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.

RealAmp was comparable to PCR in detecting malaria parasites and shows promise as a tool to detect submicroscopic infections in malaria control and elimination programs worldwide.

Abstract. We conducted contact tracing and high-risk group screening using pooled real-time polymerase chain reaction (PCR) to support malaria elimination in Thailand. PCR detected more Plasmodium infections than the local and expert microscopists. High-throughput pooling technique reduced costs and allowed prompt reporting of results.Thailand's National Malaria Control and Elimination Strategy aims to free 80% of the country from locally acquired malaria by the year 2020 (Bureau of Vector-Borne Diseases, Ministry of Public Health, Thailand, unpublished data). However, the elimination of local transmission requires rapid detection and treatment of all infections, including those infections in asymptomatic individuals who may serve as important reservoirs.1-3 Currently, malaria case detection for surveillance depends on microscopy or rapid diagnostic tests, but both methods miss low parasite densities on the order of 10 parasites/μL. Such submicroscopic infections are detectable by polymerase chain reaction (PCR) and are common in areas with low and unstable malaria. [4][5][6][7] Efforts to control and eliminate malaria from Trat province on the border with Cambodia are intensifying because of the potential spread of artemisinin resistance. The detection of submicroscopic cases may facilitate containment efforts and help preserve artemisinin-based combination therapies for effective malaria treatment (Bureau of Vector-Borne Diseases, Ministry of Public Health, Thailand, unpublished data).Real-time PCR is a highly sensitive tool for detecting and speciating Plasmodia. Pooling samples before analysis facilitates the large-scale application of this technique for surveillance by reducing cost and analysis time. 7,8 As Thailand aims for malaria elimination, including elimination of artemisininresistant parasites, improvements in case detection are necessary. We aimed to determine if pooled real-time PCR could be integrated with the existing active case detection systems in Thailand and if so, if it would be more effective than microscopy for identifying low-density parasitemias.A single index case, with mixed P. falciparum-P. vivax infection, was identified during hospitalization for severe malaria in July of 2011 through passive case detection. This infection was likely acquired during frequent forest exposure. Two weeks after this identification, 187 residents in Bo Rai district, Trat province, Thailand (Figure 1) were contacted over 3 days according to the policies of the National Malaria Control Program of Thailand, which includes contact tracing (Case Investigation Survey) and high-risk group screening (Special Case Detection). For contact tracing, we screened neighbors within 1 km of the index case. For high-risk group screening, we screened soldiers from Khao Lan and Ban Sapanhin army camps and residents of Takang and Ban Samoh villages, which have a high proportion of Burmese Mon migrants.We administered a questionnaire to collect demographic information and risk factors for malaria, such as history of fever a...

BackgroundAsymptomatic carriage of Plasmodium falciparum and Plasmodium vivax is common in both low-and high-transmission settings and represents an important reservoir of infection that needs to be targeted if malaria elimination is to succeed.MethodsMass blood examinations (475 individuals) were conducted in two villages in Mae Hong Son, an area of endemic but low-transmission malaria in the north-west of Thailand. The microscopist at the local malaria clinic did not detect any infections. Pools of four samples were screened by real-time PCR; individual members of all of the positive pools were then re-examined by expert microscopy and by a second species-specific PCR reaction.ResultsEight subjects were found to be positive by both PCR and expert microscopy and one was found to be positive by PCR alone. The slides contained asexual stage parasites of P. vivax, P. falciparum and Plasmodium malariae, but no gametocytes. The local clinic was notified within two to eight days of the survey.ConclusionA combination of pooling, real-time PCR and expert microscopy provides a feasible approach to identifying and treating asymptomatic malaria infections in a timely manner.

