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      Validation of a New Test for Schistosoma haematobium Based on Detection of Dra1 DNA Fragments in Urine: Evaluation through Latent Class Analysis

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          Abstract

          Background

          Diagnosis of urogenital schistosomiasis in chronically infected adults is challenging but important, especially because long term infection of the bladder and urinary tract can have dire consequences. We evaluated three tests for viable infection: detection of parasite specific DNA Dra1 fragments, haematuria and presence of parasite eggs for sensitivity (Se) and specificity (Sp).

          Methods

          Over 400 urine specimens collected from adult volunteers in an endemic area in Western Nigeria were assessed for haematuria then filtered in the field, the filter papers dried and later examined for eggs and DNA. The results were stratified according to sex and age and subjected to Latent Class analysis.

          Conclusions

          Presence of Dra1 in males (Se = 100%; Sp = 100%) exceeded haematuria (Se = 87.6%: Sp = 34.7%) and detection of eggs (Se = 70.1%; Sp = 100%). In females presence of Dra1 was Se = 100%: Sp = 100%, exceeding haematuria (Se = 86.7%: Sp = 77.0%) and eggs (Se = 70.1%; Sp = 100%). Dra1 became undetectable 2 weeks after praziquantel treatment. We conclude detection of Dra1 fragment is a definitive test for the presence of Schistosoma haematobium infection.

          Author Summary

          The definitive test for schistosomiasis has been to detect parasite eggs in excreta. This is effective in children but as people age, it is difficult to find the eggs in spite of suspicion of infection. It also implies that adults have thrown off the infection, and therefore the infection is mainly one of children. However this is not the case as adults with chronic infections can develop severe bladder infection that can progress to bladder cancer. In clinical situations a sensitive test for urogenital schistosomiasis is important. We have recently shown that it is possible to detect schistosome species-specific DNA in urine, even in the absence of eggs in excreta. Here we use latent class analysis to compare sensitivity and specificity of three test procedures, haematuria, eggs in urine and specific DNA in urine in the absence of a gold standard. We show that the latter test is extremely promising for the diagnosis of urogenital schistosomiasis and should also be useful in field studies as it will be able to reveal infections in people frequently presumed to be uninfected. This new test indicated the presence of schistosomes in 10.7% of adults who did not pass eggs in urine specimens.

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          Most cited references 24

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          Relationship between schistosomiasis and bladder cancer.

          Carcinoma of the urinary bladder is the most common malignancy in the Middle East and parts of Africa where schistosomiasis is a widespread problem. Much evidence supports the association between schistosomiasis and bladder cancer: this includes the geographical correlation between the two conditions, the distinctive patterns of gender and age at diagnosis, the clinicopathological identity of schistosome-associated bladder cancer, and extensive evidence in experimentally infected animals. Multiple factors have been suggested as causative agents in schistosome-associated bladder carcinogenesis. Of these, N-nitroso compounds appear to be of particular importance since they were found at high levels in the urine of patients with schistosomiasis-associated bladder cancer. Various strains of bacteria that can mediate nitrosation reactions leading to the formation of N-nitrosamines have been identified in the urine of subjects with schistosomiasis at higher intensities of infection than in normal subjects. In experimental schistosomiasis, the activities of carcinogen-metabolizing enzymes are increased soon after infection but are reduced again during the later chronic stages of the disease. Not only could this prolong the period of exposure to activated N-nitrosamines, but also inflammatory cells, stimulated as a result of the infection, may induce the endogenous synthesis of N-nitrosamines as well as generating oxygen radicals. Higher than normal levels of host cell DNA damage are therefore anticipated, and they have indeed been observed in the case of alkylation damage, together with an inefficiency in the capacity of relevant enzymes to repair this damaged DNA. In experimental schistosomiasis, it was also found that endogenous levels of host cell DNA damage were related to the intensity of infection. All of these factors could contribute to an increased risk of bladder cancer in patients with schistosomiasis, and in particular, the gene changes observed may have potential for use as biomarkers in the early detection of bladder cancer that may assist in alleviating the problem.
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            Diagnosis of Schistosoma haematobium by detection of specific DNA fragments from filtered urine samples.

