Candida spp. in vitro susceptibility profile to four antifungal agents. Resistance surveillance study in Venezuelan strains

Medical Mycology March 2009, 47, 137143 Candida spp. in vitro susceptibility profile to four antifungal agents. Resistance surveillance study in Venezuelan strains MARÍA MERCEDES PANIZO, VERA REVIÁKINA, MARIBEL DOLANDE & SOFÍA SELGRAD Mycology Department, National Institute of Hygiene Rafael Rangel, Caracas, Bolivarian Republic of Venezuela The aim of this study was to determine in vitro susceptibility profiles of Venezuelan strains of Candida spp. to four antifungal agents. One hundred and forty five (145) isolates were recovered during a 1-year period (June 2006 to June 2007) from clinical specimens of patients with severe Candida spp. infections in 15 hospitals. In vitro susceptibilities to amphotericin B, fluconazole, itraconazole and voriconazole were determined by modified Etest. Non Candida albicans Candida spp. were the most frequently isolated yeasts (72.4%) in comparison with C. albicans (27.6%). Candida spp. strains showed MIC ranges between B0.002 and 0.5 mg/ml to amphotericin B. While none were found to be resistant to voriconazole, 5.5% and 27.6% of the test strains were resistant to fluconazole and itraconazole, respectively. C. albicans remains the most susceptible of the yeasts studied to fluconazole and itraconazole (PB0.05) when compared with non C. albicans Candida spp. C. krusei showed the greater cross-resistance to azoles, followed by C. glabrata, C. tropicalis and C. parapsilosis, while C. albicans isolates did not demonstrate this characteristic. It is very important to carry out the correct species identification of clinical yeast isolates because they show up variations in both distribution and susceptibility profiles according to the hospital, patient s underlying disease, clinical specimen analyzed, and the geographical region in which the studies were conducted. The Mycology Department of the INHRR is the national reference center responsible for antifungal resistance surveillance, performing the susceptibility tests with isolates recovered from hospitalized patients in public health centres which do not have mycological diagnosis laboratories. Keywords in vitro antifungal susceptibility testing, Candida spp., resistance, surveillance Introduction Candida species are the main cause of fungal invasive infections and their incidence has increased considerably during the last 30 years. Invasive candidiasis is the Received 29 October 2007; Received in final revised form 5 March 2008; Accepted 19 April 2008 Correspondence: María Mercedes Panizo, Departamento de Micología, Instituto Nacional de Higiene Rafael Rangel, Ciudad Universitaria, Los Chaguaramos (detrás del Hospital Universitario de Caracas), Caracas 1060, República Bolivariana de Venezuela. Tel: 58 212 5371991. Mobile: 58 416 6060257 E-mail: merceval @cantv.net; mmpanizo@gmail.com most frequent fungal infection in the immunocompromised host whose immune system condition influences the severity and clinical manifestations of this infection. However, there has been an increase of this form of the disease observed in critically ill non-immunocompromised hosts. Candida albicans is the most frequent specie associated with the latter infections, but other species within the genus, e.g., C. tropicalis, C. parapsilosis, C. glabrata and C. krusei, have been reported as etiologic agents [14]. The correct species identification leads to the establishment of the definitive etiological diagnosis, allows the prediction of some species potential pathogenicity, 2009 ISHAM DOI: 10.1080/13693780802144339

