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.

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%.

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.

Phenotypic and Molecular Evaluation of Echinocandin Susceptibility of Candida glabrata, Candida bracarensis, and Candida nivariensis Strains Isolated during 30 Years in Argentina.

The echinocandin susceptibilities of 122 Candida glabrata complex strains (including 5 Candida nivariensis and 3 Candida bracarensis strains) were evaluated by microdilution and compared with the results from a molecular tool able to detect FKS mutations. No echinocandin resistance was detected. The PCR results coincide with the MIC data in 99.25% of the cases (1 C. glabrata strain was misidentified as resistant) but were 20 h faster. C. nivariensis FKS genes were sequenced and showed differences with C. glabrataFKS genes.

Identification of Candida parapsilosis Sensu Lato in Pediatric Patients and Antifungal Susceptibility Testing.

A total of 59 Candida parapsilosis sensu stricto and 1 Candida orthopsilosis recovered from catheters and blood cultures of pediatric patients from the northeastern region of Argentina were studied. Susceptibility to azoles, amphotericin B, and echinocandins was tested by the broth microdilution method. According to CLSI clinical breakpoints, >91% of the strains were azole susceptible, whereas 15% showed high amphotericin B MICs.

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.

Aspergillus fumigatus Intrinsic Fluconazole Resistance Is Due to the Naturally Occurring T301I Substitution in Cyp51Ap.

Aspergillus fumigatus intrinsic fluconazole resistance has been demonstrated to be linked to the CYP51A gene, although the precise molecular mechanism has not been elucidated yet. Comparisons between A. fumigatus Cyp51Ap and Candida albicans Erg11p sequences showed differences in amino acid residues already associated with fluconazole resistance in C. albicans The aim of this study was to analyze the role of the natural polymorphism I301 in Aspergillus fumigatus Cyp51Ap in the intrinsic fluconazole resistance phenotype of this pathogen. The I301 residue in A. fumigatus Cyp51Ap was replaced with a threonine (analogue to T315 at Candida albicans fluconazole-susceptible Erg11p) by changing one single nucleotide in the CYP51A gene. Also, a CYP51A knockout strain was obtained using the same parental strain. Both mutants' antifungal susceptibilities were tested. The I301T mutant exhibited a lower level of resistance to fluconazole (MIC, 20 µg/ml) than the parental strain (MIC, 640 µg/ml), while no changes in MIC were observed for other azole- and non-azole-based drugs. These data strongly implicate the A. fumigatus Cyp51Ap I301 residue in the intrinsic resistance to fluconazole.

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.

Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans.

A rapid molecular-based assay for the detection of the Candida albicans FKS1 gene mutations responsible for resistance to echinocandin drugs was designed and evaluated. The assay consisted of a multiplexed PCR set of 5 tubes able to detect the most commonly described resistance mechanism, including FKS1 hot spot 1 and hot spot 2 mutations. The performance and specificity of the assay was evaluated using a double-blinded panel of 50 C. albicans strains. The assay showed a sensitivity of 96% and was able to detect all homozygous mutants included in the collection of strains, demonstrating that it is a robust, quick, and labor-saving method that is suitable for a routine clinical diagnostic laboratory.

Set of classical PCRs for detection of mutations in Candida glabrata FKS genes linked with echinocandin resistance.

Clinical echinocandin resistance among Candida glabrata strains is increasing, especially in the United States. Antifungal susceptibility testing is considered mandatory to guide therapeutic decisions. However, these methodologies are not routinely performed in the hospital setting due to their complexity and the time needed to obtain reliable results. Echinocandin failure in C. glabrata is linked exclusively to Fks1p and Fks2p amino acid substitutions, and detection of such substitutions would serve as a surrogate marker to identify resistant isolates. In this work, we report an inexpensive, simple, and quick classical PCR set able to objectively detect the most common mechanisms of echinocandin resistance in C. glabrata within 4 h. The usefulness of this assay was assessed using a blind collection of 50 C. glabrata strains, including 16 FKS1 and/or FKS2 mutants.

Epidemiology and antifungal susceptibilities of yeasts causing vulvovaginitis in a teaching hospital.

