Characterization of Candida parapsilosis complex strains isolated from invasive fungal infections

Transcription

1 DOI /s x ARTICLE Characterization of Candida parapsilosis complex strains isolated from invasive fungal infections E. orghi & R. Sciota & R. Iatta & C. iassoni & M. T. Montagna & G. Morace Received: 24 November 2010 / Accepted: 21 March 2011 # Springer-Verlag 2011 Abstract In the present work, we studied the distribution of Candida parapsilosis complex species and the antifungal susceptibility of clinical isolates collected during an Italian surveillance study of yeast invasive fungal infections (IFIs) in intensive care units (ICUs). Minimum inhibitory concentrations (MICs) were determined using the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method. ani digestion patterns of the secondary alcohol dehydrogenase polymerase chain reaction (PCR) products were used to identify C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. A total of 138 C. parapsilosis isolates were stored (January 2007 December 2008). The overall frequency of C. parapsilosis complex in IFIs was 22%. Of the 138 tested isolates, 95% were C. parapsilosis sensu stricto, 3.6% were C. orthopsilosis, and 1.4% were C. metapsilosis. The MIC 50 values (expressed as μg/ml) for anidulafungin, caspofungin, and micafungin for C. parapsilosis complex were 2, 1, and 2, respectively, and the MIC 90 values were 4, 2, and 4, E. orghi (*) : G. Morace Department of Public Health-Microbiology-Virology, Università degli Studi di Milano, Polo Universitario San Paolo, via di Rudinì 8, Milan, Italy Elisa.borghi@unimi.it R. Sciota : C. iassoni Specialization School in Microbiology and Virology, University of Milan, via di Rudinì 8, Milan, Italy R. Iatta : M. T. Montagna Department of iomedical Science and Human Oncology, Section of Hygiene, University of ari, via E. Orabona 4, ari, Italy respectively. The MIC 50 and MIC 90 values for itraconazole and posaconazole were 0.12 and 0.25, respectively, and for fluconazole, they were 1 and 4, respectively. This study, the most comprehensive study conducted to date to evaluate the frequency and antifungal susceptibility profiles of C. parapsilosis complex isolates from critically ill patients in Italy, highlights the low prevalence of C. orthopsilosis and C. metapsilosis in IFIs. Introduction The findings of several recent studies clearly show that invasive fungal infections (IFIs) are on the increase, not only among oncology and transplant patients, but also among patients admitted to intensive care units (ICUs). The rise in ICU IFIs can be attributed to the growing use of complex surgical procedures, invasive medical devices, and/or longterm, broad-spectrum antibiotic therapy [1, 2]. Candida species bloodstream infections (SIs) have become a focus of attention, representing the fourth most common cause of SIs in ICUs, with crude mortality rates varying from 23% to 53% according to the population studied and the different Candida species involved [3, 4]. Although Candida albicans remains the most prevalent species, the number of IFIs caused by non-albicans Candida species is increasing worldwide, with Candida parapsilosis being the most common causative agent in Europe and Latin America [5 7]. Recent studies have reported an association between C. parapsilosis candidemia and the use of indwelling devices and total parenteral nutrition, and have also highlighted the ability of this yeast to grow as biofilms [7, 8]. C. parapsilosis is responsible for a wide variety of clinical manifestations which are more common in patients admitted to neonatal or surgical ICUs [7].

