The natural occurring Y129F polymorphism in Rhizopus oryzae (R. arrhizus) Cyp51Ap accounts for its intrinsic voriconazole resistance.

Rhizopus oryzae (heterotypic synonym: R. arrhizus) intrinsic voriconazole and fluconazole resistance has been linked to its CYP51A gene. However, the amino acid residues involved in this phenotype have not yet been established. A comparison between R. oryzae and Aspergillus fumigatus Cyp51Ap sequences showed differences in several amino acid residues. Some of them were already linked with voriconazole resistance in A. fumigatus. The objective of this work was to analyze the role of two natural polymorphisms in the intrinsic voriconazole resistance phenotype of R. oryzae (Y129F and T290A, equivalent to Y121F and T289A seen in triazole-resistant A. fumigatus). We have generated A. fumigatus chimeric strains harboring different R. oryzae CYP51A genes (wild-type and mutants). These mutant R. oryzae CYP51A genes were designed to carry nucleotide changes that produce mutations at Cyp51Ap residues 129 and 290 (emulating the Cyp51Ap protein of azole susceptible A. fumigatus). Antifungal susceptibilities were evaluated for all the obtained mutants. The polymorphism T290A (alone or in combination with Y129F) had no impact on triazole MIC. On the other hand, a > 8-fold decrease in voriconazole MICs was observed in A. fumigatus chimeric strains harboring the RoCYP51Ap-F129Y. This phenotype supports the assumption that the naturally occurring polymorphism Y129F at R. oryzae Cyp51Ap is responsible for its voriconazole resistance phenotype. In addition, these chimeric mutants were posaconazole hypersusceptible. Thus, our experimental data demonstrate that the RoCYP51Ap-F129 residue strongly impacts VRC susceptibility and that it would be related with posaconazole-RoCYP51Ap interaction. LAY SUMMARY: Rhizopus oryzae is intrinsically resistant to voriconazole, a commonly used antifungal agent. In this work, we analyze the role of two natural polymorphisms present in the target of azole drugs. We established that F129 residue is responsible of the intrinsic voriconazole resistance in this species.

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.

First Clinical Isolation of an Azole-Resistant Aspergillus fumigatus Isolate Harboring a TR46 Y121F T289A Mutation in South America.

One of the most recently described Aspergillus fumigatusCYP51A-mediated azole resistance mechanisms is TR46 Y121F T289A. Clinical A. fumigatus strains harboring these substitutions have been reported worldwide, with the exception of South America. We describe the first clinical A. fumigatus strain with this resistance mechanism isolated from an Argentinian patient. The strain was isolated in 2009 (1 year after the first-described mutant in United States), demonstrating that these alleles were scattered worldwide earlier than previously thought.

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.

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.

Emergence of Triazole Resistance in Aspergillus spp. in Latin America.

PURPOSE OF REVIEW: Azole resistance in Aspergillus spp. is becoming a public health problem worldwide. However, data about this subject is lacking in Latin American countries. This review focuses in the epidemiology and molecular mechanisms of azole resistance in Aspergillus spp. emphasizing in Latin America. Data on Aspergillus fumigatus stands out because it is the most prevalent Aspergillus spp. pathogen. RECENT FINDINGS: Azole resistance in Aspergillus spp. emergence was linked with intensive use of these antifungals both in the clinical setting and in the environment (as pesticides). Reports on azole-resistant A. fumigatus strains are being constantly published in different countries. Molecular mechanisms of resistance mainly involve substitution in the azole target (CYP51A) and/or overexpression of this gene. However, several other non-CYP51A-related mechanisms were described. Moreover, intrinsically resistant cryptic Aspergillus species are starting to be reported as human pathogens. SUMMARY: After a comprehensive literature review, it is clear that azole resistance in Aspergillus spp. is emerging in Latin America and perhaps it is underestimated. All the main molecular mechanisms of azole resistance were described in patients and/or environmental samples. Moreover, one of the molecular mechanisms was described only in South America. Cryptic intrinsic azole-resistant species are also described.

SARS-CoV-2 and Aspergillus section Fumigati coinfection in an immunocompetent patient treated with corticosteroids.

BACKGROUND: Patients with severe viral pneumonia are likely to receive high-dose immunomodulatory drugs to prevent clinical worsening. Aspergillus species have been described as frequent secondary pneumonia agents in severely ill influenza patients receiving steroids. COVID-19 patients admitted to Intensive Care Unit (ICU) are receiving steroids as part of their treatment and they share clinical characteristics with other patients with severe viral pneumonias. COVID-19 patients receiving steroids should be considered a putative risk group of invasive aspergillosis. CASE REPORT: We are reporting a SARS-CoV-2/Aspergillus section Fumigati coinfection in an elderly intubated patient with a history of pulmonary embolism treated with corticosteroids. The diagnosis was made following the ad hoc definitions described for patients admitted to ICU with severe influenza, including clinical criteria (fever for 3 days refractory to the appropriate antibiotic therapy, dyspnea, pleural friction rub, worsening of respiratory status despite antibiotic therapy and need of ventilator support), a radiological criterion (pulmonary infiltrate) and a mycological criterion (several positive galactomannan tests on serum with ratio ≥0.5). In addition, Aspergillus section Fumigati DNA was found in serum and blood samples. These tests were positive 4 weeks after the patient was admitted to the ICU. The patient received voriconazole and after two month in ICU his respiratory status improved; he was discharged after 6 weeks of antifungal treatment. CONCLUSIONS: Severely ill COVID-19 patients would be considered a new aspergillosis risk group. Galactomannan and Aspergillus DNA detection would be useful methods for Aspergillus infection diagnosis as they allow avoiding the biosafety issues related to these patients.

