Hideyo Yamaguchi 1, Katsuhisa Uchida 1, Yayoi Nishiyama 1 and the Japan Antifungal Surveillance Program Group

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1 Medical Mycology Research Vol. 3 No. 1: 17-26, 2012 ISSN Original Articles Species distribution and in vitro susceptibility to three antifungal triazoles of clinical Candida isolates from a five-year nation-wide survey in Japan Hideyo Yamaguchi 1, Katsuhisa Uchida 1, Yayoi Nishiyama 1 and the Japan Antifungal Surveillance Program Group 1 Teikyo University Institute of Medical Mycology, Tokyo, Japan ABSTRACT We collected 1,486 clinical isolates of Candida species over a five-year period (2001 to 2005) in Japan and examined their species distribution and patterns of susceptibility to three antifungal azoles (fluconazole, itraconazole, and voriconazole). The most frequently isolated species was C. albicans, being followed by C. glabrata, C. tropicalis, C. parapsilosis and C. krusei, in this order, at every survey. Isolation rates of C. albicans were 58.6%, 57.3% and 61.6% in the , 2003 and 2005 surveys, respectively. Azole resistance was scarcely or not detected among isolates of C. albicans and C. parapsilosis. Overall resistance rates to fluconazole, itraconazole and voriconazole were: 4.6, 4.6 and 0.9%, respectively, among C. glabrata isolates; 2.3, 2.3 and 0.8%, respectively, among C. tropicalis isolates; and 59, 6.9 and 6.9%, respectively, among C. krusei isolates. An increase in azole resistance over time was only seen with fluconazole resistance of C. krusei. Voriconazole-resistant Candida species isolates exhibited complete cross-resistance to fluconazole and itraconazole. Keywords: antifungal susceptibility, Candida, species distribution, triazoles Introduction With the increase in size of the at-risk population, the prevalence of invasive fungal infections has significantly increased during the last 30 years 1,2). Over the time period, Candida species are the most frequently isolated fungal pathogens causing morbidity and mortality in immunocompromised and hospitalized patients 3-5). Although Candida albicans remains the most frequent cause of invasive Candida infections, longitudinal surveillance programs in the United States and Europe have detected an increase in the prevalence of invasive infections caused by non-albicans Candida, such as Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis 5-11). Antifungal susceptibility patterns vary among different Candida species and treatment failures due to drug resistance have been reported 12). The latter may be related to the increase of infections caused by nonalbicans Candida species which are less susceptible or resistant to fluconazole 13). Hence, information on the distribution and resistance profiles of various Candida species involved in infections is important to establish empirical treatment protocols, to guide the selection of antifungal therapy and to evaluate the potential impact of newer antifungal agents 14,15). Most global surveillance programs suggest that substantial differences exist in the distribution and the antifungal susceptibility of Candida species over time and geographic area 10,16,17). This underscores the need for continued surveillance in the respective areas or countries to monitor trends in pathogen distributions and drug susceptibilities

2 Species distribution and in vitro susceptibility to three antifungal triazoles of clinical Candida isolates from a five-year nation-wide survey in Japan To date, in Japan only one national surveillance study of Candida bloodstream isolates has been conducted, in which species distribution and the in vitro susceptibility of isolates from 2001 to 2002 to five existing antifungal agents, including voriconazole and micafungin, was determined 18). However, no prospective surveillance study of the activity of these agents against Candida isolates has been reported. In order to monitor changing trends in the in vitro antifungal susceptibility patterns of Candida isolates, along with those of other pathogenic yeasts and filamentous fungi, we undertook a longitudinal nationwide surveillance program involving a variety of community and academic medical institutions in Japan during 2001 to The data collected and analyzed in individual surveillances were published elsewhere 19-21). In the present study, we analyzed retrospectively the species distribution of Candida isolates and their profiles of susceptibility to three azole antifungal agents, viz., fluconazole, itraconazole and voriconazole, and resistance rates over a five-year surveillance period. In addition, cross-resistance profiles of the Candida isolates were also studied. Materials and Methods Collection of organisms. Candida and non-candida yeast isolates were collected from 14 hospitals in Japan participating