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Abstract Background Parenteral artesunate is the first-line therapy for severe malaria. Artesunate, in its current formulation, must be prepared immediately before administration by first dissolving in sodium bicarbonate solution and then diluting in saline. A novel solvent for rapid and stable single step reconstitution of artesunate was recently developed showing improved solubility and stability. This study aimed to compare the safety and pharmacokinetic properties of the currently available and newly developed parenteral formulation of artesunate in healthy Thai volunteers. Methods This was an open-label, randomized, 4 periods, 4-treatments, 24-sequence, single-dose, cross-over study in 72 male and female healthy Thai volunteers. Frequent pharmacokinetic samples were collected in all volunteers at each dose occasion. Observed concentration–time profiles were analysed with a non-compartmental approach followed by a bioequivalence evaluation. Results Both intramuscular and intravenous administrations of the new parenteral formulation of artesunate were safe and well-tolerated, with no additional safety signals compared to the currently used formulation. The pharmacokinetic properties of artesunate and its active metabolite, dihydroartemisinin, were well-characterized, and showed rapid conversion of artesunate into dihydroartemisinin. Intramuscular administration of the newly formulated artesunate resulted in almost complete bioavailability of dihydroartemisinin. The pharmacokinetic properties were similar between the old and new formulation. Conclusions The new and more easily prepared formulation of artesunate was safe and well-tolerated, with similar pharmacokinetic properties compared to the currently used formulation. Dihydroartemisinin, the active metabolite responsible for the majority of the anti-malarial effect, showed equivalent exposure after both intravenous and intramuscular administration of artesunate, suggesting that both routes of administration should generate comparable therapeutic effects. Trial registration: The study was registered to clinicaltrials.gov (#TCTR20170907002).

Abstract In preparation for mass vaccinations with R21/Matrix-M™ combined with mass administrations of dihydroartemisinin, piperaquine, and a single low dose primaquine we assessed the tolerability, safety, and potential interactions of this combination affecting immunogenicity or pharmacokinetics. 120 healthy Thai volunteers were randomised to receive either antimalarials combined with vaccinations (n = 50), vaccinations alone (n = 50), or antimalarials only (n = 20). Three rounds of vaccines and antimalarials were administered one month apart. The vaccine was well tolerated alone and in combination with the antimalarials. None of the participants failed completion of the 3-dose vaccine course. There was no significant difference in the vaccine immunogenicity or in the pharmacokinetics of piperaquine given individually or in combination. This study supports proceeding to a large trial of mass vaccinations with R21/Matrix-M™ combined with mass antimalarial administration in Bangladesh.

Abstract Brief summary In early symptomatic COVID-19 treatment, high dose oral favipiravir did not accelerate viral clearance. Background Favipiravir, an anti-influenza drug, has in vitro antiviral activity against SARS-CoV-2. Clinical trial evidence to date is inconclusive. Favipiravir has been recommended for the treatment of COVID-19 in some countries. Methods In a multicentre open-label, randomised, controlled, adaptive platform trial, low-risk adult patients with early symptomatic COVID-19 were randomised to one of ten treatment arms including high dose oral favipiravir (3.6g on day 0 followed by 1.6g daily to complete 7 days treatment) or no study drug. The primary outcome was the rate of viral clearance (derived under a linear mixed-effects model from the daily log10 viral densities in standardised duplicate oropharyngeal swab eluates taken daily over 8 days [18 swabs per patient]), assessed in a modified intention-to-treat population (mITT). The safety population included all patients who received at least one dose of the allocated intervention. This ongoing adaptive platform trial was registered at ClinicalTrials.gov (NCT05041907) on 13/09/2021. Results In the final analysis, the mITT population contained data from 114 patients randomised to favipiravir and 126 patients randomised concurrently to no study drug. Under the linear mixed-effects model fitted to all oropharyngeal viral density estimates in the first 8 days from randomisation (4,318 swabs), there was no difference in the rate of viral clearance between patients given favipiravir and patients receiving no study drug; a -1% (95% credible interval: -14 to 14%) difference. High dose favipiravir was well-tolerated. Interpretation Favipiravir does not accelerate viral clearance in early symptomatic COVID-19. The viral clearance rate estimated from quantitative measurements of oropharyngeal eluate viral densities assesses the antiviral efficacy of drugs in vivo with comparatively few studied patients.

Background: Primaquine is an 8-aminoquinoline antimalarial. It is the only widely available treatment to prevent relapses of Plasmodium vivax malaria. The 8-aminoquinolines cause dose-dependent haemolysis in glucose-6-phosphate dehydrogenase deficiency (G6PDd). G6PDd is common in malaria endemic areas but testing is often not available. As a consequence primaquine is underused. Methods: We conducted an adaptive pharmacometric study to characterise the relationship between primaquine dose and haemolysis in G6PDd. The aim was to explore shorter and safer primaquine radical cure regimens compared to the currently recommended 8-weekly regimen (0.75 mg/kg once weekly), potentially obviating the need for G6PD testing. Hemizygous G6PDd healthy adult Thai and Burmese male volunteers were admitted to the Hospital for Tropical Diseases in Bangkok. In Part 1, volunteers were given ascending dose primaquine regimens whereby daily doses were increased from 7.5 mg up to 45 mg over 15–20 days. In Part 2 conducted at least 6 months later, a single primaquine 45 mg dose was given. Results: 24 volunteers were enrolled in Part 1, and 16 in Part 2 (13 participated in both studies). In three volunteers, the ascending dose regimen was stopped because of haemolysis (n=1) and asymptomatic increases in transaminases (n=2; one was hepatitis E positive). Otherwise the ascending regimens were well tolerated with no drug-related serious adverse events. In Part 1, the median haemoglobin concentration decline was 3.7 g/dL (range: 2.1–5.9; relative decline of 26% [range: 15–40%]). Primaquine doses up to 0.87 mg/kg/day were tolerated subsequently without clinically significant further falls in haemoglobin. In Part 2, the median haemoglobin concentration decline was 1.7 g/dL (range 0.9–4.1; relative fall of 12% [range: 7–30% decrease]). The ascending dose primaquine regimens gave seven times more drug but resulted in only double the haemoglobin decline. Conclusions: In patients with Southeast Asian G6PDd variants, full radical cure treatment can be given in under 3 weeks compared with the current 8-week regimen. Funding: Medical Research Council of the United Kingdom (MR/R015252/1) and Wellcome (093956/Z/10/C, 223253/Z/21/Z). Clinical trial number: Thai Clinical Trial Registry: TCTR20170830002 and TCTR20220317004.

Background The optimal dosing of primaquine to prevent relapsing Plasmodium vivax malaria in South Asia remains unclear. We investigated the efficacy and safety of different primaquine regimens to prevent P. vivax relapse.Methods A systematic review identified P. vivax efficacy studies from South Asia published between 1 January 2000 and 23 August 2021. In a one-stage meta-analysis of available individual patient data, the cumulative risks of P. vivax recurrence at day 42 and 180 were assessed by primaquine total mg/kg dose and duration. The risk of recurrence by day 180 was also determined in a two-stage meta-analysis. Patients with a >25% drop in haemoglobin to 50 g/L between days 1 and 14 were categorised by daily mg/kg primaquine dose.Results In 791 patients from 7 studies in the one-stage meta-analysis, the day 180 cumulative risk of recurrence was 61.1% (95% CI 42.2% to 80.4%; 201 patients; 25 recurrences) after treatment without primaquine, 28.8% (95% CI 8.2% to 74.1%; 398 patients; 4 recurrences) following low total (2 to 25% drop in haemoglobin to

Transcriptomic analysis of isolates from the malaria parasite (Plasmodium falciparum) in the Greater Mekong Subregion of Southeast Asia identifies gene expression patterns that are correlated with resistance to a common anti-malaria drug, artemisinin.

