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Abstract Introduction Symptoms reported following the administration of investigational drugs play an important role in decisions for registration and treatment guidelines. However, symptoms are subjective, and interview methods to quantify them are difficult to standardise. We explored differences in symptom reporting across study sites of a multicentre antimalarial trial, with the aim of informing trial design and the interpretation of safety and tolerability data. Methods Data were derived from the IMPROV trial, a randomised, placebo-controlled double blinded trial of high dose primaquine to prevent Plasmodium vivax recurrence conducted in eight study sites in Afghanistan, Ethiopia, Indonesia and Vietnam. At each follow up visit a 13-point symptom questionnaire was completed. The number and percentage of patients with clinically relevant symptoms following the administration of primaquine or placebo, were reported by study site including vomiting, diarrhoea, anorexia, nausea, abdominal pain and dizziness. Multivariable logistic regression was used to estimate the confounder-adjusted site-specific proportion of each symptom. Results A total of 2,336 patients were included. The greatest variation between sites in the proportion of patients reporting symptoms was for anorexia between day 0 and day 13: 97.3% (361/371) of patients in Arba Minch, Ethiopia, reported the symptom compared with 4.7% (5/106) of patients in Krong Pa, Vietnam. Differences attenuated slightly after adjusting for treatment arm, age, sex, day 0 parasite density and fever; with the adjusted proportion for anorexia ranging from 4.8% to 97.0%. Differences between sites were greater for symptoms graded as mild or moderate compared to those rated as severe. Differences in symptom reporting were greater between study sites than between treatment arms within the same study site. Conclusion Despite standardised training, there was large variation in symptom reporting across trial sites. The reporting of severe symptoms was less skewed compared to mild and moderate symptoms, which are likely to be more subjective. Trialists should clearly distinguish between safety and tolerability outcomes. Differences between trial arms were much less variable across sites, suggesting that the relative difference in reported symptoms between intervention and control group is more relevant than absolute numbers and should be reported when possible. Trial registration Clinicaltrials.gov: NCT01814683; March 20th, 2013.

Abstract Background In many endemic areas, Plasmodium vivax malaria is predominantly a disease of young adults and children. International recommendations for radical cure recommend fixed target doses of 0.25 or 0.5 mg/kg/day of primaquine for 14 days in glucose-6-phosphate dehydrogenase normal patients of all ages. However, for many anti-malarial drugs, including primaquine, there is evidence that children have lower exposures than adults for the same weight-adjusted dose. The aim of the study was to develop 14-day weight-based and age-based primaquine regimens against high-frequency relapsing tropical P. vivax. Methods The recommended adult target dose of 0.5 mg/kg/day (30 mg in a 60 kg patient) is highly efficacious against tropical P. vivax and was assumed to produce optimal drug exposure. Primaquine doses were calculated using allometric scaling to derive a weight-based primaquine regimen over a weight range from 5 to 100 kg. Growth curves were constructed from an anthropometric database of 53,467 individuals from the Greater Mekong Subregion (GMS) to define weight-for-age relationships. The median age associated with each weight was used to derive an age-based dosing regimen from the weight-based regimen. Results The proposed weight-based regimen has 5 dosing bands: (i) 5–7 kg, 5 mg, resulting in 0.71–1.0 mg/kg/day; (ii) 8–16 kg, 7.5 mg, 0.47–0.94 mg/kg/day; (iii) 17–40 kg, 15 mg, 0.38–0.88 mg/kg/day; (iv) 41–80 kg, 30 mg, 0.37–0.73 mg/kg/day; and (v) 81–100 kg, 45 mg, 0.45–0.56 mg/kg/day. The corresponding age-based regimen had 4 dosing bands: 6–11 months, 5 mg, 0.43–1.0 mg/kg/day; (ii) 1–5 years, 7.5 mg, 0.35–1.25 mg/kg/day; (iii) 6–14 years, 15 mg, 0.30–1.36 mg/kg/day; and (iv) ≥ 15 years, 30 mg, 0.35–1.07 mg/kg/day. Conclusion The proposed weight-based regimen showed less variability around the primaquine dose within each dosing band compared to the age-based regimen and is preferred. Increased dose accuracy could be achieved by additional dosing bands for both regimens. The age-based regimen might not be applicable to regions outside the GMS, which must be based on local anthropometric data. Pharmacokinetic data in small children are needed urgently to inform the proposed regimens.

