Department of Zoology, University of Delhi, Delhi 110007, India.
Malar J. 2012 Jan 11;11:16. doi: 10.1186/1475-2875-11-16.
Chloroquine resistance (CQR) phenotype in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr-1 genes. Mutations at amino acid position 72-76 of pfcrt gene, here defined as pfcrt haplotype are associated with the geographic origin of chloroquine resistant parasite. Here, mutations at 72-76 and codon 220 of pfcrt gene and N86Y pfmdr-1 mutation were studied in blood samples collected across 11 field sites, inclusive of high and low P. falciparum prevalent areas in India. Any probable correlation between these mutations and clinical outcome of CQ treatment was also investigated.
Finger pricked blood spotted on Whatman No.3 papers were collected from falciparum malaria patients of high and low P. falciparum prevalent areas. For pfcrt haplotype investigation, the parasite DNA was extracted from blood samples and used for PCR amplification, followed by partial sequencing of the pfcrt gene. For pfmdr-1 N86Y mutation, the PCR product was subjected to restriction digestion with AflIII endonuclease enzyme.
In 240 P. falciparum isolates with reported in vivo CQ therapeutic efficacy, the analysis of mutations in pfcrt gene shows that mutant SVMNT-S (67.50%) and CVIET-S (23.75%) occurred irrespective of clinical outcome and wild type CVMNK-A (7.91%) occurred only in adequate clinical and parasitological response samples. Of 287 P. falciparum isolates, SVMNTS 192 (66.89%) prevailed in all study sites and showed almost monomorphic existence (98.42% isolates) in low P. falciparum prevalent areas. However, CVIETS-S (19.51%) and CVMNK-A (11.84%) occurrence was limited to high P. falciparum prevalent areas. Investigation of pfmdr-1 N86Y mutation shows no correlation with clinical outcomes. The wild type N86 was prevalent in all the low P. falciparum prevalent areas (94.48%). However, mutant N86Y was comparably higher in numbers at the high P. falciparum prevalent areas (42.76%).
The wild type pfcrt gene is linked to chloroquine sensitivity; however, presence of mutation cannot explain the therapeutic efficacy of CQ in the current scenario of chloroquine resistance. The monomorphic existence of mutant SVMNT haplotype, infer inbreeding and faster spread of CQR parasite in areas with higher P. vivax prevalance and chloroquine exposure, whereas, diversity is maintained in pfcrt gene at high P. falciparum prevalent areas.
恶性疟原虫对氯喹的耐药性（CQR）表型与 pfcr 和 pfmdr-1 基因的突变有关。pfcr 基因 72-76 位氨基酸的突变，在此定义为 pfcr 单倍型，与氯喹耐药寄生虫的地理起源有关。在此，对来自 11 个现场的血液样本进行了 pfcr 基因 72-76 和 220 位密码子以及 pfmdr-1 N86Y 突变的研究，包括印度高和低恶性疟原虫流行地区。还研究了这些突变与氯喹治疗的临床结果之间的任何可能相关性。
在高和低恶性疟原虫流行地区采集疟疾病人的指尖血斑于 Whatman No.3 滤纸上。为了研究 pfcr 单倍型，从血液样本中提取寄生虫 DNA，进行 PCR 扩增，然后对 pfcr 基因进行部分测序。对于 pfmdr-1 N86Y 突变，用 AflIII 内切酶对 PCR 产物进行酶切消化。
在 240 株报告体内氯喹治疗疗效的恶性疟原虫分离株中，pfcr 基因突变分析显示，突变 SVMNT-S（67.50%）和 CVIET-S（23.75%）无论临床结果如何均有发生，而野生型 CVMNK-A（7.91%）仅发生在充分的临床和寄生虫学反应样本中。在 287 株恶性疟原虫分离株中，SVMNTS 192（66.89%）在所有研究地点均占优势，在低恶性疟原虫流行地区几乎呈单态存在（98.42%的分离株）。然而，CVIETS-S（19.51%）和 CVMNK-A（11.84%）的发生仅限于高恶性疟原虫流行地区。pfmdr-1 N86Y 突变的研究表明与临床结果无关。野生型 N86 在所有低恶性疟原虫流行地区均占优势（94.48%）。然而，在高恶性疟原虫流行地区，突变型 N86Y 的数量相对较高（42.76%）。
野生型 pfcr 基因与氯喹敏感性有关；然而，突变的存在并不能解释在当前氯喹耐药情况下氯喹的治疗效果。在疟原虫密度较高和氯喹暴露较多的地区，突变 SVMNT 单倍型的单态存在暗示了近亲繁殖和更快的 CQR 寄生虫传播，而在高恶性疟原虫流行地区，pfcr 基因的多样性得以维持。
