Guillermo Quindós

Epidemiology, species distribution and in vitro antifungal susceptibility of fungaemia in a Spanish multicentre prospective survey

OBJECTIVES To update the knowledge of the epidemiology of fungaemia episodes in Spain, the species implicated and their in vitro antifungal susceptibilities. METHODS Episodes were identified prospectively over 13 months at 44 hospitals. Molecular methods were used to determine the cryptic species inside the Candida parapsilosis and Candida glabrata complexes. Susceptibility to amphotericin B, anidulafungin, caspofungin, fluconazole, flucytosine, itraconazole, micafungin, posaconazole and voriconazole was determined by a microdilution colorimetric method. New species-specific clinical breakpoints (SSCBPs) for echinocandins, fluconazole and voriconazole were applied. RESULTS The incidence of the 1357 fungaemia episodes evaluated was 0.92 per 1000 admissions. The incidence of Candida albicans fungaemia was the highest (0.41 episodes/1000 admissions), followed by Candida parapsilosis sensu stricto (0.22). Candida orthopsilosis was the fifth cause of fungaemia (0.02), outnumbered by Candida glabrata and Candida tropicalis. Interestingly, the incidence of fungaemia by C. parapsilosis was 11 and 74 times higher than that by C. orthopsilosis and Candida metapsilosis, respectively. Neither Candida nivariensis nor Candida bracarensis was isolated. Fungaemia was more common in non-intensive care unit settings (65.2%) and among elderly patients (46.4%), mixed fungaemia being incidental (1.5%). Overall susceptibility rates were 77.6% for itraconazole, 91.9% for fluconazole and 96.5%-99.8% for the other agents. Important resistance rates were only observed in C. glabrata for itraconazole (24.1%) and posaconazole (14.5%), and in Candida krusei for itraconazole (81.5%). CONCLUSIONS Fungaemia is more common in non-critical patients. C. albicans is the most common species, followed by C. parapsilosis and C. glabrata. Nearly 90% of yeasts are susceptible to all antifungal agents tested. Resistance rates change moderately when applying the new SSCBPs.

Prospective Multicenter Study of the Epidemiology, Molecular Identification, and Antifungal Susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis Isolated from Patients with Candidemia

ABSTRACT A 13-month prospective multicenter study including 44 hospitals was carried out to evaluate the epidemiology of Candida parapsilosis complex candidemia in Spain. Susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin was tested by the microdilution colorimetric method. A total of 364 C. parapsilosis complex isolates were identified by molecular methods: C. parapsilosis (90.7%), Candida orthopsilosis (8.2%), and Candida metapsilosis (1.1%). Most candidemias (C. parapsilosis, 76.4%; C. orthopsilosis, 70.0%; C. metapsilosis, 100%) were observed in adults. No C. orthopsilosis or C. metapsilosis candidemias occurred in neonates. C. parapsilosis was most frequent in adult intensive care unit (28.8%), surgery (20.9%), and internal medicine (19.7%) departments; and C. orthopsilosis was most frequent in hematology (28.6%), pediatrics (12.0%), and neonatology (11.5%) departments. The geographic distribution of C. orthopsilosis and C. metapsilosis was not uniform. According to CLSI clinical breakpoints, all C. orthopsilosis and C. metapsilosis isolates were susceptible to the nine agents tested. Resistance (MICs > 1 mg/liter) was observed only in C. parapsilosis: amphotericin B, posaconazole, itraconazole, and caspofungin (0.3% each), anidulafungin (1.9%), and micafungin (2.5%). Applying the new species-specific fluconazole and echinocandin breakpoints, the rates of resistance to fluconazole for C. parapsilosis and C. orthopsilosis increased to 4.8% and 0.3%, respectively; conversely, for C. parapsilosis they shifted from 1.9 to 0.6% (anidulafungin) and from 2.5 to 0.6% (micafungin). Our study confirms the different prevalence of C. parapsilosis complex candidemia among age groups: neither C. orthopsilosis nor C. metapsilosis was isolated from neonates; interestingly, C. metapsilosis was isolated only from adults and the elderly. The disparity in antifungal susceptibility among species could be important for therapy.

Candida dubliniensis, a new fungal pathogen

There is a high interest in Candida species other than Candida albicans because of the rise and the epidemiological shifts in candidiasis. These emerging Candida species are favored by the increase of immunocompromised patients and the use of new medical practices, and m. Most oropharyngeal candidiasis can be foundare observed in those HIV‐infected patients infected with human immunodeficiency virus (HIV). Candida dubliniensis is a recently described opportunistic pathogen that is closely related to C. albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. C. dubliniensis has been linked to oral candidiasis in AIDS patients, although it has recently been associated to invasive disease. C. dubliniensis shares diagnostic characteristics with C. albicans, as germ tube‐ and chlamydospore‐production, and it is generally misclassified as C. albicans by standard diagnostic procedures. Several recent studies have attempted to elucidate useful phenotypic and genotypic characteristics for separating both species. A large variety of methods have been developed with the aim of facilitating rapid and, accurate identification of this species. These have included differential chromogenic isolation platesculture media, direct immunological tests, and enhanced manual and automated biochemical and enzymatic panels. Chromogenic isolation media, as CHROMagar Candida, demonstrate better detection rates than traditional media, and allow the presumptive identification of C. dubliniensis by means of colony color (dark‐green colonies). API 20 C AUX system is considered a reference method, but ID 32 C strip, the VITEK Yeast Biochemical Card and the VITEK 2 ID‐YST system correctly identify most C. dubliniensis isolates, being VITEK 2 ID‐YSTthe latter the most accurate. Spectroscopic methods, such as Fourier transformed‐infrared spectroscopy, offer potential advantages. However, many authors consider that standard methods for differentiation of Candida species are time‐consuming, often insensitive and can fail to distinguish C. dubliniensis. To overcome these low sensitivity, poor specificity and intolerable delay,drawbacks, molecular tools have been developed to discriminate C. dubliniensis, and particularly those based on the polymerase chain reaction. But, molecular tools prove difficult and too complex for routine use in the clinical laboratory setting and new developments are necessary. Moreover, an increased resistance to antifungal drugs has been described. Although preliminary studies indicate that most strains of C. dubliniensis are susceptible to establishedantifungal agents, fluconazole‐resistant strains have been detected. Furthermore, fluconazole‐resistant strains are easily derived in vitro, showing an increased expression of multidrug resistance transporters, as MDR1.

Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species

Minimum fungicidal concentrations (MFCs) of amphotericin B were obtained for 165 bloodstream isolates (104 Candida parapsilosis, 14 C.glabrata, 13 C.tropicalis, 15 C.krusei, and 19 C.albicans) and 36 C.dubliniensis from oropharyngeal infections. Minimum inhibitory concentrations (MICs) were determined by the M27-A microdilution method. MFCs (> or =99.9% killing) were obtained following MIC determination (inoculum size, 10(4) CFU/ml) by seeding the entire volume of all clear wells. The best fungicidal activity was for C. albicans, (MFC90 1 microg/ml) and the lowest for C.parapsilosis, C.tropicalis and C.glabrata (MFC90 16 microg/ml). Although MFCs were > or =16x MIC for some isolates, including C. glabrata, the overall MFCs were > or =2x MICs. However, major differences between MICs and MFCs were observed for C.parapsilosis and C.dubliniensis (3.8% and 8.9%, respectively, were tolerant: MFC > or =32MIC). MFCs for C.tropicalis and C. glabrata were > or =2 microg/ml. By this more stringent method we found substantial differences from those previously reported between amphotericin B MIC and MFCs for Candida spp.

In vitro susceptibility of Candida dubliniensis to current and new antifungal agents.

Background: Candida dubliniensis is a recently described Candida species closely related to Candida albicans, which has been associated with oral candidiasis in HIV-infected patients. Fluconazole-resistant strains of C. dubliniensis are easily obtained in vitro and this fact could be a complication if this resistance develops during treatment with this drug. Methods: In the present study, the in vitro antifungal susceptibilities of 36 C. dubliniensis clinical isolates and culture strains to current and new antifungal agents, such as amphotericin B (AMB), amphotericin B lipid complex (ABLC), amphotericin B colloidal dispersion (ABCD), 5-fluorocytosine (5FC), fluconazole (FLC), itraconazole (ITC), ketoconazole (KTC), liposomal amphoteri- cin B (LAMB), liposomal nystatin (LNYT), LY303366 (LY), SCH56592 (SCH), and voriconazole (VRC), were determined according to the National Committee for Clinical Laboratory Standards M27-A broth microdilution method for yeasts. Results: Most isolates of C. dubliniensis were susceptible to both new and current antifungal drugs, with 75.9% isolates susceptible to KTC, 86.2% to FLC and to ITC, and ∼100% to the other antifungal agents tested. The cross-resistance phenotypes are detailed. Four isolates were resistant (MIC ≥64 μg/ml) to FLC. These 4 isolates were also resistant to KTC, and 3 of them were also resistant to ITC (MIC ≥1 μg/ml for both agents). However, these isolates were highly susceptible to 5FC and all polyene formulations (AMB, ABLC, ABCD, LAMB, and LNYT), triazole (SCH and VRC) and echinocandin (LY) antifungal agents. Conclusion: The new liposomal and lipidic formulations of AMB, LNYT, and the new triazoles and echinocandins may provide new alternatives to FLC for the treatment of infections by C. dubliniensis.

Utilidad de la detección de (1→3)-ß-D-glucano y anticuerpos anti-micelio de Candida albicans para el diagnóstico y seguimiento terapéutico de la candidiasis invasora en pacientes neutropénicos adultos

Resumen Se ha evaluado la utilidad de la deteccion dos veces por semana de s-glucano (BG) y de anticuerpos anti-micelio de Candida albicans (CAGT) para el diagnostico y el seguimiento de la candidiasis invasora (CI) en 35 episodios de pacientes neutropenicos de alto riesgo. Se diagnosticaron tres casos de CI probada y tres probables. Se alcanzaron resultados positivos para ambos marcadores en el 100% de las CI probadas y en el 66% de las CI probables. La sensibilidad, especificidad, valores predictivos positivo y negativo para la deteccion de BG y anticuerpos contra CAGT fueron 83,3%, 89,6%, 62,5% y 96,3%, y 83,3%, 86,2%, 55,5% y 96,1%, respectivamente. El porcentaje de falsos positivos para BG y anticuerpos contra CAGT fue del 10,3% y 13,8% para la deteccion de BG y anticuerpos anti-CAGT, respectivamente. Sin embargo, los pacientes con resultados falsos positivos fueron diferentes para cada prueba. Ambas pruebas se anticiparon al diagnostico clinico y radiologico, asi como al inicio de la terapia antifungica en la mayoria de los pacientes. La combinacion de ambas pruebas mejoro la especificidad y el valor predictivo positivo hasta el 100%.

Evaluation of the New Chromogenic Medium Candida ID 2 for Isolation and Identification of Candida albicans and Other Medically Important Candida Species

ABSTRACT The usefulness of Candida ID 2 (CAID2) reformulated medium (bioMérieux, France) has been compared with that of the former Candida ID (CAID; bioMérieux), Albicans ID 2 (ALB2; bioMérieux), and CHROMagar Candida (CAC; Chromagar, France) chromogenic media for the isolation and presumptive identification of clinically relevant yeasts. Three hundred forty-five stock strains from culture collections, and 103 fresh isolates from different clinical specimens were evaluated. CAID2 permitted differentiation based on colony color between Candida albicans (cobalt blue; sensitivity, 91.7%; specificity, 97.2%) and Candida dubliniensis (turquoise blue; sensitivity, 97.9%; specificity, 96.6%). Candida tropicalis gave distinguishable pink-bluish colonies in 97.4% of the strains in CAID2 (sensitivity, 97.4%; specificity, 100%); the same proportion was reached in CAC, where colonies were blue-gray (sensitivity, 97.4%; specificity, 98.7%). CAC and CAID2 showed 100% sensitivity values for the identification of Candida krusei. However, with CAID2, experience is required to differentiate the downy aspect of the white colonies of C. krusei from other white-colony-forming species. The new CAID2 medium is a good candidate to replace CAID and ALB2, and it compares well to CAC for culture and presumptive identification of clinically relevant Candida species. CAID2 showed better results than CAC in some aspects, such as quicker growth and color development of colonies from clinical specimens, detection of mixed cultures, and presumptive differentiation between C. albicans and C. dubliniensis.

Isolation of Candida dubliniensis in denture stomatitis

OBJECTIVE To describe the isolation of Candida dubliniensis from a patient with denture stomatitis and to compare with the presence of yeasts in the oral cavities of denture wearers. DESIGN One hundred and fifty-two Candida isolates were recovered through oral swabs from denture as well as the underlying mucosa from 100 patients wearing denture. For detection and identification of fungal isolates, standard phenotypic and genotypic methods were used. RESULTS Forty-five of 100 denture wearers suffered from denture stomatitis. Seventy-three Candida isolates were recovered from 38 denture wearers without denture stomatitis. In this group, Candida albicans was the predominant species (58.9%), followed by Candida tropicalis (15.1%), Candida guilliermondii (13.7%), Candida glabrata (9.6%), and Candida parapsilosis (2.7%). Seventy-nine isolates were yielded from 40 patients suffering from denture stomatitis. C. albicans was also the most frequently isolated species (58 isolates, 73.4%), followed by C. glabrata and C. tropicalis (7 isolates each, 8.9%), and Saccharomyces cerevisiae (2 isolates, 2.5%). One isolate was yielded of the following species: Candida famata, Candida krusei, C. parapsilosis and C. guilliermondii. Moreover 1 isolate was phenotypic and genotypic identified as C. dubliniensis genotype 1. CONCLUSIONS C. albicans is the predominant fungal species isolated from denture wearers. C. dubliniensis could be isolated from adults with denture stomatitis.

Value of detection of antibodies toCandida albicans germ tube in the diagnosis of systemic candidosis

To test the value of detection of anti-Candida albicans germ tube antibodies by indirect immunofluorescence assay in the diagnosis of systemic candidosis, a retrospective study was done using 126 sera from 27 patients with presumptive systemic candidosis (13 immunocompromised), 165 sera from 45 patients with aspergillosis (29 immunocompromised), 35 sera from eight patients with cryptococcosis (6 immunocompromised), and 101 sera from 101 blood donors. While 21 of 27 patients with systemic candidosis (77.8%) had anti-germ tube antibodies, these antibodies were absent in all patients with cryptococcosis and in all blood donors. They were however detected in 5 of 45 patients with aspergillosis (11.1%). Ten of 13 (76.9%) immunocompromised patients with candidosis had anti-germ tube antibodies; similar results were obtained in immunocompetent patients with candidosis (78.6%). The specificity was 96.8%, indicating a high degree of discrimination was possible between systemic candidosis and other invasive mycoses in the patients studied. Anti-germ tube responses did not appear to be significantly reduced in immunocompromised patients.

Prevalence and antifungal susceptibility patterns of new cryptic species inside the species complexes Candida parapsilosis and Candida glabrata among blood isolates from a Spanish tertiary hospital

OBJECTIVES There is scarce information on the clinical relevance and antifungal susceptibility of Candida bracarensis, Candida nivariensis, Candida orthopsilosis and Candida metapsilosis. The objective of this study was to assess the prevalence and in vitro antifungal susceptibility of these cryptic species among 173 blood isolates previously identified as Candida glabrata or Candida parapsilosis at the Hospital of Cruces (Barakaldo, Spain). The survey was extended to 518 clinical isolates from the culture collection of the Universidad del País Vasco-Euskal Herriko Unibertsitatea (UPV-EHU; Bilbao, Spain). METHODS In vitro susceptibilities to 5-fluorocytosine, amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole, micafungin, posaconazole and voriconazole were tested. RESULTS All isolates of C. glabrata were identified as C. glabrata sensu stricto. Inside the C. parapsilosis complex, 2.4% of isolates from the Hospital of Cruces and 5.8% from the UPV-EHU were C. metapsilosis or C. orthopsilosis. Of 457 isolates, 435 (95.19%) were C. parapsilosis sensu stricto, 11 (2.41%) C. metapsilosis and 11 (2.41%) C. orthopsilosis. Only seven blood isolates were C. metapsilosis (0.44%) or C. orthopsilosis (1.09%). These cryptic species were also isolated from other relevant clinical specimens. Four C. parapsilosis sensu stricto (5.6%) were susceptible dose-dependent, and one was resistant to both fluconazole and voriconazole (1.4%). Moreover, 19 isolates of C. parapsilosis sensu stricto (26.4%) were intermediately susceptible to itraconazole and higher concentrations of echinocandins were needed to inhibit this species. Most C. orthopsilosis and C. metapsilosis were susceptible to all antifungal agents tested, but one otic isolate of C. metapsilosis was resistant to fluconazole and 5-fluorocytosine. CONCLUSIONS C. metapsilosis and C. orthopsilosis are associated with human disease and show a different antifungal susceptibility profile compared with C. parapsilosis sensu stricto.