Strategies for Optimizing the Diagnostic Predictive Value of Clostridium difficile Molecular Diagnostics

Kociolek, Larry K.

doi:10.1128/jcm.00147-17

Cited by 19 publications

(18 citation statements)

References 25 publications

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“…The traditional gold standard tests for detecting toxigenic C. difficile organisms (toxigenic culture assay) and C. difficile toxin (cell-culture cytotoxicity neutralization assay) are slow, labor-intensive, and technically challenging 37 . The diagnostics currently in wide-spread use, such as enzyme-linked immunoassays (EIA) for C. difficile toxins and DNA-based qPCR assays for C. difficile toxin genes, offer greatly improved performance characteristics but have their own limitations 21 .…”

Section: Resultsmentioning

confidence: 99%

A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

et al. 2018

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There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease. Current methods for profiling the microbiome typically utilize next-generation sequencing applications that are expensive, slow, and complex. Here, we present a synthetic biology platform for affordable, on-demand, and simple analysis of microbiome samples using RNA toehold switch sensors in paper-based, cell-free reactions. We demonstrate species-specific detection of mRNAs from 10 different bacteria that affect human health and four clinically relevant host biomarkers. We develop a method to quantify mRNA using our toehold sensors and validate our platform on clinical stool samples by comparison to RT-qPCR. We further highlight the potential clinical utility of the platform by showing that it can be used to rapidly and inexpensively detect toxin mRNA in the diagnosis of Clostridium difficile infections.

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Section: Resultsmentioning

confidence: 99%

A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

et al. 2018

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“…In other words, a patient with intrinsically high levels of mef(A) in her healthy microbiome would The question of whether our RPA assay would distinguish infection from colonization is related to a larger debate in the diagnostic field: when is a molecular assay too sensitive? Molecular detection methods like qPCR or RPA are much more sensitive than culture methods, often identifying many more microbes than culture [40,63], leading some to conclude that the diagnostic utility of these methods is limited due to false positives [64]. However, there are several strategies for mitigating this risk: for example, testing only at-risk populations, as applied to testing for C. difficile or Group-A Streptococcus (S. pyogenes) [64].…”

Section: Discussionmentioning

confidence: 99%

“…Molecular detection methods like qPCR or RPA are much more sensitive than culture methods, often identifying many more microbes than culture [40,63], leading some to conclude that the diagnostic utility of these methods is limited due to false positives [64]. However, there are several strategies for mitigating this risk: for example, testing only at-risk populations, as applied to testing for C. difficile or Group-A Streptococcus (S. pyogenes) [64]. This strategy minimizes the chance of a false-positive detection by not employing the test in cases unlikely to represent true infection.…”

Section: Discussionmentioning

confidence: 99%

Rapid molecular detection of macrolide resistance

2019

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BackgroundEmerging antimicrobial resistance is a significant threat to human health. However, methods for rapidly diagnosing antimicrobial resistance generally require multi-day culture-based assays. Macrolide efflux gene A, mef(A), provides resistance against erythromycin and azithromycin and is known to be laterally transferred among a wide range of bacterial species.MethodsWe use Recombinase Polymerase Assay (RPA) to detect the antimicrobial resistance gene mef(A) from raw lysates without nucleic acid purification. To validate these results we performed broth dilution assays to assess antimicrobial resistance to erythromycin and ampicillin (a negative control).ResultsWe validate the detection of mef(A) in raw lysates of Streptococcus pyogenes, S. pneumoniae, S. salivarius, and Enterococcus faecium bacterial lysates within 7–10 min of assay time. We show that detection of mef(A) accurately predicts real antimicrobial resistance assessed by traditional culture methods, and that the assay is robust to high levels of spiked-in non-specific nucleic acid contaminant. The assay was unaffected by single-nucleotide polymorphisms within divergent mef(A) gene sequences, strengthening its utility as a robust diagnostic tool.ConclusionsThis finding opens the door to implementation of rapid genomic diagnostics in a clinical setting, while providing researchers a rapid, cost-effective tool to track antibiotic resistance in both pathogens and commensal strains.

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“…Among these methods, either the TC or CCNA has been considered to be the gold standard for the diagnosis of CDI over the past 30 years 17 , 18 . These tests use the following principles: TC detects the presence of C. difficile strains that actively produce toxin(s) (e.g., organism detection), whereas CCNA detects fecal protein toxins that have been produced in the stool (e.g., fecal toxin detection) 19 – 21 . It is noteworthy that both the TC and CCNA assays have limitations.…”

Section: Current Laboratory Diagnosismentioning

confidence: 99%

Advances in the diagnosis and treatment of Clostridium difficile infections

Peng

Ling

Stratton

et al. 2018

Emerging Microbes & Infections

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Clostridium difficile is a leading cause of antibiotic-associated diarrhea worldwide. The diagnosis of C. difficile infection (CDI) requires both clinical manifestations and a positive laboratory test for C. difficile and/or its toxins. While antibiotic therapy is the treatment of choice for CDI, there are relatively few classes of effective antibiotics currently available. Therefore, the development of novel antibiotics and/or alternative treatment strategies for CDI has received a great deal of attention in recent years. A number of emerging agents such as cadazolid, surotomycin, ridinilazole, and bezlotoxumab have demonstrated activity against C. difficile; some of these have been approved for limited clinical use and some are in clinical trials. In addition, other approaches such as early and accurate diagnosis of CDI as well as disease prevention are important for clinical management. While the toxigenic culture and the cell cytotoxicity neutralization assay are still recognized as the gold standard for the diagnosis of CDI, new diagnostic approaches such as nucleic acid amplification methods have become available. In this review, we will discuss both current and emerging diagnostic and therapeutic modalities for CDI.

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Strategies for Optimizing the Diagnostic Predictive Value of Clostridium difficile Molecular Diagnostics

Cited by 19 publications

References 25 publications

A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

Rapid molecular detection of macrolide resistance

Advances in the diagnosis and treatment of Clostridium difficile infections

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