Single-Dose Tafenoquine to Prevent Relapse of Plasmodium vivax Malaria.

BACKGROUND: Treatment of Plasmodium vivax malaria requires the clearing of asexual parasites, but relapse can be prevented only if dormant hypnozoites are cleared from the liver (a treatment termed "radical cure"). Tafenoquine is a single-dose 8-aminoquinoline that has recently been registered for the radical cure of P. vivax. METHODS: This multicenter, double-blind, double-dummy, parallel group, randomized, placebo-controlled trial was conducted in Ethiopia, Peru, Brazil, Cambodia, Thailand, and the Philippines. We enrolled 522 patients with microscopically confirmed P. vivax infection (>100 to <100,000 parasites per microliter) and normal glucose-6-phosphate dehydrogenase (G6PD) activity (with normal activity defined as ≥70% of the median value determined at each trial site among 36 healthy male volunteers who were otherwise not involved in the trial). All patients received a 3-day course of chloroquine (total dose of 1500 mg). In addition, patients were assigned to receive a single 300-mg dose of tafenoquine on day 1 or 2 (260 patients), placebo (133 patients), or a 15-mg dose of primaquine once daily for 14 days (129 patients). The primary outcome was the Kaplan-Meier estimated percentage of patients who were free from recurrence at 6 months, defined as P. vivax clearance without recurrent parasitemia. RESULTS: In the intention-to-treat population, the percentage of patients who were free from recurrence at 6 months was 62.4% in the tafenoquine group (95% confidence interval [CI], 54.9 to 69.0), 27.7% in the placebo group (95% CI, 19.6 to 36.6), and 69.6% in the primaquine group (95% CI, 60.2 to 77.1). The hazard ratio for the risk of recurrence was 0.30 (95% CI, 0.22 to 0.40) with tafenoquine as compared with placebo (P<0.001) and 0.26 (95% CI, 0.18 to 0.39) with primaquine as compared with placebo (P<0.001). Tafenoquine was associated with asymptomatic declines in hemoglobin levels, which resolved without intervention. CONCLUSIONS: Single-dose tafenoquine resulted in a significantly lower risk of P. vivax recurrence than placebo in patients with phenotypically normal G6PD activity. (Funded by GlaxoSmithKline and Medicines for Malaria Venture; DETECTIVE ClinicalTrials.gov number, NCT01376167 .).

Correction: Quantification of glucose-6-phosphate dehydrogenase activity by spectrophotometry: A systematic review and meta-analysis.

[This corrects the article DOI: 10.1371/journal.pmed.1003084.].

Quantification of glucose-6-phosphate dehydrogenase activity by spectrophotometry: A systematic review and meta-analysis.

