Resistance of Clinical Isolated Gram Negative Bacilli to Tigecycline / 现代检验医学杂志

Objective To investigate the resistance of clinical isolated gram negative bacilli to tigecycline.Methods One hun-dred and fifteen Escherichia coli isolates,110 Klebsiella pneumoniae isolates and 99 Acinetobacter calcoaceticus-Acinetobact-er baumannii complex isolates were collected from Affiliated Second Hospital of Zhejiang University College of Medicine from the year 2012.The other 393 A.calcoaceticus-A .baumannii complex isolates were collected from 15 hospitals of nine cities in Zhejiang province during September to October 2012.Species identification and susceptibility test were confirmed by VITEK-2 compact system,while the 393 A.calcoaceticus-A .baumannii complex strains isolated from Zhejiang province were identified by MALDI-TOF MS.Moreover,159 A.baumannii isolates with tigecycline MIC value ≥8 μg/ml or 4 μg/ml and part of the MIC value ≤0.5 μg/ml,1 μg/ml and 2 μg/ml which were firstly determined by VITEK 2 GN AST-16 Suscepti-bility card were then determined by Etest.Results The 393 A.calcoaceticus-A .baumannii complex strains were identified as 317 of A.baumannii,32 of A.pittii and 44 of A.nosocomialis.When using the FDA breakpoints,the resistance rate of tige-cycline against 115 E.coli isolates,110 K.pneumoniae isolates and 99 A.calcoaceticus-A .baumannii complex isolates were 0.0%,7.3% and 6.1%,respectively.Interestingly,85.7% of the tigecycline-resistant gram negative bacilli were resistant to carbapenems.However,only 10.0% of the carbapenem-resistant gram negative bacilli were resistant to tigecycline.Conclu-sion Tigecycline had a good activity against clinical isolated multi-drug resistant or even pan-drug resistant gram negative bacilli.No matter which criteria for tigecycline was referred to,the resistance rate oftigecycline was slightly lower by Etest than by GN AST-16 Susceptibility card.

Comparison of the ATB® Fungus 2 with the AFST-EUCAST for in vitro susceptibility testing of Candida spp

The increase of diseases caused by Candida spp., and the treatment failures, has underscored the need for testing the susceptibilities to antifungal agents. The commercial panel ATB® Fungus 2 was compared with the reference testing method of the European Subcommittee on Antifungal Susceptibility Testing of the Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) for the evaluation of the susceptibility of isolates of Candida spp. to three agents. The percentage of agreement was calculated based on the minimum inhibitory concentrations. There was a high correlation for AMB (100% қ = 1.0 Bhapkar coefficient p = 1.0); while it was lower with azoles (85%, қ = 0.41, p = Bhapkar coefficient 0.02 and 83.0%, қ = 0.15, Bhapkar coefficient p = 0.0006, respectively). The ATB® Fungus 2 and AFST-EUCAST are fully comparable methods for testing the susceptibility to AMB and to lesser extend comparable for ITR and FCA.

El aumento de infecciones por Candida spp. y de las fallas en los tratamientos, suscitan la necesidad de pruebas de susceptibilidad. Se comparó la marca comercial ATB® Fungus 2 con la técnica estándar del Subcomité para las Pruebas de Sensibilidad Antifúngica de la Unión Europea, de la Sociedad de Microbiología Clínica y Enfermedades Infecciosas (AFST-EUCAST) para evaluar la susceptibilidad de aislamientos de Candida spp. a tres antifúngicos. Con base en las concentraciones inhibitorias mínimas se calculó el porcentaje de acuerdo. La concordancia para anfotericina B (AMB) fue alta (100% қ = 1.0, Coeficiente de Bhapkarp = 1.0); para itraconazol (ITR) y fluconazol (FCA) fue inferior (85% қ = 0.41, Coeficiente de Bhapkarp =0.02 y 83.0 %, қ = 0.15, Coeficiente de Bhapkarp = 0.0006, respectivamente). Por lo tanto, ambas técnicas son comparables para la evaluación de la susceptibilidad a AMB; con los azoles el porcentaje de acuerdo es menor.

Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species

Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods.

