Identification of Candida Species from Blood Cultures with Fluorescent In Situ Hybridization (FISH), Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Conventional Methods

Abstract

Objectives: Rapid and accurate identification of Candida species from blood cultures is crucial to ensure effective antifungal therapy and to reduce the morbidity and mortality associated with bloodstream fungal infections. In this study, we aimed to identify Candida spp. from blood culture samples with fluorescent in situ hybridization (FISH), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and conventional methods. Materials and Methods: A total of 50 yeast positive samples out of 325 blood culture positive samples and 50 blood culture negative samples were examined by FISH, PCRRFLP and conventional methods to identify Candida spp. Results: All three methods generally were compatible for identification of single-species Candida spp. (p<0.001). But, FISH and PCR-RFLP for the identification of multi-species Candida spp. were found more compatible than conventional methods (p<0.001). Besides, FISH is cheaper and quicker than the other two methods in the identification of Candida spp. from blood culture positive samples. The rates of multi-species candidemia with FISH, PCR-RFLP and conventional methods were 20%, 6% and 4%, respectively. Conclusion: Both PCR-RFLP and FISH methods might be preferred for the rapid identification of Candida spp. from blood culture positive samples. However, FISH is a more suitable method for the detection of multi-species candidemia. Amaç: Kan kültürlerinden Candida türlerinin doğru ve hızlı identifikasyonu fungal kan dolaşımı infeksiyonlarla ilgili mortalite ve morbiditeyi azaltmak ve etkili antifungal tedavi sağlamak için önemlidir. Bu çalışmada, kan kültürü örneklerinden Candida türlerinin floresan in situ hibridizasyon (FISH), polimeraz zincir reaksiyonu-restriksiyon fragment uzunluk polimorfizmi (PCR-RFLP) ve konvansiyonel yöntemlerle identifikasyonu amaçlandı. Gereçler ve Yöntemler: Candida türlerininin identifikasyonu için 325 kan kültürü pozitif örnekten maya pozitif olan 50 örnek ile kan kültürü negatif 50 örnek FISH, PCR-RFLP ve konvansiyonel yöntemlerle incelendi Bulgular: Her üç yöntemde tek tür Candida identifikasyonunda uyumlu idi (p<0.001). Ancak birden fazla Candida türlerinin identifikasyonunda FISH ve PCR-RFLP, konvansiyonel yöntemlere göre daha duyarlı bulundu (p<0.001). Ayrıca, FISH kan kültürü pozitif örneklerden Candida türlerinin identifikasyonunda diğer iki yönteme göre daha hızlı ve ucuzdu. Birden fazla türle oluşan kandidemi oranı FISH, PCR-RFLP ve konvansiyonal yöntemle sırasıyla %20, %6 ve %4 olarak bulundu. Sonuç: Kan kültürü pozitif örneklerden Candida türlerinin hızlı identifikasyonunda PCR-RFLP ve FISH yöntemi tercih edilebilir. Ancak FISH birden fazla türle oluşan kandidemilerin belirlenmesinde daha uygun bir yöntemdir.

Keywords

• Candida spp.; FISH; PCR-RFLP; Candidemia; identification

Kan Kültürü Örneklerinden Candida Türlerinin Floresan In Situ Hibridizasyon (FISH), Polimeraz Zincir Reaksiyonu-Restriksiyon Fragment Uzunluk Polimorfizmi (PCR-RFLP) ve Konvansiyonel Yöntemlerle İdentifikasyonu

Öz

Amaç: Kan kültürlerinden Candida türlerinin doğru ve hızlı identifikasyonu fungal kan dolaşımı infeksiyonlarla ilgili mortalite ve morbiditeyi azaltmak ve etkili antifungal tedavi sağlamak için önemlidir. Bu çalışmada, kan kültürü örneklerinden Candida türlerinin floresan in situ hibridizasyon (FISH), polimeraz zincir reaksiyonu-restriksiyon fragment uzunluk polimorfizmi (PCR-RFLP) ve konvansiyonel yöntemlerle identifikasyonu amaçlandı. Gereçler ve Yöntemler: Candida türlerininin identifikasyonu için 325 kan kültürü pozitif örnekten maya pozitif olan 50 örnek ile kan kültürü negatif 50 örnek FISH, PCR-RFLP ve konvansiyonel yöntemlerle incelendi Bulgular: Her üç yöntemde tek tür Candida identifikasyonunda uyumlu idi (p < 0.001). Ancak birden fazla Candida türlerinin identifikasyonunda FISH ve PCR-RFLP, konvansiyonel yöntemlere göre daha duyarlı bulundu (p < 0.001). Ayrıca, FISH kan kültürü pozitif örneklerden Candida türlerinin identifikasyonunda diğer iki yönteme göre daha hızlı ve ucuzdu. Birden fazla türle oluşan kandidemi oranı FISH, PCR-RFLP ve konvansiyonal yöntemle sırasıyla %20, %6 ve %4 olarak bulundu. Sonuç: Kan kültürü pozitif örneklerden Candida türlerinin hızlı identifikasyonunda PCR-RFLP ve FISH yöntemi tercih edilebilir. Ancak FISH birden fazla türle oluşan kandidemilerin belirlenmesinde daha uygun bir yöntemdir.

