Population pharmacokinetic and pharmacodynamic properties of artesunate in patients with artemisinin sensitive and resistant infections in Southern Myanmar.

BACKGROUND: Artemisinins are the most effective anti-malarial drugs for uncomplicated and severe Plasmodium falciparum malaria. However, widespread artemisinin resistance in the Greater Mekong Region of Southeast Asia is threatening the possibility to control and eliminate malaria. This work aimed to evaluate the pharmacokinetic and pharmacodynamic properties of artesunate and its active metabolite, dihydroartemisinin, in patients with sensitive and resistant falciparum infections in Southern Myanmar. In addition, a simple nomogram previously developed to identify artemisinin resistant malaria infections was evaluated. METHODS: Fifty-three (n = 53) patients were recruited and received daily oral artesunate monotherapy (4 mg/kg) for 7 days. Frequent artesunate and dihydroartemisinin plasma concentration measurements and parasite microscopy counts were obtained and evaluated using nonlinear mixed-effects modelling. RESULTS: The absorption of artesunate was best characterized by a transit-compartment (n = 3) model, followed by one-compartment disposition models for artesunate and dihydroartemisinin. The drug-dependent parasite killing effect of dihydroartemisinin was described using an Emax function, with a mixture model discriminating between artemisinin sensitive and resistant parasites. Overall, 56% of the studied population was predicted to have resistant malaria infections. Application of the proposed nomogram to identify artemisinin-resistant malaria infections demonstrated an overall sensitivity of 90% compared to 55% with the traditional day-3 positivity test. CONCLUSION: The pharmacokinetic-pharmacodynamic properties of artesunate and dihydroartemisinin were well-characterized with a mixture model to differentiate between drug sensitive and resistant infections in these patients. More than half of all patients recruited in this study had artemisinin-resistant infections. The relatively high sensitivity of the proposed nomogram highlights its potential clinical usefulness.

K13 mutations and pfmdr1 copy number variation in Plasmodium falciparum malaria in Myanmar.

BACKGROUND: Artemisinin-based combination therapy has been first-line treatment for falciparum malaria in Myanmar since 2005. The wide extent of artemisinin resistance in the Greater Mekong sub-region and the presence of mefloquine resistance at the Myanmar-Thailand border raise concerns over resistance patterns in Myanmar. The availability of molecular markers for resistance to both drugs enables assessment even in remote malaria-endemic areas. METHODS: A total of 250 dried blood spot samples collected from patients with Plasmodium falciparum malarial infection in five malaria-endemic areas across Myanmar were analysed for kelch 13 sequence (k13) and pfmdr1 copy number variation. K13 mutations in the region corresponding to amino acids 210-726 (including the propeller region of the protein) were detected by nested PCR amplification and sequencing, and pfmdr1 copy number variation by real-time PCR. In two sites, a sub-set of patients were prospectively followed up for assessment of day-3 parasite clearance rates after a standard course of artemether-lumefantrine. RESULTS: K13 mutations and pfmdr1 amplification were successfully analysed in 206 and 218 samples, respectively. Sixty-nine isolates (33.5 %) had mutations within the k13 propeller region with 53 of these (76.8 %) having mutations already known to be associated with artemisinin resistance. F446I (32 isolates) and P574L (15 isolates) were the most common examples. K13 mutation was less common in sites in western border regions (29 of 155 isolates) compared to samples from the east and north (40 of 51 isolates; p < 0.0001). The overall proportion of parasites with multiple pfmdr1 copies (greater than 1.5) was 5.5 %. Seven samples showed both k13 mutation and multiple copies of pfmdr1. Only one of 36 patients followed up after artemether-lumefantrine treatment still had parasites at day 3; molecular analysis indicated wild-type k13 and single copy pfmdr1. CONCLUSION: The proportion of P. falciparum isolates with mutations in the propeller region of k13 indicates that artemisinin resistance extends across much of Myanmar. There is a low prevalence of parasites with multiple pfmdr1 copies across the country. The efficacy of artemisinin-based combination therapy containing mefloquine and lumefantrine is, therefore, expected to be high, although regular monitoring of efficacy will be important.

Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.

Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia.

BACKGROUND: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. METHODS: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. RESULTS: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas. CONCLUSIONS: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

Challenges in universal coverage and utilization of insecticide-treated bed nets in migrant plantation workers in Myanmar.

