Candida glabrata: an emerging pathogen in Brazilian tertiary care hospitals

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1 Medical Mycology January 2013, 51, Candida glabrata: an emerging pathogen in Brazilian tertiary care hospitals ARNALDO L. COLOMBO *, MARCIA GARNICA, LUIS FERNANDO ARANHA CAMARGO, CLOVIS ARNS DA CUNHA, ANTONIO CARLOS BANDEIRA #, DANIELLE BORGHI ^, TATIANA CAMPOS, ANA LUCIA SENNA $, MARIA EUGENIA VALIAS DIDIER &, VIVIANE CARVALHO DIAS & MARCIO NUCCI * Universidade Federal de S ã o Paulo, S ã o Paulo, University Hospital, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Hospital Israelita Albert Einstein, S ã o Paulo, Hospital Nossa Senhora das Gra ç as, Curitiba, #Hospital Alian ç a, Salvador, ^ Hospital Copa D Or, Rio de Janeiro, Hospital Barra D Or, Rio de Janeiro, $ Hospital Quinta D Or, Rio de Janeiro, and & Hospital Felicio Rocho, Belo Horizonte, Brazil Candida glabrata is an infrequent cause of candidemia in Brazilian public hospitals. We investigated putative differences in the epidemiology of candidemia in institutions with different sources of funding. Prospective laboratory-based surveillance of candidemia was conducted in seven private and two public Brazilian tertiary care hospitals. Among 4,363 episodes of bloodstream infection, 300 were caused by Candida spp. (6.9%). Incidence rates were significantly higher in public hospitals, i.e., 2.42 vs episodes per 1,000 admissions ( P 0.01). Patients in private hospitals were older, more likely to be in an intensive care unit and to have been exposed to fluconazole before candidemia. Candida parapsilosis was more frequently recovered as the etiologic agent in public (33% vs. 16%, P 0.001) hospitals, whereas C. glabrata was more frequently isolated in private hospitals (13% vs. 3%, P 0.001). Fluconazole resistance among C. glabrata isolates was more frequent in private hospitals (76.5% vs. 20%, P 0.02). The 30-day mortality was slightly higher among patients in public hospitals (53% vs. 43%, P 0.10). Candida glabrata is an emerging pathogen in private institutions and in this setting, fluconazole should not be considered as a safe option for primary therapy of candidemia. Keywords Candida glabrata, emergent fungal pathogen, fluconazole resistance, epidemiology Introduction Candidemia is associated with high morbidity and mortality, and its management has a great impact on hospital costs [1,2]. The epidemiology of such infections varies among different geographic regions and even between medical centers within the same region. In Brazil, epidemiologic studies conducted in public hospitals showed that the burden of candidemia in tertiary care hospitals is high, with incidence rates much higher than those reported in the Northern Hemisphere [3,4]. These epidemiologic studies Re c e ive d 19 Ja nu a r y ; Re c e ive d i n fi nal revised form 27 March 2012; Accepted 24 May Correspondence: Arnaldo L. Colombo, Division of Infectious Diseases Hospital S ã o Paulo, Escola Paulista de Medicina-Universidade Federal de S ã o Paulo, Rua Botucatu, 740 S ã o Paulo, Brasil. colomboal@ terra.com.br also demonstrated that Candida albicans, C. tropicalis and C. parapsilosis were involved in 90% of candidemia cases, but that the incidence of caused by C. glabrata was low [4]. However, a recent retrospective study conducted in Brazil revealed an increase in the incidence of candidemia due to C. glabrata, apparently related to a high level of fluconazole use in hospitals [5]. The Brazilian health system has a mixture of public and private hospitals, where the share of private spending in healthcare is about 53%. The private hospitals which are ranked best in the quality of assistance of patients have more funding to provide healthcare than public institutions. Indeed, private institutions derive their funding from private insurers, mainly on a fee-for-service basis. In contrast, public institutions have more limited funding obtained exclusively from government resources. Interestingly, an inverse correlation between healthcare funding, frequency 2013 ISHAM DOI: /