138 Panizo et al. and guides the selection of antifungal therapy [5]. On the other hand, the marked differences found in the international reports about Candida spp. distribution and antifungal profile susceptibility related to geographical regions and hospital centres, indicate that it is important to maintain continuous surveillance of candidiasis in order to follow the changes that take place in the susceptibility and prevalence of these yeasts [6]. Candida spp. antifungal resistance surveillance is essential to note trends in the incidence of Candida spp. causing severe infections, as well as the incidence of drugs resistance. The Mycology Department of the Instituto Nacional de Higiene Rafael Rangel (National Institute of Hygiene Rafael Rangel; INHRR) is a mycological diagnostic reference centre, performing antifungal susceptibility tests on clinical strains isolated from hospitalized patients in public health centres all over the country. The objective of this study was to determine the in vitro susceptibility profiles to four antifungal agents (amphotericin B, fluconazole, itraconazole and voriconazole) in Venezuelan strains of Candida spp. Materials and methods Specimens We carried out a transversal and prospective study on clinical specimens from patients with Candida spp. severe infections that were received from 15 public health centres in the Mycology Department of the INHRR between June 2006 and June 2007. Methods The yeasts were identified according to standard procedures, i.e., germ tube test, chlamydospores production, high temperature growth and carbohydrate assimilation tests following the Haley-Standard method [710]. Etest (AB Biodisk, Solna, Sweden) antifungal susceptibility tests were performed following the manufacturer s instructions with some modifications. The culture media used was Casitone agar (Difco) with 2% of glucose [11] distributed in 19 ml aliquots in tubes. One ml of an inoculum yeast suspension adjusted spectrophotometrically to the turbidity of a 0.5 McFarland standard (10 6 cells/ ml), was incorporated in the melted agar at 568C, and contents of the tubes were distributed in 90 mm petri dishes [12]. After the agar surface was firm, two Etest strips with fluconazole (0.016256 mg/ml) and amphotericin B, itraconazole, and voriconazole (0.00232 mg/ ml) were applied per dish. The dishes were incubated at 358C and read 24 h later. The susceptibility to voriconazole was not examined with all yeast isolates. The minimal inhibitory concentration (MIC) was interpreted as the lowest concentration obtained when the borders of the elliptical inhibition zone intercepted the measurement scale of the strip. Interpretive guidelines MICs interpretative criteria were susceptible (S), susceptible dose-dependent (SDD) and resistant (R), following the breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 document [13] and the conference of the antifungal subcommittee of the Clinical Laboratory Standard Institute 2005 (CLSI 2005) [14,15]. The established breakpoints were as follows: fluconazole S: B8; SDD: 1632; R: 64; itraconazole S: B0.125; SDD: 0.250.5; R: 1; and voriconazole S: B1; SDD: 2; R: 4. Amphotericin B breakpoints are not indicated because they have not been established by the NCCLS. Because of Etest measurement scale has a continuous concentration gradient, MIC values found to be in between twofold dilutions were raised to the next twofold level of the reference method in order to compare our results with those from the reference method. Off-scale MICs at the upper limit were taken to the next higher concentration and off-scale results at the lower limit were left unchanged [16]. Quality Control (QC) QC was performed by using Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 as controls in accordance with NCCLS document M27-A2 [13]. Statistical analysis The incidence percentage of Candida spp. isolates was calculated by gender, age, underlying disease and condition of the patients, as well as by clinical specimen type. The percentages of S, SDD and R to all four antifungal agents were also calculated. The analysis of variance (ANOVA) by Kruskal-Wallis was used to compare C. albicans and non-candida albicans Candida spp. antifungal susceptibility to fluconazole and itraconazole. Chi-square test was used to analyze the relationship between susceptibility and resistance to fluconazole and itraconazole of Candida spp. strains. Statistical analysis was done by use the Statgraphics program for Windows version 5.0.

