Detection of Schistosoma mansoni-derived DNA in human urine samples by loop-mediated isothermal amplification (LAMP)

Abstract

BackgroundSchistosoma 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 findingsThe 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/SignificanceThe 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.

Evaluation of serum-based real-time PCR to detect Schistosoma mansoni infection before and after treatment

“…The serum-based real-time PCR performed excellently with 96.3% sensitivity, whereas the urine-based real-time PCR showed the lowest sensitivity (33.3%) and the lowest Kappa agreement with the "gold standard" (0.119). Various other studies indicate that the diagnosis of S. mansoni infection is possible using urine as a source of DNA [32][33][34][35]. In our study, the performance of the urine-based real-time PCR was even lower than the KK method, despite the fact that the DNA in the urine was stabilized with a special reagent.…”

Development of a convenient detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65

“…As complicated body fluid, urine and genital secretion had been applied to detecting T. vaginalis on the basis of nucleic acid, and the sensitivity of which were 64-100% for urine samples and 81-100% to genital secretion swabs [45,46]. Previous studies have shown that LAMP reactions were not inhibited in spiked urine specimens [47]. Nevertheless, due to the usage of the wild type enzyme in the LAMP approach, unwanted activity of DNA polymerase in the process of setup possibly led to fail in reproducible amplification.…”

Development of a rapid detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65

“…As complicated body fluid, urine and genital secretion had been applied to detecting T. vaginalis on the basis of nucleic acid, and the sensitivity of which were 64% -100% for urine samples and 81% -100% to genital secretion swabs [45,46]. Previous studies have shown that LAMP reactions were not inhibited in spiked urine specimens [47]. Nevertheless, due to the usage of the wild type enzyme in the LAMP approach, unwanted activity of DNA polymerase in the process of setup possibly led to fail in reproducible amplification.…”