Anahtar Kelimeler

• Candida spp., FISH, PCR-RFLP, kandidemi, identifikasyon.

References

1. Bedini A, Venturelli C, Mussini C, Guaraldi G, Codeluppi M, Borghi V, et al. Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiary-care hospital. Clin Microbiol Infect 2006;12:75-80.
2. Pfaller MA, Diekema DJ. Epidemiology of invasive candi- diasis: a persistent public health problem. Clin Microbiol Rev 2007;20:133-63.
3. Yamamura DL, Rotstein C, Nicolle LE, Ioannou S. Candidemia at selected Canadian sites: results from the Fungal Disease Registry, 1992-1994. Fungal Disease Registry of the Canadian Infectious Disease Society. CMAJ 1999;160:493-9.
4. Edmond MB, Wallace SE, McClish DK, Pfaller MA, Jones RN, Wenzel RP. Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clin Infect Dis 1999;29:239-44.
5. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nation- wide surveillance study. Clin Infect Dis 2004;39:309-17.
6. Pfaller MA, Diekema DJ, Jones RN, Sader HS, Fluit AC, Hollis RJ, et al. International surveillance of bloodstream infections due to Candida species: frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole, and voriconazole of isolates collected from 1997 through 1999 in the SENTRY antimicrobial surveillance program. J Clin Microbiol 2001;39:3254-9.
7. Bassetti M, Righi E, Costa A, Fasce R, Molinari MP, Rosso R, et al. Epidemiological trends in nosocomial candidemia in intensive care. BMC Infect Dis 2006;6:21.
8. Hope W, Morton A, Eisen DP. Increase in prevalence of nosocomial non-Candida albicans candidaemia and the asso- ciation of Candida krusei with fluconazole use. J Hosp Infect 2002;50:56-65.

