Species distribution and drug susceptibilities of Candida isolates in TSARY 2010

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Species distribution and drug susceptibilities of Candida isolates in TSARY 2010

☆

Yun-Liang Yang

a,b

, Hui-Ting Chen

b,c

, Chih-Chao Lin

c

, Wen-Li Chu

c

, Hsiu-Jung Lo

c,d,

⁎

and TSARY Hospitals

a

Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC

b

Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan, ROC

c

National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan, ROC

d_{School of Dentistry, China Medical University, Taichung, Taiwan, ROC}

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 14 November 2012 Received in revised form 6 March 2013 Accepted 6 March 2013

Available online 8 April 2013 Keywords:

Antifungal Candida species Drug susceptibility

Susceptibilities to antifungal drugs of 1083 Candida isolates collected in Taiwan Surveillance of Antimicrobial Resistance of Yeasts in 2010 were determined. There were 422 (39%) C. albicans, 270 (24.9%) C. tropicalis, 258 (23.8%) C. glabrata, 87 (8%) C. parapsilosis, 18 (1.7%) C. krusei, and 28 (2.6%) of 13 other species. In the present study, we have applied species-specific clinical breakpoints for common species and epidemiological cutoff values for rare species. We found that majority of isolates were susceptible to tested drugs. A total of 15, 3, 2, and 0 isolates were not susceptible to fluconazole, voriconazole, amphotericin B, and anidulafungin, respectively. We found that three of the fourfluconazole non-susceptible C. albicans isolates were resistant to voriconazole. Hence, there is an issue of cross-resistance among azole-type drugs.

1. Introduction

The increased prevalence of fungal infections is due to the expanding number of immunocompromised patients, increased use of invasive medical devices, and extensive use of broad spectrum antibiotics (Pappas et al., 2009; Pfaller and Diekema, 2010; Yang and Lo, 2001). Hence, the epidemiology of fungal infections has become important in the last few decades. Candida species are the most frequently isolated fungal pathogens responsible for signiﬁcant morbidity and mortality (Chen et al., 2006; Chen et al., 2011; Cheng et al., 2004; Pfaller and Diekema, 2007; Warnock, 2007; Yang et al., 2010) and they are leading causes of fungal infections in the world (Chuang et al., 2010; Colombo et al., 2006; Edwards, 1995; Pfaller and Diekema, 2010; Ruan and Hsueh, 2009; Warnock, 2007; Wisplinghoff et al., 2004; Yang et al., 2010). Among the 30 to 40 Candida species causing diseases in human (Johnson, 2009; Richardson, 1991), C. albicans, C. tropicalis, C. glabrata, C. parapsilosis, and C. krusei are the ﬁve most common species causing candidiasis (Almirante et al., 2005; Colombo et al., 2006; Pfaller and Diekema, 2010; Yang et al., 2010).

Azoles (ﬂuconazole and voriconazole), echinocandins (anidula-fungin, caspo(anidula-fungin, and micafungin), and polyene (amphotericin B) are the three classes of drugs most commonly prescribed for treating systemic fungal infections. The emergence of drug-resistant fungal pathogens is a growing concern in medical settings (Sanglard and Odds, 2002; White et al., 1998; Yang and Lo, 2001).

In 1999 and again in 2002 and 2006, three national surveys, Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY), have been conducted. The drug susceptibilities of the 632, 909, and 964 Candida isolates collected in 1999, 2002, and 2006 respectively, have been determined (Yang et al., 2004; Yang et al., 2005a; Yang et al., 2008). Among them, 0.5% (1999), 2.5% (2002), and 1.8% (2006) were with amphotericin B MICs≧2 mg/L after 48-hour (h) incubation. There were 8.4%, 1.9%, and 17.1% of the isolates in 1999, 2002, and 2006, respectively, withﬂuconazole MICs ≧64 mg/L after 48 h (Yang et al., 2004; Yang et al., 2005a; Yang et al., 2008).

