JAC Advance Access originally published online on September 1, 2003
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Journal of Antimicrobial Chemotherapy (2003) 52, 679-682
© 2003 The British Society for Antimicrobial Chemotherapy
The European Confederation of Medical Mycology ECMM) survey of candidaemia in Italy: antifungal susceptibility patterns of 261 non-albicans Candida isolates from blood
Istituto di Igiene e Medicina Preventiva, Università degli Studi – IRCCS Ospedale Maggiore di Milano, via F. Sforza 35, 20122 Milano, Italy
Received 14 April 2003; returned 23 May 2003; revised 17 June 2003; accepted 26 June 2003
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Abstract |
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Objectives: To analyse the in vitro antifungal susceptibility of 261 non-albicans Candida bloodstream strains isolated during the European Confederation of Medical Mycology survey of candidaemia performed in Lombardia, Italy (September 1997–December 1999).
Methods: In vitro susceptibility to flucytosine, fluconazole, itraconazole, posaconazole and voriconazole was determined using the broth microdilution method described in the NCCLS M27-A guidelines. Etest strips were used to assess susceptibility to amphotericin B. In vitro findings were correlated with the patient’s underlying condition and previous antifungal treatment.
Results: MICs (mg/L) at which 90% of the strains were inhibited were, respectively, 2 for flucytosine, 8 for fluconazole, 0.5 for itraconazole, 0.25 for voriconazole and 0.25 for posaconazole. Amphotericin B MIC endpoints were <0.50 mg/L in all the isolates tested. Flucytosine resistance was detected in 19 isolates (7%), mainly among Candida tropicalis strains (30%). Innate or secondary fluconazole resistance was detected in 13 strains (5%). Among the 13 patients with fluconazole-resistant Candida bloodstream infection, three were HIV positive, including one treated with fluconazole for oral candidosis; the four who were HIV negative had received the azole during the 2 weeks preceding the candidaemia. Cross-resistance among fluconazole and other azoles was a rare event.
Conclusions: Resistance is still uncommon in non-albicans Candida species recovered from blood cultures. However, in fungaemias caused by C. tropicalis, Candida glabrata and Candida krusei, there is a high prevalence of resistance to fluconazole and flucytosine. Fluconazole resistance should be suspected in patients treated previously with azoles, mainly those with advanced HIV infection.
Keywords: flucytosine, fluconazole, itraconazole, posaconazole, voriconazole
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Introduction |
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Candidaemia is the most frequent fungal infection in hospitalized patients and is associated with significant mortality and prolonged hospital stay.1 Although Candida albicans continues to predominate, the emergence of non-albicans Candida species as a cause of bloodstream infection (BSI) has been reported in all surveillance programmes,1 including the large prospective survey performed in Europe by the European Confederation of Medical Mycology (ECMM) (unpublished data). This emergence is of special concern because of the virulence of these species in immunocompromised patients, and the occurrence of resistance to currently available antifungals.2
Non-albicans Candida species accounted for 41% of the episodes of Candida BSI reported during the ECMM survey of candidaemia performed in one Italian region by the Federazione Italiana di Micopatologia Umana e Animale (FIMUA).3 The in vitro susceptibility to the antifungals amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole of 261 non-albicans Candida bloodstream strains isolated during this survey is reported. Resistance was correlated with the underlying conditions associated with infection and with previous antifungal therapy.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Isolates
A total of 261 non-albicans Candida strains isolated from blood (the first isolate from each episode) during the ECMM–FIMUA survey of candidaemia were studied. Thirty-five medical centres in Lombardia, Italy, participated in the survey during the period September 1997–December 1999.
Isolates included 99 Candida parapsilosis, 80 Candida glabrata, 44 Candida tropicalis, 11 Candida guilliermondii, eight Candida pelliculosa, six Candida krusei, five Candida lusitaniae, two Candida lipolytica, and one each of Candida diversa, Candida dubliniensis, Candida famata, Candida inconspicua, Candida norvegensis and Candida utilis.
The strains received by the coordinating centre were subcultured on Chromagar Candida medium (CHROMagar Microbiology, Paris, France) to ensure viability and purity.
Yeast identification was checked by morphology evaluation on potato–carrot–ox gall agar, and analysis of the biochemical patterns by ID 32 (bioMérieux, Roma, Italy). Isolates were stored as suspensions in distilled water at room temperature until needed.
Susceptibility testing
The antifungals tested were fluconazole (Pfizer Central Research, Sandwich, UK), itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer), posaconazole (Schering-Plough Research Institute, Kenilworth, NJ, USA), flucytosine (Sigma-Chemical, Milano, Italy) and amphotericin B.
