Abstract
We report the molecular identifications and antifungal susceptibilities of the isolates causing fungemia collected in the CANDIPOP population-based study conducted in 29 Spanish hospitals. A total of 781 isolates (from 767 patients, 14 of them having mixed fungemia) were collected. The species found most frequently were Candida albicans (44.6%), Candida parapsilosis (24.5%), Candida glabrata (13.2%), Candida tropicalis (7.6%), Candida krusei (1.9%), Candida guilliermondii (1.7%), and Candida lusitaniae (1.3%). Other Candida and non-Candida species accounted for approximately 5% of the isolates. The presence of cryptic species was low. Compared to findings of previous studies conducted in Spain, the frequency of C. glabrata has increased. Antifungal susceptibility testing was performed by using EUCAST and CLSI M27-A3 reference procedures; the two methods were comparable. The rate of fluconazole-susceptible isolates was 80%, which appears to be a decrease compared to findings of previous studies, explained mainly by the higher frequency of C. glabrata. Using the species-specific breakpoints and epidemiological cutoff values, the rate of voriconazole and posaconazole in vitro resistance was low (<2%). In the case of C. tropicalis, using the EUCAST procedure, the rate of azole resistance was around 20%. There was a correlation between the previous use of azoles and the presence of fluconazole-resistant isolates. Resistance to echinocandins was very rare (2%), and resistance to amphotericin B also was very uncommon. The sequencing of the hot spot (HS) regions from FKS1 or FKS2 genes in echinocandin-resistant isolates revealed previously described point mutations. The decrease in the susceptibility to fluconazole in Spanish isolates should be closely monitored in future studies.
INTRODUCTION
The incidence of invasive fungal infections has increased in modern hospitals due to the growing number of susceptible patients (1). Fungemia, the most frequent invasive fungal infection, is caused mainly by Candida albicans, although non-albicans Candida species are becoming more prevalent (1–3). Other non-Candida yeasts showing intrinsic antifungal resistance have also been reported (4, 5). Candidemia is commonly difficult to detect in patients with invasive Candida infections; consequently, many patients receive empirical antifungal therapy. Although rapid and prompt starting of antifungal therapy is important in order to improve the outcome for the patients (6), resistance may contribute to treatment failures. Considering the Candida species-specific patterns of antifungal susceptibility, local knowledge of the species causing fungemia and the antifungal resistance rate is essential in order to optimize antifungal treatment.
The distribution of Candida spp. isolated from blood differs among studies conducted in different geographical areas. Candida albicans is the species found most frequently in different nationwide and population-based studies. In contrast, C. glabrata is the second species in terms of frequency in Northern Europe and the USA, whereas C. parapsilosis is more common in Latin America and Southern Europe (1, 7–9). Molecular identification of isolates allows a precise knowledge of the epidemiology of fungemia, since several cryptic species are indistinguishable by using conventional procedures of identification (10). Furthermore, the expanding use of antifungal agents in the clinical setting is another factor affecting the epidemiology of fungemia and also promoting the appearance of antifungal resistance.
Resistance to fluconazole is a matter of concern in the management of candidemia, since patients infected by strains or species exhibiting high fluconazole MIC values have a poor response to the treatment. Several population-based surveys of invasive candidiasis have reported rates of resistance to fluconazole between 10% and 25% (8, 11–13). A population-based study of candidemia that was carried out in Barcelona, Spain, in 2005 (7) reported a rate of fluconazole resistance below 10%, which is similar to that reported in another Spanish prospective sequential study (FUNGEMYCA study), carried out in 2009 (9). Taking into account that the isolates were collected years ago and that epidemiological changes are being detected in some European countries (8, 11), a Spanish nationwide study on fungemia was necessary. For that reason, a multicenter study involving 767 patients with fungemia from 2010 and 2011 admitted to 29 hospitals located in different geographical regions in Spain (CANDIPOP study) was carried out. The epidemiological and clinical characteristics of the patients have been reported elsewhere (14). In this study, we analyzed the species distribution and antifungal susceptibility patterns of the 781 yeast isolates collected in the CANDIPOP study. We performed molecular identification and antifungal susceptibility testing according to both EUCAST and CLSI protocols.
MATERIALS AND METHODS
Patients studied.
A total of 29 Spanish hospitals participated in the study CANDIPOP conducted from 1 May 2010 to 30 April 2011. All patients (n = 767) diagnosed with fungemia and admitted to the above-mentioned hospitals during the study period who consented to participate in the study were recruited. Fungemia was defined as the isolation of yeasts in blood cultures in patients with clinical signs and symptoms of infection.
Strains and molecular identification.
The incident isolate causing the fungemia was studied. A total of 781 isolates were collected (14 patients had polyfungal fungemia caused by two different yeast species). Isolates, which were collected and stored at each participating hospital, were further sent to the Mycology Reference Laboratory at the National Centre for Microbiology (Instituto de Salud Carlos III) in Madrid, Spain. Upon arrival, isolates were grown on Sabouraud dextrose agar and incubated at 30°C. The molecular identification of the isolates was achieved by sequencing the internal transcribed spacer 1 (ITS1) and ITS2 regions from the ribosomal DNA as previously described (15).
Antifungal susceptibility testing.
Antifungal susceptibility testing was performed on the 781 isolates according to the microdilution broth procedures CLSI M27-A3 and EUCAST (EDef 7.1). The following antifungals were used: amphotericin B and flucytosine (Sigma-Aldrich, Madrid, Spain); fluconazole, voriconazole, and anidulafungin (Pfizer Pharmaceutical Group, New York, NY); itraconazole (Janssen Pharmaceutical Research and Development, Madrid, Spain); posaconazole and caspofungin (Merck &Co., Inc., Rahway, NJ); and micafungin (Astellas Pharma, Inc., Tokyo, Japan).
(i) CLSI M27-A3 procedure.
