Abstract

INTRODUCTION:

Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.

METHODS:

Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.

RESULTS:

Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.

CONCLUSIONS:

We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Keywords:
Candida parapsilosis; In vitro antifungal susceptibility; Candidemia; Onychomycosis

INTRODUCTION

Although Candida albicans is the most common isolate from human infections, other Candida species have also been observed in the 1990s, supporting the increased frequency of non- albicans species ¹1 Nucci M, Queiroz-Telles F, Alvarado-Matute T, Tiraboschi IN, Cortes J, Zurita J, et al. Epidemiology of Candidemia in Latin America: A Laboratory-Based Survey. PLoS One 2013; 8:e59373. ²2 Sardi JC, Scorzoni L, Bernardi T, Fusco-Almeida AM, Mendes Giannini MJ. Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol 2013; 62:10-24. . Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices ³3 Cantón E, Pemán J, Quindós G, Eraso E, Miranda-Zapico I, Álvarez M, et al. Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrob Agents Chemother 2011; 55:5590-5596. ⁴4 Ataides FS, Chaul MH, El Essal FE, Costa CR., Souza LK, Fernandes OFL, et al. Antifungal susceptibility patterns of yeasts and filamentous fungi isolated from nail infection. J Eur Acad Dermatol Venereol 2012; 26:1479-1485. ⁵5 Montagna MT, Caggiano G, Lovero G, De Giglio O, Coretti C, Cuna T, et al. Epidemiology of invasive fungal infections in the intensive care unit: results of a multicenter Italian survey (AURORA Project). Infection 2013; 41:645-653. ⁶6 Silva LB, Oliveira DBC, Silva BV, Souza RA, Silva PR, Ferreira-Paim K, et al. Identification and antifungal susceptibility of fungi isolated from dermatomycoses. J Eur Acad Dermatol Venereol2014; 28:633-640. . In State of Goiás, Brazil, C. parapsilosis was found in 52.5% of nail samples and was the second most commonly isolated species (24.2%) from blood cultures ⁴4 Ataides FS, Chaul MH, El Essal FE, Costa CR., Souza LK, Fernandes OFL, et al. Antifungal susceptibility patterns of yeasts and filamentous fungi isolated from nail infection. J Eur Acad Dermatol Venereol 2012; 26:1479-1485. ⁷7 Passos XS, Costa CR, Araújo CR, Nascimento ES, Souza LK, Fernandes OLF, et al. Species distribution and antifungal susceptibility patterns of Candida spp. bloodstream isolates from a Brazilian tertiary care hospital. Mycopathologia 2007; 163:145-151. . Previous work has shown that C. parapsilosis isolates may exhibit genotypic differences, allowing the classification of new species by restriction polymorphism analysis of the secondary alcohol dehydrogenase (SADH) gene.

Candida parapsilosis forms a complex composed of three genetically distinct species: C. parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis ⁸8 Tavanti A, Davidson AD, Gow NA, Maiden MC, Odds FC. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J Clin Microbiol 2005; 43:284-292. . C. parapsilosis complex species are usually susceptible to antifungal agents; however, some reports have shown that these isolates may exhibit decreased susceptibility to azoles and echinocandins ⁹9 Miranda-Zapico I, Eraso E, Hernández-Almaraz JL, López-Soria LM, Carrillo-Muñoz AJ, Hernández-Molina J, et al. Prevalence and antifungal susceptibility patterns of new cryptic species inside the species complexes Candida parapsilosis and Candida glabrata among blood isolates from a Spanish tertiary hospital. J Antimicrob Chemother 2011; 66:2315-2322. . This difference in antifungal susceptibility among the three species has led to an increased interest in the study of C. parapsilosis complex species ¹⁰10 Földi R, Kovács R, Gesztelyi R, Kardos G, Berényi R, Juhász B, et al. Comparison of in vitro and in vivo efficacy of caspofungin against Candida parapsilosis, C. orthopsilosis, C. metapsilosis and C. albicans . Mycopathologia 2012; 174:311-318. . Moreover, antifungal susceptibility surveillance is an important major strategy for the prophylaxis and treatment of candidiasis caused by C. parapsilosis .