2 C. glabrata is emerging as pathogen in Brazil 39 of sepsis and hospital mortality has been reported [6]. In this study, we conducted a laboratory-based survey of candidemia in seven private top-ranked medical institutions and two reference public hospitals in Brazil in order to characterize differences in the epidemiology and outcomes of candidemia in medical centers with different mechanisms of funding. Materials and methods Patient selection and data collection We conducted a prospective, laboratory-based surveillance study from July 2006 to December 2007 in nine tertiary care medical centers located in five cities of the Northeastern, Southeastern and Southern regions of Brazil. All hospitals had automated blood culture systems (BACTEC [BD-Becton Dickinson, Durham, NC, USA] or BacT- ALERT [biom é rieux SA, Marcy l Etoile, France]) and provided medical care to adults and children. We selected hospitals representative of two different sources of funding for healthcare, i.e., Group I which consisted of two reference public hospitals that provide medical assistance to low-income patients, and Group II, composed of seven forprofit medical institutions that primarily provide care to patients covered by private insurances. A case of candidemia was diagnosed when a Candida sp. was isolated from a blood culture. Positive blood cultures for Candida spp. occurring 30 days after the initial positive blood cultures were defined as new episodes. Patients were followed for 30 days following the diagnosis of candidemia. Fever was defined as an axillary temperature 37.8 C, and neutropenia was classified as an absolute neutrophil count 500/mm 3. Conditions associated with candidemia were considered if present 30 days prior to the initial diagnosis of candidemia, except for surgery ( 90 days). All medical records were reviewed and monitored by a central data collection system to assure completeness and consistency. Yeast identifi cation and antifungal susceptibility testing All Candida bloodstream isolates were sent to the Special Mycology Laboratory at Universidade Federal de S ã o Paulo for species identification and antifungal susceptibility testing. Isolates were identified according to their microscopic morphology on cornmeal Tween 80 agar which was complemented by biochemical tests using the ID 32C system (biom é rieux SA). Antifungal susceptibility tests were performed using a homemade broth microdilution assay following the methods recommended by the Clinical and Laboratory Standards Institute (CLSI) [7]. The following antifungal drugs were tested: fluconazole (Pfizer Incorporated, New York, NY, USA), flucytosine (Sigma Chemical Corporation, St Louis, MO, USA), amphotericin B (Sigma) and voriconazole (Pfizer). The assays were incubated at 35 C for 24 h. The minimum inhibitory concentration (MIC) breakpoints used for fluconazole, voriconazole and flucytosine were those suggested by CLSI [7]. Due to a lack of consensus about the definition of MIC breakpoints for amphotericin B, values suggested by previous publications were employed. MICs 1 μ g/ml were considered susceptible and those which were 2 μ g/ml were considered resistant [8]. In addition, since new breakpoints for fluconazole have been recently proposed [9], we also analyzed fluconazole susceptibility of C. albicans, C. tropicalis, C. parapsilosis and C. glabrata using these new definitions which for C. albicans, C. tropicalis and C. parapsilosis were 2 μ g/ml susceptible, 4 μ g/ml susceptible dose-dependent (SDD) and 8 μ g/ml resistant, whereas 32 μg/ml was SDD and 64 μ g/ml resistant for C. glabrata isolates. Data analysis and sample size calculation Incidence rates of candidemia at each institution were calculated using the number of patients hospitalized and the number of admissions during the study period as denominators. Incidence rates were expressed as cases per 1,000 patient days and per 1,000 admissions. The sample size was determined on the basis of the expected prevalence of candidemia due to C. glabrata in the two groups of hospitals., which we hypothesized would be much higher in private hospitals than in public hospitals. Based on the prevalence rate of candidemia due to C. glabrata of 4.9% observed in our previous study [10] conducted in 11 