Abstract Background Malaria reactive case detection is the testing and, if positive, treatment of close contacts of index cases. It is included in national malaria control programmes of countries in the Greater Mekong Subregion to accelerate malaria elimination. Yet the value of reactive case detection in the control and elimination of malaria remains controversial because of the low yield, limited evidence for impact, and high demands on resources. Methods Data from the epidemiological assessments of large mass drug administration (MDA) studies in Myanmar, Vietnam, Cambodia and Laos were analysed to explore malaria infection clustering in households. The proportion of malaria positive cases among contacts screened in a hypothetical reactive case detection programme was then determined. The parasite density thresholds for rapid diagnostic test (RDT) detection was assumed to be > 50/µL (50,000/mL), for dried-blood-spot (DBS) based PCR > 5/µL (5000/mL), and for ultrasensitive PCR (uPCR) with a validated limit of detection at 0.0022/µL (22/mL). Results At baseline, before MDA, 1223 Plasmodium infections were detected by uPCR in 693 households. There was clustering of Plasmodium infections. In 637 households with asymptomatic infections 44% (278/637) had more than one member with Plasmodium infections. In the 132 households with symptomatic infections, 65% (86/132) had more than one member with Plasmodium infections. At baseline 4% of households had more than one Plasmodium falciparum infection, but three months after MDA no household had more than one P. falciparum infected member. Reactive case detection using DBS PCR would have detected ten additional cases in six households, and an RDT screen would have detected five additional cases in three households among the 169 households with at least one RDT positive case. This translates to 19 and 9 additional cases identified per 1000 people screened, respectively. Overall, assuming all febrile RDT positive patients would seek treatment and provoke reactive case detection using RDTs, then 1047 of 1052 (99.5%) Plasmodium infections in these communities would have remained undetected. Conclusion Reactive case detection in the Greater Mekong subregion is predicted to have a negligible impact on the malaria burden, but it has substantial costs in terms of human and financial resources.

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.

Abstract Background Sample size calculations for cluster randomized trials are a recognized methodological challenge for malaria research in pre-elimination settings. Positively correlated responses from the participants in the same cluster are a key feature in the estimated sample size required for a cluster randomized trial. The degree of correlation is measured by the intracluster correlation coefficient (ICC) where a higher coefficient suggests a closer correlation hence less heterogeneity within clusters but more heterogeneity between clusters. Methods Data on uPCR-detected Plasmodium falciparum and Plasmodium vivax infections from a recent cluster randomized trial which aimed at interrupting malaria transmission through mass drug administrations were used to calculate the ICCs for prevalence and incidence of Plasmodium infections. The trial was conducted in four countries in the Greater Mekong Subregion, Laos, Myanmar, Vietnam and Cambodia. Exact and simulation approaches were used to estimate ICC values for both the prevalence and the incidence of parasitaemia. In addition, the latent variable approach to estimate ICCs for the prevalence was utilized. Results The ICCs for prevalence ranged between 0.001 and 0.082 for all countries. The ICC from the combined 16 villages in the Greater Mekong Subregion were 0.26 and 0.21 for P. falciparum and P. vivax respectively. The ICCs for incidence of parasitaemia ranged between 0.002 and 0.075 for Myanmar, Cambodia and Vietnam. There were very high ICCs for incidence in the range of 0.701 to 0.806 in Laos during follow-up. Conclusion ICC estimates can help researchers when designing malaria cluster randomized trials. A high variability in ICCs and hence sample size requirements between study sites was observed. Realistic sample size estimates for cluster randomized malaria trials in the Greater Mekong Subregion have to assume high between cluster heterogeneity and ICCs. This work focused on uPCR-detected infections; there remains a need to develop more ICC references for trials designed around prevalence and incidence of clinical outcomes. Adequately powered trials are critical to estimate the benefit of interventions to malaria in a reliable and reproducible fashion. Trial registration: ClinicalTrials.govNCT01872702. Registered 7 June 2013. Retrospectively registered. https://clinicaltrials.gov/ct2/show/NCT01872702

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

Abstract Microscopy and 18S qPCR are the most common and field-friendly methods for quantifying malaria parasite density, and it is important that these methods can be interpreted as giving equivalent results. We compared results of quantitative measurement of Plasmodium falciparum parasitemia by microscopy and by 18S qPCR in a phase 2a study. Microscopy positive samples (n = 355; median 810 parasites/μL [IQR 40–10,471]) showed close agreement with 18S qPCR in mean log10/mL transformed parasitemia values by paired t test (difference 0.04, 95%CI − 0.01–0.10, p = 0.088). Excellent intraclass correlation (0.97) and no evidence of systematic or proportional differences by Passing–Bablok regression were observed. 18S qPCR appears to give equivalent parasitemia values to microscopy, which indicates 18S qPCR is an appropriate alternative method to quantify parasitemia in clinical trials.

