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BACKGROUND: Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents. OBJECTIVES: This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna. METHODS: We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created. RESULTS: Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype. CONCLUSIONS: This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance.
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Antifúngicos , Azoles , Candida , Candidiasis , Pruebas de Sensibilidad Microbiana , Antifúngicos/farmacología , Azoles/farmacología , Candida/efectos de los fármacos , Candida/genética , Candida/clasificación , Candida/aislamiento & purificación , Candidiasis/microbiología , Humanos , Farmacorresistencia Fúngica Múltiple/genética , Colombia , Anfotericina B/farmacología , Farmacorresistencia Fúngica/genética , Mutación Puntual , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Proteínas Fúngicas/genética , Análisis de Secuencia de ADN , Proteínas de Saccharomyces cerevisiaeRESUMEN
Candida tropicalis is emerging as one of the most common Candida species causing opportunistic infections in Latin America. Outbreak events caused by C. tropicalis were reported, and antifungal resistant isolates are on the rise. In order to investigate population genomics and look into antifungal resistance, we applied a short tandem repeat (STR) genotyping scheme and antifungal susceptibility testing (AFST) to 230 clinical and environmental C. tropicalis isolates from Latin American countries. STR genotyping identified 164 genotypes, including 11 clusters comprised of three to seven isolates, indicating outbreak events. AFST identified one isolate as anidulafungin-resistant and harboring a FKS1 S659P substitution. Moreover, we identified 24 clinical and environmental isolates with intermediate susceptibility or resistance to one or more azoles. ERG11 sequencing revealed each of these isolates harboring a Y132F and/or Y257H/N substitution. All of these isolates, except one, were clustered together in two groups of closely related STR genotypes, with each group harboring distinct ERG11 substitutions. The ancestral C. tropicalis strain of these isolates likely acquired the azole resistance-associated substitutions and subsequently spread across vast distances within Brazil. Altogether, this STR genotyping scheme for C. tropicalis proved to be useful for identifying unrecognized outbreak events and better understanding population genomics, including the spread of antifungal-resistant isolates.
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Candida auris is considered a public health problem due to its resistance and its tendency to cause nosocomial outbreaks. CHROMagarTMCandida Plus has recently been marketed as capable of presumptively identifying C. auris. The objective of this work was to analyze the ability of this new chromogenic medium to differentiate C. auris from other members of the C. haemulonii complex and from other yeasts commonly isolated in clinical practice. A collection of 220 strains including species of the C. haemulonii (n = 83) and C. parapsilosis (n = 80) complexes was studied. The strains were identified by molecular methods and cultured as individual or as mixed aqueous inoculum on CHROMagarTMCandida Plus plates. Colony morphotypes were evaluated at 5 time points. CHROMagarTMCandida Plus was a helpful tool for presumptive identification for C. auris. Better reading results were obtained after 48 hours of incubation at 35°C. It is able to easily differentiate C. auris from other closely related species of the C. haemulonii complex and other yeasts. This chromogenic medium would be also useful as screening and surveillance tool for C. auris colonization. However, we demonstrated that it would be a possible misidentification of C. parapsilosis as C. auris (44.3% showed similar morphotypes). To reduce false positives when it is used in a context of a C. auris outbreak, we propose to supplement the chromogenic medium with 8 µg/ml fluconazole. This modified medium was tested and it clearly differentiate C. parapsilosis from C. auris.
CHROMagarTMCandida Plus is able to differentiate C. auris from other Candida spp., including other species of the C. haemulonii complex. However, 44.3% of the tested C. parapsilosis strains would be misidentified as C. auris. We propose the addition of 8 µg/ml fluconazole to solve this issue.
