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Background Artemisinin and artemisinin-based combination therapy (ACT) partner drug resistance in Plasmodium falciparum have spread across the Greater Mekong Subregion compromising antimalarial treatment. The current 3-day artemether-lumefantrine regimen has been associated with high treatment failure rates in pregnant women. Although ACTs are recommended for treating Plasmodium vivax malaria, no clinical trials in pregnancy have been reported. Methods Pregnant women with uncomplicated malaria on the Thailand-Myanmar border participated in an open-label randomized controlled trial comparing dihydroartemisinin-piperaquine (DP), artesunate-mefloquine (ASMQ) and a 4-day artemether-lumefantrine regimen (AL+). The primary endpoint for P. falciparum infections was the PCR-corrected cure rate and for P. vivax infections was recurrent parasitaemia, before delivery or day 63, whichever was longer, assessed by Kaplan-Meier estimate. Results Between February 2010 and August 2016, 511 pregnant women with malaria (353 P. vivax, 142 P. falciparum, 15 co-infections, 1 Plasmodium malariae) were randomized to either DP (n=170), ASMQ (n=169) or AL+ (n=172) treatments. Successful malaria elimination efforts in the region resulted in premature termination of the trial. The majority of women had recurrent malaria (mainly P. vivax relapses, which are not prevented by these treatments). Recurrence-free proportions (95% confidence interval [95% CI]) for vivax malaria were 20.6% (5.1–43.4) for DP (n=125), 46.0% (30.9–60.0) for ASMQ (n=117) and 28.7% (10.0–50.8) for AL+ (n=126). DP and ASMQ provided longer recurrence-free intervals. PCR-corrected cure rates (95% CI) for falciparum malaria were 93.7% (81.6–97.9) for DP (n=49), 79.6% (66.1–88.1) for AMSQ (n=55) and 87.5% (74.3–94.2) for AL+ (n=50). Overall 65% (85/130) of P. falciparum infections had Pfkelch13 propeller mutations which increased over time and recrudescence occurred almost exclusively in them; risk ratio 9.42 (95% CI 1.30–68.29). Among the women with falciparum malaria, 24.0% (95% CI 16.8–33.6) had P. vivax parasitaemia within 4 months. Nausea, vomiting, dizziness and sleep disturbance were more frequent with ASMQ. Miscarriage, small-for-gestational-age and preterm birth did not differ significantly among the treatment groups, including first trimester exposures (n=46). Conclusions DP was well tolerated and safe, and was the only drug providing satisfactory efficacy for P. falciparum-infected pregnant woman in this area of widespread artemisinin resistance. Vivax malaria recurrences are very common and warrant chloroquine prophylaxis after antimalarial treatment in this area. Trial registration ClinicalTrials.gov identifier NCT01054248, registered on 22 January 2010.

Relapses arising from dormant liver-stage Plasmodium vivax parasites (hypnozoites) are a major cause of vivax malaria. However, in endemic areas, a recurrent blood-stage infection following treatment can be hypnozoite-derived (relapse), a blood-stage treatment failure (recrudescence), or a newly acquired infection (reinfection). Each of these requires a different prevention strategy, but it was not previously possible to distinguish between them reliably. We show that individual vivax malaria recurrences can be characterised probabilistically by combined modelling of time-to-event and genetic data within a framework incorporating identity-by-descent. Analysis of pooled patient data on 1441 recurrent P. vivax infections in 1299 patients on the Thailand–Myanmar border observed over 1000 patient follow-up years shows that, without primaquine radical curative treatment, 3 in 4 patients relapse. In contrast, after supervised high-dose primaquine only 1 in 40 relapse. In this region of frequent relapsing P. vivax, failure rates after supervised high-dose primaquine are significantly lower (∼3%) than estimated previously., Relapse, reinfection and recrudescence can all cause recurrent infection after treatment of Plasmodium vivax malaria in endemic areas, but are difficult to distinguish. Here the authors show that they can be differentiated probabilistically and thereby demonstrate the high efficacy of primaquine treatment in preventing relapse.