Among the phenomena associated with resistance, “trailing” describes the reduced but persistent growth that some isolates exhibit at drug concentrations above the MIC in broth dilution tests with azoles (Arthington-Skaggs et al., 2002; Lee et al., 2004). In TSARY 2006 (Yang et al., 2008), we have found, among the isolates with fluconazole MICs ≧64 mg/L after incubation for 48 h, 72.7% (96/132) C. tropicalis, 70% (7/10) C. albicans, and 44.4% (4/9) C. glabrata isolates exhibited the trailing phenomenon, whereas none of the C. krusei isolates did. Epidemiological cutoff values for Candida species and species-specific clinical breakpoints for flucon-azole (Pfaller et al., 2010), voriconazole (Pfaller et al., 2011a), and the echinocandins (Pfaller et al., 2011b) for common species have been established to minimize the trailing and to support a shorter time for reporting MICs. In the present study, we have conducted a follow-up TSARY in 2010. We have applied newly defined species-specific breakpoints for fluconazole, voriconazole, and echinocan-dins for common species and epidemiological cutoff values for the rare species (Pfaller and Diekema, 2012) to investigate drug susceptibilities of 1083 Candida isolates.

☆ Declaration of interest: Authrors declare that they have no conﬂict of interest. ⁎ Corresponding author. Tel.: +886-37-246-166x35516; fax: +886-37-586-457.

E-mail address:[email protected](H.-J. Lo).

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Diagnostic Microbiology and Infectious Disease

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2. Materials and methods 2.1. Organisms and media

Yeast isolates were collected from the 23 hospitals participating in TSARY from July 1 to September 30 in 2010 (Yang et al., 2013). Each hospital was asked to submit all yeast pathogens from sterile sites and thefirst ten C. albicans and 40 C. albicans yeast isolates from non-sterile sites. In principle, only one isolate was accepted from each specimen. Nevertheless, when there were multiple species isolated from one specimen, one isolate from each species was analyzed. All the collected isolates were stored frozen at−70 °C in vials containing 50% glycerol. After their arrival at the laboratory at National Health Research Institutes (NHRI), these isolates werefirst sub-cultured on sabouraud dextrose agar (SDA, BBL, Becton Dickinson Cockeysville, MD, USA) to assess the purity and identification. When there were more than one species from one frozen vial, the cells from the vial will be streaked onto CHROMagar Candida medium (BBL, Becton Dickin-son Cockeysville, MD, USA) and pure single colony of each morphotype was labeled and stored in vials containing 50% glycerol at−70 °C awaiting further analyses.

2.1.1. Identiﬁcation

The identifications of the isolates were first performed by the contributing TSARY hospitals and then reassessed in the laboratory at NHRI. The procedure for the identification was established previously (Lo et al., 2001; Yang et al., 2013). In brief, all isolates identified as C. albicans by the TSARY hospitals were subjected to germ tube assay in RPMI medium 1640 (Gibco BRL, 31800–022) containing 10% fetal bovine serum (GibcoBRL, US-628531). The germ tube-positive iso-lates failing to grow at 42 °C were identified by sequencing their ribosomal DNA (rDNA) (Leaw et al., 2007). All isolates identified as non-C. albicans by the TSARY hospitals were tested with VITEK 2 (bioMérieux, Marcy l'Etoile, France). The sequences of the internal transcribed spacer (ITS) and/or the D1/D2 regions of rDNA were used for species identification when one of the followings occurred: the identi_{fication probability of the VITEK 2 was less than 85%, the} identification of an organism was inconsistent between the hospital and the NHRI laboratory, and when uncommon species were reported. The ITS regions were ampli_{fied by the primers ITS1,}

5′-TCCGTAGGTGAACCTGCGG-3, and ITS4 5

′-TCCTCCGCTTATTGATATGC-3′, and the D1/D2 regions were ampliﬁed by the primers NL1

5′-GCATATCAATAAGCGGAGGAAAAG-3′ and NL4

5′-GGTCCGTGTTTCAA-GACGG-3′ (Leaw et al., 2007). In addition, to determine whether Candida nivariensis isolates were recovered in the present study, we have streaked out all isolates identiﬁed as C. glabrata onto CHROMagar Candida medium (Alcoba-Florez et al., 2005b). The rDNA sequences of the cells showing white color on the CHROMagar Candida medium were then determined (Alcoba-Florez et al., 2005a).