public centers, we estimated that we would require a total of 318 episodes of candidemia to detect a three-fold higher incidence in private hospitals (alpha 5% and beta 20%). The Chi-square test was used to compare proportions, and Mann-Whitney test was used to compare continuous variables. To identify predictors of poor outcome, we performed logistic regression analysis and modeled those variables with P value 0.1 by univariate analysis. P-values 0.05 were considered statistically significant. All statistical analyses were performed using SPSS for Windows (version , SPSS, Inc., Chicago, IL, USA). Results A total of 4,363 cases of bloodstream infections (BSI) were identified, 300 of which were caused by Candida spp. (6.9%). The median patient age was 56 years (range 0 96), of which 16 were neonates (5.3%), 45 were 13 years old (15%), and 133 were 60 years old (44.2%). As shown in Fig. 1, incidence rates varied widely among different hos-

3 40 Colombo et al. * * ** ** Fig. 1 Incidence rates of candidemia in private and public hospitals. Priv, private; Publ, public. * Overall incidence per 1,000 admissions: 2.42 in public vs in private hospitals, relative risk 2.63, 95% confidence interval , P **Overall incidence per 1,000 patient days: 0.32 in public vs in private hospitals, relative risk 1.61, 95% confidence interval , P pitals, and were significantly higher in public than in private hospitals, i.e., 2.42 vs episodes per 1,000 admissions (relative risk [RR] 2.63, 95% confidence interval [95% CI] , P 0.001) and 0.32 vs episodes per 1,000 patient-days (RR 1.61, 95% CI , P 0.001). Table 1 presents the characteristics of the episodes of candidemia. The median age of patients admitted to public hospitals was lower than that of patients admitted to private hospitals, with a significantly higher proportion of children (20% vs. 9%, P 0.009) and a significantly lower proportion of patients 60 years old (33% vs. 58%, P 0.001) in public facilities. A higher proportion of patients in private hospitals had solid tumor, neurological disease, diabetes mellitus, central venous catheter and previous fluconazole use. In addition, patients in private hospitals were more likely to be in an ICU, although the median APACHE II score was not significantly different between the two patient groups (16 among 53 patients in public hospitals and 15 among 51 patients in private hospitals, P 0.74). There was no significant difference in the proportion of patients receiving treatment for candidemia. However, patients in public hospitals were more likely to have received amphotericin B deoxycholate as their primary treatment (38% vs. 3%, P 0.001), while patients in private hospitals were more likely to have received a lipid formulation of amphotericin B (10% vs. 1%, P 0.002) or caspofungin (25% vs. 0.7%, P 0.001). The 30-day survival rate was slightly higher among patients in private hospitals (53% vs. 43%), but the difference was not statistically significant ( P 0.10). Table 2 presents the species distribution of the causative agents of candidemia. Candida albicans was the leading agent in both study groups. Candida parapsilosis was more likely to cause candidemia in patients admitted to public hospitals (33% vs. 16%, P 0.001), while C. glabrata was more frequent in private hospitals (13% vs. 3%, P 0.001). The same trend of species distribution was observed when we analyzed adult patients only and found 27% of blood stream infections were caused by C. parapsilosis in public vs. 16% in private hospitals ( P 0.02), and 4% were due to C. glabrata in public vs. 15% in private hospitals ( P 0.002). As shown in Table 3, using the old breakpoints, with the exception of one C. tropicalis isolate, resistance to fluconazole was limited to isolates of C. glabrata (64%) and C. krusei (100%). In addition, four C. glabrata isolates were SDD to fluconazole and all C. krusei isolates were susceptible to voriconazole. In contrast, 14% of C. glabrata isolates were SDD and 9% resistant to voriconazole. Overall, the proportion of isolates that were SDD and resistant to fluconazole was higher in patients in private hospitals than among those in public hospitals (15% vs. 