Protocol development for the use of matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) for detection of clostridium difficile toxin A and B from stool samples
Notice bibliographique
Résumé
Background: Clostridium difficile is an anaerobic, Gram-positive, spore-forming bacillus. C. difficile is important in the medical community because it is the most common cause of antibiotic-associated diarrhea and infections can lead to serious complications such as toxic megacolon and pseudomembranous colitis. Infection is mediated by toxins A and B, both of which are proteins that undergo processing within the target cell to yield a 63 kDa active domain.\n\nObjective: This study investigates the use of MALDI-TOF-MS to detect the active domains of C. difficile toxins A and B from stool samples to explore the possibility of using MALDI as a diagnositc tool for C. difficile infections.\n\nMethods: Known toxin and antigen positive and negative stool samples sent from Royal Inland Hospital were diluted 10-fold in deionized water or phosphate buffered saline (PBS) and vortexed to create a relatively homogeneous suspension. Samples were then centrifuged and the pellet removed. Proteins in the supernatant were precipitated with acetonitrile or ammonium sulfate and the solution was centrifuged again. The pellet was resuspended in deionized water or TA30 and spotted on a MALDI plate with a sinnapinic acid (SA), SDHB (a mixture of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2-hydroxy-5-methoxybenzoic acid), or CHCA (α-Cyano-4-hydroxycinnamic acid) matrix co-crystalized.\n\nResults and Discussion: MALDI analysis showed no difference between samples diluted in deionized water and those diluted in PBS. Protein precipitation with acetonitrile produced higher quality spectra than protein precipitation with ammonium sulfate. Sample co-crystalization with a SA matrix provided higher quality spectra than sample co-crystalization with a SDHB or CHCA matrix. MALDI analysis showed no peaks in the 63 kDa range in any of the samples. Because stool is a complex combination of materials, MALDI mass spectra were expected to be complicated and show vast differences between samples. Surprisingly, all ten MALDI spectra acquired were relatively similar. Similar individual ion signals were seen between 20 and 60 kDa and above 70 kDa. No individual ion signals were seen in the 63 kDa range in any of the samples, regardless of their being toxin and antigen positive or negative. This suggests that there is an open mass window for unambiguous detection of the 63 kDa active domain.\n\nConclusion: We were unable to use MALDI to detect the 63 kDa active domains of C. difficile toxins A and B from crude stool protein extracts. Further studies would be required to ascertain the possibility of using this technological tool to detect C. difficile toxins as an alternative method of diagnosis to the tests currently available. Although inconclusive, this study is a starting point for the investigation of MALDI as a diagnostic tool in a clinical setting.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,005 | 0,006 |
| Méta-épidémiologie (sens strict) | 0,004 | 0,003 |
| Méta-épidémiologie (sens large) | 0,003 | 0,003 |
| Bibliométrie | 0,003 | 0,002 |
| Études des sciences et des technologies | 0,002 | 0,001 |
| Communication savante | 0,001 | 0,001 |
| Science ouverte | 0,003 | 0,001 |
| Intégrité de la recherche | 0,002 | 0,003 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,036 | 0,033 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».