2.1.2. Antifungal susceptibility testing

The MICs of the four antifungal agents were determined by the in vitro antifungal susceptibility testing established in our laboratory (Yang et al., 2004), according to the guidelines of M27-A3 recom-mended by the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2008). RPMI medium 1640 (31800–022, Gibco BRL) was used for growth and dilution of the yeast. Strains from American Type Culture Collection (ATCC), including C. albicans (ATCC 90028), C. krusei (ATCC 6258), and C. parapsilosis (ATCC 22019), were used as the standard controls. The growth of each isolate was measured by the Biotrak II plate spectrophotometric reader (Amersham Biosciences, Biochrom, Cambridge, England) after incubation at 35 °C for 24 h.

Standard powders of amphotericin B, kindly provided by Bristol Myers Squibb, and anidulafungin,fluconazole, and voriconazole, by Pfizer, were dissolved in dimethal sulfoxide (DMSO). The final concentrations of the anidulafungin and voriconazole were from

0.0156 mg/L to 8 mg/L, amphotericin B, 0.0313–16 mg/L, and

ﬂuconazole, 0.125–64 mg/L.

The MICs were defined as the concentration of drugs capable of reducing the turbidity of cells to greater than 50% for anidulafungin, fluconazole, and voriconazole and completely inhibiting cell growth for amphotericin B. The epidemiological cutoff values for amphoter-icin B after 24-h incubation was 2 mg/L for all species (Pfaller et al., 2012). The newly defined species-specific breakpoints for the five common Candida species, C. albicans, C. krusei, C. glabrata, C. parapsilosis, and C. tropicalis were applied in the present study (Pfaller and Diekema, 2012). Forfluconazole, the clinical breakpoints of C. albicans, C. tropicalis, and C. parapsilosis are as following: MICs≤2 mg/L were considered to be susceptible,≥8 mg/L resistant, and 4 mg/ L susceptible-dose dependent (SDD); for C. glabrata, MICs≤32 mg/L were SDD,≥64 mg/L resistant. For voriconazole, the clinical break-points of C. albicans, C. tropicalis, and C. parapsilosis were MICs≤0.125 mg/L susceptible,≥1 mg/L resistant, and 0.25–0.5 mg/L intermediate. For C. krusei, MICs≤0.5 mg/L were susceptible, ≥2 mg/L resistant, and 1 mg/L intermediate. For anidulafungin, the clinical breakpoints of C. albicans, C. krusei, and C. tropicalis were MICs ≤0.25 mg/L susceptible, ≥1 mg/L resistant, and 0.5 mg/L intermediate. For C. parapsilosis and C. guilliermondii, the breakpoints were changed to

MICs ≤2 mg/L susceptible, ≥8 mg/L, resistant, and 4 mg/L,

intermediate. For C. glabrata, they were ≤0.125 mg/L susceptible, ≥0.5 mg/L resistant, and 0.25 mg/L intermediate. For the species of which clinical breakpoints have not been established, we applied epidemiological cutoff values instead (Pfaller and Diekema, 2012). The MICs of 50% and 90% of the total population were deﬁned as MIC50

and MIC90,respectively.

2.1.3. Database and analysis

The database for this study contained the characteristic informa-tion of each submitted isolate: hospital origin, locainforma-tion and type of the hospital, identification and source of the isolate. The statistical significance of the differences in frequencies and proportions was determined by the chi-square test with Mantel-Haenszel correction or fisher exact with 2-tailed correction.