Summary Background The Greater Mekong subregion is a recurrent source of antimalarial drug resistance in Plasmodium falciparum malaria. This study aimed to characterise the extent and spread of resistance across this entire region between 2007 and 2018. Methods P falciparum isolates from Myanmar, Thailand, Laos, and Cambodia were obtained from clinical trials and epidemiological studies done between Jan 1, 2007, and Dec 31, 2018, and were genotyped for molecular markers (pfkelch, pfcrt, pfplasmepsin2, and pfmdr1) of antimalarial drug resistance. Genetic relatedness was assessed using microsatellite and single nucleotide polymorphism typing of flanking sequences around target genes. Findings 10 632 isolates were genotyped. A single long pfkelch Cys580Tyr haplotype (from −50 kb to +31·5 kb) conferring artemisinin resistance (PfPailin) now dominates across the eastern Greater Mekong subregion. Piperaquine resistance associated with pfplasmepsin2 gene amplification and mutations in pfcrt downstream of the Lys76Thr chloroquine resistance locus has also developed. On the Thailand–Myanmar border a different pfkelch Cys580Tyr lineage rose to high frequencies before it was eliminated. Elsewhere in Myanmar the Cys580Tyr allele remains widespread at low allele frequencies. Meanwhile a single artemisinin-resistant pfkelch Phe446Ile haplotype has spread across Myanmar. Despite intense use of dihydroartemisinin–piperaquine in Kayin state, eastern Myanmar, both in treatment and mass drug administrations, no selection of piperaquine resistance markers was observed. pfmdr1 amplification, a marker of resistance to mefloquine, remains at low prevalence across the entire region. Interpretation Artemisinin resistance in P falciparum is now prevalent across the Greater Mekong subregion. In the eastern Greater Mekong subregion a multidrug resistant P falciparum lineage (PfPailin) dominates. In Myanmar a long pfkelch Phe446Ile haplotype has spread widely but, by contrast with the eastern Greater Mekong subregion, there is no indication of artemisinin combination therapy (ACT) partner drug resistance from genotyping known markers, and no evidence of spread of ACT resistant P falciparum from the east to the west. There is still a window of opportunity to prevent global spread of ACT resistance. Funding Thailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.

Background Plasmodium vivax is the predominant Plasmodium species in Afghanistan. National guidelines recommend the combination of chloroquine and primaquine (CQ-PQ) for radical treatment of P. vivax malaria. Artesunate in combination with the antifolates sulfadoxine-pyrimethamine (SP) has been first-line treatment for uncomplicated falciparum malaria until 2016. Although SP has been the recommended treatment for falciparum and not vivax malaria, exposure of the P. vivax parasite population to SP might still have been quite extensive because of community based management of malaria. The change in the P. vivax antifolate resistance markers between 2007 and 2017 were investigated. Methods Dried blood spots were collected (n = 185) from confirmed P. vivax patients in five malaria-endemic areas of Afghanistan bordering Tajikistan, Turkmenistan and Pakistan, including Takhar, Faryab, Laghman, Nangarhar, and Kunar, in 2007, 2010 and 2017. Semi-nested PCR, RFLP and nucleotide sequencing were used to assess the pyrimethamine resistant related mutations in P. vivax dihydrofolate reductase (pvdhfr I13L, P33L, N50I, F57L, S58R, T61I, S93H, S117N, I173L) and the sulfonamide resistance related mutations in P. vivax dihydropteroate synthase (pvdhps A383G, A553G). Results In the 185 samples genotyped for pvdhfr and pvdhps mutations, 11 distinct haplotypes were observed, which evolved over time. In 2007, wild type pvdhfr and pvdhps were the most frequent haplotype in all study sites (81%, 80/99). However, in 2017, the frequency of the wild-type was reduced to 36%, (21/58; p value ≤ 0.001), with an increase in frequency of the double mutant pvdhfr and pvdhps haplotype S58RS117N (21%, 12/58), and the single pvdhfr mutant haplotype S117N (14%, 8/58). Triple and quadruple mutations were not found. In addition, pvdhfr mutations at position N50I (7%, 13/185) and the novel mutation S93H (6%, 11/185) were observed. Based on in silico protein modelling and molecular docking, the pvdhfr N50I mutation is expected to affect only moderately pyrimethamine binding, whereas the S93H mutation does not. Conclusions In the course of ten years, there has been a strong increase in the frequency pyrimethamine resistance related mutations in pvdhfr in the P. vivax population in Afghanistan, although triple and quadruple mutations conferring high grade resistance were not observed. This suggests relatively low drug pressure from SP on the P. vivax parasite population in the study areas. The impact of two newly identified mutations in the pvdhfr gene on pyrimethamine resistance needs further investigation.

