Dataset Information

Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples.

ABSTRACT: This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen's kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

SUBMITTER: Hoffmann T

PROVIDER: S-EPMC7916106 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples.

Hoffmann Tanja T Hahn Andreas A Verweij Jaco J JJ Leboulle Gérard G Landt Olfert O Strube Christina C Kann Simone S Dekker Denise D May Jürgen J Frickmann Hagen H Loderstädt Ulrike U

Pathogens (Basel, Switzerland) 20210209 2

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays ...[more]

PMID: 33572338

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Project description:BackgroundOver one billion people are infected with soil-transmitted helminths (STH), i.e. Ascaris lumbricoides, hookworm and Trichuris trichiura. For estimating drug efficacy and monitoring anthelminthic drug resistance, accurate diagnostic methods are critical. FECPAKG2 is a new remote-diagnostic tool used in veterinary medicine, which produces an image of the stool sample that can be stored on an internet cloud. We compared for the first time FECPAKG2 with the recommended Kato-Katz method.Methodology/principal findingsTwo stool samples were collected from adolescent participants (age 15-18 years) at baseline and 14 to 21 days after treatment in the framework of a randomized clinical trial on Pemba Island, Tanzania. Stool samples were analyzed with different diagnostic efforts: i) one or ii) two Kato-Katz thick smears from the first sample, iii) two Kato-Katz thick smears from two samples and iv) FECPAKG2 from the first sample. Parameters were calculated based on a hierarchical Bayesian egg count model. Complete data for all diagnostic efforts were available from 615 participants at baseline and 231 hookworm-positive participants at follow-up. At baseline FECPAKG2 revealed a sensitivity of 75.6% (72.0-77.7) for detecting A. lumbricoides, 71.5% (67.4-95.3) for hookworm and 65.8% (64.9-66.2) for T. trichiura, which was significantly lower (all p<0.05) than any of the Kato-Katz methods and highly dependent on infection intensity. Despite that the egg counts based on FECPAKG2 were relatively lower compared to Kato-Katz by a ratio of 0.38 (0.32-0.43) for A. lumbricoides, 0.36 (0.33-0.40) for hookworm and 0.08 (0.07-0.09) for T. trichiura, the egg reduction rates (ERR) were correctly estimated with FECPAKG2.Conclusions/significanceThe sensitivity to identify any STH infection was considerably lower for FECPAKG2 compared to Kato-Katz. Following rigorous development, FECPAKG2 might be an interesting tool with unique features for epidemiological and clinical studies.

Dataset Information

Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples.

Publications

Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples.

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