3. Results

3.1. Distribution of Candida species

In the present study, we collected theﬁrst ten C. albicans and 40 non-C. albicans yeast isolates from non-sterile sites. Therefore, the prevalence of C. albicans was underestimated. Even so, it was still the most frequently isolated species in the present study, accounting for 39% of the total isolates. Candida tropicalis (270, 24.9%) and C. glabrata (258, 23.8%) were the two most frequently isolated non-C. albicans Candida species followed by C. parapsilosis (87, 8%), C. krusei (18,1.7%), C. guilliermondii (7, 0.6%), and others (21, 1.9%) (Table 1). When classiﬁed according to the sources, there were 489 (45.2%) isolates from urine, 213 (19.7%) from blood, 141 (13%) from sputum, 53 (4.9%) from catheter tip, 40 (3.7%) from wound, 33 (3%) from ascites, 28 (2.6%) from pus, 22 (2%) from bronchial washing, and 64 (5.9%) from other 23 different sites. Candida albicans was still the most common cause for candidemia (103, 48.4%), followed by C. tropicalis (41, 19.2%), C. parapsilosis (35, 16.4%), C. glabrata (22, 10.3%), C. krusei (2, 0.9%), and others (10, 4.7%).

3.2. Susceptibilities to amphotericin B and anidulafungin of Candida species

The range of amphotericin B MICs of the 1083 isolates was from 0.0131 to 2 mg/L (Table 2). The MIC50and MIC90were 0.25 and 0.5

mg/L, respectively. Only two C. albicans isolates were with amphoter-icin B MIC_{≧2 mg/L. The range of anidulafungin MICs was from 0.0156}

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to 2 mg/L (Table 2). The MIC50and MIC90were 0.0156 and 0.0625

mg/L, respectively. All isolates were susceptible to anidulafungin. 3.3. Susceptibilities to azoles of Candida species

The range ofﬂuconazole MICs of the 1083 isolates was from 0.125 to 64 mg/L (Table 2). The MIC50 and MIC90 were 0.25 and 1 mg/L,

respectively. There were two C. albicans and one C. parapsilosis with MIC ≧8 mg/L and two C. glabrata with MICs ≧64 mg/L, considered as resistant. The MIC50and MIC90of some species, such as C. glabrata (1 and

4 mg/L), C. krusei (8 and 16 mg/L), and C. guilliermondii (1 and 4 mg/L) were higher than others. The range of MICs to voriconazole was from 0.0125 to 8 mg/L (Table 2). The MIC50and MIC90were 0.0156 and

0.0313 mg/L, respectively. There were three C. albicans with 8 mg/L voriconazole MICs, considered as resistant.

4. Discussion

In the present study, we have determined the distribution of species and susceptibilities to antifungal drugs of 1083 Candida

isolates collected in TSARY 2010. The distribution of species according to body sites in the present study was similar to that of the three previous surveys in 1999, 2002, and 2006 (Yang et al., 2004; Yang et al., 2005a; Yang et al., 2008). Interestingly, the ratio of isolates from blood in the present study was higher than that in the TSARY surveys in 2002 (13.9% P = 0.0006 ) (Yang et al., 2005a) and 2006 (15% P = 0.006) (Yang et al., 2008). Candida albicans was still the most common cause for candidemia (48.4%) in the present study, consistent with other reports (Cheng et al., 2004; Peman et al., 2005; Pfaller and Diekema, 2007; Yang et al., 2010). The distribution of C. parapsilosis in human body sites was different from other species because it has a broader distribution in the environment and can be isolated from various non-human sources (Abi-Said et al., 1997; Hedderwick et al., 2000; Levin et al., 1998). In the present study, there were 87 isolates of C. parapsilosis, about 8% of the 1083 isolates. However, they comprised 16.4% of the 213 blood isolates, consistent with our previous report (Yang et al., 2005a). A major proportion (40.2%) of those 87 isolates was from blood, which may have resulted from their capability to adhere to catheters. Overall, C. parapsilosis has accounted for 3% to 27% of the fungemia cases in other studies (Pfaller et al., 1999; Pfaller et al., Table 1

Distribution of 1083 Candida isolates from different body sites.

