Molecular Diagnosis Compared to Conventional Diagnosis for Rapid Detection of Resistant Strains of Candida Spp.
Keywords:Candida Spp; Molecular identification; Antifungal activity.
Yeast Candida spp. can cause serious infections, especially in patients of the hospital environment. Variation in the virulence profile of Candida clinical isolates and high mortality rates indicate the importance of rapid and accurate identification, as well as susceptibility testing for successful candidiasis treatment. A total of 20 strains were isolated from patients from Tertiary Hospital of the Northern Zone of Ceará. PCR amplification followed by agarose gel electrophoresis (PCR-AGE) and the manual method (culturing on CHROMagar-Candida) and VITEK automated method were used to test a total of 20 fungal strains from clinics sources. Five strains of C. albicans, 2 of C. tropicalis and 4 strains of C. parapsilosis which were cultured for the determination of MIC (Minimum Inhibitory Concentration). The best sensitivity rate was demonstrated by automatic identification and PCR-AGE, where all 20 strains isolated with a sensitivity profile of 100% (20/20) were completely identified. According to the data obtained, resistance to amphotericin B (CIM ≥ 1 µg/ml) was observed in some isolates of C. tropicalis and C. parapsilosis. However, for the other antifungals, these same species presented 100% sensitivity (MIC ≤ 0.125 μg / ml; MIC ≤ 8 μg / ml). Isolates of C. albicans were sensitive to all antifungal agents with 100% sensitivity, although 75% of the isolates showed a tendency for less susceptibility to amphotericin B. In this study, we confirmed the efficacy and applicability of PCR-AGE in the identification and differentiation of yeast Candida spp. from clinical samples.
Ali, H. H. et al. (2015). Molecular identification of Candida species isolated from ears of dogs infected with Otitis externa by detecting internal transcript spacer (ITS1 and ITS4) in Sulaimania, Iraq. Adv. Anim. Vet. Sci, 3(9), 491-499.
Almeida, E. I. A. M. (2021). Development of fast methods for the detection of patulin producing fungi. Doctorate thesis.
Almirante, B., et al. (2005). Epidemiology and predictors of mortality in cases of Candida bloodstream infection: results from population-based surveillance, Barcelona, Spain, from 2002 to 2003. Journal of Clinical Microbiology, 43(4),1829-1835.
Asadzadeh, M. et al. (2015). Simple, low-cost detection of Candida parapsilosis complex isolates and molecular fingerprinting of Candida orthopsilosis strains in Kuwait by ITS region sequencing and amplified fragment length polymorphism analysis. PloS one, 10(11), e0142880.
Aubertine, C. L. et al. (2006). Comparative study of the new colorimetric VITEK 2 yeast identification card versus the older fluorometric card and of CHROMagar Candida as a source medium with the new card. Journal of clinical microbiology, 44(1), 227-228.
Azeredo, F. J. (2013). Modelagem farmacocinética/farmacodinâmica do antifúngico fluconazol.
Back-Brito et al. (2009). PCR-AGE, automated and manual methods to identify Candida strains from veterinary sources: a comparative approach. Veterinary microbiology, 139(3-4), 318-322.
Badiee, P. & Alborzi, A. (2011). Susceptibility of clinical Candida species isolates to antifungal agents by E-test, Southern Iran: A five year study. Iranian journal of microbiology, 3(4), 183.
Barry, T. G. et al. (2017). SWI5 gene as a diagnostic target for the identification of fungal and yeast species. U.S. Patent n. 9,745,636, 29 ago. 2017
Berberi, A; Noujeim, Z. & Aoun, G. (2015). Epidemiology of oropharyngeal candidiasis in human immunodeficiency virus/acquired immune deficiency syndrome patients and CD4+ counts. JIOH, 7(3), 20.
Bockelmann, W; Heller, M. & Heller, K. (2008). Identification of yeasts of dairy origin by amplified ribosomal DNA restriction analysis (ARDRA). International dairy journal, 18(10-11), 1066-1071.
Boyanton, J. R et al. (2008) DNA pyrosequencing–based identification of pathogenic Candida species by using the internal transcribed spacer 2 region. Archives of pathology & laboratory medicine, 132, (4), 667-674.
