Juan L. Rodriguez-Tudela

Epidemiology and Predictors of Mortality in Cases of Candida Bloodstream Infection: Results from Population-Based Surveillance, Barcelona, Spain, from 2002 to 2003

ABSTRACT We conducted population-based surveillance for Candida bloodstream infections in Spain to determine its incidence, the extent of antifungal resistance, and risk factors for mortality. A case was defined as the first positive blood culture for any Candida spp. in a resident of Barcelona, from 1 January 2002 to 31 December 2003. We defined early mortality as occurring between days 3 to 7 after candidemia and late mortality as occurring between days 8 to 30. We detected 345 cases of candidemia, for an average annual incidence of 4.3 cases/100,000 population, 0.53 cases/1,000 hospital discharges, and 0.73 cases/10,000 patient-days. Outpatients comprised 11% of the cases, and 89% had a central venous catheter (CVC) at diagnosis. Overall mortality was 44%. Candida albicans was the most frequent species (51% of cases), followed by Candida parapsilosis (23%), Candida tropicalis (10%), Candida glabrata (8%), Candida krusei (4%), and other species (3%). Twenty-four isolates (7%) had decreased susceptibility to fluconazole (MIC ≥ 16 μg/ml). On multivariable analysis, early death was independently associated with hematological malignancy (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.1 to 10.4). Treatment with antifungals (OR, 0.05; 95% CI, 0.01 to 0.2) and removal of CVCs (OR, 0.3; 95% CI, 0.1 to 0.9) were protective factors for early death. Receiving adequate treatment, defined as having CVCs removed and administration of an antifungal medication (OR, 0.2; 95% CI, 0.08 to 0.8), was associated with lower odds of late mortality; intubation (OR, 7.5; 95% CI, 2.6 to 21.1) was associated with higher odds. The incidence of candidemia and prevalence of fluconazole resistance are similar to other European countries, indicating that routine antifungal susceptibility testing is not warranted. Antifungal medication and catheter removal are critical in preventing mortality.

Head-to-Head Comparison of the Activities of Currently Available Antifungal Agents against 3,378 Spanish Clinical Isolates of Yeasts and Filamentous Fungi

ABSTRACT We have compared the activities of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and filamentous fungi. A total of 1,997 clinical isolates of Candida spp., 359 of other yeast species, 697 strains of Aspergillus spp., and 325 nondermatophyte non-Aspergillus spp. were included. The average geometric means of the MICs of agents that were tested against Candida spp. were 0.23 μg/ml for amphotericin B, 0.29 μg/ml for flucytosine, 0.97 μg/ml for fluconazole, 0.07 μg/ml for itraconazole, 0.04 μg/ml for voriconazole, 0.15 μg/ml for caspofungin, and 0.03 μg/ml for posaconazole. Voriconazole and posaconazole were active in vitro against the majority of isolates, with resistance to fluconazole and itraconazole, and against Cryptococcus neoformans and other Basidiomycota yeasts. Posaconazole was the most active of antifungal agents tested against Aspergillus spp., with an average geometric mean of 0.10 μg/ml. It was active against Paecilomyces spp., Penicillium spp., Scedosporium apiospermum, and some black fungi, such as Alternaria spp. Multiresistant filamentous fungi, such as Scedosporium prolificans, Scopulariopsis brevicaulis, and Fusarium solani, were also resistant to voriconazole, caspofungin, and posaconazole. Amphotericin B and posaconazole were found to be active against most of the Mucorales strains tested. Posaconazole and currently available antifungal agents exhibit a potent activity in vitro against the majority of pathogenic fungal species.

Scedosporium apiospermum: changing clinical spectrum of a therapy-refractory opportunist

Current knowledge on the opportunist Scedosporium apiospermum (teleomorph: Pseudallescheria boydii), generated over a period of more than 120 years, is reviewed. The natural environmental habitat of the fungus is unknown; nutrient-rich, brackish waters like river estuaria have been suggested. The fungus is strongly promoted by agricultural and particularly by industrial pollution.