Vulvovaginal candidiasis is one of the most common mycosis. However, the information about antifungal susceptibilities of the yeasts causing this infection is scant. We studied 121 yeasts isolated from 118 patients with vulvovaginal candidiasis. The isolates were identified by phenotypic and molecular methods, including four phenotypic methods described to differentiate Candida albicans from C. dubliniensis. Antifungal susceptibility testing was performed according to CLSI documents M27A3 and M27S4 using the drugs available as treatment option in the hospital. Diabetes, any antibacterial and amoxicillin treatment were statistically linked with vulvovaginal candidiasis, while oral contraceptives were not considered a risk factor. Previous azole-based over-the-counter antifungal treatment was statistically associated with non-C.albicans yeasts infections. The most common isolated yeast species was C. albicans (85.2 %) followed by C. glabrata (5 %), Saccharomyces cerevisiae (3.3 %), and C. dubliniensis (2.5 %). Fluconazole- and itraconazole-reduced susceptibility was observed in ten and in only one C. albicans strains, respectively. All the C. glabrata isolates showed low fluconazole MICs. Clotrimazole showed excellent potency against all but seven isolates (three C. glabrata, two S. cerevisiae, one C. albicans and one Picchia anomala). Any of the strains showed nystatin reduced susceptibility. On the other hand, terbinafine was the less potent drug. Antifungal resistance is still a rare phenomenon supporting the use of azole antifungals as empirical treatment of vulvovaginal candidiasis.

In Vitro and In Vivo Evaluation of Voriconazole-Containing Antifungal Combinations against Mucorales Using a Galleria mellonella Model of Mucormycosis.

Mucorales are resistant to most antifungals. Mucormycosis associated mortality is unacceptable and new treatment approaches are needed. The objectives of this work were (i) to evaluate the nature and intensity of the in vitro effect of three drugs combinations which included voriconazole (plus amphotericin B, posaconazole and caspofungin) against 25 strains of six different Mucorales species; (ii) to evaluate a Galleria mellonella mucormycosis model; and (iii) to establish if any in vitro⁻in vivo correlation exists. As expected, amphotericin B and posaconazole were the most active drugs when tested alone. However, species-specific differences were found. The ΣFICs varied according to the used combination. Only five strains showed synergism when voriconazole was combined with posaconazole and three strains when combined with amphotericin B. Microscopic hyphae alteration were observed for some isolates when confronted against drugs combinations. Using a Galleria mellonella mucormycosis model, better survival was seen in voriconazole plus amphotericin B and plus caspofungin combined treatments when compared with AMB alone for R. microsporus. These survival improvements were obtained using a 32-fold lower amphotericin B doses when combined with VRC than when treated with the polyene alone. These lower antifungal doses emulate the antifungal concentrations where the microscopic hyphae alterations were seen.

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.

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.

Multiplex PCR designed to differentiate species within the Candida glabrata complex.

BACKGROUND: No phenotypic methods are available to unequivocally differentiate species within the Candida glabrata complex. AIMS: To develop a new multiplex PCR method to differentiate between the three species of the C. glabrata species complex, as well as using it to study a C. glabrata collection to discover strains of the newly described species. METHODS: The method was developed based on the Internal Transcribed Spacer (ITS) sequence differences between the species. It was validated by using a blinded collection of strains and, finally, the new molecular method was used to study a collection of 192 C. glabrata species complex strains. The obtained results were compared with ITS sequencing. RESULTS: The proposed method showed 100% concordance with ITS sequencing and proved to be effective for clinical and epidemiological applications. Two Candida bracarensis and three Candida nivariensis were found out of the 192 studied strains (0.93% and 1.40% prevalence, respectively). CONCLUSIONS: A fast, inexpensive, robust and highly reproducible multiplex PCR method is presented. Its usefulness is demonstrated by studying a large collection of C. glabrata sensu lato strains.

Candida dubliniensis and Candida albicans differentiation by colony morphotype in Sabouraud-triphenyltetrazolium agar.