in the Japan Antifungal Surveillance Program (JASP). Surveillances were performed three times between June 2001 and June 2005 in 2001 through 2002 ( ), 2003 and 2005 as the first, second and third surveillances, respectively. Twelve of the 14 hospitals participated in all the three surveillances. Most institutions were academic medical centers and several were community or nonacademic medical centers distributing widely in various districts of Mainland and Kyushu Island of Japan. Each participating institution was asked to submit all pathogenic or opportunistic yeast isolates from blood and other sterile sites and the first 50 to 100 isolates from non-sterile sites that were collected within a prescribed one month (mostly June) for identification and susceptibility testing in every surveillance. Only the initial isolate from each patient was submitted for evaluation. All Candida species isolates considered pathogens from all clinical specimens, including blood, urine, normally sterile body fluids (e.g., pleural and peritoneal fluids), autopsied deep tissues, sputum, lower respiratory tract specimens (e.g., bronchoalveolar lavage and endotracheal aspirate), catheter, soft tissue and wound and related exudates were collected and evaluated. The identification of yeast isolates was performed locally in accordance with each study site s routine methods. The majority of the study sites employed one or more commercially available yeast identification systems (e.g., API, Vitek, and/or MicroScan) and the remainder used the classical biochemical and morphological methods. Unidentified isolates were sent to the central laboratory in Teikyo University Institute of Medical Mycology where they were subjected to identification by the molecular methods. Antifungal susceptibility testing. In vitro antifungal susceptibility testing to fluconazole, itraconazole and voriconazole was performed in accordance with the reference broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI) M27- A2 22). Preparations of fluconazole and voriconazole were provided by Pfizer Japan Inc. (Tokyo, Japan) and a preparation of itraconazole was the product of Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The RPMI medium 1640 (Sigma-Aldrich Co.) buffered with MOPS (Sigma-Aldrich Co.) was used for testing. The growth of each isolates was measured after incubation at 35 C for 24 and 48 h. The MIC of all the three test antifungal azoles against all Candida isolates was defined as their concentration capable of reducing the turbidity of cultures to greater than 50% relative to drug-free controls. The interpretive susceptibility criteria used for fluconazole, itraconazole and voriconazole were those specified by the CLSI 23). The MICs of 50% and 90% of the total population were defined as MIC 50 and MIC 90, respectively. All statistical analyses were performed using SPSS 13.0 for Windows (SPSS, Chicago, USA). The chisquare test was used to analyze differences in distribution among variables as well as in frequencies and proportions. Values of P 0.05 were considered statistically significant. Results Distribution of Candida species. A total of 1,

3 Medical Mycology Research Vol. 3 (No. 1) 2012 Table 1. Species distribution of Candida isolates over a five-year period, to Isolates collected and tested by year or period of: Species No. (% of total) No. (% of total) No. (% of total) No. (% of total) C. albicans 326(58.6) 223(57.3) 333(61.6) 882(59.4) C. glabrata 131(23.6) 91(23.4) 107(19.8) 329(22.1) C. tropicalis 57(10.3) 29(7.5) 46(8.5) 132(8.9) C. parapsilosis 20(3.6) 27(6.9) 27(5.0) 74(5.0) C. krusei 8(1.4) 7(1.8) 14(2.6) 29(2.0) C. guilliermondii 4(<1.0) 5(1.3) 8(1.5) 17(1.1) C. lusitaniae 6(1.1) 4(1.0) 4(<1.0) 14(<1.0) C. dubliniensis 1(<1.0) 2(<1.0) 1(<1.0) 4(<1.0) C. kefyr 2(<1.0) 0 0 2(<1.0) C. inconspicua 1(<1.0) 0 0 1(<1.0) C. rugosa 0 1(<1.0) 0 1(<1.0) C. auris sp. nov a 0 0 1(<1.0) 1(<1.0) Total 556(100) 389(100) 541(100) 1,486(100) a Described by Satoh et al. 24) as a novel species of Candida. Table 2. Trends in in vitro susceptibility to three antifungal azoles among isolates of the most frequent five Candida species over a five-year period. Species (No. of isolates: /2003/2005) C. albicans (326/223/333) C. glabrata (131/91/107) C. tropicalis (57/29/46) C. parapsilosis (20/27/27) C. krusei (8/7/14) Antifungal azoles MIC (μg/ml) against isolates collected in; MIC 50 MIC 90 MIC 50 MIC 90 MIC 50 MIC 90 Fluconazole Itraconazole Voriconazole Fluconazole Itraconazole Voriconazole Fluconazole Itraconazole Voriconazole Fluconazole Itraconazole Voriconazole Fluconazole < Itraconazole Voriconazole Candida isolates were collected and tested in three surveys between 2001 and 2005 (Table 1). Twelve different species of Candida, including a novel species Candida auris sp. nov. 