RESUMO

Determination of sensitivity to polymyxins has always been a challenge, especially in clinical laboratory routines. This study evaluated two rapid, simple, and inexpensive phenotypic methods to test polymyxin B (PMB) susceptibility in Enterobacterales and non-fermenting Gram-negative bacilli. One hundred isolates were used in the tests. The isolates were collected in three hospitals in southern and southeastern Brazil from 1995 to 2019. We compared broth microdilution (reference method) with the broth disk elution test and modified drop test, using polymyxin B -disk or PMB -powder in 2 concentrations (12 and 16 µg/ml). For the broth disk elution and modified drop test with the concentration of 12 µg/ml, categorical agreement values exceeded 90%. The modified drop test with a concentration of 12 µg/ml and broth disk elution may be excellent for initial screening of polymyxin-resistance in laboratory routines. Moreover, these methods are simple and use inexpensive supplies, and may optimize therapeutic decisions.

Assuntos

RESUMO

OBJECTIVE: We comparatively evaluated the performance of 3 phenotypic tests for the detection of carbapenemase production. MATERIALS AND METHODS: Carbapenemase production was evaluated using the modified Hodge test (MHT), the modified carbapenemase inhibition method (mCIM), and the Rapidec Carba NP test (RCNP). RESULTS: Among the 170 isolates, 79 were CP-CRE and 91 were non-CP-CRE. The CP-CRE isolates produced GES-5 (n = 66), KPC (n = 4), NDM (n = 7), NDM and OXA-48 (n = 1), and VIM (n = 1). For KPC producers, all 3 methods showed a sensitivity of 75%. The sensitivities of MHT, mCIM, and RCNP were 14.3%, 100%, and 71.4%, respectively, for NDM producers, and 1.5%, 12.1%, and 18.2% for GES-5 producers, respectively. CONCLUSION: The performance of the phenotypic tests varied depending on the type of carbapenemase. For intensive infection control, phenotypic and molecular tests are required for the detection of common and rare types of carbapenemases.

Assuntos

RESUMO

Background: The environment is the nontuberculous mycobacteria (NTM) reservoir, opportunistic pathogens of great diversity and ubiquity, which is observed in the constant description of new species capable of causing infection. Since its introduction, molecular methods are essential for species identification. Most comparative studies between molecular and conventional methods, have used isolated strains from clinical samples. Methods: The aim of this study was to evaluate the usefulness of molecular methods, especially the hsp65-PRA (PCR-Restriction Enzyme Analysis), and biochemical tests in the identification of NTM recovered from water of different origins, using the sequencing of 16S rRNA and hsp 65 genes as assessment methods of the previous ones. Species identification was performed for all 56 NTM isolates what were recovered from 32 (42.1%) positive water samples, using conventional phenotypic methods, hsp65-PRA, partial sequencing of 16S rRNA and sequencing of hsp 65 genes. Results: Phenotypic evaluation and hsp65-PRA were concordant with 23 (41.1%) isolates. Also, the PRA was concordant with 16 (28.6%) and 27 (48.2%) isolates, with the partial sequencing of 16S rRNA and sequencing of hsp 65 genes, respectively. It is considered that the 19.6% (n = 11) could not be identified. Conclusion: Identification of NTM environmental isolates to the species level, especially when they are pigmented and fast-growing, both the analysis of the restriction patterns obtained by PRA and the sequencing of the 16S rRNA and hsp 65 genes are insufficient by themselves. Although they are demanding and time-consuming, biochemical tests are very useful to support data obtained by molecular methods.

