Resistencia a los azoles en cepas de Aspergilus fumigatus sensu stricto aislados de muestras clínicas respiratorias

Introducción. La aspergilosis invasiva presenta alta mortalidad en pacientes con enfermedades crónicas e inmunocomprometidos. Aspergillus fumigatus sensu stricto (AFSS) es el principal agente etiológico y su tipificación requiere de métodos moleculares. La incidencia incrementada de AI resistentes a los antifúngicos demanda un diagnóstico certero y oportuno. Métodos. Se estudiaron 20 cepas de la micoteca del Instituto de Medicina Tropical - Universidad Nacional Mayor de San Marcos, aisladas de muestras respiratorias e identificadas como Aspergillus fumigatus sensu lato mediante el estudio macroscópico y microscópico. Las cepas fueron referidas a la Universidad Nacional del Litoral para su tipificación mediante una PCR screening para AFSS basada en secuencias del gen CYP51A y el estudio de sensibilidad antifúngica para voriconazol (VOR), itraconazol (ITC), posaconazol (POS), isavuconazol (ISA), anidulafungina (ANF), caspofungina (CSF) y anfotericina B (AMB) obteniendo la concentración inhibitoria mínima (CIM) mediante el protocolo de CLSI M38M51S-Ed3. Resultados. Las 20 cepas fueron identificadas como AFSS. Ninguna de las cepas tuvo una CIM por encima del punto de corte clínico (VOR), ni epidemiológico (ITC, ISA, AMB y CSF). POS fue la droga más potente frente a la colección de cepas evaluadas (media geométrica (GM) de CIM de 0,042 µg/ml). Conclusiones. Todos los aislamientos fueron tipificados como AFSS sensibles a los azoles según los puntos de corte clínico, posaconazol tuvo la mayor actividad antifúngica. Nuestros hallazgos aportan a incrementar la escasa información sobre la etiología y sensibilidad a los antifúngicos de uso clínico de las aspergilosis invasiva en nuestro país.

Introduction. Invasive Aspergillosis (IA) poses a significant threat to patients with chronic diseases and compromised immune systems, with Aspergillus fumigatus sensu stricto (AFSS) being the primary etiological agent. Accurate and timely diagnosis is crucial, particularly given the rising incidence of IA strains resistant to antifungals, necessitating molecular methods for typing. Methods. Twenty strains from Instituto de Medicina Tropical - Universidad Nacional Mayor de San Marcos, mycological collection, previously identified as Aspergillus fumigatus sensu lato through macroscopic and microscopic analysis, were studied. These strains were forwarded to the Universidad Nacional del Litoral for AFSS typing using a PCR screening based on CYP51A gene sequences. Antifungal susceptibility testing was performed for Voriconazole (VOR), Itraconazole (ITC), Posaconazole (POS), Isavuconazole (ISA), Anidulafungin (ANF), Caspofungin (CSF), and Amphotericin B (AMB), obtaining Minimum Inhibitory Concentrations (MICs) according to CLSI M38M51S-Ed3. Results. All 20 strains were identified as AFSS. None of the strains exhibited MICs above the clinical breakpoint (VOR) or the epidemiological cutoffs (ITC, ISA, AMB, and CSF). POS demonstrated the highest potency against the strain collection, with a geometric mean MIC of 0,042 µg/ml. Conclusions. All isolates were classified as azole-sensitive Aspergillus fumigatus sensu stricto (AFSS) based on clinical cutoff points, particularly posaconazole, which exhibited superior antifungal activity. Our findings contribute to augmenting the limited information on the etiology and clinical antifungal sensitivity of IA in our country.

Expression of ERG11, ERG3, MDR1 and CDR1 genes in Candida tropicalis

Introduction. Drug resistance to azoles is a growing problem in the Candida genus. Objective. To analyze molecularly the genes responsible for fluconazole resistance in Candida tropicalis strains. Materials and methods. Nineteen strains, with and without exposure to fluconazole, were selected for this study. The expression of MDR1, CDR1, ERG11, and ERG3 genes was analyzed in sensitive, dose-dependent sensitive, and resistant strains exposed to different concentrations of the antifungal drug. Results. MDR1, ERG11 and ERG3 genes were significantly overexpressed in the different sensitivity groups. CDR1 gene expression was not statistically significant among the studied groups. Seven of the eight fluconazole-resistant strains showed overexpression of one or more of the analyzed genes. In some dose-dependent sensitive strains, we found overexpression of CDR1, ERG11, and ERG3. Conclusion. The frequency of overexpression of ERG11 and ERG3 genes indicates that they are related to resistance. However, the finding of dose-dependent resistant/sensitive strains without overexpression of these genes suggests that they are not exclusive to this phenomenon. More basic research is needed to study other potentially involved genes in the resistance mechanism to fluconazole.

