Targeting a highly repetitive genomic sequence for sensitive and specific molecular detection of the filarial parasite Mansonella perstans from human blood and mosquitoes.
Targeting a highly repetitive genomic sequence for sensitive and specific molecular detection of the filarial parasite Mansonella perstans from human blood and mosquitoes.
复制标题
DOI：
10.1371/journal.pntd.0010615
复制
发表时间：
2022-12
影响因子：
3.8
通讯作者：
中科院分区：
文献类型：
--
作者：
持久性曼森氏菌是最被忽视的热带疾病之一，据信在非洲造成的人类感染比任何其他丝虫病原体都多。主要基于有限的感染相关的发病率的假设，这种病原体仍然研究不足，许多基本的问题，有关其致病性，分布，流行和媒介-宿主关系仍然没有答案。然而，近年来，越来越多的证据表明，曼森氏菌感染相关疾病的可能性增加，引发了研究兴趣的更新。这反过来又产生了对改进的诊断的需求，能够提供感染流行、病原体分布和媒介-宿主相互作用的更准确的图片。利用先前描述的用于发现最佳分子诊断靶点的管道，我们鉴定了重复DNA序列，并开发了相应的测定方法，该方法允许灵敏和种特异性地鉴定M。人体血液样本中的持久性有机污染物。试验还证明了利用该检测试剂盒检测M的能力。在实地收集的蚊子样本中，当测试这两种样品类型时，我们的重复靶向指数测定优于核糖体序列靶向参考测定，有助于鉴定额外的M。使用灵敏度较低的检测方法将持久性有机污染物阳性样本错误地定性为“阴性”。通过开发基于系统鉴定最佳DNA靶序列的测定法，我们的新型诊断测定法将提供具有灵敏度和特异性的测试平台的程序性努力，该平台能够准确地定位M。perstans感染和确定患病率。此外，随着识别M的存在的能力的增加。通过对蚊子样本中的持久性有机污染物进行检测，该分析将有助于确定我们对该病原体与各种地理相关蚊子物种之间存在的关系的认识，这些蚊子物种被推测为在某些条件下代表潜在的次级媒介。检测M.蚊子中的持久性有机污染物还将证明以蚊子为基础监测主要不是蚊子传播的丝虫病原体的概念验证，这一方法扩大了综合监测的机会。在世界上许多热带和亚热带地区，持久性曼森氏菌感染仍然非常普遍。然而，M。perstans在很大程度上是研究不足，由于长期以来认为，这种病原体是没有什么临床意义。然而，近年来，研究界的许多人开始提倡增加对这种病原体的研究，指出曼氏菌病相关疾病发病率的证据，曼氏菌属的潜力。混淆其他病原体的诊断检测，以及与M. perstans影响疾病进展和其他感染的治疗结果。随着人们对M.然而，需要改进诊断方法，能够为研究人员提供所需的工具，以准确有效地绘制感染图，探索病原体-病媒关系，并确定病原体流行率。为了满足这些需求，我们开发了一种新的实时PCR检测方法，靶向M. perstans基因组。当比较测试人类血液和现场收集的蚊子样本时，该指数测定优于核糖体序列靶向参考测定。因此，这种检测方法将为研究人员提供一种改进的鉴定M的工具。perstans作为各种业务研究工作的一部分。
Mansonella perstans is among the most neglected of the neglected tropical diseases and is believed to cause more human infections than any other filarial pathogen in Africa. Based largely upon assumptions of limited infection-associated morbidity, this pathogen remains understudied, and many basic questions pertaining to its pathogenicity, distribution, prevalence, and vector-host relationships remain unanswered. However, in recent years, mounting evidence of the potential for increased Mansonella infection-associated disease has sparked a renewal in research interest. This, in turn, has produced a need for improved diagnostics, capable of providing more accurate pictures of infection prevalence, pathogen distribution, and vector-host interactions. Utilizing a previously described pipeline for the discovery of optimal molecular diagnostic targets, we identified a repetitive DNA sequence, and developed a corresponding assay, which allows for the sensitive and species-specific identification of M. perstans in human blood samples. Testing also demonstrated the ability to utilize this assay for the detection of M. perstans in field-collected mosquito samples. When testing both sample types, our repeat-targeting index assay outperformed a ribosomal sequence-targeting reference assay, facilitating the identification of additional M. perstans-positive samples falsely characterized as “negative” using the less sensitive detection method. Through the development of an assay based upon the systematic identification of an optimal DNA target sequence, our novel diagnostic assay will provide programmatic efforts with a sensitive and specific testing platform that is capable of accurately mapping M. perstans infection and determining prevalence. Furthermore, with the added ability to identify the presence of M. perstans in mosquito samples, this assay will help to define our knowledge of the relationships that exist between this pathogen and the various geographically relevant mosquito species, which have been surmised to represent potential secondary vectors under certain conditions. Detection of M. perstans in mosquitoes will also demonstrate proof-of-concept for the mosquito-based monitoring of filarial pathogens not vectored primarily by mosquitoes, an approach expanding opportunities for integrated surveillance. Infection with Mansonella perstans remains exceedingly common in many of the world’s tropical and sub-tropical regions. However, M. perstans is largely understudied due to the long-held belief that this pathogen is of little clinical significance. However, in recent years, many within the research community have begun to advocate for the increased study of this pathogen, pointing to evidence of mansonellosis-associated disease morbidity, the potential for Mansonella spp. to confound diagnostic testing for other pathogens, and the possibility for co-infections with M. perstans to impact disease progression and treatment outcomes for other infections. As a result of this growing appreciation of the importance of M. perstans, there exists a need for improved diagnostic options, capable of providing researchers with the tools required to accurately and effectively map infection, explore the pathogen-vector relationship, and determine pathogen prevalence. In response to these needs, we have developed a novel real-time PCR assay targeting a highly repetitive DNA sequence within the M. perstans genome. This index assay outperformed a ribosomal-sequence targeting reference assay when comparatively testing both human blood and field-collected mosquito samples. As such, this assay will provide researchers with an improved tool for the identification of M. perstans as part of various operational research efforts.