Candida spp. in vitro susceptibility profile 139 Results One hundred and forty five (145) strains of Candida spp. were recovered from the following specimens of patients with severe infections; 62 urine, 33 blood, 15 respiratory tract secretions, 11 catheter, 11 oral scrapings, 6 vaginal secretions. In addition, 7 isolates were obtained from 2 of stools and 1 each from scrotal secretion, abdominal secretion, peritoneal fluid, cerebrospinal fluid and skin. Candida albicans was the most frequently isolated member of the genus, followed by C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. However, we noted that Candida spp. distribution in candidemias was completely different with C. parapsilosis being the most commonly isolated member of the genus, followed by C. tropicalis, C. albicans, C. pelliculosa, C. intermedia, C. glabrata and C. lusitaniae. Non-Candida albicans Candida spp. isolates (105 of 145 or 72.4%) were more frequent than C. albicans (40 of 145: 27.6% as seen in Table 1. Fifty four patients were B1 year of age (37.2%), with a mean age distribution of 18.5913.4 days (range 670 days) involving 30 male and 24 female. C. parapsilosis was the most frequently isolated yeast in this patient group, followed by C. albicans, C. tropicalis and C. glabrata. The 91 remaining patients (62.8%) had a mean age of 45.4922.4 years (range 1 to 86 years) and included 43 male and 48 female. For Candida spp., a different distribution was observed in those patients who were 60 years in that C. tropicalis was the most frequently recovered member of the genus, followed by C. albicans, C. glabrata and C. parapsilosis (Table 1). Eighty five of the patients were hospitalized in intensive care units (ICU), 40 in general medicine wards and 20 were out-patients. Neonatal sepsis was the most frequent underlying disease, followed by pneumonia, diabetes, malignancy, septicaemia, vaginal candidiasis, renal insufficiency and/or chronic obstructive uropathy, acquired immunodeficiency syndrome (AIDS) and necrotizing enterocolitis. Other diseases, included pancreatitis, idiopathic myelofibrosis, lymphadenopathic syndrome, meningitis, soft tissue infections, vulgar pemphigus, renal transplant, oral candidiasis, cerebrovascular accident, bilateral hydronephrosis, Guillain-Barre syndrome, intestinal obstruction and febrile icteric-hemorrhagic syndrome (Table 1). Information about previous treatment with antifungal and antibiotic agents were recorded for only 26 patients, with amphotericin B the most commonly used (15 of 26 patients), followed by fluconazole (7 of 26 patients). Aminoglucosides and third generation cephalosporins were the most frequently administered antibiotics. Candida spp. in vitro antifungal susceptibility to four antifungal agents and the percentages of S, SDD and R are shown in Table 2. Decreased trailing growth with the azoles was observed with the Etest modified procedure reduced yeasts overgrowth on the agar surface and facilitating the reading of results. Candida spp. showed MICs ranges of B0.0020.5 mg/ml to amphotericin B and none of the 105 strains included in the study showed voriconazole resistance. Only two C. krusei isolates recovered from oral scrapping of AIDS patients had SDD fluconazole values of 2 mg/ml. All C. albicans and C. parapsilosis strains were fluconazole susceptible. Nineteen strains (13.1%) were found to be SDD relative to fluconazole. Primarily these were isolates of C. parapsilosis and C. tropicalis recovered from blood, urine, respiratory tract secretions, catheter, vaginal secretions and stools from patients with neonatal sepsis, septicaemia, pneumonia, diabetes, renal insufficiency, renal transplant, malignancy, acute pancreatitis, vulgar pemphigus, necrotizing enterocolitis, and vaginal candidiasis. One of these strains was found to be C. intermedia, acandida specie with low frequency of recovery from clinical material. Nine Candida spp. (6.2%) strains showed fluconazole resistance, including one C. tropicalis, twoc. glabrata and one C. utilis, all of which been isolated urine samples from pneumonia, diabetes, and neonatal sepsis patients. The five remaining strains were identified