Because of the medical importance of C. parapsilosis in our area and the limited local epidemiological data on this species, we aimed to verify the distribution of C. parapsilosis sensu stricto, C. orthopsilosis , and C. metapsilosis species within a culture collection containing blood and subungual scraping samples from patients attending a tertiary hospital in Goiás, Brazil. Additionally, we assessed the in vitro antifungal susceptibility of these species to fluconazole (FLC), itraconazole (ITC), voriconazole (VOR), posaconazole (POS), amphotericin B (AMB), and caspofungin (CAS).

METHODS

Isolates

A total of 87 C. parapsilosis isolates were recovered from different clinical specimens, including blood (737) and nail specimens (136). These isolates were stored at -70°C in yeast extract peptone dextrose (YEPD) broth (Difco) with 10% glycerol and were obtained from the Laboratory of Mycology, Institute of Tropical Pathology and Public Health, Federal University of Goiás from 2007 to 2012.

Candida parapsilosis was obtained from adult patients with candidemia (n = 54) and onychomycosis (n = 33). The study was approved by the Bioethic Committee from Hospital das Clínicas in Goiânia-GO (protocol 065/2008). Confirmation of the identification of C. parapsilosis sensu lato , after subculture on YEPD agar (Difco) for 48h at 37°C, was based on colony color on CHROMagar Candida medium (Difco), micromorphology of colonies on cornmeal agar medium (Difco), and assimilation profiles on ID32C (BioMérieux, France). C. parapsilosis ATCC 22019 was included as quality control strain.

Molecular identification of the Candida parapsilosis complex species

Genomic deoxyribonucleic acid (DNA) was extracted from each isolate using high-speed cell disruption followed by phenol-chloroform extraction and ethanol precipitation, as described by Tavanti et al. ⁸8 Tavanti A, Davidson AD, Gow NA, Maiden MC, Odds FC. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J Clin Microbiol 2005; 43:284-292. .

Polymerase chain reaction (PCR) was carried out to amplify the SADH gene using the primers S1F (5′-GTTGATGCTGTTGGATTGT-3′) and SIR (5′-CAATGCCAAATCTCCCAA-3′) and conditions previously described by Tavanti et al. ⁸8 Tavanti A, Davidson AD, Gow NA, Maiden MC, Odds FC. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J Clin Microbiol 2005; 43:284-292.Amplification was performed in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA), and the amplified products were loaded onto 2% agarose gels containing ethidium bromide (0.5mg/mL). A 100-bp DNA ladder was used as a molecular size marker (Invitrogen Life Technologies, USA). Amplified products were electrophoresed on 1.5% agarose gels, visualized under ultraviolet light, and analyzed on a photo documenter (Bio-Rad).

The PCR products (fragments of 716bp) were purified with a specialized kit (Qiagen, Valencia, CA, USA), and digestion of the purified PCR product was carried out for 120 min at 37°C using the restriction enzyme Ban I (Thermo Scientific, USA). Digestion of the PCR products was subjected to electrophoresis onto 2% agarose gels (Invitrogen Life Technologies). C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96141, and C. metapsilosis ATCC 96143 were used as controls.

In vitro susceptibility testing

All isolates were evaluated using Etests (AB Biodisk, Solna, Sweden), as recommended by the manufacturer's guidelines, with Etest strips for ITC, VOR, POS, AMB, and CAS at concentrations ranging from 0.002 to 32µg/mL and for FLC at concentrations ranging from 0.016 to 256µg/mL. C. parapsilosis ATCC 22019 was included on each day of testing to verify the reproducibility of the results.

Interpretative criteria for FLC minimum inhibitory concentrations (MICs) were as follows: ≤ 2µg/mL, susceptible; 4µg/mL, susceptible-dose dependent (S-DD), and ≥8µg/mL, resistant. ITC and VOR MICs were interpreted as follows: ≤0.125µg/mL, susceptible; 0.25-0.5µg/mL, S-DD; and ≥ 1µg/mL, resistant. Caspofungin MICs were interpreted as follows: ≤2µg/mL, susceptible; 4µg/mL, intermediate; and ≥8µg/mL, resistant ¹¹11 Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard CLSI document M27-A3. 3rd ed. Wayne: CLSI; 2008. ¹²12 Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast. Approved standard CLSI document M27-S4, fourth informational supplement. Wayne: CLSI; 2012. . Isolates with MICs of greater than 1µg/mL were considered resistant to AMB and POS, as suggested by Nguyen et al. ¹³13 Nguyen MH, Clancy CJ, Yu VL, Yu YC, Morris AJ, Snydman DR, et al. Do in vitro susceptibility data predict the microbiologic response to amphotericin B? Results of a prospective study of patients with Candidafungemia. J Infect Dis 1998; 177:425-430. and Cantón et al. ³3 Cantón E, Pemán J, Quindós G, Eraso E, Miranda-Zapico I, Álvarez M, et al. Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrob Agents Chemother 2011; 55:5590-5596. .

