Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period

Introduction: In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. Methods: Candida isolates were identified by polymerase chain reaction (PCR) using speciesspecific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. Results: Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group ≥61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. Conclusions: Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.

Although ubiquitous in nature, Candida species can cause various infections primarily in hospitalized patients.With the evolution of intensive care medicine, it has become increasingly evident that critically ill patients represent a patient population susceptible to candidal infections.The increased susceptibility of these patients is largely due to the use of invasive devices, impaired immune mechanisms due to severe underlying illness, and widespread use of antibiotics 1,2 .
Although Candida albicans remains the main individual etiologic agent of candidemia, an increase in the number of cases caused by non-albicans species has been reported, including in Brazilian medical centers 1, 3-4, 7, 9-10 .The most common nonalbicans Candida species causing bloodstream infections are Candida parapsilosis and Candida tropicalis 14 .
Fluconazole is a systemic antifungal drug effective against most of the Candida species, although different degrees of susceptibility among species have been described; for example, Candida krusei has intrinsic resistance, and Candida glabrata shows less susceptibility than other Candida species 15 .Furthermore, the emergence of fluconazole resistance has been reported in C. albicans, C. tropicalis, and C. parapsilosis [16][17][18][19][20] , including that observed in a Brazilian tertiary-care hospital 13,[21][22] that justifies the correct species identification and evaluation of their susceptibilities.
In this study, we analyzed for the first time the species distribution and fluconazole susceptibility of Candida isolates in the Hospital Universitário de Londrina, Parana State, Brazil.

Candida isolates and cultures
We evaluated Candida sp.isolates obtained from patients admitted at the Hospital Universitário de Londrina (HU), a general tertiary-care hospital with 333 beds, including 43 intensive care unit (ICU) beds, in the southern Brazil from January 2005 to December 2007.The following data were recorded: sex, age, and patient location at the time of Candida detection.
Surveillance fungal cultures were obtained from several specimens during the trial.Cultures included in this study were those of the urine, blood, tracheal secretion, and superficial mycoses (nail/skin).For the urine specimens, Candida isolates were selected from significant candiduria (≥10 4 CFU/mL).The tracheal secretion isolates were colonization.The specimens were cultured using standard mycological procedures.Samples were inoculated on Sabouraud Dextrose Agar supplemented with chloramphenicol (100µg/ml).The plates were incubated at 28°C for 48 h.All the collected isolates were stored in yeast peptone dextrose (YPD) medium with 25% glycerol at -70°C and were deposited at the Candida culture collection of the Fungal Genetics Laboratory, Universidade Estadual de Londrina-Brazil.
PCR was carried out using genomic DNA obtained as described by Furlaneto-Maia et al. 26 .For the optimum PCR conditions, a reaction volume of 20µL contained 0.8µL (2.5µM) of each deoxynucleoside triphosphate, 0.6µL (50mM) magnesium chloride, 0.5µL (20pmol/µL) of each primer, 10x Taq buffer and 1U of Taq polymerase (Invitrogen), and 2 l (5ng/µL) DNA template.Negative controls were performed with sterile deionised water in place of the template DNA.Reaction mixtures were subjected to an initial denaturing cycle of 5min at 96°C, followed by 34 cycles of 94°C for 1 min (denaturation), 60°C for 1 min (annealing), and 72°C for 1 min (elongation).The sizes of amplified DNA fragments were identified by comparison with molecular size marker DNA (100 bp DNA ladder).The identification of C. dubliniensis was based on phenotypic characteristics 27 .
In vitro antifungal susceptibility testing A total of 201 samples of Candida spp.were tested.The in vitro antifungal susceptibility test was performed according to the EUCAST-AFST reference procedure (EDef 7.1) 28 , using fluconazole (Sigma-Aldrich) as antifungal drug.Fluconazole powder was dissolved in sterile distilled water, in a stock solution at 6400ug/mL, and stored at -20°C overnight.
Serial dilutions of fluconazole were performed in RPMI-1640 medium, supplemented with glucose 2% (w/v) and buffered with 0.165M morpholinepropanesulfonic acid (MOPS-Sigma-Aldrich).The final ranges of the drug dilutions tested were 0.125 to 64 mg/L.
Flat-bottom microdilution plates containing 100uL of the twofold serial dilutions of fluconazole were inoculated with 100uL of the inocula.The final concentration of cells was 0.5 to 2.5×10 5 cells/mL.The microdilution plates were incubated at 37°C for 24h.Each sample was tested in triplicate.Minimal inhibitory concentration (MIC) endpoints were determined spectrophotometrically at 590 nm.MIC 90 was defined as the concentration capable of reducing the growth of 90% of the total population.C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 standard strains were used as quality controls of the tests 28 .Interpretative breakpoints proposed by EUCAST for fluconazole (susceptible ≤2 mg/L and resistant ≥8 mg/L) were used 28 .
Ethical considerations This study was approved by the Ethics Committee of Hospital Universitário da Universidade Estadual de Londrina-PR.

