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Background: Polymorphisms of Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multi-drug resistance 1 (pfmdr1) and Plasmodium falciparum kelch 13-propeller (pfk13) genes are accepted as valid molecular markers of quinoline antimalarials and artemisinins. This study investigated the distribution patterns of these genes in P. falciparum isolates from the areas along the Thai-Myanmar border during the two different periods of antimalarial usage in Thailand. Results: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect pfcrt mutations at codons 76, 220, 271, 326, 356, and 371 as well as pfmdr1 mutation at codon 86. The prevalence of pfcrt mutations was markedly high (96.4–99.7%) in samples collected during both periods. The proportions of mutant genotypes (number of mutant/total isolate) at codons 76, 220, 271, 326, 356 and 371 in the isolates collected during 1993–1998 (period 1) compared with 2002–2008 (period 2) were 97.9% (137/140) vs. 97.1% (401/413), 97.9% (140/143) vs. 98.8% (171/173), 97.2% (139/143) vs. 97.1% (333/343), 98.6% (140/142) vs. 99.7% (385/386), 96.4% (134/139) vs. 98.2% (378/385) and 97.8% (136/139) vs. 98.9% (375/379), respectively. Most isolates carried pfmdr1 wild-type at codon 86, with a significant difference in proportions genotypes (number of wild type/total sample) in samples collected during period 1 [92.9% (130/140)] compared with period 2 [96.9% (379/391)]. Investigation of pfmdr1 copy number was performed by real-time PCR. The proportions of isolates carried 1, 2, 3 and 4 or more than 4 copies of pfmdr1 (number of isolates carried correspondent copy number/total isolate) were significantly different between the two sample collecting periods (65.7% (90/137) vs. 87.8% (390/444), 18.2% (25/137) vs. 6.3%(28/444), 5.1% (7/137) vs. 1.4% (6/444) and 11.0% (15/137) vs. 4.5% (20/444), for period 1 vs. period 2, respectively). No pfk13 mutation was detected by nested PCR and nucleotide sequencing in all samples with successful analysis (n = 68). Conclusions: The persistence of pfcrt mutations and pfmdr1 wild-types at codon 86, along with gene amplification in P. falciparum, contributes to the continued resistance of chloroquine and mefloquine in P. falciparum isolates in the study area. Regular surveillance of antimalarial drug resistance in P. falciparum, incorporating relevant molecular markers and treatment efficacy assessments, should be conducted. [ABSTRACT FROM AUTHOR]

A 3-day artesunate-mefloquine combination therapy has been using as first-line treatment for acute uncomplicated Plasmodium falciparum malaria in Thailand since 1995 on the background of mefloquine resistance. The aim of the present study was to assess sensitivity of P. falciparum isolates (n = 44) in an area along the Thai-Myanmar border (year 2009) to artesunate, mefloquine, chloroquine and quinine, including their correlation with clinico-parasitological response. Twenty, 19, and 5 isolates were collected from patients with ‘Adequate Clinical and Parasitological Response (ACPR)’, ‘Late Parasitological Failure (LPF)’ and ‘re-infection’, respectively. The IC 50 of artesunate and mefloquine were significantly higher in patients with LPF compared with ACPR and re-infection. The proportion of isolates with declined artesunate or mefloquine sensitivity in the LPF group (47.4%) was significantly higher than the ACPR group (5.0%). A weak but statistical significant correlation ( r = 0.384, p = 0.01) was observed between IC 50 values of artesunate and parasite clearance time (PCT). There was no significant relationship between in vitro sensitivity of parasite isolates to chloroquine or quinine and clinical response. In vitro susceptibility of P. falciparum isolates to artesunate and mefloquine may be used as a useful reliable tool to predict clinico-pathological response following a 3-day artesunate-mefloquine combination therapy. [ABSTRACT FROM AUTHOR]