Abstract Background Plasmodium vivax malaria elimination can only be achieved by the deployment of 8-aminoquinolines (primaquine and tafenoquine) in combination with ACT to kill both blood and liver-stage parasites. However, primaquine and the other 8-aminoquinolines cause dose-dependent haemolysis in subjects with G6PD deficiency, an X-linked disorder of red blood cells that is very common in populations living in tropical and subtropical areas. In order to inform safer use of 8-aminoquinolines in the Greater Mekong Subregion, a multi-centre study was carried out to assess the prevalence of G6PD deficiency and to identify the main G6PD variants in samples collected in Cambodia, Lao PDR, Myanmar, Thailand and Vietnam. Methods Blood samples were collected in the five countries during National Malaria Surveys or during Population Surveys. During Population Surveys samples were characterized for G6PD phenotype using the Fluorescent Spot Test. Samples were then genotyped for a panel of G6PD mutations. Results G6PD deficiency was found to be common in the region with an overall mean prevalence of deficient or mutated hemizygous males of 14.0%, ranging from a mean 7.3% in Thailand, 8.1% in Lao PDR, 8.9% in Vietnam, 15.8% in Myanmar and 18.8% in Cambodia. Mahidol and Viangchan mutations were the most common and widespread variants found among the nine investigated. Conclusions Owing to the high prevalence of G6PD deficiency in the Greater Mekong Subregion, strategies for vivax malaria elimination should include point-of-care G6PD testing (both qualitative and quantitative) to allow safe and wide treatment with 8-aminoquinolines.

Abstract Background Despite the well-documented clinical efficacy of artemisinin-based combination therapy (ACT) against malaria, the population-level effects of ACT have not been studied thoroughly until recently. An ideal case study for these population-level effects can be found in Vietnam’s gradual adoption of artemisinin in the 1990s. Methods and results Analysis of Vietnam’s national annual malaria reports (1991–2014) revealed that a 10% increase in artemisinin procurement corresponded to a 32.8% (95% CI 27.7–37.5%) decline in estimated malaria cases. There was no consistent national or regional effect of vector control on malaria. The association between urbanization and malaria was generally negative and sometimes statistically significant. Conclusions The decline of malaria in Vietnam can largely be attributed to the adoption of artemisinin-based case management. Recent analyses from Africa showed that insecticide-treated nets had the greatest effect on lowering malaria prevalence, suggesting that the success of interventions is region-specific. Continuing malaria elimination efforts should focus on both vector control and increased access to ACT.

Abstract Background Prophylaxis for high-risk populations, such as forest workers, could be one component for malaria elimination in the Greater Mekong Sub-region. A study was conducted to assess the malaria incidence in forest rangers and the feasibility of malaria prophylaxis for rangers sleeping in forest camps. Methods Forest rangers deployed in the Bu Gia Map National Park, Vietnam were invited to participate in the study. Plasmodium infections were cleared using presumptive treatment, irrespective of malaria status, with a 3-day course dihydroartemisinin/piperaquine (DP) and a 14-day course of primaquine. Before returning to the forest, study participants were randomly allocated to a 3-day course of DP or placebo. Fifteen days after returning from their forest deployment the participants were tested for Plasmodium infections using uPCR. Results Prior to treatment, 30 of 150 study participants (20%) were found to be infected with Plasmodium. Seventeen days (median) after enrolment the rangers were randomized to DP or placebo 2 days before returning to forest camps where they stayed between 2 and 20 days (median 9.5 days). One ranger in the DP-prophylaxis arm and one in the placebo arm were found to be infected with Plasmodium falciparum 15 days (median) after returning from the forest. The evaluable P. falciparum isolates had molecular markers indicating resistance to artemisinins (K13-C580Y) and piperaquine (plasmepsin), but none had multiple copies of pfmdr1 associated with mefloquine resistance. Conclusion Anti-malarial prophylaxis in forest rangers is feasible. The findings of the study highlight the threat of multidrug-resistant malaria. Trial registration NCT02788864