Infectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of Salamanca-Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of Salamanca, Salamanca, Spain.
Department of Medical and Surgical Sciences, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain.
PLoS One. 2019 Mar 26;14(3):e0214125. doi: 10.1371/journal.pone.0214125. eCollection 2019.
Schistosoma mansoni is the main species causing hepatic and intestinal schistosomiasis in Sub-Saharan Africa, and it is the only species in South America. Adult stages of the parasite reside in the mesenteric venous plexus of infected hosts, and eggs are shed in feces. Collecting patient stool samples for S. mansoni diagnostic purposes is difficult in large-scale field trials. Urine samples would be an alternative approach for molecular S. mansoni detection since they have several advantages over stool samples, including better handling, management and storage. Additionally, loop-mediated isothermal amplification (LAMP) technology is a powerful molecular diagnostic tool for infectious diseases, particularly under field conditions in developing countries. The present study aimed to assess the effectiveness of our previously developed LAMP assay (SmMIT-LAMP) for S. mansoni-specific detection in clinical urine samples.
METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity of SmMIT-LAMP in urine was established in simulated fresh human urine samples artificially spiked with genomic DNA from S. mansoni. LAMP for 120 min instead of 60 min improved the sensitivity, reaching values of 0.01 fg/μL. A set of well-defined frozen stored human urine samples collected from Sub-Saharan immigrant patients was selected from a biobank to evaluate the diagnostic validity of SmMIT-LAMP. The set included urine samples from patients with microscopy-confirmed infections with S. mansoni, S. haematobium and other nonschistosome parasites, as well as urine samples from patients with microscopy-negative eosinophilia without a confirmed diagnosis. The SmMIT-LAMP was incubated for 60 and 120 min. A longer incubation time was shown to increase the LAMP-positive results in patient urine samples. We also tested urine samples from mice experimentally infected with S. mansoni, and LAMP-positive results were obtained from the third week after infection. A real-time LAMP assay was also performed with three individual urine samples.
CONCLUSIONS/SIGNIFICANCE: The SmMIT-LAMP could effectively detect S. mansoni DNA in mouse urine samples and produced promising results for human clinical samples. The detection of S. mansoni DNA in mouse urine samples from the third week after infection indicates that early diagnosis of active S. mansoni infection is possible using urine as a source of DNA. Further studies are still needed, but our method could be used as a promising molecular tool applicable to urine samples to diagnose human intestinal schistosomiasis caused by S. mansoni.
曼氏血吸虫是撒哈拉以南非洲地区引起肝肠血吸虫病的主要物种，也是南美洲唯一的物种。寄生虫的成虫栖息在受感染宿主的肠系膜静脉丛中，虫卵随粪便排出。采集患者粪便样本进行曼氏血吸虫诊断在大规模现场试验中较为困难。尿液样本是一种替代方法，用于分子曼氏血吸虫检测，因为它们相对于粪便样本具有几个优势，包括更好的处理、管理和储存。此外，环介导等温扩增（LAMP）技术是一种用于传染病的强大分子诊断工具，特别是在发展中国家的现场条件下。本研究旨在评估我们之前开发的 LAMP 检测方法（SmMIT-LAMP）在临床尿液样本中用于曼氏血吸虫特异性检测的有效性。
方法/主要发现：在人工用曼氏血吸虫基因组 DNA 污染的模拟新鲜人尿液样本中，确定了 SmMIT-LAMP 在尿液中的灵敏度。与 60 分钟相比，120 分钟的 LAMP 孵育时间提高了灵敏度，达到 0.01 fg/μL。从生物库中选择了一组来自撒哈拉以南移民患者的冷冻储存的人尿液样本，用于评估 SmMIT-LAMP 的诊断有效性。该组包括显微镜确认感染曼氏血吸虫、埃及血吸虫和其他非血吸虫寄生虫的患者的尿液样本，以及显微镜检查阴性嗜酸性粒细胞增多但未确诊的患者的尿液样本。SmMIT-LAMP 孵育 60 和 120 分钟。较长的孵育时间显示增加了患者尿液样本中 LAMP 阳性结果。我们还测试了用曼氏血吸虫感染的小鼠的尿液样本，并且在感染后的第三周获得了 LAMP 阳性结果。还对三个单独的尿液样本进行了实时 LAMP 检测。
结论/意义：SmMIT-LAMP 可有效检测小鼠尿液样本中的曼氏血吸虫 DNA，并在人临床样本中取得了有希望的结果。从感染后的第三周开始，从感染曼氏血吸虫的小鼠尿液样本中检测到了曼氏血吸虫 DNA，这表明可以使用尿液作为 DNA 来源来早期诊断活动性曼氏血吸虫感染。还需要进一步研究，但我们的方法可作为一种有前途的分子工具，适用于尿液样本，用于诊断由曼氏血吸虫引起的人类肠血吸虫病。
