Dual action effects of ethyl-p-methoxycinnamate against dengue virus infection and inflammation via NF-κB pathway suppression.

Dengue virus (DENV) infection can lead to severe outcomes through a virus-induced cytokine storm, resulting in vascular leakage and inflammation. An effective treatment strategy should target both virus replication and cytokine storm. This study identified Kaempferia galanga L. (KG) extract as exhibiting anti-DENV activity. The major bioactive compound, ethyl-p-methoxycinnamate (EPMC), significantly reduced DENV-2 infection, virion production, and viral protein synthesis in HepG2 and A549 cells, with half-maximal effective concentration (EC50) values of 22.58 µM and 6.17 µM, and impressive selectivity indexes (SIs) of 32.40 and 173.44, respectively. EPMC demonstrated efficacy against all four DENV serotypes, targeting the replication phase of the virus life cycle. Importantly, EPMC reduced DENV-2-induced cytokines (IL-6 and TNF-α) and chemokines (RANTES and IP-10), as confirmed by immunofluorescence and immunoblot analyses, indicating inhibition of NF-κB activation. EPMC's role in preventing excessive inflammatory responses suggests it as a potential candidate for dengue treatment. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness for EPMC were predicted using SwissADME and ProTox II servers, showing good drug-like properties without toxicity. These findings highlight KG extract and EPMC as promising candidates for future anti-dengue therapeutics, offering a dual-action approach by inhibiting virus replication and mitigating inflammatory reactions.

Distribution patterns of molecular markers of antimalarial drug resistance in Plasmodium falciparum isolates on the Thai-Myanmar border during the periods of 1993-1998 and 2002-2008.

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.

Antiproliferative and Anti-Inflammatory Activities of Deprungsith Formulation and Its Bioactive Compounds Against Mild Psoriasis and Potential of Metabolic Herb-Drug Interactions.

Psoriasis is an incurable, chronic and auto-immune skin disorder with a global prevalence rate of approximately 2-3%. The study investigated the antipsoriasis activities of Deprungsith formulation and its bioactive components and their potential for inhibitory activities on human cytochrome P450 (CYP450). HaCaT and peripheral blood mononuclear cells (PBMCs) from healthy volunteers (n = 9) and psoriasis patients (n = 10) were exposed to Deprungsith formulation (Thai traditional medicine for psoriasis consisting of 16 plants), ethyl p-methoxycinnamate (EPMC), ligustilide and cyclosporin for 24 and 48 h. The antiproliferative, cell apoptosis and cell cycle arrest activities were evaluated using MTT assay and flow cytometry, respectively. The pro-inflammatory cytokine mRNA expression levels were measured using real-time polymerase chain reaction (RT-PCR). The CYP450 inhibitory effect was investigated using a bioluminescent-based CYP450 assay. Deprungsith formulation and the bioactive compounds inhibited HaCaT cells and PBMCs with weak to moderate potencies. EPMC and ligustilide combination produced an additive effect. Most substances arrested cell transition at sub-G1 and S phases, leading to early and late apoptosis induction. With prolonged exposure (48 h), all test substances decreased PBMCs necrosis. The mRNA expression of all pro-inflammatory cytokines was downregulated. Deprungsith formulation, EPMC, ligustilide and ferulic acid inhibited CYP1A2, CYP2C9, CYP2D6 and CYP3A4 activities with weak to moderate potencies. Deprungsith formulation and bioactive components induced cell apoptosis by inhibiting cell transition at specific cell cycle phases, which was correlated with the mRNA downregulation of interleukin (IL-6, IL-12p19, IL-23) and tumor necrosis factor (TNF-α). There is a low risk of potential adverse drug reactions and toxicity due to CYP450 interaction when Deprungsith formulation is concurrently administered with modern medicines.

Monitoring antimalarial drug-resistance markers in Somalia.

The use of an effective antimalarial drug is the cornerstone of malaria control. However, the development and spread of resistant Plasmodium falciparum strains have placed the global eradication of malaria in serious jeopardy. Molecular marker analysis constitutes the hallmark of the monitoring of Plasmodium drug-resistance. This study included 96 P. falciparum PCR-positive samples from southern Somalia. The P. falciparum chloroquine resistance transporter gene had high frequencies of K76T, A220S, Q271E, N326S, and R371I point mutations. The N86Y and Y184F mutant alleles of the P. falciparum multidrug resistance 1 gene were present in 84.7 and 62.4% of the isolates, respectively. No mutation was found in the P. falciparum Kelch-13 gene. This study revealed that chloroquine resistance markers are present at high frequencies, while the parasite remains sensitive to artemisinin (ART). The continuous monitoring of ART-resistant markers and in vitro susceptibility testing are strongly recommended to track resistant strains in real time.