BACKGROUND: The radical cure of Plasmodium vivax and P. ovale requires treatment with primaquine or tafenoquine to clear dormant liver stages. Either drug can induce haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, necessitating screening. The reference diagnostic method for G6PD activity is ultraviolet (UV) spectrophotometry; however, a universal G6PD activity threshold above which these drugs can be safely administered is not yet defined. Our study aimed to quantify assay-based variation in G6PD spectrophotometry and to explore the diagnostic implications of applying a universal threshold. METHODS AND FINDINGS: Individual-level data were pooled from studies that used G6PD spectrophotometry. Studies were identified via PubMed search (25 April 2018) and unpublished contributions from contacted authors (PROSPERO: CRD42019121414). Studies were excluded if they assessed only individuals with known haematological conditions, were family studies, or had insufficient details. Studies of malaria patients were included but analysed separately. Included studies were assessed for risk of bias using an adapted form of the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. Repeatability and intra- and interlaboratory variability in G6PD activity measurements were compared between studies and pooled across the dataset. A universal threshold for G6PD deficiency was derived, and its diagnostic performance was compared to site-specific thresholds. Study participants (n = 15,811) were aged between 0 and 86 years, and 44.4% (7,083) were women. Median (range) activity of G6PD normal (G6PDn) control samples was 10.0 U/g Hb (6.3-14.0) for the Trinity assay and 8.3 U/g Hb (6.8-15.6) for the Randox assay. G6PD activity distributions varied significantly between studies. For the 13 studies that used the Trinity assay, the adjusted male median (AMM; a standardised metric of 100% G6PD activity) varied from 5.7 to 12.6 U/g Hb (p < 0.001). Assay precision varied between laboratories, as assessed by variance in control measurements (from 0.1 to 1.5 U/g Hb; p < 0.001) and study-wise mean coefficient of variation (CV) of replicate measures (from 1.6% to 14.9%; p < 0.001). A universal threshold of 100% G6PD activity was defined as 9.4 U/g Hb, yielding diagnostic thresholds of 6.6 U/g Hb (70% activity) and 2.8 U/g Hb (30% activity). These thresholds diagnosed individuals with less than 30% G6PD activity with study-wise sensitivity from 89% (95% CI: 81%-94%) to 100% (95% CI: 96%-100%) and specificity from 96% (95% CI: 89%-99%) to 100% (100%-100%). However, when considering intermediate deficiency (<70% G6PD activity), sensitivity fell to a minimum of 64% (95% CI: 52%-75%) and specificity to 35% (95% CI: 24%-46%). Our ability to identify underlying factors associated with study-level heterogeneity was limited by the lack of availability of covariate data and diverse study contexts and methodologies. CONCLUSIONS: Our findings indicate that there is substantial variation in G6PD measurements by spectrophotometry between sites. This is likely due to variability in laboratory methods, with possible contribution of unmeasured population factors. While an assay-specific, universal quantitative threshold offers robust diagnosis at the 30% level, inter-study variability impedes performance of universal thresholds at the 70% level. Caution is advised in comparing findings based on absolute G6PD activity measurements across studies. Novel handheld quantitative G6PD diagnostics may allow greater standardisation in the future.

Pharmacogenetic assessment of tafenoquine efficacy in patients with Plasmodium vivax malaria.

Plasmodium vivax has the largest geographic range of human malaria species and is challenging to manage and eradicate due to its ability to establish a dormant liver stage, the hypnozoite, which can reactivate leading to relapse. Until recently, the only treatment approved to kill hypnozoites was the 8-aminoquinoline, primaquine, requiring daily treatment for 14 days. Tafenoquine, an 8-aminoquinoline single-dose treatment with activity against P. vivax hypnozoites, has recently been approved by the US Food and Drug Administration and Australian Therapeutic Goods Administration for the radical cure of P. vivax malaria in patients 16 years and older. We conducted an exploratory pharmacogenetic analysis (GSK Study 208099) to assess the role of host genome-wide variation on tafenoquine efficacy in patients with P. vivax malaria using data from three GSK clinical trials, GATHER and DETECTIVE Part 1 and Part 2. Recurrence-free efficacy at 6 and 4 months and time to recurrence up to 6 months postdosing were analyzed in 438 P. vivax malaria patients treated with tafenoquine. Among the approximately 10.6 million host genetic variants analyzed, two signals reached genome-wide significance (P value ≤ 5 × 10). rs62103056, and variants in a chromosome 12 intergenic region, were associated with recurrence-free efficacy at 6 and 4 months, respectively. Neither of the signals has an obvious biological rationale and would need replication in an independent population. This is the first genome-wide association study to evaluate genetic influence on response to tafenoquine in P. vivax malaria.

Performance of the Access Bio/CareStart rapid diagnostic test for the detection of glucose-6-phosphate dehydrogenase deficiency: A systematic review and meta-analysis.