RESULTS

Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto (two restriction sites), five (5.7%) were identified as C. orthopsilosis (no restriction sites), and four (4.6%) were identified as C. metapsilosis (four restriction sites). In blood samples, 47 (54.1%), five (5.7%), and two (2.3%) isolates were C. parapsilosis, C. metapsilosis, and C. orthopsilosis , respectively. In nail samples, C. parapsilosis and C. metapsilosis represented 31 (35.6%) and two (2.3%) isolates, respectively. Ban I restriction profiles of SADH fragments used for distinguishing among the three species of the C. parapsilosis complex are shown in Figure 1 .

Figure 1:
Agarose gel electrophoresis of secondary alcohol dehydrogenase ( SADH ) gene polymerase chain reaction (PCR) products from Candida parapsilosis sensu lato after digestion with Ban I. Candida parapsilosis sensu stricto : lanes 1-3, 5-8, 10-14, 16; Candida orthopsilosis : lanes 9 and 15; Candida metapsilosis: lane 4. Lane M: molecular size marker 100-bp.

According to the interpretative criteria for resistance used for the antifungal drugs described in the Material and Methods, we found that very few isolates were resistant to azoles and AMB. One C. parapsilosis sensu stricto isolate was resistant to AMB and ITC. Moreover, 10.2% of C. parapsilosis sensu stricto isolates weare resistant to CAS. Two C. parapsilosis sensu stricto isolates and one C. metapsilosis isolate met the criterion for S-DD to FLC.

Susceptible-dose dependent strains were detected in the three species. C. orthopsilosis met the criterion for S-DD to ITC; C. metapsilosisand C. parapsilosis sensu stricto met the criterion for S-DD to ITC and FLC; and isolates of all three species exhibited intermediate susceptibility to CAS. The profiles of the in vitro susceptibility of C. parapsilosiscomplex species to azoles, AMB, and CAS, including MIC ranges, MIC ₅₀ , MIC ₉₀ , susceptibility intermediate or S-DD, and resistance, are summarized in Table 1 .