BACKGROUND: High coverage of the bed nets can reduce mortality and morbidity of mosquito-borne diseases including malaria. Although the migrant workers are at high risk of malaria, there are many hidden challenges in universal coverage and utilization of the insecticide-treated nets (ITNs) in this populations. METHODS: Cross sectional study was conducted in 170 migrant workers in palm oil plantation sites in Tanintharyi Region and 175 in rubber plantation sites in Mon State. A multistage stratified cluster sampling was applied to select the participants. During household visit, face-to-face interviews using structured pre-coded, pre tested questionnaires and direct observation on installation of the bed nets was conducted. Two focus group discussions in each site were done by sample stratified purposive sampling method mainly focused on effective utilization of bed nets. RESULTS: Among them, 332 (96.2%) had a bed net and 284 (82.3%) had an ITN, while 204 (59.1%) had unused extranets. Among the ITNs users, 28.9% reported problems including insecticide smell (56.9%), dizziness (20.2%), headache (12.8%) and itchiness (9.2%). More than 75% received ITNs from health authorities and NGOs free-of-charge. More than 70% wanted to buy a net but they were unaffordable for 64% of them. On observation, only five families could show no bed net, but 80% showed 1-3 ITNs. Consistent utilization in all seasons was noted in 189 (53.1%), that was higher in palm oil plantation than rubber plantation workers (p = 0.0001) due to the nature of the work at night. Perceived malaria risk was also significantly higher ITNs consistent users than non-users (p = 0.0004) and better willingness to buy an ITN by themselves (p = 0.0005). They said that effectiveness of the ITNs was reduced after 6 months and 2-3 times washing. They wished to receive more durable smooth nets with small holes in lace. Misuses of the ITNs such as use the nets for animals and fishing, were also noted. CONCLUSION: There should be efforts to improve effective utilization of ITNs by continuous mass free distribution, durability monitoring, surveillance of insecticide resistance of the vector and behaviour change interventions in migrant plantation workers.

Field evaluation of HRP2 and pan pLDH-based immunochromatographic assay in therapeutic monitoring of uncomplicated falciparum malaria in Myanmar.

BACKGROUND: Malaria rapid diagnostic tests (RDT) are used for diagnostic purpose in malaria-endemic areas where reliable microscopy is not available. Persistence of the antigenaemia causes over-diagnosis and may limit the usefulness of the RDT in monitoring treatment. In this study, the usefulness of histidine-rich protein-2 (HRP2) and pan-specific or species-specific Plasmodium lactate dehydrogenase (pLDH) in treatment monitoring of uncomplicated falciparum malaria was carried out in an endemic setting in Myanmar. METHODS: A prospective longitudinal, single-arm, cohort study was done by microscopy to confirm Plasmodium falciparum mono-infected cases. After direct treatment with an artemether-lumefantrine combination, patients were followed up on day 3, 7, 14, 21, 28 and any other day of recurrent fever. Blood film examination and RDT were carried out on day 0 and all follow-up days. RESULTS: Out of 77 recruited falciparum cases, 63 became adequate clinical and parasitological response (ACPR) cases, and 60.3% of them were still positive for HRP2 up to day 28. Eleven out of 12 treatment failure cases (91.6%) were detected by pan pLDH. The mean duration required to become negative of HRP2 was 20 days (SD ± 6.03) and that of pan pLDH was six days with or without gametocytes and 3.7 days without gametocytes. CONCLUSION: Although treatment monitoring cannot be performed by HRP2, it can be assessed by pan pLDH-based assay after day 3 if a gametocidal drug has been administered and after day 7 if the presence of gametocytes was not excluded. The pan pLDH-based assay was a suitable test to monitor the treatment response of uncomplicated falciparum malaria patients.

Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar.

BACKGROUND: Plasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries. METHODS: A non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days) was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia), parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope), and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels. RESULTS: The median (range) parasite clearance half-life and time were 4.8 (2.1-9.7) and 60 (24-96) hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours) in approximately 1/3 of infections. Fourteen of 52 participants (26.9%) had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics. CONCLUSIONS: A subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread to this location from its site of origin in western Cambodia. Resistance containment efforts are underway in Myanmar. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000896077.