BACKGROUND: To reduce the risk of drug-induced haemolysis, all patients should be tested for glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd) prior to prescribing primaquine (PQ)-based radical cure for the treatment of vivax malaria. This systematic review and individual patient meta-analysis assessed the utility of a qualitative lateral flow assay from Access Bio/CareStart (Somerset, NJ) (CareStart Screening test for G6PD deficiency) for the diagnosis of G6PDd compared to the gold standard spectrophotometry (International Prospective Register of Systematic Reviews [PROSPERO]: CRD42019110994). METHODS AND FINDINGS: Articles published on PubMed between 1 January 2011 and 27 September 2019 were screened. Articles reporting performance of the standard CSG from venous or capillary blood samples collected prospectively and considering spectrophotometry as gold standard (using kits from Trinity Biotech PLC, Wicklow, Ireland) were included. Authors of articles fulfilling the inclusion criteria were contacted to contribute anonymized individual data. Minimal data requested were sex of the participant, CSG result, spectrophotometry result in U/gHb, and haemoglobin (Hb) reading. The adjusted male median (AMM) was calculated per site and defined as 100% G6PD activity. G6PDd was defined as an enzyme activity of less than 30%. Pooled estimates for sensitivity and specificity, unconditional negative predictive value (NPV), positive likelihood ratio (LR+), and negative likelihood ratio (LR-) were calculated comparing CSG results to spectrophotometry using a random-effects bivariate model. Of 11 eligible published articles, individual data were available from 8 studies, 6 from Southeast Asia, 1 from Africa, and 1 from the Americas. A total of 5,815 individual participant data (IPD) were available, of which 5,777 results (99.3%) were considered for analysis, including data from 3,095 (53.6%) females. Overall, the CSG had a pooled sensitivity of 0.96 (95% CI 0.90-0.99) and a specificity of 0.95 (95% CI 0.92-0.96). When the prevalence of G6PDd was varied from 5% to 30%, the unconditional NPV was 0.99 (95% CI 0.94-1.00), with an LR+ and an LR- of 18.23 (95% CI 13.04-25.48) and 0.05 (95% CI 0.02-0.12), respectively. Performance was significantly better in males compared to females (p = 0.027) but did not differ significantly between samples collected from capillary or venous blood (p = 0.547). Limitations of the study include the lack of wide geographical representation of the included data and that the CSG results were generated under research conditions, and therefore may not reflect performance in routine settings. CONCLUSIONS: The CSG performed well at the 30% threshold. Its high NPV suggests that the test is suitable to guide PQ treatment, and the high LR+ and low LR- render the test suitable to confirm and exclude G6PDd. Further operational studies are needed to confirm the utility of the test in remote endemic settings.

A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms.

BACKGROUND: Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. METHODS: We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. RESULTS: We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. CONCLUSIONS: No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).

Use of mobile technology-based participatory mapping approaches to geolocate health facility attendees for disease surveillance in low resource settings.

BACKGROUND: Identifying fine-scale spatial patterns of disease is essential for effective disease control and elimination programmes. In low resource areas without formal addresses, novel strategies are needed to locate residences of individuals attending health facilities in order to efficiently map disease patterns. We aimed to assess the use of Android tablet-based applications containing high resolution maps to geolocate individual residences, whilst comparing the functionality, usability and cost of three software packages designed to collect spatial information. RESULTS: Using Open Data Kit GeoODK, we designed and piloted an electronic questionnaire for rolling cross sectional surveys of health facility attendees as part of a malaria elimination campaign in two predominantly rural sites in the Rizal, Palawan, the Philippines and Kulon Progo Regency, Yogyakarta, Indonesia. The majority of health workers were able to use the tablets effectively, including locating participant households on electronic maps. For all households sampled (n = 603), health facility workers were able to retrospectively find the participant household using the Global Positioning System (GPS) coordinates and data collected by tablet computers. Median distance between actual house locations and points collected on the tablet was 116 m (IQR 42-368) in Rizal and 493 m (IQR 258-886) in Kulon Progo Regency. Accuracy varied between health facilities and decreased in less populated areas with fewer prominent landmarks. CONCLUSIONS: Results demonstrate the utility of this approach to develop real-time high-resolution maps of disease in resource-poor environments. This method provides an attractive approach for quickly obtaining spatial information on individuals presenting at health facilities in resource poor areas where formal addresses are unavailable and internet connectivity is limited. Further research is needed on how to integrate these with other health data management systems and implement in a wider operational context.

The challenges of introducing routine G6PD testing into radical cure: a workshop report.

