Ex vivo and in vitro antiplasmodial activity and toxicity of Caesalpinia decapetala (Roth) Alston (Fabaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is among the most prevalent and devastating parasitic diseases globally with most cases reported in Sub-Saharan Africa. One of the major reasons for the high malaria prevalence is the ever-increasing emergence of resistant strains of malaria-causing parasites to the currently used antimalarial drugs. This, therefore, calls for the search for antimalarial compounds with alternative modes of action. Plants used in traditional medicine for the treatment of malaria offer possible sources of such compounds. Caesalpinia decapetala has been used traditionally for the treatment of various diseases including malaria. However, the antiplasmodial activity of the plant has never been reported. AIM OF THE STUDY: To determine the ex vivo and in vitro antiplasmodial activities of the extracts of the roots, stem bark and leaves of Caesalpinia decapetala. METHODOLOGY: The roots, stem bark and leaves of Caesalpinia decapetala (Roth) Alston (Caesalpiniaceae) were collected and air-dried under a shade then extracted consecutively with dichloromethane and methanol (1:1 (v/v) (4 × 0.8 L). The extracts were tested for antiplasmodial activities against four strains of Plasmodium falciparum (W2, DD2, 3D7, and D6) and fresh P. falciparum field isolates using the SYBR green I assay. The mean fifty percent inhibition concentration (IC50) was determined for each assay. An acute oral toxicity test was done based on the Organization for Economic Cooperation and Development (OECD 425) guidelines using Swiss albino mice. RESULTS: The leaves and stem bark extracts showed good antiplasmodial activities with IC50 values of 4.54 and 4.86 µg/mL, respectively, when tested against the fresh field isolates ex vivo. Similarly, the roots extract showed an IC50 value of 6.49 µg/mL when tested against field isolates ex vivo. The roots extract showed the highest antiplasmodial activities among the samples when tested against W2 (IC50 = 6.12 µg/mL), DD2 (IC50 = 8.17 µg/mL), and D6 (IC50 = 16.02 µg/mL) strains of P. falciparum whereas the leaves showed the highest activity (IC50 = 9.3 µg/mL) when tested against the 3D7 strain of P. falciparum. No mortality was observed for the mice treated with 2000 mg/kg of the leaves and stem bark extracts. The mouse treated with 2000 mg/kg of the roots extracts regained weight by day 12 of the observation period. CONCLUSION: Caesalpinia decapetala has the potential to suppress the growth of P. falciparum thereby contributing to combating the recurrent emergence of antimalarial drug resistance.

In vitro and in vivo anti-eczema effect of Artemisia annua aqueous extract and its component profiling.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. belongs to the Asteraceae family and has a long history of clinical application in China. It has been widely used for centuries to treat fever, malaria, jaundice and some skin diseases (such as scabies and sores). Modern pharmacological studies have shown that it has anti-inflammatory, immunomodulatory, antimalarial and antibacterial effects. AIM OF STUDY: This study aimed to investigate the anti-eczema effect of A. annua aqueous extract (AAE), profile its potential bioactive components and try to explore its possible underlying mechanisms. MATERIALS AND METHODS: The MTT assay was employed to assess the cytotoxicity of AAE. The anti-eczema effect of AAE was evaluated using both an in vitro 3D epidermal inflammation model and an in vivo guinea pig itching model. The bioactive components of AAE were characterized by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry coupled with the UNIFI platform. RESULTS: In this study, we found that AAE is safe for primary human skin keratinocytes at concentrations ranging from 31.3 µg/mL to 250 µg/mL. Further investigations indicate that AAE can increase the itching threshold, inhibit the expression of the inflammatory cytokine TSLP, and promote the expression of FLG mRNA. Additionally, the utilization of UPLC-QTOF/MS and UNIFI platform enabled us to identify 61 potential bioactive components of AAE, with sesquiterpenes and phenolic acids being the most abundant components. CONCLUSIONS: In this study, the anti-inflammatory and anti-itch effects of the A. annua extract were revealed, along with sesquiterpenes and phenolic acids were identified as potential bioactive components according to literature. The AAE extract holds potential for utilization in the treatment of eczema.