Susceptibility to amphotericin B was determined using Etest strips (AB Biodisk, Solna, Sweden) on Antibiotic medium 3 (Difco Laboratories, Detroit, MI, USA).
Susceptibility to flucytosine and azoles was determined using a broth microdilution method described by the NCCLS.4 Testing was conducted in RPMI 1640 without sodium bicarbonate (Sigma-Chemical), buffered to pH 7.0 with 0.165 M MOPS (Sigma-Chemical) and supplemented with 2% glucose and 0.03% L-glutamine (Sigma-Chemical). C. parapsilosis ATCC 22019 was used as the quality control strain.
The interpretative breakpoints suggested in the NCCLS document were adopted.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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MICs for C. parapsilosis ATCC 22019 were within the expected range.
Amphotericin B and flucytosine were very active against the 261 non-albicans Candida isolates from blood.
Testing for susceptibility to amphotericin B, performed using Etest strips, showed MIC endpoints <0.50 mg/L in all the isolates tested.
MICs of flucytosine, at which 50% (MIC50) and 90% (MIC90) of the strains were inhibited, were 
0.25 and 2 mg/L, respectively. Resistance (MIC 
32 mg/L) was detected in 19 isolates, including 13 C. tropicalis, two C. glabrata, two C. krusei, one C. parapsilosis and one C. pelliculosa. Overall, the rate of flucytosine resistance (7.3%) was higher compared with that reported in other studies.5,6 The high prevalence of resistance among C. tropicalis (13/44, 30%) and C. krusei (2/6) is noteworthy and consistent with the relatively poor susceptibility of these species observed in the first studies on this drug.7 A large recent investigation confirms the data on C. krusei, but reports resistance of only 8% among C. tropicalis.6 None of the patients infected by flucytosine-resistant Candida was HIV positive and none had received flucytosine in the previous 2 weeks. Surgery (12/19) and cancer (9/19) were the main underlying conditions associated with resistance. However, there is a frequent association of surgery and cancer with C. tropicalis, which has a high level of resistance to flucytosine (71% and 58% of C. tropicalis BSI occurred in surgical and cancer patients, respectively);3 thus these conditions together may be responsible for the resistance. Resistance among C. tropicalis isolates occurred in patients hospitalized in 11 different institutions, ruling out the presence of a unique flucytosine-resistant strain infecting all patients.
The patterns of in vitro susceptibility to azoles are reported in a continuous fashion (Table 1).
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In addition to the eight isolates belonging to intrinsically fluconazole-resistant species (C. krusei, C. inconspicua and C. norvegensis), three C. glabrata and two C. tropicalis isolates showed resistance to this azole (MIC
64 mg/L). Overall the frequency of resistance to fluconazole was low (13/261, 5%). In particular the rate among C. glabrata isolates (3/80, 3.75%) was lower than that reported in other surveillance studies, in which it ranged from 7% to 40%.1 Resistance occurred in 4% (10/251) of HIV-negative and in 30% (3/10) of HIV-positive patients with non-albicans Candida BSI included in our series (P = 0.14). Two patients had undergone vascular surgery and four surgery of the alimentary tract. Two patients had a solid tumour and one leukaemia. Resistance occurred in patients hospitalized in nine different institutions. Four of the HIV-negative patients and one HIV-positive subject received fluconazole during the 2 weeks preceding candidaemia.
Overall, 52% and 85% of isolates were inhibited by 0.06 and 0.25 mg/L of itraconazole, respectively. Elevated MICs (1 mg/L) were detected in seven isolates, that is two C. tropicalis strains, both fluconazole-resistant, and five C. glabrata strains, two of which were susceptible to a specific dose of fluconazole. None of the patients infected by these strains received azole antifungals during the 14 day period prior to candidaemia.
As reported in other studies,8,9 both voriconazole and posaconazole were very active against non-albicans Candida isolates (MIC90, 0.25 mg/L). C. glabrata and C. tropicalis were the least susceptible species.
Cross-resistance among fluconazole and other triazoles was a rare event. In most cases only an increase in the MIC endpoints of the other azoles was observed: three fluconazole-resistant C. glabrata strains were inhibited by itraconazole at 0.5 mg/L and by voriconazole and posaconazole at 1–2 mg/L. Out of the two C. tropicalis isolates, one was inhibited by itraconazole, voriconazole and posaconazole at concentrations of 1–2 mg/L, the other at concentrations 16 mg/L. Intrinsically, fluconazole-resistant Candida species showed no cross resistance: MICs of itraconazole, voriconazole and posaconazole for C. krusei, C. norvegensis and C. inconspicua isolates were 0.5 mg/L.