24), were isolated, of which C. albicans was the most common with an overall isolation rate of 59.4%, with being followed by C. glabrata (22.1%), C. tropicalis (8.9%), C. parapsilosis (5.0%) and C. krusei (2.0%). Other 7 species, including C. guilliermondii, C. lusitaniae and C. dubliniensis, showed lower frequencies and accounted for 2.7% of the total isolates. No appreciable change in the isolation rate of C. albicans or any non-albicans Candida species was noted at three surveys over the five-year period. Trends in the antifungal susceptibilities in terms of the MIC 50 and MIC 90 among the most frequent Candida species over the entire surveillance period. Table 2 shows MIC 50 and MIC 90 values of the three azole antifungal agents, viz., fluconazole, itraconazole and voriconazole, against isolates of the five most frequently Candida species collected at three surveys in , 2003 and Both MIC 50 and MIC 90 of all the three azoles against C. albicans were lower than corresponding values against C. glabrata, C. tropicalis, C. parapsilosis and C. krusei and maintained almost constant levels over the fiveyear period. By contrast, MIC 50 and MIC 90 of the three azoles against C. krusei showing the highest values among the five Candida species appeared to be gradually elevated, reaching a 4- or 8-fold increase during the surveillance period. For C. glabrata, C. tropicalis and C. parapsilosis, although MIC 50 and/or MIC 90 values of fluconazole and voriconazole also increased 4- or 8-fold, no global shift in itraconazole MIC distribution was observed over time

4 Species distribution and in vitro susceptibility to three antifungal triazoles of clinical Candida isolates from a five-year nation-wide survey in Japan Table 3. Trends in in vitro resistance to three antifungal azoles among isolates of the most frequent five Candida species over a five-year period, using the cutoff values for resistance (R) according to CLSI M27- S3 23). Species Antifungal No. of isolates (%R) (No. of isolates: /2003/2005) azoles Total C. albicans Fluconazole 2(0.6) 0 0 2(0.2) Itraconazole 2(0.6) 0 0 2(0.2) (326/223/333) Voriconazole 2(0.6) 0 0 2(0.2) C. glabrata Fluconazole 5(3.8) 2(2.2) 8(7.5) 15(4.6) Itraconazole 4(3.1) 1(1.1) 10(9.3) 15(4.6) (131/91/107) Voriconazole 1(0.3) 0 2(1.9) 3(0.9) C. tropicalis Fluconazole 1(1.8) 0 2(4.3) 3(2.3) Itraconazole 1(1.8) 1(3.5) 1(2.2) 3(2.3) (57/29/46) Voriconazole 0 0 1(2.2) 1(0.8) C. parapsilosis Fluconazole Itraconazole (20/27/27) Voriconazole C. krusei Fluconazole 0 4(57) 13(93) 17(59) Itraconazole 0 0 2(14) 2(6.9) (8/7/14) Voriconazole 0 0 2(14) 2(6.9) All species Fluconazole 8(1.5) 6(1.6) 23(4.4) 37(2.6) Itraconazole 7(1.3) 2(0.5) 13(2.5) 22(1.5) (542/377/527) Voriconazole 3(0.6) 0 5(0.9) 8(0.6) Trends in the antifungal resistance among the most frequent Candida species over the entire surveillance period. Overall, 37 (2.6%), 22 (1.5%) and 8 (0.6%) isolates of the five commonest species (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei) were resistant to fluconazole, itraconazole and voriconazole, respectively (Table 3). The numbers of isolates of specific species resistant to fluconazole, itraconazole and voriconazole were as follows: C. albicans, each two (0.2%) isolates; C. glabrata, 15 (4.6%), 15 (4.6%) and three (0.9%) isolates, respectively; C. tropicalis, three (2.3%), three (2.3%) and one (0.8%) isolates, respectively; and C. krusei, 17 (59%), two (6.9%) and two (6.9%) isolates, respectively. None of C. parapsilosis isolates was resistant to any antifungal azole. There was not a substantial change in the rates of resistance to any azoles over time among the isolates of C. albicans, C. glabrata and C. tropicalis. However, between 2001 and 2005, the proportion of C. krusei isolates to itraconazole and voriconazole increased from 0% to 14% and, in particular, the proportion of the fluconazole resistant isolated steadily increased from 0% to 93%, although not a statistical significance. four major Candida species (C. albicans, C. glabrata, C. tropicalis and C. krusei) to the other two antifungal azoles. It is noted that all (two) of two fluconazole-resistant C. albicans isolates were also resistant to both itraconazole and voriconazole. By contrast, only a small proportion (two of 17) of fluconazole-resistant isolates of C. krusei was resistant to itraconazole and voriconazole. In respect with fluconazole-resistant isolates of C. tropicalis and C. glabrata, more than half of the former isolates (two of three) and the latter (10 of 15) were resistant to itraconazole, while smaller numbers of C. tropicalis isolates (one of three) and those of C. glabrata isolates (three of 15) were resistant to voriconazole. Data on susceptibility patterns for 22 itraconazole-resistant isolates showed that all of two C. albicans isolates and two C. krusei isolates were also resistant to both fluconazole and voriconazole, and that 10 and three of 15 C. glabrata isolates and two and one of three C. tropicalis isolates were resistant to fluconazole and voriconazole, respectively. All of eight voriconazole-resistant isolated consisting of two C. albicans isolates, three C. glabrata isolates, one C. tropicalis isolate and two C. krusei isolates were resistant to both fluconazole and itraconazole. Cross-resistance to antifungal azoles among Candida isolates. Table 4 summarizes susceptibilities of each of fluconazole-resistant isolates, of the Discussion The continual ARTEMIS DISK global antifungal