Genomic complexity of parasites and vectors challenge malaria control in Southeast Asia.

Southeast Asia is a uniquely complex region of malaria transmission that maintains an astounding level of species diversity among potential malaria vectors and also generates drug resistant and quickly diverging populations of malaria parasites. All 5 human malaria species circulate in Southeast Asia with over 50 Anopheles species that vary in their ability to transmit these pathogens. The intricate relationships of these parasites and vectors is not well understood. Human activity in Southeast Asian countries has created an increasingly fragmented landscape, bringing humans and mosquitoes into more frequent contact, sustaining malaria transmission in a region where few control tools are effective. Genomic shifts at the species, population, and individual level in parasites and vectors introduce variation that has produced drug- and insecticide-resistance. The goal of this review is to highlight genomic studies of Southeast Asian malaria parasites and vectors that demonstrate how diversity in these organisms present unique challenges and opportunities for global malaria control and eradication efforts.

The voltage-gated sodium channel, para, limits Anopheles coluzzii vector competence in a microbiota dependent manner.

The voltage-gated sodium channel, para, is a target of DDT and pyrethroid class insecticides. Single nucleotide mutations in para, called knockdown resistant or kdr, which contribute to resistance against DDT and pyrethroid insecticides, have been correlated with increased susceptibility of Anopheles to the human malaria parasite Plasmodium falciparum. However, a direct role of para activity on Plasmodium infection has not yet been established. Here, using RNA-mediated silencing, we provide in vivo direct evidence for the requirement of wild-type (wt) para function for insecticide activity of deltamethrin. Depletion of wt para, which is susceptible to insecticide, causes deltamethrin tolerance, indicating that insecticide-resistant kdr alleles are likely phenocopies of loss of para function. We then show that normal para activity in An. coluzzii limits Plasmodium infection prevalence for both P. falciparum and P. berghei. A transcriptomic analysis revealed that para activity does not modulate the expression of immune genes. However, loss of para function led to enteric dysbiosis with a significant increase in the total bacterial abundance, and we show that para function limiting Plasmodium infection is microbiota dependent. In the context of the bidirectional "enteric microbiota-brain" axis studied in mammals, these results pave the way for studying whether the activity of the nervous system could control Anopheles vector competence.

Randomized field trial to assess the safety and efficacy of dihydroartemisinin-piperaquine for seasonal malaria chemoprevention in school-aged children in Bandiagara, Mali.

BACKGROUND: Owing to the increased cases of malaria in older children, the World Health Organization has recently recommended extending seasonal malaria chemoprevention (SMC) to children >5 years of age and using other effective drugs for malaria. We herein report the safety and efficacy of dihydroartemisinin-piperaquine (DHA-PQ) for SMC in school-aged children in Mali. METHOD: This randomized controlled trial included 345 participants aged 6-15 years randomized to receive DHA-PQ, sulfadoxine-pyrimethamine plus amodiaquine (SP-AQ), or no chemoprevention (albendazole) at a 1:1:1 ratio. Four rounds of SMC were conducted from September to December 2021. The participants were assessed 7 days after each round for safety and efficacy of the interventions. RESULTS: Abdominal pain (11.8% vs. 29.2%), headache (11.2% vs. 19.2%) and vomiting (5.7% vs. 15.2%) were frequently reported in the DHA-PQ and SP-AQ arms. On Day 120 of follow-up, the incidence of clinical malaria was 0.01 episodes/person-month in the DHA-PQ and SP-AQ arms and 0.17 episodes/person-month in the control arm (p <0.0001). Gametocytes were detected in 37 participants in all arms. CONCLUSION: Children in DHA-PQ arm reported less adverse events compared to SP-AQ arm. Both drugs were effective against clinical malaria and infection.

Hiding in plain sight: an epitope-based strategy for a subunit malaria vaccine.

