斯威士兰疟原虫分离株中的抗疟药物耐药标志物：天然寄生虫分离株中 pfmdr1-86F 的鉴定。
Markers of anti-malarial drug resistance in Plasmodium falciparum isolates from Swaziland: identification of pfmdr1-86F in natural parasite isolates.
机构信息
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St, London WC1E 7HT, UK.
出版信息
Malar J. 2010 Mar 3;9:68. doi: 10.1186/1475-2875-9-68.
BACKGROUND
The development of Plasmodium falciparum resistance to chloroquine (CQ) has limited its use in many malaria endemic areas of the world. However, despite recent drug policy changes to adopt the more effective artemisinin-based combination (ACT) in Africa and in the Southern African region, in 2007 Swaziland still relied on CQ as first-line anti-malarial drug.
METHODS
Parasite DNA was amplified from P. falciparum isolates from Swaziland collected in 1999 (thick smear blood slides) and 2007 (filter paper blood spots). Markers of CQ and sulphadoxine-pyrimethamine (SP) resistance were identified by probe-based qPCR and DNA sequencing.
RESULTS
Retrospective microscopy, confirmed by PCR amplification, found that only six of 252 patients treated for uncomplicated malaria in 2007 carried detectable P. falciparum. The pfcrt haplotype 72C/73V/74I/75E/76T occurred at a prevalence of 70% (n = 64) in 1999 and 83% (n = 6) in 2007. Prevalence of the pfmdr1-86N allele was 24% in 1999 and 67% in 2007. A novel substitution of phenylalanine for asparagine at codon 86 of pfmdr1 (N86F) occurred in two of 51 isolates successfully amplified from 1999. The pfmdr1-1246Y allele was common in 1999, with a prevalence of 49%, but was absent among isolates collected in 2007. The 86N/184F/1246D pfmdr1 haplotype, associated with enhanced parasite survival in patients treated with artemether-lumefantrine, comprised 8% of 1999 isolates, and 67% among 2007 isolates. The pfdhfr triple-mutant 16C/51I/59R/108N/164I haplotype associated with pyrimethamine resistance was common in both 1999 (82%, n = 34) and 2007 (50%, n = 6), as was the wild-type 431I/436S/437A/540K/581A/613A haplotype of pfdhps (100% and 93% respectively in 1999 and 2007). The quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA, associated with high-level resistance to SP, was rare (9%) among 1999 isolates and absent among 2007 isolates.
CONCLUSIONS
The prevalence of pfcrt and pfmdr1 alleles reported in this study is consistent with a parasite population under sustained CQ drug pressure. The low prevalence of dhps-437G and dhps-540E mutations (ISGEAA) and the rarity of quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA suggest that SP retains some efficacy in Swaziland. Anti-malarial policy changes in neighbouring countries may have had an impact on the prevalence of molecular markers of anti-malarial resistance in Swaziland, and it is hoped that this new information will add to understanding of the regional anti-malarial resistance map.
背景
恶性疟原虫对氯喹（CQ）的耐药性发展限制了其在世界许多疟疾流行地区的使用。然而，尽管最近的药物政策发生了变化，在非洲和南部非洲地区采用更有效的青蒿素为基础的联合疗法（ACT），但 2007 年斯威士兰仍依赖 CQ 作为一线抗疟药物。
方法
从 1999 年（厚涂片血样）和 2007 年（滤纸血斑）采集的斯威士兰恶性疟原虫分离株中扩增寄生虫 DNA。通过探针 qPCR 和 DNA 测序鉴定 CQ 和磺胺多辛-乙胺嘧啶（SP）耐药的标记物。
结果
回顾性显微镜检查，通过 PCR 扩增证实，2007 年治疗 252 例无并发症疟疾的患者中只有 6 例携带可检测的恶性疟原虫。1999 年 pfcrt 72C/73V/74I/75E/76T 单倍型的流行率为 70%（n = 64），2007 年为 83%（n = 6）。1999 年 pfmdr1-86N 等位基因的流行率为 24%，2007 年为 67%。pfmdr1 86 位密码子的苯丙氨酸替代天冬酰胺（N86F）的新型取代在 1999 年成功扩增的 51 个分离株中有 2 个发生。pfmdr1-1246Y 等位基因在 1999 年很常见，流行率为 49%，但在 2007 年分离株中不存在。与青蒿素-咯萘啶治疗的寄生虫存活增强相关的 86N/184F/1246D pfmdr1 单倍型在 1999 年的分离株中占 8%，在 2007 年的分离株中占 67%。与乙胺嘧啶耐药相关的 pfdhfr 三重突变 16C/51I/59R/108N/164I 单倍型在 1999 年（82%，n = 34）和 2007 年（50%，n = 6）都很常见，野生型 431I/436S/437A/540K/581A/613A pfdhps 单倍型在 1999 年和 2007 年分别为 100%和 93%。与磺胺多辛-乙胺嘧啶高度耐药相关的 pfdhfr/pfdhps-CIRNI/ISGEAA 五重突变单倍型在 1999 年的分离株中很少见（9%），在 2007 年的分离株中不存在。
结论
本研究报告的 pfcrt 和 pfmdr1 等位基因的流行率与持续受到 CQ 药物压力的寄生虫种群一致。dhps-437G 和 dhps-540E 突变（ISGEAA）的低流行率和五重突变单倍型 pfdhfr/pfdhps-CIRNI/ISGEAA 的罕见性表明 SP 在斯威士兰仍具有一定的疗效。邻国的抗疟药物政策变化可能对斯威士兰抗疟药物耐药性的分子标记物的流行率产生了影响，希望这些新信息将有助于了解区域抗疟耐药性地图。
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