5%, P 0.001), mainly due to the higher proportion of C. glabrata candidemia in private hospitals. Consequently, the MIC-50 for fluconazole was significantly higher among C. glabrata isolates from private vs. public hospitals (MIC-50 of 64 μ g/ml, range 2 64 vs. 16 μg/ml, range 2 64, P 0.048). The proportion of isolates that were SDD and resistant to voriconazole was not significantly different in public (1%) vs. private (3%) hospitals ( P 0.24). Applying the new breakpoints for fluconazole, we observed only one C. parapsilosis (public institution) and two C. tropicalis strains (private institutions) that were considered SDD to fluconazole. As shown in Table 4, older age, higher APACHE II score, admission to an ICU, diabetes mellitus, liver disease, chronic renal failure, mechanical ventilation, dialysis, and receipt of corticosteroids were significantly associated with higher 30-day mortality rates. In contrast, better 30-day survival rates were observed more frequently in candidemia due to C. parapsilosis and in patients who received fluconazole as primary treatment. By multivariate analysis,

4 C. glabrata is emerging as pathogen in Brazil 41 Table 1 Characteristics of candidemic patients in public (2) and private institutions (7). Overall n 300 Public n 174 Private n 126P value * Gender, male: female 151:150 88:86 62: Age (years), median (range) 56 (0 96) 49 (0 89) 68 (0 96) year 20 (6) 16 (9) 4 (3) years 45 (15) 34 (20) 11 (9) years 131 (44) 58 (33) 73 (58) APACHE II score **, median (range) 16 (2 38) 16 (2 38) 15 (4 34) 0.74 Duration (days) of hospitalization 19.5 (0 312) 21 (0 244) 17 (0 312) 0.32 before candidemia, median (range) Admission to an ICU 146 (48) 72 (41) 74 (59) Cancer 101 (34) 54 (31) 47 (37) 0.24 Hematological malignancy 28 (9) 20 (11) 8 (6) 0.13 Solid tumor 73 (24) 34 (19) 39 (31) 0.02 Cardiac disease 97 (32) 51 (29) 46 (37) 0.15 Neurological disease 84 (28) 38 (22) 46 (37) Lung disease 77 (26) 43 (25) 34 (27) 0.67 Diabetes mellitus 64 (21) 30 (17) 34 (27) 0.03 Renal failure 92 (31) 47 (27) 45 (36) 0.08 Chronic renal failure 42 (14) 25 (14) 17 (14) 0.88 Surgery 149 (49) 81 (46) 68 (54) 0.15 Abdominal surgery 89 (30) 26 (26) 43 (34) 0.12 Parenteral nutrition 77 (26) 38 (22) 39 (31) 0.07 Dialysis 47 (16) 23 (13) 24 (19) 0.16 Neutropenia 28 (9) 16 (9) 12 (9) 0.91 Central venous catheter 247 (82) 135 (78) 112 (89) 0.01 Antimicrobial use 272 (91) 160 (92) 112 (89) 0.36 Corticosteroid use 134 (45) 77 (44) 57 (45) 0.91 Previous use of fluconazole 48 (16) 22 (12) 26 (21) 0.05 Received antifungal therapy 241 (80) 135 (78) 106 (84) 0.11 Primary treatment 242 (80) 136 (78) 106 (84) 0.17 Fluconazole 130/242 (54) 65/136 (48) 65/106 (61) Amphotericin B deoxycholate 55/242 (23) 52/136 (38) 3/106 (3) Lipid formulation of amphotericin B 13/242 (5) 2/136 (1) 11/106 (10) Caspofungin 28/242 (12) 1/136 (0.7) 27/106 (25) day survival 141 (47) 75 (43) 66 (53) 0.10 Note. Numbers are no. (%) of patients, unless otherwise indicated. APACHE, Acute Physiologic and Chronic Health Evaluation; *P -values are for the comparison between public and private hospitals; * * Data are available for 53 patients in public hospitals and 51 patients in private hospitals. higher APACHE II score (odds ratio [OR] 1.11, 95% confidence interval (95% CI) , P 0.001), mechanical ventilation (OR 3.00, 95% CI , P 0.03) and liver disease (OR 4.29, 95% CI , P 0.048) were associated with significantly higher 30-day mortality rates. Since the APACHE II score was available for only 104 patients, we ran another multivariate analysis excluding this variable. Multivariate predictors of 30-day mortality using this model were older age (OR 1.03, 95% CI , P 0.001), neutropenia (OR 4.46, 95% CI , P 0.003), mechanical ventilation (OR 4.20, 95% CI , P 0.001), liver disease (OR 3.16, 95% CI , P 0.01), admission to a public hospital (OR 2.81, 95% CI , P 0.001), and receipt of corticosteroids (OR 1.81, 95% CI , P 0.03). Discussion In the present study, we evaluated the epidemiology of candidemia in two types of institutions receiving different sources of funding. We observed that the incidence rates, age, co-morbid conditions, Candida species distribution, antifungal susceptibility patterns, and treatment practices of candidemia differed significantly between these two types of hospitals. We confirmed the high incidence of candidemia in public hospitals reported previously [10], and observed that the incidence in private hospitals was much lower, closer to that reported in studies from the Northern Hemisphere. While a comparison of the incidences of candidemia between these studies is limited by the fact that the reported rates were not risk-adjusted, it is tempting to speculate that

