Impact of Short-Time Urine Freezing on the Sensitivity of an Established Schistosoma Real-Time PCR Assay

Kenguele, Hilaire Moundounga; Adegnika, Ayôla Akim; Nkoma, Anne-Marie; Ateba-Ngoa, Ulysse; Mbong, Mirabeau; Zinsou, Jeannot Fréjus; Lell, Bertrand; Verweij, Jaco J.

doi:10.4269/ajtmh.14-0005

Cited by 19 publications

(12 citation statements)

References 17 publications

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“…This seems to be logical since after centrifugation to remove and retain supernatants, both potential free S . haematobium DNA and parasite eggs-and therefore containing DNA- found in patients´ urine samples should be concentrated at the bottom of the tube, thus improving the sensitivity of the DNA molecular detection methods, as previously described [ 68 ]. When using the pellets, the simple rapid-heating method allowed us to obtain a very good-quality detectable DNA that did not compromise LAMP amplification and all the S .…”

Section: Discussionmentioning

confidence: 99%

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

et al. 2015

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BackgroundUrogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis.Methodology/Principal FindingsA LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%).Conclusions/SignificanceWe have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas.

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Section: Discussionmentioning

confidence: 99%

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

et al. 2015

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“…This range largely reflects the quality and quantity of the microscopy procedures used in the different studies [96][97][98]. For the detection of DNA in urine, the sensitivity further increases by the use of concentration procedures such as sedimentation or filtration, the latter with potential application for population-based surveillance in more remote endemic regions [99,100].…”

Section: Dna Detection Tests For the Detection And Quantification Ofmentioning

confidence: 99%

New diagnostic tools in schistosomiasis

Utzinger

Becker

Lieshout

et al. 2015

Clinical Microbiology and Infection

226

225

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Schistosomiasis is a water-based parasitic disease that affects over 250 million people. Control efforts have long been in vain, which is one reason why schistosomiasis is considered a neglected tropical disease. However, since the new millennium, interventions against schistosomiasis are escalating. The initial impetus stems from a 2001 World Health Assembly resolution, urging member states to scale-up deworming of school-aged children with the anthelminthic drug praziquantel. Because praziquantel is safe, efficacious and inexpensive when delivered through the school platform, diagnosis before drug intervention was deemed unnecessary and not cost-effective. Hence, there was little interest in research and development of novel diagnostic tools. With the recent publication of the World Health Organization (WHO) Roadmap to overcome the impact of neglected tropical diseases in 2020, we have entered a new era. Elimination of schistosomiasis has become the buzzword and this has important ramifications for diagnostic tools. Indeed, measuring progress towards the WHO Roadmap and whether local elimination has been achieved requires highly accurate diagnostic assays. Here, we introduce target product profiles for diagnostic tools that are required for different stages of a schistosomiasis control programme. We provide an update of the latest developments in schistosomiasis diagnosis, including microscopic techniques, rapid diagnostic tests for antigen detection, polymerase chain reaction (PCR) assays and proxy markers for morbidity assessments. Particular emphasis is placed on challenges and solutions for new technologies to enter clinical practice.

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“…A 9-mL aliquot of each sample was centrifuged for 5 min at 3000 rpm. Next, two aliquots (200 μL) of the mixed pellet were collected; one was transferred into a 2-ml screw-capped tube without beads and one into a 2-ml screw-capped tube with ceramic beads (Roche), and frozen at − 20 °C (Kenguele et al 2014 ). For microscopy, the remaining milliliter of urine was examined by the urine filtration (25-mm diameter and 12-μm pore size filter, Whatman Nuclepore), and results were expressed as corrected number of eggs/10 mL for the volume examined.…”

Section: Methodsmentioning

confidence: 99%

Improved detection of DNA Schistosoma haematobium from eggs extracted by bead beating in urine

et al. 2018

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Diagnosis of Schistosoma haematobium relies primarily on microscopical analysis of urine. The method is time consuming and requires some expertise. Genus-specific real-time PCRs have been developed, but we still observed low sensitivity. In the present study, in order to achieve a more sensitive DNA detection of eggs of S. haematobium in urine samples, we wanted to develop a novel protocol of DNA extraction using mechanic disruption of eggs by bead beating as supplementary step. We tested Schistosoma spp. internal transcribed spacer 2 real-time PCR after both methods with and without bead beating. First, we preliminary assessed the DNA detection after bead beating using dilution of 2, 10, 50, and 90 eggs/10 mL, and the Ct value analysis showed significant improved DNA detection per each point of egg concentration using the novel supplementary step. Twenty microscopy positive and five microscopy negative urine samples were used to validate the procedure. All urines came from imported cases and admitted at center for tropical medicine, and were examined by microscopy. PCR results after novel method with bead beating showed 100% to be positive for S. haematobium, compared with 85% positive by our standard extraction procedure. Results confirmed mechanic disruption of eggs by bead beating before DNA extraction to be highly effective method for the detection of S. haematobium DNA in urine.Electronic supplementary materialThe online version of this article (10.1007/s00436-018-6137-7) contains supplementary material, which is available to authorized users.

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Impact of Short-Time Urine Freezing on the Sensitivity of an Established Schistosoma Real-Time PCR Assay

Cited by 19 publications

References 17 publications

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

New diagnostic tools in schistosomiasis

Improved detection of DNA Schistosoma haematobium from eggs extracted by bead beating in urine

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