Introducción. La farmacorresistencia a los azoles es un problema creciente en el género Candida. Objetivo. Analizar molecularmente los genes responsables de la resistencia a fluconazol en cepas de Candida tropicalis. Materiales y métodos. Para este estudio, se seleccionaron 19 cepas, con exposición a fluconazol y sin ella. Se analizó la expresión de los genes MDR1, CDR1, ERG11 y ERG3 en cepas sensibles, sensibles dependiente de la dosis, y resistentes, previamente expuestas a diferentes concentraciones del fármaco antifúngico. Resultados. Se encontró que los genes MDR1, ERG11 y ERG3 estaban significativamente sobreexpresados en los diferentes grupos de sensibilidad. La expresión del gen CDR1 no fue estadísticamente significativa entre los grupos estudiados. Siete de las ocho cepas resistentes a fluconazol mostraron sobreexpresión de uno o más de los genes analizados. En algunas cepas sensibles dependientes de la dosis, se encontró sobreexpresión de CDR1, ERG11 y ERG3. Conclusión. La sobreexpresión de los genes ERG11 y ERG3 indica que están relacionados con la resistencia de las cepas de Candida. Sin embargo, el hallazgo de cepas resistentes o sensibles según la dosis, sin sobreexpresión de estos genes, sugiere que pueden existir otros genes involucrados en este fenómeno. Se necesitan más investigaciones básicas que contribuyan al estudio de otros genes potencialmente involucrados en el mecanismo de resistencia al fluconazol.

In vitro sensitivity of Malassezia furfur isolates from HIV-positive and negative patients to antifungal agents / Sensibilidad in vitro a antifúngicos de aislamientos de Malassezia furfur de pacientes positivos y negativos para HIV

Introduction. Malassezia is a lipophilic and lipid-dependent yeast genus belonging to the skin microbiota of humans and other animals. However, due to dysbiosis processes or other factors in the host, this yeast can cause different pathologies, ranging from skin diseases, such as seborrheic dermatitis, to fungemia. Isolation of Malassezia furfur has been reported in HIV-positive patients with or without skin lesions. Due to its opportunistic nature and its variable resistance to antifungal compounds, it is relevant to know the Malassezia sensitivity profiles. Objective. To determine the sensitivity to different antifungal agents, of clinical isolates of M. furfur obtained from HIV-positive or negative patients, with or without seborrheic dermatitis. Materials and methods. Assessment of isolates sensitivity to itraconazole, voriconazole, fluconazole, and amphotericin B was performed by two techniques: (1) Broth microdilution using Clinical and Laboratory Standards Institute (CLSI) protocol M27-A3 with modifications; and (2) agar tests using Etest®. Results. Isolates obtained from HIV patients showed an increase in the minimum inhibitory concentration of fluconazole, voriconazole, and amphotericin B, compared with those of non-HIV patients. Itraconazole was the antifungal with the lowest minimum inhibitory concentration (MIC) in most isolates. Conclusion. We observed differences in the sensitivity profiles of M. furfur isolates according to the context of the patient. High MIC of antifungals like fluconazole, commonly used for treating pathologies caused by Malassezia, were identified.

Introducción. Malassezia es un género de levaduras lipofílicas que dependen de los lípidos y hacen parte de la microbiota de la piel de humanos y otros animales. No obstante, debido a procesos de disbiosis u otros factores en el huésped, esta levadura puede llegar a causar diferentes enfermedades: desde cutáneas (como dermatitis seborreica) hasta fungemias. Se han reportado aislamientos de Malassezia furfur en pacientes positivos para HIV, con lesiones cutáneas o sin ellas. Por su carácter oportunista y sensibilidad variable a los compuestos antifúngicos, es relevante conocer los perfiles de sensibilidad. Objetivo. Determinar la sensibilidad a diferentes antifúngicos de aislamientos clínicos de M. furfur obtenidos de pacientes positivos o negativos para HIV, con dermatitis seborreica o sin ella. Materiales y métodos. La sensibilidad de los aislamientos a itraconazol, voriconazol, fluconazol y anfotericina B, se determinó mediante dos técnicas: microdilución en caldo según el protocolo M27-A3 del Clinical & Laboratory Standards Institute (CLSI), con modificaciones, y pruebas en agar mediante Etest®. Resultados. Los aislamientos obtenidos de pacientes con HIV mostraron aumento de la concentración inhibitoria mínima a fluconazol, voriconazol y anfotericina B, en comparación con los de pacientes sin HIV. Por otro lado, al evaluar la mayoría de los aislamientos, el itraconazol fue el antifúngico con la menor concentración inhibitoria mínima. Conclusión. Se evidencian diferencias en los perfiles de sensibilidad de los aislamientos de M. furfur, según el contexto del paciente, y elevadas concentraciones inhibitorias mínimas de antifúngicos como el fluconazol, usados comúnmente para el tratamiento de las enfermedades causadas por Malassezia spp.