Background: There is no generally accepted methodology for in vivo assessment of antiviral activity in SARS-CoV-2 infections. Ivermectin has been recommended widely as a treatment of COVID-19, but whether it has clinically significant antiviral activity in vivo is uncertain. Methods: In a multicentre open label, randomized, controlled adaptive platform trial, adult patients with early symptomatic COVID-19 were randomized to one of six treatment arms including high-dose oral ivermectin (600 µg/kg daily for 7 days), the monoclonal antibodies casirivimab and imdevimab (600 mg/600 mg), and no study drug. The primary outcome was the comparison of viral clearance rates in the modified intention-to-treat population. This was derived from daily log10 viral densities in standardized duplicate oropharyngeal swab eluates. This ongoing trial is registered at https://clinicaltrials.gov/ (NCT05041907). Results: Randomization to the ivermectin arm was stopped after enrolling 205 patients into all arms, as the prespecified futility threshold was reached. Following ivermectin, the mean estimated rate of SARS-CoV-2 viral clearance was 9.1% slower (95% confidence interval [CI] –27.2% to +11.8%; n=45) than in the no drug arm (n=41), whereas in a preliminary analysis of the casirivimab/imdevimab arm it was 52.3% faster (95% CI +7.0% to +115.1%; n=10 (Delta variant) vs. n=41). Conclusions: High-dose ivermectin did not have measurable antiviral activity in early symptomatic COVID-19. Pharmacometric evaluation of viral clearance rate from frequent serial oropharyngeal qPCR viral density estimates is a highly efficient and well-tolerated method of assessing SARS-CoV-2 antiviral therapeutics in vivo. Funding: ‘Finding treatments for COVID-19: A phase 2 multi-centre adaptive platform trial to assess antiviral pharmacodynamics in early symptomatic COVID-19 (PLAT-COV)’ is supported by the Wellcome Trust Grant ref: 223195/Z/21/Z through the COVID-19 Therapeutics Accelerator. Clinical trial number: NCT05041907.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network. It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented. For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations. We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent. We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines. Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

The emergence of artemisinin resistance is a major obstacle to the global malaria eradication/elimination programs. Artemisinin is a very fast-acting antimalarial drug and is the most important drug in the treatment of severe and uncomplicated malaria. For the treatment of acute uncomplicated falciparum malaria, artemisinin derivatives are combined with long half-life partner drugs and widely used as artemisinin-based combination therapies (ACTs). Some ACTs have shown decreased efficacy in the Southeast Asian region. Fortunately, artemisinin has an excellent safety profile and resistant infections can still be treated successfully by modifying the ACT. This review describes the pharmacological properties of ACTs, mechanisms of artemisinin resistance and the potential changes needed in the treatment regimens to overcome resistance. The suggested ACT modifications are extension of the duration of the ACT course, alternating use of different ACT regimens, and addition of another antimalarial drug to the standard ACTs (Triple-ACT). Furthermore, a malaria vaccine (e.g., RTS,S vaccine) could be added to mass drug administration (MDA) campaigns to enhance the treatment efficacy and to prevent further artemisinin resistance development. This review concludes that artemisinin remains the most important antimalarial drug, despite the development of drug-resistant falciparum malaria.

Abstract Background Mass drug administration (MDA) has received growing interest to accelerate the elimination of multi-drug resistant malaria in the Greater Mekong Subregion. Targeted MDA, sometimes referred to as focal MDA, is the practice of delivering MDA to high incidence subpopulations only, rather than the entire population. The potential effectiveness of delivering targeted MDA was demonstrated in a recent intervention in Kayin State, Myanmar. Policymakers and funders need to know what resources are required if MDA, targeted or otherwise, is to be included in elimination packages beyond existing malaria interventions. This study aims to estimate the programmatic cost and the unit cost of targeted MDA in Kayin State, Myanmar. Methods We used financial data from a malaria elimination initiative, conducted in Kayin State, to estimate the programmatic costs of the targeted MDA component using a micro-costing approach. Three activities (community engagement, identification of villages for targeted MDA, and conducting mass treatment in target villages) were evaluated. We then estimated the programmatic costs of implementing targeted MDA to support P. falciparum malaria elimination in Kayin State. A costing tool was developed to aid future analyses. Results The cost of delivering targeted MDA within an integrated malaria elimination initiative in eastern Kayin State was approximately US$ 910,000. The cost per person reached, distributed among those in targeted and non-targeted villages, for the MDA component was US$ 2.5. Conclusion This cost analysis can assist policymakers in determining the resources required to clear malaria parasite reservoirs. The analysis demonstrated the value of using financial data from research activities to predict programmatic implementation costs of targeting MDA to different numbers of target villages.

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr, dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination.

IntroductionUnderstanding 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.MethodsIn 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.ResultsMicroscopy 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).ConclusionPfs230 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.

Abstract Background Plasmodium malariae is a widely spread but neglected human malaria parasite, which causes chronic infections. Studies on genetic polymorphisms of anti-malarial drug target genes in P. malariae are limited. Previous reports have shown polymorphisms in the P. malariae dihydrofolate reductase gene associated with pyrimethamine resistance and linked to pyrimethamine drug pressure. This study investigated polymorphisms of the P. malariae homologous genes, chloroquine resistant transporter and multidrug resistant 1, associated with chloroquine and mefloquine resistance in Plasmodium falciparum. Methods The orthologous P. malariae crt and mdr1 genes were studied in 95 patients with P. malariae infection between 2002 and 2016 from Thailand (N = 51) and Myanmar (N = 44). Gene sequences were analysed using BioEdit, MEGA7, and DnaSP programs. Mutations and gene amplifications were compared with P. falciparum and Plasmodium vivax orthologous genes. Protein topology models derived from the observed pmcrt and pmmdr1 haplotypes were constructed and analysed using Phyre2, SWISS MODEL and Discovery Studio Visualization V 17.2. Results Two non-synonymous mutations were observed in exon 2 (H53P, 40%) and exon 8 (E278D, 44%) of pmcrt. The topology model indicated that H53P and E278D were located outside of the transmembrane domain and were unlikely to affect protein function. Pmmdr1 was more diverse than pmcrt, with 10 non-synonymous and 3 synonymous mutations observed. Non-synonymous mutations were located in the parasite cytoplasmic site, transmembrane 11 and nucleotide binding domains 1 and 2. Polymorphisms conferring amino acid changes in the transmembrane and nucleotide binding domains were predicted to have some effect on PmMDR1 conformation, but were unlikely to affect protein function. All P. malariae parasites in this study contained a single copy of the mdr1 gene. Conclusions The observed polymorphisms in pmcrt and pmmdr1 genes are unlikely to affect protein function and unlikely related to chloroquine drug pressure. Similarly, the absence of pmmdr1 copy number variation suggests limited mefloquine drug pressure on the P. malariae parasite population, despite its long time use in Thailand for the treatment of falciparum malaria.