Recent data suggest that approaches to developing a subunit blood-stage malaria vaccine may be misdirected. While antigenic polymorphism is recognized as a challenge, efforts to counter this have primarily involved enhancing the quantity and quality of antibody with potent adjuvants, identifying conserved target proteins, or combining multiple antigens to broaden the immune response. However, paradoxically, evidence has emerged that narrowing, rather than broadening, the immune response may be required to obtain an immune response protective against multiple Plasmodium strains. Non-immunodominant, conserved epitopes are crucial. The evidence comes from studying the immune response to red cell surface-expressed antigens but should also be applicable to merozoite surface antigens. Strategies to define the targets of these highly focused immune responses are provided.

Review of intervention products for use in the prevention and control of anemia.

Anemia remains a major public health problem, especially in low- and middle-income countries. The World Health Organization recommends several interventions to prevent and manage anemia in vulnerable population groups, including young children, menstruating adolescent girls and women, and pregnant and postpartum women. Daily iron supplementation reduces the risk of anemia in infants, children, and pregnant women, and intermittent iron supplementation reduces anemia risk in menstruating girls and women. Micronutrient powders reduce the risk of anemia in children. Fortifying wheat flour with iron reduces the risk of anemia in the overall population, whereas the effect of fortifying maize flour and rice is still uncertain. Regarding non-nutrition-related interventions, malaria treatment and deworming have been reported to decrease anemia prevalence. Promising interventions to prevent anemia include vitamin A supplementation, multiple micronutrient supplementation for pregnant women, small-quantity lipid-based supplements, and fortification of salt with iodine and iron. Future research could address the efficacy and safety of different iron supplementation formulations, identify the most bioavailable form of iron for fortification, examine adherence to supplementation regimens and fortification standards, and investigate the effectiveness of integrating micronutrient, helminth, and malaria control programs.

Sequence-dependent heterochromatin formation in the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum represses transcription of the gene encoding AP2-G, which is the master regulator of germ cell differentiation, via heterochromatin condensation following histone H3 lysine 9 trimethylation (H3K9me3). Although H3K9me3-marked heterochromatin is typically constitutive and its establishment depends on the RNA interference (RNAi) pathway in fission yeast centromeres, malaria parasites lack molecular members essential for RNAi. We developed a strategy to assess heterochromatin establishment on artificial chromosomes introduced into P. falciparum. We show that a particular DNA sequence in the AP2-G promoter is able to induce de novo H3K9me3 nucleosome deposition. In addition, we also found that the AP2-G promoter contains a distinct element required in maintenance of the repression memory. Thus, we speculate that malaria parasites have evolutionarily acquired a sequence-dependent establishment system of non-constitutive, i.e. facultative, H3K9me3-marked heterochromatin.

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.

Improved estimates of foetal growth are associated with perinatal outcomes: A latent modelling approach in a population-based birth cohort.

Background: We aimed to estimate latent foetal growth conditions and explore their determinants among maternal characteristics and ultrasound biometric parameters. We additionally investigated the influence of foetal growth conditions on perinatal variables. Methods: We used data from live-born singletons in the Maternal and Child Health and Nutrition in Acre, Brazil (MINA-Brazil Study) population-based birth cohort. Maternal and perinatal characteristics were assessed in medical records from the maternity hospital and interviews with participants from July 2015 to June 2016. A sub-sample went through ultrasound examinations during the antenatal period, with assessment of foetal head and abdominal circumferences, and femur length. We estimated latent foetal growth conditions with a structural equation modelling framework, informed by the child's birth weight z-scores (BWZ) and birth length z-scores (BLZ) according to gestational age. Odds ratios and 95% confidence intervals (CIs) for the occurrence of perinatal events were estimated according to linear predictions of the latent variable. Results: We included 1253 participants. Latent foetal growth conditions explained 88.3% of BWZ and 53.7% of BLZ variation. Maternal elevated blood pressure, primiparity, smoking, malaria, and insufficient gestational weight gain negatively impacted foetal growth conditions. In the subsample (n = 499), ultrasound biometric parameters assessed at 28 weeks were positively associated with the latent variable, with the largest contribution from foetal abdominal circumference. Each standardised unit of predicted foetal growth conditions halved the chance for preterm birth (95% CI = 0.26, 0.74) and longer hospital stay (>3 days) (95% CI = 0.28, 0.88). Conversely, BWZ and BLZ were not independently associated with these perinatal variables in separate logistic regression models. Conclusions: Latent foetal growth conditions jointly encompassing weight gain and linear growth during gestation were negatively influenced by a scenario of dual burden of maternal morbidities, with perinatal implications.