Analysis of resistance and sensitivity of 1 200 strains of vulvovaginal candidiasis in China to five common antifungal drugs / 中华妇产科杂志

Objective:To test the antibiotic susceptibility of vulvovaginal candidiasis pathogenic strains to 5 antifungal drugs commonly used in clinic.Methods:A total of 1 200 vulvovaginal candida patients from 23 gynecological and family planning outpatient departments in China were enrolled. Their vaginal secretions were collected for candida strain isolation and species identification. According to Clinical and Laboratory Standards Institute (CLSI) M27-S3, the sensitivity of 1 200 strains to clotrimazole, fluconazole, miconazole, itraconazole and nystatin was tested.Results:(1) The sensitivity and resistance of 1 200 vulvovaginal candidiasis pathogens to 5 antifungal drugs were statistically different ( χ2=3 513.201, P<0.01). (2) All strains had higher sensitivity to nystatin [99.92% (1 199/1 200)], followed by miconazole [92.25% (1 107/1 200)] and clotrimazole [87.17% (1 046/1 200)]. All strains had higher resistance to fluconazole [69.17% (830/1 200)], while itraconazole was 50.83% (610/1 200). (3) There was no significant difference between candida albicans and non-candida albicans in drug sensitivity to nystatin ( P=0.315) and miconazole ( P=0.425). (4) Candida albicans and non-candida albicans showed different sensitivity to clotrimazole, fluconazole and itraconazole, respectively. Compared with non-candida albicans, candida albicans showed higher sensitivity to clotrimazole [susceptibility rate: 73.01% (165/226) vs 90.45% (881/974); P<0.001] and higher resistance to fluconazole [resistance rate: 50.88% (115/226) vs 73.41% (715/974); P<0.001]. Although the drug sensitivity of itraconazole was not high, the susceptibility rate of candida albicans to itraconazole was slightly higher than that of non-candida albicans [37.68% (367/974) vs 23.89% (54/226)], and the drug resistance rate was lower [49.28% (480/974) vs 57.52% (130/226)]. Conclusions:The sensitivity of 1 200 strains of candida to 5 antifungal drugs is significantly different, the sensitivity rate of nystatin, miconazole and clotrimazole are higher, but the resistance rate of fluconazole and itraconazole are higher. The sensitivity of candida albicans and non-candida albicans to the same drug is also significantly different. It is suggested that in clinical diagnosis and treatment, we should pay attention to the identification of candida and drug sensitivity test, so as to select antifungal drugs rationally.

Retrospective analysis of 201 clinical isolates of Aspergillus fumigatus from a hospital in Nanjing: clinical characteristics of infected patients and azole resistance / 中华皮肤科杂志

Objective:To summarize clinical characteristics of patients with Aspergillus fumigatus infection in a hospital in Nanjing, to preliminarily assess azole resistance in clinical isolates of Aspergillus fumigatus, and to investigate risk factors for the emergence of azole-resistant Aspergillus fumigatus. Methods:Clinical isolates of Aspergillus fumigatus were collected from inpatients in Department of Laboratory, Nanjing Drum Tower Hospital from March 2017 to February 2021. Clinical data on these infected patients were analyzed, azole sensitivity testing and mutation analysis of the cyp51A gene and its promoter region were performed for these Aspergillus fumigatus isolates. Results:A total of 201 strains of Aspergillus fumigatus were collected, and mainly isolated from sputum specimens. Among the infected patients, there were 131 males and 70 females, and their age were 64.2 ± 15.8 years. The patients were mainly collected from department of respiratory medicine (79 cases), department of intensive medicine (34 cases), department of rheumatology (19 cases), etc. Among these patients, common underlying diseases included interstitial pneumonia (32 cases), malignant tumors (18 cases), pneumonia (13 cases), trauma (12 cases), systemic lupus erythematosus (8 cases), etc. Drug susceptibility testing showed that 6 (2.99%) strains of Aspergillus fumigatus were resistant to itraconazole and posaconazole, and 3 patients infected with azole-resistant Aspergillus fumigatus had used antifungal drugs before testing. Sequencing was performed on the cyp51A gene and its promoter region in the 6 strains of azole-resistant Aspergillus fumigatus, and showed TR34/L98H/S297T/F495I mutation in 5 strains and TR34/L98H mutation in 1 strain. Conclusion:Compared with previously published data about azole resistance in China during 2010 -2015, the resistance of Aspergillus fumigatus to azoles in Nanjing Drum Tower Hospital did not increase from 2017 to 2021, and the mechanism of azole resistance was mostly associated with TR34/L98H/S297T/F495I mutation in the cyp51A gene and its promoter region.

Mechanisms underlying the resistance of Candida albicans to echinocandin antifungals / 中华皮肤科杂志

With the application of echinocandin antifungals, more and more resistant Candida albicans strains have been detected. It has been reported that mechanisms underlying the resistance of Candida albicans to echinocandin antifungals mainly involve FKS, MSH2 and ERG3 gene mutations, biofilm formation, cellular stress response, compensatory increases in chitin, sphingolipid synthesis, etc. This review focuses on echinocandin resistance-related genes and underlying mechanisms in Candida albicans, which will help to overcome and prevent echinocandin resistance in clinical practice, and explore new therapeutic targets and drugs for Candida albicans infection.