5 Medical Mycology Research Vol. 3 (No. 1) 2012 Table 4. In vitro azole cross-resistance among isolates of the four Candida species including one or more resistant isolate(s) collected over a five-year period, using the cutoff values according to CLSI M27-S3 23). Isolates with Fluconazole-susceptibility a Itraconazole-susceptibility a Voriconazole-susceptibility a Species resistance to: S SDD R S SDD R S SDD R Fluconazole C. albicans (n=37) C. glabrata C. tropicalis C. krusei All species Itraconazole C. albicans (n=22) C. glabrata C. tropicalis C. krusei All species Voriconazole C. albicans (n=8) C. glabrata C. tropicalis C. krusei All species a S, susceptible; S-DD, susceptible-dose dependent; R, resistant surveillance studies (ARTEMIS studies) have reported the longitudinal surveillance data on the species distribution and susceptibilities to fluconazole and voriconazole of clinical isolates of the major Candida species in various geographical regions of the world including the Asia-Pacific region 17,25,26). However, in the ARTEMIS studies isolates are not collected from any Japanese institution, and no such survey has been conducted in Japan. In this study, we examined prevalence and in vitro susceptibility of the most important and frequent pathogenic Candida species, whose isolates were collected nationwide in Japan over a five-year period, 2001 to The results demonstrate that the species distribution among the collected Candida isolates is quite average in comparison to that for the Asia- Pacific region in the ARTEMIS study, 1997 to ). It looks likely to occur a steady increase in the isolation of C. krusei in Japan. The present study determined the trend of susceptibilities to three antifungal azoles (fluconazole, itraconazole and voriconazole) for the more common Candida species (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei) from 2001 to C. albicans remains quite susceptible to all of the three azoles. Pfaller et al. showed a relatively stable C. albicans MIC 50 of 0.25μg/ml over a 10-year period, between 1992 and ,27). Not only our MIC 50 of 0.25μg/ml for fluconazole but also the values of 0.016μg/ml and 0.008μg/ml for itraconazole and voriconazole, respectively, are basically identical to those reported by several different research groups 17,18,27,28,30). In comparison, our data show that lower susceptibilities to fluconazole, itraconazole and/or voriconazole were for all of the four non-albicans Candida species, among which C. krusei has the highest MICs and C. glabrata to the next as reported previously 13,17,18,27-31). Moreover, it also appears that there is a continuous decrease in the extent of azole susceptibility (i.e., an increase in MIC 50 and/or MIC 90 values) for all the nonalbicans Candida species. As expected from the MIC data, resistance to fluconazole, itraconazole and/or voriconazole was not detected among 74 isolates of C. parapsilosis and rarely detected among 882 C. albicans isolates and 132 C. tropicalis isolates. We found only two (0.2%) C. albicans resistant isolates which exhibit resistance to all the three azoles and three (2.3%), three (2.3%) and one (0.8%) C. tropicalis isolates resistant to fluconazole, itraconazole and voriconazole, respectively. These results indicate that C. parapsilosis has the lowest resistance rate to all the three azoles among major Candida species, followed by C. albicans and C. tropicalis in this order. All the values of resistance rate to individual azoles for the isolates of C. albicans, C. tropicalis and C. parapsilosis obtained in our surveys, to 2005, appear to be considerably lower than those for the isolates of comparable Candida species from Japan and the Asia-Pacific region collected in the Japan Invasive Mycosis Surveillance study (JIMS study), 2001 to ) and the ARTEMIS study, 1997 to ), respectively. Although there is no explanation for such discrepancy in azole resistance data between these two studies and ours, the

