Poonuch Muhamad

Molecular analysis of pfatp6 and pfmdr1 polymorphisms and their association with in vitro sensitivity in Plasmodium falciparum isolates from the Thai-Myanmar border.

The association between pfatp6, pfmdr1 polymorphisms (gene mutation and amplification) and in vitro susceptibility to mefloquine (MQ), artesunate (AS), quinine (QN), and chloroquine (CQ) was investigated in a total of sixty-three Plasmodium falciparum isolates collected from the Thai-Myanmar border. The mutations of pfatp6 at codons R37K, G639D, S769N, and I898I and of pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Pfatp6 and pfmdr1 gene copy numbers were analyzed by quantitative real time-polymerase chain reaction (qRT-PCR). In vitro susceptibility test was successful in 58 culture-adapted isolates. Median (range) IC(50) values for MQ, AS, QN, and CQ were 28.96 (3.4-100.5), 1.74 (0.8-5.57), 223.9 (14.99-845.47), and 69.93 (9.6-183.18) nM, respectively. There was a significant positive correlation (R(2) = 0.58) of parasite susceptibility to MQ, AS, and QN. Twelve isolates showed marked decline in susceptibility to AS [median (range) IC(50) = 3.78 (3.07-5.57) nM]. Almost all isolates carried wild-type pfatp6 and pfmdr1 alleles at the investigated codons, while only three isolates (5%) carried pfmdr1 mutation alleles at codon 86. Mutation at codon 86 was associated with a significant increase in the susceptibility of parasite isolates to MQ and QN. All of the sixty-three isolates carried only one pfatp6 copy number. Thirty-three out of the 58 isolates showed increase in pfmdr1 gene copies, which was associated with reduced in vitro susceptibility to MQ, AS, and QN. No association between mutation or amplification of pfatp6 gene and in vitro susceptibility of P. falciparum isolates was found.

Monitoring of clinical efficacy and in vitro sensitivity of Plasmodium vivax to chloroquine in area along Thai Myanmar border during 2009-2010

BackgroundIn Thailand, the proportion of Plasmodium vivax infection has become equal to Plasmodium falciparum. Reports of a trend of gradual decline of in vitro sensitivity of P. vivax to chloroquine in some areas of the country, together with accumulating evidences of chloroquine resistance P. vivax in other parts of the world, emphasize the need for closely and continuously monitoring clinical efficacy in conjunction with in vitro sensitivity of P. vivax isolates.MethodsThe study was conducted at Mae Tao clinic for migrant workers, Tak Province during March 2008 - August 2009. A total of 130 patients (17 Thais and 113 Burmeses; 64 males and 66 females) with mono-infection of P. vivax malaria, aged between 15-60 years and weighing more than 40 kg, were included in the study. Patients received treatment with chloroquine (2,000 mg chloroquine phosphate over three days) and the anti-relapse drug primaquine (15 mg for 14 days). In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition assay.ResultsAll patients showed satisfactory response to treatment. The cure rate was virtually 100% within the follow-up period of 42 days. Neither recurrence of P. vivax parasitaemia nor appearance of P. falciparum occurred during the investigation period. In vitro data showed a stable sensitivity of chloroquine in this area since 2006. Geometric mean and median (95% CI) values of IC50 for chloroquine were 100.1 and 134.7 (1.1-264.9) nM, respectively.ConclusionIn vivo results suggest that the standard regimen of chloroquine was still very effective for the treatment of blood infections with P. vivax in the Thai-Myanmar border area. In vitro sensitivity data however, raise the possibility of potential advent of resistance in the future. Regular monitoring of the chloroquine sensitivity of P. vivax is essential to facilitate the early recognition of treatment failures and to expedite the formulation of appropriate changes to the drug policy.

Four years' monitoring of in vitro sensitivity and candidate molecular markers of resistance of Plasmodium falciparum to artesunate-mefloquine combination in the Thai-Myanmar border.

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.

Plasmodium vivax Drug Resistance Genes; Pvmdr1 and Pvcrt-o Polymorphisms in Relation to Chloroquine Sensitivity from a Malaria Endemic Area of Thailand

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 μl) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.

Polymorphisms of Molecular Markers of Antimalarial Drug Resistance and Relationship with Artesunate-Mefloquine Combination Therapy in Patients with Uncomplicated Plasmodium falciparum Malaria in Thailand

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.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

OBJECTIVE To compare the applicability of the SYBR Green-I assay with the standard schizont maturation assay, for determination of sensitivity of Plasmodium vivax (P. vivax) to chloroquine and a new antifolate WR 99210. METHODS The study was conducted at Mae Tao Clinic for migrant workers, Tak Province during April 2009 to July 2010. A total of 64 blood samples (1 mL blood collected into sodium heparinized plastic tube) were collected from patients with mono-infection with P. vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-I assays. RESULTS A total of 30 out of 64 blood samples collected from patients with P. vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays. The failure rates of the schizont maturation inhibition assay (50%) and the SYBR Green-I assay (54%) were similar (P=0.51). The median IC10s, IC50s and IC90s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P. vivax tested. Based on the cut-off of 100 nM, the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates, respectively. The strength of agreement between the two methods was very poor for both chloroquine and WR99210. CONCLUSIONS On the basis of this condition and its superior sensitivity, the microscopic method appears better than the SYBR Green-I Green assay for assessing in vitro sensitivity of fresh P. vivax isolates to antimalarial drugs.