9. Clancy CJ, Staley B, Nguyen MH. In vitro susceptibility of breakthrough Candida bloodstream isolates correlates with daily and cumulative doses of fluconazole. Antimicrob Agents Chemother 2006;50:3496-8.
10. Kempf VA, Mändle T, Schumacher U, Schäfer A, Autenrieth IB. Rapid detection and identification of pathogens in blood cultures by fluorescence in situ hybridization and flow cytometry. Int J Med Microbiol 2005;295:47-55.
11. Rigby S, Procop GW, Haase G, Wilson D, Hall G, Kurtzman C, et al. Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albi- cans directly from blood culture bottles. J Clin Microbiol 2002;40:2182-6.
12. Mycology. In: Forbes BA, Sahm DF, Weissfeld AS, editors. Bailey and Scott's diagnostic microbiology. 10th ed. St. Louis: Mosby; 1998. p. 870-961.
13. Bautista-Muñoz C, Boldo XM, Villa-Tanaca L, Hernández- Rodríguez C. Identification of Candida spp. by randomly amplified polymorphic DNA analysis and differentiation between Candida albicans and Candida dubliniensis by direct PCR methods. J Clin Microbiol 2003;41:414-20.
14. Maaroufi Y, Heymans C, De Bruyne JM, Duchateau V, Rodriguez-Villalobos H, Aoun M, et al. Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay. J Clin Microbiol 2003;41:3293-8.
15. Chang HC, Leaw SN, Huang AH, Wu TL, Chang TC. Rapid identification of yeasts in positive blood cultures by a mul- tiplex PCR method. J Clin Microbiol 2001;39:3466-71.
16. Leaw SN, Chang HC, Sun HF, Barton R, Bouchara JP, Chang TC. Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. J Clin Microbiol 2006;44:693-9.
17. Mirhendi H, Makimura K, Zomorodian K, Maeda N, Ohshima T, Yamaguchi H. Differentiation of Candida albi- cans and Candida dubliniensis using a single-enzyme PCR- RFLP method. Jpn J Infect Dis 2005;58:235-7.
18. Mirhendi H, Makimura K, Khoramizadeh M, Yamaguchi H. A one-enzyme PCR-RFLP assay for identification of six medically important Candida species. Jpn J Med Mycol 2006;47:225-9.
19. Amann RI. In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid probes. In: Akkermans ADL, Van Elsas JD, De Bruijn FJ, editors. Molecular microbial ecology manual. Dordrecht: Kluwer Academic Publishers; 1995. p. 1-15.
20. Kempf VA, Trebesius K, Autenrieth IB. Fluorescent in situ hybridization allows rapid identification of microorgan- isms in blood cultures. J Clin Microbiol 2000;38:830-8.
21. Oliveira K, Haase G, Kurtzman C, Hyldig-Nielsen JJ, Stender H. Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with pep- tide nucleic acid probes. J Clin Microbiol 2001;39:4138-41.
22. probeBase. Available from: http://www.microbial-ecology. net/probebase/
23. PNA FISH. Available from: http://www.advandx.com/ products/pna_fish.php.
24. BACTfish. Available from: http://www.bactfish.com/ product_range/diagnostic_kits/sepsis_kit/sepsis_kit.html
25. Terlecka JA, du Cros PA, Orla Morrissey C, Spelman D. Rapid differentiation of Candida albicans from non-albicans species by germ tube test directly from BacTAlert blood culture bottles. Mycoses 2007;50:48-51.
26. Noordhoek GT, van Embden JD, Kolk AH. Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories. J Clin Microbiol 1996;34:2522-5.
27. van Deventer AJ, Goessens WH, van Belkum A, van Vliet HJ, van Etten EW, Verbrugh HA. Improved detection of Candida albicans by PCR in blood of neutropenic mice with systemic candidiasis. J Clin Microbiol 1995;33:625-8.
28. Wolff A, Perch-Nielsen IR, Poulsen CR, El-Ali J, Bang DD. Removal of PCR inhibitors using dielectrophoresis for sample preparation in a microfabricated system. 7th lnternational Conference on Miniaturized Chemical and Biochemical Analysis Systems. October 5-9 2003; Squaw Valley, California USA: 1137-40.
29. Fredricks DN, Relman DA. Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J Clin Microbiol 1998;36:2810-6.
30. Kirchgesser M, Alberdi MB, Bollwein M, Miedl B, Malmberg W, Reischl U. MagNA pure LC DNA isolation kit III (bac- teria, fungi)-automated isolation of bacterial DNA from various sample materials. Biochemica 2001;4:4-6. Available from: http://www. Roche-applied-science.com/PROD_ INF/BIOCHEMI/no4_01/PDF/p4_6.pdf. 23.06.2008.
31. Mankes K, Forrest G, Jabra-Rizk MA, Johnson JK, Venezia RA. Outcomes of peptide nucleic acid fluorescence in situ hybridization probes for rapid identification of Candida albi- cans. [Abstract 276]. 43rd Annual Meeting of IDSA, October 6-9, 2005. San Francisco, USA. Available from: http://www. advandx.com/uploads/documents/idsa2005candidap- nauniversityofmarylandmedicalcenter.pdf.
32. Ahmad S, Khan Z, Mustafa AS, Khan ZU. Seminested PCR for diagnosis of candidemia: comparison with culture, antigen detection, and biochemical methods for species identification. J Clin Microbiol 2002;40:2483-9.
33. Haase G, Peltroche-Llacsahuang H, Chapin K, Castellone T, Oliveira K, Stender H, et al. Evaluation of dual color S. aureus/CNS PNA FISH for simultaneous identifica- tion of Staphylococcus aureus and coagulase-negative Staphylococci directly from positive blood culture bottles. ASM 2006;P.C-059. Available from: http://www.advandx. com/uploads/documents/asm2006postersacnsfinal.pdf.
34. Perry-O'Keefe H, Rigby S, Oliveira K, Sİrensen D, Stender H, Coull J, et al. Identification of indicator microorgan- isms using a standardized PNA FISH method. J Microbiol Methods 2001;47:281-92.
35. Sonnex C, Lefort W. Microscopic features of vaginal can- didiasis and their relation to symptomatology. Sex Transm Infect 1999;75:417-9.
36. Linhares LM, Witkin SS, Miranda SD, Fonseca AM, Pinotti JA, Ledger WJ. Differentiation between women with vulvo- vaginal symptoms who are positive or negative for Candida species by culture. Infect Dis Obstet Gynecol 2001;9:221-5.
37. Hospenthal DR, Murray CK, Beckius ML, Green JA, Dooley DP. Persistence of pigment production by yeast isolates grown on CHROMagar Candida medium. J Clin Microbiol 2002;40:4768-70.