Abstract Background Understanding the genetic structure of natural populations provides insight into the demographic and adaptive processes that have affected those populations. Such information, particularly when integrated with geospatial data, can have translational applications for a variety of fields, including public health. Estimated effective migration surfaces (EEMS) is an approach that allows visualization of the spatial patterns in genomic data to understand population structure and migration. In this study, we developed a workflow to optimize the resolution of spatial grids used to generate EEMS migration maps and applied this optimized workflow to estimate migration of Plasmodium falciparum in Cambodia and bordering regions of Thailand and Vietnam. Methods The optimal density of EEMS grids was determined based on a new workflow created using density clustering to define genomic clusters and the spatial distance between genomic clusters. Topological skeletons were used to capture the spatial distribution for each genomic cluster and to determine the EEMS grid density; i.e., both genomic and spatial clustering were used to guide the optimization of EEMS grids. Model accuracy for migration estimates using the optimized workflow was tested and compared to grid resolutions selected without the optimized workflow. As a test case, the optimized workflow was applied to genomic data generated from P. falciparum sampled in Cambodia and bordering regions, and migration maps were compared to estimates of malaria endemicity, as well as geographic properties of the study area, as a means of validating observed migration patterns. Results Optimized grids displayed both high model accuracy and reduced computing time compared to grid densities selected in an unguided manner. In addition, EEMS migration maps generated for P. falciparum using the optimized grid corresponded to estimates of malaria endemicity and geographic properties of the study region that might be expected to impact malaria parasite migration, supporting the validity of the observed migration patterns. Conclusions Optimized grids reduce spatial uncertainty in the EEMS contours that can result from user-defined parameters, such as the resolution of the spatial grid used in the model. This workflow will be useful to a broad range of EEMS users as it can be applied to analyses involving other organisms of interest and geographic areas.

Introduction: RTS,S/AS01 is currently the most advanced malaria vaccine but provides incomplete, short-term protection. It was developed for use within the expanded program on immunizations (EPI) for African children. Another use could be adding mass RTS,S/AS01 vaccination to the integrated malaria elimination strategy in the Greater Mekong Subregion (GMS), where multidrug-resistant P.falciparum strains have emerged and spread. Prior to evaluating RTS,S/AS01 in large-scale trials we assessed whether the vaccine, administered with and without antimalarial drugs, is safe and immunogenic in Asian populations. Methods: An open-label, randomized, controlled phase 2 trial was conducted in healthy, adult Thai volunteers. Seven vaccine regimens with and without antimalarial drugs (dihydroartemisinin-piperaquine plus a single low dose primaquine) were assessed. Antibody titres against the PfCSP full-length (NANP) 6, PfCSP anti-C–term, PfCSP full-length (N + C-Terminal) were measured by standard enzyme-linked immunosorbent assays. Liquid chromatography was used to measure piperaquine, primaquine and carboxy-primaquine concentrations. Results: 193 volunteers were enrolled and 186 study participants completed the 6 months follow-up period. One month after the last vaccination all study participants had seroconverted to the PfCSP (NANP)6, and the PfCSP Full Length (N + C-Terminal). More than 90% had seroconverted to the Pfanti-C-Term CSP. There was no indication that drug concentrations were influenced by vaccine regimens or the antibody levels by the drug regimens. Adverse events were similarly distributed between the seven treatment groups. No serious adverse events attributable to the study interventions were detected. Conclusion: This study found that RTS,S/AS01 with and without dihydroartemisinin-piperaquine plus a single low dose primaquine was safe and immunogenic in a healthy, adult Asian population.

Background: National Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures. Methods: Samples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs. Results: GenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs. Conclusions: GenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community. Funding: The GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Abstract Background Adding 8-aminoquinoline to the treatment of falciparum, in addition to vivax malaria, in locations where infections with both species are prevalent could prevent vivax reactivation. The potential risk of haemolysis under a universal radical cure policy using 8-aminoquinoline needs to be weighed against the benefit of preventing repeated vivax episodes. Estimating the frequency of sequential Plasmodium vivax infections following either falciparum or vivax malaria episodes is needed for such an assessment. Methods Quarterly surveillance data collected during a mass drug administration trial in the Greater Mekong Subregion in 2013–17 was used to estimate the probability of asymptomatic sequential infections by the same and different Plasmodium species. Asymptomatic Plasmodium infections were detected by high-volume ultrasensitive qPCR. Quarterly surveys of asymptomatic Plasmodium prevalence were used to estimate the probability of a P. vivax infection following Plasmodium falciparum and P. vivax infections. Results 16,959 valid sequential paired test results were available for analysis. Of these, 534 (3%) had an initial P. falciparum monoinfection, 1169 (7%) a P. vivax monoinfection, 217 (1%) had mixed (P. falciparum + P. vivax) infections, and 15,039 (89%) had no Plasmodium detected in the initial survey. Participants who had no evidence of a Plasmodium infection had a 4% probability to be found infected with P. vivax during the subsequent survey. Following an asymptomatic P. falciparum monoinfection participants had a 9% probability of having a subsequent P. vivax infection (RR 2.4; 95% CI 1.8 to 3.2). Following an asymptomatic P. vivax monoinfection, the participants had a 45% probability of having a subsequent P. vivax infection. The radical cure of 12 asymptomatic P. falciparum monoinfections would have prevented one subsequent P. vivax infection, whereas treatment of 2 P. vivax monoinfections may suffice to prevent one P. vivax relapse. Conclusion Universal radical cure could play a role in the elimination of vivax malaria. The decision whether to implement universal radical cure for P. falciparum as well as for P. vivax depends on the prevalence of P. falciparum and P. vivax infections, the prevalence and severity of G6PD deficiency in the population and the feasibility to administer 8-aminoquinoline regimens safely. Trial registration ClinicalTrials.gov Identifier: NCT01872702, first posted June 7th 2013, https://clinicaltrials.gov/ct2/show/NCT01872702. This study was registered with ClinicalTrials.gov under NCT02802813 on 16th June 2016. https://clinicaltrials.gov/ct2/show/NCT02802813

Melioidosis and scrub typhus are endemic diseases in Malaysia. However, unlike melioidosis, scrub typhus is more difficult to be diagnosed and in Malaysia its true disease burden is unknown. We report a case of severe melioidosis and scrub typhus co-infection in a patient living in a small village in Peninsular Malaysia whom presented with fever and headache. This case highlights the importance of considering scrub typhus as a diagnostic possibility in cases of acute undifferentiated febrile illness in rural areas such as that of in Peninsular Malaysia.

The authors wish to make the following correction to this paper [...]

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Rickettsial infections are among the leading etiologies of acute febrile illness in Southeast Asia. However, recent data from Malaysia are limited. This prospective study was conducted in Teluk Intan, Peninsular Malaysia, during January to December 2016. We recruited 309 hospitalized adult patients with acute febrile illness. Clinical and biochemistry data were obtained, and patients were stratified into mild and severe infections based on the sepsis-related organ failure (qSOFA) scoring system. Diagnostic assays including blood cultures, real-time PCR, and serology (IFA and MAT) were performed. In this study, pathogens were identified in 214 (69%) patients, of which 199 (93%) patients had a single etiology, and 15 (5%) patients had >1 etiologies. The top three causes of febrile illness requiring hospitalization in this Malaysian study were leptospirosis (68 (32%)), dengue (58 (27%)), and rickettsioses (42 (19%)). Fifty-five (18%) patients presented with severe disease with a qSOFA score of ≥2. Mortality was documented in 38 (12%) patients, with the highest seen in leptospirosis (16 (42%)) followed by rickettsiosis (4 (11%)). While the significance of leptospirosis and dengue are recognized, the impact of rickettsial infections in Peninsular Malaysia remains under appreciated. Management guidelines for in-patient care with acute febrile illness in Peninsular Malaysia are needed.

