Qianyun Lin1,2, Nira R Pollock3,4, Alice Banz5, Aude Lantz5, Hua Xu2, Limei Gu2, Dale N Gerding6, Kevin W Garey7, Anne J Gonzales-Luna7, Mingwei Zhao8, Linan Song8, David C Duffy8, Ciaran P Kelly2, Xinhua Chen2. 1. Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, China. 2. Divisions of Gastroenterology, Massachusetts. 3. Infectious Diseases, Beth Israel Deaconess Medical Center, Massachusetts. 4. Department of Laboratory Medicine, Boston Children's Hospital, Massachusetts. 5. bioMerieux, Marcy L'Etoile, France. 6. Department of Veterans Affairs, Edward Hines Jr Veterans Affairs Hospital, Hines, Illinois. 7. Department of Pharmacy Practice and Translational Research, University of Houston College of Pharmacy, Texas. 8. Quanterix, Billerica, Massachusetts.
Abstract
BACKGROUND: Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A-negative, toxin B-positive (A-B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. METHODS: An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). RESULTS: There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23-17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). CONCLUSIONS: We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.
BACKGROUND: Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A-negative, toxin B-positive (A-B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. METHODS: An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). RESULTS: There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23-17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). CONCLUSIONS: We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.
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