5 42 Colombo et al. Table 2 Etiologic agents of candidemia in public (2) and private hospitals (7). Overall n 300 (%) Public n 174 (%) Private n 126 (%)P value * Candida albicans 103 (34) 61 (35) 42 (33) 0.78 C. parapsilosis 78 (26) 58 (33) 20 (16) C. tropicalis 72 (24) 35 (20) 37 (29) 0.06 C. glabrata 22 (7) 5 (3) 17 (13) C. krusei 10 (3) 5 (3) 5 (4) 0.75 C. guilliermondii 7 (2) 6 (3) 1 (1) 0.25 Other ** * P -values are for the comparison between public and private hospitals. * * Other species were once case each of Candida lusitaniae, C. pelliculosa, C. famata, and C. spp. in public hospitals and one case each of C. lusitaniae, C. famata, C. rugosa and C. pelliculosa in private hospitals. different standards of practice between public and private Brazilian hospitals may account for these differences. Private hospitals in Brazil have standards closer to those of medical centers in developed countries. Another finding that may be explained by these arguments is the lower proportion of candidemia due to C. parapsilosis in private hospitals, with rates closer to those reported in the Northern Hemisphere [10 13]. Candidemia due to C. parapsilosis is thought to be acquired from external sources, and public hospitals would not have optimal conditions available to prepare intravenous solutions and manage intravascular catheters. Patients in private hospitals were older, a fact that likely influenced the proportion of patients with comorbidities that are more frequent in older patients, such as solid tumors and diabetes mellitus. A higher prevalence of these degenerative diseases in the elderly has also been observed in other investigations [14,15]. Another finding observed in the present study was the higher proportion of ICU patients with candidemia in private hospitals, despite the fact that the median APACHE II score was similar in the two groups. This difference is probably related to the shortage of ICU beds in public hospitals, as shown by the similar proportion of patients on mechanical ventilation in the two groups. The proportion of candidemia due to C. glabrata was significantly higher in private hospitals. In addition, C. glabrata isolates from private hospitals were less susceptible to fluconazole. In this regard, the epidemiology of candidemia in private institutions in Brazil was similar to that reported in studies from centers in the United States [16]. Candidemic patients in private hospitals were more likely to have received fluconazole prior to developing candidemia. These data indicate that C. glabrata is an emerging pathogen in private hospitals in Brazil, possibly because of a higher use of fluconazole, as reported elsewhere [17,18]. Of interest, a total of three isolates representative of C. tropicalis (2) and C. parapsilosis (1) were considered to be non-susceptible to fluconazole according to the new breakpoints. Once those isolates exhibit MIC values Table 3 In vitro susceptibility of Candida species to four antifungal agents. MIC * (μg/ml) Species ( N ) Antifungal agent Range MIC 50 MIC 90 Resistant, n (%) * * Candida albicans (103) Amphotericin B Fluconazole Flucytosine Voriconazole C. parapsilosis (78) Amphotericin B Fluconazole Flucytosine (3) Voriconazole C. tropicalis (78) Amphotericin B Fluconazole (3) 5-Flucytosine Voriconazole C. glabrata (22) Amphotericin B Fluconazole (64) 5-Flucytosine Voriconazole (9) C. krusei (10) Amphotericin B Fluconazole (100) 5-Flucytosine Voriconazole *MIC, minimum inhibitory concentration. * *Resistance was defined using the following MIC breakpoints: amphotericin B: 2 μg/ml, fluconazole: 64 μg/ml, flucytosine: 32 μg/ml, voriconazole: 4 μg/ml.