The only currently available drug that effectively removes malaria hypnozoites from the human host is primaquine. The use of 8-aminoquinolines is hampered by haemolytic side effects in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. Recently a number of qualitative and a quantitative rapid diagnostic test (RDT) format have been developed that provide an alternative to the current standard G6PD activity assays. The WHO has recently recommended routine testing of G6PD status prior to primaquine radical cure whenever possible. A workshop was held in the Philippines in early 2015 to discuss key challenges and knowledge gaps that hinder the introduction of routine G6PD testing. Two point-of-care (PoC) test formats for the measurement of G6PD activity are currently available: qualitative tests comparable to malaria RDT as well as biosensors that provide a quantitative reading. Qualitative G6PD PoC tests provide a binomial test result, are easy to use and some products are comparable in price to the widely used fluorescent spot test. Qualitative test results can accurately classify hemizygous males, heterozygous females, but may misclassify females with intermediate G6PD activity. Biosensors provide a more complex quantitative readout and are better suited to identify heterozygous females. While associated with higher costs per sample tested biosensors have the potential for broader use in other scenarios where knowledge of G6PD activity is relevant as well. The introduction of routine G6PD testing is associated with additional costs on top of routine treatment that will vary by setting and will need to be assessed prior to test introduction. Reliable G6PD PoC tests have the potential to play an essential role in future malaria elimination programmes, however require an improved understanding on how to best integrate routine G6PD testing into different health settings.

Protocol for spatial prediction of soil transmitted helminth prevalence in the Western Pacific region using a meta-analytical approach.

BACKGROUND: Soil transmitted helminth (STH) infections are estimated to impact 24% of the world's population and are responsible for chronic and debilitating morbidity. Disadvantaged communities are among the worst affected and are further marginalized as infection prevalence fuels the poverty cycle. Ambitious targets have been set to eliminate STH infections, but accurate epidemiological data will be required to inform appropriate interventions. This paper details the protocol for an analysis that aims to produce spatial prediction mapping of STH prevalence in the Western Pacific Region (WPR). METHODS: The protocol follows the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocol (PRISMA-P) guidelines. The study design will combine the principles of systematic review, meta-analysis, and geospatial analysis. Systematic searches will be undertaken in PubMed, Scopus, ProQuest, Embase, and Web of Science for studies undertaken post 2000, to identify surveys that enable the prevalence of human STH infection within the WPR to be calculated. Covariate data for multivariable analysis will be obtained from publicly accessible sources. Survey data will be geolocated, and STH prevalence and covariates will be linked to produce a spatially referenced dataset for analysis. Bayesian model-based geostatistics will be used to generate spatially continuous estimates of STH prevalence mapped to a resolution of 1 km2. A separate geospatial model will be constructed for each STH species. Predictions of prevalence will be made for unsampled locations and maps will be overlaid for each STH species to obtain co-endemicity maps. DISCUSSION: This protocol facilitates study replication and may be applied to other infectious diseases or alternate geographies. Results of the subsequent analysis will identify geographies with high STH prevalence's and can be used to inform resource allocation in combating this neglected tropical disease. TRIAL REGISTRATION: Open Science Framework: osf.io/qmxcj.

First malaria in pregnancy followed in Philippine real-world setting: proof-of-concept of probabilistic record linkage between disease surveillance and hospital administrative data.