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BACKGROUND: Candida auris is an emerging multidrug-resistant yeast that can cause invasive infections and healthcare-associated outbreaks. Here, we describe 34 cases of pediatric C. auris bloodstream infections (BSIs) identified during July 2014-October 2017 in 2 hospitals in Colombia. METHODS: We conducted a retrospective review of microbiology records for possible C. auris cases in 2 hospitals in Barranquilla and Cartagena. BSIs that occurred in patients aged <18 years confirmed as C. auris were included in this analysis. RESULTS: We identified 34 children with C. auris BSIs. Twenty-two (65%) patients were male, 21% were aged <28 days, 47% were aged 29-365 days, and 32% were aged >1 year. Underlying conditions included preterm birth (26%), being malnourished (59%), cancer (12%), solid-organ transplant (3%), and renal disease (3%). Eighty-two percent had a central venous catheter (CVC), 82% were on respiratory support, 56% received total parenteral nutrition (TPN), 15% had a surgical procedure, and 9% received hemodialysis. Preinfection inpatient stay was 22 days (interquartile range, 19-33 days), and in-hospital mortality was 41%. CONCLUSIONS: Candida auris affects children with a variety of medical conditions including prematurity and malignancy, as well as children with CVCs and those who receive TPN. Mortality was high, with nearly half of patients dying before discharge. However, unlike most other Candida species, C. auris can be transmitted in healthcare settings, as suggested by the close clustering of cases in time at each of the hospitals.Candida auris is an emerging multidrug-resistant yeast that can cause invasive infections and healthcare-associated outbreaks. This report describes 34 cases of pediatric C. auris bloodstream infections, identified in two hospitals in Colombia, South America.
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Candidiasis Invasiva , Nacimiento Prematuro , Sepsis , Antifúngicos/uso terapéutico , Candida , Candidiasis Invasiva/tratamiento farmacológico , Niño , Colombia/epidemiología , Femenino , Humanos , Recién Nacido , Masculino , Pruebas de Sensibilidad Microbiana , Embarazo , Nacimiento Prematuro/tratamiento farmacológico , Estudios Retrospectivos , Sepsis/tratamiento farmacológicoRESUMEN
BACKGROUND: Candida auris is an emerging multidrug-resistant yeast that causes outbreaks in healthcare settings around the world. In 2016, clinicians and public health officials identified patients with C. auris bloodstream infections (BSI) in Colombian healthcare facilities. To evaluate potential risk factors and outcomes for these infections, we investigated epidemiologic and clinical features of patients with C. auris and other Candida species BSI. METHODS: We performed a retrospective case-case investigation in four Colombian acute care hospitals, defining a case as Candida spp. isolated from blood culture during January 2015-September 2016. C. auris BSI cases were compared to other Candida species BSI cases. Odds ratio (OR), estimated using logistic regression, was used to assess the association between risk factors and outcomes. RESULTS: We analyzed 90 patients with BSI, including 40 with C. auris and 50 with other Candida species. All had been admitted to the intensive care unit (ICU). No significant demographic differences existed between the two groups. The following variables were independently associated with C. auris BSI: ≥ 15 days of pre-infection ICU stay (OR: 5.62, CI: 2.04-15.5), evidence of severe sepsis (OR: 3.70, CI 1.19-11.48), and diabetes mellitus (OR 5.69, CI 1.01-31.9). CONCLUSION: Patients with C. auris BSI had longer lengths of ICU stay than those with other candidemias, suggesting that infections are acquired during hospitalization. This is different from other Candida infections, which are usually thought to result from autoinfection with host flora.
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Candida/aislamiento & purificación , Candidemia/epidemiología , Candidiasis/diagnóstico , Diagnóstico Diferencial , Adulto , Antifúngicos/uso terapéutico , Candidemia/diagnóstico , Candidemia/tratamiento farmacológico , Candidiasis/tratamiento farmacológico , Candidiasis/epidemiología , Colombia/epidemiología , Infección Hospitalaria/diagnóstico , Infección Hospitalaria/epidemiología , Complicaciones de la Diabetes/microbiología , Brotes de Enfermedades , Femenino , Humanos , Control de Infecciones , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Sepsis/complicaciones , Sepsis/microbiología , Resultado del Tratamiento , Adulto JovenRESUMEN
