Detection of Clostridium Difficile Toxins on Patients With Hematolojical Malignency

Year 2020, , 596 - 602, 09.11.2020

Gül Durmaz , Meltem Olga Akay , Mehdi Meskini Heydarlou

https://doi.org/10.20515/otd.571859

Abstract

Clostridium difficile, the member of human gastrointestinal system microbiotia, is a gram- positive, spore-forming anaerobic bacilli. It is present in the gastrointestinal tract in asymptomatic carriers, though; it can cause a variety of clinical features such as: mild diarrhea, non-pseudomembranous colitis, pseudomembranous colitis (PMC), and fulminant colitis/toxic megacolon. Following the use age of broad spectrum antibiotics, C.difficile strains gain benefit of ecological advantage and start to multiply and produce toxins leading to diarrhea. In the last 20 years, nosocomial and community acquired C.difficile infections showed significant increasing incidence and mortality rates. Our study is aimed to investigate toxigenic C.difficile in diarrheal stool specimens collected from in-patients of Eskişehir Osmangazi University Hospital Hematology and Oncology departments who receive broad spectrum antibiotics for prophylactic or therapeutic purposes. Toxigenic C.difficile was investigated by immunodiagnostic and molecular tests. During the period between January-June 2015, 82 diarrheal specimens were collected in suitable stool containers and sent to Microbiology laboratory within an hour. EIA method (TECH LAB, Inc.) was used to detect the presence of glutamate dehydrogenase enzyme (GDH) and toxins A plus B. Also, real-time PCR (BD GeneOhm™ Cdiff Assay) was used to detect toxin B gene. Fifty six samples were negative in all three tests performed. However; nine specimens were positive in at least two diagnostic tests performed. Three samples were positive by EIA (GDH, toxins A and B) while these specimens were negative by the molecular test. The negative result could be false negative due to tcdB gene mutation, or it could be due to toxin B-/binary toxin producing strain. According to our results, GDH EIA is a reliable initial screening test to be applied in high risk patients. Though, it would be an appropriate approach to verify GDH EIA positive results by performing either immuno diagnostic tests which detect toxin A and B or toxin B gene based molecular tests. Our study is aimed to investigate toxigenic C.difficile in diarrheal stool specimens collected from in-patients of Eskişehir Osmangazi University Hospital Hematology and Oncology departments who receive broad spectrum antibiotics for prophylactic or therapeutic purposes. Toxigenic C.difficile was investigated by immunodiagnostic and molecular tests. During the period between January-June 2015, 82 diarrheal specimens were collected in suitable stool containers and sent to Microbiology laboratory within an hour. EIA method (TECH LAB, Inc.) was used to detect the presence of glutamate dehydrogenase enzyme and toxins A plus B. Also, real-time PCR (BD GeneOhm™ Cdiff Assay) was used to detect toxin B gene. Fifty six samples were negative in all three tests performed. However; nine specimens were positive in at least two diagnostic tests performed. Three samples were positive by EIA (GDH, toxins A and B) while these specimens were negative by the molecular test. The negative result could be false negative due to tcdB gene mutation, or it could be due to toxin B-/binary toxin producing strain. According to our results, GDH EIA is a reliable initial screening test to be applied in high risk patients. Though, it would be an appropriate approach to verify GDH EIA positive results by performing either immuno diagnostic tests which detect toxin A and B or toxin B gene based molecular tests.

Keywords

Clostridium difficile; ToxinA; Toxin B; Diarrhea; Diagnostic tests.

Supporting Institution

ESOGÜ BAP

Project Number

2014-567

Hematolojik Malignansili Hastalarda Gelişen Diyare Olgularında Clostridium difficile Toksinlerinin Araştırılması