Purpose A successful pregnancy relies on the interplay of various biological systems. Deviations from the norm within a system or intersystemic interactions may result in pregnancy-associated complications and adverse pregnancy outcomes. Systems biology approaches provide an avenue of unbiased, in-depth phenotyping in health and disease. The molecular signature in pregnancy (MSP) cohort was established to characterise longitudinal, cross-omic trajectories in pregnant women from a resource constrained setting. Downstream analysis will focus on characterising physiological perturbations in uneventful pregnancies, pregnancy-associated complications and adverse outcomes.Participants First trimester pregnant women of Karen or Burman ethnicity were followed prospectively throughout pregnancy, at delivery and until 3 months post partum. Serial high-frequency sampling to assess whole blood transcriptomics and microbiome composition of the gut, vagina and oral cavity, in conjunction with assessment of gene expression and microbial colonisation of gestational tissue, was done for all cohort participants.Findings to date 381 women with live born singletons averaged 16 (IQR 15–18) antenatal visits (13 094 biological samples were collected). At 5% (19/381) the preterm birth rate was low. Other adverse events such as maternal febrile illness 7.1% (27/381), gestational diabetes 13.1% (50/381), maternal anaemia 16.3% (62/381), maternal underweight 19.2% (73/381) and a neonate born small for gestational age 20.2% (77/381) were more often observed than preterm birth.Future plans Results from the MSP cohort will enable in-depth characterisation of cross-omic molecular trajectories in pregnancies from a population in a resource-constrained setting. Moreover, pregnancy-associated complications and unfavourable pregnancy outcomes will be investigated at the same granular level, with a particular focus on population relevant needs such as effect of tropical infections on pregnancy. More detailed knowledge on multiomic perturbations will ideally result in the development of diagnostic tools and ultimately lead to targeted interventions that may disproportionally benefit pregnant women from this resource-limited population.Trial registration number NCT02797327.

BackgroundElectrocardiographic QT interval prolongation is the most widely used risk marker for ventricular arrhythmia potential and thus an important component of drug cardiotoxicity assessments. Several antimalarial medicines are associated with QT interval prolongation. However, interpretation of electrocardiographic changes is confounded by the coincidence of peak antimalarial drug concentrations with recovery from malaria. We therefore reviewed all available data to characterise the effects of malaria disease and demographic factors on the QT interval in order to improve assessment of electrocardiographic changes in the treatment and prevention of malaria.Methods and findingsWe conducted a systematic review and meta-analysis of individual patient data. We searched clinical bibliographic databases (last on August 21, 2017) for studies of the quinoline and structurally related antimalarials for malaria-related indications in human participants in which electrocardiograms were systematically recorded. Unpublished studies were identified by the World Health Organization (WHO) Evidence Review Group (ERG) on the Cardiotoxicity of Antimalarials. Risk of bias was assessed using the Pharmacoepidemiological Research on Outcomes of Therapeutics by a European Consortium (PROTECT) checklist for adverse drug events. Bayesian hierarchical multivariable regression with generalised additive models was used to investigate the effects of malaria and demographic factors on the pretreatment QT interval. The meta-analysis included 10,452 individuals (9,778 malaria patients, including 343 with severe disease, and 674 healthy participants) from 43 studies. 7,170 (68.6%) had fever (body temperature ≥ 37.5°C), and none developed ventricular arrhythmia after antimalarial treatment. Compared to healthy participants, patients with uncomplicated falciparum malaria had shorter QT intervals (-61.77 milliseconds; 95% credible interval [CI]: -80.71 to -42.83) and increased sensitivity of the QT interval to heart rate changes. These effects were greater in severe malaria (-110.89 milliseconds; 95% CI: -140.38 to -81.25). Body temperature was associated independently with clinically significant QT shortening of 2.80 milliseconds (95% CI: -3.17 to -2.42) per 1°C increase. Study limitations include that it was not possible to assess the effect of other factors that may affect the QT interval but are not consistently collected in malaria clinical trials.ConclusionsAdjustment for malaria and fever-recovery-related QT lengthening is necessary to avoid misattributing malaria-disease-related QT changes to antimalarial drug effects. This would improve risk assessments of antimalarial-related cardiotoxicity in clinical research and practice. Similar adjustments may be indicated for other febrile illnesses for which QT-interval-prolonging medications are important therapeutic options.

BackgroundMass administrations of antimalarial drugs (MDA) have reduced the incidence and prevalence of P. falciparum infections in a trial in the Greater Mekong Subregion. Here we assess the impact of the MDA on P. vivax infections.MethodsBetween May 2013 and July 2017, four villages in each Myanmar, Vietnam, Cambodia and Lao PDR were selected based on high prevalence of P. falciparum infections. Eight of the 16 villages were randomly assigned to receive MDA consisting of three-monthly rounds of three-day courses of dihydroartemisinin-piperaquine and, except in Cambodia, a single low-dose of primaquine. Cross-sectional surveys were conducted at quarterly intervals to detect Plasmodium infections using ultrasensitive qPCR. The difference in the cumulative incidence between the groups was assessed through a discrete time survival approach, the difference in prevalence through a difference-in-difference analysis, and the difference in the number of participants with a recurrence of P. vivax infection through a mixed-effect logistic regression.Results3,790 (86%) residents in the intervention villages participated in at least one MDA round, of whom 2,520 (57%) participated in three rounds. The prevalence of P. vivax infections fell from 9.31% to 0.89% at month 3 but rebounded by six months to 5.81%. There was no evidence that the intervention reduced the cumulative incidence of P.vivax infections (95% confidence interval [CI] Odds ratio (OR): 0.29 to 1.36). Similarly, there was no evidence of MDA related reduction in the number of participants with at least one recurrent infection (OR: 0.34; 95% CI: 0.08 to 1.42).ConclusionMDA with schizontocidal drugs had a lasting effect on P. falciparum infections but only a transient effect on the prevalence of P. vivax infections. Radical cure with an 8-aminoquinoline will be needed for the rapid elimination of vivax malaria.

Abstract Background The increase in multidrug resistant Plasmodium falciparum infections threatens the malaria elimination goals in countries within the Greater Mekong Sub-region. A multi-pronged approach assuring access to basic malaria control measures, including insecticide-treated bed nets and early diagnosis and treatment was followed by mass drug administrations (MDA) in southern Savannakhet Province, Laos. The main objective of this study was to evaluate the effectiveness and safety of mass drug administrations as well as their effects on the dynamic of asymptomatic P. falciparum infections in 4 malaria endemic villages. Methods Two villages were randomized to early MDA consisting of 3 rounds of a 3-day course of dihydroartemisinin–piperaquine with a single low dose of primaquine. In the other 2 villages MDA was deferred by 1 year. A total of 1036 residents were enrolled in early MDA villages and 883 in control villages (deferred-MDA). Tri-monthly parasitaemia surveys using uPCR were conducted for a year in the 4 villages. Results Eighty-four percent (872/1036) of the residents participated in the MDAs, of whom 90% (781/872) completed 3 rounds of MDA (9 doses). In intervention villages, the prevalence of asymptomatic P. falciparum infections decreased by 85% after MDA from 4.8% (95% CI 3.4–6.4) at baseline (month 0 or M0) to 0.7% (95% CI 0.3–1.6) at month 12. In control villages there was a decrease of 33% in P. falciparum prevalence between M0: 17.5% (95% CI 15.9–20.3) and M12: 11.6% (95% CI 9.3–14.2). In bivariate and multivariate analyses P. falciparum infections were significantly reduced with early MDA (adjusted incidence rate ratios (AIRR): 0.08, CI 0.01–0.091) and completion of 3 MDA rounds (AIRR: 0.06; CI 0.01–0.66). A quarter of participants (226/872) reported adverse events of which 99% were mild. Conclusion The study found a significant reduction in P. falciparum prevalence and incidence following MDA. MDA was safe, well tolerated, feasible, and achieved high population coverage and adherence. MDAs must be integrated in multi-pronged approaches such as vector control and preventive measures with a focus on specific risk groups such as mobile, migrant population and forest goers for a sustained period to eliminate the remaining parasite reservoirs. Trial registration ClinicalTrials.gov Identifier: NCT01872702