DISCUSSION

Within the genus Candida , C. parapsilosis is the second most commonly isolated species from blood cultures in several casuistics ⁵5 Montagna MT, Caggiano G, Lovero G, De Giglio O, Coretti C, Cuna T, et al. Epidemiology of invasive fungal infections in the intensive care unit: results of a multicenter Italian survey (AURORA Project). Infection 2013; 41:645-653. ¹⁴14 Dzierzanowska-Fangrat K, Romanowska E, Gryniewicz-Kwiatkowska O, Migdał M, Witulska K, Ryżko J, et al. Candidaemia in a Polish tertiary paediatric hospital, 2000 to 2010. Mycoses 2014; 57:105-109. ¹⁵15 Kazak E, Akın H, Ener B, Sığırlı D, Özkan Ö, Gürcüoğlu E, et al. An investigation of Candida species isolated from blood cultures during 17 years in a university hospital. Mycoses 2014; 57:623-629. . Moreover, this species represents frequent cause of onychomycosis in several countries, including Brazil ⁴4 Ataides FS, Chaul MH, El Essal FE, Costa CR., Souza LK, Fernandes OFL, et al. Antifungal susceptibility patterns of yeasts and filamentous fungi isolated from nail infection. J Eur Acad Dermatol Venereol 2012; 26:1479-1485. , Chile ¹⁶16 Fich F, Abarzúa-Araya A, Pérez M, Nauhm Y, León E. Candida parapsilosis and Candida guillermondii : emerging pathogens in nail candidiasis. Indian J Dermatol 2014; 59:24-29. , México ¹⁷17 Jimenez-Gonzalez C, Mata-Marin JA, Arroyo-Anduiza CI, Ascencio-Montiel IJ, Fuentes-Allen JL, Gaytan-Martinez J. Prevalence and etiology of onychomycosis in the HIV-infected Mexican population. Eur J Dermatol 2013; 23:378-381. , and Iran ¹⁸18 Razzaghi-Abyaneh M, Sadeghi G, Zeinali E, Alirezaee M, Shams-Ghahfarokhi M, Amani A, et al. Species distribution and antifungal susceptibility of Candida spp. isolated from superficial candidiasis in outpatients in Iran. J Mycol Med 2014; 24:e43-50. . Although all 87 isolates of C. parapsilosis studied in our work have been identified by phenotypic methods, we were not able to differentiate among the C. parapsilosis complex species, which are phenotypically identical. Amplification of the SADH gene followed by BanI restriction enzyme digestion has been used as a rapid and reliable method with high discriminative power for C. parapsilosis complex species ⁶6 Silva LB, Oliveira DBC, Silva BV, Souza RA, Silva PR, Ferreira-Paim K, et al. Identification and antifungal susceptibility of fungi isolated from dermatomycoses. J Eur Acad Dermatol Venereol2014; 28:633-640. ¹⁹19 Ge YP, Lu GX, Shen YN, Liu WD. In vitro evaluation of phospholipase, proteinase, and esterase activities of Candida parapsilosis and Candida metapsilosis . Mycopathol 2011; 172:429-438. ²⁰20 Bonfietti LX, Martins MA, Szeszs MW, Pukiskas SB, Purisco SU, Pimentel FC, et al. Prevalence, distribution and antifungal susceptibility profiles of Candida parapsilosis, Candida orthopsilosis and Candida metapsilosisbloodstream isolates. J Med Microbiol2012; 61:1003-1008. ²¹21 Romeo O, Delfino D, Costanzo B, Cascio A, Criseo G. Molecular characterization of Italian Candida parapsilosis isolates reveals the cryptic presence of the newly described species Candida orthopsilosis in blood cultures from newborns. Diagn Microbiol Infect Dis 2012; 72:234-238. . Data have shown that these three species exhibit different prevalence rates, virulence, and in vitro antifungal susceptibility ³3 Cantón E, Pemán J, Quindós G, Eraso E, Miranda-Zapico I, Álvarez M, et al. Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrob Agents Chemother 2011; 55:5590-5596. ¹⁰10 Földi R, Kovács R, Gesztelyi R, Kardos G, Berényi R, Juhász B, et al. Comparison of in vitro and in vivo efficacy of caspofungin against Candida parapsilosis, C. orthopsilosis, C. metapsilosis and C. albicans . Mycopathologia 2012; 174:311-318. ¹⁹19 Ge YP, Lu GX, Shen YN, Liu WD. In vitro evaluation of phospholipase, proteinase, and esterase activities of Candida parapsilosis and Candida metapsilosis . Mycopathol 2011; 172:429-438. ²²22 Bertini A, De Bernardis F, Hensgens LA, Sandini S, Senesi S, Tavanti A. Comparison of Candida parapsilosis, Candida orthopsilosis , and Candida metapsilosis adhesive properties and pathogenicity. Int J Med Microbiol2013; 303:98-103. ²³23 Tosun I, Akyuz Z, Guler NC, Gulmez D, Bayramoglu G, Kaklikkaya N, et al. Distribution, virulence attributes and antifungal susceptibility patterns of Candida parapsilosis complex strains isolated from clinical samples. Med Mycol 2013; 51:483-492. ²⁴24 Treviño-Rangel RJ, González JG, González GM. Aspartyl proteinase, phospholipase, esterase and hemolysin activities of clinical isolates of the Candida parapsilosis species complex. Med Mycol2013; 51:331-335. . In vitro studies have also shown that C. parapsilosis sensu stricto is more pathogenic than C. orthopsilosis , which is, in turn, is more pathogenic than C. metapsilosis ²⁵25 Gago S, García-Rodas R, Cuesta I, Mellado E, Alastruey-Izquierdo A. Candida parapsilosis , Candida orthopsilosis , and Candida metapsilosis virulence in the non-conventional host Galleria mellonella . Virulence 2014; 5:278-285. .