(16). This procedure was carried out at the Clinical Microbiology Department of Gregorio Marañón Hospital, Madrid, Spain. The following ranges of antifungal concentrations were studied: fluconazole, 0.062 to 64 mg/liter; amphotericin B, voriconazole, posaconazole, caspofungin, micafungin, and anidulafungin, (0.0017 to 2 mg/liter). MICs were obtained after 24 h of incubation at 35°C. Cryptococcus spp. and non-Candida isolates showing poor growth were incubated for 72 h. The MIC for amphotericin B was defined as the lowest drug concentration that prevents any discernible growth. The MIC for azoles and echinocandins was considered the lowest drug concentration allowing a reduction of an approximately 50% in growth relative to that of the drug-free growth control.
(ii) EUCAST procedure.
(17). This procedure was carried out at the Mycology Reference Laboratory at the National Centre for Microbiology. The following range of concentrations was studied: amphotericin B, caspofungin, micafungin and anidulafungin, 0.03 to 16 mg/liter; flucytosine and fluconazole, 0.125 to 64 mg/liter; voriconazole, itraconazole, and posaconazole, 0.015 to 8 mg/liter. The optical densities of the plates were measured after 24 h of incubation at 35°C. The MIC was defined as the antifungal concentration that produced 90% (amphotericin B) or 50% (remaining antifungal agents) of growth inhibition relative to that of the drug-free growth control. For strains that showed poor growth, such as some C. neoformans isolates or other nonfermentative species, antifungal susceptibility testing was repeated with shaking and incubation at 30°C for 48 h before determining the optical density.
Amplification of hot spot (HS) regions from FKS genes.
We searched for mutations at the FKS genes (which encode the enzyme β-1,3-glucan synthase) in isolates showing resistance to one or more echinocandins according to the CLSI or EUCAST procedures. Briefly, we amplified the HS1 and HS2 regions of C. albicans (18), C. glabrata (19), and C. tropicalis (20) as previously described. PCR products were sequenced using the same oligonucleotides used in the PCR using the BigDye kit (Applied Biosystems). Sequences were analyzed using SeqMan II and Ediseq software programs (DNAStar, Madison, WI).
Data analysis.
Distribution of MIC values was assessed using SPSS software (IBM SPSS Statistics 21). Antifungal activity was expressed as the MIC90, MIC50, geometric mean MIC, mode, and range of MICs (minimum and maximum). Resistant strains were defined according to the species-specific breakpoints proposed by the EUCAST 6.1 (valid since 11 March 2013) and 5.0 (valid until 1 March 2013), and CLSI M27-S4 documents (17). Antifungal susceptibility testing results for caspofungin should be interpreted with caution due to the interlaboratory variation reported, due possibly to potency variation (21, 22). All isolates showing high MICs were retested and confirmed.
(i) CLSI M27-A3 procedure.
Isolates were classified according to the species-specific breakpoints included in the M27-S4 document for C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, C. krusei, and C. guilliermondii. We used the epidemiological cutoff values (ECVs) to classify as non-wild type isolates for other species-drug combinations in the absence of clinical breakpoints (23). Cryptococcus neoformans isolates were considered fluconazole resistant if they showed an MIC of ≥16 mg/liter (24). In order to make comparable the rates of resistance obtained both by CLSI and EUCAST, we tentatively used the C. albicans breakpoints for classifying the remaining Candida and non-Candida isolates (n = 19) as susceptible or resistant to fluconazole and voriconazole; Rhodotorula species, C. neoformans, and Trichosporon species isolates were considered intrinsically resistant to echinocandins (see Tables 4 and 5).
TABLE 4.
Rates of resistance to amphotericin B, fluconazole, voriconazole, and posaconazole for isolates of species with available breakpoints proposed by the CLSI M27-S3 document and the EUCAST procedurea
| Species (nb) | Category | % of isolates in category for antifungal agent |
|||||
|---|---|---|---|---|---|---|---|
| Fluconazole |
Voriconazole |
Posaconazole |
|||||
| CLSI | EUCAST | CLSI | EUCAST | CLSI | EUCAST | ||
| C. albicans (348) | SDD/I | 0 | 0.3 | 1.2 | 0 | NA | NA |
| R | 1.4 | 0.9 | 0.3 | 0.86 | 0.9 | 0.28 | |
| C. parapsilosis complex (200)c | SDD/I | 3 | 4 | 0.5 | NA | NA | NA |
| R | 2.5 | 0 | 0.5 | 0.5 | 1.5 | ||
| C. glabrata complex (103)d | SDD/I | 90.4 | 94.2 | NA | NA | NA | NA |
| R | 9.6 | 5.8 | 10.2 | NA | 2.9 | NA | |
| C. tropicalis (59) | SDD/I | 1.7 | 0 | 1.7 | 0 | NA | NA |
| R | 1.7 | 22 | 0 | 25.4 | 0 | 18.6 | |
| C. kruseie (15) | SDD/I | NA | NA | 0 | NA | NA | NA |
| R | 100 | 100 | 0 | NA | 6.7 | NA | |
| Overallf | SDD/I | 12.5 | 15.5 | 0.64 | NA | NA | NA |
| R/N-WT | 6 | 6.9 | 1.66 | 9.2 | 1.1 | 12.67 | |
The overall resistance rate also included non-wild-type isolates. NA, not applicable. SDD, susceptible, dose-dependent category, available only for fluconazole and voriconazole, as proposed in the CLSI M27-S4 document. I, intermediate category, available only for EUCAST and fluconazole (MIC = 4 mg/liter, except for C. glabrata, for which MIC > 0.002 to 32 mg/liter). N-WT, non-wild type.
n, no. of isolates.
Isolates of C. metapsilosis (n = 7) and C. orthopsilosis (n = 2) were considered to be C. parapsilosis for analysis of the rate of resistance.
The C. nivariensis isolate was considered to be C. glabrata for analysis of the rate of resistance.
Although EUCAST or CLSI do not provide fluconazole breakpoints for C. krusei, all the strains were considered resistant.