BACKGROUND: Although the Philippines targets malaria elimination by 2030, it remains to be a disease that causes considerable morbidity in provinces that report malaria. Pregnant women residing in endemic areas are a vulnerable population, because in addition to the risk of developing severe malaria, their pregnancy is not followed through, and the outcome of their pregnancy is unknown. This study determined the utility of real-world data integrated with disease surveillance data set as real-world evidence of pregnancy and delivery outcomes in areas endemic for malaria in the Philippines. METHODS: For the period of 2015 to 2019, electronic data sets of malaria surveillance data and Ospital ng Palawan hospital admission log of pregnant women residing in the four selected barangays of Rizal, Palawan were merged using probabilistic linkage. The source data for record linkage were first and last names, birth date, and address as the mutual variable. The data used for characteristics of the pregnant women from the hospital data set were admission date, discharge date, admitting and final diagnosis and body weight on admission. From the malaria surveillance data these were date of consultation, and malaria parasite species. The Levenshtein distance formula was used for a fuzzy string-matching algorithm. Chi-square test, and Mann-Whitney U test were used to compare the means of the two data sets. RESULTS: The prevalence of pregnant women admitted to the tertiary referral hospital, Ospital ng Palawan, was estimated to be 8.34/100 overall, and 11.64/100 from the four study barangays; that of malaria during pregnancy patients was 3.45/100 and 2.64/100, respectively. There was only one true-positive matched case from 238 women from the hospital and 54 women from the surveillance data sets. The overall Levenshstein score was 97.7; for non-matched cases, the mean overall score was 36.6 (35.6-37.7). The matched case was a minor who was hospitalized for severe malaria. The outcome of her pregnancy was detected from neither data set but from village-based records. CONCLUSIONS: This proof-of-concept study demonstrated that probabilistic record linkage could match real-world data in the Philippines with further validation required. The study underscored the need for more integrated and comprehensive database to monitor disease intervention impact on pregnancy and its outcome in the Philippines.

Risk mapping and socio-ecological drivers of soil-transmitted helminth infections in the Philippines: a spatial modelling study.

Background: The Philippines reports a high prevalence of soil-transmitted helminth (STH) infections despite the implementation of nationwide mass drug administration since 2006. The spatial variation of STH infections in the Philippines was last described using the 2005-2007 national STH and schistosomiasis survey. This study aimed to identify sociodemographic and environmental factors that drive STH transmission and predict high-risk areas in the Philippines. Methods: Epidemiological data on STH for students aged 5-16 years were obtained from the 2015 Philippines National Prevalence survey, while environmental data were extracted from satellite images and publicly available sources. Model-based geostatistics, implemented in a Bayesian framework, was used to identify sociodemographic and environmental correlates and predict high-risk areas for STH across the Philippines. The best-fitting model with the lowest deviance information criterion (DIC) was used to interpret the findings of the model and predict STH infection risk for the entire country. Risk maps were developed for each STH infection using the posterior means derived from the model. Findings: The prevalence of Ascaris lumbricoides (20.0%) and Trichuris trichiura (29.3%) was higher in the Visayas Island than in the Luzon and Mindanao Islands. Hookworm prevalence was highest in Mindanao Island (1.3%). Risk of A. lumbricoides was positively associated with males (odds ratio [OR]: 1.197; 97.5% Credible Interval [CrI]: 1.114, 1.286) and temperature (OR: 1.148; 97.5% CrI: 1.033, 1.291), while normalized difference vegetation index (OR: 0.354; 97.5% CrI: 0.138, 0.930) and soil pH (OR: 0.606; 97.5% CrI: 0.338, 0.949) were negatively associated with the transmission. T. trichiura risk was positively associated with males (OR: 1.261; 97.5% CrI: 1.173, 1.341), temperature (OR: 1.153; 97.5% CrI: 1.001, 1.301), and rainfall (OR: 1.004; 97.5% CrI: 1.011, 1.069). Hookworm risk was positively associated with males (OR: 2.142; 97.5% CrI: 1.537, 2.998), while children aged ≤12 years (OR: 0.435; 97.5% CrI: 0.252, 0.753) had a negative association with risk compared to those over 12 years. Focal areas of high risk were identified for A. lumbricoides and T. trichiura in the Visayas Island, and hookworm in the Mindanao Island. Interpretation: The spatial distribution of all three STH infections has considerably decreased since a previous national risk-mapping exercise. The high-risk areas identified in the study can be used to strategically target deworming and health education activities to further reduce the burden of STH and support progress toward elimination. Funding: The Australian Centre for the Control and Elimination of Neglected Tropical Diseases and the Australian National Health and Medical Research Council.

Review of key knowledge gaps in glucose-6-phosphate dehydrogenase deficiency detection with regard to the safe clinical deployment of 8-aminoquinoline treatment regimens: a workshop report.

The diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency is a crucial aspect in the current phases of malaria control and elimination, which will require the wider use of 8-aminoquinolines for both reducing Plasmodium falciparum transmission and achieving the radical cure of Plasmodium vivax. 8-aminoquinolines, such as primaquine, can induce severe haemolysis in G6PD-deficient individuals, potentially creating significant morbidity and undermining confidence in 8-aminoquinoline prescription. On the other hand, erring on the side of safety and excluding large numbers of people with unconfirmed G6PD deficiency from treatment with 8-aminoquinolines will diminish the impact of these drugs. Estimating the remaining G6PD enzyme activity is the most direct, accessible, and reliable assessment of the phenotype and remains the gold standard for the diagnosis of patients who could be harmed by the administration of primaquine. Genotyping seems an unambiguous technique, but its use is limited by cost and the large range of recognized G6PD genotypes. A number of enzyme activity assays diagnose G6PD deficiency, but they require a cold chain, specialized equipment, and laboratory skills. These assays are impractical for care delivery where most patients with malaria live. Improvements to the diagnosis of G6PD deficiency are required for the broader and safer use of 8-aminoquinolines to kill hypnozoites, while lower doses of primaquine may be safely used to kill gametocytes without testing. The discussions and conclusions of a workshop conducted in Incheon, Korea in May 2012 to review key knowledge gaps in G6PD deficiency are reported here.

Analytical approaches for antimalarial antibody responses to confirm historical and recent malaria transmission: an example from the Philippines.

Background: Assessing the status of malaria transmission in endemic areas becomes increasingly challenging as countries approach elimination. Serology can provide robust estimates of malaria transmission intensities, and multiplex serological assays allow for simultaneous assessment of markers of recent and historical malaria exposure. Methods: Here, we evaluated different statistical and machine learning methods for analyzing multiplex malaria-specific antibody response data to classify recent and historical exposure to Plasmodium falciparum and Plasmodium vivax. To assess these methods, we utilized samples from a health-facility based survey (n = 9132) in the Philippines, where we quantified antibody responses against 8 P. falciparum and 6 P. vivax-specific antigens from 3 sites with varying transmission intensity. Findings: Measurements of antibody responses and seroprevalence were consistent with the 3 sites' known endemicity status. Among the models tested, a machine learning (ML) approach (Random Forest model) using 4 serological markers (PfGLURP R2, Etramp5.Ag1, GEXP18, and PfMSP119) gave better predictions for P. falciparum recent infection in Palawan (AUC: 0.9591, CI 0.9497-0.9684) than individual antigen seropositivity. Although the ML approach did not improve P. vivax infection predictions, ML classifications confirmed the absence of recent exposure to P. falciparum and P. vivax in both Occidental Mindoro and Bataan. For predicting historical P. falciparum and P. vivax transmission, seroprevalence and seroconversion rates based on cumulative exposure markers AMA1 and MSP119 showed reliable trends in the 3 sites. Interpretation: Our study emphasizes the utility of serological markers in predicting recent and historical exposure in a sub-national elimination setting, and also highlights the potential use of machine learning models using multiplex antibody responses to improve assessment of the malaria transmission status of countries aiming for elimination. This work also provides baseline antibody data for monitoring risk in malaria-endemic areas in the Philippines. Funding: Newton Fund, Philippine Council for Health Research and Development, UK Medical Research Council.

Prevalence and temporal changes of mutations linked to antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Palawan, Philippines.

OBJECTIVE: This study provides 2016 data on the prevalence of key single nucleotide polymorphisms (SNPs) associated with antimalarial drug resistance in Palawan, Philippines. Findings were combined with historical data to model temporal changes in the prevalence of these SNPs in Plasmodium isolates. METHODS: Plasmodium isolates were genotyped using drug resistance markers pfmdr1, pfcrt, pfdhfr, pfdhps, kelch-13, pvmdr1, pvdhfr, and pvdhps. Temporal trends in the probability of mutations were estimated as a function of time using a binomial generalised linear model. RESULTS: All samples sequenced for Plasmodium falciparum chloroquine markers pfmdr1 and pfcrt had wild-type alleles. Varying mutation patterns were observed for the sulphadoxine/pyrimethamine markers pfdhps and pfdhfr; complete quintuplet mutations were not found. No SNPs were observed for the artemisinin marker kelch-13. For Plasmodium vivax, differing patterns were detected for pvmdr1, pvdhfr, and pvdhps. CONCLUSIONS: The study findings suggest that the current drugs remain effective and that there is limited importation and establishment of resistant parasites in the area. Clear temporal trends were recognised, with prominent decreases in the proportions of pfcrt and pfmdr mutations detected within the past 15 years, consistent with a change in antimalarial drug policy. Continuous surveillance of antimalarial drug resistance is important to support malaria elimination efforts.