In our study, C. parapsilosis sensu stricto was the dominant species among blood isolates, followed by C. orthopsilosis and C. metapsilosis . Similar to our results, some researchers have shown that C. parapsilosis sensu stricto is the most frequently found species in patients with candidemia ³3 Cantón E, Pemán J, Quindós G, Eraso E, Miranda-Zapico I, Álvarez M, et al. Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrob Agents Chemother 2011; 55:5590-5596. ¹⁵15 Kazak E, Akın H, Ener B, Sığırlı D, Özkan Ö, Gürcüoğlu E, et al. An investigation of Candida species isolated from blood cultures during 17 years in a university hospital. Mycoses 2014; 57:623-629. ²⁶26 Lockhart SR, Messer SA, Pfaller MA, Diekema DJ. Geographic distribution and antifungal susceptibility of the newly described species Candida orthopsilosis and Candida metapsilosis in comparison to the closely related species Candida parapsilosis . J Clin Microbiol2008; 46:2659-664. ²⁷27 Cebeci Güler N, Tosun I, Bayramoğlu G, Buruk K, Aydın F. Genotypic identification and distribution patterns of Candida parapsilosis complex species ( C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis ) isolated from clinical samples. Mikrobiyol Bul 2011; 45:723-728. . The high frequency of this species in blood samples in critically ill patients may be explained by several factors, including the high affinity of these species for vascular devices and medical instrumentation and their capacity for colonization on the hands of healthcare workers ²⁸28 González GM, Treviño-Rangel RJ, Palma-Nicolás JP, Martínez C, González JG, Ayala J, et al.. Species distribution and antifungal susceptibility of bloodstream fungal isolates in paediatric patients in Mexico: a nationwide surveillance study. J Antimicrob Chemother2013; 68:2847-2851. ²⁹29 Delfino D, Scordino F, Pernice I, Lo Passo C, Galbo R, David A, et al. Potential association of specific Candida parapsilosis genotypes, bloodstream infections and colonization of health workers' hands. Clin Microbiol Infect 2014; 20:946-951. .

Several studies have found variations in the frequencies of C. orthopsilosis and C. metapsilosis in candidemia cases. In Spain, among the 171 strains identified as part of the C. parapsilosis complex, 2.4% were shown to be C. orthopsilosis , and 1.2% were shown to be C. metapsilosis ³⁰30 Marcos-Zambrano LJ, Escribano P, Sánchez C, Muñoz P, Bouza E, Guinea J. Antifungal resistance to fluconazole and echinocandins is not emerging in yeast isolates causing fungemia in a Spanish tertiary care center. Antimicrob Agents Chemother2014; 58:4565-4572. . In Brazil, a study by Ruiz et al. ³¹31 Ruiz LS, Khouri S, Hahn RC, Silva EG, Oliveira VK, Gandra R, et al. Candidemia by species of the Candida parapsilosis complex in children's hospital: prevalence, biofilm production and antifungal susceptibility. Mycopathol 2013; 175:231-239. showed frequencies of 10.2% for C. orthopsilosis and 6.1% for C. metapsilosis in strains isolated from blood. Moreover, some studies have shown that C. metapsilosis does not cause bloodstream infections ³²32 Blanco-Blanco MT, Gómez-García AC, Hurtado C, Galán-Ladero MA, Lozano MC, García-Tapias A, et al. Candida orthopsilosis fungemias in a Spanish tertiary care hospital: incidence, epidemiology and antifungal susceptibility. Rev Iberoam Micol 2014; 31:145-148. ³³33 Taj-Aldeen SJ, Kolecka A, Boesten R, Almaslamani M, Chandra P, Meis JF, et al. Epidemiology of candidemia in Qatar, the Middle East: performance of MALDI-TOF MS for the identification of Candida species, species distribution, outcome, and susceptibility pattern. Infection 2014; 42:393-404. . A possible explanation for low frequency or absence of C. metapsilosis as the cause of candidemia is its poor ability to express virulence factors ³⁴34 Orsi CF, Colombari B, Blasi E, Alolaqi A, Almaslamani M, Chandra P, et al. Candida metapsilosis as the least virulent member of the C. parapsilosis complex. Med Mycol2010; 48:1024-1033. .

