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Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples.

ABSTRACT: BACKGROUND:Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples. METHODS:A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar's ?(2) test, with consideration of a P-value of <0.05 as indication of significant difference. RESULTS:In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture. CONCLUSION:Single PCR method amplifying a short (100bp) target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.

SUBMITTER: Moghaddassani H

PROVIDER: S-EPMC3279873 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples.

Moghaddassani H H Mirhendi H H Hosseini M M Rokni Mb M Mowlavi Gh G Kia Eb E

Iranian journal of parasitology 20110601 2

<h4>Background</h4>Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples.<h4>Methods</h4>A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confir ...[more]

PMID: 22347284

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Project description:Detecting infections of Strongyloides stercoralis is arduous and has low sensitivity. Clinically this is a major problem because chronic infections may disseminate in the host and lead to a life threatening condition. Epidemiologically, S. stercoralis is often missed in surveys as it is difficult to identify by standard stool examination procedures. We present, for the first time, evidence that the infection can be detected in filtered urine samples collected and processed in the field and subsequently assayed for the presence of parasite DNA. Urine specimens (∼40mL) were collected from 125 test and control individuals living in rural and peri-urban regions of Northern Argentina. From the same individuals, fresh stool specimens were processed using three different copropological methods. Urine specimens were filtered in the field through a 12.5cm Whatman No. 3 filter. The filters were dried and packed individually in sealable plastic bags with desiccant and shipped to a laboratory where DNA was recovered from the filter and PCR-amplified with primers specific to a dispersed repetitive sequence. Prevalence of S. stercoralis infection by stool culture and direct examination was 35/125 (28%), In contrast, PCR-based detection of parasite-specific trans-renal DNA in urine indicated that 56/125 (44.8%) carried the parasite. Of the patients that tested positive for urine-based parasite DNA, approximately half also tested positive in their stool specimens. There were 6.4% of cases where parasite larvae were seen in the stool but no DNA was amplified from the urine. As proof of principle, DNA amplification from urine residue reveals significantly more cases of S. stercoralis infection than the current standard stool examination techniques. Additional work is required to establish the relative utility, sensitivity and specificity of urine-based analysis compared to parasitological and nucleic acid detection from stool for clinical and epidemiological detection for S. stercoralis infection.

Project description:Strongyloidiasis is a neglected tropical disease caused by the soil-transmitted nematode by Strongyloides stercoralis, that affects approximately 600 million people worldwide. In immunosuppressed individuals disseminated strongyloidiasis can rapidly lead to fatal outcomes. There is no gold standard for diagnosing strongyloidiasis, and infections are frequently misdiagnosed. A better understanding of the molecular biology of this parasite can be useful for example for the discovery of potential new biomarkers. Interestingly, recent evidence showed the presence of small RNAs in Strongyloididae, but no data was provided for S. stercoralis. In this study, we present the first identification of miRNAs of both L1 and iL3 larval stages of S. stercoralis. For our purpose, the aims were: (i) to analyse the miRNome of L1 and iL3 S. stercoralis and to identify potential miRNAs of this nematode, (ii) to obtain the mRNAs profiles in these two larval stages and (iii) to predict potential miRNA target sites in mRNA sequences. Total RNA was isolated from L1 and iL3 collected from the stool of 5 infected individuals. For the miRNAs analysis, we used miRDeep2 software and a pipeline of bio-informatic tools to construct a catalog of a total of 385 sequences. Among these, 53% were common to S. ratti, 19% to S. papillosus, 1% to Caenorhabditis elegans and 44% were novel. Using a differential analysis between the larval stages, we observed 6 suggestive modulated miRNAs (STR-MIR-34A-3P, STR-MIR-8397-3P, STR-MIR-34B-3P and STR-MIR-34C-3P expressed more in iL3, and STR-MIR-7880H-5P and STR-MIR-7880M-5P expressed more in L1). Along with this analysis, we obtained also the mRNAs profiles in the same samples of larvae. Multiple testing found 81 statistically significant mRNAs of the total 1553 obtained (FDR < 0.05; 32 genes expressed more in L1 than iL3; 49 genes expressed more in L3 than iL1). Finally, we found 33 predicted mRNA targets of the modulated miRNAs, providing relevant data for a further validation to better understand the role of these small molecules in the larval stages and their valuein clinical diagnostics.

Project description:BACKGROUND:Strongyloidiasis is a gut infection with Strongyloides stercoralis which is common world wide. Chronic infection usually causes a skin rash, vomiting, diarrhoea or constipation, and respiratory problems, and it can be fatal in people with immune deficiency. It may be treated with ivermectin or albendazole or thiabendazole. OBJECTIVES:To assess the effects of ivermectin versus benzimidazoles (albendazole and thiabendazole) for treating chronic strongyloides infection. SEARCH METHODS:We searched the Cochrane Infectious Diseases Group Specialized Register (24 August 2015); the Cochrane Central Register of Controlled Trials (CENTRAL), published in the Cochrane Library; MEDLINE (January 1966 to August 2015); EMBASE (January 1980 to August 2015); LILACS (August 2015); and reference lists of articles. We also searched the metaRegister of Controlled Trials (mRCT) using 'strongyloid*' as a search term, reference lists, and conference abstracts. SELECTION CRITERIA:Randomized controlled trials of ivermectin versus albendazole or thiabendazole for treating chronic strongyloides infection. DATA COLLECTION AND ANALYSIS:Two review authors independently extracted data and assessed risk of bias in the included trials. We used risk ratios (RRs) with 95% confidence intervals (CIs) and fixed- or random-effects models. We pooled adverse event data if the trials were sufficiently similar in their adverse event definitions. MAIN RESULTS:We included seven trials, enrolling 1147 participants, conducted between 1994 and 2011 in different locations (Africa, Southeast Asia, America and Europe).In trials comparing ivermectin with albendazole, parasitological cure was higher with ivermectin (RR 1.79, 95% CI 1.55 to 2.08; 478 participants, four trials, moderate quality evidence). There were no statistically significant differences in adverse events (RR 0.80, 95% CI 0.59 to 1.09; 518 participants, four trials, low quality evidence).In trials comparing ivermectin with thiabendazole, there was little or no difference in parasitological cure (RR 1.07, 95% CI 0.96 to 1.20; 467 participants, three trials, low quality evidence). However, adverse events were less common with ivermectin (RR 0.31, 95% CI 0.20 to 0.50; 507 participants; three trials, moderate quality evidence).In trials comparing different dosages of ivermectin, taking a second dose of 200 μg/kg of ivermectin was not associated with higher cure in a small subgroup of participants (RR 1.02, 95% CI 0.94 to 1.11; 94 participants, two trials).Dizziness, nausea, and disorientation were commonly reported in all drug groups. There were no reports of serious adverse events or death. AUTHORS' CONCLUSIONS:Ivermectin results in more people cured than albendazole, and is at least as well tolerated. In trials of ivermectin with thiabendazole, parasitological cure is similar but there are more adverse events with thiabendazole.

Dataset Information

Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples.

Publications

Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples.

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