An open dataset of Plasmodium vivax genome variation in 1,895 worldwide samples.

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.

A molecular barcode and web-based data analysis tool to identify imported Plasmodium vivax malaria.

Traditionally, patient travel history has been used to distinguish imported from autochthonous malaria cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative method to identify, and map imported cases. Using machine learning approaches incorporating hierarchical fixation index and decision tree analyses applied to 799 P. vivax genomes from 21 countries, we identified 33-SNP, 50-SNP and 55-SNP barcodes (GEO33, GEO50 and GEO55), with high capacity to predict the infection's country of origin. The Matthews correlation coefficient (MCC) for an existing, commonly applied 38-SNP barcode (BR38) exceeded 0.80 in 62% countries. The GEO panels outperformed BR38, with median MCCs > 0.80 in 90% countries at GEO33, and 95% at GEO50 and GEO55. An online, open-access, likelihood-based classifier framework was established to support data analysis (vivaxGEN-geo). The SNP selection and classifier methods can be readily amended for other use cases to support malaria control programs.

Report of the 2018 annual meeting of the Asia Pacific Malaria Elimination Network Vector Control Working Group: harnessing skills and knowledge for malaria elimination across the Asia Pacific.

The 2018 Asia Pacific Malaria Elimination Network's Vector Control Working Group (APMEN VCWG) annual meeting took place 3-5 September 2018 in Bangkok, Thailand. It was designed to be a forum for entomology and public health specialists from APMEN country programmes (over 90 participants from 30 countries) to discuss current progress and challenges related to planning, implementing, and sustaining effective vector control (VC) strategies for malaria elimination across the region, and to suggest practical and applicable solutions to these moving forward. The meeting was organised as a joint collaboration between the VCWG host institution-Faculty of Tropical Medicine, Mahidol University, Thailand-and leading partner institutions within the VCWG: Malaria Consortium and the Malaria Elimination Initiative at the University of California, San Francisco, Global Health Group (UCSF Global Health Group), under the leadership of the APMEN Director and VCWG Co-Chairs from ministries of health in Malaysia and India. This report provides an introduction to the role and nature of the VCWG, highlights key themes and topics presented and discussed at the meeting, and outlines the future objectives and focal areas for the VCWG and APMEN at large.

The seasonal dynamics and biting behavior of potential Anopheles vectors of Plasmodium knowlesi in Palawan, Philippines.

BACKGROUND: A small number of human cases of the zoonotic malaria Plasmodium knowlesi have been reported in Palawan Island, the Philippines. Identification of potential vector species and their bionomics is crucial for understanding human exposure risk in this setting. Here, we combined longitudinal surveillance with a trap-evaluation study to address knowledge gaps about the ecology and potential for zoonotic spillover of this macaque malaria in Palawan Island. METHODS: The abundance, diversity and biting behavior of human-biting Anopheles mosquitoes were assessed through monthly outdoor human landing catches (HLC) in three ecotypes representing different land use (forest edge, forest and agricultural area) across 8 months. Additionally, the host preference and biting activity of potential Anopheles vectors were assessed through comparison of their abundance and capture time in traps baited with humans (HLC, human-baited electrocuting net-HEN) or macaques (monkey-baited trap-MBT, monkey-baited electrocuting net-MEN). All female Anopheles mosquitoes were tested for the presence of Plasmodium parasites by PCR. RESULTS: Previously incriminated vectors Anopheles balabacensis and An. flavirostris accounted for > 95% of anophelines caught in longitudinal surveillance. However, human biting densities were relatively low (An. balabacensis: 0.34-1.20 per night, An. flavirostris: 0-2 bites per night). Biting densities of An. balabacensis were highest in the forest edge, while An. flavirostris was most abundant in the agricultural area. The abundance of An. balabacensis and An. flavirostris was significantly higher in HLC than in MBT. None of the 357 female Anopheles mosquitoes tested for Plasmodium infection were positive. CONCLUSIONS: The relatively low density and lack of malaria infection in Anopheles mosquitoes sampled here indicates that exposure to P. knowlesi in this setting is considerably lower than in neighboring countries (i.e. Malaysia), where it is now the primary cause of malaria in humans. Although anophelines had lower abundance in MBTs than in HLCs, An. balabacensis and An. flavirostris were caught by both methods, suggesting they could act as bridge vectors between humans and macaques. These species bite primarily outdoors during the early evening, confirming that insecticide-treated nets are unlikely to provide protection against P. knowlesi vectors.