Additional file 3. Synonymous mutations in Pvpm4 gene Thailand, Lao PDR and Cambodia.

Background Relapse of Plasmodium vivax infection is the main cause of vivax malaria in many parts of Asia. However at the individual patient level, recurrence of a blood stage infection following treatment within the endemic area can be either a relapse (from dormant liver-stage parasites), a recrudescence (blood-stage treatment failure), or a reinfection (following a new mosquito inoculation). Each requires a different prevention strategy, but previously they could not be distinguished reliably. Time-of-event and genetic data provide complimentary information about the cause of P. vivax recurrence, but the optimum approach to genotyping and analysis remains uncertain. Methods Individual-level data from two large drug trials in acute vivax malaria patients (Vivax History: VHX; Best Primaquine Dose: BPD) conducted on the Thailand-Myanmar border with follow-up of one year were pooled (n=1299). A total of 710 isolates from both acute and recurrent P. vivax episodes were genotyped using 3-9 highly polymorphic microsatellite markers. These pooled data were analyzed using a novel population statistical model incorporating an assessment of genetic relatedness, treatment drug administered, and the time-to-recurrence. Results 99% of genotyped recurrences in individuals who did not receive primaquine (n=365) were estimated to be relapses. In comparison, 14% of genotyped recurrences (n=121) were estimated to be relapses following high-dose supervised primaquine. By comparing episodes across individuals (90194 comparisons), the false-positive rate of relapse identification using genetic data alone was estimated to be 2.2%. We estimated the true failure rate after high-dose primaquine (7mg/kg total dose) to be 2.6% in this epidemiological context, substantially lower the reinfection unadjusted estimate of 12%. Simulation studies show that 9 highly polymorphic microsatellite markers suffice to discriminate between recurrence states. Drug exposures reflected by plasma carboxy-primaquine concentrations were not predictive of treatment failure, but did identify non-adherence. Conclusion Using this novel statistical model, relapse of P. vivax malaria could be distinguished reliably from reinfection. This showed that in this population supervised high-dose primaquine could avert up to 99% of relapses. In low transmission settings, microsatellite genotyping combined with time-to-event data can accurately discriminate between the different causes of recurrent P. vivax malaria. Author summary One hundred years ago, Plasmodium vivax, the most globally diverse cause of human malaria, was present across most of the old-world, throughout tropical and temperate climes. Its main evolutionary advantage over Plasmodium falciparum, responsible for the most deadly type of human malaria, is its ability to stay dormant in the liver, emerging weeks to years later causing recurring illness and continuing transmission. The dormant liver-stage parasites are called hypnozoites. A recurrent infection can either be hypnozoite-derived (a relapse), a blood-stage treatment failure (recrudescence), or from a new mosquito bite (a reinfection). At a population level, each requires a different preventative strategy but no widely applicable methodology existed to discriminate between the three possible states: relapse, recrudescence and reinfection. Parasite genetic data alone cannot resolve the different possibilities. We developed a novel probabilistic framework which uses both epidemiological and genetic data to determine the most likely cause of vivax recurrence at the individual level. We applied this method to the largest available pooled clinical trial data from Southeast Asia incorporating information from highly polymorphic microsatellite markers, drug treatments received and time-to-recurrent illness. This analysis provides a tool for estimating probabilities of relapse, recrudescence and reinfection and, importantly, shows that hypnozoiticidal high-dose primaquine radical cure is much more effective than previously thought.