For CLSI, resistance rates were calculated including only the isolates from species with species-specific breakpoints; tentative resistance rates (non-wild-type isolates) were calculated by using the ECVs for the following species-drug combinations: fluconazole (C. lusitaniae, C. guilliiermondii, C. dubliniensis, and C. kefyr), voriconazole (C. glabrata, C. lusitaniae, C. guilliiermondii, C. dubliniensis, and C. kefyr), and posaconazole (C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, C. krusei, C. lusitaniae, C. guilliermondii, C. dubliniensis, and C. kefyr); for the remaining Candida and non-Candida isolates, we tentatively used the C. albicans breakpoints. For EUCAST, tentative fluconazole breakpoints (S < 0.002; I = 0.004; R > 32, in mg/liter) were used for C. guilliermondii; isolates were considered tentatively resistant to voriconazole or posaconazole if showing an MIC > 0.12 mg/liter or > 0.06 mg/ml, respectively; for tentative breakpoints used for non-Candida yeasts, see Materials and Methods.
TABLE 5.
Rates of resistance to caspofungin, micafungin, and anidulafungin of isolates of species with available breakpoints proposed by the CLSI M27-S3 document and the EUCAST procedurea
| Species (nb) | Category | % of isolates in category for antifungal agent |
|||
|---|---|---|---|---|---|
| Caspofungin, CLSIf | Micafungin, CLSI | Anidulafungin |
|||
| CLSI | EUCAST | ||||
| C. albicans (348) | I | 0.3 | 0.3 | 0 | NA |
| R | 0 | 0 | 0 | 0.29 | |
| C. parapsilosis complex (200)c | I | 0 | 0 | 0 | 100 |
| R | 0 | 0 | 0 | 0 | |
| C. glabrata complex (103)d | I | 34.6 | 0 | 0 | NA |
| R | 1.0 | 1.0 | 1.0 | 0.97 | |
| C. tropicalis (59) | I | 1.7 | 0 | 0 | NA |
| R | 0 | 3.3 | 3.3 | 3.39 | |
| C. krusei (15) | I | 6.7 | 0 | 0 | NA |
| R | 0 | 0 | 0 | 0 | |
| C. guilliermondii (13) | I | 0 | 0 | 0 | NA |
| R | 0 | 0 | 0 | ||
| Globale | I | 5.1 | 0 | 0 | 27.1 |
| R | 2.2 | 1.9 | 1.8 | 3.1e | |
The overall resistance rate also included the non-wild-type isolates. I, intermediate category according to the CLSI and EUCAST procedures. NA, not applicable.
n, no. of isolates.
Isolates of C. metapsilosis (n = 7) and C. orthopsilosis (n = 2) were considered to be C. parapsilosis for analysis of the rate of resistance.
The C. nivariensis isolate was considered to be C. glabrata for analysis of the rate of resistance.
For CLSI, tentative rates of resistance to echinocandins (non-wild-type isolates) were calculated by using the ECVs for C. lusitaniae, C. guilliermondii, C. dubliniensis, and C. kefyr; In order to make comparable the rate of resistance obtained both by CLSI and EUCAST, we tentatively used the C. albicans breakpoints (R > 4 mg/liter) for classifying the remaining Candida and non-Candida isolates (n = 19) as susceptible or resistant to fluconazole and voriconazole; Rhodotorula species, C. neoformans, and Trichosporon species isolates were considered intrinsically resistant to echinocandins. For EUCAST, the isolates were considered resistant if showing an MIC > 0.06 mg/liter, except for C. albicans (R > 0.03 mg/liter) and species from the C. parapsilosis complex (R > 4).
(ii) EUCAST procedure.
The following breakpoints were used to classify isolates as resistant (R) or intermediate (I): for amphotericin, R, >1 mg/liter; for fluconazole, sensitive [S], ≤2 mg/liter; R, >4 mg/liter, for all Candida spp. with the exception of C. glabrata and tentatively for C. guilliermondii (S, ≤0.002 mg/liter; I, 0.004 to 32 mg/liter; R, >32 mg/liter); for voriconazole, R, >0.12 mg/liter; for posaconazole, R, >0.06 mg/liter; for anidulafungin against C. albicans, R, > 0.03 mg/liter]; against C. glabrata, C. tropicalis, and C. krusei, R, >0.06 mg/liter; against C. parapsilosis, C. orthopsilosis, C. metapsilosis, and C. guilliermondii, S, ≤0.002 mg/liter; I, 0.004 to 4 mg/liter,; R, >4 mg/liter. MIC values of echinocandins were interpreted according to breakpoints published recently in the EUCAST webpage (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Antifungal_breakpoints_v_6.1.pdf). There are no species-specific breakpoints for C. guilliermondii and fluconazole or anidulafungin. According to the MIC distribution of both agents and the presence of a polymorphism in the FKS genes (M instead L in the second amino acid of HS1 of C. guilliermondii and A instead P in the last amino acid of the HS1 of C. parapsilosis), we used the C. glabrata (for fluconazole) or C. parapsilosis (for anidulafungin) breakpoints as tentative epidemiological cutoff values (ECOFFs) for both drugs (http://www.eucast.org/antifungal_susceptibility_testing_afst/rationale_documents_for_antifungals/). For non-Candida species, the Candida albicans breakpoints were used as tentative ECOFFs to classify them as non-wild type.
(iii) Impact of the use of antifungal agents on the promotion of antifungal resistance.
We also evaluated the impact of the use of azoles or echinocandins in the previous month with diagnosis of fungemia on the proportion of episodes caused by resistant or non-wild-type isolates (defined by the CLSI or EUCAST procedures). The chi-square test was used to study the correlation between previous antifungal use and the presence of resistant isolates.
RESULTS
Species distribution.
We studied a total of 781 clinical isolates from 767 patients, with 14 of them having candidemia caused by two different species. As shown in Table 1, C. albicans was the most frequently isolated species, accounting for 44.6% of episodes, followed by C. parapsilosis (24.5%), C. glabrata (13.2%), and C. tropicalis (7.6%). Other Candida species, such as C. krusei, C. guilliermondii, or C. lusitaniae, represented approximately 2% of the isolates each. We observed differences in species distribution among the 5 participating Spanish regions. For example, the frequency of C. albicans was higher in Madrid and Barcelona; in contrast, the frequency of C. parapsilosis was lower in both cities. Differences in the frequencies of C. tropicalis and C. glabrata were also observed (14).
