Department of Pharmacology & Therapeutics, Makerere University, Kampala, Uganda.
Makerere University Biomedical Research Center, Kampala, Uganda.
PLoS One. 2022 May 5;17(5):e0268095. doi: 10.1371/journal.pone.0268095. eCollection 2022.
In Uganda, Artemether-Lumefantrine and Artesunate are recommended for uncomplicated and severe malaria respectively, but are currently threatened by parasite resistance. Genetic and epigenetic factors play a role in predisposing Plasmodium falciparum parasites to acquiring Pfkelch13 (K13) mutations associated with delayed artemisinin parasite clearance as reported in Southeast Asia. In this study, we report on the prevalence of mutations in the K13, pfmdr-2 (P. falciparum multidrug resistance protein 2), fd (ferredoxin), pfcrt (P. falciparum chloroquine resistance transporter), and arps10 (apicoplast ribosomal protein S10) genes in Plasmodium falciparum parasites prior to (2005) and after (2013) introduction of artemisinin combination therapies for malaria treatment in Uganda. A total of 200 P. falciparum parasite DNA samples were screened. Parasite DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany) procedure. The PCR products were sequenced using Sanger dideoxy sequencing method. Of the 200 P. falciparum DNA samples screened, sequencing for mutations in K13, pfmdr-2, fd, pfcrt, arps10 genes was successful in 142, 186, 141, 128 and 74 samples respectively. Overall, we detected six (4.2%, 6/142; 95%CI: 1.4-7.0) K13 single nucleotide polymorphisms (SNPs), of which 3.9% (2/51), 4.4% (4/91) occurred in 2005 and 2013 samples respectively. All four K13 SNPs in 2013 samples were non-synonymous (A578S, E596V, S600C and E643K) while of the two SNPs in 2005 samples, one (Y588N) is non-synonymous and the other (I587I) is synonymous. There was no statistically significant difference in the prevalence of K13 (p = 0.112) SNPs in the samples collected in 2005 and 2013. The overall prevalence of SNPs in pfmdr-2 gene was 39.8% (74/186, 95%CI: 25.1-50.4). Of this, 4.2% (4/95), 76.9% (70/91) occurred in 2005 and 2013 samples respectively. In 2005 samples only one SNP, Y423F (4.2%, 4/95) was found while in 2013, Y423F (38.5%, 35/91) and I492V (38.5%, 35/91) SNPs in the pfmdr-2 gene were found. There was a statistically significant difference in the prevalence of pfmdr-2 SNPs in the samples collected in 2005 and 2013 (p<0.001). The overall prevalence of arps10 mutations was 2.7% (2/72, 95%CI: 0.3-4.2). Two mutations, V127M (4.5%: 1/22) and D128H (4.5%: 1/22) in the arps10 gene were each found in P. falciparum parasite samples collected in 2013. There was no statistically significant difference in the prevalence of arps10 SNPs in the samples collected in 2005 and 2013 (p = 0.238). There were more pfmdr-2 SNPs in P. falciparum parasites collected after introduction of Artemisinin combination therapies in malaria treatment. This is an indicator of the need for continuous surveillance to monitor emergence of molecular markers of artemisinin resistance and its potential drivers in malaria affected regions globally.
在乌干达，青蒿琥酯-咯萘啶和青蒿素分别被推荐用于治疗无并发症和严重疟疾，但目前都受到寄生虫耐药性的威胁。遗传和表观遗传因素在导致恶性疟原虫寄生虫获得与东南亚报道的青蒿素寄生虫清除延迟相关的 Pfkelch13（K13）突变方面起着作用。在这项研究中，我们报告了在青蒿素联合疗法引入疟疾治疗之前（2005 年）和之后（2013 年）在乌干达，恶性疟原虫寄生虫中 K13、pfmdr-2（恶性疟原虫多药耐药蛋白 2）、fd（铁氧还蛋白）、pfcrt（恶性疟原虫氯喹耐药转运蛋白）和 arps10（质体核糖体蛋白 S10）基因中的突变的流行率。总共筛选了 200 个恶性疟原虫寄生虫 DNA 样本。使用 QIAamp DNA 迷你试剂盒（Qiagen，GmbH，德国）程序提取寄生虫 DNA。使用 Sanger 双脱氧测序法对 PCR 产物进行测序。在所筛选的 200 个恶性疟原虫 DNA 样本中，成功对 K13、pfmdr-2、fd、pfcrt、arps10 基因的突变进行测序的分别有 142、186、141、128 和 74 个样本。总体而言，我们检测到 6 个（4.2%，6/142；95%CI：1.4-7.0）K13 单核苷酸多态性（SNP），其中 2005 年和 2013 年样本中分别为 3.9%（2/51）和 4.4%（4/91）。2013 年样本中所有 4 个 K13 SNP 均为非同义突变（A578S、E596V、S600C 和 E643K），而 2005 年样本中的 2 个 SNP 中，一个（Y588N）为非同义突变，另一个（I587I）为同义突变。2005 年和 2013 年采集的样本中 K13 SNP 的流行率没有统计学差异（p=0.112）。pfmdr-2 基因 SNP 的总体流行率为 39.8%（74/186，95%CI：25.1-50.4）。其中，2005 年和 2013 年样本中分别为 4.2%（4/95）和 76.9%（70/91）。2005 年样本中仅发现一个 SNP，Y423F（4.2%，4/95），而 2013 年，pfmdr-2 基因中的 Y423F（38.5%，35/91）和 I492V（38.5%，35/91）SNP 也被发现。2005 年和 2013 年采集的样本中 pfmdr-2 SNP 的流行率有统计学差异（p<0.001）。arps10 基因突变的总体流行率为 2.7%（2/72，95%CI：0.3-4.2）。2013 年，恶性疟原虫寄生虫样本中分别发现 2 个突变，V127M（4.5%：1/22）和 D128H（4.5%：1/22）。在 2005 年和 2013 年采集的样本中，arps10 SNP 的流行率没有统计学差异（p=0.238）。在青蒿素联合疗法引入疟疾治疗后，恶性疟原虫寄生虫中的 pfmdr-2 SNP 更多。这表明需要持续监测，以监测全球疟疾流行地区青蒿素耐药性及其潜在驱动因素的分子标志物的出现。
