National surveillance of species distribution in blood isolates of Candida species in Japan and their susceptibility to six antifungal agents including voriconazole and micafungin

Shunji Takakura1,*, Naoko Fujihara1, Takashi Saito2, Toyoichiro Kudo1, Yoshitsugu Iinuma1, Satoshi Ichiyama1 and the Japan Invasive Mycosis Surveillance Study Group

1 Departments of Clinical Laboratory Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 6068507; 2 KKR Takamatsu Hospital, Takamatsu, Kagawa 7600018, Japan

Received 7 August 2003; returned 23 September 2003; revised 21 October 2003; accepted 5 November 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The aim of this study was to evaluate species distribution and antifungal susceptibility of Candida blood isolates in Japan.

Methods: In a 1 year surveillance programme, 535 Candida blood isolates were collected. Identification of species was followed by examination with the broth microdilution method, as described in NCCLS M27-A2, of antifungal susceptibility to six agents, including voriconazole and micafungin, with readings after 24 and 48 h of incubation.

Results: The overall species distribution was: 41% Candida albicans, 23% Candida parapsilosis, 18% Candida glabrata, 12% Candida tropicalis and 2% Candida krusei. The concentrations of fluconazole necessary to inhibit 90% of the isolates (MIC90) at 24/48 h were 0.25/1 mg/L for C. albicans, 0.5/2 mg/L for C. parapsilosis, 4/32 mg/L for C. glabrata and 4/>128 mg/L for C. tropicalis. Percentages of fluconazole resistance were 1.8% for C. albicans, 0.8% for C. parapsilosis, 5.2% for C. glabrata and 3.2% for C. tropicalis, taking the tendency of trailing growth of C. tropicalis into account. MIC90 of voriconazole was 0.5 mg/L, although 35% of isolates less susceptible (>=16 mg/L) to fluconazole showed resistance (>=2 mg/L). Micafungin was very active against all species (MIC90, 0.03 mg/L) except for C. parapsilosis (MIC90, 2 mg/L).

Conclusions: These data suggest that, in Japan, the species distribution of Candida bloodstream infections and the fluconazole resistance rate are similar to those reported previously in North America and Europe. Voriconazole and micafungin appear to have strong in vitro activity against Candida blood isolates, although continuing surveillance and further clinical research are needed.

Keywords: candidaemia, triazoles, echinocandins


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bloodstream infections (BSIs) caused by Candida species have been reported to be on the increase in recent decades, possibly because of rapid changes in medical practice, with a consequent rise in related mortality and prolonged hospitalizations.15 In Japan, as in the USA, Candida species rank as the fourth most common cause of BSIs.1,6 The proliferation of large-scale surveillance programmes of Candida BSIs has provided useful information regarding resistance trends, the distribution of species in various countries and types of infections.3,4,711 From the results of these studies, it appears that substantial differences exist in species distribution and antifungal susceptibility profiles,4,9,10 and it is probably important to obtain such information for every country. However, no large-scale, nationwide surveillance study has been conducted in Japan.

Appropriate susceptibility testing is mandatory before proper treatment can be initiated. The establishment by the NCCLS of the reference method (M27-A) for broth dilution antifungal susceptibility testing of yeasts has resulted in significant progress in this field.12 In a recently revised document, M27-A2, two important but controversial points are mentioned.13 The first concerns spectrophotometric determination of MIC endpoints in broth microdilution modification, and the second the impact of reading MICs after 24 h of incubation (M27-A2 recommends 48 h). The former may reflect some difficulty with visual determination of ‘a prominent decrease in turbidity’ in the microdilution format, and the latter, the problematic effect of trailing growth of some strains leading to overestimation of MICs. Spectrophotometric reading seems to be effective for maintaining objectivity, resulting in improved inter-laboratory reproducibility through avoiding the overestimation of MICs. However, there have been few reports on comparisons of 24 and 48 h endpoint readings.11,14,15

Of several new triazoles and echinocandin agents, voriconazole1618 and micafungin (FK463)19,20 appear to be especially effective against yeast isolates with primary or secondary resistance to fluconazole and/or itraconazole. These agents thus are promising candidates for the treatment of patients with Candida BSIs caused by strains or species resistant to former azoles, or who cannot be expected to tolerate therapy with a full dose of amphotericin B. Micafungin, an echinocandin recently approved in Japan, inhibits fungal 1–3 ß-D glucan synthesis and has been reported to show good in vitro activity against most Candida blood isolates from cancer patients.21 However, no large-scale surveillance of the activity of this agent against Candida blood isolates has yet been conducted.