Abstract

Clostridium difficile, asemptomatik taşıyıcılık, ılımlı diyare, psödomembransız kolit, psödomemranöz kolit (PMK), fulminant kolit/toksik megakolon gibi çok çeşitli klinik tablolara neden olabilen insan gastrointestinal sistem mikrobiyota üyesi, gram pozitif, sporlu anaerob çomakçıktır. Geniş spektrumlu antibiyotik kullanımı sonrası kolonda bulunan C.difficile suşları ekolojik avantaj kazanıp çoğalmakta ve toksin sentezleyerek ishale neden olmaktadır. Son 20 yılda gerek hastane, gerekse toplum kaynaklı C.difficile enfeksiyonlarının insidans ve mortalitesinde belirgin bir artış olduğu göze çarpmaktadır. Bu çalışmada Eskişehir Osmangazi Üniversitesi Hastanesi Hematoloji ve Onkoloji kliniklerinde terapötik veya proflaktik amaçlı geniş spektrumlu antibiyotik kullanımı sonrasında ishal gelişen hastaların dışkısında toksijenik C.difficile varlığının iki farklı immündiagnostik testin yanısıra moleküler test kullanılarak araştırılması amaçlanmıştır. Ocak- Haziran 2015 tarihleri arasında Hematoloji ve Onkoloji kliniklerinde yatan ve diyare gelişen 82 hastanın dışkı örnekleri incelenmiştir. Dışkı örnekleri kapaklı, sızdırmaz transport dışkı kaplarında bir saat içinde Mikrobiyoloji laboratuvarına ulaştırılmıştır. Glutamat dehidrogenaz enzimi (GDH) ve toksin A ve B saptamada EIA (enzim immunoassay) yöntemi (TECH LAB, Inc.), toksin B genini saptamada ise real-time PCR yöntemi (BD GeneOhm™ Cdiff Assay) kullanılmıştır. Elli altı örnekte her üç testle negatif sonuç elde edilmiş olup, dokuz hastada da yapılan testlerden en az iki tanesi pozitif olarak bulunmuştur. Üç hastada ise EIA (GDH, toksin A ve B) testleri pozitif iken moleküler yöntemle negatif sonuç alındı. Moleküler yöntem ile saptanan ve yalancı negatiflik olarak kabul ettiğimiz bu durumun tcdB geninde gerçekleşen mutasyon veya sadece toksin B-/binary toksin sentezleyen suş nedeniyle olduğu düşünülmüştür. GDH EIA testinin riskli hasta gruplarına uygulanacak güvenirlilikte bir tarama testi olduğu ve GDH pozitifliği durumunda ise toksin A ve B saptayan immündiagnostik testler veya toksin B genini saptayan moleküler testlerden biriyle sonucun doğrulanmasının uygun bir yaklaşım olacağı sonucuna varıldı. Bu çalışmada Eskişehir Osmangazi Üniversitesi Tıp Fakültesi Hastanesi Hematoloji ve Onkoloji kliniklerinde terapötik veya proflaktik amaçlı geniş spekturumlu antibiyotik kullanımı sonrasında ishal gelişen hastaların dışkısında toksijenik C.difficile varlığının iki farklı immündiagnostik testin yanısıra moleküler test kullanılarak araştırılması amaçlanmıştır. Ocak-Haziran 2015 tarihleri arasında Hematoloji ve Onkoloji kliniklerde yatan ve diyaresi olan 82 hastanın dışkı örnekleri incelenmiştir. Dışkı örnekleri kapaklı, sızdırmaz transport dışkı kaplarında bir saat içinde Mikrobiyoloji laboratuvarına ulaştırılmıştır. Glutamat dehidrogenaz enzimi (GDH) ve toksin A ve B saptamada EIA (enzim immunoassay) yöntemi (TECH LAB, Inc.), toksin B genini saptamada ise Real-Time PCR yöntemi (BD GeneOhm™ CdiffAssay) kullanılmıştır. Elli altı örnekte her üç testle negatif sonuç elde edilmiş olup, dokuz hastada da yapılan testlerden en az iki tanesi pozitif olarak bulunmuştur. Üç hastada ise EIA (GDH, toksin A ve B) testleri pozitif iken moleküler yöntemle negatif sonuç alındı. Moleküler yöntem ile saptanan ve yalancı negatiflik olarak kabul ettiğimiz bu durumun tcdB geninde gerçekleşen mutasyon veya sadece toksin B-/binary toksin sentezleyen suş nedeniyle olduğu düşünülmüştur. GDH EIA testinin riskli hasta gruplarına uygulanacak güvenirlilikte bir tarama testi olduğu ve GDH pozitifliği durumunda ise toksin A ve B saptayan immündiagnostik testler veya toksin B genini saptayan moleküler testlerden biriyle sonucun doğrulanmasının uygun bir yaklaşım olacağı sonucuna varıldı.

Keywords

Clostridium difficile; Toksin A; Toksin B; Diyare; Tanısal test.

