Identification of Candida albicans and Candida dubliniensis Species Isolated from Bronchoalveolar Lavage Samples Using Genotypic and Phenotypic Methods

Kianipour, Sahar; Ardestani, Mohammad Emami; Dehghan, Parvin

Identification of Candida albicans and Candida dubliniensis Species Isolated from Bronchoalveolar Lavage Samples Using Genotypic and Phenotypic Methods

Document Type : Original Article

Authors

¹ Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

² Department of Internal Medicine, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: Candida dubliniensis is a newly diagnosed species very similar to Candida albicans phenotypically and first discovered in the mouth of people with AIDS in 1995. Among the different phenotypic and genotypic methods, a cost-effective method should be selected which makes it possible to differentiate these similar species. Materials and Methods: Polymerase chain reaction (PCR)-restriction fragment length polymorphism with MspI enzyme and the Duplex-PCR method were done by DNA extraction using boiling. The sequencing of the amplified ribosomal region was used to confirm the C. dubliniensis species. Direct examination and colony count of the yeasts were applied for bronchoalveolar lavage (BAL) samples and the growth rate of the yeasts were studied at 45°C. To understand the ability formation of chlamydoconidia in yeast isolates, they were separately cultured on the sunflower seed agar, wheat flour agar, and corn meal agar media. Results: Fifty-nine (49.2%) yeast colonies were identified from the total of 120 BAL specimens. Twenty-nine isolated yeasts; including 17 (58.6%) of C. albicans/dubliniensis complex and 12 (41.4%) of nonalbicans isolates produced pseudohypha or blastoconidia in direct smear with a mean colony count of 42000 CFU/mL. C. albicans with the frequency of 15 (42.9%) were the most common isolated yeasts, whereas C. dubliniensis was identified in two nonHIV patients. Conclusion: Sequencing of the replicated gene fragment is the best method for identifying the yeasts, but the determination of the species by phenotypic methods such as the creation of chlamydoconidia in sunflower seeds agar and wheat flour agar media can be cost-effective, have sensitivity and acceptable quality.