BACKGROUND: Candida dubliniensis is a germ tube and chlamydoconidia producing Candida species that may be misidentified as Candida albicans. Molecular-based methods are the most reliable techniques for C. albicans and C. dubliniensis differentiation. However, accurate, quick and inexpensive phenotypic tests are needed to be used in low-complexity mycology laboratories. AIMS: To evaluate colony morphotypes on Sabouraud-triphenyltetrazolium agar as a tool for C. dubliniensis and C. albicans differentiation. METHODS: The morphology of 126 C. albicans and C. dubliniensis strains was evaluated and compared with their identification by molecular methods. RESULTS: The method showed 100% sensitivity and specificity when color and the presence or absence of large white mycelial halo was evaluated. CONCLUSIONS: Colony morphotype on Sabouraud-triphenyltetrazolium agar should be considered as a new tool to differentiate C. dubliniensis and C. albicans.

Molecular tools for cryptic Candida species identification with applications in a clinical laboratory.

Candida spp. includes more than 160 species but only 20 species pose clinical problems. C. albicans and C. parapsilosis account for more than 75% of all the fungemias worldwide. In 1995 and 2005, one C. albicans and two C. parapsilosis-related species were described, respectively. Using phenotypic traits, the identification of these newly described species is inconclusive or impossible. Thus, molecular-based procedures are mandatory. In the proposed educational experiment we have adapted different basic molecular biology techniques designed to identify these species including PCR, multiplex PCR, PCR-based restriction endonuclease analysis and nuclear ribosomal RNA amplification. During the classes, students acquired the ability to search and align gene sequences, design primers, and use bioinformatics software. Also, in the performed experiments, fungal molecular taxonomy concepts were introduced and the obtained results demonstrated that classic identification (phenotypic) in some cases needs to be complemented with molecular-based techniques. As a conclusion we can state that we present an inexpensive and well accepted group of classes involving important concepts that can be recreated in any laboratory.

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

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)

Multiplex PCR designed to differentiate species within the Candida glabrata complex / PCR multiplex diseñada para diferenciar las especies del complejo Candida glabrata

Background. No phenotypic methods are available to unequivocally differentiate species within the Candida glabrata complex. Aims. To develop a new multiplex PCR method to differentiate between the three species of the C. glabrata species complex, as well as using it to study a C. glabrata collection to discover strains of the newly described species. Methods. The method was developed based on the Internal Transcribed Spacer (ITS) sequence differences between the species. It was validated by using a blinded collection of strains and, finally, the new molecular method was used to study a collection of 192 C. glabrata species complex strains. The obtained results were compared with ITS sequencing. Results. The proposed method showed 100% concordance with ITS sequencing and proved to be effective for clinical and epidemiological applications. Two Candida bracarensis and three Candida nivariensis were found out of the 192 studied strains (0.93% and 1.40% prevalence, respectively). Conclusions. A fast, inexpensive, robust and highly reproducible multiplex PCR method is presented. Its usefulness is demonstrated by studying a large collection of C. glabrata sensu lato strains (AU)

Antecedentes. No hay métodos fenotípicos disponibles para diferenciar las especies del complejo Candida glabrata. Objetivos. Diseñar un método de PCR multiplex para diferenciar las tres especies del complejo C. glabrata y usarlo para estudiar una colección de cepas identificadas anteriormente como C. glabrata. Métodos. El método fue desarrollado con base en las diferencias de la secuencia internal transcribed spacer (ITS) entre las especies. El método se validó mediante el uso de una colección de cepas incógnitas y se utilizó posteriormente para estudiar una colección de 192 cepas. Los resultados se compararon con las secuencias ITS. Resultados. El método propuesto mostró 100% de concordancia con la secuenciación de las regiones ITS y demostró ser eficaz clínica y epidemiológicamente. Se identificaron dos aislamientos de Candida bracarensis y tres de Candida nivariensis dentro de las 192 cepas identificadas fenotípicamente como C. glabrata (prevalencia de 0,93% y 1,40%, respectivamente). Conclusiones. Presentamos un método de PCR múltiplex rápido, económico y fiable. La utilidad de la metodología queda demostrada con el estudio de una gran colección de cepas de C. glabrata sensu lato (AU)