Our surveillance examined the species distribution of Candida BSI isolates in Japan and their susceptibility profiles to various antifungal agents including voriconazole and micafungin. Our report also deals with practical issues concerning the impact of reading time (24 versus 48 h) on antifungal susceptibility test results.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design

As part of the work by the Japan Invasive Mycosis Surveillance (JIMS) Study Group, set up by us and participated in by 156 institutions throughout Japan, the study presented here focuses on BSIs caused by Candida species. In this JIMS study, conducted during November 2001–October 2002, all isolates of Candida species from blood cultures (one isolate per patient) were collected consecutively at the participating institutions. Demographic and clinical data recorded on a standardized form were also collected from 75 of the participating institutions. These data included age, sex, underlying disease, predisposing factors [coexisting illness, major surgery and presence of a central venous catheter (CVC)]. The institutions that contributed isolate and patient data to the study are listed on the Kyoto University Hospital website (http://www.kuhp.kyoto-u. ac.jp/~ict/jimslist.htm).

Organism identification

All fungal isolates from blood cultures were first identified at the participating institutions with the routine method in use at their laboratory. Species identification was confirmed at our laboratory with the Vitek YBC system, or with API 20C AUX or API 32ID (BioMérieux, St Louis, MO, USA), as recommended by the manufacturer, if the identification procedures at the participating institutions did not include any of these methods. When necessary, germ tube analysis and morphological analysis on cornmeal-Tween 80 agar were also performed at our laboratory. Candida dubliniensis was identified tentatively by using the API 20C AUX profile and differential growth at 35 and 43°C.22 The isolates were subcultured onto Sabouraud dextrose agar and CHROMagar Candida medium (Becton Dickinson Japan, Tokyo, Japan) to ensure viability and purity. Isolates were stored in Microbank vials (Pro-Lab Diagnostic, Toronto, ON, Canada) at –80°C until needed.

Susceptibility testing

Antifungal susceptibility testing of isolates of Candida species was performed in exact accordance with the reference broth microdilution method described by the NCCLS, document M27-A2.13 Standard powders of amphotericin B (Sigma, St Louis, MO, USA), flucytosine (Sigma), fluconazole (ICN Pharmaceuticals, Tokyo, Japan), itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer, Inc., New York, NY, USA) and micafungin (Fujisawa Pharmaceutical, Osaka, Japan), were obtained from the respective manufacturers and diluted in distilled water or dimethyl sulphoxide. Frozen microdilution panels, containing serial two-fold dilutions made in RPMI 1640 medium (Invitrogen, Tokyo, Japan) buffered to pH 7.0 with 0.165 M MOPS buffer (Wako Pure Chemical Industries, Osaka, Japan) for all antifungal agents, were prepared in a single lot at the laboratory of the BML Incorporation, Tokyo, Japan, and stored at –80°C until use. The final concentrations of the antifungal agents were flucytosine and fluconazole 0.125–128 mg/L, amphotericin B and itraconazole 0.016–16 mg/L, voriconazole 0.031–64 mg/L and micafungin 0.008–16 mg/L. The final concentration of the solvent did not exceed 1% in any of the wells. Drug-free growth control wells and drug- and yeast-free sterility check wells were included. The inoculum in each well was adjusted to a final size of 0.5 x 103–2.5 x 103 cfu/mL. After 24 and 48 h of incubation at 35°C in ambient air, growth reduction was estimated visually and calculated spectrophotometrically; for the latter, optical densities (ODs) of the wells were determined at 630 nm (MR5000; Dynatech Laboratories, Chantilly, VA, USA) after plate agitation for 2 min. The background OD of the drug- and yeast-free sterility check well was subtracted from the ODs of all the wells, which were then divided by the OD of the drug-free growth control well to determine the percentage of growth relative to that of the drug-free growth control. The MICs of flucytosine, fluconazole, itraconazole and voriconazole were defined as the lowest concentrations at which a prominent decrease in turbidity (>=50% reduction in spectrophotometrically determined growth) relative to that in drug-free, growth-control wells was observed.13 The MICs of amphotericin B and micafungin were defined as the lowest concentrations resulting in complete inhibition of growth (>=95% reduction in spectrophotometrically determined growth). An MIC endpoint estimated by visual reading was given priority, but when the endpoint was hard to determine, the one determined spectrophotometrically was referred to. MIC50 and MIC90 were defined as the respective concentrations of each antifungal agent necessary to inhibit 50% and 90% of the isolates. Quality control for each measurement was ensured by testing the strains recommended by the NCCLS,13 that is, Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019. The interpretive susceptibility criteria used for flucytosine, fluconazole and itraconazole were the same as those specified by the NCCLS.13 Although voriconazole has not been assigned any interpretive breakpoints, we used a resistance breakpoint of 2 mg/L for purposes of comparison on the basis of preliminary pharmacokinetic data and previously reported findings.10,23