TABLE 1.
Species distribution of the 781 isolates identified after amplification and further sequencing of the ITS1-5.8S-ITS2 region
| Species | No. (%) of isolates |
|---|---|
| Candida albicans | 348 (44.6) |
| Candida parapsilosis | 191 (24.5) |
| Candida glabrata | 103 (13.2) |
| Candida tropicalis | 59 (7.6) |
| Candida krusei | 15 (1.9) |
| Candida guilliermondii | 13 (1.7) |
| Candida lusitaniae | 10 (1.3) |
| Candida orthopsilosis | 7 (0.9) |
| Cryptococcus neoformans | 5 (0.6) |
| Candida kefyr | 4 (0.5) |
| Candida dubliniensis | 4 (0.5) |
| Candida lipolytica | 4 (0.5) |
| Dipodascus capitatus | 3 (0.4) |
| Trichosporon asahii | 3 (0.4) |
| Candida metapsilosis | 2 (0.3) |
| Pichia anomala | 2 (0.3) |
| Rhodotorula mucilaginosa | 2 (0.3) |
| Candida intermedia | 1 (0.1) |
| Kodamea ohmeri | 1(0.1) |
| Lodderomyces elongisporus | 1(0.1) |
| C. nivariensis | 1(0.1) |
| Pichia fabianii | 1(0.1) |
| Metschnikowia pulcherrima | 1(0.1) |
| Total | 781 (100) |
The use of identification based on molecular biology techniques (ITS sequencing) allowed a proper detection of cryptic and rare species, such as C. dubliniensis (normally misidentified as C. albicans), C. orthopsilosis and C. metapsilosis (normally misidentified as C. parapsilosis), and C. nivariensis (normally misidentified as C. glabrata). A total of 20 strains were misidentified by local methods compared to identification by ITS sequencing. The most frequent discrepancies occurred in the C. parapsilosis complex (C. orthopsilosis [6 out of 7 strains] and C. metapsilosis [2 out of 2 strains] were confused with C. parapsilosis) and C. albicans complex (C. dubliniensis [3 out of 4 strains] was misidentified as C. albicans). Finally, 2 out of 13 C. guilliermondii strains were misidentified as C. famata. We found that the frequency of these cryptic species was low. The remaining isolates comprised a group of 16 rare species and accounted for around 5% of the isolates.
Antifungal susceptibility testing results.
We performed antifungal susceptibility testing on the 781 isolates using EUCAST and CLSI protocols in two different institutions. The antifungal activities of the agents studied are shown in Table 2 and Table 3. We first focused on the susceptibility profile of the most prevalent species (C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, and C. krusei). As shown in Table 2, all isolates were susceptible to amphotericin B, C. krusei being the species showing the lowest susceptibility by both EUCAST and CLSI methods (4- to 5-fold-decreased susceptibility compared to that of C. albicans). CLSI tended to provide higher amphotericin B MICs than EUCAST. In the case of the echinocandins, the EUCAST method provided slightly higher MIC values than CLSI for caspofungin (geometric mean [GM] around 2 to 3 times higher), although the different ranges of concentrations of echinocandins studied by CLSI or EUCAST may explain the differences. Concerning fluconazole, MIC values obtained by the two methods were comparable, with the exception of those for C. glabrata and C. tropicalis. The intrinsic diminished susceptibility to azoles of C. glabrata is well known, but this feature was more pronounced when antifungal susceptibility was assessed using the CLSI method (GM for fluconazole, 10 mg/liter; mode, 8 mg/liter) than with the EUCAST method (geometric mean, 3.07 mg/liter; mode, 2 mg/liter). We observed the opposite with C. tropicalis, which showed a lower susceptibility to fluconazole with the EUCAST method than with the CLSI method. The antifungal resistance of rare species causing fungemia is shown in Tables S1 and S2 in the supplemental material.