Preliminary Investigation of the Contribution of CYP2A6, CYP2B6, and UGT1A9 Polymorphisms on Artesunate-Mefloquine Treatment Response in Burmese Patients with Plasmodium falciparum Malaria

CYP2A6, CYP2B6, and UGT1A9 genetic polymorphisms and treatment response after a three-day course of artesunate-mefloquine was investigated in 71 Burmese patients with uncomplicated Plasmodium falciparum malaria. Results provide evidence for the possible link between CYP2A6 and CYP2B6 polymorphisms and plasma concentrations of artesunate/dihydroartemisinin and treatment response. In one patient who had the CYP2A6*1A/*4C genotype (decreased enzyme activity), plasma concentration of artesunate at one hour appeared to be higher, and the concentration of dihydroartemisinin was lower than for those carrying other genotypes (415 versus 320 ng/mL). The proportion of patients with adequate clinical and parasitologic response who had the CYP2B6*9/*9 genotype (mutant genotype) was significantly lower compared with those with late parasitologic failure (14.0% versus 19.0%). Confirmation through a larger study in various malaria-endemic areas is required before a definite conclusion on the role of genetic polymorphisms of these drug-metabolizing enzymes on treatment response after artesunate-based combination therapy can be made.

SYBR Green I and Taqman Quantitative Real-Time Polymerase Chain Reaction Methods for the Determination of Amplification of Plasmodium falciparum Multidrug Resistance-1 Gene (PFMDR1)

abstract:  The pfmdr1 gene, which encodes P-glycoprotein homolog 1, has been shown to be a reliable marker of resistance for Plasmodium falciparum related to artesunate and mefloquine combination therapy. The aims of this study are to investigate the copy number of pfmdr1 in P. falciparum isolates collected from the 4 malaria-endemic areas of Thailand (Kanchanaburi, Mae Hongson, Ranong, and Tak) along the Thailand–Myanmar (Burma) border (Thai–Myanmar border) by using SYBR Green I and the standard method TaqMan real-time polymerase chain reaction (RT-PCR) and to compare the efficiency (sensitivity and specificity) of SYBR Green I with TaqMan RT-quantitative (q)PCR methods in determining pfmdr1 gene copy number. Ninety-six blood samples were collected onto filter paper from patients with uncomplicated falciparum malaria who attended malaria clinics in the Kanchanaburi (n  =  45), Mae Hongson (n  =  18), Ranong (n  =  11), and Tak (n  =  22) provinces in Thailand. Parasite genomic DNA was extracted from dried blood spots by using QIAcube™ automated sample preparation. Pfmdr1 gene copy number was determined by TaqMan (63 samples) and SYBR Green I (96 samples) real-time PCR. Seventy-one (74.0%), 14 (14.6%), 10 (10.4%), and 1 (1%) isolates carried 1, 2, 3, and 4 pfmdr1 gene copies, respectively. Forty-three of 48 (89.6%), 6 of 11 (54.5%), and 3 of 4 (75.0%) samples, respectively, showed agreement with results of 1, 2, and 3 pfmdr1 gene copies as determined by both methods. The efficiency of SYBR Green I in identifying pfmdr1 gene copy number was found to be significantly correlated with that of TaqMan. Considering its simplicity and relatively low cost, SYBR Green I RT-qPCR is therefore a promising alternative technique for the determination of pfmdr1 copy number.

Genetic polymorphisms of candidate markers and in vitro susceptibility of Plasmodium falciparum isolates from Thai-Myanmar border in relation to clinical response to artesunate-mefloquine combination.

The genetic polymorphisms of the candidate markers of antimalarial drug resistance pfcrt, pfmdr1, pfatp6, and pfmrp1 were investigated in relationship with in vitro susceptibility of Plasmodium falciparum isolates and clinical response following artesunate (AS)-mefloquine (MQ) combination in 21 and 27 samples obtained from patients with recrudescence and adequate clinical response, respectively. MQ (21.0 vs. 49.9nM) and AS (1.6 vs. 2.8nM) IC50 values (concentrations that inhibit parasite growth by 50%) were significantly higher in isolates collected from patients with recrudescence. Furthermore, a significantly higher MQ IC50 was found in isolates from patients with recrudescence that carried pfmrp1 mutations at amino acid residues 191Y, 437A, and 876V. For AS sensitivity, a significant association was also detected in isolates from patients with recrudescence that carried gene mutations at amino acid residues 437A and 876V. MQ IC50 of the isolates with recrudescence which carried ≥4 pfmdr1 gene copies was significantly higher than that carrying only one gene copy. In addition, a significantly higher proportion of isolates carrying one gene copy was detected in the group with adequate clinical response compared with recrudescence. Results from this limited sample size suggested the potential link between pfmdr1 gene copy number and pfmrp1 gene mutation, in vitro parasite susceptibility, and AS-MQ treatment response.