6 C. glabrata is emerging as pathogen in Brazil 43 Table 4 Factors associated with 30-day mortality by univariate analysis of 300 patients with candidemia Survived n 142 Died n 158P value Gender, male: female 71:71 79: Age (years), median (range) 41 (0 89) 63 (0 98) APACHE II score *, median (range) 13 (2 29) 21 (4 38) Type of hospital, public: private 76:66 99: Admission to an ICU 46 (32) 100 (63) Cancer 47 (33) 54 (34) 0.87 Hematological malignancy 15 (11) 13 (8) 0.48 Solid tumor 32 (22) 41 (26) 0.51 Cardiac disease 38 (27) 59 (37) Neurological disease 44 (31) 40 (25) 0.26 Lung disease 31 (22) 46 (29) 0.13 Diabetes mellitus 23 (16) 41 (26) 0.04 Liver disease 9 (6) 26 (16) Renal failure 39 (27) 53 (33) 0.27 Chronic renal failure 26 (18) 16 (10) 0.04 Surgery 77 (54) 72 (45) 0.12 Abdominal surgery 43 (30) 46 (29) 0.80 Parenteral nutrition 34 (24) 43 (27) 0.54 Mechanical ventilation 28 (20) 81 (51) Dialysis 15 (11) 32 (20) 0.02 Neutropenia 9 (6) 19 (12) 0.09 Receipt of corticosteroids 50 (32) 84 (53) Candida species C. albicans 45 (31) 58 (36) 0.38 C. parapsilosis 46 (32) 32 (20) 0.01 C. tropicalis 31 (22) 41 (26) 0.42 C. glabrata 7 (5) 15 (9) 0.13 Primary treatment Fluconazole 78 (55) 52 (33) Amphotericin B deoxycholate 26 (18) 29 (18) 0.99 Lipid formulation of 5 (3) 8 (5) 0.52 amphotericin B Caspofungin 14 (10) 14 (9) 0.75 Note. Numbers are no. (%) of patients, unless indicated otherwise. APACHE, Acute Physiologic and Chronic Health Evaluation. * Data are available for 53 patients in public hospitals and 51 patients in private hospital. substantially higher than the epidemiological cutoff of the two species, they may represent strains starting to express mechanisms of resistance but may still respond to higher doses of fluconazole [9]. Otherwise, the exposure to azoles will likely increase the MICs. Further studies are necessary to consolidate the best clinical approach when facing patients infected by such strains. As expected, amphotericin B deoxycholate was the most common drug used to treat candidemic episodes in public hospitals. However, we were surprised that fluconazole was the leading agent for primary treatment of candidemia in private institutions, given the availability of echinocandins and lipid formulations of amphotericin B in these hospitals. In our analysis of prognostic factors, having a higher APACHE II score, being on mechanical ventilation and having liver disease were associated with higher 30-day mortality. However, because data on the APACHE II score were only available for one-third of patients, we ran another multivariate analysis without the APACHE II score. In this analysis, factors related to the host s immune status (older age, neutropenia and receipt of corticosteroids) were identified as prognostic variables. Surprisingly, being treated in a private hospital was associated with lower 30-day mortality despite the fact that patients in private hospitals were older, more likely to be admitted to an ICU and less likely to be infected by C. parapsilosis. Reasons for these differences may be multifactorial, including the shortage of ICU beds in public hospitals. Finally, considering that patients treated in these two types of hospitals have substantial differences in their economic and social backgrounds, it is possible that these differences might have an impact on patient outcomes. In conclusion, we showed that in Brazil there are substantial differences in candidemia treated in medical centers with different mechanisms of funding, and that C. glabrata is an emerging pathogen in private institutions. In this setting, fluconazole should not be considered a safe option for primary therapy of candidemia before the reliable identification of the pathogen. Acknowlegements We are very grateful to United Medical and Gilead that provided unconditional funding to partially support this study. Declaration of interest : Dr Colombo and Dr Nucci have received research and educational grants from Astellas, MSD and Pfizer in the last two years. The other authors report no conflicts of interest. 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