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Abstract: Malaria is one of the major causes of morbidity and mortality worldwide. The major factor which has aggravated the situation is the emergence of multidrug resistant Plasmodium falciparum malaria. To successfully deal with the problem, thorough understanding of the molecular bases for reduced parasite sensitivity to existing antimalarial drugs is of considerable importance. The objective of this work was to broaden the insight into the molecular mechanisms of resistance of P. falciparum to quinoline-containing antimalarials and artemisinin derivatives. Polymorphisms of the candidate genes pfmdr1 and pfcrt were investigated in relation to the susceptibility (in vitro sensitivity) of P. falciparum isolates to chloroquine (CQ), mefloquine (MQ), quinine (QN) and the artemisinin derivative – artesunate (AS). A total of 26 P. falciparum isolates were successful cultured. In vitro sensitivity results indicate the increase in susceptibility of P. falciparum strains in Thailand to CQ, while the susceptibility to MQ and QN was markedly declined. The pattern of cross-resistance was observed between MQ vs QN vs AS. Only one point mutation in the pfmdr1 gene, i.e., N86Y was observed with low prevalence of 7.7% (2/26). In contrast, the mutations at positions 76T, 220S, 271E, 326S, 356T and 371I in the pfcrt gene were identified in almost all isolates (25 isolates, 96.2%). The association between polymorphisms of the pfmdr1 and susceptibility of the parasite to MQ and QN was observed (increased susceptibilities to MQ and QN in isolates with mutations). Moreover, the correlation between pfmdr1 gene amplification and susceptibility of the parasite to MQ, QN and AS was observed (decreased susceptibilities to MQ, QN and AS in isolates with increased pfmdr1 copy number). [Copyright &y& Elsevier]

Abstract: The treatment and control of malaria is becoming increasingly difficult due to resistance of Plasmodium falciparum strains resistance to commonly used antimalarials. Combination therapy is currently the strategy for combating multi-drug resistant falciparum malaria, through exploiting phamacodynamic synergistic effect and delaying the emergence of drug resistance. The objective of the present study was to investigate antimalarial activity of inhibitors of cytochrome P450 (CYP) enzyme including their interactions with the antimalarial mefloquine against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) P. falciparum clones in vitro. Results showed IC50 (drug concentration which produces 50% schizont maturation inhibition) values [mean (range)] of mefloquine against K1 and 3D7 clones to be 8.6 (8.0–9.3) and 12.1 (10.5–13.8) nM, respectively. The corresponding values for the IC50 of quinidine were 32.2 (31.9–32.5) and 28.7 (28.4–29.0) nM, and for ketoconazole were 3.9 (3.7–4.1) and 4.8 (4.6–5.1) μM, respectively. Analysis of isobologram revealed a trend of decreasing of fraction IC50 (FIC), which indicates synergistics of the either quinidine or ketoconazole with mefloquine for both chloroquine-resistant and chloroquine-sensitive clones. [Copyright &y& Elsevier]

The emergence of drug-resistant Plasmodium vivax poses problems for malaria control and elimination in some parts of the world, especially in developing countries where individuals are routinely exposed to the infection. The aim of this study was to determine the single nucleotide polymorphisms (SNPs) in dihydropteroate synthase ( pvdhps ) and dihydrofolate reductase ( pvdhfr ) genes associated with sulfadoxine-pyrimethamine (SP) drug resistance among P . vivax isolates collected in Palawan, Philippines. Genetic polymorphisms of pvdhps and pvdhfr were analysed by nested PCR. Analysis at specific codons I 13 P 33 F 57 S 58 T 61 S 117 I 173 associated with pyrimethamine resistance in the pvdhfr gene revealed that most of the samples (66/87, 75.9%) carried double mutation at positions I 13 P 33 F 57 R 58 T 61 N 117 I 173 , while only 18.4% (16/87) of the isolates carried the wild-type haplotype (I 13 P 33 F 57 S 58 T 61 S 117 I 173 ). For the pvdhps gene, the codons involved in sulfadoxine resistance S 382 A 383 K 512 A 553 V 585 were investigated. Single mutation at position S 382 G 383 K 512 A 553 V 585 was most observed in 68.0% (68/100) of the samples, whereas wild-type haplotype was found in 26.0% (26/100) of samples. The pvdhps and pvdhfr combination S 382 A 383 K 512 A 553 V 585 /I 13 P 33 F 57 S 58 T 61 S 117 I 173 (wild-type), S 382 G 383 K 512 A 553 V 585 /I 13 P 33 F 57 R 58 T 61 N 117 I 173 , and S 382 A 383 K 512 A 553 V 585 -I 13 P 33 F 57 R 58 T 61 N 117 I 173 were the most frequently observed combination haplotypes from the three study sites. The information on molecular markers associated with antifolate drug-resistance could help better understanding ofthe molecular epidemiology and situation of SP resistant P. vivax malaria in the country. Continuous surveillance of these genetic markers is necessary to monitor the evolution of SP resistance in the Philippines. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study was to find evidence for a homologous protein of the mammalian cytochrome P450 family member CYP2B1/B2 in Plasmodium falciparum at the nucleic acid level. Prior research had demonstrated enzyme activity in the parasite comparable to mammalian CYP1A, 2A, 2B and 2E enzymes and presence of CYP enzymes by spectrophotometric and electrophoretic analyses. In recent years, the transcriptome/proteome data of P. falciparum and other Plasmodium spp. have been published and we performed an in silico analysis to identify putative cytochrome P450 family members in the parasite. This analysis failed to identify homologs to CYP1A, 2A, 2B and 2E enzymes in Plasmodium. A prior study had also claimed the presence of a conserved CYP2B1/B2 gene in the parasite by using Northern analysis with a rat CYP2B1/B2 probe. We have repeated this analysis by cloning a rat CYP2B1/B2 cDNA and using it as a hybridization probe against total RNA extracted from P. falciparum K1 and 3D7 clones but did not obtain positive results. This is consistent with the transcriptome/proteome sequence data and suggests that the genus Plasmodium contains either only highly diverged CYP proteins which are not easily identified by their primary sequence or that they have been functionally replaced by other enzymes. It is suggested that further studies are performed that allow isolation and identification of such proteins through their functional activities. [Copyright &y& Elsevier]

Abstract: 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) IC50 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) IC50 =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. [Copyright &y& Elsevier]