Abstract Background Research funders, regulatory agencies, and journals are increasingly expecting that individual-level data from health research will be shared. Broad consent to such sharing is considered appropriate, feasible and acceptable in low- and middle-income settings, but to date limited empirical research has been conducted to inform the design of such processes. We examined stakeholder perspectives about how best to seek broad consent to sharing data from the Mahidol Oxford Tropical Medicine Research Unit, which implemented a data sharing policy and broad consent to data sharing in January 2016. Methods Between February and August 2017 qualitative data were collected at two sites, Bangkok and the Thai-Myanmar border town of Mae Sot. We conducted eighteen semi-structured interviews. We also conducted four focus group discussions with a total of nineteen people. Descriptive and thematic coding informed analysis of aspects of data sharing that are considered most important to inform participants about, and the best ways to explain complex and abstract topics relating to data sharing. Results The findings demonstrated that clinical trial participants prioritise information about the potential benefits and harms of data sharing. Stakeholders made multiple suggestions for clarifying information provided about data sharing on such topics. There was significant variation amongst stakeholders’ perspectives about how much information should be provided about data sharing, and it was clear that effective information provision should be responsive to the study, the study population, the individual research participant and the research context. Conclusions Effectively communicating about data sharing with research participants is challenging in practice, highlighting the importance of robust and effective data sharing governance in this context. Broad consent should incorporate effective and efficient explanations of data sharing to promote informed decision-making, without impeding research participants’ understandings of key aspects of the research from which data will be shared. Further work is required to refine both the development of core information about data sharing to be provided to all research participants, and appropriate solutions for context specific-challenges arising when explaining data sharing.

Abstract Background Plasmodium malariae is characterized by its long asymptomatic persistence in the human host. The epidemiology of P. malariae is incompletely understood and is hampered by the limited knowledge of genetic polymorphisms. Previous reports from Africa have shown heterogeneity within the P. malariae circumsporozoite protein (pmcsp) gene. However, comparative studies from Asian countries are lacking. Here, the genetic polymorphisms in pmcsp of Asian isolates have been characterized. Methods Blood samples from 89 symptomatic P. malariae-infected patients were collected, from Thailand (n = 43), Myanmar (n = 40), Lao PDR (n = 5), and Bangladesh (n = 1). pmcsp was amplified using semi-nested PCR before sequencing. The resulting 89 pmcsp sequences were analysed together with 58 previously published pmcsp sequences representing African countries using BioEdit, MEGA6, and DnaSP. Results Polymorphisms identified in pmcsp were grouped into 3 populations: Thailand, Myanmar, and Kenya. The nucleotide diversity and the ratio of nonsynonymous to synonymous substitutions (dN/dS) in Thailand and Myanmar were higher compared with that in Kenya. Phylogenetic analysis showed clustering of pmcsp sequences according to the origin of isolates (Asia vs. Africa). High genetic differentiation (Fst = 0.404) was observed between P. malariae isolates from Asian and African countries. Sequence analysis of pmcsp showed the presence of tetrapeptide repeat units of NAAG, NDAG, and NAPG in the central repeat region of the gene. Plasmodium malariae isolates from Asian countries carried fewer copies of NAAG compared with that from African countries. The NAPG repeat was only observed in Asian isolates. Additional analysis of 2 T-cell epitopes, Th2R and Th3R, showed limited heterogeneity in P. malariae populations. Conclusions This study provides valuable information on the genetic polymorphisms in pmcsp isolates from Asia and advances our understanding of P. malariae population in Asia and Africa. Polymorphisms in the central repeat region of pmcsp showed association with the geographical origin of P. malariae isolates and can be potentially used as a marker for genetic epidemiology of P. malariae population.

Abstract 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

Abstract Background Genetic diversity of the three important antigenic proteins, namely thrombospondin-related anonymous protein (TRAP), apical membrane antigen 1 (AMA1), and 6-cysteine protein (P48/45), all of which are found in various developmental stages of Plasmodium parasites is crucial for targeted vaccine development. While studies related to the genetic diversity of these proteins are available for Plasmodium falciparum and Plasmodium vivax, barely enough information exists regarding Plasmodium malariae. The present study aims to demonstrate the genetic variations existing among these three genes in P. malariae by analysing their diversity at nucleotide and protein levels. Methods Three surface protein genes were isolated from 45 samples collected in Thailand (N = 33), Myanmar (N = 8), and Lao PDR (N = 4), using conventional polymerase chain reaction (PCR) assay. Then, the PCR products were sequenced and analysed using BioEdit, MEGA6, and DnaSP programs. Results The average pairwise nucleotide diversities (π) of P. malariae trap, ama1, and p48/45 were 0.00169, 0.00413, and 0.00029, respectively. The haplotype diversities (Hd) of P. malariae trap, ama1, and p48/45 were 0.919, 0.946, and 0.130, respectively. Most of the nucleotide substitutions were non-synonymous, which indicated that the genetic variations of these genes were maintained by positive diversifying selection, thus, suggesting their role as a potential target of protective immune response. Amino acid substitutions of P. malariae TRAP, AMA1, and P48/45 could be categorized to 17, 20, and 2 unique amino-acid variants, respectively. For further vaccine development, carboxyl terminal of P48/45 would be a good candidate according to conserved amino acid at low genetic diversity (π = 0.2–0.3). Conclusions High mutational diversity was observed in P. malariae trap and ama1 as compared to p48/45 in P. malariae samples isolated from Thailand, Myanmar, and Lao PDR. Taken together, these results suggest that P48/45 might be a good vaccine candidate against P. malariae infection because of its sufficiently low genetic diversity and highly conserved amino acids especially on the carboxyl end.

Abstract Background Most evidence on the association between malaria in pregnancy and adverse pregnancy outcomes focuses on falciparum malaria detected at birth. We assessed the association between the number and timing of falciparum and vivax malaria episodes during pregnancy on small-for-gestational-age (SGA) and preterm birth. Methods We analysed observational data collected from antenatal clinics on the Thailand-Myanmar border (1986–2015). We assessed the effects of the total number of malaria episodes in pregnancy on SGA and the effects of malaria in pregnancy on SGA, very preterm birth, and late preterm birth, by the gestational age at malaria detection and treatment using logistic regression models with time-dependent malaria variables (monthly intervals). World Health Organisation definitions of very preterm birth (≥28 and