TABLE 2.
Antifungal activities of amphotericin B, flucytosine, caspofungin, micafungin, and anidulafungin against the 781 isolates collecteda
| Species (nb) | Statistic | Antifungal activity (in mg/liter) |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Amphotericin B |
Flucytosine, EUCAST | Caspofungin |
Micafungin |
Anidulafungin |
||||||
| EUCAST | CLSI | EUCAST | CLSI | EUCAST | CLSI | EUCAST | CLSI | |||
| C. albicans (348) | GM | 0.051 | 0.15 | 0.16 | 0.31 | 0.09 | 0.03 | 0.007 | 0.03 | 0.005 |
| Mode | 0.03 | 0.12 | 0.12 | 0.25 | 0.12 | 0.03 | 0.007 | 0.03 | 0.007 | |
| MIC90 | 0.12 | 0.12 | 0.5 | 0.5 | 0.12 | 0.03 | 0.03 | 0.03 | 0.007 | |
| Range | ≤0.03–0.25 | 0.015–1 | ≤0.12–32 | 0.12–2 | 0.015–0.5 | ≤0.03–1 | 0.0017–1 | ≤0.03–0.25 | 0.0017–0.25 | |
| C. parapsilosis (191) | GM | 0.14 | 0.14 | 0.49 | 1.36 | 0.23 | 0.84 | 0.45 | 0.93 | 0.89 |
| Mode | 0.12 | 0.12 | 0.12 | 1 | 0.25 | 1 | 0.5 | 1 | 1 | |
| MIC90 | 0.25 | 0.25 | 0.25 | 2 | 0.5 | 2 | 1 | 2/2 | 2 | |
| Range | ≤0.03–1 | 0.03–1 | ≤0.12–0.5 | 0.5–4 | 0.003–1 | 0.25–4 | 0.0017–1 | 0.12–4 | 0.0017–2 | |
| C. glabrata (103) | GM | 0.106 | 0.19 | 0.133 | 0.44 | 0.15 | 0.031 | 0.016 | 0.031 | 0.032 |
| Mode | 0.12 | 0.25 | 0.12 | 0.5 | 0.12 | 0.03 | 0.015 | 0.03 | 0.03 | |
| MIC90 | 0.25 | 0.5 | 0.12 | 1 | 0.25 | 0.03 | 0.03 | 0.03 | 0.06 | |
| Range | ≤0.03–0.5 | 0.03–1 | ≤0.12–≥64 | 0.25–1 | 0.007–>2 | ≤0.03–1 | 0.007–2 | ≤0.03–0.5 | 0.007–1 | |
| C. tropicalis (59) | GM | 0.079 | 0.22 | 0.154 | 0.41 | 0.12 | 0.034 | 0.021 | 0.034 | 0.014 |
| Mode | 0.06 | 0.25 | 0.12 | 0.5 | 0.12 | 0.03 | 0.015 | 0.03 | 0.015 | |
| MIC90 | 0.12 | 0.5 | 0.12 | 0.5 | 0.25 | 0.03 | 0.06 | 0.03 | 0.03 | |
| Range | ≤0.03–0·5 | 0.03–1 | ≤0.12–32 | 0.12 −2 | 0.015–0.5 | ≤0.03–2 | 0.003–1 | ≤0.03–1 | 0.0017–2 | |
| C. krusei (15) | GM | 0.236 | 0.5 | 3.48 | 0.87 | 0.24 | 0.08 | 0.097 | 0.034 | 0.053 |
| Mode | 0.25 | 0.5 | 4 | 1 | 0.25 | 0.06 | 0.12 | 0.03 | 0.06 | |
| MIC90 | 0.5 | 1 | 4 | 1 | 0.25 | 0.12 | 0.25 | 0.03 | 0.12 | |
| Range | ≤0.12–0.5 | 0.12–2 | 2–4 | 0.5–2 | 0.12–0.5 | 0.06–0.12 | 0.003–0.25 | ≤0.03–0.06 | 0.007–0.12 | |
| C. guilliermondii (13) | GM | 0.067 | 0.17 | 0.127 | 0.9 | 0.28 | 0.33 | 0.21 | 0.5 | 1 |
| Mode | 0.12 | 0.12 | 0.12 | 1 | 0.25 | 0.25 | 0.25 | 0.5 | 1 | |
| MIC90 | 0.12 | 0.5 | 0.25 | 2 | 0.5 | 0.5 | 1 | 1 | 2 | |
| Range | ≤0.03–0.12 | 0.06–0.5 | ≤0.12–0.25 | 0.5–2 | 0.25–0.5 | 0.25–0.5 | 0.003–1 | 0.25–1 | 0.5–2 | |
| C. lusitaniae (10) | GM | 0.06 | 0.1 | 0.287 | 0.92 | 0.16 | 0.05 | 0.039 | 0.036 | 0.047 |
| Mode | 0.03 | 0.06 | 0.12 | 1 | 0.25 | 0.06 | 0.06 | 0.03 | 0.06 | |
| MIC90 | 0.25 | 0.25 | >64 | 1 | 0.25 | 0.06 | 0.06 | 0.06 | 0.06 | |
| Range | ≤0.03–0.25 | 0.06–0.5 | ≤0.12–≥64 | 0.5–1 | 0.06–0.25 | ≤0.03–0.06 | 0.003–0.06 | ≤0.03–0.12 | 0.015–0.06 | |
| Other (781) | GM | 0.12 | 0.17 | 0.66 | 1.86 | 0.27 | 0.52 | 0.11 | 0.47 | 0.14 |
| Mode | 0.03 | 0.12 | 0.12 | 0.25 | 0.12 | 0.03 | 0.062 | 0.03 | 0.03 | |
| MIC90 | 0.5 | 1 | 64 | >16 | >2 | >16 | 2 | >16 | >2 | |
| Range | ≤0.03–2 | 0.01–>2 | ≤0.12–≥64 | ≤0.12–≥16 | 0.03–>2 | ≤0.03–≥16 | 0.0017–2 | 0.03–≥16 | 0.0017–>2 | |
“Other” includes the remaining Candida and non-Candida species. GM, geometric mean.
n, no. of isolates.
TABLE 3.