DEEP INFECTIONS CAUSED BY SCEDOSPORIUM PROLIFICANS : A REPORT ON 16 CASES IN SPAIN AND A REVIEW OF THE LITERATURE

Scedosporium prolificans, a mold morphologically similar to Scedosporium apiospermum, may cause asymptomatic colonization or localized or disseminated infection following trauma, surgery, and immunosuppression. S. prolificans is normally resistant to available antifungal agents, and prognosis depends largely on the hosts immune status, extent of infection, and feasibility of surgical debridement. We report on 16 patients with deep S. prolificans infections, focusing on predisposing factors, clinical characteristics, outcome, postmortem findings, and antifungal susceptibility testing to 6 antifungal agents. Between 1989 and 1994, 16 cases of deep infections by S. prolificans were documented in 6 clinical centers in Spain (15 adults and 1 child: male/female = 0.77). Fifteen patients had underlying hematologic malignancy (14 with neutropenia) and 1 had a prosthetic cardiac valve. Syndromes included disseminated infection in 14 patients (1 with prosthetic valve endocarditis) and fungal pneumonia and meningoencephalitis in 1 patient each. S. prolificans was isolated from 2 specimens in 14 patients and from 1 specimen in 2 patients (blood, n = 12; respiratory tract, n = 4; CNS, n = 4; and skin biopsy, n = 3). Antifungal susceptibility testing by a micromethod with RPMI-2% glucose medium was performed in 8 isolates, all of which were resistant to amphotericin B, flucytosine, ketoconazole, fluconazole, itraconazole, and miconazole. All patients received antifungal therapy (amphotericin B, n = 9; amphotericin B+ flucytosine, n = 1; amphotericin B+ itraconazole, n = 2; liposomal amphotericin B+ itraconazole, n = 1; amphotericin B+ fluconazole, n = 1 and 2 underwent surgical procedures. Two patients survived coinciding with hematologic recovery and 14 (87.5%) patients died in a median time of 4 days after the first positive culture (range, 0-60 d). Necropsy was performed in 10 patients, and disseminated infection was found in 9. In conclusion, S. prolificans is an emerging multiresistant fungal pathogen that may cause asymptomatic colonization, localized infection related to trauma or surgery, and rapidly fatal disseminated infection in immunocompromised hosts, particularly those with neutropenia. This mycosis underscores the urgent need for new antifungal agents.

Epidemiology, risk factors, and prognosis of Candida parapsilosis bloodstream infections: case-control population-based surveillance study of patients in Barcelona, Spain, from 2002 to 2003.

ABSTRACT Candida parapsilosis has emerged as an important yeast species causing fungemia. We describe the incidence and epidemiology of C. parapsilosis fungemia. Data from active population-based surveillance in Barcelona, Spain, from January 2002 to December 2003 were analyzed. We focused on 78 episodes of C. parapsilosis fungemia, and we compared them with 175 Candida albicans controls. C. parapsilosis accounted for 23% of all fungemias. The annual incidences were 1 episode per 105 patients, 1.2 episodes per 104 discharges, and 1.7 episodes per 105 patient days. All isolates but one (99%) were fluconazole susceptible. Seventy-two isolates (92%) were inpatient candidemias. Forty-two episodes (51%) were considered catheter-related fungemia, 35 (45%) were considered primary fungemia, and 3 (4%) were considered secondary fungemia. Risk factors for candidemia were vascular catheterization (97%), prior antibiotic therapy (91%), parenteral nutrition (54%), prior surgery (46%), prior immunosuppressive therapy (38%), malignancy (27%), prior antifungal infection (26%), transplant recipient (16%), neutropenia (12%), and prior colonization (11%). Multivariate analysis of the differential characteristics showed that the factors that independently predicted the presence of C. parapsilosis fungemia were neonate patients (odds ratio [OR], 7.5; 95% confidence interval [CI], 2.1 to 26.8; P = 0.002), transplant recipients (OR, 9.2; 95% CI, 1.9 to 43.3; P = 0.005), patients with a history of prior antifungal therapy (OR, 5.4; 95% CI, 1.8 to 15.9; P = 0.002), and patients who received parenteral nutrition (OR, 2.2; 95% CI, 1.09 to 4.6; P = 0.028). The overall mortality rate was lower than that associated with C. albicans candidemia (23% versus 43%; P < 0.01). In summary, C. parapsilosis was responsible for 23% of all candidemias and was more frequent in neonates, in transplant recipients, and in patients who received parenteral nutrition or previous antifungal therapy, mainly fluconazole. The mortality rate was lower than that associated with C. albicans fungemia.

Fungal cell gigantism during mammalian infection

The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 µm in diameter and capsules resistant to stripping with γ-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20–50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

Epidemiological Cutoffs and Cross-Resistance to Azole Drugs in Aspergillus fumigatus

ABSTRACT Antifungal susceptibility testing of molds has been standardized in Europe and in the United States. Aspergillus fumigatus strains with resistance to azole drugs have recently been detected and the underlying molecular mechanisms of resistance characterized. Three hundred and ninety-three isolates, including 32 itraconazole-resistant strains, were used to define wild-type populations, epidemiological cutoffs, and cross-resistance between azole drugs. The epidemiological cutoff for itraconazole, voriconazole, and ravuconazole for the wild-type populations of A. fumigatus was ≤1 mg/liter. For posaconazole, the epidemiological cutoff was ≤0.25 mg/liter. Up till now, isolates susceptible to itraconazole have not yet displayed resistance to other azole drugs. Cross-resistance between azole drugs depends on specific mutations in cyp51A. Thus, a substitution of glycine in position 54 of Cyp51A confers cross-resistance between itraconazole and posaconazole. A substitution of methionine at position 220 or a duplication in tandem of a 34-bp fragment in the cyp51A promoter combined with a substitution of leucine at position 98 for histidine confers cross-resistance to all azole drugs tested. The results obtained in this study will help to develop clinical breakpoints for azole drugs and A. fumigatus.

Aspergillus Section Fumigati: Antifungal Susceptibility Patterns and Sequence-Based Identification

ABSTRACT This study analyzed 28 Aspergillus strains belonging to the section Fumigati that were isolated from clinical samples in Spain. All isolates sporulated slowly and were unable to grow at 48°C. Phylogenetic analysis based on sequencing of partial sequences of the β-tubulin and rodlet A genes was used to classify the 28 strains into six different clades (Neosartorya hiratsukae, Neosartorya pseudofischeri, Aspergillus viridinutans, Aspergillus lentulus, Aspergillus fumigatiaffinis, and Aspergillus fumisynnematus). Antifungal susceptibility testing showed heterogeneous patterns and grouped the strains together by species. Most A. lentulus and A. fumigatiaffinis isolates showed high MICs of amphotericin B (geometric mean [GM] MICs, ≥4.5 μg/ml), itraconazole (GM MICs, ≥6 μg/ml), voriconazole (GM MICs, ≥3 μg/ml), and ravuconazole (GM MICs, ≥3 μg/ml); N pseudofischeri and A. viridinutans showed high MICs of itraconazole (GM MICs, ≥8 μg/ml), voriconazole (GM MICs, ≥3.33 μg/ml), and ravuconazole (GM MICs, ≥2 μg/ml); and N. hiratsukae and A. fumisynnematus were susceptible to all the antifungals tested. In conclusion, a number of different species whose morphological features resemble those of Aspergillus fumigatus could succeed in producing invasive infections in the susceptible host. In addition, some of them showed high MICs for most of the antifungals available for the treatment of patients infected with these organisms. The epidemiology and clinical relevance of these species should therefore be addressed.

Interlaboratory Comparison of Results of Susceptibility Testing with Caspofungin against Candida and Aspergillus Species

ABSTRACT Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual “prominent growth reduction” (MIC2) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode ± one dilution range across all 17 laboratories. MIC2 at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.

Environmental Study of Azole-Resistant Aspergillus fumigatus and Other Aspergilli in Austria, Denmark, and Spain

ABSTRACT A single mechanism of azole resistance was shown to predominate in clinical and environmental Aspergillus fumigatus isolates from the Netherlands, and a link to the use of azoles in the environment was suggested. To explore the prevalence of azole-resistant A. fumigatus and other aspergilli in the environment in other European countries, we collected samples from the surroundings of hospitals in Copenhagen, Innsbruck, and Madrid, flowerbeds in an amusement park in Copenhagen, and compost bags purchased in Austria, Denmark, and Spain and screened for azole resistance using multidish agars with itraconazole, voriconazole, and posaconazole. EUCAST method E.DEF 9.1 was used to confirm azole resistance. The promoter and entire coding sequence of the cyp51A gene were sequenced to identify azole-resistant A. fumigatus isolates. A. fumigatus was recovered in 144 out of 185 samples (77.8%). Four A. fumigatus isolates from four Danish soil samples displayed elevated azole MICs (8%), and all harbored the same TR/L98H mutation of cyp51A. One A. lentulus isolate with voriconazole MIC of 4 mg/liter was detected in Spain. No azole-resistant aspergilli were detected in compost. Finally, A. terreus was present in seven samples from Austria. Multi-azole-resistant A. fumigatus is present in the environment in Denmark. The resistance mechanism is identical to that of environmental isolates in the Netherlands. No link to commercial compost could be detected. In Spain and Austria, only Aspergillus species with intrinsic resistance to either azoles or amphotericin B were found.