BackgroundThe emergence and spread of multidrug-resistant Plasmodium falciparum in the Greater Mekong Subregion (GMS) threatens global malaria elimination efforts. Mass drug administration (MDA), the presumptive antimalarial treatment of an entire population to clear the subclinical parasite reservoir, is a strategy to accelerate malaria elimination. We report a cluster randomised trial to assess the effectiveness of dihydroartemisinin-piperaquine (DP) MDA in reducing falciparum malaria incidence and prevalence in 16 remote village populations in Myanmar, Vietnam, Cambodia, and the Lao People's Democratic Republic, where artemisinin resistance is prevalent.Methods and findingsAfter establishing vector control and community-based case management and following intensive community engagement, we used restricted randomisation within village pairs to select 8 villages to receive early DP MDA and 8 villages as controls for 12 months, after which the control villages received deferred DP MDA. The MDA comprised 3 monthly rounds of 3 daily doses of DP and, except in Cambodia, a single low dose of primaquine. We conducted exhaustive cross-sectional surveys of the entire population of each village at quarterly intervals using ultrasensitive quantitative PCR to detect Plasmodium infections. The study was conducted between May 2013 and July 2017. The investigators randomised 16 villages that had a total of 8,445 residents at the start of the study. Of these 8,445 residents, 4,135 (49%) residents living in 8 villages, plus an additional 288 newcomers to the villages, were randomised to receive early MDA; 3,790 out of the 4,423 (86%) participated in at least 1 MDA round, and 2,520 out of the 4,423 (57%) participated in all 3 rounds. The primary outcome, P. falciparum prevalence by month 3 (M3), fell by 92% (from 5.1% [171/3,340] to 0.4% [12/2,828]) in early MDA villages and by 29% (from 7.2% [246/3,405] to 5.1% [155/3,057]) in control villages. Over the following 9 months, the P. falciparum prevalence increased to 3.3% (96/2,881) in early MDA villages and to 6.1% (128/2,101) in control villages (adjusted incidence rate ratio 0.41 [95% CI 0.20 to 0.84]; p = 0.015). Individual protection was proportional to the number of completed MDA rounds. Of 221 participants with subclinical P. falciparum infections who participated in MDA and could be followed up, 207 (94%) cleared their infections, including 9 of 10 with artemisinin- and piperaquine-resistant infections. The DP MDAs were well tolerated; 6 severe adverse events were detected during the follow-up period, but none was attributable to the intervention.ConclusionsAdded to community-based basic malaria control measures, 3 monthly rounds of DP MDA reduced the incidence and prevalence of falciparum malaria over a 1-year period in areas affected by artemisinin resistance. P. falciparum infections returned during the follow-up period as the remaining infections spread and malaria was reintroduced from surrounding areas. Limitations of this study include a relatively small sample of villages, heterogeneity between villages, and mobility of villagers that may have limited the impact of the intervention. These results suggest that, if used as part of a comprehensive, well-organised, and well-resourced elimination programme, DP MDA can be a useful additional tool to accelerate malaria elimination.Trial registrationClinicalTrials.gov NCT01872702.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency may affect the clinical presentation of dengue due to the altered redox state in immune cells. We aimed to determine the association between G6PD deficiency and severity of dengue infection in paediatric patients in Myanmar. A cross-sectional study was conducted among paediatric patients aged 2-13 years with dengue in Yankin Children Hospital, Myanmar. One hundred and ninety-six patients positive for dengue infection, as determined via PCR or ELISA, were enrolled. Dengue severity was determined according to the 2009 WHO classification guidelines. Spectrophotometric assays determined G6PD levels. The adjusted median G6PD value of males in the study population was used to define various cut-off points according to the WHO classification guidelines. G6PD genotyping for Mahidol, Kaiping and Mediterranean mutations was performed for 128 out of 196 samples by real-time multiplex PCR. 51 of 196 (26.0%) patients had severe dengue. The prevalence of G6PD phenotype deficiency (< 60% activity) in paediatric patients was 14.8% (29/196), specifically, 13.6% (14/103) in males and 16.2% (15/93) in females. Severe deficiency (< 10% activity) accounted for 7.1% (14/196) of our cohort, occurring 11.7% (12/103) in males and 2.2% (2/93) in females. Among 128 samples genotyped, the G6PD gene mutations were detected in 19.5% (25/128) of patients, with 20.3% (13/ 64) in males and 18.8% (12/64) in females. The G6PD Mahidol mutation was 96.0% (24/25) while the G6PD Kaiping mutation was 4.0% (1/25). Severe dengue was not associated with G6PD enzyme deficiency or presence of the G6PD gene mutation. Thus, no association between G6PD deficiency and dengue severity could be detected. Trial registration: The study was registered following the WHO International Clinical Trials Registry Platform (WHO-ICTRP) on Thai Clinical Trials Registry (TCTR) website, registration number # TCTR20180720001.

Abstract 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.

Background : No universal demarcation of gestational age distinguishes miscarriage and stillbirth or extreme preterm birth (exPTB). This study provides a synopsis of outcome between 22 to

BACKGROUND:Meningitis caused by Mycobacterium tuberculosis is a major cause of morbidity and mortality worldwide. We evaluated the performance of cerebrospinal fluid (CSF) testing with the GeneXpert MTB/RIF assay versus traditional approaches for diagnosing tuberculosis meningitis (TBM). METHODS:Patients were adults (n = 37) presenting with suspected TBM to the Hospital Nacional Dos de Mayo, Lima, Peru, during 12 months until 1st January 2015. Each participant had a single CSF specimen that was divided into aliquots that were concurrently tested for M. tuberculosis using GeneXpert, Ziehl-Neelsen smear and culture on solid and liquid media. Drug susceptibility testing used Mycobacteria Growth Indicator Tube (MGIT 960) and the proportions method. RESULTS:81% (30/37) of patients received a final clinical diagnosis of TBM, of whom 63% (19/30, 95% confidence intervals, CI: 44-80%) were HIV-positive. 22% (8/37, 95%CI: 9.8-38%), of patients had definite TBM. Because definite TBM was defined by positivity in any laboratory test, all laboratory tests had 100% specificity. Considering the 30 patients who had a clinical diagnosis of TBM: diagnostic sensitivity was 23% (7/30, 95%CI: 9.9-42%) for GeneXpert and was the same for all culture results combined; considerably greater than 7% (2/30, 95%CI: 0.82-22%) for microscopy; whereas all laboratory tests had poor negative predictive values (20-23%). Considering only the 8 patients with definite TBM: diagnostic sensitivity was 88% (7/8, 95%CI: 47-100%) for GeneXpert; 75% (6/8, 95%CI: 35-97%) for MGIT culture or LJ culture; 50% (4/8, 95%CI 16-84) for Ogawa culture and 25% (2/8, 95%CI: 3.2-65%) for microscopy. GeneXpert and microscopy provided same-day results, whereas culture took 20-56 days. GeneXpert provided same-day rifampicin-susceptibility results, whereas culture-based testing took 32-71 days. 38% (3/8, 95%CI: 8.5-76%) of patients with definite TBM with data had evidence of drug-resistant TB, but 73% (22/30) of all clinically diagnosed TBM (definite, probable, and possible TBM) had no drug-susceptibility results available. CONCLUSIONS:Compared with traditional culture-based methods of CSF testing, GeneXpert had similar yield and faster results for both the detection of M. tuberculosis and drug-susceptibility testing. Including use of the GeneXpert has the capacity to improve the diagnosis of TBM cases.

In severe falciparum malaria cytoadherence of parasitised red blood cells (PRBCs) to vascular endothelium (causing sequestration) and to uninfected red cells (causing rosette formation) contribute to microcirculatory flow obstruction in vital organs. Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated falciparum malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum isolates from Thailand were investigated. Trophozoite stages of P. falciparum-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of P. falciparum isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0-38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts P. falciparum rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria.