as C. krusei and had been recovered from two oral scraping specimens of AIDS patients, and three oral scraping and urine samples of cancer and pneumonia patients. Twenty-two Candida spp. isolates (15.2%) were found to be SDD with respect to itraconazole, with C. parapsilosis being the most common within this group. An additional 40 strains were resistant to this antifungal (27.6%), of which C. glabrata was the most common. We found statistically significant differences when comparing the susceptibility of C. albicans and non- Candida albicans Candida spp. to fluconazole and itraconazole (P B0.05), with C. albicans being found to be the most susceptible to these antifungal agents. When analyzing susceptibility and resistance patterns of Candida spp. isolates with respect to fluconazole and itraconazole we found that it was dependent upon the Candida species. Seventy three of 98 (74.5%) Candida spp. were fluconazole and itraconazole susceptible, 16 of 98 (16.3%) were fluconazole susceptible but itraconazole resistant, and 9 of 98 (9.2%) were resistant to both antifungal agents, which implies the presence of cross-resistance. C. krusei (80%) had the higher crossresistance, followed by C. glabrata (18.2%), C. tropicalis

140 Panizo et al. Table 1 Demographic and clinical data for Candida spp. severe infections patients (n145). Characteristics Candida species n (%) Ca Cp Ct Cg Ck Candida sp.* Total (%) Gender Male 22 (30.1) 17 (23.3) 16 (22) 9 (12.3) 5 (6.8) 4 (5.5) 73 (50.3) Female 18 (25) 22 (30.6) 13 (18.1) 13 (18.1) 6 (8.3) 72 (49.7) Age (years) B1 year 11 (20.4) 24 (44.4) 6 (11.1) 4 (7.4) 9 (16.7) 54 (37.2) 19 3 (60) 1 (20) 1 (20) 5 (3.4) 1019 3 (33.3) 3 (33.3) 2 (22.2) 1 (11.1) 9 (6.2) 2029 3 (37.5) 1 (12.5) 2 (25) 1 (12.5) 1 (12.5) 8 (5.5) 3039 3 (21.4) 3 (21.4) 2 (14.3) 3 (21.4) 3 (21.4) 14 (9.7) 4049 5 (31.3) 3 (18.8) 3 (18.8) 4 (25) 1 (6.3) 16 (11) 5059 4 (40) 4 (40) 2 (20) 10 (6.9) ]60 8 (27.6) 4 (13.8) 9 (31) 7 (24.1) 1 (3.4) 29 (20) Patient condition Ambulatory 5 (25) 4 (20) 3 (15) 5 (25) 2 (10) 1 (5) 20 (13.8) Medicine ward hospitalization 16 (40) 5 (12.5) 7 (17.5) 9 (22.5) 2 (5) 1 (2.5) 40 (27.6) ICUs hospitalization 19 (22.4) 30 (35.3) 19 (22.4) 7 (8.2) 1 (1.2) 9 (10.6) 85 (58.6) Site of isolation Urine 19 (30.6) 13 (21) 13 (21) 13 (21) 1 (1.6) 3 (4.8) 62 (42.8) Blood 3 (9.1) 16 (48.5) 7 (21.2) 1 (3) 6 (18.2) 33 (22.8) Bronchoalveolar lavage/sputum 10 (66.7) 1 (6.7) 1 (6.7) 2 (13.3) 1 (6.7) 15 (10.3) Catheter 1 (9.1) 4 (36.4) 6 (54.5) 11 (7.6) Oral scrapings 5 (45.5) 1 (9.1) 2 (18.2) 3 (27.3) 11 (7.6) Vaginal secretions 1 (16.7) 1 (16.7) 4 (66.7) 6 (4.1) Other isolation sites 1 (14.3) 4 (57.1) 1 (14.3) 1 (14.3) 7 (4.8) Underlying disease Neonatal sepsis 9 (19) 21 (44.7) 6 (12.8) 4 (8.5) 7 (15) 47 (32.4) Pneumonia 14 (37.8) 6 (16.2) 11 (29.7) 4 (10.8) 2 (5.4) 37 (25.5) Diabetes 2 (20) 2 (20) 2 (20) 3 (30) 1 (10) 10 (7) Malignancy 2 (25) 2 (25) 3 (37.5) 1 (12.5) 8 (5.5) Septicaemia 1 (12.5) 2 (25) 4 (50) 1 (12.5) 8 (5.5) Vaginal candidiasis 1 (16.7) 1 (16.7) 4 (66.7) 6 (4.1) Renal insufficiency and/or chronic obstructive uropathy 1 (20) 2 (40) 2 (40) 5 (3.4) AIDS 3 (60) 2 (40) 5 (3.4) Necrotizing enterocolitis 1 (20) 2 (40) 2 (40) 5 (3.4) Other diseases 6 (42.9) 3 (21.4) 2 (14.3) 3 (21.4) 14 (9.7) Total 40 (27.6) 39 (26.9) 29 (20) 22 (15.2) 5 (3.4) 10 (6.9) 145 nnumber of isolates; Ca: Candida albicans; Cp: Candida parapsilosis; Ct: Candida tropicalis; Cg: Candida glabrata; Ck: Candida krusei. *Candida sp. includes: C. intermedia (4), C. pelliculosa (3), C. lusitaniae (2) and C. utilis (1). (5%) and C. parapsilosis (3.7%). C. albicans did not show cross-resistance to the azoles. Discussion The results of this study demonstrated a remarkable increase in the recovery of non-candida albicans Candida spp. from clinical specimens as has been noted previously in the literature [6,1620]. However, variations of Candida spp. distribution was found to be dependent on the hospital, type of clinical specimen and the geographical region in which the study was conducted. Therefore, the results of this investigation are comparable with Argentinean studies [21] and those international surveillance programs containing data of North America, Europe and Latin America [16,17], but different from those from investigations in Canada [6], India [20] and the ARTEMIS global antifungal susceptibility program [18]. In our results, the distribution for Candida spp. isolated from patients with candidemia is different to those reported by international surveillance programs. C. parapsilosis, C. tropicalis, and C. glabrata were the causal agents of these infections in neonates. In contrast, C. albicans was responsible for more than 50% of candidemias, followed by C. glabrata as the