Candida auris is a pathogenic yeast that causes invasive infections with high mortality. Infections most often occur in intensive care units of health care facilities. It is crucial to trace the source and prevent further spread of C. auris during an outbreak setting; therefore, genotyping of C. auris is required. To enable fast and cost-effective genotyping, we developed a short tandem repeat (STR) typing assay for C. auris STRs in C. auris were identified, and from an initial selection of 23 STRs, 12 were used to develop a STR typing assay. Having shown that the STR typing assay was reproducible and specific, a robust set of 444 C. auris isolates was investigated to identify genotypic diversity. In concordance with whole-genome sequencing (WGS) analysis, we identified five major different C. auris clusters of South American, South Asian, African, East Asian, and Iranian origin. Overall, a total of 40 distinct genotypes were identified, with the largest variety in the South Asian clade. Comparison with WGS demonstrated that isolates with <20 single nucleotide polymorphisms (SNPs) are mostly not differentiated by STR analysis, while isolates with 30 or more SNPs usually have differences in one or more STR markers. Altogether, a highly reproducible and specific STR typing assay for C. auris was developed; this assay distinguishes the five different C. auris clades in identical fashion to WGS, while most isolates differing by >30 SNPs, as determined via WGS, are also separated. This new C. auris-specific genotyping technique is a rapid, reliable, and cost-effective alternative to WGS analysis to investigate outbreaks.IMPORTANCECandida auris is an emerging fungal pathogen now recognized as a threat to public health. The pathogen has spread worldwide and causes mainly hospital-associated outbreaks. To track and trace outbreaks and to relate them to new introductions from elsewhere, whole-genome sequencing and amplified fragment length polymorphism (AFLP) have been used for molecular typing. Whole-genome sequencing is costly and available only at a few centers, and AFLP is a complicated technique and hard to interpret. We describe a novel simple STR genotyping technique based on short tandem repeats in the C. auris genome. We also show that the performance of this STR-based genotyping technique has proven comparable to that of WGS. Overall, this work provides a novel, rapid, reliable, and cost-effective method of molecular outbreak investigations of C. auris.
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Candida/genética , Candida/aislamiento & purificación , Candidiasis/microbiología , Repeticiones de Microsatélite , Candida/clasificación , Sitios Genéticos , Marcadores Genéticos , Genoma Fúngico , Genómica/métodos , Genotipo , Humanos , Tipificación Molecular , FilogeniaRESUMEN
Invasive Candidiasis (IC) and candidemia (as its most frequent manifestation) have become the main cause of opportunistic mycosis at hospital settings. This study, made by members of the Colombian Association of Infectious Diseases (ACIN), was aimed at providing a set of recommendations for the management, follow-up and prevention of IC / candidemia and mucous membrane candida infection in adult, pediatric and neonatal patients in a hospital setting, including the hemato-oncological and critical care units. All the data obtained through an exhaustive search were reviewed and analyzed in a comprehensive manner by all the members of the group, and the recommendations issued are being made after a careful review of the scientific literature available and the consensus of all specialists involved; the emergence of Candida Spp. problem is highlighted and a correct orientation to health professionals regarding the management of patients with candidiasis is provided in a rational and practical way, emphasizing patient evaluation, diagnostic strategies, prophylaxis, empirical treatment, directed treatment and preventative therapy.
La Candidiasis Invasora (CI) y la candidemia, como su manifestación más frecuente, se ha convertido en la principal causa de micosis oportunista a nivel hospitalario. Este manuscrito realizado por miembros de la Asociación Colombiana de Infectología (ACIN), tuvo como objetivo proporcionar un conjunto de recomendaciones para manejo, seguimiento y prevención de la CI/candidemia y de la infección candidiásica de mucosas, en población adulta, pediátrica y neonatal, en un entorno hospitalario, incluyendo las unidades hemato-oncológicas y unidades de cuidado crítico. Todos los datos obtenidos mediante una búsqueda exhaustiva, fueron revisados y analizados de manera amplia por todos los miembros del grupo, y las recomendaciones emitidas se elaboraron luego de la evaluación de la literatura científica disponible, y el consenso de todos los especialistas involucrados, reconociendo el problema de la emergencia de las infecciones por Candida Spp. y brindando una correcta orientación a los profesionales de la salud sobre el manejo de pacientes con enfermedad candidiásica, de una forma racional y práctica, enfatizando en la evaluación del paciente, estrategias de diagnóstico, profilaxis, tratamiento empírico, tratamiento dirigido y terapia preventiva.