Background: A markedly high failure rate of three-day artesunate-mefloquine was observed in the area along the Thai-Myanmar border. Methods: Identification of Plasmodium falciparum isolates with intrinsic resistance to each component of the artesunate-mefloquine combination was analysed with integrated information on clinico-parasitological response, together with systemic drug exposure (area under blood/plasma concentration-time curves (AUC)) of dihydroartemisinin and mefloquine, and in vitro sensitivity of P. falciparum in a total of 17 out of 29 P. falciparum isolates from patients with acute uncomplicated falciparum malaria. Analysis of the contribution of in vitro parasite sensitivity and systemic drug exposure and relationship with pfmdr1 copy number in the group with sensitive response was performed in 21 of 69 cases. Results: Identification of resistance and/or reduced intrinsic parasitocidal activity of artesunate and/or mefloquine without pharmacokinetic or other host-related factors were confirmed in six cases: one with reduced sensitivity to artesunate alone, two with resistance to mefloquine alone, and three with reduced sensitivity to artesunate combined with resistance to mefloquine. Resistance and/or reduced intrinsic parasitocidal activity of mefloquine/ artesunate, together with contribution of pharmacokinetic factor of mefloquine and/or artesunate were identified in seven cases: two with resistance to mefloquine alone, and five with resistance to mefloquine combined with reduced sensitivity to artesunate. Pharmacokinetic factor alone contributed to recrudescence in three cases, all of which had inadequate whole blood mefloquine levels (AUC0-7days). Other host-related factors contributed to recrudescence in one case. Amplification of pfmdr1 (increasing of pfmdr1 copy number) is a related molecular marker of artesunate-mefloquine resistance and seems to be a suitable molecular marker to predict occurrence of recrudescence. Conclusions: Despite the evidence of a low level of a decline in sensitivity of P. falciparum isolates to artemisinins in areas along the Thai-Myanmar border, artemisinin-based combination therapy (ACT) would be expected to remain the key anti-malarial drug for treatment of multidrug resistance P. falciparum. Continued monitoring and active surveillance of clinical efficacy of ACT, including identification of true artemisinin resistant parasites, is required for appropriate implementation of malaria control policy in this area. [ABSTRACT FROM AUTHOR]

Duffy binding–like domain (DBL) and cysteine-rich interdomain region (CIDR) domain genes of Plasmodium falciparum erythrocyte membrane protein 1 (Pf EMP1) encode malaria virulence proteins. The variants of these genes have been reported to be associated with severe/complicated malaria. The present study investigated the prevalence and distribution patterns of DBLα0.6/9, DBLα1.1, DBLα1 not var3 genes, DBLα2/α1.1/2/4/7, DBLβ12 & DBLβ3/5, DBLε8, CIDRα1.4, and CIDRα1.6 of P. falciparum isolates along the Thai-Myanmar border. The association between Pf EMP1 variants and parasite density was also investigated. Two hundred and thirteen finger-prick dried blood spot (DBS) or whole blood samples were collected in 2007 and 2015, from patients with acute uncomplicated P. falciparum in Tak, Kanchanaburi, and Ranong provinces. Analysis of the variant genes was performed using polymerase chain reaction (PCR). The DBLs variant which was found at the highest and lowest frequencies in the three provinces were DBLα1 not var3 (72.77%), and DBLε8 (17.37%). The two CIDR domain variants were found at relatively lower frequencies compared with DBL domain variants (9.9% and 30.1%). P. falciparum isolates carrying the four Pf EMP1 variants, i.e., DBLα0.6/9, DBLα1.1, DBLα2/α.1.1/2/4/7, and DBLε8 were found to be significantly associated with low parasitemia. Both DBLα0.6/9 and DBLα2/α1.1/2/4/7 variant genes which were present at high frequencies in this border area could be potential candidate markers for predicting P. falciparum hyperparasitemia and in this border area. Furthermore, the information could be exploited as candidate proteins for the development of an effective malaria vaccine in specific malaria-endemic areas. [ABSTRACT FROM AUTHOR]

