A multiplex real-time PCR method for presumptive identification of NAP1 clone of Clostridium difficile from stools
Bibliographic record
Abstract
Background: Clostridium difficile infection (CDI) is the leading cause of nosocomial diarrhea in adults and paediatric patients. There has been significant morbidiity and mortality related to CDI due to the presence of hypervirulent strain (NAP1) associated with unregulated production of toxin. NAP1 has been responsible for a number of outbreaks in many countries. Rapid detection for the presence of NAP1 is essential for appropriate patient management and to minimize nosocomial transmission. Currently, pulsed-field gel electrophoresis (PFGE) and repetitive sequence-based (REP-PCR) methods which require bacterial culture have been used to identify NAP1. A 18 bp deletion in the tcdC gene and the presence of cdtA gene (binary toxin) have been shown to be associated with NAP1. We describe a rapid multiplex real-time PCR method for presumptive identification of NAP1. Methods: Ninety blinded frozen stool samples previously tested for toxin A/B by CD TOX A/B® II EIA (TechLab, Inverness Medical) were used. A fragment of tcdC flanking the 18 bp deletion (surrogate marker for tcdA/tcdB), cdtA, and 16S rDNA (internal control) genes were amplified using Multiplex QuantiTechTM (Qiagen, Inc.) and detected by TaqMan probes. ProGastroTM (Prodesse, Inc.) kit that detects tcdB was used for comparison. DNA from stools were extracted using easyMAG (BioMerieux Inc) and tested accodring to manufacturers’ specifications. Five !l of the same DNA was used for the in-house method. Both real-time PCR amplifications were done in a Rotor-Gene 6000 (Corbette, Inc.). PCR products from both tcdC and cdtA positive samples were separated on agarose gels to detect tcdC deletion. Results: DNA from 85 specimens were positive for tcdC (toxigenic) and 53 of those showed the presence of cdtA gene (presumptive NAP1). DNA from none of the specimens were positive for cdtA only.The tcdB detection by ProGastroCDTM was concordant with the detection of tcdC. Gel electrophoresis of all tcdC and cdtA positive PCR products detected the 18 bp deletion in the tcdC amplicon and were confirmed as NAP1 by PFGE. All cdtA negative DNA samples were negative for 18 bp deletion in the tcdC. Conclusion: Detection of toxigenic strains by the in-house method is comparable to the commercial assay and it presumptively identified the presence of NAP1. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.002 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".