Assuntos

RESUMO

Rare nonfermenting Gram-negative bacilli, such as Chryseobacterium indologenes and Elizabethkingia meningoseptica, have clinical importance in nosocomial infections and cystic fibrosis (CF), and their identification is a challenge to microbiology laboratories. Thus, the objective of this study was to verify the performance of phenotypic and mass spectrometry (matrix-assisted desorption ionization-time of flight mass spectrometry, MALDI-TOF MS) methods to identify C. indologenes and E. meningoseptica. In this context, the results obtained with phenotypic methods-namely manual biochemical and automated VITEK 2 (bioMérieux, Marcy l'Etoile, France) and Phoenix tests (Becton Dickinson (BD), San Diego, CA, USA)-and by MALDI-TOF MS-namely MALDI-TOF VITEK MS (MALDI-MS; bioMérieux) and MALDI-TOF BioTyper (MALDI-BD; BD)-of 22 isolates (blood cultures of patients with nosocomial infection (n = 15) and from patients with CF (n = 7)), initially identified as C. indologenes and E. meningoseptica, were compared. As result, using the manual phenotypic method, it was possible to identify the species level in 18/22; no identification was found in 4/22. There was a low agreement level between manual and VITEK 2 automated phenotypic methods when considering the genus level. The greatest agreement for genus-level identification occurred in MALDI-TOF MS equipment (15/22). When comparing all methods to identify the 22 isolates, there was agreement of 4/22 at the genus level and of 4/22 at the species level. In conclusion, there is low agreement level among identification methods of C. indologenes and E. meningoseptica. Although MALDI-TOF MS equipment shows a higher agreement level among them, results present low levels of confidence.

RESUMO

ABSTRACT The present study aimed to genotypically and phenotypically characterize clinical isolates of carbapenem-resistant Enterobacteriaceae collected from inpatients at the University Hospital of Santa Maria, during seven months. Among the clinical isolates subjected to the modified Hodge test (MHT), 62.5% were positive, indicating possible production of carbapenemase. Polymerase chain reaction (PCR) demonstrated that blaKPC was the most frequently found gene (31%), followed by blaIMP (12.5%). Combined use of the methods is needed to identify carbapenem resistance in enterobacteria to prevent their spread and control the infections caused by these organisms.

RESUMO Objetivou-se caracterizar fenotípica e genotipicamente isolados clínicos de enterobactérias resistentes aos carbapenêmicos (CRE) provenientes do Hospital Universitário de Santa Maria (RS). Entre os isolados clínicos submetidos ao teste modificado de Hodge (MHT), 62,5% apresentaram positividade, indicando possível produção de carbapenemase. A reação em cadeia da polimerase (PCR) demonstrou que o blaKPC foi o gene mais encontrado (31%), seguido de blaIMP (12,5%). O uso conjunto de distintas metodologias faz-se necessário para identificar a resistência aos carbapenêmicos produzida pelas enterobactérias, de modo a auxiliar o controle de infecção prevenindo a disseminação desses microrganismos.

RESUMO

Se investigó el desempeño de diversos métodos fenotípicos para la detección de carbapenemasas KPC en 44 aislamientos de Klebsiella pneumoniae con sensibilidad disminuida a los carbapenems, 30 productores y 14 no productores de KPC. Se determinó la sensibilidad a imipenem, meropenem y ertapenem por dilución en agar y difusión por discos. Se evaluaron los siguientes métodos fenotípicos: sinergia entre discos de carbapenems y discos con los inhibidores amoxicilina/ácido clavulánico (AMC ) y ácido aminofenilborónico (APB); inhibición por "discos combinados" (según una técnica de predifusión diseñada a tal fin en nuestro laboratorio), comparando el efecto de los carbapenems solos o asociados con los inhibidores mencionados; y el test de Hodge modificado, para el cual se propone una lectura cuantificada. El método de Hodge detectó todos los aislamientos KPC positivos con los tres carbapenems evaluados, mientras que fue negativo para todos los aislamientos KPC negativos con imipenem y meropenem, y produjo dos resultados falsos positivos con ertapenem. Cuantificando la lectura de este método se pudieron discriminar objetivamente los resultados verdaderos positivos (≥ 8 mm) de los falsos positivos (< 5 mm). Se observó sinergia entre carbapenems y APB con todos los aislamientos KPC positivos, aunque esto requirió ajustar la distancia entre discos. En los aislamientos KPC negativos no se observó sinergia entre carbapenems y APB. Empleando el método con discos combinados con imipenem o meropenem más APB se detectaron la mayoría de los aislamientos KPC positivos y no se observaron falsos positivos. Por el contrario, las combinaciones carbapenem más AMC no fueron sensibles ni específicas.(AU)

