Dataset Information

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

ABSTRACT:

Background

In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study.

Methodology

Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months.

Principal findings

Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic "gold standard", the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100).

Conclusions

The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments.

SUBMITTER: Schunk M

PROVIDER: S-EPMC4567332 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

Schunk Mirjam M Kebede Mekonnen Seleshi S Wondafrash Beyene B Mengele Carolin C Fleischmann Erna E Herbinger Karl-Heinz KH Verweij Jaco J JJ Geldmacher Christof C Bretzel Gisela G Löscher Thomas T Zeynudin Ahmed A

PloS one 20150911 9

<h4>Background</h4>In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study.<h4>Methodology</h4> ...[more]

PMID: 26360049

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Project description:BACKGROUND: Schistosomiasis constitutes a major public health problem, and 200 million people are estimated to be infected with schistosomiasis worldwide. In Brazil, schistosomiasis has been reported in 19 states, showing areas of high and medium endemicity and a wide range of areas of low endemicity (ALE). Barra Mansa in Rio de Janeiro state has an estimated prevalence of 1%. ALE represent a new challenge for the helminth control because about 75% of infected individuals are asymptomatic and infections occur with a low parasite load (<100 eggs per gram of feces), causing a decrease in sensitivity of stool parasitological techniques, which are a reference for the laboratory diagnosis of this helminth. The objective of this study was to evaluate the performance of a TaqMan quantitative polymerase chain reaction (qPCR) technique in serum and feces DNA samples using the techniques of Kato-Katz (KK), Hoffman, Pons and Janer (HH) as references, during an epidemiological survey using fecal samples and sera from randomized residents from an ALE. METHODS: A cross-sectional study conducted from April to December 2011 using a probabilistic sampling that collected 572 fecal and serum samples. The laboratory diagnostic techniques used were: KK, HH and qPCR (feces and serum). RESULTS: We obtained the following results using the different diagnostic techniques: KK and HH, 0.9% (n =5); qPCR-feces, 9.6% (n =55); and qPCR-serum, 1.4% (n =8). The qPCR-feces presented the highest positivity, whereas the techniques of HH and KK were the least sensitive to detect infections (0.8%). Compared to HH and KK, qPCR-feces showed a statistically significant difference in positivity (p <0.05), although with poor agreement. CONCLUSION: The positivity rate presented by the qPCR approach was far higher than that obtained by parasitological techniques. The lack of adequate surveillance in ALE of schistosomiasis indicates a high possibility of these areas being actually of medium and high endemicity. This study presents a control perspective, pointing to the possibility of using combined laboratory tools in the diagnosis of schistosomiasis in ALE.

Project description:BackgroundTo detect acute schistosomiasis, low-intensity infections, or to verify the success of treatment with praziquantel, highly sensitive test methods are required. The aim of this study was therefore to demonstrate the performance of Schistosoma mansoni specific DNA detection in serum and urine using real-time polymerase chain reaction (PCR) in an endemic area before and after treatment.MethodsThe study pursued a 1-week and 20-weeks longitudinal design with a treatment intervention among 36 study participants aged 18 to 70 years in the community of Kayenze, a fishing village in Ilemela district on the southern shore of Lake Victoria in north-western Tanzania between February and June 2018. Blood, urine and stool samples were collected from each participant to diagnose Schistosoma mansoni infection before and two times after treatment with praziquantel using serum- and urine based real-time PCR, point-of-care circulating cathodic antigen (POC-CCA) rapid diagnostic test and the microscopic Kato-Katz (KK) method. Kappa coefficient (κ) was used to estimate the agreement between these diagnostic tests compared to a combined "gold standard" of positive results by serum-based real-time PCR and/or positive egg counts determined by KK. Kendall's Tau rank correlation was used to examine the relationship between cycle threshold (Ct)-values and egg counts and the Wilcoxon signed-rank test was used to compare the median Ct-values of the different examination time points.ResultsBy using the combined "gold standard" of the parasitological Kato-Katz test and/or serum-based real-time PCR, a S. mansoni prevalence of 77.1% could be determined at baseline. In terms of sensitivity, serum-based real-time PCR (96.3%) and POC-CCA assay (77.8%) showed the highest results. The detection of DNA from urine samples showed the lowest sensitivity (33.3%). Treatment with praziquantel resulted in a significantly reduced prevalence of S. mansoni. No infection could be detected by Kato-Katz, with the POC-CCA test only 33.3%. The analysis of the median Ct values over time (which were determined by the serum-based real-time PCR) showed that the Ct decreases significantly shortly after treatment (from 30.3 to 28) and increases above baseline level (34.9) three months later.ConclusionsThe data presented here show that the serum-based real-time PCR exhibits excellent diagnostic accuracy, in contrast to the use of urine as sample material for S. mansoni DNA detection. However, as circulating DNA does not necessarily reflect the persistence of living worms in schistosomiasis, this method is less well suited to verify the success of treatment with praziquantel.

Project description:BackgroundAccurate diagnosis followed by timely treatment is an effective strategy for the prevention of complications together with reducing schistosomiasis transmission. Recombinase Polymerase Amplification (RPA) is a simple, rapid, sensitive, and specific isothermal method with low resource needs. This research aimed at the development and optimisation of a real-time (RT) and a lateral flow (LF) RPA assay for the detection of Schistosoma mansoni.MethodologyRecombinase Polymerase Amplification reactions were performed at full- (as recommended) and half-volumes (to reduce costs), with RT or LF detection systems targeting the S. mansoni mitochondrial minisatellite region. The specificity was assessed using gDNA from other Schistosoma species, helminths co-endemic with S. mansoni, human stool, and urine, and Biomphalaria snail hosts. The analytical sensitivity was evaluated using serial dilutions of gDNA, synthetic copies of the target, and single eggs. The ability of both assays to detect the S. mansoni DNA in human urine and stool samples was also tested. The long-term stability of the RT-RPA reagents was evaluated by storing the reaction components in different temperature conditions for up to 3 weeks.ResultsThe RT- and the LF-RPA (SmMIT- and SmMIT-LF-RPA, respectively) presented similar results when used full- and half-volumes, thus the latter was followed in all experiments. The SmMIT-RPA was 100% specific to S. mansoni, able to detect a single egg, with a limit of detection (LOD) of down to 1 fg of gDNA and one synthetic copy of the target. The assay was able to detect S. mansoni DNA from stool containing 1 egg/g and in spiked urine at a concentration of 10 fg/μl. SmMIT-RPA reagents were stable for up to 3 weeks when kept at 19°C, and 2 weeks when stored at 27°C. The SmMIT-LF-RPA cross-reacted with Clinostomidae, presented the LOD of 10 fg and one synthetic copy of the target, being able to detect a single egg and 1 egg/g in a stool sample. The LOD in spiked urine samples was 10 pg/μl.ConclusionThe half-volume SmMIT-RPA is a promising method to be used in the field. It is specific, sensitive, robust, and tolerant to inhibitors, with a long-term stability of the reaction components and the real-time visualisation of results.

Dataset Information

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

Background

Methodology

Principal findings

Conclusions

Publications

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

Similar Datasets

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