Molecular surveillance of Plasmodium vivax dhfr and dhps mutations in isolates from Afghanistan

<p>Abstract</p> <p>Background</p> <p>Analysis of dihydrofolate reductase (<it>dhfr</it>) and dihydropteroate synthase (<it>dhps</it>) mutations in <it>Plasmodium vivax </it>wild isolates has been considered to be a valuable molecular approach for mapping resistance to sulphadoxine-pyrimethamine (SP). The present study investigates the frequency of SNPs-haplotypes in the <it>dhfr </it>and <it>dhps </it>genes in <it>P. vivax </it>clinical isolates circulating in two malaria endemic areas in Afghanistan.</p> <p>Methods</p> <p><it>P. vivax </it>clinical isolates (n = 171) were collected in two different malaria endemic regions in north-west (Herat) and east (Nangarhar) Afghanistan in 2008. All collected isolates were analysed for SNP-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the <it>pvdhfr </it>and 383 and 553 of the <it>pvdhps </it>genes using PCR-RFLP methods.</p> <p>Results</p> <p>All 171 examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 4.1% and 12.3% of Afghan isolates, respectively. Based on the size polymorphism of <it>pvdhfr </it>genes at repeat region, type B was the most prevalent variant among Herat (86%) and Nangarhar (88.4%) isolates. Mixed genotype infections (type A/B and A/B/C) were detected in only 2.3% (2/86) of Herat and 1.2% (1/86) of Nangarhar isolates, respectively. The combination of <it>pvdhfr </it>and <it>pvdhps </it>haplotypes among all 171 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were wild-type (86%) and single mutant haplotype I₁₃P₃₃F₅₇S₅₈T₆₁<b>N </b>₁₁₇I_173/A₃₈₃A₅₅₃(6.4%).</p> <p>Double (I₁₃P₃₃S₅₇<b>R</b>₅₈T₆₁<b>N</b>₁₁₇I₁₇₃/A₃₈₃A₅₅₃) and triple mutant haplotypes (I₁₃P₃₃S₅₇<b>R </b>₅₈T₆₁<b>N</b>₁₁₇I₁₇₃/<b>G</b>₃₈₃A₅₅₃) were found in 1.7% and 1.2% of Afghan isolates, respectively. This triple mutant haplotype was only detected in isolates from Herat, but in none of the Nangarhar isolates.</p> <p>Conclusion</p> <p>The present study shows a limited polymorphism in <it>pvdhfr </it>from Afghan isolates and provides important basic information to establish an epidemiological map of drug-resistant vivax malaria, and updating guidelines for anti-malarial policy in Afghanistan. The continuous usage of SP as first-line anti-malarial drug in Afghanistan might increase the risk of mutations in the <it>dhfr </it>and <it>dhps </it>genes in both <it>P. vivax </it>and <it>Plasmodium falciparum </it>isolates, which may lead to a complete SP resistance in the near future in this region. Therefore, continuous surveillance of <it>P. vivax </it>and <it>P. falciparum </it>molecular markers are needed to monitor the development of resistance to SP in the region.</p

Detection of high levels of mutations involved in anti-malarial drug resistance in Plasmodium falciparum and Plasmodium vivax at a rural hospital in southern Ethiopia

<p>Abstract</p> <p>Background</p> <p>In Ethiopia, malaria is caused by <it>Plasmodium falciparum </it>and <it>Plasmodium vivax</it>, and anti-malarial drug resistance is the most pressing problem confronting control of the disease. Since co-infection by both species of parasite is common and sulphadoxine-pyrimethamine (SP) has been intensively used, resistance to these drugs has appeared in both <it>P. falciparum </it>and <it>P. vivax </it>populations. This study was conducted to assess the prevalence of anti-malarial drug resistance in <it>P. falciparum </it>and <it>P. vivax </it>isolates collected at a rural hospital in southern Ethiopia.</p> <p>Methods</p> <p>A total of 1,147 patients with suspected malaria were studied in different months across the period 2007-2009. <it>Plasmodium falciparum dhfr </it>and <it>dhps </it>mutations and <it>P. vivax dhfr </it>polymorphisms associated with resistance to SP, as well as <it>P. falciparum pfcrt </it>and <it>pfmdr1 </it>mutations conferring chloroquine resistance, were assessed.</p> <p>Results</p> <p>PCR-based diagnosis showed that 125 of the 1147 patients had malaria. Of these, 52.8% and 37.6% of cases were due to <it>P. falciparum </it>and <it>P. vivax </it>respectively. A total of 10 cases (8%) showed co-infection by both species and two cases (1.6%) were infected by <it>Plasmodium ovale</it>. <it>Pfdhfr </it>triple mutation and <it>pfdhfr/pfdhps </it>quintuple mutation occurred in 90.8% (95% confidence interval [CI]: 82.2%-95.5%) and 82.9% (95% CI: 72.9%-89.7%) of <it>P. falciparum </it>isolates, respectively. <it>Pfcrt </it>T76 was observed in all cases and <it>pfmdr1 </it>Y86 and <it>pfmdr1 </it>Y1246 in 32.9% (95% CI: 23.4%-44.15%) and 17.1% (95% CI: 10.3-27.1%), respectively. The <it>P. vivax dhfr </it>core mutations, N117 and R58, were present in 98.2% (95% CI: 89.4-99.9%) and 91.2% (95% CI: 80.0-96.7%), respectively.</p> <p>Conclusion</p> <p>Current molecular data show an extraordinarily high frequency of drug-resistance mutations in both <it>P. falciparum </it>and <it>P. vivax </it>in southern Ethiopia. Urgent surveillance of the emergence and spread of resistance is thus called for. The level of resistance indicates the need for implementation of entire population access to the new first-line treatment with artemether-lumefantrine, accompanied by government monitoring to prevent the emergence of resistance to this treatment.</p

Plasmodium falciparum: gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of Pyrimethamine

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22–46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed