25, P = 0.004) and with plasma lactate (rs = 0.38, P <0.0001), the strongest predictor of death in the series. Plasma angiopoietin-2 (Ang-2) concentrations were markedly elevated suggesting widespread endothelial activation; the median (IQR) Ang-2 concentration was 21.9 ng/mL (13.4-29.4 ng/mL) in patients that died versus 14.9 ng/mL (9.8-29.3 ng/mL) in survivors (P = 0.035). Ang-2 concentrations correlated with estimated parasite biomass (rs = 0.35, P <0.001) and plasma lactate (rs = 0.37, P <0.0001). Microvascular obstruction and Ang-2 concentrations were not significantly correlated with each other (rs = 0.17, P = 0.06), but were independently associated with plasma lactate (P <0.001 and P = 0.002, respectively). CONCLUSIONS: Microvascular obstruction and systemic endothelial activation are independently associated with plasma lactate, the strongest predictor of death in adults with falciparum malaria. This supports the hypothesis that the two processes make an independent contribution to the pathogenesis and clinical manifestations of the disease.

The clinical implications of thrombocytopenia in adults with severe falciparum malaria: a retrospective analysis.

BACKGROUND: Thrombocytopenia is a common finding in adults with severe falciparum malaria, but its clinical and prognostic utility is incompletely defined. METHODS: Clinical and laboratory data from 647 adults with severe falciparum malaria were analysed retrospectively to determine the relationship between a patient's platelet count on admission to hospital and their subsequent clinical course. RESULTS: On admission, 614 patients (94.9%) were thrombocytopenic (platelet count <150 × 10(9)/L) and 328 (50.7%) had a platelet count <50 × 10(9)/L. The admission platelet count was inversely correlated with parasite biomass (estimated from plasma PfHRP2 concentrations, rs = -0.28, P = 0.003), the degree of microvascular sequestration (measured with orthogonal polarizing spectral imaging, rs = -0.31, P = 0.001) and disease severity (the number of World Health Organization severity criteria satisfied by the patient, rs = -0.21, P <0.001). Platelet counts were lower on admission in the patients who died (median: 30 (interquartile range 22 to 52) × 10(9)/L versus 50 (34 to 78) × 10(9)/L in survivors; P <0.001), but did not predict outcome independently from other established laboratory and clinical prognostic indices. The 39 patients (6%) with profound thrombocytopenia (platelet count <20 × 10(9)/L) were more likely to die (odds ratio: 5.00, 95% confidence interval: 2.56 to 9.75) than patients with higher platelet counts, but these high-risk patients could be identified more rapidly with simple bedside clinical assessment. The admission platelet count did not reliably identify the 50 patients (7.7%) with major bleeding during the study. CONCLUSIONS: Thrombocytopenia is a marker of disease severity in adults with falciparum malaria, but has limited utility in prognostication, triage and management.

Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker.

BACKGROUND: Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of molecular surveillance in remote areas through analysis of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by determining the relative prevalence of P falciparum parasites carrying K13-propeller mutations. METHODS: We did this cross-sectional survey at malaria treatment centres at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between January, 2013, and September, 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estimated prevalence of mutations of the K13 propeller region across Myanmar. FINDINGS: Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations. INTERPRETATION: Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided. FUNDING: Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.

Plasma concentration of parasite DNA as a measure of disease severity in falciparum malaria.

In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples.