Evidence suggests that the PfKelch13 mutations that confer artemisinin resistance in falciparum malaria have multiple independent origins across the Greater Mekong subregion, which has motivated a regional malaria elimination agenda. We aimed to use molecular genotyping to assess antimalarial drug resistance selection and spread in the Greater Mekong subregion. In this observational study, we tested Plasmodium falciparum isolates from Myanmar, northeastern Thailand, southern Laos, and western Cambodia for PfKelch13 mutations and for Pfplasmepsin2 gene amplification (indicating piperaquine resistance). We collected blood spots from patients with microscopy or rapid test confirmed uncomplicated falciparum malaria. We used microsatellite genotyping to assess genetic relatedness. As part of studies on the epidemiology of artemisinin-resistant malaria between Jan 1, 2008, and Dec 31, 2015, we collected 434 isolates. In 2014-15, a single long PfKelch13 C580Y haplotype (-50 to +31·5 kb) lineage, which emerged in western Cambodia in 2008, was detected in 65 of 88 isolates from northeastern Thailand, 86 of 111 isolates from southern Laos, and 14 of 14 isolates from western Cambodia, signifying a hard transnational selective sweep. Pfplasmepsin2 amplification occurred only within this lineage, and by 2015 these closely related parasites were found in ten of the 14 isolates from Cambodia and 15 of 15 isolates from northeastern Thailand. C580Y mutated parasites from Myanmar had a different genetic origin. Our results suggest that the dominant artemisinin-resistant P falciparum C580Y lineage probably arose in western Cambodia and then spread to Thailand and Laos, outcompeting other parasites and acquiring piperaquine resistance. The emergence and spread of fit artemisinin-resistant P falciparum parasite lineages, which then acquire partner drug resistance across the Greater Mekong subregion, threatens regional malaria control and elimination goals. Elimination of falciparum malaria from this region should be accelerated while available antimalarial drugs still remain effective. The Wellcome Trust and the Bill and Melinda Gates Foundation.

Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of molecular surveillance in remote areas through analysis of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by determining the relative prevalence of P falciparum parasites carrying K13-propeller mutations. We did this cross-sectional survey at malaria treatment centres at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between January, 2013, and September, 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estimated prevalence of mutations of the K13 propeller region across Myanmar. Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations. Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided. Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.

The epidemiology of malaria in “low-transmission” areas has been underestimated. Molecular detection methods have revealed higher prevalences of malaria than conventional microscopy or rapid diagnostic tests, but these typically evaluate finger-prick capillary blood samples (∼5 μl) and therefore cannot detect parasite densities of <200/ml. Their use underestimates true parasite carriage rates. To characterize the epidemiology of malaria in low-transmission settings and plan elimination strategies, more sensitive quantitative PCR (qPCR) is needed to identify and quantify low-density malaria parasitemias. A highly sensitive “high-volume” quantitative PCR (qPCR) method based on Plasmodium sp. 18S RNA was adapted for blood sample volumes of ≥250 μl and scaled for high throughput. The methods were validated by assessment of the analytical sensitivity and specificity, diagnostic sensitivity, and specificity, efficiency, precision, analytical and diagnostic accuracies, limit of detection, root cause analysis of false positives, and robustness. The high-volume qPCR method based on Plasmodium sp. 18S RNA gave high PCR efficiency of 90 to 105%. Concentrations of parasite DNA from large volumes of blood gave a consistent analytical detection limit (LOD) of 22 parasites/ml (95% CI, 21.79 to 74.9), which is some 2,500 times more sensitive than conventional microscopy and 50 times more sensitive than currently used PCR methods from filter paper blood spots. The diagnostic specificity was 99.75%. Using automated procedures it was possible to process 700 blood samples per week. A very sensitive and specific high-throughput high-volume qPCR method for the detection of low-density parasitemias (>20 parasites/ml) was developed and validated.

The importance of the submicroscopic reservoir of Plasmodium infections for malaria elimination depends on its size, which is generally considered small in low transmission settings. The precise estimation of this reservoir requires more sensitive parasite detection methods. The prevalence of asymptomatic, sub-microscopic malaria was assessed by a sensitive, high blood volume quantitative real-time polymerase chain reaction method in three countries of the Greater Mekong Sub-region. Cross-sectional surveys were conducted in three villages in western Cambodia, four villages along the Thailand–Myanmar border and four villages in southwest Vietnam. Malaria parasitaemia was assessed by Plasmodium falciparum/pan malaria rapid diagnostic tests (RDTs), microscopy and a high volume ultra-sensitive real-time polymerase chain reaction (HVUSqPCR: limit of detection 22 parasites/mL). All villagers older than 6 months were invited to participate. A census before the surveys identified 7355 residents in the study villages. Parasite prevalence was 224/5008 (4 %) by RDT, 229/5111 (5 %) by microscopy, and 988/4975 (20 %) when assessed by HVUSqPCR. Of these 164 (3 %) were infected with P. falciparum, 357 (7 %) with Plasmodium vivax, 56 (1 %) with a mixed infection, and 411 (8 %) had parasite densities that were too low for species identification. A history of fever, male sex, and age of 15 years or older were independently associated with parasitaemia in a multivariate regression model stratified by site. Light microscopy and RDTs identified only a quarter of all parasitaemic participants. The asymptomatic Plasmodium reservoir is considerable, even in low transmission settings. Novel strategies are needed to eliminate this previously under recognized reservoir of malaria transmission.

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. 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. 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. 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. Thailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.

In Southeast Asia, Plasmodium knowlesi, a parasite of long-tailed macaques (Macaca fascicularis), is an important cause of human malaria. Plasmodium cynomolgi also commonly infects these monkeys, but only one naturally acquired symptomatic human case has been reported previously. Malariometric studies involving 5422 subjects (aged 6 months to 65 years) were conducted in 23 villages in Pailin and Battambang, western Cambodia. Parasite detection and genotyping was conducted on blood samples, using high-volume quantitative PCR (uPCR). Asymptomatic malaria parasite infections were detected in 1361 of 14732 samples (9.2%). Asymptomatic infections with nonhuman primate malaria parasites were found in 21 individuals living close to forested areas; P. cynomolgi was found in 11, P. knowlesi was found in 8, and P. vivax and P. cynomolgi were both found in 2. Only 2 subjects were female, and 14 were men aged 20–40 years. Geometric mean parasite densities were 3604 parasites/mL in P. cynomolgi infections and 52488 parasites/mL in P. knowlesi infections. All P. cynomolgi isolates had wild-type dihydrofolate reductase genes, in contrast to the very high prevalence of mutations in the human malaria parasites. Asymptomatic reappearance of P. cynomolgi occurred in 2 subjects 3 months after the first infection. Asymptomatic naturally acquired P. cynomolgi and P. knowlesi infections can both occur in humans. NCT01872702.