Enhanced Health Facility Surveys to Support Malaria Control and Elimination across Different Transmission Settings in the Philippines.

Following substantial progress in malaria control in the Philippines, new surveillance approaches are needed to identify and target residual malaria transmission. This study evaluated an enhanced surveillance approach using rolling cross-sectional surveys of all health facility attendees augmented with molecular diagnostics and geolocation. Facility surveys were carried out in three sites representing different transmission intensities: Morong, Bataan (pre-elimination), Abra de Ilog, Occidental Mindoro (stable medium risk), and Rizal, Palawan (high risk, control). Only one rapid diagnostic test (RDT)-positive infection and no PCR confirmed infections were found in Bataan and Occidental Mindoro, suggesting the absence of transmission. In Palawan, the inclusion of all health facility attendees, regardless of symptoms, and use of molecular diagnostics identified 313 infected individuals in addition to 300 cases identified by routine screening of febrile patients with the RDT or microscopy. Of these, the majority (313/613) were subpatent infections and only detected using molecular methods. Simultaneous collection of GPS coordinates on tablet-based applications allowed real-time mapping of malaria infections. Risk factor analysis showed higher risks in children and indigenous groups, with bed net use having a protective effect. Subpatent infections were more common in men and older age-groups. Overall, malaria risks were not associated with participants' classification, and some of the non-patient clinic attendees reported febrile illnesses (1.9%, 26/1,369), despite not seeking treatment, highlighting the widespread distribution of infection in communities. Together, these data illustrate the utility of health facility-based surveys to augment surveillance data to increase the probability of detecting infections in the wider community.

Eco-bio-social determinants of dengue vector breeding: a multicountry study in urban and periurban Asia.

OBJECTIVE: To study dengue vector breeding patterns under a variety of conditions in public and private spaces; to explore the ecological, biological and social (eco-bio-social) factors involved in vector breeding and viral transmission, and to define the main implications for vector control. METHODS: In each of six Asian cities or periurban areas, a team randomly selected urban clusters for conducting standardized household surveys, neighbourhood background surveys and entomological surveys. They collected information on vector breeding sites, people's knowledge, attitudes and practices surrounding dengue, and the characteristics of the study areas. All premises were inspected; larval indices were used to quantify vector breeding sites, and pupal counts were used to identify productive water container types and as a proxy measure for adult vector abundance. FINDINGS: The most productive vector breeding sites were outdoor water containers, particularly if uncovered, beneath shrubbery and unused for at least one week. Peridomestic and intradomestic areas were much more important for pupal production than commercial and public spaces other than schools and religious facilities. A complex but non-significant association was found between water supply and pupal counts, and lack of waste disposal services was associated with higher vector abundance in only one site. Greater knowledge about dengue and its transmission was associated with lower mosquito breeding and production. Vector control measures (mainly larviciding in one site) substantially reduced larval and pupal counts and "pushed" mosquito breeding to alternative containers. CONCLUSION: Vector breeding and the production of adult Aedes aegypti are influenced by a complex interplay of factors. Thus, to achieve effective vector management, a public health response beyond routine larviciding or focal spraying is essential.