Control of an outbreak of post-transplant cutaneous mucormycosis by removing the vehicle: An intervention study of contiguous cohorts.

BACKGROUND: An outbreak of post-kidney transplant cutaneous mucormycosis (PK-CM), a severe and even fatal complication in immunocompromised patients, occurred in our institution. The objective of this study was to compare the usual fixation of sterile wound dressings with non-sterile elastic bandages and fixation with sterile bandages processed at our centralized sterile services department with regard to PK-CM prevention. METHODS: We conducted a before-and-after trial in a private tertiary care hospital. The pre-intervention cohort (n = 16) included all patients who had undergone kidney transplant (KT) during the outbreak (June 2010-April 2011), and the post-intervention cohort (n = 49) included all patients who had undergone KT between May 2011 and October 2013. From May 2011, only bandages sterilized by the centralized sterile services department were used to fix wound dressings of KT patients. RESULTS: Differences in age (50.2 years vs 51.3 years), sex (43.8% male vs 59.2% female), weight (63.3 kg vs 69.7 kg), hemodialysis vintage (55.6 months vs 47.8 months), and other risk factors were not observed between the pre- and post-intervention cohorts, respectively. PK-CM incidence dropped from 25% in the pre-intervention cohort (4/16) to 0% in the post-intervention cohort (0/49). Relative risk was 0 (P = .003). CONCLUSIONS: With regard to KT patients and sterile wound dressing fixation with non-sterile bandages versus the use of autoclaved bandages, fixation with autoclaved bandages proved to be effective for cutaneous mucormycosis outbreak control and prevention in our institution.

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.

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.

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)

SARS-CoV-2 and Aspergillus section Fumigati coinfection in an immunocompetent patient treated with corticosteroids / Coinfección de SARS-CoV-2 y Aspergillus sección Fumigati en un paciente inmunocompetente tratado con corticosteroides

BACKGROUND: Patients with severe viral pneumonia are likely to receive high-dose immunomodulatory drugs to prevent clinical worsening. Aspergillus species have been described as frequent secondary pneumonia agents in severely ill influenza patients receiving steroids. COVID-19 patients admitted to Intensive Care Unit (ICU) are receiving steroids as part of their treatment and they share clinical characteristics with other patients with severe viral pneumonias. COVID-19 patients receiving steroids should be considered a putative risk group of invasive aspergillosis. CASE REPORT: We are reporting a SARS-CoV-2/Aspergillus section Fumigati coinfection in an elderly intubated patient with a history of pulmonary embolism treated with corticosteroids. The diagnosis was made following the ad hoc definitions described for patients admitted to ICU with severe influenza, including clinical criteria (fever for 3 days refractory to the appropriate antibiotic therapy, dyspnea, pleural friction rub, worsening of respiratory status despite antibiotic therapy and need of ventilator support), a radiological criterion (pulmonary infiltrate) and a mycological criterion (several positive galactomannan tests on serum with ratio ≥0.5). In addition, Aspergillus section Fumigati DNA was found in serum and blood samples. These tests were positive 4 weeks after the patient was admitted to the ICU. The patient received voriconazole and after two month in ICU his respiratory status improved; he was discharged after 6 weeks of antifungal treatment. CONCLUSIONS: Severely ill COVID-19 patients would be considered a new aspergillosis risk group. Galactomannan and Aspergillus DNA detection would be useful methods for Aspergillus infection diagnosis as they allow avoiding the biosafety issues related to these patients

ANTECEDENTES: Los pacientes con neumonía viral grave reciben altas dosis de fármacos inmunomoduladores para prevenir el empeoramiento clínico. Los pacientes con influenza grave que reciben esteroides tienen neumonías secundarias causadas por Aspergillus con una frecuencia relativamente alta. Los pacientes con COVID-19 ingresados en la unidad de cuidados intensivos (UCI) reciben dicha medicación como parte de su tratamiento, y comparten con otro tipo de pacientes muchas de las características clínicas de otras neumonías virales graves. Estos pacientes deberían considerarse como un grupo de riesgo de aspergilosis invasiva. CASO CLÍNICO: Se presenta un caso de coinfección por SARS-CoV-2 y Aspergillus de la sección Fumigati en un paciente intubado de edad avanzada con antecedentes de embolia pulmonar y tratado con corticosteroides. El diagnóstico siguió las definiciones ad hoc descritas para pacientes ingresados en la UCI con gripe grave. El paciente cumplía varios criterios clínicos (fiebre durante 3 días refractaria al tratamiento antibiótico apropiado, disnea, fricción pleural, empeoramiento del estado respiratorio a pesar del tratamiento antibiótico y la necesidad de soporte respiratorio), el criterio radiológico (infiltrado pulmonar) y un criterio micológico (test de galactomanano positivo en suero, (ratio ≥0,5). Además, se detectó ADN de Aspergillus de la sección Fumigati en muestras de suero y sangre del paciente. Estas pruebas fueron positivas 4 semanas después de que el paciente ingresara en la UCI. El paciente recibió tratamiento con voriconazol, y después de 2 meses en la UCI mejoró su estado pulmonar; fue dado de alta después de 6 semanas de tratamiento antifúngico. CONCLUSIONES: Los pacientes gravemente enfermos con COVID-19 deberían considerarse un nuevo grupo de riesgo para la aspergilosis. La detección de galactomanano y ADN de Aspergillus son métodos útiles para el diagnóstico de infección por este hongo al evitar los problemas de bioseguridad en estos pacientes