6 Species distribution and in vitro susceptibility to three antifungal triazoles of clinical Candida isolates from a five-year nation-wide survey in Japan data reported herein demonstrate that all of the three major Candida species (C. albicans, C. tropicalis and C. parapsilosis) regarded as intrinsically fluconazolesusceptible ones remain to be quite susceptible not only to fluconazole but also to both itraconazole and voriconazole with substantially low rates of azole resistance. Moreover, there may be no trend in increasing azole resistance among isolates of these three common Candida species over the surveillance period from 2001 to Contrary to an azole-susceptible group of Candida species (C. albicans, C. tropicalis and C. parapsilosis), C. glabrata and C. krusei, particularly the latter, are known to be intrinsically resistant to fluconazole 33), and have been reported to have higher resistance rates to fluconazole in almost all previous surveillance studies. The overall resistance rates to fluconazole, itraconazole and voriconazole for C. glabrata isolates (4.6%, 4.6% and 0.9%, respectively) and for C. krusei isolates (59%, 6.9% and 6.9%, respectively) observed in the present study are average or low compared to those reported in the ARTEMIS studies 17,25,26), the JIMS study 18) and a considerable number of other local and global antifungal surveillance studies 13,28,29,33-39). However, regarding fluconazole, there may be continuous increases in the rate of resistance among isolates of C. glabrata and C. krusei, particular the latter. We found in this study that C. krusei has the highest resistance rate to fluconazole among Candida species tested as reported in almost all previous surveillance studies. Our data also show that, compared to the rate of fluconazole resistance, that of voriconazole resistance is extremely low among isolates of C. glabrata and/or C. krusei. These results are consistent with those reported by previous investigators 17,18,25,26,34,36-39). In regard to the resistance rate to itraconazole among Candida species, much more controversial data have been reported in the literature. There are reports from local, national or international surveillance studies showing that C. glabrata is the most resistant to itraconazole among major Candida species 11,35,40-46), and that resistance rates among C. glabrata and most other Candida species to itraconazole are higher than those to fluconazole 18,42,47,48). However, somewhat different from these data, our findings suggest that the resistance rates among C. glabrata and all Candida species tested to itraconazole are almost equal to or rather low compared to those to fluconazole. It is highly possible that clinical isolates of Candida species exhibit cross-resistance among antifungal azole agents because they are considered to be subject to the same mechanisms of resistance, such as the upregulation of the CDR and MDR efflux pumps and alterations in the gene for the target enzyme ERG ). The results of the present study examining whether or not and to what extent clinical isolates of each of the four major Candida species singly resistant to fluconazole, itraconazole, and voriconazole were basically consistent with previous findings 44,49,50). It should be noted that less than one-forth isolates of Candida species resistant to fluconazole are also resistant to voriconazole, whereas all of the voriconazole-resistant ones exhibit resistance to fluconazole and itraconazole. The excellent activity of voriconazole against most Candida species isolates, including a large proportion of fluconazole-resistant ones, may be attributed to enhanced binding of this azole to the target molecule compared to that of fluconazole 52). The results of this study demonstrate that the rate of resistance to antifungal azoles among Candida species isolates from 2001 to 2005 surveys is generally low, with being the lowest for voriconazole resistance. This suggests the sustained usefulness of azoles, in particular voriconazole, for the treatment and prevention of invasive Candida infections. However, our data do not represent its current and future states. Therefore, continuous surveillance of anti- Candida activities and their trends, as well as of Candida species distribution, remains important. Acknowledgments Other members of the Japan Antifungal Surveillance Program Group are Katsuko Okuzumi (Dokkyo University Hospital, Mibu, Tochigi-ken, Japan), Toyoko Oguri (Juntendo University Hospital, Tokyo, Japan), Keiko Adachi (Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan), Sayoko Kawakami (Teikyo University Tokyo Hospital, Tokyo, Japan), Yoshiki Misawa (The University of Tokyo Hospital, Tokyo, Japan), Tosaka Masakazu (Kumamoto University Hospital, Kumamoto, Japan), Chieko Kawashima (Ashikaga Red Cross Hospital, Ashikaga, Japan), Yoshiko Hori (Okayama Saiseikai Genera Hospital, Okayama, Japan), Toshimi Kitazawa (Kitkohkai General Hospi

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