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Recién Nacido , Adulto , Candidemia , Candidiasis Invasiva , Micosis , Manejo de Atención al Paciente , Colombia , Infecciones Fúngicas Invasoras , Neutropenia/diagnósticoRESUMEN
OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections (SSTIs) represent a major clinical problem in Colombia. The aim of this study was to evaluate the risk factors associated with MRSA SSTI in Colombia. METHODS: A multicenter cohort study with nested case-control design was performed. Patients with an SSTI with at least 48h of inpatient care were included. Patients with an MRSA SSTI were considered the case group and patients with either a non-MRSA SSTI or with an Methicillin-susceptible S. aureus (MSSA) SSTI were the control groups. A multivariate logistic regression approach was used to evaluate risk factors associated with MRSA SSTI with two different statistical models. RESULTS: A total 1134 patients were included. Cultures were positive for 498 patients, of which 52% (n=259) were Staphylococcus aureus. MRSA was confirmed in 68.3% of the S. aureus cultures. In the first model, independent risk factors for MRSA SSTI were identified as the presence of abscess (P<0.0001), cellulitis (P=0.0007), age 18-44 years (P=0.001), and previous outpatient treatment in the previous index visit (P=0.003); surgical site infection was a protective factor (P=0.008). In the second model, the main risk factor found was previous outpatient treatment in the previous index visit (P=0.013). CONCLUSIONS: Community-acquired SSTIs in Colombia are commonly caused by MRSA. Therefore, clinicians should consider MRSA when designing the initial empirical treatment for purulent SSTI in Colombia, although there seems to be low awareness of this fact.
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Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones de los Tejidos Blandos/microbiología , Infecciones Cutáneas Estafilocócicas/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/administración & dosificación , Estudios de Casos y Controles , Estudios de Cohortes , Colombia/epidemiología , Femenino , Humanos , Masculino , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Persona de Mediana Edad , Factores de Riesgo , Infecciones de los Tejidos Blandos/tratamiento farmacológico , Infecciones de los Tejidos Blandos/epidemiología , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Infecciones Cutáneas Estafilocócicas/epidemiología , Adulto JovenRESUMEN
Candida auris is an emerging multidrug-resistant fungus that causes hospital-associated outbreaks of invasive infections with high death rates. During 2015-2016, health authorities in Colombia detected an outbreak of C. auris. We conducted an investigation to characterize the epidemiology, transmission mechanisms, and reservoirs of this organism. We investigated 4 hospitals with confirmed cases of C. auris candidemia in 3 cities in Colombia. We abstracted medical records and collected swabs from contemporaneously hospitalized patients to assess for skin colonization. We identified 40 cases; median patient age was 23 years (IQR 4 months-56 years). Twelve (30%) patients were <1 year of age, and 24 (60%) were male. The 30-day mortality was 43%. Cases clustered in time and location; axilla and groin were the most commonly colonized sites. Temporal and spatial clustering of cases and skin colonization suggest person-to-person transmission of C. auris. These cases highlight the importance of adherence to infection control recommendations.
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Candida , Candidiasis/epidemiología , Candidiasis/microbiología , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/microbiología , Infección Hospitalaria , Brotes de Enfermedades , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Candida/efectos de los fármacos , Candidemia/epidemiología , Candidemia/microbiología , Candidiasis/tratamiento farmacológico , Candidiasis/historia , Niño , Preescolar , Colombia/epidemiología , Enfermedades Transmisibles Emergentes/historia , Farmacorresistencia Fúngica , Femenino , Historia del Siglo XXI , Humanos , Lactante , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mortalidad , Evaluación del Resultado de la Atención al Paciente , Vigilancia en Salud Pública , Estaciones del Año , Adulto JovenRESUMEN
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%.
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Anfotericina B/farmacología , Anidulafungina/farmacología , Antifúngicos/farmacología , Candida/efectos de los fármacos , Caspofungina/farmacología , Viabilidad Microbiana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Factores de TiempoRESUMEN
Background: Candida auris is a multidrug-resistant yeast associated with hospital outbreaks worldwide. During 2015-2016, multiple outbreaks were reported in Colombia. We aimed to understand the extent of contamination in healthcare settings and to characterize the molecular epidemiology of C. auris in Colombia. Methods: We sampled patients, patient contacts, healthcare workers, and the environment in 4 hospitals with recent C. auris outbreaks. Using standardized protocols, people were swabbed at different body sites. Patient and procedure rooms were sectioned into 4 zones and surfaces were swabbed. We performed whole-genome sequencing (WGS) and antifungal susceptibility testing (AFST) on all isolates. Results: Seven of the 17 (41%) people swabbed were found to be colonized. Candida auris was isolated from 37 of 322 (11%) environmental samples. These were collected from a variety of items in all 4 zones. WGS and AFST revealed that although isolates were similar throughout the country, isolates from the northern region were genetically distinct and more resistant to amphotericin B (AmB) than the isolates from central Colombia. Four novel nonsynonymous mutations were found to be significantly associated with AmB resistance. Conclusions: Our results show that extensive C. auris contamination can occur and highlight the importance of adherence to appropriate infection control practices and disinfection strategies. Observed genetic diversity supports healthcare transmission and a recent expansion of C. auris within Colombia with divergent AmB susceptibility.