Comparison of drug susceptibility of and drug resistance mutations in fluconazole-resistant Candida albicans strains from superficial and deep infections / 中华皮肤科杂志

Objective:To compare the in vitro susceptibility of fluconazole-resistant Candida albicans strains from superficial and deep infections to 8 antifungal drugs, and to compare drug resistance mutations in these strains. Methods:According to the Clinical and Laboratory Standards Institute (CLSI) protocol M27-A4, 26 deep infection-derived and 33 superficial infection-derived drug-resistant Candida albicans strains were tested for in vitro susceptibility to 8 antifungal drugs (fluconazole, voriconazole, itraconazole, posaconazole, amphotericin B, fluorocytosine, terbinafine, and micafungin) alone or in combination. DNA was extracted from all drug-resistant strains, and mutations in 3 drug resistance genes, including ERG3, ERG11 and FUR1, were detected by PCR. Normally distributed measurement data with homogeneous variance were compared between two groups by using two-independent-sample t test, non-normally distributed measurement data with non-homogeneous variance were compared using Mann-Whitney U test, and enumeration data were compared using chi-square test. Results:The minimum inhibitory concentrations (MICs) of fluconazole, itraconazole, voriconazole, posaconazole and fluorocytosine all significantly differed between the superficial infection group and deep infection group (all P < 0.05) , while there was no significant difference in the MIC of amphotericin B or micafungin between the two groups (both P > 0.05) . The MIC of terbinafine was >64 μg/ml in 96.6% of the above strains, so could not be compared between groups. As combination drug susceptibility testing revealed, the combination of terbinafine with azoles (fluconazole, voriconazole, itraconazole or posaconazole) showed synergistic inhibitory effects against 15 Candida albicans strains (7 strains from deep infections, 8 strains from superficial infections) , with fractional inhibitory concentration (FIC) indices being 0.033 to 0.187; no marked synergistic effect was observed in the combinations between fluorocytosine and azoles, between fluorocytosine and amphotericin B, or between amphotericin B and fluconazole, with the FIC indices being 0.56 to 1.125. The missense mutation V351A in the ERG3 gene was identified in all the 33 (100%) superficial infection-derived strains, as well as in 13 (50%) deep infection-derived strains, and the mutation A353T in the ERG3 gene was identified in 4 (15%) deep infection-derived strains; as for the ERG11 gene, missense mutations identified in the superficial infection-derived strains included I437V (32 strains, 97%) , Y132H (23 strains, 70%) , T123I (16 strains, 48%) , K128T (6 strains, 18%) , D116E (5 strains, 15%) , A114S (4 strains, 12%) , E266D (2 strains, 6%) , G448E (2 strains, 6%) , and G465S (2 strains, 6%) , while missense mutations identified in the deep infection-derived strains included I437V (23 strains, 88%) , E266D (13 strains, 50%) , E260G (5 strains, 19%) , and V488I (4 strains, 15%) ; the missense mutation R101C in the FUR1 gene was identified in 11 (33%) superficial infection-derived strains, but not identified in deep infection-derived strains. Conclusion:The drug susceptibility and drug resistance mutations differed to some extent between superficial infection- and deep infection-derived fluconazole-resistant Candida albicans strains.

Primer reporte de Candida auris en Perú

Candida auris es un hongo emergente, con gran relevancia en corto tiempo, como problema de salud pública mundial. Se reporta por primera vez en el Perú la presencia de Candida auris en 3 pacientes adultos internados en un hospital nacional de alta complejidad en el último trimestre del año 2020, durante la pandemia COVID-19. Los pacientes fueron hospitalizados en UCI; sin embargo, solo en 2 pacientes se aisló dicho germen durante su internamiento en UCI. Los pacientes tuvieron varias comorbilidades y tiempos prolongados de hospitalización desde su admisión hasta tener el primer cultivo positivo a C. auris. Todos los pacientes adquirieron una infección nosocomial bacteriana en algún momento de su hospitalización y recibieron antibióticos de amplio espectro. Todas las cepas aisladas fueron resistentes a fluconazol. El equipo de control de infecciones del hospital reforzó las medidas de contención y el Ministerio de Salud del Perú emitió una alerta epidemiológica.

Candida auris is an emerging fungus that has gained great relevance as a global public health problem in a short time. The presence of Candida auris in 3 adult patients admitted to a national hospital of high complexity in the last quarter of 2020 in the midst of the COVID-19 pandemic is reported for the first time in Peru. The patients were hospitalized in the ICU, however, this germ was isolated in only 2 patients while they were hospitalized in the ICU. The patients had various comorbidities and long hospitalization times from admission to having their first culture positive for C. auris. All patients acquired a bacterial nosocomial infection at some point during their hospitalization and received broad-spectrum antibiotics. All isolates were resistant to fluconazole. The hospital's infection control team reinforced containment measures and the Ministry of Health of Peru issued an epidemiological alert.