2 C. parapsilosis has recently been reclassified as separate, closely related species: C. parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis [9]. Several authors have reported that the prevalence rate of C. parapsilosis complex species varies according to region: C. orthopsilosis is more common in South America (10.9% of all C. parapsilosis complex isolates) than in Europe (3.5% to 5.7%), whereas the opposite is true for C. metapsilosis (6.9% vs. 3%) [10, 11]. However, few microbiology laboratories currently distinguish between these species, and much of the published data is derived from studies that analyzed yeasts from different body sites and tissues; therefore, it is unclear whether these findings are a true reflection of the prevalence of IFIs attributable to C. orthopsilosis and C. metapsilosis [12]. A different antifungal susceptibility profile has been observed for the new species C. orthopsilosis and C. metapsilosis, which could be of clinical relevance, suggesting the need for regional epidemiological surveillance of these species [12, 13]. In the present study, we used a large Italian collection of presumed C. parapsilosis isolates, collected as part of an Italian surveillance study (GISIA3), to screen for C. orthopsilosis and C. metapsilosis. A further objective of the study was to determine the susceptibility of the clinical isolates to the available antifungal compounds, especially to the echinocandins: anidulafungin, caspofungin, and micafungin. Materials and methods Clinical isolates A total of 138 isolates of C. parapsilosis were tested in this study. The isolates were obtained from a recent study on yeast isolates from ICU wards, involving 13 hospitalassociated microbiology laboratories, distributed uniformly within Italy. Isolates of the same species originating from the same patient were included only if they had been isolated from different specimens (i.e., blood and spinal fluid) or from the same specimen but at least 15 days apart. The isolates were identified using standard procedures, i.e., morphology on cornmeal agar plates, germ-tube production in serum, and biochemical analysis using the Vitek system, API 20CAUX, or AT 32 C panels (io-merieux, Rome, Italy). Antifungal susceptibility testing Fluconazole, voriconazole, and anidulafungin (Pfizer Pharmaceuticals, Groton, CT, USA), caspofungin (Merck & Co, Inc., Whitehouse Station, NJ, USA), posaconazole (Schering-Plough Corporation, Kenilworth, NJ, USA), and micafungin (Astellas Pharma Inc., Tokyo, Japan) were obtained as standard powders from their manufacturers. and itraconazole were purchased from Sigma-Aldrich, Milan, Italy. Caspofungin, micafungin, and fluconazole were dissolved in sterile water, whereas all other drugs were dissolved in dimethyl sulfoxide. roth microdilution testing was performed in accordance with the guidelines of the Clinical Laboratory Standards Institute (CLSI) document M27-A3 [14] using RPMI 1640 medium, an inoculum of cells/ ml, and incubation at 35 C. The minimum inhibitory concentration (MIC), defined as the lowest concentration of drug that caused a significant diminution (>50% inhibition) of growth below control levels, was determined both visually and spectrophotometrically after incubation for 24 h. C. parapsilosis ATCC was included as the control organism in all experiments. The antifungal susceptibility testing was repeated three times for each isolate in order to confirm the data. We used the recently revised clinical breakpoints (CPs) to define C. parapsilosis complex isolates resistant to echinocandins and fluconazole (MIC values >4 μg/ml) and to voriconazole and posaconazole (MIC values >2 μg/ml) [15, 16]. The MIC values were considered to be in essential agreement (EA) between two different tests when they were within two dilutions. DNA extraction efore nucleic acid extraction, all C. parapsilosis complex strains were cultured on Sabouraud dextrose agar to verify viability, and were then grown overnight at 35 C in YPD medium. Yeast genomic DNA was extracted using the PrepMan Ultra Sample Preparation Reagent (Applied iosystems, CA, USA), according to the manufacturer s protocol. riefly, 1 ml of overnight culture was centrifuged for 2 min, and the pellet was resuspended in 100 μl of PrepMan reagent. After incubation for 10 min at 100 C, the sample was centrifuged again to recover supernatant. DNA was quantified spectrophotometrically. C. parapsilosis complex genotyping ani digestion patterns of the secondary alcohol dehydrogenase (SADH) polymerase chain reaction (PCR) products were used to identify the three species [9]. riefly, a 716-bp fragment of the SADH gene was amplified, purified, and digested with ani. Digestion patterns were used to differentiate between the three species, since the C. parapsilosis sensu stricto, C. metapsilosis, and C. orthopsilosis SADH amplicons contain 1, 3, and 0 ani restriction sites, respectively. C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96139, and C. metapsilosis ATCC were included as controls in all experiments.