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Anfotericina B/farmacología , Antifúngicos/farmacología , Candida/clasificación , Candida/efectos de los fármacos , Candidiasis/epidemiología , Candidiasis/microbiología , Farmacorresistencia Fúngica , Candida/genética , Candida/aislamiento & purificación , Portador Sano/epidemiología , Portador Sano/microbiología , Colombia/epidemiología , Microbiología Ambiental , Humanos , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Tipificación Molecular , Técnicas de Tipificación Micológica , Secuenciación Completa del GenomaRESUMEN
PURPOSE: CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9. METHODS: We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting. RESULTS: The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient. CONCLUSION: We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.
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Ascomicetos , Proteínas Adaptadoras de Señalización CARD/genética , Predisposición Genética a la Enfermedad , Heterocigoto , Infecciones Fúngicas Invasoras , Mutación , Feohifomicosis/epidemiología , Feohifomicosis/etiología , Factores de Edad , Edad de Inicio , Ascomicetos/genética , Ascomicetos/inmunología , Biomarcadores , Preescolar , Colombia/epidemiología , Biología Computacional/métodos , Análisis Mutacional de ADN , Femenino , Humanos , Inmunohistoquímica , Inmunofenotipificación , Imagen por Resonancia Magnética , Linaje , Feohifomicosis/diagnóstico , Feohifomicosis/inmunología , Fenotipo , Tomografía Computarizada por Rayos X , Secuenciación del ExomaRESUMEN
Multiple Erg11 amino acid substitutions were identified in clinical isolates of Candida auris originating from India and Colombia. Elevated azole MICs were detected in Saccharomyces cerevisiae upon heterologous expression of C. aurisERG11 alleles that encoded for Y132F or K143R substitutions; however, expression of alleles encoding I466M, Y501H, or other clade-defined amino acid differences yielded susceptible MICs. Similar to other Candida species, specific C. aurisERG11 mutations resulted directly in reduced azole susceptibility.
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Antifúngicos/farmacología , Azoles/farmacología , Candida/efectos de los fármacos , Candida/genética , Mutación/genética , Sistema Enzimático del Citocromo P-450/genética , Farmacorresistencia Fúngica/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
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Candida/clasificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Técnicas de Tipificación Micológica/métodos , Secuencia de Bases , Candida/genética , Candidiasis/microbiología , Enfermedades Transmisibles Emergentes/microbiología , Cartilla de ADN , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Humanos , Reacción en Cadena de la Polimerasa Multiplex/instrumentación , Análisis de Secuencia de ADN , Especificidad de la Especie , Levaduras/genéticaRESUMEN
Candida auris has simultaneously emerged on five continents as a fungal pathogen causing nosocomial outbreaks. The challenges in the treatment of C. auris infections are the variable antifungal susceptibility profiles among clinical isolates and the development of resistance to single or multiple classes of available antifungal drugs. Here, the in vitro susceptibility to echinocandin antifungal drugs was determined and FKS1 sequencing was performed on 106 C. auris clinical isolates. Four isolates were identified to be resistant to all tested echinocandins (MIC ≥ 4 mg/liter) and harbored an S639F mutation in FKS1 hot spot region 1. All remaining isolates were FKS1 wild type (WT) and echinocandin susceptible, with micafungin being the most potent echinocandin (MIC50 = 0.125 mg/liter). Antifungal susceptibility testing with caspofungin was challenging due to the fact that all FKS1 WT isolates exhibited an Eagle effect (also known as the paradoxical growth effect), which occurred at various intensities. To assess whether the Eagle effect resulted in pharmacodynamic resistance, 8 representative isolates were evaluated for their in vivo drug response in a murine model of invasive candidiasis. All isolates were susceptible to caspofungin at a human therapeutic dose, except for those harboring the S639F mutation. The data suggest that only isolates carrying mutations in FKS1 are echinocandin resistant and that routine in vitro testing of C. auris isolates for susceptibility to caspofungin by the broth microdilution method should be viewed cautiously or avoided.