Buitrón, R. et al. (2009). Candida glabrata: an emergent opportunist in vulvovaginitis. Cirugia y cirujanos, 77(6), 423-427.
Calvo, B. et al. (2016). First report of Candida auris in America: clinical and microbiological aspects of 18 episodes of candidemia. Journal of Infection, 73(4), 369-374.
Canuto, M. M. & Rodero, F. G. (2002) Antifungal drug resistance to azoles and polyenes. The Lancet infectious diseases, 2(9), 550-563.
Castro, M. da C. A. (2017). Ocorrência de espécies do complexo Candida parapsilosis em amostras biológicas de pacientes e profissionais de hospitais do Natal/RN. Masters dissertation.
Černáková, L. et al. (2021). Candida auris: a quick review on identification, current treatments, and challenges. International Journal of Molecular Sciences. 22(9), 4470
Cirak, M. Y.; Kalkanci, A. & Kustimur, S. (2003) Use of molecular methods in identification of Candida species and evaluation of fluconazole resistance. Memórias do Instituto Oswaldo Cruz, 98(8), 1027-1032.
Córdoba, S. et al. (2011) Species distribution and susceptibility profile of yeasts isolated from blood cultures: results of a multicenter active laboratory-based surveillance study in Argentina. Revista argentina de microbiología, 43, (3), 176-185.
Crocco, E. I. et al. (2004) Identificação de espécies de Candida e susceptibilidade antifúngica in vitro: estudo de 100 pacientes com candidíases superficiais Identification of Candida species and antifungal susceptibility in vitro: a study on 100 patients with. An Bras Dermatol, 79(6), 689-697.
Da silva, A. U. A. et al. (2020). Factors associated with the isolation of Candida Spp. from the oral microbiota of people living with HIV. Clinical Nursing Studies, 8(3).
Diekema, D. J. et al. (2002) Epidemiology of candidemia: 3-year results from the emerging infections and the epidemiology of Iowa organisms study. Journal of Clinical Microbiology, 40(4), 1298-1302.
Doi, A. M. et al. (2016). Epidemiology and microbiologic characterization of nosocomial candidemia from a Brazilian national surveillance program. PloS one, 11(1), e0146909.
Du, H., et al. (2020) Candida auris: Epidemiology, biology, antifungal resistance, and virulence. PLoS pathogens, 16(10), e1008921.
Espinel-Ingroff, A. et al. (1998). Comparison of RapID yeast plus system with API 20C system for identification of common, new, and emerging yeast pathogens. Journal of clinical microbiology, 36(4), 883-886.
Favalessa, O. C; Martins, M. A. & Hahn, R. C. (2010). Aspectos micológicos e suscetibilidade in vitro de leveduras do gênero Candida em pacientes HIV-positivos provenientes do Estado de Mato Grosso. Revista da Sociedade Brasileira de Medicina Tropical, 43(6), 673-677.
Fujita, S. et al. (2001) Multiplex PCR using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains. Journal of clinical microbiology, 39(10), 3617-3622.
Furlaneto, M. C. et al. (2011). Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period. Revista da Sociedade Brasileira de Medicina Tropical, 44(5), 595-599.
Gardes, M. et al. (1991). Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Canadian Journal of Botany, 69(1), 180-190.
Girmenia, C. et al. (2000). In vitro activity of fluconazole and voriconazole against isolates of Candida albicans from patients with haematological malignancies. Journal of Antimicrobial Chemotherapy, 46(3), 479-484.
Gokce, G; Cerikcioglu, N. & Yagci, A. (2007). Acid proteinase, phospholipase, and biofilm production of Candida species isolated from blood cultures. Mycopathologia, 164(6), 265.
Graf, Barbara et al. (2000) Evaluation of the VITEK 2 system for rapid identification of yeasts and yeast-like organisms. Journal of clinical microbiology, 38(5), 1782-1785.
Greene, C. E. & Chandler, F. W. (1998). Candidiasis, torulopsosis, and rhodotorulosis. Infectious diseases of the dog and cat, 3, 627-633.
Guimarães, T. et al. Epidemiology and predictors of a poor outcome in elderly patients with candidemia. International Journal of Infectious Diseases, 16(6), e442-e447.
