Detection of toxigenic Clostridium difficile by Loop-Mediated Isothermal Amplification (LAMP)
Bibliographic record
Abstract
Background: Clostridium difficile-associated disease (CDAD) is a leading cause of nosocomial diarrhea in adults. Therefore, rapid and accurate reporting of toxigenic C. difficile is essential for improving patient outcomes and minimizing hospital-acquired disease. The PCR-based in-house and commercial methods available now to detect toxigenic C. difficile are expensive or require nucleic acid purification. We describe a multiplex real-time loop-mediated isothermal amplification method (LAMP) to detect toxigenic C. difficile without DNA purification and to presumptively identify the NAP1 strain directly from diarrheal stools. Methods & Materials: Five-hundred and eighteen stools submitted for routine PCR testing of C. difficile was used. The DNA was extracted by mixing and boiling 100 μl of 1:10 dilution of stools with 100 μl of lysis solution for 15 min. Two microliters of the clear supernatant was used for LAMP reactions. LAMP method amplified and detected tcdC, cdtA, and λ DNA (IC). Primers used for amplification were designed using Genbank and PrimerExplorer V4 (Eiken Chemical Co., Ltd. Japan). DNA amplification was done at 59 0C for 60 min using Rotorgene 6500 (Qiagen) and a standard LAMP reaction. Amplification was detected by displacement of a fluorescent probe annealed to the quencher-labeled primer: Detection of Amplification using Reduced Quenching (DARQ). The limit of detection (LOD) was determined by using a C. difficile negative stool specimen spiked with known number of colony forming units of C. difficile ATCC43255. Results: Out of 518 specimens 200 tested were positive and 307 were negative for tcdC by both methods. There were 11 discrepant specimens that were all negative by LAMP but positive by PCR with high CT values (>35). The test performance characteristics of LAMP method as compared to the PCR was as follows: sensitivity, 95%; specificity, 100%; NPV, 97%; PPV, 100%. The LOD was estimated to be 750 genome equivalents. The presence of both tcdC and cdtA presumptively identified 65 specimens to have the NAP1 strain by both methods. Conclusion: The turn-around-time for LAMP-DARQ was 90 min as compared to 3.5 hours for the in-house PCR. The estimated cost per test for LAMP-DARQ was Cdn $4.00 and 60% cheaper than the PCR. The LAMP-DARQ is a cost-effective, sensitive, faster method than PCR to detect toxigenic C. difficile.
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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 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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".