Keywords

References

1.	Miceli MH, Díaz JA, Lee SA. Emerging opportunistic yeast infections. Lancet Infect Dis 2011;11:142-51.
2.	Wu JQ, Zhu LP, Ou XT, Xu B, Hu XP, Wang X, et al. Epidemiology and risk factors for non-Candida albicans candidemia in non-neutropenic patients at a Chinese teaching hospital. Med Mycol 2011;49:552-5. [PUBMED]
3.	Al Mosaid A, Sullivan DJ, Coleman DC. Differentiation of Candida dubliniensis from Candida albicans on Pal's agar. J Clin Microbiol 2003;41:4787-9. [PUBMED]
4.	Khan ZU, Ahmad S, Mokaddas E, Chandy R. Simplified sunflower (Helianthus annuus) seed agar for differentiation of Candida dubliniensis from Candida albicans. Clin Microbiol Infect 2004;10:590-2. [PUBMED]
5.	Livério HO, Ruiz LDS, Freitas RS, Nishikaku A, Souza AC, Paula CR, et al. Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients. Rev Inst Med Trop Sao Paulo 2017;59:e14.
6.	Neppelenbroek KH, Seó RS, Urban VM, Silva S, Dovigo LN, Jorge JH, et al. Identification of Candida species in the clinical laboratory: A review of conventional, commercial, and molecular techniques. Oral Dis 2014;20:329-44.
7.	Dehghan P, Shadzi S, Zadeh AH. Growth rate and identification of yeasts in three different media: Flour, sprout of cereals and commercial media. J Res Med Sci 2002;7:1-5.
8.	Brandt ME, Lockhart SR. Recent taxonomic developments with Candida and other opportunistic yeasts. Curr Fungal Infect Rep 2012;6:170-7. [PUBMED]
9.	Mohammadi R, Mirhendi H, Rezaei-Matehkolaei A, Ghahri M, Shidfar MR, Jalalizand N, et al. Molecular identification and distribution profile of Candida species isolated from Iranian patients. Med Mycol 2013;51:657-63. [PUBMED]
10.	Ahmad S, Khan Z, Asadzadeh M, Theyyathel A, Chandy R. Performance comparison of phenotypic and molecular methods for detection and differentiation of Candida albicans and Candida dubliniensis. BMC Infect Dis 2012;12:230. [PUBMED]
11.	Fischer J, van Koningsbruggen-Rietschel S, Rietschel E, Vehreschild MJ, Wisplinghoff H, Krönke M, et al. Prevalence and molecular characterization of azole resistance in Aspergillus spp. Isolates from German cystic fibrosis patients. J Antimicrob Chemother 2014;69:1533-6.
12.	Holding KJ, Dworkin MS, Wan PC, Hanson DL, Klevens RM, Jones JL, et al. Aspergillosis among people infected with human immunodeficiency virus: Incidence and survival. Adult and adolescent spectrum of HIV disease project. Clin Infect Dis 2000;31:1253-7. [PUBMED]
13.	Silva GA, Bernardi TL, Schaker PD, Menegotto M, Valente P. Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification. Braz Arch Biol Technol 2012;55:319-27.
14.	Korabecna M. The variability in the fungal ribosomal DNA (ITS1, ITS2, and 5.8 S rRNA gene): Its biological meaning and application in medical mycology. Commun Curr Res Educ Top Trends Appl Microbiol 2007;2:783-7.
15.	Kali A, Charles MP, Noyal MJ, Sivaraman U, Kumar S, Easow JM, et al. Prevalence of candida co-infection in patients with pulmonary tuberculosis. Australas Med J 2013;6:387-91.
16.	Chadeganipour M, Shadzi S, Dehghan P, Bijary J. The incidence of opportunistic fungi in patients suspected of tuberculosis. Mycoses 2000;43:269-72. [PUBMED]
17.	Yamazaki T, Kume H, Murase S, Yamashita E, Arisawa M. Epidemiology of visceral mycoses: Analysis of data in annual of the pathological autopsy cases in Japan. J Clin Microbiol 1999;37:1732-8. [PUBMED]
18.	Hajialiakbar V, Emami M, Eskandari A. Frequency of fungal pathogens in bronchoalveolar lavage and biopsy specimens of patients hospitalized in Chamran and Amir al momenin Hospitals. EBNESINA 2007;10:21-5.
19.	Djahromi S, Khaksar A. Fungal isolates of the respiratory tract. Mycoses 2002;45:6.
20.	Khodavaisy S, Alialy M, Mahdavi OS, Habibi MR, Amri P, Monadi M, et al. The study on fungal colonization of respiratory tract in patients admitted to intensive care units of Sari and Babol hospitals. Med J Mashad Univ Med Sci. 2011;177-184.
21.	BAUM GL. The significance of Candida albicans in human sputum. N Engl J Med 1960;263:70-3. [PUBMED]
22.	Haghdoost AA, Mostafavi E, Mirzazadeh A, Navadeh S, Feizzadeh A, Fahimfar N, et al. Modelling of HIV/AIDS in Iran up to 2014. J AIDS HIV Res 2011;3:231-9.
23.	Chavasco JK, Paula CR, Hirata MH, Aleva NA, Melo CE, Gambale W, et al. Molecular identification of Candida dubliniensis isolated from oral lesions of HIV-positive and HIV-negative patients in São paulo, Brazil. Rev Inst Med Trop Sao Paulo 2006;48:21-6. [PUBMED]
24.	Meis JF, Ruhnke M, De Pauw BE, Odds FC, Siegert W, Verweij PE, et al. Candida dubliniensis candidemia in patients with chemotherapy-induced neutropenia and bone marrow transplantation. Emerg Infect Dis 1999;5:150-3.
25.	Coleman DC, Sullivan DJ, Bennett DE, Moran GP, Barry HJ, Shanley DB, et al. Candidiasis: The emergence of a novel species, Candida dubliniensis. AIDS 1997;11:557-67.
26.	Sullivan DJ, Moran GP, Coleman DC. Candida dubliniensis: Ten years on. FEMS Microbiol Lett 2005;253:9-17. [PUBMED]
27.	Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC, et al. Hidden killers: Human fungal infections. Sci Transl Med 2012;4:165rv13.
28.	Masur H, Rosen PP, Armstrong D. Pulmonary disease caused by Candida species. Am J Med 1977;63:914-25. [PUBMED]
29.	Peters BM, Yano J, Noverr MC, Fidel PL Jr. Candida vaginitis: When opportunism knocks, the host responds. PLoS Pathog 2014;10:e1003965. [PUBMED]
30.	Hainer BL, Gibson MV. Vaginitis: Diagnosis and treatment. Am Fam Physician 2011;83:807-15. [PUBMED]
31.	Morrell M, Fraser VJ, Kollef MH. Delaying the empiric treatment of Candida bloodstream infection until positive blood culture results are obtained: A potential risk factor for hospital mortality. Antimicrob Agents Chemother 2005;49:3640-5. [PUBMED]
32.	Mohammadi F, Javaheri MR, Nekoeian S, Dehghan P. Identification of Candida species in the oral cavity of diabetic patients. Curr Med Mycol 2016;2:1-7.
33.	Arvanitis M, Anagnostou T, Fuchs BB, Caliendo AM, Mylonakis E. Molecular and nonmolecular diagnostic methods for invasive fungal infections. Clin Microbiol Rev 2014;27:490-526. [PUBMED]
34.	Shin JH, Ranken R, Sefers SE, Lovari R, Quinn CD, Meng S, et al. Detection, identification, and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus PCR coupled with electrospray ionization/mass spectrometry. J Clin Microbiol 2013;51:136-41. [PUBMED]
35.	Sullivan DJ, Westerneng TJ, Haynes KA, Bennett DE, Coleman DC. Candida dubliniensis sp. Nov.: Phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 1995;141(Pt 7):1507-21.
36.	Moore-Landecker E. Fundamentals of the Fungi. USA; Prentice Hall; 1996.
37.	Atlas RM. Handbook Of Microbiological Media.USA; CRC press; 2010.
38.	Caplice N, Moran GP. Candida albicans exhibits enhanced alkaline and temperature induction of Efg1-regulated transcripts relative to Candida dubliniensis. Genom Data 2015;6:130-5. [PUBMED]
39.	Stokes C, Moran GP, Spiering MJ, Cole GT, Coleman DC, Sullivan DJ, et al. Lower filamentation rates of Candida dubliniensis contribute to its lower virulence in comparison with Candida albicans. Fungal Genet Biol 2007;44:920-31.

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