Candida auris osteomielitis: Case report / Osteomielitis por Candida auris: reporte de caso

Abstract Introduction: Candida auris is an opportunistic yeast associated with multiple infections, which was first reported in 2009 in Tokyo, Japan. Provided that it has great antifungal resistance to azoles and amphotericin B, its treatment options are limited, and therefore an empiric therapy using echinocandins such as micafungin should be considered. Case presentation: A rare case of a 48-year-old male patient with osteomyelitis caused by C. auris was reported in the city of Popayán, Colombia. The patient had a history of femoral head fracture, paraplegia due to firearm-related injury and neurogenic bladder, and reported having experienced abundant purulent foul-smelling secretions through trochanteric right ulcer for 15 days. MRI images revealed myositis and bone intensity alterations, which allowed diagnosing him with osteomyelitis. Due to repeated isolations of C. haemulonii in several bone samples, antifungal management was initiated. However, since no improvement in the patient's condition was observed, a culture was sent to the Colombian National Institute of Health to identify the pathogen considering the repeated isolations of C. haemulonii and its apparent resistance to antifungals. C. auris was finally confirmed as the pathogen. Conclusion: Osteomyelitis by C. auris is a rare entity, which must be considered when treating patients with predisposing risk factors such as long hospital stays, bearing in mind that this is an inpatient-associated opportunistic infection.

Resumen Introducción. Candida auris es una levadura oportunista asociada a múltiples infecciones que, en 2009, fue descrita por primera vez en Tokio, Japón. Dado que tiene una gran resistencia antifúngica a los azoles y a la anfotericina B, su manejo es limitado, por lo que se debe considerar iniciar un tratamiento empírico con equinocandinas como la micafungina. Presentación de caso. Caso inusual de osteomielitis por C. auris en un hombre de 48 años de Popayán, Colombia, con antecedentes de fractura de cabeza de fémur, paraplejia por herida con arma de fuego y vejiga neurogénica. El paciente tenía cuadro clínico de 15 días de evolución consistente en salida abundante de líquido purulento fétido en úlcera derecha por presión trocantérica. Mediante resonancia magnética se identificaron miositis y alteraciones de intensidad ósea, por lo que fue diagnosticado con osteomielitis. Debido a la identificación de aislamientos repetidos de Candida haemulonii en varias muestras óseas, se inició manejo antifúngico; sin embargo, ya que no se observó ninguna mejora en la condición del paciente, el cultivo fue enviado al Instituto Nacional de Salud para confirmar la identificación del patógeno debido a aislamientos repetidos de C. haemulonii y su aparente resistencia a los antifúngicos. Finalmente, el patógeno identificado fue C. auris. Conclusión. La osteomielitis por C. auris es una entidad inusual cuyo diagnóstico debe ser considerado en pacientes con factores de riesgo predisponente, como aquellos con larga estancia hospitalaria, ya que esta es una infección oportunista asociada a pacientes hospitalizados.

Análisis cuantitativo de la expresión de genes de resistencia a fluconazol en cepas de Candida albicans aisladas al ingreso de adultos mayores a una unidad de cuidados intensivos de Manizales, Colombia / Quantitative analysis of the expression of fluconazole-resistant genes in strains of Candida albicans isolated from elderly people at their admission in an intensive care unit in Manizales, Colombia

Introducción. Las infecciones oportunistas asociadas con Candida albicans han tenido gran repercusión en la salud pública por la mortalidad que generan en determinados grupos poblacionales. Aunque existen tratamientos farmacológicos disponibles, es evidente el aumento de la resistencia desarrollada por el agente patógeno, por lo que la determinación de los mecanismos de resistencia de las cepas presentes en las áreas hospitalarias es importante, ya que permitiría plantear mejores esquemas de tratamiento. Objetivo. Analizar la expresión de los genes ERG11, CDR1 y MDR1 en cepas de C. albicans aisladas de adultos mayores a su ingreso en la unidad de cuidados intensivos del Hospital Santa Sofía de Manizales, Colombia. Materiales y métodos. Se seleccionaron 29 muestras (21 resistentes y 8 sensibles) y se conformaron dos grupos de trabajo, uno de muestras con exposición al fluconazol y el otro sin esta. El ARN extraído se cuantificó mediante reacción en cadena de la polimerasa con transcriptasa inversa en tiempo real (RT-qPCR). Resultados. Se encontraron diferencias significativas en la expresión del gen MDR1 en el grupo de cepas de C. albicans resistentes. Dos de las cepas resistentes (104 y 62-2) expuestas al antifúngico presentaron valores muy elevados en la expresión de este gen. La expresión del ERG11 y del CDR1 no fue significativa en los grupos estudiados. Conclusión. El aumento de sobreexpresión del gen MDR1 indica que este puede ser el responsable de la resistencia; sin embargo, algunas cepas resistentes no sobreexpresaron los genes analizados, lo que indica que puede haber otros genes involucrados en la resistencia de las cepas estudiadas.