Validación del inóculo por densitometría para las pruebas de susceptibilidad a los antifúngicos en especies del género Fusarium / Validation of the inoculum by densitometry for antifungal susceptibility testing in Fusarium species

El objetivo de este trabajo fue validar la preparación del inóculo por densitometría para las pruebas de susceptibilidad a los antifúngicos en especies del género Fusarium. Se emplearon 15 aislamientos clínicos de Fusarium spp. para preparar los inóculos por espectrofotometría y contaje de unidades formadoras de colonias en cámara de Neubauer, siguiendo los protocolos establecidos por los documentos de referencia M38-A2 del Instituto de Estándares Clínicos y de Laboratorio (CLSI) y E.DEF 9.1 del Comité Europeo para Pruebas de Susceptibilidad a los Antimicrobianos (EUCAST), respectivamente. En paralelo se determinaron las lecturas por densitometría para ambos procedimientos. Se estableció un rango de 0,5-0,7 unidades McFarland para la preparación del inóculo por densitometría según el CLSI, y un rango de 0,2-0,8 unidades McFarland para la metodología descrita por el EUCAST. Con este estudio, se logró validar la preparación del inóculo para las pruebas de susceptibilidad en Fusarium spp., utilizando la densitometría como método alternativo de los procedimientos descritos internacionalmente, con considerables ventajas para ser implementado en los laboratorios de microbiología clínica. La variabilidad en cuanto a la capacidad de esporulación y tamaño de las conidias, sobre todo en especies poco frecuentes de Fusarium, sugiere la necesidad de validar el inóculo por especie.

The purpose of this work was to validate the preparation of the inoculum by densitometry for antifungal susceptibility testing in Fusarium species. Fifteen clinical isolates of Fusarium spp. were used to prepare the inocula by spectrophotometry and counting of colony forming units in a Neubauer chamber, according to the protocols established by the reference documents M38-A2 of the Clinical and Laboratory Standards Institute (CLSI), and E.DEF 9.1 of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), respectively. Densitometry readings were determined in parallel for both procedures. A range of 0.5-0.7 McFarland units was established for inocula preparation by densitometry according to the CLSI, and a range of 0.2-0.8 McFarland units was established for the methodology described by EUCAST. This study allowed validating the preparation of the inocula for antifungal susceptibility testing in Fusarium spp., using densitometry as an alternative method for other procedures described internationally, with considerable advantages that can be implemented at clinical microbiology laboratories. The variability regarding sporulation capacity and conidia size, especially in less frequent Fusarium species, suggests the need of validating inocula per species.

In Vitro Fluconazole and Voriconazole Susceptibilities of Candida Bloodstream Isolates in Korea: Use of the CLSI and EUCAST Epidemiological Cutoff Values

BACKGROUND: At present, the clinical breakpoints (CBPs) of both fluconazole and voriconazole are available only for 3 common Candida species in the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. Epidemiological cutoff values (ECVs) were recently applied to both methods to detect the emergence of acquired resistance (i.e., non-wild-type isolates) among 5 common Candida species. METHODS: We performed a nationwide study to determine the fluconazole and voriconazole susceptibility of Candida bloodstream isolates (BSIs) using both the CLSI and EUCAST methods. A total of 423 BSIs of 5 Candida species were collected from 8 hospitals. The azole susceptibilities were assessed on the basis of the species-specific CBPs and ECVs. RESULTS: Of the 341 BSIs of 3 common Candida species (i.e., C. albicans, C. tropicalis, and C. parapsilosis), 0.3% and 0.9%, 0.0% and 1.5% of isolates were categorized as fluconazole and voriconazole resistant according to the CLSI and EUCAST CBPs, respectively. Of 423 total BSIs, 1.4% and 2.6% had fluconazole minimum inhibitory concentrations (MICs) exceeding the ECVs according to the CLSI and EUCAST, respectively; 1.0% and 2.1% had voriconazole MICs exceeding the ECVs according to the CLSI and EUCAST, respectively. Categorical agreement between the methods using ECVs was 98.3% for fluconazole and 98.3% for voriconazole. CONCLUSIONS: The EUCAST and CLSI methods using ECVs provide highly concordant results. Moreover, non-wild-type isolates with possibly acquired azole resistance were rare among the BSIs of 5 common Candida species in Korea.