We evaluated phenotypic methods for the detection of KPC carbapenemases in 44 clinical isolates of K. pneumoniae having reduced susceptibility to carbapenems, 30 of which were KPC-positive and 14 KPC-negative. Both the agar dilution and disk diffusion methods were performed for imipenem, meropenem and ertapenem. The following phenotypic methods were assayed: the double disk synergy test, using boronic acid or clavulanic acid as inhibitors, "combined" disks of carbapenem plus inhibitor (boronic acid, clavulanic acid and both boronic plus clavulanic acid), by using a pre-diffusion technique and the modified Hodge test. The double disk diffusion test using boronic acid could detect all KPC-positive isolates, but adjustment of disk distance was necessary for achieving such performance. The simulation of combined disks by our pre-diffusion technique detected all KPCpositive strains for all 3 carbapenems when using boronic acid as inhibitor, clavulanic acid was less susceptible and specific as compared with boronic acid. The modified Hodge test using any carbapenem was clearly positive for all KPC-producing isolates. This test was negative for all KPC-negative strains when imipenem or meropenem were used, but 2/14 isolates yielded a weak positive result when using ertapenem. By measuring the enhanced growth of E. coli ATCC 25922 observed in this test, we could objectively discriminate true-positive (≥ 8 mm) from false-positive results (< 5 mm). Our results show that the use of phenotypic methods is effective for the rapid detection of KPC producers in K. pneumoniae isolates with reduced susceptibility to carbapenems.(AU)

Assuntos

RESUMO

Se investigó el desempeño de diversos métodos fenotípicos para la detección de carbapenemasas KPC en 44 aislamientos de Klebsiella pneumoniae con sensibilidad disminuida a los carbapenems, 30 productores y 14 no productores de KPC. Se determinó la sensibilidad a imipenem, meropenem y ertapenem por dilución en agar y difusión por discos. Se evaluaron los siguientes métodos fenotípicos: sinergia entre discos de carbapenems y discos con los inhibidores amoxicilina/ácido clavulánico (AMC ) y ácido aminofenilborónico (APB); inhibición por "discos combinados" (según una técnica de predifusión diseñada a tal fin en nuestro laboratorio), comparando el efecto de los carbapenems solos o asociados con los inhibidores mencionados; y el test de Hodge modificado, para el cual se propone una lectura cuantificada. El método de Hodge detectó todos los aislamientos KPC positivos con los tres carbapenems evaluados, mientras que fue negativo para todos los aislamientos KPC negativos con imipenem y meropenem, y produjo dos resultados falsos positivos con ertapenem. Cuantificando la lectura de este método se pudieron discriminar objetivamente los resultados verdaderos positivos (≥ 8 mm) de los falsos positivos (< 5 mm). Se observó sinergia entre carbapenems y APB con todos los aislamientos KPC positivos, aunque esto requirió ajustar la distancia entre discos. En los aislamientos KPC negativos no se observó sinergia entre carbapenems y APB. Empleando el método con discos combinados con imipenem o meropenem más APB se detectaron la mayoría de los aislamientos KPC positivos y no se observaron falsos positivos. Por el contrario, las combinaciones carbapenem más AMC no fueron sensibles ni específicas.

We evaluated phenotypic methods for the detection of KPC carbapenemases in 44 clinical isolates of K. pneumoniae having reduced susceptibility to carbapenems, 30 of which were KPC-positive and 14 KPC-negative. Both the agar dilution and disk diffusion methods were performed for imipenem, meropenem and ertapenem. The following phenotypic methods were assayed: the double disk synergy test, using boronic acid or clavulanic acid as inhibitors, "combined" disks of carbapenem plus inhibitor (boronic acid, clavulanic acid and both boronic plus clavulanic acid), by using a pre-diffusion technique and the modified Hodge test. The double disk diffusion test using boronic acid could detect all KPC-positive isolates, but adjustment of disk distance was necessary for achieving such performance. The simulation of combined disks by our pre-diffusion technique detected all KPCpositive strains for all 3 carbapenems when using boronic acid as inhibitor, clavulanic acid was less susceptible and specific as compared with boronic acid. The modified Hodge test using any carbapenem was clearly positive for all KPC-producing isolates. This test was negative for all KPC-negative strains when imipenem or meropenem were used, but 2/14 isolates yielded a weak positive result when using ertapenem. By measuring the enhanced growth of E. coli ATCC 25922 observed in this test, we could objectively discriminate true-positive (≥ 8 mm) from false-positive results (< 5 mm). Our results show that the use of phenotypic methods is effective for the rapid detection of KPC producers in K. pneumoniae isolates with reduced susceptibility to carbapenems.