Introduction: Opportunistic infections associated with Candida albicans have had a great impact on public health due to the mortality they generate in certain population groups. Although pharmacological treatments are available, the resistance developed by the pathogen has become increasingly evident. For this reason, determining the mechanisms of resistance associated with the strains found in different hospital areas is important since it would help improving treatment plans. Objective: To analyze the expression of ERG11, CDR1, and MDR1 genes in strains of C. albicans isolated from elderly patients at admittance in the intensive care unit of Hospital Santa Sofía in Manizales, Colombia. Materials and methods: A total of 29 samples (21 resistant and 8 sensitive) were selected and distributed in two working groups: with and without exposure to fluconazole. The extracted RNA was quantified by real-time reverse transcription polymerase chain reaction (RT-qPCR). Results: Significant differences were found in the expression of the MDR1 gene in the group of resistant C. albicans strains. Two of the resistant strains (104 and 62-2) exposed to the antifungal showed very high values in the expression of this gene. The expression of ERG11 and CDR1 was not significant among the groups studied. Conclusion: The increased overexpression of the MDR1 gene indicates that it may be responsible for the resistance. However, some resistant strains did not overexpress any of the genes analyzed, which indicates that there may be other genes involved in the resistance of the strains under study.

Synergic effect of simvastatin in combination with amphotericin B against environmental strains of Cryptococcus neoformans from northeastern Brazil: a prospective experimental study

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds' droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.

Situación de la resistencia antifúngica de especies del género Candida en Perú / Situation of Anti-Fungal Resistance of Species of the Genus Candida in Peru

RESUMEN Actualmente asistimos a un cambio constante y continuo del panorama epidemiológico de la resistencia de levaduras a antifúngicos, relacionado con el tipo de huésped, la etiología de la enfermedad, el microorganismo involucrado y sus mecanismos de resistencia. Esta situación epidemiológica hace más complicado el manejo de los pacientes con infecciones del torrente sanguíneo por especies del género Candida. Observándose una alta mortalidad, incremento en el uso de antifúngicos, así como el desarrollo de métodos fiables para realizar estudios de sensibilidad. Por tanto, es importante la vigilancia local y regional para conocer el perfil de sensibilidad y la distribución de especies de Candida, con la finalidad de instaurar una terapia antifúngica adecuada.

ABSTRACT We are currently witnessing a constant and ongoing change of the epidemiological pictures of yeast resistance to antifungal agents, related to the type of host, etiology of the disease, the microorganism involved, and its mechanisms of resistance. This epidemiological situation complicates management of patients with infections of the bloodstream due to species of the genus Candida, exhibiting a high mortality and an increase in the use of anti-fungal agents, as well as the need to develop reliable methods to conduct sensitivity studies. Therefore, local and regional monitoring is important in order to know the sensitivity profile and the distribution of Candida species so as to start an adequate anti-fungal therapy.

A single-center 18-year experience with oral candidiasis in Brazil: a retrospective study of 1, 534 cases

Abstract Despite the large number of published studies about oral candidiasis and associated risk factors, reports of large single-center retrospective studies on the prevalence of oral candidiasis, risk factors, and the oral candidiasis types diagnosed more frequently in oral diagnostic reference centers are scarce. The objective of the present study was to retrospectively survey the demographic and clinical profiles of 1,534 patients diagnosed with candidiasis and treated at the Center for Diagnosis of Oral Diseases (CDOD), Pelotas Dental School, Federal University of Pelotas between 1997 and 2014. Using a retrospective, cross-sectional, epidemiological design, data on race, gender, age, systemic diseases, oral candidiasis type and location, symptoms, and harmful habits such as smoking and alcohol consumption were collected. The statistical analysis was performed using STATA version 13.1. Risk factors for chronic atrophic candidiasis (CAC) were evaluated using Poisson regression with robust variance (p ≤ 0.05). The majority of patients with oral candidiasis seen at the CDOD over the 18-year period of analysis were Caucasian women, aged 51-60 years, nonsmokers, and nondrinkers, with no systemic disease, and who wore some form of dental prostheses. CAC was the single most common clinical type of candidiasis detected, and the most frequently affected oral site was the palate. These data from a large single-center in Brazil agree with previous evidence about the clinical and demographic profiles of patients with oral candidiasis.

Production of farnesol in Candida albicans biofilms of resistant and standard strains in different media / 中华皮肤科杂志