Guinea, J. (2014). Global trends in the distribution of Candida species causing candidemia. Clinical Microbiology and Infection, 20, 5-10.
Guo, Y. et al. (2016). A real-time PCR assay based on 5.8 S rRNA gene (5.8 S rDNA) for rapid detection of Candida from whole blood samples. Mycopathologia, 181(5-6), 405-413.
Hazen, K. C. (1995) New and emerging yeast pathogens. Clinical microbiology reviews, 8(4), 462-478.
Higashi, C. M. et al. (2015) Comparação do sistema de identificação automatizado Vitek 2 e PCR-ITS para caracterização das espécies dos isolados clínicos de Candida spp. Semina: Ciências Biológicas e da Saúde, 36(1) 233-242.
Irinyi, L. et al. (2015). International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database—the quality controlled standard tool for routine identification of human and animal pathogenic fungi. Medical mycology, 53(4), 313-337.
Jackson, C. J. et al. (1999) Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions. Journal of clinical microbiology, 37, (4), 931-936.
Junqueira, J. C. et al. Oral colonization by yeasts in HIV-positive patients in Brazil. Revista do Instituto de Medicina Tropical de Sao Paulo, 54(1), 17-24.
Kaplan, J. E. et al. (2000). Epidemiology of human immunodeficiency virus-associated opportunistic infections in the United States in the era of highly active antiretroviral therapy. Clinical infectious diseases, 30, S5-S14.
Kiraz, N. et al. (2010). Correlation between broth microdilution and disk diffusion methods for antifungal susceptibility testing of caspofungin, voriconazole, amphotericin B, itraconazole and fluconazole against Candida glabrata. Journal of microbiological methods, 82, (2), 136-140.
Koehler, P; Tacke, D. & Cornely, O. A. (2014) Our 2014 approach to candidaemia. Mycoses, 57(10), 581-583.
Kumar, S. et al. (2013). Application of chromagar Candida for identification of clinically important Candida species and their antifungal susceptibility pattern. Int J Biol Med Res, 4(4), 600-3606.
Lazo, J. S. et al. Goodman & Gilman: As bases farmacológicas da terapêutica. In: Goodman & Gilman: as bases farmacológicas da terapêutica. 2006.
Li, Yu-Ye et al. (2013). Asymptomatic oral yeast carriage and antifungal susceptibility profile of HIV-infected patients in Kunming, Yunnan Province of China. BMC infectious diseases, 13(1), 46.
Lima, V. D. et al. (2015). AIDS incidence and AIDS-related mortality in British Columbia, Canada, between 1981 and 2013: a retrospective study. The Lancet HIV, 2(3), e92-e97.
Luo, G. & Mitchell, T. G. (2002) Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR. Journal of clinical microbiology, 40(8), 2860-2865.
Makimura, K. M. S. Y; Murayama, S. Y. & Yamaguchi, H. (1994). Detection of a wide range of medically important fungi by the polymerase chain reaction. Journal of Medical Microbiology, 40(5), 358-364.
Massonet, C. et al. (2004). Comparison of VITEK 2 with ITS2-fragment length polymorphism analysis for identification of yeast species. Journal of clinical microbiology, 42(5), 2209-2211.
Maubon, D. et al. (2014). Resistance of Candida spp. to antifungal drugs in the ICU: where are we now? Intensive care medicine, 40(9), 1241-1255.
Meletiadis, J. et al. (2011). Comparative evaluation of three commercial identification systems using common and rare bloodstream yeast isolates. Journal of clinical microbiology, 49(7), 2722-2727.
Menezes, E. A. et al. (2012). Identificação molecular e suscetibilidade antifúngica de Candida parapsilosis isoladas no Ceará, Brasil. Jornal Brasileiro de Patologia e Medicina Laboratorial, 48(6), 415-420.
Menezes, E. A. et al. (2016) Suscetibilidade a antifúngicos e fatores de virulência de Candida spp. isoladas em Russas, Ceará. RBAC, 48(1), 33-8.
Menezes, E. A; Mendes, L. G. & Cunha, F. A. (2009). Resistência a antifúngicos de Candida tropicalis isoladas no Estado do Ceará. Rev Soc Bras Med Trop, 2, 354-355.
