Wahab Abdul, Shaukat Ayaz, Ali Qasim, Hussain Mubashir, Khan Taj Ali, Khan M Azmat Ullah, Rashid Imran, Saleem Mushtaq A, Evans Mike, Sargison Neil D, Chaudhry Umer
University of Science and Technology, Kohat, Khyber Pakhtunkhwa, Pakistan.
University of Central Punjab, Lahore, Punjab, Pakistan.
Infect Genet Evol. 2020 Aug;82:104305. doi: 10.1016/j.meegid.2020.104305. Epub 2020 Apr 2.
Various PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have recently developed for the first time a novel high throughput 'haemoprotobiome' metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species. The different mock pools demonstrate the accurate detection ability and to show the proportions of each of the species being present. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. The deep amplicon sequencing showed that P. vivax was present in 69.8%, P. falciparum in 29.5% and mixed infection in 0.7% patients examined. The immunochromatographic assay showed that P. vivax was present in 65.6%, P. falciparum in 27.4%, mixed infection 0.7% patients and 6.32% malaria-positive cases were negative in immunochromatographic assay, but positive in the deep amplicon sequencing. Overall, metabarcoded DNA sequencing demonstrates better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.
已经描述了多种基于聚合酶链反应（PCR）的疟疾诊断方法，但大多数方法依赖于使用疟原虫物种特异性探针和引物；因此，只能鉴定出所检测的物种，而关于实际循环物种多样性的可用数据有限。需要灵敏的诊断工具及其使用平台，以检测临床病例和无症状感染中有助于疾病传播的疟原虫物种。我们最近首次开发了一种新型的高通量“血源微生物组”元条形码DNA测序方法，并将其应用于家畜血源原生动物寄生虫（泰勒虫属和巴贝斯虫属）的定量分析。在此，我们描述一种使用Illumina MiSeq平台的新型高通量方法，以展示源自人类疟疾患者的元条形码DNA样本中疟原虫物种的比例。使用恶性疟原虫和间日疟原虫阳性对照基因组DNA（gDNA）制备寄生虫模拟DNA池，以评估该检测方法对这两种物种各自的检测阈值。不同的模拟池展示了准确的检测能力，并显示了每种物种的存在比例。然后，我们将该检测方法应用于疟疾阳性的人类样本，以展示巴基斯坦旁遮普省和阿富汗 - 巴基斯坦部落地区疟原虫群落的物种组成。将深度扩增子测序方法的诊断性能与该地区广泛使用的免疫层析检测法进行了比较。深度扩增子测序显示，在所检测的患者中，间日疟原虫占69.8%，恶性疟原虫占29.5%，混合感染占0.7%。免疫层析检测法显示，间日疟原虫占65.6%，恶性疟原虫占27.4%，混合感染占0.7%，6.32%的疟疾阳性病例在免疫层析检测中为阴性，但在深度扩增子测序中为阳性。总体而言，元条形码DNA测序显示出更好的诊断性能，大大提高了疟原虫感染的估计流行率。使用元条形码DNA的下一代测序方法在疟原虫感染的诊断、监测、治疗和控制以及研究寄生虫生物学方面具有潜在应用价值。