Antifungal activities of fluconazole, itraconazole, voriconazole, and posaconazole against the 781 isolates collecteda
| Species (nb) | Statistic | Antifungal activity (mg/liter) |
||||||
|---|---|---|---|---|---|---|---|---|
| Fluconazole |
Itraconazole, EUCAST | Voriconazole |
Posaconazole |
|||||
| EUCAST | CLSI | EUCAST | CLSI | EUCAST | CLSI | |||
| C. albicans (348) | GM | 0.21 | 0.39 | 0.016 | 0.016 | 0.009 | 0.015 | 0.006 |
| Mode | 0.25 | 0.25 | 0.015 | 0.015 | 0.007 | 0.015 | 0.007 | |
| MIC90 | 0·25 | 1 | 0·015 | 0.015 | 0.03 | 0·015 | 0.015 | |
| Range | ≤0.12–≥64 | 0.12–>64 | ≤0.015–≥8 | ≤0.015–≥8 | 0.0017–>2 | ≤0.015–8 | 0.0017–>2 | |
| C. parapsilosis (191) | GM | 0.48 | 0.64 | 0.018 | 0.017 | 0.008 | 0.016 | 0.008 |
| Mode | 0.5 | 0.5 | 0.015 | 0.015 | 0.007 | 0.015 | 0.007 | |
| MIC90 | 1 | 2 | 0.03 | 0.03 | 0.015 | 0.015 | 0.031 | |
| Range | ≤0.12–64 | 0.06–64 | ≤0.015–0.25 | ≤0.015–0.5 | 0.0017–0.25 | ≤0.015–0·12 | 0.0017–>2 | |
| C. glabrata (103) | GM | 3.074 | 10 | 0.127 | 0.12 | 0.11 | 0.124 | 0.25 |
| Mode | 2 | 8 | 0.06 | 0.12 | 0.12 | 0.12 | 0.25 | |
| MIC90 | 16 | 64 | 1 | 0.5 | 0.5 | 0.5 | 1 | |
| Range | 0.5–≥64 | 1–>64 | ≤0.015–≥8 | ≤0.015–8 | 0.003–>2 | ≤0.015–8 | 0.015–>2 | |
| C. tropicalis (59) | GM | 1.83 | 0.71 | 0.057 | 0.13 | 0.026 | 0.047 | 0.023 |
| Mode | 0.5 | 1 | 0.015 | 0.03 | 0.03 | 0.015 | 0.03 | |
| MIC90 | >64 | 1 | 8 | >8 | 0.06 | 8 | 0.06 | |
| Range | ≤0.12–≥64 | 0.12–8 | ≤0.015–≥8 | ≤0.015–≥8 | 0.003–0.25 | ≤0.015–≥8 | 0.0017–0.12 | |
| C. krusei (15) | GM | 33.5 | 38.5 | 0.066 | 0.3 | 0.15 | 0.048 | 0.1 |
| Mode | 32 | 32 | 0.12 | 0.5 | 0.12 | 0.06 | 0.12 | |
| MIC90 | 64 | 64 | 0.25 | 0.5 | 0.25 | 0.12 | 1 | |
| Range | 16–64 | 16–64 | ≤0.015–0.25 | ≤0.015–1 | 0.03–0.25 | ≤0.015–0.12 | 0.015–2 | |
| C. guilliermondii (13) | GM | 3.22 | 5.51 | 0.22 | 0.12 | 0.061 | 0.075 | 0.054 |
| Mode | 4 | 8 | 0.12 | 0.12 | 0.031 | 0.06 | 0.12 | |
| MIC90 | 16 | 32 | 1 | 0.25 | 0.5 | 0.25 | 0.25 | |
| Range | ≤0.12–32 | 0.5–64 | 0.06–2 | 0.06–0.25 | 0.007–0.5 | ≤0.015–0.25 | 0.003–0.25 | |
| C. lusitaniae (10) | GM | 0.45 | 1.19 | 0.02 | 0.023 | 0.01 | 0.018 | 0.009 |
| Mode | 0.25 | 0.5 | 0.015 | 0.015 | 0.007 | 0.015 | 0.003 | |
| MIC90 | 32 | 32 | 0.12 | 0.5 | 0.12 | 0.06 | 0.03 | |
| Range | ≤0.12–64 | 0.5–64 | ≤0.015–0.25 | ≤0.015–0.5 | 0.003–0.25 | ≤0.015–0.06 | 0.003–0.031 | |
| Other (781) | GM | 1.92 | 1 | 0.066 | 0.07 | 0.017 | 0.061 | 0.029 |
| Mode | 0.5 | 1 | 0.015 | 0.015 | 0.007 | 0.015 | 0.015 | |
| MIC90 | >64 | 64 | 2 | 4 | 1/2 | 2 | 0.5/0.5 | |
| Range | ≤0.12–≥64 | 0.06–>64 | ≤0.015–4 | ≤0.015–≥8 | 0.0017–>2 | ≤0.015–≥8 | 0.0017–>2 | |
“Other” includes the remaining Candida and non-Candida species. GM, geometric mean.
n, no. of isolates.
The rate of resistance or the proportion of non-wild-type isolates for each antifungal for the most frequent Candida spp. is shown in Tables 4 and 5. Although overall fluconazole resistance was low regardless of the method used, sharp differences were found among the species. Resistance in C. albicans or C. parapsilosis was low, whereas the rate of fluconazole resistance or the proportion of non-wild-type isolates was higher in the case of C. glabrata (6 to 10%) or C. guilliermondii (23%). For C. tropicalis, we found differences in the rate of fluconazole resistance obtained by the CLSI procedure (1.7%) or the EUCAST method (22%). High rates of resistance in C. tropicalis for the newest triazoles, voriconazole (25.4%) and posaconazole (18.6%), when MICs were obtained by the EUCAST method were also observed. In contrast, the rate of resistance for the new triazoles was low when antifungal susceptibility testing was assessed using the CLSI protocol. When we analyzed the overall susceptibility to fluconazole in different geographical regions, the rate of resistance or non-wild-type isolates ranged from 3.7% to 9.2% regardless the method used (Table 6).
TABLE 6.
Percentages of fluconazole resistance found in the isolates studieda
| City | % of isolates in category |
|||
|---|---|---|---|---|
| CLSI |
EUCAST |
|||
| SDD | R | I | R | |
| Madrid | 14.4 | 5.9 | 17.4 | 5.1 |
| Valencia | 15 | 7.5 | 14.3 | 9.2 |
| Bilbao | 9.5 | 8.3 | 10.4 | 5.8 |
| Seville | 10.2 | 3.7 | 11.4 | 8.8 |
| Barcelona | 12.1 | 6.0 | 12.1 | 9 |
| Overall | 12.5 | 6.0 | 14.8 | 6.8 |
Overall and per-city percentages are shown. Resistance rates were calculated as described in Materials and Methods. SDD, susceptible, dose dependent.
Only one Candida isolate showed acquired resistance to amphotericin B. As expected, resistance to echinocandins was also very infrequent. Only 1 isolate of C. albicans, 2 of C. tropicalis, and 1 of C. glabrata were resistant to one or more echinocandins. After sequencing the HS regions from the FKS1 or FKS2 genes of the isolates, the following previously described point mutations conferring resistance were found: C. albicans, F641S mutation in the FKS1 gene; C. glabrata, deletion of the F659 amino acid from FKS2; and C. tropicalis, F641L and S645F substitutions in FKS1 of each isolate.
Impact of previous use of antifungal agents on rate of resistance.