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Antifúngicos/farmacología , Candida/efectos de los fármacos , Candida/genética , Farmacorresistencia Fúngica/genética , Equinocandinas/farmacología , Glucosiltransferasas/genética , Animales , Candida/aislamiento & purificación , Candidiasis/tratamiento farmacológico , Candidiasis/microbiología , Candidiasis Invasiva/tratamiento farmacológico , Candidiasis Invasiva/microbiología , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad MicrobianaRESUMEN
OBJECTIVES: Invasive candidiasis has a high impact on morbidity and mortality in hospitalised patients. Accurate and timely methods for identification of Candida spp. and determination of echinocandin susceptibility have become a priority for clinical microbiology laboratories. METHODS: This study was performed to compare matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) identification with sequencing of the D1/D2 region of the rRNA gene complex 28 subunit in 147 Candida spp. isolates obtained from patients with candidaemia. Antimicrobial susceptibility testing was performed by broth microdilution (BMD) and Etest. Sequencing of the FKS1 and FKS2 genes was performed. RESULTS: The most common species isolated were Candida albicans (40.8%), followed by Candida parapsilosis (23.1%) and Candida tropicalis (17.0%). Overall agreement between the results of identification by MALDI-TOF/MS and molecular identification was 99.3%. Anidulafungin and caspofungin susceptibility by the BMD method was 98.0% and 88.4%, respectively. Susceptibility to anidulafungin and caspofungin by Etest was 93.9% and 98.6%, respectively. Categorical agreement between Etest and BMD was 91.8% for anidulafungin and 89.8% for caspofungin, with lower agreements in C. parapsilosis for anidulafungin (76.5%) and C. glabrata for caspofungin (40.0%). No mutations related to resistance were found in the FKS genes, although 54 isolates presented synonymous polymorphisms in the hotspots sequenced. CONCLUSIONS: MALDI-TOF/MS is a good alternative for routine identification of Candida spp. isolates. DNA sequencing of the FKS genes suggested that the isolates analysed were susceptible to echinocandins; alternatively, unknown resistance mechanisms or limitations related to antifungal susceptibility tests may explain the resistance found in a few isolates.
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Antifúngicos/farmacología , Candida/efectos de los fármacos , Candida/genética , Candidemia/epidemiología , Equinocandinas/farmacología , Anidulafungina/farmacología , Cultivo de Sangre , Candida/aislamiento & purificación , Caspofungina/farmacología , Colombia , Pruebas Antimicrobianas de Difusión por Disco , Genes de ARNr , Secuenciación de Nucleótidos de Alto Rendimiento , Hospitales/estadística & datos numéricos , Humanos , Pruebas de Sensibilidad Microbiana , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii, Candida haemulonii, and Candida lusitaniae Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species.
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Candida/clasificación , Candida/aislamiento & purificación , Candidiasis/diagnóstico , Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Humanos , Factores de TiempoRESUMEN
INTRODUCTION: Carbapenems resistance is a growing phenomenon and a threat to public health because of the reduced therapeutic options for resistant infections. METHODS: A retrospective case-control study was conducted in 2 tertiary-care hospitals in Medellín, Colombia. Fifty patients infected with ertapenem-resistant enterobacteriaceae were compared with a control group consisting of 100 patients with infections caused by ertapenem susceptible enterobacteriaceae. A multivariate logistic regression model was used to identify factors that best explain ertapenem-resistant enterobacteriaceae infections. RESULTS: The factors associated with ertapenem-resistant enterobacteriaceae infections were prior exposure to carbapenems (adjusted OR 3.43; 95% IC 1.08-10.87) and prior exposure to cefepime (adjusted OR 6.46; 95% IC 1.08-38.38). CONCLUSION: Prior exposure to antibiotics is the factor that best explains the ertapenem-resistant enterobacteriaceae infection in this population, highlighting the importance of antimicrobial stewardship programs in hospitals.