Correction: Correction: Asymptomatic Plasmodium vivax malaria in the Brazilian Amazon: Submicroscopic parasitemic blood infects Nyssorhynchus darlingi.

[This corrects the article DOI: 10.1371/journal.pntd.0011429.].

Preparations for sustainability and transition from Global Fund support in the Eastern Mediterranean Region.

Background: HIV, tuberculosis and malaria (HTM) services receive financial support from the Global Fund and need to plan for sustainability and transition from external funding. Aim: To recommend actions for addressing key sustainability and transition issues in 15 countries receiving Global Fund grants in the WHO Eastern Mediterranean (EMR) Region. Methods: We reviewed documents, interviewed key informants, and conducted case studies in Sudan and Tunisia to highlight key considerations for sustainability and transition from Global Fund that is tailored to the EMR and the health system building blocks. Sustainability considerations should align with the health system building blocks, including governance, financing, service delivery, workforce and health product management, with the addition of considerations for key and vulnerable populations because of their particular importance for HIV and tuberculosis services. Conclusion: While hoping for economic growth and reduction of the burden of HTM, EMR countries need to prepare for transition from Global Fund support. Proactive steps that are tailored to the health system building blocks and address the needs of key and vulnerable populations should progressively increase national capabilities as well as resources dedicated to HTM.

Measuring the Impact of Genetic Heterogeneity and Chromosomal Inversions on the Efficacy of CRISPR-Cas9 Gene Drives in Different Strains of Anopheles gambiae.

The human malaria vector Anopheles gambiae is becoming increasingly resistant to insecticides, spurring the development of genetic control strategies. CRISPR-Cas9 gene drives can modify a population by creating double-stranded breaks at highly specific targets, triggering copying of the gene drive into the cut site ("homing"), ensuring its inheritance. The DNA repair mechanism responsible requires homology between the donor and recipient chromosomes, presenting challenges for the invasion of laboratory-developed gene drives into wild populations of target species An. gambiae species complex, which show high levels of genome variation. Two gene drives (vas2-5958 and zpg-7280) were introduced into three An. gambiae strains collected across Africa with 5.3-6.6% variation around the target sites, and the effect of this variation on homing was measured. Gene drive homing across different karyotypes of the 2La chromosomal inversion was also assessed. No decrease in gene drive homing was seen despite target site heterology, demonstrating the applicability of gene drives to wild populations.

Patterns of genetic differentiation imply distinct phylogeographic history of the mosquito species Anopheles messeae and Anopheles daciae in Eurasia.

Detailed knowledge of phylogeography is important for control of mosquito species involved in the transmission of human infectious diseases. Anopheles messeae is a geographically widespread and genetically diverse dominant vector of malaria in Eurasia. A closely related species, An. daciae, was originally distinguished from An. messeae based on five nucleotide substitutions in its ribosomal DNA (rDNA). However, the patterns of phylogeographic history of these species in Eurasia remain poorly understood. Here, using internal transcribed spacer 2 (ITS2) of rDNA and karyotyping for the species identification we determined the composition of five Anopheles species in 28 locations in Eurasia. Based on the frequencies of 11 polymorphic chromosomal inversions used as genetic markers, a large-scale population genetics analysis was performed of 1932 mosquitoes identified as An. messeae, An. daciae and their hybrids. The largest genetic differences between the species were detected in the X sex chromosome suggesting a potential involvement of this chromosome in speciation. The frequencies of autosomal inversions in the same locations differed by 13%-45% between the species demonstrating a restricted gene flow between the species. Overall, An. messeae was identified as a diverse species with a more complex population structure than An. daciae. The clinal gradients in frequencies of chromosomal inversions were determined in both species implicating their possible involvement in climate adaptations. The frequencies of hybrids were low ~1% in northern Europe but high up to 50% in south-eastern populations. Thus, our study revealed critical differences in patterns of phylogeographic history between An. messeae and An. daciae in Eurasia. This knowledge will help to predict the potential of the malaria transmission in the northern territories of the continent.