Statistical methods

MICs were converted into ordinal variables (log2 MICs) before statistical analyses. Before that, the low off-scale MICs were left unchanged, and the high off-scale MICs were converted into the next highest concentration. Relationships between categorical variables were analysed with the {chi}2 test, between categorical and ordinal variables with the Kruskal–Wallis test, and between categorical and continuous variables with the unpaired Student’s t-test. Scheffe’s F test was used for post-hoc comparisons. All reported P values were two-tailed and a P value of <0.05 was considered significant. All of the analyses were performed with StatView version 5.0 (SAS Institute, Cary, NC, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Species distribution of the isolates and clinical background of the Candida BSI cases

During the study period, a total of 535 Candida clinical blood isolates (one isolate per patient) were collected from 105 participating institutions. At 51 institutions, Candida species were not isolated from blood cultures. The number of isolates obtained ranged from 1–19 per institution, with a median number of four. The overall species distribution is shown in Table 1. Candida albicans was the most prevalent species, followed by C. parapsilosis, Candida glabrata, Candida tropicalis and C. krusei. These five major species accounted for 95% of all isolates. Other species identified included seven Candida guilliermondii, four Candida famata and three Candida lusitaniae. Nine isolates were unidentified as to species level. One isolate, which was positive for germ tube analysis, did not assimilate XYL and MDG on API 20C AUX or show growth at 43°C. These phenotypic analyses tentatively identified it as C. dubliniensis. Complete sets of clinical background data were obtained from 325 patients (60.7% of the total number). However, the species distribution of the isolates obtained from patients was not rendered significantly different by the presence or not of background data ({chi}2 test, P = 0.26). Notable features of the cases were high age distribution and high prevalence of CVC placement. The patients were aged 0–96 years with a median age of 69, and CVC placement was reported in 85.2% of the cases.


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Table 1. Species distribution of 535 Candida blood isolates in the course of a 1 year surveillance programme in Japan
 
Susceptibility to amphotericin B and flucytosine

The MIC ranges, MIC50 and MIC90 at 24 and 48 h, are summarized in Table 2. None of the isolates showed an amphotericin B MIC level higher than 2 mg/L at 48 h. MICs within 0.125–0.5 mg/L were observed in 88% of the isolates, with C. albicans showing a lower and C. krusei a higher MIC, both with statistical significance, than the other species (Kruskal–Wallis test and Scheffe’s F test, P < 0.001). All isolates of C. lusitaniae, which has been reported to be occasionally resistant to amphotericin B,24 had relatively low MICs. Only 2.0% of the isolates were resistant to flucytosine (MIC of >=32 mg/L) at 48 h, with the highest resistance among species recorded for C. tropicalis (8.1%), followed by C. krusei (7.7%), C. parapsilosis (2.4%) and C. albicans (0.9%). It is especially noteworthy that none of the C. glabrata isolates showed a flucytosine MIC higher than 0.5 mg/L.


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Table 2. Antifungal susceptibilities of 535 Candida blood isolates from Japan as determined by the NCCLS microdilution method after 24 and 48 h of incubation
 
Susceptibility to fluconazole and itraconazole

Fluconazole resistance (>=64 mg/L) was identified in 0.6% of all isolates at 24 h and in 7.8% at 48 h (Table 2). Other than C. krusei, which is considered to be intrinsically fluconazole resistant irrespective of MIC, fluconazole resistance rates were 1.8% for C. albicans, 0.8% for C. parapsilosis and 5.2% for C. glabrata. Although 35.5% of C. tropicalis isolates appeared to have a fluconazole MIC of >=64 mg/L at 48 h, the resistance rate was estimated to be 3.2%, calculated from 24 h results, because most of the isolates with high MICs showed extensive trailing growth at 48 h. Other than these five species, 13% of the other isolates were resistant at 48 h. Itraconazole resistance (MICs >= 1 mg/L at 48 h in 18.4% of isolates) occurred more frequently than resistance to fluconazole. None of the fluconazole-resistant C. albicans was susceptible to itraconazole. Candida species other than these five also showed a high resistance rate to itraconazole of 34.8% at 48 h. MICs of fluconazole and itraconazole for the tentatively identified C. dubliniensis isolate were <=0.125 and <=0.016 mg/L, respectively.

