Search

Background: 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. [ABSTRACT FROM AUTHOR]

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas. [ABSTRACT FROM AUTHOR]