Fernández-Soto Pedro, Sánchez-Hernández Alicia, Gandasegui Javier, Bajo Santos Cristina, López-Abán Julio, Saugar José María, Rodríguez Esperanza, Vicente Belén, Muro Antonio
Unidad de Investigación Enfermedades Infecciosas y Tropicales (e-INTRO), Instituto de Investigación Biomédica de Salamanca-Centro de Investigación de Enfermedades Tropicales de la Universidad de Salamanca (IBSAL-CIETUS), Facultad de Farmacia, Universidad de Salamanca, Salamanca, Spain.
Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
PLoS Negl Trop Dis. 2016 Jul 14;10(7):e0004836. doi: 10.1371/journal.pntd.0004836. eCollection 2016 Jul.
Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples.
METHODOLOGY/PRINCIPAL FINDINGS: Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests.
CONCLUSIONS/SIGNIFICANCE: Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting.
粪类圆线虫是人类类圆线虫病的主要病原体，是一种全球分布的线虫，但主要流行于热带和亚热带地区。慢性感染通常在临床上无症状，但在免疫功能低下的患者中可能导致严重的超感染综合征或播散性类圆线虫病。诊断该疾病使用的技术多种多样，但确诊是通过对粪便样本进行寄生虫学检查以鉴定寄生虫的形态来完成的。到目前为止，尚未在尿液样本中测试任何分子方法作为粪便样本诊断类圆线虫病的替代方法。本研究旨在评估一种新的环介导等温扩增（LAMP）检测法在成熟的Wistar大鼠实验感染模型中的应用，该模型使用粪便样本，并且首次使用尿液样本。LAMP检测法也在患者粪便样本中进行了临床评估。
方法/主要发现：在28天的时间里，每天从皮下感染不同感染剂量委内瑞拉类圆线虫第三期幼虫的大鼠中获取粪便和尿液样本。通过在感染后第1天至28天每天计算每克粪便中的虫卵数量来确定寄生虫感染的动态变化。基于委内瑞拉类圆线虫18S rRNA基因中的一段DNA序列设计了一组用于LAMP检测的引物。所建立的LAMP检测法（即Strong-LAMP）能够灵敏地检测从每组感染大鼠获得的粪便和尿液样本中的委内瑞拉类圆线虫DNA，并且对经寄生虫学和实时PCR检测先前确诊为类圆线虫病患者的粪便样本中的粪类圆线虫DNA扩增也有效。
结论/意义：我们的Strong-LAMP检测法是研究粪便和尿液样本中类圆线虫病实验感染模型的一种有用的分子工具。经过进一步验证后，Strong-LAMP也可能潜在地应用于临床环境中类圆线虫病的有效诊断。