Applying the COM-B behaviour change model to a pilot study delivering volatile pyrethroid spatial repellents and insecticide-treated clothing to forest-exposed populations in Mondulkiri Province, Cambodia.

BACKGROUND: Southeast Asia is making tremendous progress towards their 2030 malaria elimination goal but needs new interventions to stop forest malaria. This study trials two new vector control tools, a volatile pyrethroid spatial repellent (VPSR) and insecticide-treated clothing (ITC), amongst forest-exposed populations in Mondulkiri Province Cambodia to inform their potential use for eliminating forest malaria. METHODS: 21 forest-exposed individuals were given a questionnaire on their perceptions of malaria and preventive practices used, after which they trialed two products sequentially. Clothes was treated with ITC by the study team. Mixed methods were used to understand their experience, attitudes, and preferences regarding the products trialed. Quantitative data was summarized and qualitative insights were analysed using thematic analysis, applying the Capability, Opportunity, and Motivation Behaviour Change (COM-B) model and Behaviour Change Wheel Framework to identify intervention functions to support tailored product rollout amongst these populations. RESULTS: Study participants reported a need for protection from mosquito bites in outdoor and forest-exposed settings and perceived both products trialed to be effective for this purpose. The VPSR product was preferred when travel was not required, whereas ITC was preferred for ease of use when going to the forest, especially in rainy conditions. COM-B analysis identified that key enablers for use of both products included their perceived efficacy and ease of use, which required no skill or preparation. For barriers to use, the odour of ITC was sometimes perceived as being toxic, as well as its inability to protect uncovered skin from mosquito bites, while the perceived usefulness of the VPSR product trialed was limited by its water sensitivity in rainy forest settings. Intervention components to encourage appropriate and sustained use of these products include education about how to use these products and what to expect, persuasion to use them from community leaders and targeted channels, and enablement to facilitate convenient and affordable access. CONCLUSION: The rollout of VPSRs and ITC amongst forest-exposed populations can be useful for eliminating malaria in Southeast Asia. Study findings can be applied to increase product uptake among forest exposed populations in Cambodia, while manufacturers can aim to develop products that are rainproof, easy to use in forest settings, and have favourable odour profiles to target users.

Maize pollen diet enhances malaria mosquito longevity and infectivity to Plasmodium parasites in Ethiopia.

Although larval diet quality may affect adult mosquito fitness, its impact on parasite development is scarce. Plant pollen from Zea mays, Typha latifolia, and Prosopis juliflora was ultraviolet-sterilized and examined for effects on larval development, pupation rate, adult mosquito longevity, survival and infectivity. The control larvae were fed Tetramin fish food as a comparator food. Four treatment and two control groups were used for each pollen diet, and each experimental tray had 25 larvae. Female An. arabiensis were starved overnight and exposed to infectious blood using a membrane-feeding system. The Kaplan-Meier curves and log-rank test were used for analysis. The Z. mays pollen diet increased malaria mosquito survival and pupation rate (91.3%) and adult emergence (85%). Zea mays and Tetramin fish food had comparable adulthood development times. Adults who emerged from larvae fed Z. mays pollen had the longest average wing length (3.72 mm) and were more permissive to P. vivax (45%) and P. falciparum (27.5%). They also survived longer after feeding on infectious blood and had the highest number of P. vivax oocysts. Zea mays pollen improved larval development, adult mosquito longevity, survival and infectivity to Plasmodium. Our findings suggest that malaria transmission in Z. mays growing villages should be monitored.

Differences in catalase levels between malaria-infected individuals and uninfected controls: a systematic review and meta-analysis.

