Antony Hiasindh Ashmi, Das Sindhusuta, Parija Subhash Chandra, Padhi Sanghamitra
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry 605006, India.
Department of Microbiology, Maharaja Krishna Chandra Gajapati Medical College (MKCG Medical College), Odisha 760004, India.
Genom Data. 2016 Apr 18;8:85-90. doi: 10.1016/j.gdata.2016.04.010. eCollection 2016 Jun.
Due to the widespread resistance of Plasmodium falciparum to chloroquine drug, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment. This study aims to evaluate the extent of chloroquine resistance in P. falciparum infection after the introduction of ACT. This study was carried out based on the mutation analysis in P. falciparum chloroquine resistant transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes. Identification of these molecular markers plays a significant role in monitoring and assessment of drug resistance as well as in designing an effective antimalarial drug policy in India.
Sixty blood samples were collected from patients infected with P. falciparum from JIPMER, Puducherry and MKCG Medical College, Odisha. Polymerase chain reaction-restriction fragment length polymorphism was performed, targeting the point mutation of K76T in pfcrt and N86Y in pfmdr1 gene. The PCR products were sequenced, genotyped and further analysed for amino acid changes in these codons.
The frequency of pfcrt mutation at 76th position was dominant for mutant T allele with 56.7% and wild type K, 43.3%. Majority of pfmdr1 86 allele were wild type, with N (90%) and mutant, Y (10%). Additionally, we found three haplotypes for CQ resistance, SVMNT, CVIET and CVIKT in association with the pfcrt gene. However, a poorly studied SNP in pfmdr1 gene (Y184F) associated with CQ resistance showed high frequency (70%) in P. falciparum isolates.
The point mutation K76T of pfcrt is high in P. falciparum suggesting a sustained high CQ resistance even after five years of the introduction of ACTs for antimalarial therapy. The present study suggests a strong association of CQ resistance with pfcrt T76, but not with the pfmdr1 Y86 mutation. However, sequence analysis showed that Y184F mutation on pfmdr1 gene was found to be associated with high resistance. Also, a new pfcrt haplotype 'CVIKT' associated with CQ resistance was found to be present in Indian strains of P. falciparum. The data obtained from this study helps in continuous monitoring of drug resistance in malaria and also suggests the need for careful usage of CQ in Plasmodium vivax malarial treatment.
由于恶性疟原虫对氯喹广泛耐药，以青蒿素为基础的联合疗法（ACT）已被推荐为一线治疗方法。本研究旨在评估引入ACT后恶性疟原虫感染中氯喹耐药的程度。本研究基于对恶性疟原虫氯喹抗性转运蛋白（pfcrt）和恶性疟原虫多药耐药1（pfmdr1）基因的突变分析进行。鉴定这些分子标记在监测和评估耐药性以及制定印度有效的抗疟药物政策方面发挥着重要作用。
从位于本地治里的贾瓦哈拉尔·尼赫鲁医学院（JIPMER）和奥里萨邦的MKCG医学院的恶性疟原虫感染患者中采集了60份血样。进行聚合酶链反应-限制性片段长度多态性分析，针对pfcrt基因中K76T的点突变和pfmdr1基因中N86Y的点突变。对PCR产物进行测序、基因分型，并进一步分析这些密码子中的氨基酸变化。
第76位pfcrt突变中，突变型T等位基因占主导，为56.7%，野生型K为43.3%。pfmdr1 86等位基因多数为野生型，N（90%）和突变型Y（10%）。此外，我们发现与pfcrt基因相关的三种氯喹抗性单倍型，即SVMNT、CVIET和CVIKT。然而，pfmdr1基因中一个研究较少的与氯喹抗性相关的单核苷酸多态性（Y184F）在恶性疟原虫分离株中显示出高频率（70%）。
恶性疟原虫中pfcrt的K76T点突变率很高，这表明即使在引入ACT进行抗疟治疗五年后，氯喹抗性仍持续处于较高水平。本研究表明氯喹抗性与pfcrt T76密切相关，但与pfmdr1 Y86突变无关。然而，序列分析表明pfmdr1基因上的Y184F突变与高抗性相关。此外，在印度恶性疟原虫菌株中发现了一种与氯喹抗性相关的新的pfcrt单倍型“CVIKT”。本研究获得的数据有助于持续监测疟疾的耐药性，也表明在间日疟原虫疟疾治疗中需要谨慎使用氯喹。