Candida spp. in vitro susceptibility profile 141 Table 2 Candida spp. in vitro susceptibility to four antifungal agents (n145). Species Antifungal agents Ranges* Geometric mean* Modal MIC* 10 n (% of isolates) S SDD R All Candida spp. (n145) Amphotericin B B0.0020.5 0.009 0.002 Fluconazole 0.25256 3.1 1.5 117 (80.4) 19 (13.1) 9 (6.2) Itraconazole 0.00864 0.2 0.032 83 (57.2) 22 (15.2) 40 (27.6) Voriconazole 1 0.0042 0.06 0.047 103 (98) 2 (2) Candida albicans (n40) Amphotericin B 0.0020.094 2.1 0.002 Fluconazole 0.256 0.08 1 40 (100) Itraconazole 0.0081 0.05 0.032 38 (95) 1 (2.5) 1 (2.5) Voriconazole 2 0.0040.5 0.03 0.012 & 0.023 24 (100) Candida parapsilosis (n39) Amphotericin B 0.0020.094 0.009 0.006 & 0.008 Fluconazole 0.532 2.34 1 36 (92.3) 3 (7.7) Itraconazole 0.01232 0.14 0.125 24 (61.5) 10 (25.6) 5 (12.8) Voriconazole 3 0.0080.19 0.047 0.047 32 (100) Candida tropicalis (n29) Amphotericin B B 0.0020.125 0.009 0.002 Fluconazole 0.25128 3.4 1.5 22 (76) 6 (21) 1 (3) Itraconazole 0.0162 0.23 0.125 14 (48.3) 6 (20.7) 9 (31) Voriconazole 4 0.0640.5 0.07 0.047 21 (100) Candida glabrata (n22) Amphotericin B 0.0020.38 0.01 0.002 Fluconazole 0.564 8.3 4 11 (50) 9 (41) 2 (9) Itraconazole 0.04732 8.4 32 2 (9.1) 2 (8.1) 18 (81.8) Voriconazole 5 0.0320.75 0.15 0.125 & 0.25 14 (100) Candida krusei (n5) 8 Amphotericin B 0.0020.5 0.023 0.002 Fluconazole 1256 84.4 256 5 (100) Itraconazole 0.12564 4 2 1 (20) 4 (80) Voriconazole 6 0.382 1.03 2 2 (50) 2 (50) Candida sp. (n10) 9 Amphotericin B 0.0020.047 0.006 0.002 Fluconazole 0.75256 4.26 1.5 8 (80) 1 (10) 1 (10) Itraconazole 0.02332 0.31 0.023 4 (40) 3 (30) 3 (30) Voriconazole 7 0.0080.75 0.037 0.023 10 (100) nnumber of isolates; *MICs in mg/ml; 1 n105; 2 n24; 3 n32; 4 n21; 5 n14; 6 n4; 7 n10; 8 All C. krusei were considered fluconazole resistant independent of the MIC result; 9 Candida sp. includes: C. intermedia (4), C. pelliculosa (3), C. lusitaniae (2) and C. utilis (1); 10 The most frequent MIC. second causal agent in North America and the third one in Europe. In Latin America, C. parapsilosis was ranked the second most common causal agent of candidemia, C. glabrata was the fourth, and C. tropicalis was involved in 10 to 20% of these infections. These differences persist when comparing our results with those reported in other studies relative to the age groups and the Candida spp. distribution in candidemias [4,6,1620,2224]. C. albicans and C. parapsilosis neonatal candidemia were previously described by Kao et al. [25] and Saiman et al. [26], among others. The differences are related to the preferential use of amphotericin B as opposed to fluconazole by the neonatologists for the treatment of candidemias [27]. On the other hand, C. parapsilosis horizontal transmission in the neonatal ICUs, as well as the relationship of this specie with parenteral nutrition, surgical material contamination, and inadequate catheter handling has been previously demonstrated. This is probably the cause for the prevalence of this specie in patients with neonatal sepsis, indicating failures associated with control of nosocomial infections [2224,28,29]. Underlying diseases observed in this study are comparable to those observed by other investigators. Our findings show that most of the severe infections caused by Candida spp. were associated with neonates, pneumonia and diabetic patients. In contrast, Candida spp. infections occurred in a low percentage of patients with AIDS and malignancies [3,4,6,20]. Our susceptibility results for amphotericin B, fluconazole, itraconazole and voriconazole are comparable to those reported in the United States, Canada, Europe and Latin America, with slight differences that were dependent on the underlying disease and the Candida species involved in the infection [3,4,6,1618,22,23]. In our investigation, MIC ranges for amphotericin B stayed below 0.5 mg/ml for all the Candida spp. None of the 105 strains assayed with voriconazole showed resistance to this antifungal agent. All the C. albicans and C. parapsilosis strains were susceptible to

142 Panizo et al. fluconazole. Fluconazole resistance was mainly associated with C. krusei, which was primarily isolated from AIDS and malignancy patients. In addition, other Candida species, such as C. tropicalis, C. glabrata and C. utilis, isolated from non-immunosuppressed patients showed fluconazole resistance. These results are comparable to those reported in the literature. C. glabrata, followed by C. tropicalis, C. parapsilosis, C. krusei and other Candida species were resistant to itraconazole, and these results are slightly different to those reported in the United States, Europe and Latin America. In the latter geographical areas C. glabrata is the most resistant to itraconazole, followed by C. krusei and C. tropicalis [3,4,6,1618,22,23]. According to recently published data [16], an increase in triazoles resistance has not been detected in international surveillance studies. Therefore, more studies are required to enable the adequate interpretation of the epidemiological changes that point to an increase of non-c. albicans Candida spp. isolates over C. albicans. Probably, the observed variations are due to differences in the use of antifungal agents and different nosocomial infections control practices among hospitals. The influence of the geographical localization in which investigations are performed, the underlying diseases of patients, and predisposing conditions such as prolonged stays in ICUs and medical wards, should be taken into account. On the other hand, Candida spp. severe infections are difficult to characterize and, at the moment, there is only a consensus for their definitions in immunocompromised patients, while there are no strict definitions for them in non-immunocompromised hosts. Our results confirm that Candida spp. severe infections should not be considered as rare and restricted to neutropenic, AIDS, and cancer patients. All patients, particularly those critically ill and with long ICUs hospitalization periods, can present candidemia episodes and disseminated candidiasis [3,4,6,16,18,25,30]. In relation to the Etest modified technique, previous studies carried out in the Mycology Department of the INHRR, proved that the incorporation of the inoculums in the agar notably diminishes yeast overgrowth on the agar surface, improving results interpretation. This technique helps to obtain homogeneous yeast development without altering the test results, and the elliptical configuration of the strip. The partial oxygen reduction (condition obtained inside the agar) contributes to decrease the trailing effect [12]. Candida spp. display multiple mechanisms for azoles antifungal resistance [31,32]. Although Candida spp. percentage of cross-resistance to azoles obtained in this study is low compared to the findings of other investigations [3133], it should not be rejected and should be kept under observation. None of the C. albicans isolates in our study showed cross-resistance to fluconazole and itraconazole, but other Candida species did present this characteristic. Finally, it is very important to carry out the correct species identification of clinical yeast isolates, because they show variations in their distribution and antifungal susceptibility profiles according to the hospital, type of clinical specimen, underlying patient diseases, and the geographical region where the studies are performed. The Mycology Department of the INHRR is a national reference centre for mycological diagnosis, responsible of the antifungal resistance surveillance, and performs in vitro susceptibility tests on clinical isolates submitted from public health centres that do not have mycological diagnosis laboratory. 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