Voriconazole susceptibility and azole cross-resistance

MICs of voriconazole were relatively low for all of the species, with an MIC90 of 0.5 mg/L (Table 2). Voriconazole was more potent (median, 16 times) than fluconazole, whereas relative potencies were greater for C. glabrata (median, 32 times) and C. krusei (median, 128 times). All C. krusei isolates showed relatively low MICs, with none having a voriconazole MIC higher than 1 mg/L. For all species, with the exception of C. tropicalis, the resistance rate to voriconazole (>=2 mg/L) was lower than that to fluconazole. These rates were 1.4% for C. albicans, 0.8% for C. parapsilosis, 4.2% for C. glabrata and 0% for C. krusei. The voriconazole resistance rate for C. tropicalis was estimated as 4.8%, calculated from 24 h results, because the high voriconazole MICs for some C. tropicalis isolates at 48 h were also affected by trailing growth. Overall, 37 (34.6%) of the 107 isolates less susceptible to fluconazole (>=16 mg/L) had voriconazole MICs of 2 mg/L or higher. This ratio varied substantially among species: 83.9% for C. tropicalis, 50.0% for C. albicans and C. parapsilosis, 7.8% for C. glabrata and 0% for C. krusei. Among the 48 fluconazole-resistant isolates (including all C. krusei), 30 (62.5%) showed resistance to voriconazole.

Micafungin susceptibility

Micafungin was very active against all Candida species (Table 2), except for C. parapsilosis, which had a micafungin MIC50 and MIC90 of 1 and 2 mg/L, respectively. C. guilliermondii also showed relatively high MICs (MIC50, 0.5 mg/L). However, none of the isolates had an MIC higher than 4 mg/L. Micafungin MICs of fluconazole- or voriconazole-resistant isolates (median, 0.016 mg/L; range, <=0.008–1 mg/L) were not higher than those for susceptible isolates (median, 0.031 mg/L; range, <=0.008–4 mg/L). Interestingly, the difference between the 24 and 48 h MICs of micafungin was the smallest among the six agents tested.

Impact of time of reading on fluconazole susceptibility testing: 24 versus 48 h readings

As seen in Table 2, the most prominent differences in 24 and 48 h resistance rates were demonstrated by the azoles, with a difference between fluconazole MIC readings at 24 and 48 h of more than two dilutions for C. tropicalis and C. glabrata (Table 3). Moreover, there was a significant difference in dissociation among the five species (Kruskal–Wallis test, P <0.001). Twenty isolates (32.3%) of C. tropicalis were found to be susceptible (<=8 mg/L) at 24 h but resistant (>=64 mg/L) at 48 h, but none of C. parapsilosis and C. krusei showed such discrepancy. ODs of growth control wells of C. tropicalis were comparable with those of other species either at 24 h (unpaired Student’s t-test, P = 0.81) or 48 h (unpaired Student’s t-test, P = 0.54), indicating that the MIC dissociation of some C. tropicalis isolates was due to trailing growth rather than caused by slower growth during the first 24 h.


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Table 3. Differences in fluconazole MIC and growth control between readings at 24 and 48 h for five major species and all isolatesa
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The work reported here is the first nationwide, large-scale surveillance study conducted in Japan to obtain data on species distribution and antifungal susceptibility of Candida BSI isolates. Furthermore, this is, to the best of our knowledge, the first report concerning micafungin activity against a large number of Candida BSI isolates.

Patient background data could not be obtained for 40% of the cases included in our study. However, species distribution was almost equal for the isolates with or without patients’ background data. In addition, the JIMS study group comprised data from institutions with a low (<=1 case per year) as well as high incidence (19 cases per year) of Candida BSIs. Therefore, our cases can be considered as roughly representing the overall Japanese Candida BSIs.

Five major species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei) were found to account for 95% of all Candida BSIs in Japan. Although non-albicans Candida species represent more than half of all isolates, C. albicans was still the most common cause of Candida BSIs. According to the SENTRY study report, the largest international surveillance programme to date, the proportion of C. albicans tends to decrease with an increase in age.10 This may be why older patients are more likely to be infected by less virulent non-albicans species than younger patients. The comparatively high age (median, 69 years old) of the cases enrolled in our study may thus explain our results for species distribution. As previously found in Europe, Canada and Latin America,25,26 C. parapsilosis was the second most commonly occurring species in Japan. This may be due to the fact that the majority (86%) of our cases had a CVC installed, because C. parapsilosis is considered to cause catheter-related BSIs more often than other Candida species.2729 C. dubliniensis is frequently isolated from HIV-positive patients with oropharyngeal candidiasis and tends to be fluconazole resistant.20,3032 Susceptibility to all six agents was good in the tentatively identified C. dubliniensis isolate from a non-HIV 69-year-old woman enrolled in this study.