In Vitro Fluconazole and Voriconazole Susceptibilities of Candida Bloodstream Isolates in Korea: Use of the CLSI and EUCAST Epidemiological Cutoff Values

BACKGROUND: At present, the clinical breakpoints (CBPs) of both fluconazole and voriconazole are available only for 3 common Candida species in the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. Epidemiological cutoff values (ECVs) were recently applied to both methods to detect the emergence of acquired resistance (i.e., non-wild-type isolates) among 5 common Candida species. METHODS: We performed a nationwide study to determine the fluconazole and voriconazole susceptibility of Candida bloodstream isolates (BSIs) using both the CLSI and EUCAST methods. A total of 423 BSIs of 5 Candida species were collected from 8 hospitals. The azole susceptibilities were assessed on the basis of the species-specific CBPs and ECVs. RESULTS: Of the 341 BSIs of 3 common Candida species (i.e., C. albicans, C. tropicalis, and C. parapsilosis), 0.3% and 0.9%, 0.0% and 1.5% of isolates were categorized as fluconazole and voriconazole resistant according to the CLSI and EUCAST CBPs, respectively. Of 423 total BSIs, 1.4% and 2.6% had fluconazole minimum inhibitory concentrations (MICs) exceeding the ECVs according to the CLSI and EUCAST, respectively; 1.0% and 2.1% had voriconazole MICs exceeding the ECVs according to the CLSI and EUCAST, respectively. Categorical agreement between the methods using ECVs was 98.3% for fluconazole and 98.3% for voriconazole. CONCLUSIONS: The EUCAST and CLSI methods using ECVs provide highly concordant results. Moreover, non-wild-type isolates with possibly acquired azole resistance were rare among the BSIs of 5 common Candida species in Korea.

Estudio comparativo de medición entre lecturas visuales y espectrofotométricas en pruebas de susceptibilidad in vitro de aislamientos de candida / Comparative study of measure visual and spectrophotometer lectures in a in vitro antifungal susceptibility testing in candida isolates

Se realizó un estudio de susceptibilidad in vitro frente a itraconazol, ketoconazol y clotrimazol de 144 cepas de Candida, conservadas y previamente identificadas, aisladas de la cavidad oral de pacientes infectados por el virus de inmunodeficiencia humana (VIH) con cuadros clínicos de candidiasis orofaríngea (COF). El estudio se llevó a cabo mediante dos metodologías; la primera, utilizando los requerimientos del Clinical and Laboratory Standard Institute (CLSI), en el cual está establecida la lectura visual para determinar los patrones de susceptibilidad; y la segunda, mediante la propuesta de la European Committee on Antibiotic Susceptibility Testing, el cual tiene fijado la lectura espectrofotométrica para eliminar las posibles subjetividades de la metodología del CLSI. Los resultados obtenidos mediante ambas lecturas no mostraron diferencias mayores a dos diluciones en los valores de concentración mínima inhibitoria, y demostraron que ambos métodos se correlacionan y es importante para aquellos laboratorios de pocos recursos económicos.

A study of in vitro susceptibility was realized opposite to itraconazol, ketoconazol and clotrimazol of 144 strains of Candida, conserved and previously identified, isolated of the oral cavity of patients infected by the virus of human immunodeficiency (VIH) with clinical pictures of oropharingeal candidiasis (COF). The study was realized by means of two methodologies; the first one, using the requests of the Clinical and Laboratory Standard Institute (CLSI), in which the visual reading is established to determine the patterns of susceptibility; and the second one, by means of the proposal of the European Committee on Antibiotic Susceptibility Testing, which has fixed the spectrophotometric to eliminate the possible subjectivities of the methodology of the CLSI. The results obtained by means of both readings did not show differences bigger than two dilutions in the values of minimal inhibitory concentration, demonstrating that both methods are correlated and it is important for those laboratories of few economic resources.

Comparison of Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing Breakpoints for beta-Lactams in Enterobacteriaceae Producing Extended-Spectrum beta-Lactamases and/or Plasmid-Mediated AmpC beta-Lacta / 대한임상미생물학회지