Assuntos

RESUMO

A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100 percent specificity, but only the DD tests presented 100 percent sensitivity. The sensitivity of the other tests ranged from 82.2 percent (OAS)-98.3 percent (AD). The LA test showed the second lowest sensitivity (86.4 percent). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

Assuntos

RESUMO

C. albicans é o mais frequente agente responsável pela candidíase e está também associada a doençasimunossupressoras como, por exemplo, diabetes. Entretanto, outras espécies não-albicans como C.dubliniensis inicialmente associada com candidíase da orofaringe de pacientes infectados pelo vírus HIV,onde ainda é mais prevalente, tem sido reconhecida como causa de infecção em pacientes com câncer,com fibrose cística e pacientes com diversas patologias e diferentes graus de imunocomprometimento.Utilizando-se métodos fenotípicos para identificação de Candida spp. é possível discriminar rotineiramenteisolados orais em laboratórios de microbiologia. Um dos problemas que interferem na realização de identificaçãodas espécies do gênero Candida em estudos epidemiológicos mais amplos é a dificuldade da rápidaidentificação, a partir de meios de triagem de baixo custo. Dessa forma, alguns métodos fenotípicos sãoapresentados com o objetivo de contribuir na busca de testes alternativos que possam com segurança, seremaplicados às rotinas de laboratórios.

C. albicans is the frequent agent for the oral candidosis and it is also associated to immunosuppressivediseases as, for example, diabetes. However, other non-albicans species such as C. dubliniensis initiallyassociated with candidosis in orofaringe patients infected by the virus HIV, where is still more prevalent,it has been recognized as infection cause in patients with cancer, cystic fibrosis and patients with severalpathologies. Phenotypics methods for Candida spp. are easy-to-use procedures for routine discriminationof oral isolates in the clinical microbiology laboratory. One of the problems that interfere in the identificationaccomplishment of the species of Candida in more wide epidemic studies is the difficulty of the fast identification,starting from screen methods of low cost. In that way, some phenotypics methods are presented withthe objective of contributing in the search of alternative tests to be applied as routine in laboratories.

RESUMO

La detección de resistencia a meticilina es complicada debido a la heterogeneidad de su expresión fenotípica; dificultando su detección en el laboratorio, lo que ha conducido al desarrollo de varias técnicas para incrementar su expresión in vitro. A fin de evaluar cuatro técnicas para la detección de resistencia a meticilina: método de difusión del disco con oxacilina (OX, 1 ug) y cefoxitin (FOX, 30 ug); screening test, concentración inhibitoria mínima (CIM) y detección de PBP2a, utilizando la presencia del gen mecA como método de referencia, se procesaron 286 cepas de S. aureus. Se determinó la sensibilidad (SEN), especificidad (ESP), valor predictivo positivo (VPP), valor predictivo negativo (VPN) y eficiencia (EFC) de cada uno de los métodos. Se obtuvo un total de 50 cepas resistentes a oxacilina, PBP2a positivos (mecA positivos). La sensibilidad del disco de OX fue de 99.14 por ciento y la de FOX fue de 100 por ciento. La SEN, VPP, VPN y EFC de los otros métodos fue de 100 por ciento. Todas, a excepción del método de difusión del disco de OX (ESP de 99.14), resultaron 100 por ciento específicos.

Detecting methicillin resistance is complicated due to the heterogeneity of its phenotypic expression, making its detection difficult in the laboratory; this has led to the development of several techniques to increase its expression in vitro. Four techniques for detecting methicillin resistance were evaluated: the disk diffusion method with oxacillin (OX, 1 ug) and cephoxitin (FOX, 30 ug); the screening test, minimum inhibitory concentration (MIC) and detection of PBP2a, using the presence of mecA gen as a reference method; 286 strains of S. aureus, were processed. The sensibility (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV) and efficiency (EFC) of each method were determined. A total of 50 oxacillin resistant, PBP2a positive (mecA positive) strains were obtained. Sensibility of the OX disk was 99.14 percent; and of the FOX disk was 100 percent. The SEN, PPV, NVP and EFC of the other methods were 100 percent. All the tests, except the OX disk diffusion method (99.14 percent of ESP), were 100 percent specific.

Assuntos