In Cambodian villages, 3-month mass drug administration with high coverage using dihydroartemisinin-piperaquine was safe and was followed by the absence of clinical Plasmodium falciparum cases for at least 1 year, despite the presence of multidrug-resistant parasites., Background The increase in multidrug-resistant Plasmodium falciparum in Southeast Asia suggests a need for acceleration of malaria elimination. We evaluated the effectiveness and safety of mass drug administration (MDA) to interrupt malaria transmission. Methods Four malaria-endemic villages in western Cambodia were randomized to 3 rounds of MDA (a 3-day course of dihydroartemisinin with piperaquine-phosphate), administered either early in or at the end of the study period. Comprehensive malaria treatment records were collected during 2014–2017. Subclinical parasite prevalence was estimated by ultrasensitive quantitative polymerase chain reaction quarterly over 12 months. Results MDA coverage with at least 1 complete round was 88% (1999/2268), ≥2 rounds 73% (1645/2268), and all 3 rounds 58% (1310/2268). Plasmodium falciparum incidence in intervention and control villages was similar over the 12 months prior to the study: 39 per 1000 person-years (PY) vs 45 per 1000 PY (P = .50). The primary outcome, P. falciparum incidence in the 12 months after MDA, was lower in intervention villages (1.5/1000 PY vs 37.1/1000 PY; incidence rate ratio, 24.5 [95% confidence interval], 3.4–177; P = .002). Following MDA in 2016, there were no clinical falciparum malaria cases over 12 months (0/2044 PY) in all 4 villages. Plasmodium vivax prevalence decreased markedly in intervention villages following MDA but returned to approximately half the baseline prevalence by 12 months. No severe adverse events were attributed to treatment. Conclusions Mass drug administrations achieved high coverage, were safe, and associated with the absence of clinical P. falciparum cases for at least 1 year. Clinical Trials Registration NCT01872702.

Background Cambodia has seen a marked reduction in the incidence of Plasmodium falciparum over the past decade without a corresponding decline in Plasmodium vivax incidence. It is unknown to what extent local transmission is sustained by a chain of clinical and sub-clinical infections or by continued re-introduction via migration. Using an ultrasensitive molecular technique, 20 villages in western Cambodia were surveyed to detect the low season prevalence of P. falciparum and P. vivax and local treatment records were reviewed. Methods During March to May 2015 cross-sectional surveys were conducted in 20 villages in Battambang, western Cambodia. Demographic and epidemiological data and venous blood samples were collected from 50 randomly selected adult volunteers in each village. Blood was tested for Plasmodium infections by rapid diagnostic test (RDT), microscopy and high volume (0.5 ml packed red blood cell) quantitative polymerase chain reaction (uPCR). Positive samples were analysed by nested PCR to determine the Plasmodium species. Malaria case records were collected from the Provincial Health Department and village malaria workers to determine incidence and migration status. Results Among the 1000 participants, 91 (9.1%) were positive for any Plasmodium infection by uPCR, seven (0.7%) by microscopy, and two (0.2%) by RDT. uPCR P. vivax prevalence was 6.6%, P. falciparum 0.7%, and undetermined Plasmodium species 1.8%. Being male (adjusted OR 2.0; 95% CI 1.2-3.4); being a young adult Plasmodium vivax prevalence determined by uPCR significantly correlated with vivax malaria incidences in both 2014 and 2015 (p = 0.001 and 0.002, respectively), whereas no relationship was observed in falciparum malaria (p = 0.36 and p = 0.59, respectively). Discussion There was heterogeneity in the malaria parasite reservoir between villages, and Plasmodium prevalence correlated with subsequent malaria incidence. The association was attributable chiefly to P. vivax infections, which were nine-fold more prevalent than P. falciparum infections. In the absence of a radical cure with 8-aminoquinolines, P. vivax transmission will continue even as P. falciparum prevalence declines. Migration was associated with over a third of incident cases of clinical malaria. Trial registration clinicaltrials.gov (NCT01872702). Registered 4 June 2013

Additional file 5. Comparison of Pvkelch12 mutations of this study to previous publications.

Additional file 1: Figure S1. Malaria prevalence by uPCR and incidence per 1000 in 2015 in 16 villages.

Additional file 1. Details of PCR and RFLP conditions.

Additional file 1. Details of PCR and RFLP conditions.

Additional file 5. Comparison of Pvkelch12 mutations of this study to previous publications.