Similar to samples obtained from blood, we observed a predominance of C. parapsilosis sensu stricto among isolates from nail samples, followed by C. metapsilosis . However, C. orthopsilosis was not foundMohammadi et al. ³⁵35 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 Mycol2013; 51:657-663. have shown that C. parapsilosis sensu stricto is the dominant species, followed by C. orthopsilosis . In contrast, similar to our results, Ge et al. ¹⁹19 Ge YP, Lu GX, Shen YN, Liu WD. In vitro evaluation of phospholipase, proteinase, and esterase activities of Candida parapsilosis and Candida metapsilosis . Mycopathol 2011; 172:429-438. have found that C. parapsilosis sensu stricto and C. metapsilosis are the agents responsible for onychomycosis. Among C. parapsilosis complex species, the two species newly identified as C. orthopsilosis and C. metapsilosis have been less frequently isolated from patients with onychomycosis ⁶6 Silva LB, Oliveira DBC, Silva BV, Souza RA, Silva PR, Ferreira-Paim K, et al. Identification and antifungal susceptibility of fungi isolated from dermatomycoses. J Eur Acad Dermatol Venereol2014; 28:633-640. ³⁶36 Treviño-Rangel RJ, Garza-Gonzále E, González JG, Bocanegra-García V, Llaca JM, González GM. Molecular characterization and antifungal susceptibility of the Candida parapsilosis species complex of clinical isolates from Monterrey, Mexico. Med Mycol2012; 50:781-784. .

Antifungal resistance or tolerance of C. parapsilosis complex species is a growing concern in the context of current and new antifungal agents; this has been a major topic of discussion and concern among infectious disease physicians. According to some researchers, C. parapsilosis sensu stricto isolates are less susceptible to some antifungals used for the treatment of candidiasis, such as AMB, FLC, ITC, and CAS, when compared with the susceptibility of C. orthopsilosis and C. metapsilosis isolates ⁹9 Miranda-Zapico I, Eraso E, Hernández-Almaraz JL, López-Soria LM, Carrillo-Muñoz AJ, Hernández-Molina J, et al. Prevalence and antifungal susceptibility patterns of new cryptic species inside the species complexes Candida parapsilosis and Candida glabrata among blood isolates from a Spanish tertiary hospital. J Antimicrob Chemother 2011; 66:2315-2322. ³¹31 Ruiz LS, Khouri S, Hahn RC, Silva EG, Oliveira VK, Gandra R, et al. Candidemia by species of the Candida parapsilosis complex in children's hospital: prevalence, biofilm production and antifungal susceptibility. Mycopathol 2013; 175:231-239. ³⁷37 Dizbay M, Fidan I, Kalkanci A, Sari N, Yalcin B, Kustimur S, et al. High incidence of Candida parapsilosis candidaemia in non-neutropenic critically ill patients: epidemiology and antifungal susceptibility. Scand J Infect Dis2010; 42:114-120. ³⁸38 Ranque S, Lachaud L, Gari-Toussaint M, Michel-Nguyen A, Mallié M, Gaudart J, et al. Interlaboratory reproducibility of Etest amphotericin B and caspofungin yeast susceptibility testing and comparison with the CLSI method. J Clin Microbiol2012; 50:2305-2309. . Among the azole derivatives analyzed in our study, ITC was less active. Consistent with this, Ruiz et al. ³¹31 Ruiz LS, Khouri S, Hahn RC, Silva EG, Oliveira VK, Gandra R, et al. Candidemia by species of the Candida parapsilosis complex in children's hospital: prevalence, biofilm production and antifungal susceptibility. Mycopathol 2013; 175:231-239. found resistance in one C. parapsilosis sensu strictoisolate and S-DD in 100% of C. orthopsilosis isolates and 66.6% of C. metapsilosis isolates in response to ITC. Figueiredo-Carvalho et al. ³⁹39 Figueiredo-Carvalho MH, Barbedo LS, Oliveira MM, Brito-Santos F, Almeida-Paes R, Zancopé-Oliveira RM. Comparison of commercial methods and the CLSI broth microdilution to determine the antifungal susceptibility of Candida parapsilosis complex bloodstream isolates from three health institutions in Rio de Janeiro, Brazil. Mycopathol 2014; 178:27-35. verified that C. orthopsilosisisolates are less susceptible to ITC than the other species in the C. parapsilosis complex.