We observed that the proportion of episodes caused by fluconazole-resistant or non-wild-type isolates was higher among the patients who previously received azoles (14.3% versus 3.5%; P < 0.01), an effect that remained when the fluconazole dose-dependent isolates were also considered resistant (29.3% versus 16.4%; P < 0.01). Likewise, the rate of resistance to echinocandins was also higher among the patients who previously received echinocandins (10% versus 1.8%; P < 0.01). The four patients infected by the isolates showing FKS mutations had received antifungal treatment with echinocandins for more than 21 days in the 5 weeks prior to the isolation of the yeast from the blood culture, and in fact, one of these cases was considered a breakthrough infection during prophylaxis with micafungin.
DISCUSSION
At the best of our knowledge, CANDIPOP is the first population-based study on fungemia conducted in Spain that involved the largest cities of the country. The species distribution and antifungal susceptibility profiles of the isolates collected in five different geographical regions were studied.
We found that C. albicans was still the most prevalent species but represented less than 50% of the episodes. Regarding the non-albicans Candida isolates, the study confirms that the epidemiology of fungemia in Spain has a characteristic pattern, with a high proportion of cases caused by C. parapsilosis and a low proportion of C. glabrata cases. This pattern clearly differs from that found in Northern Europe and North America (8, 12) and is similar to findings of single-center studies carried out in Italy (25) or Greece (26) or in population-based studies conducted in Latin America (27, 28). The reasons that may explain the differences are not clear but may involve differences in climate, antifungal policies, or other circumstances that may facilitate the presence of some species in the microbiota of patients living in different geographical areas. Interestingly, our study showed differences in the species distribution in the different participating cities. As an example, Madrid and Barcelona showed similar patterns of species distribution, whereas the other three cities had another pattern (14). It suggests specific local patterns of species distribution that should be considered when empirical antifungal therapy is starting.
The results can be compared only with those of the two previous studies conducted in Spain 10 years ago (7, 9). We found a slightly lower proportion of cases caused by C. albicans and a higher proportion of cases caused by C. glabrata than in the study by Almirante or the study by Pemán. This may reflect a change in the epidemiology of the infection; however, the different patterns of species distribution among the participating cities may explain the differences. We can consider CANDIPOP to be the first population-based study involving the largest cities of the country, thus becoming a comparator for future studies of fungemia in Spain. Nevertheless, the trend of the frequency of isolation of C. glabrata should be closely monitored in the coming years.
The number of cases caused by rare species (defined as those found at a frequency below 1%) was low and represented less than 6% of all isolates collected. However, this heterogeneous group involved a relatively high number of species, such as Pichia anomala, Candida intermedia, Kodamea ohmeri, Lodderomyces elongisporus, Pichia fabianii, Metschnikowia pulcherrima, Rhodotorula mucilaginosa, Dipodascus capitatus, and Trichosporon spp., which have been very rarely described as causing fungemia (4, 5). Although they represented a low proportion of isolates, the guidelines for the management of patients with candidemia usually do not add specific information for the treatment of these infections (29, 30). Therefore, the management of patients infected by rare species is mostly unknown and is based on single case reports. C. neoformans fungemia was infrequent, and only 5 patients with cryptococcemia were found. However, this underrepresents the real number of cases of cryptococcosis, since we included only strains isolated from the blood and not other type of clinical samples, such as cerebrospinal fluid, where C. neoformans is more frequently isolated.
In addition to the species distribution, this study allowed us to estimate the rate of antifungal resistance in Spain. Overall, 20% of isolates showed diminished susceptibility to fluconazole (resistant or dose-dependent/intermediate isolates, and non-wild-type isolates). The percentage is higher than the 7% of isolates with reduced susceptibility to fluconazole reported by Almirante et al. (7). Although we cannot exclude that the rate of acquired fluconazole resistance may have risen, other factors, such as the increase in the frequency of isolation C. glabrata, the inclusion of non-Candida isolates (which were excluded in the Barcelona's study), and the fact the fluconazole breakpoints proposed by the CLSI have been lowered, may contribute to this effect. In the analysis of resistance per species, we observed a low frequency of fluconazole resistance in the most frequent species (<2% in C. albicans and <3.5% in C. parapsilosis). This is in agreement with recent reports of studies conducted in Spain (9), Latin America (27), North America (12), and Denmark (8), thus emphasizing that the changing epidemiology of fungemia is contributing more to fluconazole resistance than secondary acquired resistance. In fact, different rates of fluconazole resistance were observed among the different participating cities that are in line with the different patterns of species distribution observed.
Both CLSI and EUCAST procedures were used in the study, and the rates of antifungal resistance observed by using each method were similar for most of species. However, we found a higher rate of fluconazole resistance for C. tropicalis when the MICs were obtained by EUCAST. In isolates showing resistance by EUCAST and susceptibility by CLSI, a strong trailing effect was observed (data not shown), and growth inhibition was below 50% compared to that for the control well without fluconazole. The molecular basis of this trailing effect is not known, but it did not seem to have a clinical impact, since patients infected by resistant isolates as determined by the EUCAST method had mortality similar to that of patients infected by susceptible isolates (data not shown). Moreover, there was not a correlation between the obtaining of a resistant C. tropicalis isolate and the prophylactic treatment (data not shown), indicating that trailing is an in vitro effect without clinical impact. In any case, this effect led to a discrepancy between the two methods for C. tropicalis.
Resistance to echinocandins was below 2% regardless the method used. Recently, EUCAST has updated the breakpoints to detect resistance to anidulafungin and micafungin. All resistant Candida species strains detected by the EUCAST and CLSI methods contained already-known mutations in the FKS genes (31–34). In our study, the main antifungal administered both in prophylaxis and as empirical treatment was fluconazole (14), which might explain why the rate of resistance to echinocandins still remains low. However, taking into account the recent ESCMID clinical-guideline recommendation to use an echinocandin for the treatment of most of cases of candidiasis (30), epidemiological studies to assess echinocandin resistance in the future are warranted. We found a high number of C. glabrata isolates intermediate to caspofungin when MICs were obtained by the CLSI method. However, this was not the case for anidulafungin and micafungin. We do not think that these isolates may have a reduced susceptibility to echinocandins, and a potential explanation may be the variability in the potencies of different stocks of pure solutions of caspofungin, as detailed in the CLSI M27-S4 supplement (21, 22, 35). For this reason, neither EUCAST nor CLSI procedures recommend the use of caspofungin for antifungal susceptibility testing.
Our rates of fluconazole and echinocandin resistance are in agreement with recent reported data including antifungal susceptibilities of a large number of isolates collected all over the world in the SENTRY program and collected during the same period in which CANDIPOP was conducted (3).
In conclusion, our study shows that the epidemiology of fungemia in Spain has a characteristic pattern showing a high proportion of cases caused by C. parapsilosis. However, an increase in the frequency of C. glabrata has been detected compared to results of previous studies conducted in Spain. Resistance to azoles and echinocandins was low, although overall susceptibility to fluconazole has decreased, probably due to the emergence of C. glabrata. The decrease in susceptibility to fluconazole in Spanish isolates should be closely monitored in future studies.
Supplementary Material
ACKNOWLEDGMENTS
M.C.-E. has received grant support from Astellas Pharma, bioMérieux, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Schering Plough, Soria Melguizo SA, Ferrer International, the European Union, the ALBAN program, the Spanish Agency for International Cooperation, the Spanish Ministry of Culture and Education, The Spanish Health Research Fund, The Instituto de Salud Carlos III, The Ramon Areces Foundation, and The Mutua Madrileña Foundation. He has been an advisor/consultant to the Panamerican Health Organization, Astellas Pharma, Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough. He has been paid for talks on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, Astellas Pharma, and Schering Plough. J.G. has received grant support from Astellas Pharma, MICOLAB, Fundación Mutua Madrileña, and the Spanish Health Research Fund (FIS). He has been paid for talks on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, Astellas Pharma, Hickma Pharmaceutica, and United Medical. J.P. has received grant support from Astellas Pharma, Pfizer, and the Spanish Health Research Fund (FIS). He has been paid for talks on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Astellas Pharma. E.M.-M. has been paid for talks on behalf of Astellas Pharma and Merck Sharp and Dohme.
J.G. (MS09/00055) and P.E. (CD09/00230) are supported by the Fondo de Investigación Sanitaria (FIS). This work received funding from Gilead, MSD, Astellas, Pfizer, Fundación SEIMC-GESIDA, the European Regional Development Fund (ERDF) (A way to achieve Europe), and the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015).
Members of the CANDIPOP project are as follows: Jesús Guinea, Patricia Muñoz, and Belén Padilla (Hospital General Universitario Gregorio Marañón), Manuel Cuenca-Estrella and Óscar Zaragoza, (Spanish National Center for Microbiology), José Ramón Paño, Julio García Rodríguez, Ana Méndez, and Carlos García Cerrada (Hospital La Paz, Madrid, Spain), Jesús Fortún, Pilar Martín, and Elia Gómez (Hospital Ramón y Cajal, Madrid), Pablo Ryan and Carolina Campelo (Hospital Infanta Leonor, Madrid), Ignacio de los Santos and Buenaventura Buendía (Hospital Universitario La Princesa, Madrid), Beatriz Pérez and Mercedes Alonso (Hospital Niño Jesús, Madrid), José María Aguado and Francisca Sanz (Hospital 12 de Octubre, Madrid), Paloma Merino and Fernando González (Hospital Clínico, Madrid), Miguel Górgolas and Ignacio Gadea (Fundación Jiménez Díaz, Madrid), Juan Emilio Losa and Alberto Delgado-Iribarren (Hospital de Alcorcón, Madrid), Antonio Ramos, Yolanda Romero, and Isabel Sánchez (Hospital Majadahonda, Madrid), Jesús Rodríguez-Baño and Ana Isabel Suarez (Hospital Universitario Virgen Macarena, Seville, Spain), Ana Loza, Estrella Martín-Mazuelos, and Ana Isabel Aller (Hospital Universitario Virgen de Valme, Seville), Maite Ruiz and José Garnacho-Montero (Hospital Universitario Virgen del Rocío, Seville), Carlos Ortiz (Hospital Sagrado Corazón, Seville), Mónica Chávez and Fernando L. Maroto (Hospital San Juan de Dios de Aljarafe, Seville), Miguel Salavert and Javier Pemán (Hospital Universitario la Fe, Valencia, Spain), José Blanquer and David Navarro (Hospital Clínico Universitario Valencia), Vicente Abril and Concepción Gimeno (Consorcio Hospital General Universitario de Valencia), Juan José Camarena and Rafael Zaragoza (Hospital Universitario Dr. Peset, Valencia), Silvia Hernáez and Guillermo Ezpeleta (Hospital de Basurto, Bilbao, Spain), Elena Bereciartua, José Miguel Montejo, and José L. Hernández (Hospital Universitario de Cruces, Bilbao), Rosa Ana Rivas and Rafael Ayarza (Hospital de Galdakano, Bilbao), Benito Almirante, Isabel Ruiz Camps, and Ana M. Planes (Hospital Universitari Vall d′Hebron, Barcelona, Spain), José Mensa and Manel Almela (Hospital Clínic-IDIBAPS Barcelona), Mercè Gurgui and Ferran Sánchez-Reus (Hospital de la Santa Creu i Sant Pau, Barcelona), Joaquín Martinez-Montauti and Montserrat Sierra (Hospital de Barcelona, Barcelona), Juan Pablo Horcajada, Luisa Sorli and Julià Gómez (Hospital del Mar, Barcelona), and Amadeu Gené and Mireia Urrea (Hospital Sant Joan de Déu, Esplugues de Llobregat-Barcelona). Study collaborators are Mireia Puig-Asensio, Maricela Valerio, Mario Fernández-Ruiz, Ana Díaz-Martín, Francesc Puchades, and Alessandra Mularoni.
Footnotes
Published ahead of print 23 December 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02155-13.
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