C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei C. guilliermondii Other 12 spp Total

Urine 132 137 (50.7)a _{200 (77.5)}_⁎ _{14 (16.1)}_⁎ _{3 (16.7)} ₀ _{3 (14.3)} _{489 (45.2)} Blood 103 41 (15.2) 22 (8.5)⁎ 35 (40.2)⁎ 2 (11.1) 2 (28.6) 8 (38.1) 213 (19.7) Sputum 69 48 (17.8)⁎ 11 (4.3)⁎ 1 (1.1)⁎ 7 (38.9)⁎ 2 (28.6) 3 (14.3) 141 (13) Tip 24 14 (5.2) 5 (1.9)⁎ 7 (8) 1 (5.6) 0 2 (9.5) 53 (4.9) Wound 17 7 (2.6) 4 (1.6) 9 (10.3)⁎ 0 2 (28.6) 1 (4.8) 40 (3.7) Ascites 23 3 (1.1) 6 (2.3) 0 0 0 1 (4.8) 33 (3) Pus 17 5 (1.9) 1 (0.4) 4 (4.6) 1 (5.6) 0 0 28 (2.6) Bronchial washing 11 7 (2.6) 2 (0.8) 0 1 (5.6) 0 1 (4.8) 22 (2) other 23 sites 26 8 (3) 7 (2.7) 17 (19.5) 3 (16.7) 1 (14.3) 2 (9.5) 64 (5.9) Total 422 270 258 87 18 7 21 1083 a

Number of isolate (percentage in the same species).

⁎ P ≦ 0.05, comparison of percentage for each species/source group vs. percentage in total population.

Table 2

Antifungal susceptibilities of 1083 Candida spp.

MIC (mg/L) C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei C. guilliermondii Others Total

Total 422 270 258 87 18 7 21 1083 Amphotericin B 0.5 402 264 236 86 17 7 20 1032 1 18 6 22 1 1 0 1 49 2 2 0 0 0 0 0 0 2 MIC50 0.25 0.25 0.5 0.25 0.25 0.125 0.125 0.25 MIC90 0.5 0.5 0.5 0.25 0.5 0.5 0.25 0.5 Anidulafungin ≦ 0.125 422 270 256 11 18 1 18 996 0.25 0 0 2 9 0 1 2 14 0.5 0 0 0 42 0 3 1 46 1 0 0 0 20 0 2 0 22 ≧2 0 0 0 5 0 0 0 5 MIC50 0.0156 0.0156 0.0313 0.5 0.0313 0.5 0.0313 0.0156 MIC90 0.0156 0.0313 0.0313 1 0.0625 1 0.25 0.0625 Fluconazole ≦ 2 418 269 232 86 1 6 17 1029 4 2* 1* 15 0 0 1* 4* 23 8 0 0 0 1# 9 0 0 10 16 2# 0 7 0 8 0 0 17 32 0 0 2* 0 0 0 0 2 ≧64 0 0 2# 0 0 0 0 2 MIC50 0.125 0.25 1 0.25 8 1 0.5 0.25 MIC90 0.25 0.25 4 0.5 16 4 4 1 Voriconazole ≦ 0.125 419 270 248 87 18 7 21 1070 0.25 0 0 8 0 0 0 0 8 1 3# 0 2 0 0 0 0 5 MIC50 0.0156 0.0156 0.0156 0.0156 0.125 0.0313 0.0156 0.0156 MIC90 0.0156 0.0313 0.0313 0.0156 0.125 0.0625 0.0625 0.0313

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2000; Warnock, 2007; Yang et al., 2004). The majority of C. glabrata (77.5%) was isolated from urine samples, consistent with previous reports that C. glabrata is second only to C. albicans as a cause of candiduria (Yang et al., 2006; Yang et al., 2011).