Suppression of Cholangiocarcinoma Cell Growth and Proliferation by Atractylodes lancea (Thunb) DC. through ERK-Signaling Cascade.

OBJECTIVE: The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules (ERK, p-ERK, cyclin D, and eIF4B) and the growth and proliferation of CCA cells. MATERIALS AND METHODS: The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR. RESULTS: The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The IC50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors (salirasib and XMD8-92). Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell. CONCLUSION: The ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA.

Genetic diversity and distribution patterns of PfEMP1 in Plasmodium falciparum isolates along the Thai-Myanmar border.

Duffy binding-like domain (DBL) and cysteine-rich interdomain region (CIDR) domain genes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) 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, DBLß12 & DBLß3/5, DBLε8, CIDRα1.4, and CIDRα1.6 of P. falciparum isolates along the Thai-Myanmar border. The association between PfEMP1 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 PfEMP1 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.

Pretreatment gametocyte carriage in symptomatic patients with Plasmodium falciparum and Plasmodium vivax infections on the Thai-Myanmar border.

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.

Cytotoxicity, Cell Cycle Arrest, and Apoptosis Induction Activity of Ethyl-p-methoxycinnamate in Cholangiocarcinoma Cell.

OBJECTIVE: To investigate cytotoxic activity of ethyl-p-methoxycinnamate (EPMC) including its effect on p-glycoprotein (multidrug resistance-1: mdr-1 gene) in human cholangiocarcinoma cell. METHODS: Cytotoxic activity of EPMC against human cholangiocarcinoma (CL-6), fibroblast (OUMS-36T-1F), and colon cancer (Caco-2) cell lines were assessed using MTT assay. Selectivity index (SI) was determined as the ratio of IC50 (concentration that inhibits cell growth by 50%) of EPMC in OUMS-36T-1F and that in CL-6 cell. Cell cycle arrest and apoptosis in CL-6 cells were investigated by flow cytometry and fluorescent microscopy. Effect of EPMC on mdr-1 gene expression in CL-6 and Caco-2 was determined by real-time PCR. RESULTS: The median (95% CI) IC50 values of EPMC in CL-6, OUMS-36T-1F, and Caco-2 were 245.5 (243.1-266.7), 899.60 (855.8-966.3) and 347.0 (340.3-356.9) µg/ml, respectively. The SI value of the compound for the CL-6 cell was 3.70. EPMC at IC50 inhibited CL-6 cell division and induced apoptosis compared to untreated control. EPMC exposure did not induce mdr-1 gene expression in both CL-6 and Caco-2 cells. CONCLUSION: The results suggest the potential role of EPMC in cholangiocarcinoma with a low possibility of drug resistance induction.

K13 propeller domain mutations and pfmdr1 amplification in isolates of Plasmodium falciparum collected from Thai-Myanmar border area in 2006-2010.

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.

Bioactive constituents isolated from Atractylodes lancea (Thunb.) DC. rhizome exhibit synergistic effect against cholangiocarcinoma cell.

BACKGROUND: Cholangiocarcinoma (CCA) is the primary type of bile duct cancer with high morbidity and mortality, particularly in patients with advanced-stage disease. Treatment of CCA remains unsatisfactory due to the lack of sensitive and specific diagnostic tool for early detection as well as effective chemotherapeutics. PURPOSE: To investigate cytotoxic interactions between the three major constituents of the rhizomes of Atractylodes lancea (Thunb.) DC., ie, ß-eudesmol (BE), atractylodin (AT), and hinesol (HS), against CCA cell line. METHODS: Cytotoxic activities against the human CCA cells CL-6 of the dual (BE:AT, BE:HS, and AT:HS) and triple (BE:AT:HS) combinations were evaluated using MTT assay. The cytotoxic interaction of each dual combination was assessed at five concentration ratios (10:0, 7:3, 5:5, 3:7, and 0:10) using isobologram analysis. For triple combination, the concentration ratio used in the experiment was 1:1.5:2.5 (BE:AT:HS) and analysis of the interaction was performed using polygonogram analysis at the concentrations that inhibit cell growth by 50% and 90%, respectively. RESULTS: The BE:AT combination produced the additive effect with sum fractional inhibitory concentration of 0.967±0.02 (mean ± SD). The BE:HS and AT:HS combinations produced a synergistic effect with sum fractional inhibitory concentrations of 0.685±0.08 and 0.767±0.09, respectively. The mixture of the three compounds produced synergistic interaction with combination index values of 0.519±0.10 and 0.65±0.17 (mean ± SD) at the concentrations that inhibit cell growth at the 50% and 90% leveled, respectively. CONCLUSION: Results obtained would guide further development of Atractylodes lancea (Thunb.) DC. as potential anti-CCA chemotherapeutics concerning the appropriate pharmaceutical dosage form.