            Definitive diagnosis of Schistosoma haematobium infection in adult patients is a clinically important challenge. Chronically infected adults pass few eggs in the urine, which are often missed when current diagnostic methods are used. In the work presented here, we report on an alternative diagnostic method based on presence of the S. haematobium-specific Dra 1, 121 bp repeat fragment in human urine. A novel method of collecting the urine specimens in the field and filtering them through heavy Whatman No. 3 paper was introduced. After drying, the samples remained viable for several months at room temperature. To test the potential use of this method, 89 urine specimens from school children in Kollo District, Niger, were examined. In all, 52 of 89 (58.4%) were positive for hematuria, 4 of 89 (49.4%) were positive for eggs, and 51 of 89 (57.3%) showed parasite-specific DNA. These were compared with 60 filtered urine specimens obtained from random samples of adults from two study sites in Nigeria, one endemic and one non-endemic for S. haematobium. In the 30 patients from the endemic site, all 10 samples with detectable eggs and 7 of the 20 egg-negative samples were DNA positive. It was concluded that the urine filter paper method was sufficiently sensitive to detect low and cryptic infections, that DNA detection was more sensitive than egg detection, and that the filtration method facilitated specimen collection and transport from the field.
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              PCR detection of Plasmodium falciparum in human urine and saliva samples

              Background Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections. Methods Urine and saliva were obtained from 47 thick film positive and 4 negative individuals one day after collection of blood slides and filter paper blood spots. P. falciparum DNA was extracted from blood, urine and saliva, in separate groups, using the Chelex method or Qiagen DNEasy® kit (urine and saliva only). Blood, urine and saliva extracts were subjected to PCR in separate batches. Amplicons from the various sample types were examined for MSP2 polymorphisms and restriction fragment patterns on DHFR amino acid codon 59. Results and discussion Malaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/μl. Regularly matching polymorphic MSP2 genotypes were found between the corresponding urine, saliva and peripheral blood amplicons of each individual, with different inter-individual polymorphic genotypes. Amplicon yields were significantly dependent on DNA extraction method, parasite density and primer set (p < 0.001). A Qiagen® kit extraction had more than 2× higher amplicon yield than the Chelex method, for both urine and saliva. Amplicon yields were 1.6 fold higher from saliva than urine. For each unit increase in log parasite density, the probability of amplicon enhanced 1.8 fold. Highest amplicon yields were obtained from the primer set with the shortest PCR product. Conclusion P. falciparum infection is detectable by PCR on human urine and saliva samples. Subject to further refinement of extraction technique and amplicon yields, large-scale malaria parasite screening and epidemiological surveys could be possible without the need to collect blood and use of needles or sharps.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                January 2012
                3 January 2012
                : 6
                : 1
                Affiliations
                [1 ]Department of Molecular Microbiology and Immunology, Johns Hopkins University, Baltimore, Maryland, United States of America
                [2 ]MRC Centre of Outbreak Analysis and Modeling, Department of Infectious Disease Epidemiology, Imperial College London, St. Mary's Campus, London, United Kingdom
                [3 ]Department of Zoology, Obafemi Awolowo University, Ile Ife, Nigeria
                [4 ]Department of Statistics, London School of Economics, London, United Kingdom
                Natural History Museum, London, United Kingdom
                Author notes

                Conceived and designed the experiments: CS OI SA. Performed the experiments: OI CS. Analyzed the data: AK IM OI. Contributed reagents/materials/analysis tools: AK. Wrote the paper: AK OI CS. Latent Class modelling: AK IM.

                Article
                PNTD-D-11-00647
                10.1371/journal.pntd.0001464
                3250497
                22235360
                Ibironke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 6
                Categories
                Research Article
                Biology
                Microbiology
                Parasitology
                Quantitative Parasitology

                Infectious disease & Microbiology

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