Mirhendi, S. H. & Makimura, K. (2003) PCR-detection of Candida albicans in blood using a new primer pair to diagnosis of systemic candidiasis. Iranian Journal of Public Health, 32(1), 1-5.
Moretti, A. et al. Diffuse cutaneous candidiasis in a dog. Diagnosis by PCR-REA. Rev Iberoam Micol, 21(3), 139-142.
Morschhäuser, J. (2016). The development of fluconazole resistance in Candida albicans–an example of microevolution of a fungal pathogen. Journal of Microbiology, 54(3), 192-201.
Nadăş, G. C. et al. (2014) Comparative identification of Candida species isolated from animals using phenotypic and PCR-RFLP methods. Bulletin of the Veterinary Institute in Pulawy, 58(2), 219-222.
Nunes, E. B. et al. (2011). Perfil de sensibilidade do gênero Candida a antifúngicos em um hospital de referência da Região Norte do Brasil. Revista Pan-Amazônica de Saúde, 2(4), 23-30.
Odds, F. C. & BERNAERTS, R. I. A. (1994). CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species. Journal of clinical microbiology, 32(8), 1923-1929.
Oliveira, G. S.; Ribeiro, E. T. & BARONI, F. A. (2006). An evaluation of manual and mechanical methods to identify Candida spp. from human and animal sources. Revista do Instituto de Medicina Tropical de São Paulo, 48(6), 311-315.
Ortega, M. et al. (2010). Candida spp. bloodstream infection: influence of antifungal treatment on outcome. Journal of antimicrobial chemotherapy, 65(3), 562-568.
Paramythiotou, E. et al. (2014). Invasive fungal infections in the ICU: how to approach, how to treat. Molecules, 19(1), 1085-1119.
Passos, X. S. et al. Species distribution and antifungal susceptibility patterns of Candida spp. bloodstream isolates from a Brazilian tertiary care hospital. Mycopathologia, 163(3), 145-151.
Paulique, N. C. et al. (2017). Manifestações bucais de pacientes soropositivos para HIV/AIDS. Archives of health investigation, 6, (6).
Peay, K. G; Kennedy, P. G. & Bruns, T. D. (2008). Fungal community ecology: a hybrid beast with a molecular master. AIBS Bulletin, 58(9), 799-810.
Pfaller, M. A. & Diekema, D. J. (2002) Role of sentinel surveillance of candidemia: trends in species distribution and antifungal susceptibility. Journal of Clinical microbiology, 40(10), 3551-3557.
Pfaller, M. A. et al. (2002). Trends in antifungal susceptibility of Candida spp. isolated from pediatric and adult patients with bloodstream infections: SENTRY Antimicrobial Surveillance Program, 1997 to 2000. Journal of clinical microbiology, 40(3), 852-856.
Pfaller, M. A; Houston, A. & Coffmann, S. (1996). Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata. Journal of clinical microbiology, 34(1), 58-61.
Pires, E. G. et al. (2015). Agreement between RAPD, API20C AUX, CHROMagar Candida and microculture on oral Candida identification. Brazilian Journal of Oral Sciences, 14(2), 149-153.
Playford, E. G; Lipman, J. & Sorrell, T. C. (2010). Management of invasive candidiasis in the intensive care unit. Drugs,70(7), 823-839.
Radford, S. A. et al. (1998). Molecular epidemiological study ofAspergillus fumigatus in a bone marrow transplantation unit by PCR amplification of ribosomal intergenic spacer sequences. Journal of clinical microbiology, 36(5), 1294-1299.
Rex, J. H. et al. (2000). Practice guidelines for the treatment of candidiasis. Clinical infectious diseases, 30(4), 662-678.
Riederer, K. M. et al. (2002). Utility of a pre-optimized kit for random amplified polymorphic DNA in typing Candida albicans. Canadian journal of microbiology, 48(4), 369-373.
Rocha, F. G. C. W; Martins, G. B. & Medrado, A. R. A. P. (2017). Ocorrência de candidíase oral em pacientes portadores de câncer de cabeça e pescoço submetidos aos tratamentos antineoplásicos. Revista de Ciências Médicas e Biológicas, 16(3), 318-322.