Objective To compare the production of farnesol between Candida albicans (C.albicans) biofilms formed by resistant and standard strains in different media,and to investigate the changing trend of farnesol production in different phases of biofilm formation and the features of farnesol production by resistant C.albicans.Methods Fluconazole-resistant C.albicans strains were induced in vitro.Standard strains and fluconazole-resistant strains of C.albicans were separately inoculated onto different media,including RPMI 1640 medium,yeast extract peptone dextrose (YPD) medium,yeast nitrogen base (YNB) + 0.5％ glucose medium,RPMI 1640 + 10％ fetal calf serum (FCS),so as to form C.albicans biofilms.Morphological changes of C.albicans biofilms at 24 hours were observed under an inverted microscope,and gas chromatography-mass spectrometry (GC/MS)was performed to detect the level of farnesol at 1.5,3,6,12,24,36 and 48 hours.Results There were no obvious differences in the morphology of C.albicans biofilms between the resistant and standard strains when they were cultured in the same medium,while the morphology of C.albicans biofilms markedly differed between the 2 kinds of strains in the different media.Three-factor analysis of variance showed that the production of farnesol in the C.albicans biofilms changed over time (F =70.628,P ＜ 0.001).Concretely speaking,during the formation of resistant and standard C.albicans biofilms,the production of farnesol gradually increased in the RPMI 1640,YPD and YNB + 0.5％ glucose media until the biofilms matured,then showed a decreasing trend.However,the time to peak levels of farnesol was different between the 2 kinds of strains in these media.Moreover,the levels of farnesol in the 2 kinds of strains both slowly increased in the RPMI 1640 + 10％ FCS medium within 12-48 hours.Culture media also significantly affected the production of farnesol (F =176.665,P ＜ 0.001),and the levels of farnesol in the resistant and standard C.albicans biofilms were both higher in the YNB + 0.5％ glucose medium.When resistant and standard strains were separately cultured in the RPMI 1640 media and the YPD media,the level of farnesol was significantly higher in the resistant strains than in the standard stains (RPMI 1640 media at 36 hours:1.157 ± 0.064 vs.0.250 ± 0.075,P ＜ 0.05;YPD media at 6 hours:0.262 ± 0.036 vs.0.055 ± 0.062,P ＜ 0.05;YPD media at 12 hours:0.730 ± 0.030 vs.0.482 ± 0.024,P ＜ 0.05).However,when they were separately cultured in the YNB + 0.5％ glucose media,the farnesol level was significantly higher in the standard stains than in the resistant strains (36 hours:2.950 ± 0.677 vs.0.523 ± 0.266,P =0.020).Conclusion The media markedly affect the production of farnesol in the C.albicans biofilms,and there is a certain difference in the production of farnesol between resistant and standard C.albicans strains.

Sensibilidad in vitro de cepas cubanas de Aspergillus spp. de origen clínico y ambiental / In vitro susceptibility of Cuban Aspergillus spp. strains of clinical and environmental origin

Introducción. En Cuba se desconoce el comportamiento de la sensibilidad de Aspergillus spp. a los antifúngicos recomendados para el tratamiento de la aspergilosis: la anfotericina B, el itraconazol, el voriconazol y las equinocandinas. La influencia del ambiente puede condicionar la aparición de resistencia en estos microorganismos. Objetivo. Evaluar la sensibilidad in vitro de cepas de Aspergillus spp. a la anfotericina B, el itraconazol y el voriconazol, y la relación de los patrones de sensibilidad con su origen. Materiales y métodos. Se determinaron las concentraciones inhibitorias mínimas de la anfotericina B, el itraconazol y el voriconazol para 60 cepas de Aspergillus spp. de origen clínico y ambiental mediante el método M38-A2 del Clinical and Laboratory Standard Institute. Resultados. Se encontraron 21 cepas resistentes a la anfotericina B (principalmente en muestras clínicas y ambientes hospitalarios) y tres cepas resistentes al itraconazol (en ambientes interiores y exteriores no hospitalarios). No se hallaron cepas resistentes al voriconazol. No se encontró relación entre el origen de las cepas y su sensibilidad. Conclusiones. Se sugiere la posible existencia de factores ambientales o interacciones con genotipos resistentes que pueden dar origen a fenotipos resistentes en Cuba. Este es el primer reporte del país de cepas de Aspergillus spp. resistentes in vitro. Los resultados ameritan ampliar el estudio para incluir análisis moleculares y filogenéticos.

Introduction: The behavior of antifungal susceptibility of Aspergillus spp. in Cuba remains unknown. The antifungals recommended to treat aspergillosis are amphotericin B, itraconazole, voriconazole and echinocandins. The influence of the environment may set off the emergence of drug-resistance in these microorganisms. Objective: To evaluate in vitro susceptibility of Aspergillus spp. strains to amphotericin B, itraconazole and voriconazol, and the relationship between susceptibility patterns and their origin. Materials and methods: Minimum inhibitory concentrations of amphotericin B, itraconazole and voriconazole were determined for 60 Aspergillus spp. strains of clinical and environmental origin using the M38-A2 method of the Clinical and Laboratory Standards Institute. Results: We found 21 amphotericin B resistant strains (mainly from clinical samples and hospital environments), as well as three itraconazole resistant strains (from non-hospital outdoor and indoor environments). No voriconazole resistance was found. No relationship was found between strain origin and susceptibility. Conclusions: Results suggest the possible existence of environmental factors or interactions with resistant genotypes which may give rise to resistant phenotypes in our country. This is the first report of in vitro Aspergillus spp. resistant strains in Cuba. These studies should be broadened and include molecular and phylogenetic analyses.