Isolates identification
In this study, a total of 208 Candida isolates were obtained from individual patients, including: C. albicans (n=75), C. tropicalis (n=69), C. krusei (n=4), and Candida sp.(n=59) in CHROMagar® Candidayeast differential medium.In our experiments, C. albicans colonies appeared green, C. krusei appeared dry and light pink with a whitish border, and C. tropicalis appeared dark blue to metallic blue.Further identification carried out using PCR method revealed high accuracy in specificity.The PCR amplification of rDNAs from the three Candida species, C. albicans, C. tropicalis, and C. krusei, resulted in amplification of a single DNA fragment of the expected size (data not shown).
The employment of species-specific primer corresponding to the intergenic spacer region (ITS) sequence from C. parapsilosis (CP) e C. glabrata (CGL) allowed the identification of C. parapsilosis complex (n=40) and C. glabrata (n=17) isolates incompletely identified by CHROMagar® Candida characteristics.Two isolates remained not identified at the species level.The identity of C. parapsilosis (formerly C. parapsilosis group I) isolates was confirmed by the employment of primers corresponding to URA3 gene as previously described 25  In this study, we have employed the tobacco agar medium for the differentiation of C. dubliniensis from C. albicans 27 .On this medium at 28°C, only one isolate produced yellowish-brown colonies with hyphal fringes (C.dubliniensis), whereas the remaining isolates formed smooth, white-to cream-colored colonies (C.albicans).

Sex-and age-specific distribution of Candida isolates
As shown in Table 1, the majority (86%) of isolates from urine specimen were obtained from patients at ICU. Prevalence was similar in women (45.5%) and in men (54.5%).When urine samples were considered, prevalence was high in the age group ≥61 years than that in younger ones.
Considering bloodstream infection, 90% of the patients were from intensive care units.For blood samples, prevalence was high in neonates (35%) followed by advanced ages (22.5%).
Isolates from tracheal secretion and superficial (nail and skin) lesions represented 17.8% and 9.6% of the isolates obtained in this 3-year survey, respectively.Concerning sex, for tracheal secretion samples, prevalence was higher in men (66%).On the other hand for nail and skin samples, it was higher in women (71.4%) than in men (28.6%).For both body-site samples, prevalence was significantly higher in the age group ≥61 years than that in younger ones (Table 1).
Species distribution In a total of 208 Candida cultures, C. albicans represented 36%, while non-albicans Candida accounted for the majority (64%) of these isolates obtained from distinct clinical samples.Table 2 shows the species distribution of Candida isolates with regard to clinical samples.Urine was the most common source of the Candida, accounting for 53.4% of the total isolates, with the following colony counting: 10 4 CFU/mL (20.7%), 10 5 -10 6 CFU/mL (37.7%), and 10 7 CFU/mL (20.7%).Most C. albicans strains were isolated from urine samples (45%).Distribution of identified non-albicans species from urine was: C. tropicalis -36.1%; C. glabrata -12.6%; C. parapsilosis -3.6%; C. krusei -1.8%; and C. dubliniensis -0.9%, as shown in Table 2.In this study, we found a low rate of C. dubliniensis isolates among a total of 75 isolates previously identified as C. albicans.Of the isolates obtained from blood cultures, 75% were non-albicans Candida species, the most common being C. tropicalis, followed by C. parapsilosis (Table 2).Prevalence of non-albicans Candida species was also observed from nail/skin infection specimens, the most common being C. parapsilosis (45%) and C. tropicalis (35%).Most C. parapsilosis strains were isolated from tracheal secretion samples (46%).