BACKGROUND: In 2010, the Clinical and Laboratory Standards Institute (CLSI) revised breakpoints for cephalosporins and carbapenems and indicated that extended-spectrum beta-lactamase (ESBL) testing is no longer necessary for Enterobacteriaceae. We compared the results of the CLSI 2010 and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) MIC breakpoints for Enterobacteriaceae producing ESBL and/or plasmid-mediated AmpC beta-lactamase (PABL). METHODS: A total of 94 well-characterized clinical isolates of Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Shigella spp., Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, and Serratia marcescens were analyzed. Of them, 57 were ESBL producers, 24 were PABL producers, and 13 were ESBL plus PABL co-producers. Broth microdilution MIC tests were performed for cefotaxime, ceftazidime, aztreonam, cefepime, and imipenem. RESULTS: Among the 94 isolates containing ESBL and/or PABL, the number of isolates that were susceptible to cefotaxime, ceftazidime, aztreonam, cefepime, and imipenem according to the CLSI 2010 vs. the EUCAST breakpoints were 4 (4.3%) vs. 4 (4.3%); 26 (27.7%) vs. 8 (8.5%); 37 (39.4%) vs. 14 (14.9%); 71 (75.5%) vs. 31 (33.0%); and 76 (80.9%) vs. 90 (95.7%), respectively. Of the 18 isolates that were not susceptible to imipenem according to the CLSI 2010 breakpoints, 13 isolates (72.2%) were P. mirabilis. CONCLUSION: The CLSI 2010 MIC breakpoints without tests to detect ESBL and/or PABL for Enterobacteriaceae could be unreliable. Thus, special tests for ESBLs and AmpC beta-lactamases are required to detect the resistance mechanisms involved.

Estandarización del inóculo por desintometría para las pruebas de susceptibilidad antifungica en especies de fusarium / Inoculum standadization by densitometry for the antifungal susceptibility testing of fusarium species

Las especies del género Fusarium han emergido como patógenos oportunistas en las últimas décadas. La aparición de cepas resistentes y la introducción de nuevos antifúngicos, hace necesaria la realización de las pruebas de susceptibilidad en los laboratorios de microbiología clínica. El objetivo de este estudio fue estandarizar la preparación del inóculo por densitometría para las pruebas de susceptibilidad a los antifúngicos en especies de Fusarium. Se utilizaron 15 aislamientos clínicos de Fusarium spp. y se prepararon los inóculos por espectrofotometría y por contaje de unidades formadoras de conidias en cámara de Neubauer, siguiendo los protocolos establecidos por el CLSI y EUCAST respectivamente, determinando en paralelo sus lecturas por densitometría para ambos procedimientos. Las lecturas densitométricas a través del uso del Densimat®, permitieron establecer un intervalo de 0,5 ­ 0,7 unidades Mc Farland para la preparación del inóculo por la metodología descrita por el CLSI y un rango de 0,2 ­ 0,8 unidades Mc Farland para la metodología según el EUCAST. Con este estudio, pionero en Venezuela, se logró estandarizar la preparación del inóculo óptimo en las pruebas de susceptibilidad para Fusarium spp. utilizando la densitometría como método alternativo, comparable y sustitutivo de los procedimientos descritos internacionalmente, con considerables ventajas (útil, disponible y reproducible) para ser implementado en los laboratorios de microbiología clínica. La variabilidad en cuanto a la capacidad de esporulación y tamaño de las conidias, sobre todo en las especies poco frecuentes de Fusarium, sugiere la necesidad de estandarizar el inóculo por especie.

Fusarium species have emerged like opportunistic pathogens in the last decades. The appearance of new resistant strains and the introduction of new antifungal agents, make necessary susceptibility tests in clinical microbiology laboratories. The objective of this study was to standardize inoculum preparation by densitometry for antifungal susceptibility testing in Fusarium species. Fifteen clinical isolates of Fusarium spp. were used, and the inocula were prepared by spectrophotometry and by conidia forming count in Neubauer chamber, following the establishment protocols of CLSI and EUCAST, respectively, determining in parallel their densitometry readings for both procedures. Densitometry readings by Densimat® allowed to establish an interval of 0.5 - 0.7 Mc Farland units for the inoculum preparation by the CLSI methodology, and a rank of 0.2 - 0.8 Mc Farland units for the methodology according to the EUCAST. With this study, pioneer in Venezuela, it was achieved the standardization of the optimal inoculums preparation for the susceptibility tests in Fusarium spp., using densitometry like an alternative, comparable and substitute method of the described internationally standard procedures, with considerable advantages (useful, available and reproducible) to be applied in clinical microbiology laboratories. The variability as far as both sporulation capability and conidia's size, mainly in less frequent species of Fusarium, suggests the needs to standardize the inoculums by species.