Abstract
BackgroundHuman schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples’ storage or conditions for handling and DNA preservation and extraction methods.Methodology/Principal FindingsWe evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patientś urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template.Conclusions/SignificanceLong-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used.
Highlights
Human schistosomiasis -caused by different species of digenetic trematode worms of the genus Schistosoma- is a severe debilitating parasitic disease that remains as a major public health problem in developing countries in tropical and subtropical areas, especially in Sub-Saharan Africa
We evaluate different DNA extraction methods from human urine samples in order to assess the polymerase chain reaction (PCR) effectiveness for Schistosoma spp. detection in a larger series of patients urine samples after long-term frozen storage from an endemic area for schistosomiasis
In PCR tests using whole urine samples only positive results were obtained by using a starting volume of urine of 500 mL and performing DNA extraction with an equal volume of Chelex-100H resin at 5% or 20%
Summary
Human schistosomiasis -caused by different species of digenetic trematode worms of the genus Schistosoma- is a severe debilitating parasitic disease that remains as a major public health problem in developing countries in tropical and subtropical areas, especially in Sub-Saharan Africa. Detection of parasite eggs cannot be carried out in the acute phase of schistosomiasis because production and elimination of eggs begins at two months of infection To overcome these limitations with microscopic diagnostic, immunological methods to determine both circulating antigen or antibody levels are usually applied to patients with schistosomiasis clinical signs when parasites cannot be directly detected. These methods present greater sensitivity than parasitological techniques, serology-based analyses currently continue to present problems, such as obtaining schistosome antigens, inability to discriminate between active and past infection and high level of crossreactivity. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples’ storage or conditions for handling and DNA preservation and extraction methods
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