3 Results etween January 2007 and December 2008, 138 isolates of C. parapsilosis complex were stored for an Italian survey of yeast invasive infections among ICU patients. In this study, the overall incidence of C. parapsilosis complex infections was 22%. The highest proportion of isolates was collected from general ICUs (37.9%), followed by surgical (26.3%) and neonatal ICUs (14.6%); a considerably lower proportion of isolates were collected from medical ICUs (8.8%), oncology (6.6%) and burn (3.6%) ICUs, and transplant ICUs (2.2%). The median age of patients with C. parapsilosis isolates was 47 years (range 0 98 years), with the highest percentage of isolates (43.8%) recovered from patients aged at least 60 years. Newborns provided 17.7% of the isolates. Most of the C. parapsilosis complex isolates were associated with SIs (79.1%) and central venous catheterrelated infections (19.4%); the remaining two isolates were obtained from pleural fluids. Of the 138 isolates presumed to be C. parapsilosis, 95% were C. parapsilosis sensu stricto, 3.6% were C. orthopsilosis, and 1.4% were C. metapsilosis. Table 1 summarizes the MIC distributions for the available antifungal drugs for C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. According to the recently revised CPs, none of our isolates were resistant to echinocandins, posaconazole, or voriconazole [15, 16]. MIC 90 values for anidulafungin, caspofungin, and micafungin for C. parapsilosis complex were 4, 2, and 4 μg/ml, respectively, whereas the MIC 90 values for posaconazole and voriconazole were 0.25 and 0.12 μg/ml, respectively. Although 90% of isolates were fluconazole-susceptible (MIC 90 value 4 μg/ml), 13 isolates were categorized as resistant to fluconazole, with MIC values of between 8 and 32 μg/ml. In order to confirm these data, three independent assays were conducted. Overall, the MIC values were within two dilutions, giving an essential agreement of Table 1 Minimum inhibitory concentration (MIC) distribution of antifungal drugs for Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis using the Clinical and Laboratory Standards institute (CLSI) reference broth microdilution method Species (n) Antifungal agent No. of isolates for which the MIC (μg/ml) was: < C. parapsilosis sensu Anidulafungin stricto (131) Caspofungin Micafungin Fluconazole Itraconazole Posaconazole Voriconazole C. orthopsilosis (5) Anidulafungin 1 4 Caspofungin 5 Micafungin Fluconazole Itraconazole 3 2 Posaconazole 5 Voriconazole C. metapsilosis (2) Anidulafungin 2 Caspofungin 2 Micafungin Fluconazole 1 1 Itraconazole 1 1 Posaconazole 2 Voriconazole 1 1

4 100%; the MIC value repeated two times has been used in the analysis. Discussion The worldwide epidemiological shift in candidemia can be attributed to the association of non-albicans Candida species, in particular, Candida glabrata and Candida krusei, with fluconazole resistance, and to the enhanced biofilm capabilities of C. parapsilosis. Notably, compared with other Candida species, C. parapsilosis has frequently demonstrated high echinocandin MIC values, that, before the recently revised breakpoints, were interpreted as a reduced in vitro susceptibility to the echinocandins [17 19]. In fact, a large proportion of our isolates (24.6%) showed anidulafungin MICs of 4 μg/ml, and half of them (53%) presented the same MIC value for micafungin. These data are in accordance with recent reports [18, 19], although we found a greater number of isolates showing MIC values of 4 μg/ml for anidulafungin and micafungin. This difference could be attributable to the fact that the present study only utilized isolates obtained from the sterile specimens of critically ill patients. It is difficult to predict whether these findings also apply to the in vivo setting, because of the significant tendency of C. parapsilosis, particularly C. parapsilosis sensu stricto, to produce biofilms. However, according to several authors, anidulafungin has significant activity against sessile C. albicans cells within biofilms [20 22]. Although data on the antimicrobial effectiveness of the echinocandins against C. parapsilosis biofilms are limited, we should not exclude the fact that in vivo anidulafungin may be a good choice despite its higher MIC. A detailed molecular characterization of the Fks genes (FKS1, FKS2, and FKS3) of these higher-mic isolates would be of great interest. A naturally occurring amino acid substitution in an Fks1pconserved region of the C. parapsilosis complex has been reported and linked to the high echinocandin MIC values [23]. It will also be interesting to investigate whether these clinical isolates possess other glucan synthase mutations that could explain the high MIC values. According to previous studies [11 13], C. orthopsilosis and C. metapsilosis appear to be more susceptible to the echinocandins than C. parapsilosis sensu stricto, particularly to anidulafungin and micafungin. The same authors reported the opposite for fluconazole, suggesting reduced susceptibility of C. orthopsilosis and C. metapsilosis to fluconazole. We observed only one C. metapsilosis isolate resistant to fluconazole (MIC 16 μg/ml). However, because of the small number of isolates belonging to the newly identified species, our study may not provide an entirely accurate picture of the antifungal susceptibility patterns of C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. To our knowledge, our study is the most comprehensive study conducted to date to evaluate the frequency and antifungal susceptibility profiles of C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis in critically ill patients in Italy. The findings are in agreement with the results of the ARTEMIS DISK Global Antifungal Surveillance study, even though the authors reported higher C. orthopsilosis and C. metapsilosis frequencies in our country [12]. Although rare, these two new species may prove to be problematic for individual patients, and their in vitro susceptibility profiles may be useful when selecting an initial antifungal treatment regimen. Acknowledgments This work was sponsored by Pfizer Italia, Srl. We would like to thank Arianna Tavanti (Department of iology, University of Pisa, Italy) for supplying the ATCC strains. Collection of the C. parapsilosis strains was part of an Italian surveillance study (GISIA3). Members of the GISIA3 group (Gruppo Italiano per lo Studio In vitro degli Antifungini) included: G. Amato, M.M. Piccirillo, L. Degl Innocenti (Cardarelli Hospital, Naples, Italy); S. Andreoni, M.R. Fanello (Maggiore della Carità Hospital, Novara, Italy); C. Farina, S. Perin (San Carlo Hospital, Milan, Italy); R. Fontana, G. Lo Cascio, L. Maccarano (G Rossi Hospital, Verona, Italy); G. Gesu, G. Lombardi, G. Ortisi (Niguarda Ca Granda Hospital, Milan, Italy); F. Luzzaro, G. rigante (Circolo Hospital, Varese, Italy); E. Manso, Am. Calvo, C. Cutrini (Torrette Hospital, Ancona, Italy); M. Mussap, O. Soro (San Martino Hospital, Genoa, Italy); A. Nanetti, E. Tangorra (University of ologna, ologna, Italy); P. Nicoletti, P. Pecile, C. ertellini (Careggi Hospital, Florence, Italy); R. Rigoli, L. Campion (Ca Foncello Hospital, Treviso, Italy); M. Tronci, G. Parisi, (Forlanini Hospital, Rome, Italy); M.T. Montagna, R. Iatta, T. Cuna (Università degli Studi di ari, Italy); G. Morace, E. orghi, C. iassoni, M. La Francesca, R. Sciota (Università degli Studi di Milan, Italy); S. La Face, I. Mancuso (Pfizer Italia, Srl, Rome, Italy). References 1. St-Germain G, Laverdière M, Pelletier R et al (2008) Epidemiology and antifungal susceptibility of bloodstream Candida isolates in Quebec: Report on 453 cases between 2003 and Can J Infect Dis Med Microbiol 19: van Hal SJ, Marriott DJ, Chen SC et al; Australian Candidaemia Study (2009) Candidemia following solid organ transplantation in the era of antifungal prophylaxis: the Australian experience. Transpl Infect Dis 11: Eggimann P, Garbino J, Pittet D (2003) Epidemiology of Candida species infections in critically ill non-immunosuppressed patients. Lancet Infect Dis 3: Shorr AF, Lazarus DR, Sherner JH et al (2007) Do clinical features allow for accurate prediction of fungal pathogenesis in bloodstream infections? Potential implications of the increasing prevalence of non-albicans candidemia. Crit Care Med 35: Tortorano AM, iraghi E, Astolfi A et al; FIMUA Candidemia Study Group (2002) European Confederation of Medical Mycology (ECMM) prospective survey of candidaemia: report from one Italian region. J Hosp Infect 51:

5 6. Almirante, Rodríguez D, Cuenca-Estrella M et al (2006) Epidemiology, risk factors, and prognosis of Candida parapsilosis bloodstream infections: case control population-based surveillance study of patients in arcelona, Spain, from 2002 to J Clin Microbiol 44: Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20: Shin JH, Kee SJ, Shin MG et al (2002) iofilm production by isolates of Candida species recovered from nonneutropenic patients: comparison of bloodstream isolates with isolates from other sources. J Clin Microbiol 40: Tavanti A, Davidson AD, Gow NA et al (2005) Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J Clin Microbiol 43: Gonçalves SS, Amorim CS, Nucci M et al (2010) Prevalence rates and antifungal susceptibility profiles of the Candida parapsilosis species complex: results from a nationwide surveillance of candidaemia in razil. Clin Microbiol Infect 16: doi: /j x 11. Gomez-Lopez A, Alastruey-Izquierdo A, Rodríguez D et al; arcelona Candidemia Project Study Group (2008) Prevalence and susceptibility profile of Candida metapsilosis and Candida orthopsilosis: results from population-based surveillance of candidemia in Spain. Antimicrob Agents Chemother 52: Lockhart SR, Messer SA, Pfaller MA et al (2008) Geographic distribution and antifungal susceptibility of the newly described species Candida orthopsilosis and Candida metapsilosis in comparison to the closely related species Candida parapsilosis. J Clin Microbiol 46: Szabó Z, Szilágyi J, Tavanti A et al (2009) In vitro efficacy of 5 antifungal agents against Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis as determined by time kill methodology. Diagn Microbiol Infect Dis 64: Clinical and Laboratory Standards Institute (CLSI) (2008) Reference method for broth dilution antifungal susceptibility testing of yeasts, 3rd edn. Approved standard M A3. CLSI, Wayne, PA 15. Clinical and Laboratory Standards Institute (CLSI) (2008) Reference method for broth dilution antifungal susceptibility testing of yeasts Third Informational Supplement, M27-S3. CLSI, Wayne, PA 16. Pfaller MA, Castanheira M, Messer SA et al (2011) Echinocandin and triazole antifungal susceptibility profiles for Candida spp., Cryptococcus neoformans, and Aspergillus fumigatus: application of new CLSI clinical breakpoints and epidemiologic cutoff values to characterize resistance in the SENTRY Antimicrobial Surveillance Program (2009). Diagn Microbiol Infect Dis 69: Pfaller MA, Diekema DJ, Jones RN et al (2002) Trends in antifungal susceptibility of Candida spp. isolated from pediatric and adult patients with bloodstream infections: SENTRY Antimicrobial Surveillance Program, 1997 to J Clin Microbiol 40: Pfaller MA, oyken L, Hollis RJ et al (2010) Wild-type MIC distributions and epidemiological cutoff values for the echinocandins and Candida spp. J Clin Microbiol 48: Silva AP, Miranda IM, Lisboa C et al (2009) Prevalence, distribution, and antifungal susceptibility profiles of Candida parapsilosis, C. orthopsilosis, and C. metapsilosis in a tertiary care hospital. J Clin Microbiol 47: Jacobson MJ, Piper KE, Nguyen G et al (2008) In vitro activity of anidulafungin against Candida albicans biofilms. Antimicrob Agents Chemother 52: Pemán J, Cantón E, Valentín A (2008) Activity of anidulafungin against Candida biofilms [in Spanish]. Rev Iberoam Micol 25: Katragkou A, Chatzimoschou A, Simitsopoulou M et al (2008) Differential activities of newer antifungal agents against Candida albicans and Candida parapsilosis biofilms. Antimicrob Agents Chemother 52: Garcia-Effron G, Katiyar SK, Park S et al (2008) A naturally occurring proline-to-alanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis accounts for reduced echinocandin susceptibility. Antimicrob Agents Chemother 52: