Antifungal activity and killing kinetics of anidulafungin, caspofungin and amphotericin B against Candida auris.

BACKGROUND: Candida auris is an emerging MDR pathogen. It shows reduced susceptibility to azole drugs and, in some strains, high amphotericin B MICs have been described. For these reasons, echinocandins were proposed as first-line treatment for C. auris infections. However, information on how echinocandins and amphotericin B act against this species is lacking. OBJECTIVES: Our aim was to establish the killing kinetics of anidulafungin, caspofungin and amphotericin B against C. auris by time-kill methodology and to determine if these antifungals behave as fungicidal or fungistatic agents against this species. METHODS: The susceptibility of 50 C. auris strains was studied. Nine strains were selected (based on echinocandin MICs) to be further studied. Minimal fungicidal concentrations, in vitro dose-response and time-kill patterns were determined. RESULTS: Echinocandins showed lower MIC values than amphotericin B (geometric mean of 0.12 and 0.94 mg/L, respectively). Anidulafungin and caspofungin showed no fungicidal activity at any concentration (maximum log decreases in cfu/mL between 1.34 and 2.22). On the other hand, amphotericin B showed fungicidal activity, but at high concentrations (≥2.00 mg/L). In addition, the tested polyene was faster than echinocandins at killing 50% of the initial inoculum (0.92 versus >8.00 h, respectively). CONCLUSIONS: Amphotericin B was the only agent regarded as fungicidal against C. auris. Moreover, C. auris should be considered tolerant to caspofungin and anidulafungin considering that their MFC:MIC ratios were mostly ≥32 and that after 6 h of incubation the starting inoculum was not reduced in >90%.

In Vitro Activity of Combinations of Zinc Chelators with Amphotericin B and Posaconazole against Six Mucorales Species.

Mucormycosis is an emerging disease with high mortality rates. Few antifungal drugs are active against Mucorales. Considering the low efficacy of monotherapy, combination-therapy strategies have been described. It is known that fungi are susceptible to zinc deprivation, so we tested the in vitro effect of the zinc chelators clioquinol, phenanthroline, and N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine combined with amphotericin B or posaconazole against 25 strains of Mucorales. Clioquinol-posaconazole was the most active combination, although results were strain dependent.

Molecular Confirmation of the Linkage between the Rhizopus oryzae CYP51A Gene Coding Region and Its Intrinsic Voriconazole and Fluconazole Resistance.

Rhizopus oryzae is the most prevalent causative agent of mucormycosis, an increasingly reported opportunistic fungal infection. These Mucorales are intrinsically resistant to Candida- and Aspergillus-active antifungal azole drugs, such as fluconazole (FLC) and voriconazole, respectively. Despite its importance, the molecular mechanisms of its intrinsic azole resistance have not been elucidated yet. The aim of this work was to establish if the Rhizopus oryzaeCYP51 genes are uniquely responsible for intrinsic voriconazole and fluconazole resistance in these fungal pathogens. Two CYP51 genes were identified in the R. oryzae genome. We classified them as CYP51A and CYP51B based on their sequence similarity with other known fungal CYP51 genes. Later, we obtained a chimeric Aspergillus fumigatus strain harboring a functional R. oryzae CYP51A gene expressed under the regulation of the wild-type A. fumigatusCYP51A promoter and terminator. The mutant was selected after transformation by using a novel procedure taking advantage of the FLC hypersusceptibility of the A. fumigatusCYP51A deletion mutant used as the recipient strain. The azole susceptibility patterns of the A. fumigatus transformants harboring R. oryzae CYP51A mimicked exactly the azole susceptibility patterns of this mucormycete. The data presented in this work demonstrate that the R. oryzae CYP51A coding sequence is uniquely responsible for the R. oryzae azole susceptibility patterns.

Single-tube classical PCR for Candida auris and Candida haemulonii identification / PCR múltiple para la identificación de Candida auris y Candida haemulonii

Background: Candida auris and Candida haemulonii are emerging and multiresistant pathogens. C. auris has produced hospital outbreaks and is misidentified by phenotypic-based methods. The only reliable identification methods are DNA sequencing and MALDI-TOF. Aims: To develop a classical-PCR method capable of rapidly and accurately identify C. auris and C. haemulonii. Methods: A multiplex PCR was carried out in one tube that included an internal control and oligonucleotides that specifically hybridize to the ITS2 region of C. auris and C. haemulonii. The usefulness of the new method was verified by testing a collection of 50 strains of 20 different species (previously identified by ITS sequencing). The selection of species was made in order to emulate the C. auris panel used by the CDC to validate diagnostic tools. In addition, other yeast species not included in the aforementioned panel were incorporated based on reported identification errors. Results: The results obtained with the proposed protocol were in total agreement with those obtained by ITS sequencing. Conclusions: We present a PCR method able to unequivocally identify C. auris and differentiate it from C. haemulonii. It is inexpensive, fast and it could be a useful tool to reduce the chances of a C. auris outbreak

Antecedentes: Candida auris y Candida haemulonii son patógenos emergentes y multirresistentes. C. auris ha sido responsable de brotes hospitalarios y no se puede identificar por métodos fenotípicos. Los únicos métodos de identificación confiables incluyen la secuenciación y el MALDI-TOF. Objetivos: Desarrollar un método de PCR clásica capaz de identificar rápidamente C. auris y C. haemulonii. Métodos: Se llevó a cabo una PCR múltiple en un tubo que incluyó un control interno y oligonucleótidos que hibridan específicamente con la región ITS2 de C. auris y C. haemulonii. Para comprobar la utilidad del método se utilizó una colección de 50 aislamientos de 20 especies diferentes (identificadas por secuenciación del ITS). La selección de especies se hizo con el fin de emular el panel de especies que ofrece el CDC para la correcta identificación de C. auris. Además, se incluyeron especies que son confundidas con C. auris y no están incluidas en el citado panel. Resultados: Los resultados obtenidos con el protocolo propuesto estuvieron en total acuerdo con los obtenidos por la secuenciación del ITS. Conclusiones: El método que presentamos es capaz de identificar inequívocamente C. auris y diferenciarla de C. haemulonii. Es barato, rápido y podría ser una herramienta útil para reducir la posibilidad de brotes por C. auris

Single-tube classical PCR for Candida auris and Candida haemulonii identification.

BACKGROUND: Candida auris and Candida haemulonii are emerging and multiresistant pathogens. C. auris has produced hospital outbreaks and is misidentified by phenotypic-based methods. The only reliable identification methods are DNA sequencing and MALDI-TOF. AIMS: To develop a classical-PCR method capable of rapidly and accurately identify C. auris and C. haemulonii. METHODS: A multiplex PCR was carried out in one tube that included an internal control and oligonucleotides that specifically hybridize to the ITS2 region of C. auris and C. haemulonii. The usefulness of the new method was verified by testing a collection of 50 strains of 20 different species (previously identified by ITS sequencing). The selection of species was made in order to emulate the C. auris panel used by the CDC to validate diagnostic tools. In addition, other yeast species not included in the aforementioned panel were incorporated based on reported identification errors. RESULTS: The results obtained with the proposed protocol were in total agreement with those obtained by ITS sequencing. CONCLUSIONS: We present a PCR method able to unequivocally identify C. auris and differentiate it from C. haemulonii. It is inexpensive, fast and it could be a useful tool to reduce the chances of a C. auris outbreak.

Molecular Confirmation of the Relationship between Candida guilliermondii Fks1p Naturally Occurring Amino Acid Substitutions and Its Intrinsic Reduced Echinocandin Susceptibility.

Candida guilliermondii shows intrinsic reduced echinocandin susceptibility. It harbors two polymorphisms (L633M and T634A) in the Fks1p hot spot 1 region. Our objective was to confirm that the reduced echinocandin susceptibility of C. guilliermondii is due to those naturally occurring substitutions. We constructed a Saccharomyces cerevisiae mutant in which a region of the FKS1 gene (including hot spot 1) was replaced with that from C. guilliermondii The chimeric mutants showed 32-fold increases in echinocandin MIC values, confirming the hypothesis.

First itraconazole resistant Aspergillus fumigatus clinical isolate harbouring a G54E substitution in Cyp51Ap in South America / Primer aislamiento clínico en Sudamérica de una cepa de Aspergillus fumigatus resistente a itraconazol con la sustitución G54E en Cyp51Ap

Background. A 27-year-old male rural worker was admitted with a fungal keratitis due to an injury involving plant detritus. Materials and methods. Specimens were collected for microscopy examination and culture. The isolate was identified by morphological and molecular criteria. Susceptibility testing was performed using CLSI methods. CYP51A gene was PCR amplified and sequenced. Results. An Aspergillus fumigatus strain resistant to itraconazole (MIC>8μg/ml) was isolated. The isolate was susceptible to amphotericin B, posaconazole, voriconazole and caspofungin. CYP51A sequencing showed two mutations leading on the G54E substitution. The patient received natamycin as treatment. Conclusions. This is the first report in South America of a clinical A. fumigatus strain carrying the substitution G54E at Cyp51Ap associated with itraconazole resistance. Considering the patient was azole-naive, this resistant isolate may have been acquired from the environment (AU)

Antecedentes. Un trabajador rural de 27años de edad fue hospitalizado con una queratitis fúngica debido a un traumatismo con un resto vegetal. Materiales y métodos. Se tomaron las muestras para los exámenes de microscopía y cultivo. El aislamiento se identificó mediante criterios morfológicos y moleculares. Se realizaron pruebas de sensibilidad a los antifúngicos siguiendo el documento del CLSI. Se amplificó y secuenció el gen CYP51A de la cepa. Resultados. Se aisló una cepa de Aspergillus fumigatus resistente a itraconazol (CIM>8μg/ml). El aislamiento resultó sensible a la anfotericina B, el posaconazol, el voriconazol y la caspofungina. La secuenciación del gen CYP51 reveló 2 mutaciones que generan la sustitución G54E. El paciente fue tratado con natamicina oftálmica. Conclusiones. Este es el primer caso informado en Sudamérica de una cepa clínica de A. fumigatus con la sustitución G54E en el Cyp51Ap, asociada con resistencia al itraconazol. Teniendo en cuenta que el paciente no había recibido nunca antes tratamiento alguno con azoles, podría haber adquirido esta cepa resistente del ambiente (AU)

First itraconazole resistant Aspergillus fumigatus clinical isolate harbouring a G54E substitution in Cyp51Ap in South America.

BACKGROUND: A 27-year-old male rural worker was admitted with a fungal keratitis due to an injury involving plant detritus. MATERIALS AND METHODS: Specimens were collected for microscopy examination and culture. The isolate was identified by morphological and molecular criteria. Susceptibility testing was performed using CLSI methods. CYP51A gene was PCR amplified and sequenced. RESULTS: An Aspergillus fumigatus strain resistant to itraconazole (MIC>8µg/ml) was isolated. The isolate was susceptible to amphotericin B, posaconazole, voriconazole and caspofungin. CYP51A sequencing showed two mutations leading on the G54E substitution. The patient received natamycin as treatment. CONCLUSIONS: This is the first report in South America of a clinical A. fumigatus strain carrying the substitution G54E at Cyp51Ap associated with itraconazole resistance. Considering the patient was azole-naive, this resistant isolate may have been acquired from the environment.

Prevalencia y sensibilidad a los antifúngicos de Candida albicans y sus especies relacionadas, Candida dubliniensis y Candida africana aisladas de muestras vulvovaginales en un hospital de Argentina / Prevalence and antifungal susceptibility of Candida albicans and its related species Candida dubliniensis and Candida africana isolated from vulvovaginal samples in a hospital of Argentina

Candida africana taxonomical status is controversial. It was proposed as a separate species within the Candida albicans species complex; however, phylogenetic analyses suggested that it is an unusual variety of C. albicans. The prevalence of C. albicans-related species (Candida dubliniensis and C. africana) as vulvovaginal pathogens is not known in Argentina. Moreover, data on antifungal susceptibility of isolates causing vulvovaginal candidiasis is scarce. The aims of this study were to establish the prevalence of C. dubliniensis and C. africana in vaginal samples and to evaluate the antifungal susceptibilities of vaginal C. albicans species complex strains. We used a molecular-based method coupled with a new pooled DNA extraction methodology to differentiate C. dubliniensis and C. africana in a collection of 287 strains originally identified as C. albicans isolated from an Argentinian hospital during 2013. Antifungal susceptibilities to fluconazole, clotrimazole, itraconazole, voriconazole, nystatin, amphotericin B and terbinafine were evaluated by using the CLSI M27-A3 and M27-S4 documents. Of the 287 isolates, 4 C. dubliniensis and one C. africana strains (1.39% and 0.35% prevalence, respectively) were identified. This is the first description of C. africana in Argentina and its identification was confirmed by sequencing the ITS2 region and the hwp1 gene. C. dubliniensis and C. africana strains showed very low MIC values for all the tested antifungals. Fluconazole-reduced-susceptibility and azole cross-resistance were observed in 3.55% and 1.41% of the C. albicans isolates, respectively. These results demonstrate that antifungal resistance is still a rare phenomenon in this kind of isolates.

La clasificación taxonómica de Candida africana está en discusión, es considerada una nueva especie dentro del complejo C. albicans o una variedad inusual de C. albicans. La prevalencia de las especies relacionadas a C. albicans (C. dubliniensis y C. africana) como agentes de vulvovaginitis en Argentina se desconoce. Los objetivos de este trabajo fueron determinar la prevalencia de C. dubliniensis y C. africana en muestras vaginales y evaluar la sensibilidad a los antifúngicos de aislamientos vaginales de las especies del complejo C. albicans. Para diferenciar C. dubliniensis y C. africana utilizamos un método molecular asociado a un nuevo método de extracción de ADN. Se utilizó una colección de 287 cepas originalmente identificadas como C. albicans aisladas durante 2013 en un hospital de Argentina. Se evaluó la sensibilidad a fluconazol, clotrimazol, itraconazol, voriconazol, nistatina, anfotericina B y terbinafina utilizando los documentos M27-A3 y M27-S4 del CLSI. De los 287 aislamientos, se identificaron 4 C. dubliniensis y 1 C. africana (1,39 y 0,35% de prevalencia, respectivamente). Esta es la primera descripción de C. africana en Argentina. Su identificación fue confirmada por secuenciación de la región ITS2 y del gen hwp1. Las cepas identificadas como C. dubliniensis y C. africana mostraron valores de CIM muy bajos para todos los antifúngicos probados. En los aislamientos de C. albicans, la sensibilidad reducida al fluconazol y la resistencia cruzada a todos los azoles se observó en el 3,55% y el 1,41%, respectivamente. Estos resultados demuestran que la resistencia a los antifúngicos es todavía un fenómeno raro en este tipo de aislamientos.

Prevalence and antifungal susceptibility of Candida albicans and its related species Candida dubliniensis and Candida africana isolated from vulvovaginal samples in a hospital of Argentina.

Candida africana taxonomical status is controversial. It was proposed as a separate species within the Candida albicans species complex; however, phylogenetic analyses suggested that it is an unusual variety of C. albicans. The prevalence of C. albicans-related species (Candida dubliniensis and C. africana) as vulvovaginal pathogens is not known in Argentina. Moreover, data on antifungal susceptibility of isolates causing vulvovaginal candidiasis is scarce. The aims of this study were to establish the prevalence of C. dubliniensis and C. africana in vaginal samples and to evaluate the antifungal susceptibilities of vaginal C. albicans species complex strains. We used a molecular-based method coupled with a new pooled DNA extraction methodology to differentiate C. dubliniensis and C. africana in a collection of 287 strains originally identified as C. albicans isolated from an Argentinian hospital during 2013. Antifungal susceptibilities to fluconazole, clotrimazole, itraconazole, voriconazole, nystatin, amphotericin B and terbinafine were evaluated by using the CLSI M27-A3 and M27-S4 documents. Of the 287 isolates, 4 C. dubliniensis and one C. africana strains (1.39% and 0.35% prevalence, respectively) were identified. This is the first description of C. africana in Argentina and its identification was confirmed by sequencing the ITS2 region and the hwp1 gene. C. dubliniensis and C. africana strains showed very low MIC values for all the tested antifungals. Fluconazole-reduced-susceptibility and azole cross-resistance were observed in 3.55% and 1.41% of the C. albicans isolates, respectively. These results demonstrate that antifungal resistance is still a rare phenomenon in this kind of isolates.