In conclusion, as seen in other studies,1,5,10 resistance to antifungal agents is a rare finding in non-albicans Candida species isolated from blood. However, in fungaemias caused by C. tropicalis, C. glabrata and C. krusei these species are characterized by a high prevalence of resistance to fluconazole and flucytosine. In addition, fluconazole resistance should be suspected in breakthrough infections previously treated with azoles. In clinical practice, long-term use of fluconazole is required mainly in advanced stages of AIDS, in empirical treatment of candidosis in cancer and surgical patients, and as prophylaxis. As cross-resistance to other azoles is rare, fluconazole resistance does not necessarily rule out the use of newer azoles, such as voriconazole and posaconazole as potential treatment options for serious Candida infections.
Geographic variations of Candida species as the cause of BSIs and differences in the prevalence of resistance demonstrate the importance of local surveillance studies.2 Because the current limitations of in vitro susceptibility testing do not yet allow routine application of the tests, careful mycological identification at the species level of Candida isolates recovered from blood, together with information on the prevalence of more resistant species and on overall antifungal susceptibility patterns obtained during local surveillance studies, are needed to help clinicians make treatment decisions.
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Acknowledgements |
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The study was in part supported by a grant from Ministero dell’ Università e della Ricerca Scientifica e Tecnologica (COFIN 2001–065221).
We thank Pfizer Italiana SpA for the research grant to Anna Lisa Rigoni, the Janssen Research Foundation, Pfizer Central Research, and the Schering-Plough Research Institute, respectively, for providing itraconazole, voriconazole and posaconazole.
The participants in the FIMUA–ECMM candidaemia study group were A. Astolfi, C. Ossi, S. Perin, C. Bonaccorso, L. Garlaschi, G. Viola, M. Saudelli, S. Frugoni, A. Mauri, R. Passerini (Milano); C. Farina (Bergamo); M. Tejada (S. Donato M); C. Cavanna (Pavia); A. Raballo (Mantova); A. Grossi (Treviglio); C. Bezzi (Magenta); M. Spinelli (Como); S. Bramati (Monza); G. Pinsi (Brescia); L. Colombo (Desio); L. Ferrari (Cremona); P. Troupioti (Sondalo); G. L. Lombardi (Varese); M. Arghittu (Melegnano); C. Agrappi (Legnano); L. Sturla (Gallarate); G. Gialluca (Rho); M.G. Musmanno (Saronno); A. Ceraminiello (Lodi); P. Casella (Vimercate); P. Porri (Voghera).
The results of this study were presented in part at the 8th Congress of the European Confederation of Medical Mycology, Budapest, Hungary, 25–27 August 2002
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Footnotes |
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* Corresponding author. Tel: +39-02-55033487/+39-02-50320600; Fax: +39-02-50320597; E-mail: annamaria.tortorano{at}unimi.it
The FIMUA-ECMM candidaemia study group are listed in the Acknowledgements.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Pfaller, M. A. & Diekema, D. J. (2002). Role of sentinel surveillance of candidemia: trends in species distribution and antifungal susceptibility. Journal of Clinical Microbiology 40, 3551–7.
2 . Perea, S. & Patterson, T. F. (2002). Antifungal resistance in pathogenic fungi. Clinical Infectious Diseases 35, 1073–80.[CrossRef][Web of Science][Medline]
3 . Tortorano, A. M., Biraghi, E., Astolfi, A. et al. (2002). European Confederation of Medical Mycology (ECMM) prospective survey of candidaemia: report from one Italian region. Journal of Hospital Infection 51, 297–304.[CrossRef][Web of Science][Medline]
4 . National Commitee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A. NCCLS, Villanova, PA, USA.
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St-Germain, G., Laverdière, M., Pelletier, R. et al. (2001) Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada. Journal of Clinical Microbiology 39, 949–53.
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Pfaller, M. A., Messer, S. A., Boyken, L. et al. (2002). In vitro activities of 5-fluorocytosine against 8,803 clinical isolates of Candida spp.: global assessment of primary resistance using National Committee for Clinical laboratory Standards susceptibility testing methods. Antimicrobial Agents and Chemotherapy 46, 3518–21.
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Pfaller, M. A., Messer, S. A., Hollis, R. J. et al. (2002). In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrobial Agents and Chemotherapy 46, 1723–7.
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