According to the new breakpoints, majority of isolates collected in TSARY 2010 were susceptible to all tested drugs. A total of 15, 3, and 2 isolates were not susceptible tofluconazole, voriconazole, and amphotericin B, respectively. Previously, we have found an associ-ation between fluconazole susceptibility and genetic relatedness among C. tropicalis isolates (Chou et al., 2007; Wang et al., 2007). We have reported that two dominant C. tropicalis clones, DST140 and DST98, with fluconazole MICs ≧64 mg/L circulated in Taiwan (Li et al., 2009). Recently, we have found that genetically related C. tropicalis withfluconazole MICs ≧64 mg/L were isolated from the human hosts and environmental samples (Yang et al., 2012). In the present study, of the 270 C. tropicalis isolates, none was resistant to fluconazole and only one (0.4%) was SDD. Thus, we are in the process of genotyping the C. tropicalis isolates collected in TSARY 2010 to further assess the association of DST140 and DST98 with fluconazole resistance.

When comparisons were made among species, there were distinct variations in thefluconazole susceptibility. Only one C. krusei was with MICsb8 mg/L. In contrast, only one C. parapsilosis was with MICs ≧8 mg/L. These results were consistent with previous reports that C. krusei had high and C. parapsilosis had lowfluconazole-resistant rates (Akova et al., 1991; Pfaller et al., 2000; Pfaller et al., 2004; Yang et al., 2005b; Yang et al., 2008). It has been reported that continuous exposure to azoles seems to have a major impact on selecting fluconazole-resistant Candida species, especially C. glabrata ( Kon-toyiannis, 2002). In the present study, 1.6% of the C. glabrata isolates

were with ﬂuconazole MICs ≧32 mg/L. Therefore, our previous

observation that low proportion of patients receiving prior ﬂucona-zole treatments (Cheng et al., 2005) may explain the low ﬂuconazole-resistant C. glabrata in Taiwan than other areas (Fleck et al., 2007; Pfaller and Diekema, 2007).

In the present study, we found that three of the four isolates with ﬂuconazole MICs ≧4 mg/L were resistant to voriconazole. As reported previously (Yang et al., 2004a), thisﬁnding emphasizes the important issue of cross-resistance phenomenon among azole drugs.

Acknowledgments

We thank all 23 TSARY participating hospitals. They are Asia East Memorial Hospital, Buddhist Tzu-Chi General Hospital, Cathay General Hospital, Chang Gung Memorial Hospital at Kaohsiung, Chang-Hwa Christian Hospital, Cheng Ching Hospital, Chiayi Christian Hospital, Chung Shan Medical Dental College Hospital, Hua-Lien Hospital, DOH, the Executive Yuan, Kaohsiung Military Hospital, Kuan-Tien General Hospital, Lo-Hsu Foundation Inc. Lo-Tung Poh Ai Hospital, Miin Sheng General Hospital, National Cheng Kung Univer-sity Hospital, Shalu Tung's Memorial Hospital, Show Chwan Memorial Hospital, Sin-Lau Christian hospital, St. Mary Hospital, Taipei Municipal Hoping Hospital, Tri Service General Hospital, Veterans General Hospital-Taichung, Veterans General Hospital-Kaohsiung, and Zen Ai General Hospital. We thank Bristol Myers Squibb for providing amphotericin B and Pfizer for providing anidulafungin, fluconazole, and voriconazole. This work was supported in part by grants 00A1-ID-PP-04-014 and 01A1-IV-PP-04-014 from the National Health Research Institutes. Authors declare that they have no con_flict of interest.

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