Inconsistent catalase (CAT) research necessitates a comprehensive review of CAT levels among patients with malaria to achieve better therapeutic strategies. This study aimed to systematically review and meta-analyze available literature on CAT levels in nonpregnant and pregnant individuals with malaria compared with those in uninfected controls, with the goal of providing a robust evidence base for future research and potential interventions. Following PRISMA guidelines, a systematic literature search across six databases was conducted to examine CAT levels in patients with malaria. Data was extracted independently by two reviewers, and study quality was assessed using the Joanna Briggs Institute (JBI) critical appraisal checklist. The standardized mean difference of CAT levels was calculated with heterogeneity assessment. Subgroup and sensitivity analyses were conducted to explore heterogeneity and assess the robustness of the findings. Publication bias was visually and statistically assessed and corrected, if necessary. Statistical analyses were performed using Stata software, with a significance level set at P < 0.05. Nineteen studies were included in the review. These studies, published from before 2000 to 2023, primarily from Africa and Asia, focused on different Plasmodium species and age groups. Results of qualitative synthesis among nonpregnant individuals consistently showed lower CAT levels in malaria-infected individuals, although some studies reported higher levels. No significant differences in CAT levels were found between malaria-infected and uninfected individuals, as demonstrated by a meta-analysis overall (P = 0.05, Hedges' g: - 0.78, 95% confidence interval (CI): (- 1.56)-0.01, I2: 98.47, 15 studies), but subgroup analyses showed significant differences in CAT levels in studies conducted in Africa (P = 0.02, Hedges' g: - 0.57, 95% CI: - 1.02-(0.11), I2: 91.81, 7 studies), and in studies that specifically focused on children (P = 0.03, Hedges' g: - 0.57, 95% CI: - 1.07-(- 0.07), I2: 87.52, 4 studies). Pregnant women showed variations in CAT levels across trimesters. This study provides valuable insights into the association between malaria infection and CAT enzyme levels, particularly in nonpregnant individuals. Furthermore, well-designed studies are essential to decoding the intricacies of this relationship, which could have significant implications for understanding disease processes and improving patient care.

Weekly primaquine for radical cure of patients with Plasmodium vivax malaria and glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND: The World Health Organization recommends that primaquine should be given once weekly for 8-weeks to patients with Plasmodium vivax malaria and glucose-6-phosphate dehydrogenase (G6PD) deficiency, but data on its antirelapse efficacy and safety are limited. METHODS: Within the context of a multicentre, randomised clinical trial of two primaquine regimens in P. vivax malaria, patients with G6PD deficiency were excluded and enrolled into a separate 12-month observational study. They were treated with a weekly dose of 0.75 mg/kg primaquine for 8 weeks (PQ8W) plus dihydroartemisinin piperaquine (Indonesia) or chloroquine (Afghanistan, Ethiopia, Vietnam). G6PD status was diagnosed using the fluorescent spot test and confirmed by genotyping for locally prevalent G6PD variants. The risk of P. vivax recurrence following PQ8W and the consequent haematological recovery were characterized in all patients and in patients with genotypically confirmed G6PD variants, and compared with the patients enrolled in the main randomised control trial. RESULTS: Between July 2014 and November 2017, 42 male and 8 female patients were enrolled in Afghanistan (6), Ethiopia (5), Indonesia (19), and Vietnam (20). G6PD deficiency was confirmed by genotyping in 31 patients: Viangchan (14), Mediterranean (4), 357A-G (3), Canton (2), Kaiping (2), and one each for A-, Chatham, Gaohe, Ludhiana, Orissa, and Vanua Lava. Two patients had recurrent P. vivax parasitaemia (days 68 and 207). The overall 12-month cumulative risk of recurrent P. vivax malaria was 5.1% (95% CI: 1.3-18.9) and the incidence rate of recurrence was 46.8 per 1000 person-years (95% CI: 11.7-187.1). The risk of P. vivax recurrence was lower in G6PD deficient patients treated with PQ8W compared to G6PD normal patients in all treatment arms of the randomised controlled trial. Two of the 26 confirmed hemizygous males had a significant fall in haemoglobin (>5g/dl) after the first dose but were able to complete their 8 week regimen. CONCLUSIONS: PQ8W was highly effective in preventing P. vivax recurrences. Whilst PQ8W was well tolerated in most patients across a range of different G6PD variants, significant falls in haemoglobin may occur after the first dose and require clinical monitoring. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov (NCT01814683).