Artemisinin-based combination therapy (ACT) is the most effective and widely used treatment for uncomplicated malaria and is a cornerstone for malaria control and prevention globally. Resistance to artemisinin derivatives has been confirmed in the Greater Mekong Subregion (GMS) and manifests as slow parasite clearance in patients and reduced ring stage susceptibility to artemisinins in survival assays. The gene mutations associated with artemisinin-resistant parasites are now widespread in the GMS. We genotyped 277 samples collected during an observational study from 2012 to 2016 from eight provinces in Thailand to identify mutations. The results were combined with previously reported genotyping results from Thailand to construct a map illustrating the evolution of mutations from 2007 to 2016 in that country. Different mutant alleles were found in strains with different geographical origins. The artemisinin resistance-conferring Y493H and R539T mutations were detected mainly in eastern Thailand (bordering Cambodia), while P574L was found only in western Thailand and R561H only in northwestern Thailand. The C580Y mutation was found across the entire country and was nearing fixation along the Thai-Cambodia border. Overall, the prevalence of artemisinin resistance mutations increased over the last 10 years across Thailand, especially along the Thai-Cambodia border. Molecular surveillance and therapeutic efficacy monitoring should be intensified in the region to further assess the extent and spread of artemisinin resistance.

Background Integrated drug efficacy surveillance (iDES) was formally introduced nationally across Thailand in fiscal year 2018 (FY2018), building on a history of drug efficacy monitoring and interventions. According to the National Malaria Elimination Strategy for Thailand 2017–2026, diagnosis is microscopically confirmed, treatment is prescribed, and patients are followed up four times to ensure cure. Methods Routine patient data were extracted from the malaria information system for FY2018–FY2020. Treatment failure of first-line therapy was defined as confirmed parasite reappearance within 42 days for Plasmodium falciparum and 28 days for Plasmodium vivax. The primary outcome was the crude drug efficacy rate, estimated using Kaplan–Meier methods, at day 42 for P. falciparum treated with dihydroartemisinin–piperaquine plus primaquine, and day 28 for P. vivax treated with chloroquine plus primaquine; day 60 and day 90 efficacy were secondary outcomes for P. vivax. Results The proportion of patients with outcomes recorded at day 42 for P. falciparum malaria and at day 28 for P. vivax malaria has been increasing, with FY2020 follow-up rates of 61.5% and 57.2%, respectively. For P. falciparum malaria, day 42 efficacy in FY2018 was 92.4% (n = 249), in FY2019 93.3% (n = 379), and in FY2020 98.0% (n = 167). Plasmodium falciparum recurrences occurred disproportionally in Sisaket Province, with day 42 efficacy rates of 75.9% in FY2018 (n = 59) and 49.4% in FY2019 (n = 49), leading to an update in first-line therapy to pyronaridine–artesunate at the provincial level, rolled out in FY2020. For P. vivax malaria, day 28 efficacy (chloroquine efficacy) was 98.5% in FY2018 (n = 2048), 99.1% in FY2019 (n = 2206), and 99.9% in FY2020 (n = 2448), and day 90 efficacy (primaquine efficacy) was 94.8%, 96.3%, and 97.1%, respectively. Conclusions In Thailand, iDES provided operationally relevant data on drug efficacy, enabling the rapid amendment of treatment guidelines to improve patient outcomes and reduce the potential for the spread of drug-resistant parasites. A strong case-based surveillance system, integration with other health system processes, supporting biomarker collection and molecular analyses, and cross-border collaboration may maximize the potential of iDES in countries moving towards elimination.

Background: Resistance to anti-malarials is a major threat to the control and elimination of malaria. Sulfadoxinepyrimethamine (SP) anti-malarial treatment was used as a national policy for treatment of uncomplicated falciparum malaria in Thailand from 1973 to 1990. In order to determine whether withdrawal of this antifolate drug has led to restoration of SP sensitivity, the prevalence of genetic markers of SP resistance was assessed in historical Thai samples.Methods: Plasmodium falciparum DNA was collected from the Thailand-Myanmar, Thailand-Malaysia and Thailand-Cambodia borders during 2008-2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated.Results: Mutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr-pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr-pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand-Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr-pfdhps haplotypes. In particular, the prevalence of pfdhfr-pfdhps, septuple mutation was observed in the Thailand-Myanmar (50%, 73/146) and Thailand-Cambodia (65%, 26/40) border. In Thailand-Malaysia border, majority of the pfdhfr-pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008-2016. Within the pfdhfr-pfdhps haplotypes, during 2008-2013 the pfdhps A/S436F mutation was observed only in Thailand-Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (φ = 0.213, p-value = 0.001) or pfdhps K540E/N (φ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification. Conclusions:Despite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation.

Background Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs. Methods A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay. Results There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated. Conclusions The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.