In this study, C. parapsilosis complex isolates were not resistant to FLC; however, S-DD criteria were met for two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate. Similarly, FLC resistance in C. metapsilosis and C. parapsilosis sensu stricto has been observed by Chen et al. ⁴⁰40 Chen YC, Lin YH, Chen KW, Lii J, Teng HJ, Li SY. Molecular epidemiology and antifungal susceptibility of Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis in Taiwan. Diagn Microbiol Infect Dis2010; 68:284-292. Moreover, Gomez-Lopez et al ⁴¹41 Gomez-Lopez A, Alastruey-Izquierdo A, Rodriguez D, Almirante B, Pahissa A, Rodriguez-Tudela JL, et al. Prevalence and susceptibility profile of Candida metapsilosis and Candida orthopsilosis : results from population-based surveillance of candidemia in Spain. Antimicrob Agents Chemother200852:1506-1509. found an MIC of greater than 4µg/mL FLC for these species, while Silva et al. ⁶6 Silva LB, Oliveira DBC, Silva BV, Souza RA, Silva PR, Ferreira-Paim K, et al. Identification and antifungal susceptibility of fungi isolated from dermatomycoses. J Eur Acad Dermatol Venereol2014; 28:633-640. found an MIC of 16µg/mL FLC for one C. parapsilosis sensu stricto isolate. According to Van Asbeck et al. ⁴²42 Van Asbeck E, Clemons KV, Martinez M, Tong AJ, Stevens DA. Significant differences in drug susceptibility among species in the Candida parapsilosis group. Diagn Microbiol Infect Dis2008; 62:106-109. , the differences in FLC susceptibility may also reflect the different affinities of azoles for the key ergosterol-synthesizing enzyme 14-α-demethylase or for other enzymes in this pathway.

Interestingly, all isolates of the C. parapsilosis complex were susceptible to the new triazole antifungal agents tested in this study (i.e., VOR and POS). These results are consistent with several studies demonstrating the greater efficacy of these new triazoles against C. parapsilosis complex isolates ⁶6 Silva LB, Oliveira DBC, Silva BV, Souza RA, Silva PR, Ferreira-Paim K, et al. Identification and antifungal susceptibility of fungi isolated from dermatomycoses. J Eur Acad Dermatol Venereol2014; 28:633-640. ⁹9 Miranda-Zapico I, Eraso E, Hernández-Almaraz JL, López-Soria LM, Carrillo-Muñoz AJ, Hernández-Molina J, et al. Prevalence and antifungal susceptibility patterns of new cryptic species inside the species complexes Candida parapsilosis and Candida glabrata among blood isolates from a Spanish tertiary hospital. J Antimicrob Chemother 2011; 66:2315-2322. ³⁹39 Figueiredo-Carvalho MH, Barbedo LS, Oliveira MM, Brito-Santos F, Almeida-Paes R, Zancopé-Oliveira RM. Comparison of commercial methods and the CLSI broth microdilution to determine the antifungal susceptibility of Candida parapsilosis complex bloodstream isolates from three health institutions in Rio de Janeiro, Brazil. Mycopathol 2014; 178:27-35. ⁴¹41 Gomez-Lopez A, Alastruey-Izquierdo A, Rodriguez D, Almirante B, Pahissa A, Rodriguez-Tudela JL, et al. Prevalence and susceptibility profile of Candida metapsilosis and Candida orthopsilosis : results from population-based surveillance of candidemia in Spain. Antimicrob Agents Chemother200852:1506-1509. ⁴³43 Fournier P, Schwebel C, Maubon D, Vesin A, Lebeau B, Foroni L, et al. Antifungal use influences Candida species distribution and susceptibility in the intensive care unit. J Antimicrob Chemother2011; 6:2880-2886. .

In this study, we considered an isolate resistant to AMB when the MIC was greater than 1µg/mL. Thus, in our study, only one C. parapsilosis sensu stricto isolate was classified in this category. Although Lockhart et al. ²⁶26 Lockhart SR, Messer SA, Pfaller MA, Diekema DJ. Geographic distribution and antifungal susceptibility of the newly described species Candida orthopsilosis and Candida metapsilosis in comparison to the closely related species Candida parapsilosis . J Clin Microbiol2008; 46:2659-664. reported MICs of more than 1µg/mL for these three species, they found that the proportion of AMB-resistant C. parapsilosis sensu stricto is higher than those of C. orthopsilosis and C. metapsilosis.