Background&objectives: Changes in parasite biology, particularly the gametocytogenesis process, could be one of the important contributing factors for worldwide malaria resurgence. The present study investigated the prevalence rates of pretreatment gametocyte carriage and density in Plasmodium falciparum and P. vivax infections in the low malaria-endemic area on the Thai-Myanmar border., Methods: One hundred and twenty-six blood samples were collected from patients with signs and symptoms of malaria who attended malaria clinics. Malaria positive cases detected by microscopic examination were confirmed by species-specific nested-PCR in 97 (29 and 68 samples for P. falciparum and P. vivax, respectively)., Results: The proportion of P. vivax and P. falciparum-infected samples was 70.1: 29.9%. The density in P. falciparum positive samples [median (95%CI): 10,340 (5280-19,200) μ/l] was significantly higher than P. vivax positive samples [4508 (3240-6120) μ/l]. Sixteen out of twenty-nine (55.2%) and 36 out of 68 (52.9%) P. falciparum- and P. vivax-infected samples, respectively, were gametocyte-positive. Gametocyte density in the P. falciparum-infected[124 (69-253) /μl] was significantly higher than that of the P. vivax-infected [54 (45-70)/μl] samples. A significant correlation between gametocyte density and pretreatment parasitemia was only detected in P. falciparum-infected, but not P. vivax-infected samples., Interpretation & Conclusion: The observed high prevalence rates of pretreatment gametocyte carriage of both malaria species, which serves as a large malaria reservoir, particularly in P. falciparum infection, could have a significant impact on malaria control in the endemic populations., Competing Interests: None

The K13 propeller domain mutation and pfmdr1 amplification have been proposed as useful molecular markers for detection and monitoring of artemisinin resistant Plasmodium falciparum Welch, 1897. Genomic DNA isolates of P. falciparum was extracted from 235 dried blood spot or whole blood samples collected from patients with uncomplicated falciparum malaria residing in areas along the Thai-Myanmar border during 2006-2010. Nested polymerase chain reaction (PCR) and sequencing were performed to detect mutations in K13 propeller domain of P. falciparum at codon 427-709. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR. High prevalence of pfmdr1 multiple copies was observed (42.5% of isolates). The presence of K13 mutations was low (40/235, 17.2%). Seventeen mutations had previously been reported and six mutations were newly detected. The C580Y was found in two isolates (0.9%). The F446I, N458Y and P574L mutations were commonly detected. Seven isolates had both K13 mutation and pfmdr1 multiple copies. It needs to be confirmed whether parasites harbouring both K13 mutation and pfmdr1 multiple copies and/or the observed new mutations of K13 propeller domain are associated with clinical artemisinin resistance.

Objective: To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum (P. falciparum) isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border., Methods: Dried blood spot samples were collected from 172 falciparum malaria patients prior received treatment. The samples were extracted using chelex to obtain parasite DNA. PCR-RFLP was employed to detect pfcrt mutation at codons 76, 220, 271, 326, 356 and 371, and the pfmdr1 mutation at codon 86. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR., Results: Mutant alleles of pfcrt and wild type allele of pfmdr1 were found in almost all samples. Pfmdr1 gene copy number in isolates collected from all areas ranged from 1.0 to 5.0 copies and proportion of isolates carrying>1 gene copies was 38.1%. The distribution and patterns of pfcrt and pfmdr1 mutations were similar in P. falciparum isolates from all areas. However, significant differences in both number of pfmdr1 copies and prevalence of isolates carrying>1 gene copies were observed among isolates collected from different areas. The median pfmdr1 copy number in P. falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0, respectively and more than half of the isolates carried>1 gene copies., Conclusions: The observation of pfmdr1 wild type and increasing of gene copy number may suggest declining of artesunate-mefloquine treatment efficacy in P. falciparum isolates in this border area., (Copyright © 2013 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.)