Background: Individuals in conflict-affected areas rarely get appropriate care for chronic or non-infectious diseases. The prevalence of gestational diabetes mellitus (GDM) is increasing worldwide, and new evidence shows conclusively that the negative effects of hyperglycemia occur even at mild glucose elevations and that these negative effects can be attenuated by treatment. Scientific literature on gestational diabetes in refugee camp settings is critically limited. Methods: A 75 g 2-hour glucose tolerance test was administered to 228 women attending the antenatal care (ANC) clinic in Maela refugee camp on the Thai–Myanmar border. Prevalence of GDM was determined using the HAPO trial cut-offs [≥92 mg/dL (fasting),≥180 (1 hour), and≥153 (2 hour)] and the WHO criteria [≥126 mg/dL (fasting), and 140 mg/dL (2 hour)]. Results: From July 2011 to March 2012, the prevalence of GDM was 10.1% [95% confidence interval (CI): 6.2–14.0] when the cut-off determined by the HAPO trial was applied. Applying the older WHO criteria yielded a prevalence of 6.6% (95% CI 3.3–9.8). Age, parity, and BMI emerged as characteristics that may be significantly associated with GDM in this population. Other risk factors that are commonly used in screening guidelines were not applicable in this diabetes-naïve population. Discussion: The prevalence of GDM is lower in this population compared with other populations, but still complicates 10% of pregnancies. New evidence regarding gestational diabetes raises new dilemmas for healthcare providers in resource-poor settings. Efforts to identify and treat patients at risk for adverse outcomes need to be balanced with awareness of the risks and burdens associated with over diagnosis and unnecessary interventions. Screening approaches based on risk factors or using higher cut-off values may help minimize this burden and identify those most likely to benefit from intervention.

Murine typhus is a rarely diagnosed cause of acute febrile illness in Malaysia, and its true disease burden is unknown. We report a case of an acute murine typhus infection in a patient living in a small city in Peninsular Malaysia, presenting with fever, rash, and headache. Unresponsive to the initial empirical treatment for leptospirosis, he showed a rapid response to doxycycline when murine typhus was diagnosed later. This case highlights the importance of considering murine typhus as a diagnostic in cases of acute febrile illness in urban and sub-urban areas, such as that of in Peninsular Malaysia.

[This corrects the article DOI: 10.1371/journal.pntd.0004024.].

An effective antibody response can assist drug treatment to contribute to better parasite clearance in malaria patients. To examine this, sera were obtained from two groups of adult patients with acute falciparum malaria, prior to drug treatment: patients who (1) have subsequent recrudescent infection, or (2) were cured by Day 28 following treatment. Using a Plasmodium falciparum antigen library, we examined the antibody specificities in these sera. While the antibody repertoire of both sera groups was extremely broad and varied, there was a differential antibody profile between the two groups of sera. The proportion of cured patients with antibodies against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 was higher than the proportion of patients with recrudescent infection. The presence of these antibodies was associated with higher odds of treatment cure. Sera containing all six antibodies impaired the invasion of P. falciparum clinical isolates into erythrocytes. These results suggest that antibodies specific against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 in P. falciparum infections could assist anti-malarial drug treatment and contribute to the resolution of the malarial infection.

Orientia tsutsugamushi is the causative agent of scrub typhus, a disease transmitted by Leptotrombidium mites which is responsible for a severe and under-reported public health burden throughout Southeast Asia. Here we use multilocus sequence typing (MLST) to characterize 74 clinical isolates from three geographic locations in the Lao PDR (Laos), and compare them with isolates described from Udon Thani, northeast Thailand. The data confirm high levels of diversity and recombination within the natural O. tsutsugamushi population, and a rate of mixed infection of ~8%. We compared the relationships and geographical structuring of the strains and populations using allele based approaches (eBURST), phylogenetic approaches, and by calculating F-statistics (FST). These analyses all point towards low levels of population differentiation between isolates from Vientiane and Udon Thani, cities which straddle the Mekong River which defines the Lao/Thai border, but with a very distinct population in Salavan, southern Laos. These data highlight how land use, as well as the movement of hosts and vectors, may impact on the epidemiology of zoonotic infections.

Estimating gestational age in resource-limited settings is prone to considerable inaccuracy because crown-rump length measured by ultrasound before 14 weeks gestation, the recommended method for estimating gestational age, is often unavailable. Judgements regarding provision of appropriate obstetric and neonatal care are dependent on accurate estimation of gestational age. We determined the accuracy of the Dubowitz Gestational Age Assessment, a population-specific symphysis-fundal height formula, and ultrasound biometry performed between 16 and 40 weeks gestation in estimating gestational age using pre-existing data from antenatal clinics of the Shoklo Malaria Research Unit on the Thai-Myanmar border, where malaria is endemic. Two cohorts of women who gave birth to live singletons were analysed: 1) 250 women who attended antenatal care between July 2001 and May 2006 and had both ultrasound crown-rump length (reference) and a Dubowitz Gestational Age Assessment; 2) 975 women attending antenatal care between April 2007 and October 2010 who had ultrasound crown-rump length, symphysis-fundal measurements, and an additional study ultrasound (biparietal diameter and head circumference) randomly scheduled between 16 and 40 weeks gestation. Mean difference in estimated newborn gestational age between methods and 95% limits of agreement (LOA) were determined from linear mixed-effects models. The Dubowitz method and the symphysis-fundal height formula performed well in term newborns, but overestimated gestational age of preterms by 2.57 weeks (95% LOA: 0.49, 4.65) and 3.94 weeks (95% LOA: 2.50, 5.38), respectively. Biparietal diameter overestimated gestational age by 0.83 weeks (95% LOA: -0.93, 2.58). Head circumference underestimated gestational age by 0.39 weeks (95% LOA: -2.60, 1.82), especially if measured after 24 weeks gestation. The results of this study can be used to quantify biases associated with alternative methods for estimating gestational age in the absence of ultrasound crown-rump length to inform critical clinical judgements in this population, and as a point of reference elsewhere.

The association between malaria during pregnancy and low birth weight (LBW) is well described. This manuscript aims to quantify the relative contribution of malaria to small-for-gestational-age (SGA) infants and preterm birth (PTB) in pregnancies accurately dated by ultrasound on the Thai-Myanmar border at the Shoklo Malaria Research Unit.From 2001 to 2010 in a population cohort of prospectively followed pregnancies, we analyzed all singleton newborns who were live born, normal, weighed in the first hour of life and with a gestational age (GA) between 28+0 and 41+6 weeks. Fractional polynomial regression was used to determine the mean birthweight and standard deviation as functions of GA. Risk differences and factors of LBW and SGA were studied across the range of GA for malaria and non-malaria pregnancies. From 10,264 newborns records, population centiles were created. Women were screened for malaria by microscopy a median of 22 [range 1-38] times and it was detected and treated in 12.6% (1,292) of pregnancies. Malaria was associated with LBW, PTB, and SGA compared to those without malaria. Nearly two-thirds of PTB were classified as LBW (68% (539/789)), most of which 83% (447/539) were not SGA. After GA 39 weeks, 5% (298/5,966) of non-LBW births were identified as SGA. Low body mass index, primigravida, hypertension, smoking and female sex of the newborn were also significantly and independently associated with LBW and SGA consistent with previous publications.Treated malaria in pregnancy was associated with an increased risk for LBW, PTB, and SGA, of which the latter are most important for infant survival. Using LBW as an endpoint without adjusting for GA incorrectly estimated the effects of malaria in pregnancy. Ultrasound should be used for dating pregnancies and birth weights should be expressed as a function (or adjusted for GA) of GA in future malaria in pregnancy studies.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzyme defect and an important problem in areas with Plasmodium vivax infection because of the risk of haemolysis following administration of primaquine to treat the liver forms of the parasite. We undertook a genotypic survey of 713 male individuals across nine provinces of Afghanistan in which malaria is found, four in the north and five in the east. RFLP typing at nucleotide position 563 detected 40 individuals with the Mediterranean mutation 563C>T, an overall prevalence of 5.6%. This varied according to self-reported ethnicity, with prevalence in the Pashtun/Pashai group of 33/369 (8.9%) compared to 7/344 individuals in the rest of the population (2.0%; pT in exon 11, and C93T in intron XI) in a subset of 82 individuals wild-type at C563 revealed a mixture of 3 haplotypes in the background population and was consistent with data from the 1000 Genomes Project and published studies. By comparison individuals with G6PD deficiency showed a highly skewed haplotype distribution, with 95% showing the CT haplotype, a finding consistent with relatively recent appearance and positive selection of the Mediterranean variant in Afghanistan. Overall, the data confirm that the Mediterranean variant of G6PD is common in many ethnic groups in Afghanistan, indicating that screening for G6PD deficiency is required in all individuals before radical treatment of P. vivax with primaquine.