Although Trabasso et al. ⁴⁴44 Trabasso P, Matsuzawa T, Fagnani R, Muraosa Y, Tominaga K, Resende MR, et al. Isolation and drug susceptibility of Candida parapsilosis sensu latoand other species of C. parapsilosis complex from patients with blood stream infections and proposal of a novel LAMP identification method for the species. Mycopathol 2015; 179:53-62. observed some fluctuations in MIC values between the three C. parapsilosis complex species, they also showed that these isolates were susceptible to echinocandin, with MIC values within those of the reference strain or the proposed MIC values (CLSI). However, 19.5% (17/87) of C. parapsilosis complex isolates studied in our work were less susceptible to CAS, with resistance detected in eight isolates of C. parapsilosis sensu stricto ( Table 1 ). Several studies have shown that the MIC of CAS in C. parapsilosis sensu stricto is higher than that of the two species within the complex ²⁶26 Lockhart SR, Messer SA, Pfaller MA, Diekema DJ. Geographic distribution and antifungal susceptibility of the newly described species Candida orthopsilosis and Candida metapsilosis in comparison to the closely related species Candida parapsilosis . J Clin Microbiol2008; 46:2659-664. ⁴⁰40 Chen YC, Lin YH, Chen KW, Lii J, Teng HJ, Li SY. Molecular epidemiology and antifungal susceptibility of Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis in Taiwan. Diagn Microbiol Infect Dis2010; 68:284-292. ⁴¹41 Gomez-Lopez A, Alastruey-Izquierdo A, Rodriguez D, Almirante B, Pahissa A, Rodriguez-Tudela JL, et al. Prevalence and susceptibility profile of Candida metapsilosis and Candida orthopsilosis : results from population-based surveillance of candidemia in Spain. Antimicrob Agents Chemother200852:1506-1509. However, the reason for the high MIC values for CAS is unclear ⁴⁵45 Amran F, Aziz MN, Ibrahim HM, Atiqah, NH, Parameswari S, Hafiza MR, et al. In vitro antifungal susceptibilities of Candidaisolates from patients with invasive candidiasis in Kuala Lumpur Hospital, Malaysia. J Med Microbiol2011; 60:1312-1316. . Recent works have described a functional point mutation in the glucan synthase ( FKS ) gene of the C. parapsilosis complex species, which may explain this observation ⁴⁶46 Beyda ND, Lewis RE, Garey KW. Echinocandin resistance in Candida species: mechanisms of reduced susceptibility and therapeutic approaches. Ann Pharmacother 2012; 46:1086-1096. ⁴⁷47 Fekkar A, Dannaoui E, Meyer I, Imbert S, Brossas JY, Uzunov M, et al. Emergence of echinocandin-resistant Candida spp. in a hospital setting: a consequence of 10 years of increasing use of antifungal therapy? Eur J Clin Microbiol Infect Dis 2014; 33:1489-1496. . FKS gene mutations have been shown to be associated with resistance to CAS, as demonstrated by increases in MICs in mutant isolates compared with those of wild-type isolates ⁴⁸48 Pfaller MA, Castanheira M, Diekema DJ, Messer SA, Moet GJ, Jones RN. Comparison of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Etest methods with the CLSI broth microdilution method for echinocandin susceptibility testing of Candida species. J Clin Microbiol2010; 48:1592-1599. ⁴⁹49 Axner-Elings M, Botero-Kleiven S, Jensen RH, Arendrup MC. Echinocandin susceptibility testing of Candida isolates collected during a 1-year period in Sweden. J Clin Microbiol201149:2516-2521. ⁵⁰50 Garcia-Effron G, Canton E, Pemán J, Dilger A, Romá E, Perlin DS. Epidemiology and echinocandin susceptibility of Candida parapsilosis sensu lato species isolated from bloodstream infections at a Spanish university hospital. J Antimicrob Chemother2012; 67:2739-2748. .

In summary, our study reported the first molecular identification of C. parapsilosis complex species in State of Goiás, Brazil. We provided evidence showing that although C. parapsilosis sensu stricto was the dominant species among the three C. parapsilosis complex species, the other two species ( C. metapsilosis and C. orthopsilosis ) have clinical importance as pathogens in candidemia and onychomycosis. Moreover, we observed important differences in antifungal susceptibility profiles among the three species, mainly with regard to CAS.

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