He Wenqiao, Sendor Rachel, Potlapalli Varun R, Kashamuka Melchior M, Tshefu Antoinette K, Phanzu Fernandine, Kalonji Albert, Ngasala Billy, Thwai Kyaw Lay, Juliano Jonathan J, Lin Jessica T, Parr Jonathan B
Division of Infectious Diseases and Institute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
Department of Epidemiology, School of Public Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, Southern Medical University, Guangzhou, China.
medRxiv. 2023 Oct 31:2023.10.31.23297819. doi: 10.1101/2023.10.31.23297819.
spp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species, () and (). These species are thought to differ in clinical symptomatology and latency, but existing diagnostic assays have limited ability to detect and distinguish them. In this study, we developed a new duplex assay for the detection and differentiation of and that can be used to improve our understanding of these parasites.
Repetitive sequence motifs were identified in available and genomes and used for assay development and validation. We evaluated the analytical sensitivity and specificity of the best-performing assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), then validated its performance using 55 spp. samples and 40 non-ovale samples from the DRC. and prevalence among symptomatic individuals sampled across three provinces of the DRC were estimated.
The best-performing and targets had 9 and 8 copies within the reference genomes, respectively. Our duplex assay had 100% specificity and 95% confidence lower limits of detection of 4.2 and 41.2 parasite genome equivalents/μl for and , respectively. Species was determined in 80% of all spp.-positive field samples and 100% of those with >10 parasites/μl. Most spp. field samples from the DRC were found to be infections.
We identified promising multi-copy targets for molecular detection and differentiation of and and used them to develop a new duplex real-time PCR assay that performed well when applied to diverse field samples. Though low-density infections are not reliably detected, the assay is highly specific and can be used for high-throughput studies of spp. epidemiology among symptomatic cases in malaria-endemic countries like the DRC.
疟原虫感染在多个非洲国家呈地方流行，由两种不同的非重组物种，即卵形疟原虫（Plasmodium ovale）和间日疟原虫（Plasmodium vivax）引起。这些物种被认为在临床症状和潜伏期方面存在差异，但现有的诊断检测方法检测和区分它们的能力有限。在本研究中，我们开发了一种新的双重检测方法用于检测和区分卵形疟原虫和间日疟原虫，该方法可用于增进我们对这些寄生虫的了解。
在可用的卵形疟原虫和间日疟原虫基因组中鉴定重复序列基序，并用于检测方法的开发和验证。我们使用来自坦桑尼亚和刚果民主共和国（DRC）的一组样本评估了性能最佳的检测方法的分析灵敏度和特异性，然后使用来自刚果民主共和国的55份卵形疟原虫样本和40份非卵形疟原虫样本验证其性能。估计了在刚果民主共和国三个省份采样的有症状个体中的卵形疟原虫和间日疟原虫患病率。
性能最佳的卵形疟原虫和间日疟原虫靶标在参考基因组中分别有9个和8个拷贝。我们的双重检测方法具有100%的特异性，对于卵形疟原虫和间日疟原虫，检测限的95%置信下限分别为4.2和41.2个寄生虫基因组当量/微升。在所有卵形疟原虫阳性现场样本的80%以及寄生虫数>10个/微升的样本的100%中确定了物种。发现来自刚果民主共和国的大多数卵形疟原虫现场样本为间日疟原虫感染。
我们确定了用于卵形疟原虫和间日疟原虫分子检测和区分的有前景的多拷贝靶标，并使用它们开发了一种新的双重实时PCR检测方法，该方法应用于不同的现场样本时表现良好。尽管低密度的卵形疟原虫感染无法可靠检测，但该检测方法具有高度特异性，可用于在刚果民主共和国等疟疾流行国家对有症状病例中的卵形疟原虫流行病学进行高通量研究。