Susceptibility tests
Susceptibility tests for fluconazole were performed on 205 isolates of Candida species.Table 3 shows the MICs at which 50% (MIC50) and 90% (MIC90) of the isolates tested were inhibited for fluconazole.The majority of Candida isolates were considered susceptible to fluconazole (MIC of ≤2 mg/L), with the exception of C. glabrata isolates that are intrinsically less susceptible to fluconazole (Table 3).MIC readings for quality control strains (ATCC 90028 and ATCC 22019) were within the limits described in the (EDef 7.1) 28 .
The authors declare that there is no conflict of interest.

DISCUSSION
Infection represents a frequent complication among patients admitted to tertiary hospitals.In particular, the incidence of candidiasis has been increasing during the past years.The largest multicentric study conducted in Latin America reveals a large burden of candidemia in Brazilian tertiary-care hospitals 1 .
In this study, the isolate identification in species level by CHROMagar® Candida medium was in agreement to what was found in molecular identification.The PCR approach for species identification that was employed in this study is a reliable and sensitive method for the diagnosis of the most commonly encountered clinical-relevant Candida species.Furthermore, the employment of phenotypic method based on differential growth on tobacco agar 27 allowed the identification of one C. dubliniensis isolate (from urine sample).Candida dubliniensis is a newly emerging opportunistic pathogen that shares many phenotypic similarities with C. albicans.These similarities pose problems in the identification of isolates and have previously led to misidentification of these species 29 .
The frequency of non-albicans species from distinct clinical specimens observed here was greater than C. albicans, which is consistent with the results of previous studies in Brazilian tertiary hospitals 21,30 .C. tropicalis was the most frequent species isolated from candidemia (35%), which frequency was higher than that observed in the Brazilian multicenter study (16% to 29%) 1 .The second-most frequent species were C. albicans and C. parapsilosis.These data confirm the increasing importance of non-albicans species as agents of fungemia in Brazil.Besides, the low frequency of C. glabrata and C. krusei is in agreement with a previous report that consolidates the concept that candidemia due to these species is rare in Brazil 1 .
An increase of candiduria among hospitalized patients has been reported [31][32][33][34] .Although the significance of Candida isolated from urine of patients is still unclear (reviewed in Kauffman 35 ), the overall mortality associated with ICU candiduria can reach 50% 35 .In this study, identification of yeasts obtained from urine revealed C. albicans to be the most common species, followed by C. tropicalis and much less commonly by other species.Studies conducted in Brazilian medical centers have also shown increased rates of isolation of C. tropicalis from urine 30,34,[37][38] .
Candida parapsilosis was the species most often isolated from superficial mycoses (nail and skin), followed by C. tropicalis.This is in agreement with other studies that identified C. parapsilosis as the prevalent species in the processes of onychomycosis [39][40] .C. parapsilosis is a common inhabitant of normal skin, and this presumably serves as a reservoir of infection for the nails.Recently, we showed the capability of C. parapsilosis cells to adhere and grow as biofilm on human nail surfaces 41 .
Candida parapsilosis was also prevalent in tracheal secretion specimens, followed by C. albicans.In a multicenter study of immunocompetent patients receiving mechanical ventilation performed in France, Azoulay found that C. albicans was the most common species and that the Candida colonization of the respiratory tract may predispose to bacterial ventilator-associated pneumonia 42 .
In the present study, most of the isolates were susceptible to fluconazole.As expected, high resistance rate (64.7%) was observed in C. glabrata, which is an intrinsically less susceptible species.This resistance rate was similar to that observed by Bruder-Nascimento et al. 21who also evaluated the resistance of Candida isolates obtained from distinct clinical specimens in a Brazilian tertiary hospital.
Although much has been reported regarding the epidemiology of Candida infections, the present work is the first report about the distribution of Candida species in invasive and non-invasive candidiasis in our hospital.These data suggest the need for continuous surveillance of candidiasis and antifungal susceptibility trends to adopt treatment strategies applicable to particular healthcare institutions.

TABLE 1 -
Characteristics of patients with candidiasis according to specimen type of yeast isolation.male, F: female, ICU: intensive care unit, ND: not determined, a number of isolates according to specimen sample, b values in parentheses are percentages, c ages are given in years.
. None of the isolates were C. orthopsilosis (formerly C. parapsilosis group II) or C. metapsilosis (formerly C. parapsilosis group III).