Project Number

2014-567

References

• 1. Snyder, M.L., Further studies on Bacillus difficilis (Hall and O'Toole). The Journal of Infectious Diseases, 1937: p. 223-231.
• 2. Poxton, I., J. McCoubrey, and G. Blair, The pathogenicity of Clostridium difficile. Clinical microbiology and infection, 2001. 7(8): p. 421-427.
• 3. Lyerly, D.M. and T.D. Wilkins, Commercial latex test for Clostridium difficile toxin A does not detect toxin A. Journal of clinical microbiology, 1986. 23(3): p. 622-623.
• 4. Al-Barrak, A., et al., An outbreak of toxin A negative, toxin B positive Clostridium difficile-associated diarrhea in a Canadian tertiary-care hospital. Canada communicable disease report= Releve des maladies transmissibles au Canada, 1999. 25(7): p. 65.
• 5. O'Connor, J.R., S. Johnson, and D.N. Gerding, Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain. Gastroenterology, 2009. 136(6): p. 1913-1924.
• 6. Eckert, C., et al., Prevalence and pathogenicity of binary toxin–positive Clostridium difficile strains that do not produce toxins A and B. New microbes and new infections, 2015. 3: p. 12-17.
• 7. Wiström, J., et al., Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: a prospective study. Journal of antimicrobial chemotherapy, 2001. 47(1): p. 43-50.
• 8. Redelings, M.D., F. Sorvillo, and L. Mascola, Increase in Clostridium difficile–related mortality rates, United States, 1999–2004. Emerging infectious diseases, 2007. 13(9): p. 1417.
• 9. Elixhauser, A. and M. Jhung, Clostridium difficile-associated disease in US hospitals, 1993–2005. 2008.
• 10. Garzotto, A.R., et al., Risk factors associated with Clostridium difficile infection in adult oncology patients. Supportive Care in Cancer, 2015. 23(6): p. 1569-1577.
• 11. Warny, M., et al., Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. The Lancet, 2005. 366(9491): p. 1079-1084.
• 12. Cohen, S., et al., Society for Healthcare Epidemiology of America; Infectious Diseases Society of America. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol, 2010. 31(5): p. 431-455.
• 13. O'donoghue, C. and L. Kyne, Update on Clostridium difficile infection. Current opinion in gastroenterology, 2011. 27(1): p. 38-47.
• 14. Garey, K.W., et al., Prevalence of diarrhea at a university hospital and association with modifiable risk factors. Annals of Pharmacotherapy, 2006. 40(6): p. 1030-1034.
• 15. Planche, T. and M.H. Wilcox, Diagnostic pitfalls in Clostridium difficile infection. Infectious Disease Clinics, 2015. 29(1): p. 63-82.
• 16. Bartlett, J.G., Antibiotic-associated diarrhea. New England journal of medicine, 2002. 346(5): p. 334-339.
• 17. Poutanen, S.M. and A.E. Simor, Clostridium difficile-associated diarrhea in adults. Canadian Medical Association Journal, 2004. 171(1): p. 51-58.
• 18. Freeman, J., et al., Comparison of the efficacy of ramoplanin and vancomycin in both in vitro and in vivo models of clindamycin-induced Clostridium difficile infection. Journal of Antimicrobial Chemotherapy, 2005. 56(4): p. 717-725.
• 19. Ticehurst, J.R., et al., Effective detection of toxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin. Journal of clinical microbiology, 2006. 44(3): p. 1145-1149.
• 20. Carroll, K.C., Tests for the diagnosis of Clostridium difficile infection: the next generation. Anaerobe, 2011. 17(4): p. 170-174.
• 21. Stamper, P.D., et al., Comparison of a commercial real-time PCR assay for tcdB detection to a cell culture cytotoxicity assay and toxigenic culture for direct detection of toxin-producing Clostridium difficile in clinical samples. Journal of clinical microbiology, 2009. 47(2): p. 373-378.
• 22. Kvach, E.J., et al., Comparison of BD GeneOhm Cdiff real-time PCR assay with a two-step algorithm and a toxin A/B enzyme-linked immunosorbent assay for diagnosis of toxigenic Clostridium difficile infection. Journal of clinical microbiology, 2010. 48(1): p. 109-114.
• 23. Cohen, S.H., Y.J. Tang, and J. Silva Jr, Analysis of the pathogenicity locus in Clostridium difficile strains. The Journal of infectious diseases, 2000. 181(2): p. 659-663.
• 24. Rupnik, M., et al., Characterization of polymorphisms in the toxin A and B genes of Clostridium difficile. FEMS microbiology letters, 1997. 148(2): p. 197-202.
• 25. Perelle, S., et al., Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196. Infection and immunity, 1997. 65(4): p. 1402-1407.