BackgroundG6PD deficiency is common in malaria endemic regions and is estimated to affect more than 400 million people worldwide. Treatment of malaria patients with the anti-malarial drug primaquine or other 8-aminoquinolines may be associated with potential haemolytic anaemia. The aim of the present study was to investigate the prevalence of G6PD variants in Thai population who resided in malaria endemic areas (western, northern, north-eastern, southern, eastern and central regions) of Thailand, as well as the Burmese population who resided in areas along the Thai-Myanmar border.MethodsThe ten common G6PD variants were investigated in dried blood spot samples collected from 317 Thai (84 males, 233 females) and 183 Burmese (11 males, 172 females) populations residing in malaria endemic areas of Thailand using PCR-RFLP method.ResultsFour and seven G6PD variants were observed in samples collected from Burmese and Thai population, with prevalence of 6.6% (21/317) and 14.2% (26/183), respectively. Almost all (96.2%) of G6PD mutation samples collected from Burmese population carried G6PD Mahidol variant; only one sample (3.8%) carried G6PD Kaiping variant. For the Thai population, G6PD Mahidol (8/21: 38.1%) was the most common variant detected, followed by G6PD Viangchan (4/21: 19.0%), G6PD Chinese 4 (3/21: 14.3%), G6PD Canton (2/21: 9.5%), G6PD Union (2/21: 9.5%), G6PD Kaiping (1/21: 4.8%), and G6PD Gaohe (1/21: 4.8%). No G6PD Chinese 3, Chinese 5 and Coimbra variants were found. With this limited sample size, there appeared to be variation in G6PD mutation variants in samples obtained from Thai population in different regions particularly in the western region.ConclusionsResults indicate difference in the prevalence and distribution of G6PD gene variants among the Thai and Burmese populations in different malaria endemic areas. Dosage regimen of primaquine for treatment of both Plasmodium falciparum and Plasmodium vivax malaria may need to be optimized, based on endemic areas with supporting data on G6PD variants. Larger sample size from different malaria endemic is required to obtain accurate genetic mapping of G6PD variants in Burmese and Thai population residing in malaria endemic areas of Thailand.

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BackgroundThe decline in efficacy of artesunate (AS) and mefloquine (MQ) is now the major concern in areas along the Thai-Cambodian and Thai-Myanmar borders.MethodsThe correlation between polymorphisms of pfatp6, pfcrt, pfmdr1 and pfmrp1 genes and in vitro sensitivity of Plasmodium falciparum isolates to the artemisinin-based combination therapy (ACT) components AS and MQ, including the previously used first-line anti-malarial drugs chloroquine (CQ) and quinine (QN) were investigated in a total of 119 P. falciparum isolates collected from patients with uncomplicated P. falciparum infection during 2006–2009.ResultsReduced in vitro parasite sensitivity to AS [median (95% CI) IC50 3.4 (3.1-3.7) nM] was found in 42% of the isolates, whereas resistance to MQ [median (95% CI) IC50 54.1 (46.8-61.4) nM] accounted for 58% of the isolates. Amplification of pfmdr1 gene was strongly associated with a decline in susceptibility of P. falciparum isolates to AS, MQ and QN. Significant difference in IC50 values of AS, MQ and QN was observed among isolates carrying one, two, three, and ≥ four gene copies [median (95% CI) AS IC50: 1.6 (1.3-1.9), 1.8 (1.1-2.5), 2.9 (2.1-3.7) and 3.1 (2.5-3.7) nM, respectively; MQ IC50: 19.2 (15.8-22.6), 37.8 (10.7-64.8), 55.3 (47.7-62.9) and 63.6 (49.2-78.0) nM, respectively; and QN IC50: 183.0 (139.9-226.4), 256.4 (83.7-249.1), 329.5 (206.6-425.5) and 420.0 (475.2-475.6) nM, respectively]. The prevalence of isolates which were resistant to QN was reduced from 21.4% during the period 2006–2007 to 6.3% during the period 2008–2009. Pfmdr1 86Y was found to be associated with increased susceptibility of the parasite to MQ and QN. Pfmdr1 1034C was associated with decreased susceptibility to QN. Pfmrp1 191Y and 1390I were associated with increased susceptibility to CQ and QN, respectively.ConclusionHigh prevalence of CQ and MQ-resistant P. falciparum isolates was observed during the four-year observation period (2006–2009). AS sensitivity was declined, while QN sensitivity was improved. Pfmdr1 and pfmrp1 appear to be the key genes that modulate multidrug resistance in P. falciparum.

The recent reports of high failure rates and decline in in vitro sensitivity of Plasmodium falciparum to artemisinin-based combination therapies (ACTs) suggest the possibility of clinical artemisinin resistance along the Thai-Cambodian and Thai-Myanmar borders. The study investigated cellular mechanisms of action and resistance of P. falciparum to artesunate (stage specific activity, interaction with hemozoin, and anti-oxidant levels) in the two paired P. falciparum isolates (MSF046 and MSF060) collected before treatment with a 3-day artesunate-mefloquine and at the time of recrudescence. In addition, the link of these cellular mechanisms to the polymorphisms of the candidate artemisinin-resistant genes (pfatp6, pfcrt, pfmdr1, pfmrp1, and K13 propeller) was also investigated. Morphological change was observed in both pairs of the primary and recrudesced P. falciparum isolates during 12-48 h of exposure to artesunate (at IC). A marked decrease in parasite viability was found in the recrudesced isolates of both MSF046 and MSD060. The extent of the reduction (% change of baseline) in total glutathione concentrations was significantly lower in recrudesced (32.1 and 1.7%) compared with primary (45.5 and 53.7%) isolates of both MSF046 and MSF060. The extent of reduction of hemozoin content in MSF046 was significantly higher in the recrudesced (76.8%) isolate compared with the primary isolate (99.5%). For MSF060 on the other hand, increase in hemozoin content was found in the recrudesced isolate and the extent of such increase was significantly higher in recrudesced (93.1%) than the primary isolate (87.5%). Polymorphism of K13 (N458Y) together with pfmdr1 copy number correlated well with sensitivity of both isolates to artesunate. Results of this preliminary study suggests possible role of glutathione-dependent detoxification system as well as heme degradation as cellular mechanisms of action and resistance of artemisinins.

Abstract. The aim of this study was to investigate the association between genetic polymorphisms of Plasmodium falciparum chloroquine resistance transporter ( pfcrt ), P. falciparum multidrug resistance 1 ( pfmdr1 ), and P. falciparum ATPase ( pfatp6 ) and clinical outcome after a three-day mefloquine-artesunate combination therapy in 134 patients with uncomplicated Plasmodium falciparum malaria in an area with multidrug resistance along the Thailand-Myanmar border. Analysis of gene mutation and amplification were performed by nested real-time polymerase chain reaction and SYBR Green I real-time polymerase chain reaction, respectively. The mutation for pfcrt (codons 76, 220, 271, 326, 356, and 371) was found in all isolates (100%), whereas no mutation of pfmdr1 (codon 86) and pfatp6 (codons 37, 693, 769, 898) was found. The Pfmdr1 copy number was significantly higher in isolates with recrudescence (median number = 2.44) compared with a sensitive response (median number = 1.44). The gene copy number was also found to be significantly higher in paired isolates collected before treatment and at the time of recrudescence. All isolates carried one pfatp6 gene copy.