Sardi, J. C. O. et al. (2013). Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. Journal of medical microbiology, 62(1), 10-24.
Schoch, C. L. et al. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences, 109(16), 6241-6246.
Shokohi, T. et al. (2010). Identification of Candida species using PCR-RFLP in cancer patients in Iran. Indian journal of medical microbiology, 28, (2), 147.
Silveira, E. S. et al. (2017). Freqüência de micoses nas amostras de pacientes com hiv+/aids do hospital universitário da fundação universidade federal do rio grande. VITTALLE-Revista de Ciências da Saúde, 17(2), 27-34.
Sims, C. R. et al. (2006). Correlation between microdilution, E-test, and disk diffusion methods for antifungal susceptibility testing of posaconazole against Candida spp. Journal of clinical microbiology, 44, (6), 2105-2108.
Singaravelu, K; Gácser, A. & Nosanchuk, J. D. (2014). Genetic determinants of virulence–Candida parapsilosis. Revista iberoamericana de micología, 31(1), 16-21.
Sobrinho, K. N. et al. (2017). Alterações em mucosa bucal de pacientes portadores de diabetes mellitus. Revista de Odontologia da Universidade Cidade de São Paulo, 26(3), 204-211.
Spampinato, C. & Leonardi, D. (2013). Candida infections, causes, targets, and resistance mechanisms: traditional and alternative antifungal agents. BioMed research international, 2013.
Stefaniuk, E. et al. (2016) Usefulness of CHROMagar Candida medium, biochemical methods–API ID32C and VITEK 2 compact and two MALDI-TOF MS systems for Candida spp. identification. Pol J Microbiol, 65(1),111-14.
ST-Germain, G. et al. (2001). Prevalence and Antifungal Susceptibility of Candida Isolates from Blood and Other Normally Sterile Sites: Results of a 2-Year (1996 to 1998) Multicenter Surveillance Study in Quebec, Canada. Journal of Clinical Microbiology, 39, (3), 949-953.
Swinne, D. et al. (2005). In vitro activities of voriconazole, fluconazole, itraconazole and amphotericin B against non Candida albicans yeast isolates. Rev Iberoam Micol, 22, (1), 24-28.
Tamura, N. K. et al. (2007). Fatores de virulência de Candida spp isoladas de cateteres venosos e mãos de servidores hospitalares. Rev Soc Bras Med Trop, 40(1), 91-93.
Vandeputte, P. et al. (2005). Mechanisms of azole resistance in a clinical isolate of Candida tropicalis. Antimicrobial agents and chemotherapy, 49(11), 4608-4615.
Vieira, A. J. H. & Dos Santos, J. I. (2017). Mecanismos de resistência de Candida albicans aos antifúngicos anfotericina B, fluconazol e caspofungina. Brazilian Journal of Clinical Analyses, 49(3), 235-239.
Wahlquist, S. P. et al. (1991). Use of pooled formalin-preserved fecal specimens to detect Giardia lamblia. Journal of clinical microbiology, 29(8), 1725-1726.
Weinstein, R. A. et al. (2001). Prophylactic antifungal therapy in the intensive care unit. Clinical Infectious Diseases, 32(8), 1191-1200.
White, T. C; Marr, K. A. & Bowden, R. A. (1998) Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clinical microbiology reviews, 11(2), 382-402.
Wingard, J. R. (1995). Importance of Candida species other than C. albicans as pathogens in oncology patients. Clinical infectious diseases, 20(1), 115-125.
Yang, Y. et al. (2008). Host factors do not influence the colonization or infection by fluconazole resistant Candida species in hospitalized patients. Journal of negative results in biomedicine, 7(1), 12.
Yapar, N. (2014). Epidemiology and risk factors for invasive candidiasis. Therapeutics and clinical risk management, 10, 95.
How to Cite
Copyright (c) 2023 Rafael Carneiro Bastos; Erivan Ferreira Alves; Andréa Maria Neves; Carlos Franciney Moreira Vasconcelos; Erika Helena Salles de Brito; Raquel Oliveira dos Santos Fontenelle
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
1) Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
2) Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
3) Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.