Cytotoxic activities and effects of atractylodin and ß-eudesmol on the cell cycle arrest and apoptosis on cholangiocarcinoma cell line.

Cholangiocarcinoma (CCA) is the cancer of bile duct with high mortality rate particularly in Thailand. The clinical efficacy of the standard chemotherapeutics remains unsatisfactory, and therefore, discovery and development of the new alternative drugs with high efficacy and tolerability is needed. The aim of the study was to investigate cytotoxic activity as well as the underlying mechanisms through which atractylodin and ß-eudesmol exert their activities on CCA cell growth inhibition, cell cycle arrest, and cell apoptosis. Effects of the compounds on cell cytotoxicity, cell cycle arrest, and cell apoptosis were analyzed using MTT assay, BD Cycletest™ Plus DNA kit, and FITC Annexin V Apoptosis Detection Kit I, respectively. The cytotoxic activities of both compounds were concentration- and time-dependent. The IC50 [mean (SD)] of atractylodin and ß-eudesmol were 41.66 (2.51) and 39.33 (1.15) µg/ml respectively. Both promoted cell cycle arrest at G1 phase, and induced cell apoptosis through activation of caspase-3/7. The highest activity was observed at 48 h of exposure. Results suggest that these mechanisms are at least in part, explain the cell cytotoxic and anti-CCA activity of atractylodin and ß-eudesmol shown in vitro and in vivo models.

Antimalarial Activity of Piperine.

Malaria remains a public health problem in tropical and subtropical regions. Resistance of Plasmodium falciparum to artemisinins in Southeast Asia is a great concern for disease control and research on discovery and development of new alternative antimalarial drugs is urgently required. In a previous study, the fruit of Piper chaba Hunt. was demonstrated to exhibit promising antimalarial activity against the asexual stage of 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) P. falciparum clones. The aim of the present study was to further investigate the antimalarial activity of piperine, the major isolated constituent of Piper chaba Hunt. fruits against both P. falciparum clones. The antimalarial activity was determined using SYBR green-I-based assay and morphological change was observed under the light microscope with Giemsa staining. The median IC50 (concentration that inhibits parasite growth by 50%) values of piperine against 3D7 and K1 P. falciparum were 111.5 and 59 µM, respectively. A marked change in parasite morphology was observed within 48 hours of piperine exposure. Results of real-time PCR showed no effect of piperine on modulating the expression of the three genes associated with antimalarial drug resistance in P. falciparum, i.e., pfcrt, pfmdr1, and pfmrp1. Piperine could be a promising candidate for further development as an antimalarial drug based on its antimalarial potency and low risk of resistance development.

In vitro sensitivity of antimalarial drugs and correlation with clinico-parasitological response following treatment with a 3-day artesunate-mefloquine combination in patients with falciparum malaria along the Thai-Myanmar border.

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 IC50 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 IC50 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.

Patients' adherence and clinical effectiveness of a 14-day course of primaquine when given with a 3-day chloroquine in patients with Plasmodium vivax at the Thai-Myanmar border.

Primaquine is the only antimalarial drug available for eradicating the hypnozoite stage of Plasmodium vivax to prevent the disease from recurring. However, one limitation of its clinical use is the long treatment course of 14 days, which may result in poor patients' adherence and low treatment efficacy. The aim of the current study was to assess patients' adherence and the clinical effectiveness of the unsupervised standard 14-day primaquine regimen (daily dose of 15mg base/kg body weight daily for 14 days) when given together with 3-day chloroquine (25mg base/kg body weight over 3 days). The study was conducted in 85 patients with P. vivax malaria in a malaria endemic area along the Thai-Myanmar border. Patients' adherence to primaquine therapy was assessed based on primaquine concentrations in finger-prick dried blood spot (DBS) samples alongside patients' self-reporting on drug administration and pill counting methods. Results suggest high rate of patients' adherence to this 14-day primaquine regimen (95-98% based on primaquine concentrations in DBS on days 3, 7, and 14 of treatment, and 100% based on patients' self-reporting and pill counting methods. Clinical effectiveness was 100% during the 42-day follow-up.

Polymorphic patterns of pfcrt and pfmdr1 in Plasmodium falciparum isolates along the Thai-Myanmar border.

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.

Identification of resistance of Plasmodium falciparum to artesunate-mefloquine combination in an area along the Thai-Myanmar border: integration of clinico-parasitological response, systemic drug exposure, and in vitro parasite sensitivity.

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.

Polymorphic patterns of pfcrt and pfmdr1 in Plasmodium falciparum isolates along the Thai-Myanmar border

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.