The profiles of the susceptibility of all species to amphotericin B and flucytosine were consistent with those reported previously.3,8,10,11,33 However, the MIC distribution of amphotericin B was concentrated in too narrow a range to allow us to determine the precise susceptibility profile for this agent without modification of the test medium.34 Almost all isolates examined in this study showed an MIC of <=1 mg/L, and only two C. glabrata had an MIC of 2 mg/L.

Our findings suggest that fluconazole resistance of C. albicans, C. parapsilosis and C. tropicalis is still extremely rare in Japan. The prominently high rate of fluconazole resistance for C. tropicalis at 48 h seen in our study is different from rates reported previously by some large-scale surveillance programmes.3,7,8,10 Because the increase in fluconazole MICs of some C. tropicalis isolates between 24 and 48 h was exceptionally large without evidence of slower growth after the first 24 h, it seems reasonable to assume that this result is due to trailing growth of the isolates resulting in overestimation of resistance. Therefore, the resistance rate of C. tropicalis isolates to fluconazole observed in this study was assumed to be 3.2% on the basis of 24 h MIC values. This rate is comparable with that mentioned in previous reports.3,10

Resistance rates of most species to itraconazole were slightly higher than those to fluconazole. The difference was most obvious for C. glabrata as previously reported.8,11,25 This does not necessarily indicate that itraconazole has relatively less clinical efficacy, because the NCCLS interpretative breakpoint for itraconazole was decided entirely on the basis of clinical outcomes for mucosal candidiasis12,15 and no clinical research data are available as yet for Candida BSIs treated with this agent.

MICs of voriconazole were generally much lower than those of fluconazole. Although isolates with less susceptibility to fluconazole tended to show higher voriconazole MICs, all C. krusei isolates and 60% of the isolates of other species with reduced fluconazole susceptibility were susceptible to voriconazole. This cross-resistance between fluconazole and voriconazole found in our study has also been reported by others,1618,35 but voriconazole was more active than fluconazole in our study, especially against C. glabrata and C. krusei.

In earlier studies of the activity of echinocandins against Candida isolates, some isolates of C. parapsilosis and C. guilliermondii were found to have high caspofungin MICs (>16 mg/L),33 and some isolates of species other than C. parapsilosis showed high micafungin MICs (>8 mg/L).21,36 In our study, micafungin was very active against all Candida species, even against azole-resistant isolates. Only two isolates of C. parapsilosis showed micafungin MICs of 4 mg/L.

Arthington-Skaggs et al.37 reported that ~5% of all C. albicans isolates display trailing growth when tested against azoles. Our results show that more than 30% of C. tropicalis isolates may also feature trailing growth. MICs for these isolates read at 48 h are thus at risk of overestimation. In contrast, for isolates showing slower growth by the first 24 h, MICs read at 24 h may be underestimated. Therefore, drug-free control wells should be carefully checked before the reading at 24 h. In view of these potential problems, some objective criteria to define the growth as sufficient before reading would be necessary to improve the accuracy of MIC results either at 24 h or 48 h.

In summary, species distribution of Candida BSIs in Japan was found to be comparable with those reported previously, except for a slightly higher prevalence of C. parapsilosis. Reduced susceptibility to azoles was relatively rare in most species other than C. glabrata and C. krusei. Voriconazole and micafungin were found to be very active against almost all species in spite of some cross-resistance of voriconazole with fluconazole. To improve the clinical outcomes of Candida BSIs and to prevent antifungal resistance, continual surveillances and further research into the clinical efficacy of new antifungal agents will be needed.


    Acknowledgements
 
We express our appreciation to all the Japan Invasive Mycosis Surveillance (JIMS) Study participants, and Izumi Sone and Koshu Kinoshita for their clerical assistance.

This study was supported in part by a Grant-in-Aid (No. 14572182) for Scientific Research from the Japan Society for the Promotion of Science, and research grants from Pfizer Pharmaceuticals and Fujisawa Pharmaceuticals. The work was presented in part at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA, 14–17 September 2003, abstract M-1004.


    Footnotes
 
* Corresponding author. Tel: +81-75-751-4914; Fax: +81-75-751-3233; E-mail: stakakr{at}kuhp.kyoto-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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