BACKGROUND:Accuracy of rapid diagnostic tests for dengue infection has been repeatedly estimated by comparing those tests with reference assays. We hypothesized that those estimates might be inaccurate if the accuracy of the reference assays is not perfect. Here, we investigated this using statistical modeling. METHODS/PRINCIPAL FINDINGS:Data from a cohort study of 549 patients suspected of dengue infection presenting at Colombo North Teaching Hospital, Ragama, Sri Lanka, that described the application of our reference assay (a combination of Dengue IgM antibody capture ELISA and IgG antibody capture ELISA) and of three rapid diagnostic tests (Panbio NS1 antigen, IgM antibody and IgG antibody rapid immunochromatographic cassette tests) were re-evaluated using bayesian latent class models (LCMs). The estimated sensitivity and specificity of the reference assay were 62.0% and 99.6%, respectively. Prevalence of dengue infection (24.3%), and sensitivities and specificities of the Panbio NS1 (45.9% and 97.9%), IgM (54.5% and 95.5%) and IgG (62.1% and 84.5%) estimated by bayesian LCMs were significantly different from those estimated by assuming that the reference assay was perfect. Sensitivity, specificity, PPV and NPV for a combination of NS1, IgM and IgG cassette tests on admission samples were 87.0%, 82.8%, 62.0% and 95.2%, respectively. CONCLUSIONS:Our reference assay is an imperfect gold standard. In our setting, the combination of NS1, IgM and IgG rapid diagnostic tests could be used on admission to rule out dengue infection with a high level of accuracy (NPV 95.2%). Further evaluation of rapid diagnostic tests for dengue infection should include the use of appropriate statistical models.

Plasmodium vivax infections seldom kill directly but do cause indirect mortality by reducing birth weight and causing abortion. Cytoadherence and sequestration in the microvasculature are central to the pathogenesis of severe Plasmodium falciparum malaria, but the contribution of cytoadherence to pathology in other human malarias is less clear.The adherence properties of P. vivax infected red blood cells (PvIRBC) were evaluated under static and flow conditions.P. vivax isolates from 33 patients were studied. None adhered to immobilized CD36, ICAM-1, or thrombospondin, putative ligands for P. falciparum vascular cytoadherence, or umbilical vein endothelial cells, but all adhered to immobilized chondroitin sulphate A (CSA) and hyaluronic acid (HA), the receptors for adhesion of P. falciparum in the placenta. PvIRBC also adhered to fresh placental cells (N = 5). Pre-incubation with chondroitinase prevented PvIRBC adherence to CSA, and reduced binding to HA, whereas preincubation with hyaluronidase prevented adherence to HA, but did not reduce binding to CSA significantly. Pre-incubation of PvIRBC with soluble CSA and HA reduced binding to the immobilized receptors and prevented placental binding. PvIRBC adhesion was prevented by pre-incubation with trypsin, inhibited by heparin, and reduced by EGTA. Under laminar flow conditions the mean (SD) shear stress reducing maximum attachment by 50% was 0.06 (0.02) Pa but, having adhered, the PvIRBC could then resist detachment by stresses up to 5 Pa. At 37 °C adherence began approximately 16 hours after red cell invasion with maximal adherence at 30 hours. At 39 °C adherence began earlier and peaked at 24 hours.Adherence of P. vivax-infected erythrocytes to glycosaminoglycans may contribute to the pathogenesis of vivax malaria and lead to intrauterine growth retardation.

The Plasmodium vivax that was once prevalent in temperate climatic zones typically had an interval between primary infection and first relapse of 7-10 months, whereas in tropical areas P.vivax infections relapse frequently at intervals of 3-6 weeks. Defining the epidemiology of these two phenotypes from temporal patterns of illness in endemic areas is difficult or impossible, particularly if they overlap.A prospective open label comparison of chloroquine (CQ) alone versus CQ plus unobserved primaquine for either 5 days or 14 days was conducted in patients presenting with acute vivax malaria in Kolkata. Patients were followed for 15 months and primary and recurrent infections were genotyped using three polymorphic antigen and up to 8 microsatellite markers.151 patients were enrolled of whom 47 (31%) had subsequent recurrent infections. Recurrence proportions were similar in the three treatment groups. Parasite genotyping revealed discrete temporal patterns of recurrence allowing differentiation of probable relapse from newly acquired infections. This suggested that 32 of the 47 recurrences were probable relapses of which 22 (69%) were genetically homologous. The majority (81%) of probable relapses occurred within three months (16 homologous, 10 heterologous) and six genetically homologous relapses (19%) were of the long latency (8-10 month interval) phenotype.With long follow-up to assess temporal patterns of vivax malaria recurrence, genotyping of P.vivax can be used to assess relapse rates. A 14 day unobserved course of primaquine did not prevent relapse. Genotyping indicates that long latency P.vivax is prevalent in West Bengal, and that the first relapses after long latent periods are genetically homologous.Controlled-Trials.com ISRCTN14027467.

BACKGROUND: The artemisinin-based combination treatment (ACT) of dihydroartemisinin (DHA) and piperaquine (PQP) is a promising novel anti-malarial drug effective against multi-drug resistant falciparum malaria. The aim of this study was to show non-inferiority of DHA/PQP vs. artesunate-mefloquine (AS+MQ) in Asia. METHODS AND FINDINGS: This was an open-label, randomised, non-inferiority, 63-day follow-up study conducted in Thailand, Laos and India. Patients aged 3 months to 65 years with Plasmodium falciparum mono-infection or mixed infection were randomised with an allocation ratio of 2:1 to a fixed-dose DHA/PQP combination tablet (adults: 40 mg/320 mg; children: 20 mg/160 [DOSAGE ERROR CORRECTED] mg; n = 769) or loose combination of AS+MQ (AS: 50 mg, MQ: 250 mg; n = 381). The cumulative doses of study treatment over the 3 days were of about 6.75 mg/kg of DHA and 54 mg/kg of PQP and about 12 mg/kg of AS and 25 mg/kg of MQ. Doses were rounded up to the nearest half tablet. The primary endpoint was day-63 polymerase chain reaction (PCR) genotype-corrected cure rate. Results were 87.9% for DHA/PQP and 86.6% for AS+MQ in the intention-to-treat (ITT; 97.5% one-sided confidence interval, CI: >-2.87%), and 98.7% and 97.0%, respectively, in the per protocol population (97.5% CI: >-0.39%). No country effect was observed. Kaplan-Meier estimates of proportions of patients with new infections on day 63 (secondary endpoint) were significantly lower for DHA/PQP than AS+MQ: 22.7% versus 30.3% (p = 0.0042; ITT). Overall gametocyte prevalence (days 7 to 63; secondary endpoint), measured as person-gametocyte-weeks, was significantly higher for DHA/PQP than AS+MQ (10.15% versus 4.88%; p = 0.003; ITT). Fifteen serious adverse events were reported, 12 (1.6%) in DHA/PQP and three (0.8%) in AS+MQ, among which six (0.8%) were considered related to DHA/PQP and three (0.8%) to AS+MQ. CONCLUSIONS: DHA/PQP was a highly efficacious drug for P. falciparum malaria in areas where multidrug parasites are prevalent. The DHA/PQP combination can play an important role in the first-line treatment of uncomplicated falciparum malaria. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN81306618.