Relationship between ERG4 gene overexpression and azole resistance in clinical Candida albicans strains / 中华皮肤科杂志

Objective To explore the relationship between ERG4 gene overexpression and azole resistance in clinical Candida albicans strains. Methods The National Committee for Clinical Laboratory Standards (NCCLS)M27-A2 broth microdilution method was conducted to evaluate antifungal susceptibility of 34 clinical Candida albicans isolates in vitro. Total RNA was extracted from these Candida albicans strains and transcribed into cDNA. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of ERG4 gene. Statistical analysis was carried out by a two-sample t-test. Results The expression level of ERG4 mRNA was significantly higher in fluconazole-resistant than in -sensitive Candida albicans strains (4.20 ± 2.56 vs. 1.72 ± 1.33, t = 3.99, P < 0.05), higher in itraconazole-resistant than in -sensitive Candida albicans strains (3.60 ± 2.47 vs. 1.66 ± 1.61, t = 3.71, P < 0.05), and higher in voriconazole-resistant than in -sensitive Candida albicans strains (3.99 ± 2.72 vs. 2.07 ± 1.58, t = 2.91, P <0.05). Further more, increased ERG4 mRNA expression was also observed in isolates cross-resistant to all the three azole antifungal agents compared with those susceptible to all of them (4.49 ± 2.73 vs. 1.69 ± 1.82, t = 3.81, P < 0.05). Conclusions The overexpression of ERG4 gene may be associated with cross resistance to fluconazole, itraconazole and voriconazole in clinical Candida albicans strains, but its exact role is expected to be investigated through downregulation of the ERG4 gene.

Analysis on dynamic monitoring of nosocomial deep fungal infections in tumor hospital during 2010-2012 / 国际检验医学杂志

Objective To investigate the dynamic changes and drug resistance of fungal infection in our hospital during 2010 -2012 for guiding the clinical reasonable use of anti-fungal drugs .Methods The related clinical data and the drug sensitivity tests re-sults of fungal infection in the hospitalized patients from 2010 to 2012 were retrospectively analyzed by the WHONET 5 .6 soft-ware .Results Candida albicans was the major pathogen causing candida infection for these 3 years .The isolated specimens were mainly sputum and blood .In the departments of internal medicine ,general surgery and integrated Chinese and Western medicine ,the number of detected candida were more than that of the other departments .Five kinds of candida showed high sensitivity to ampho-tericin B and high resistance to itraconazole .Conclusion The distribution of pathogens causing nosocomial fungal infection has changed and the drug resistance rate is continuously rising .Therefore the dynamic monitoring and the study of fungal infection should be strengthened for reducing the occurrence of fungal infection in the patients with tumor .

Advance of resistance mechanisms of echinocandins against Candida and antifungal activity in vitro / 中华检验医学杂志

Although echinocandins,a new generation of antifungal drugs,shows good fungicidal activity against distinct species of Candida,with the extending usage,the activity of echinocandins is gradually to decline,which is caused by different resistance mechanisms.Also,a number of factors may influence its results of susceptibility in vitro (e.g.,human serum,culture temperature in vitro,the pH of the culture medium,and others).Paradoxical effect of echinocandins has its own characteristics and need a further study.In China,there are a few reports about the instances of drug resistance because of the limited clinic application.The study on the echinocandins in prevalence rate of drug resistance and antifungal activity under different status has important clinical significance.

Proteomic analysis of plasma membrane from fluconazole-resistant Candida albicans / 中华皮肤科杂志

Objective To identify and analyze plasma membrane proteins differentially expressed between fluconazole-sensitive and-resistant C.albicans strains.Methods Two C.albicans strains from a same parent,including the fluconazole-sensitive C.albicans strain CA-3 and fluconazole-resistant C.albicans strain CA-16,served as the subject of this study.Plasma membrane proteins were isolated from both of the C.albicans strains,and subjected to two-dimensional polyacrylamide gel electrophoresis analysis for the screening of differentially expressed proteins,which were then identified by using matrix assisted laser desorption/ionization time-of-flight mass spectrometry.The resultant data were searched against a protein database for C.albicans.Results Twentytwo proteins were identified to be differentially expressed between the fiuconazole-resistant and-sensitive C.albicans strain.Of them,6 proteins (Adh1p,Csp37p,Pgk1p,Pgk1p and 2 unnamed proteins,i.e.,gi227305312and gi53954641) were highly expressed,while 16 proteins (Aco1p,Aco1p,Hsp78p,Gut2p,Sdh12p,Ilv2p,Ndh51p,Ndh51p,Atp1p,Pda1p,Srb1p,Idh1p,Tdh1p,Cyt1p,Cox4p,Cox13p) were lowly expressed in the fluconazole-resistant C.albicans strain compared with the fluconazole-sensitive strain.Conclusion The plasma membrane proteins differentially expressed between fluconazole-sensitive and-resistant C.albicans strain are mainly implicated in energy metabolism and mitochondrial function.

In vitro activities of antifungal agents alone and in combination against fluconazole-susceptible and -resistant strains of Candida dubliniensis

In the present study we used two groups of Candida dubliniensis strains: one containing fluconazole-susceptible clinical isolates and another containing fluconazole-resistant laboratory derivative from the former to examine the changes on susceptibility accompanying the development of resistance to fluconazole. Our findings confirmed the ability of C. dubliniensis isolates to become resistant to fluconazole and indicated that this resistance was crossed with ketoconazole, itraconazole, ravuconazole and terbinafine. We also tested combinations of terbinafine, amphotericin B, itraconazole and voriconazole against both groups of isolates in a checkerboard assay. Surprisingly, most combinations evidenced indifferent interactions, and the best synergism appeared when terbinafine and itraconazole were combined against the fluconazole-resistant group.