Disk Carbapenemase Test for the Rapid Detection of KPC-, NDM-, and Other Metallo-β-Lactamase-Producing Gram-Negative Bacilli

BACKGROUND: Rapid detection of carbapenemase-producing gram-negative bacilli (GNB) is required for optimal treatment of infected patients. We developed and assessed a new disk carbapenemase test (DCT). METHODS: Paper disks containing 0.3 mg of imipenem and bromothymol blue indicator were developed, and the performance of the DCT were evaluated by using 742 strains of GNB with or without carbapenemases. RESULTS: The paper disks were simple to prepare, and the dried disks were stable at -20℃ and at 4℃. The DCT detected 212 of 215 strains (98.6% sensitivity with 95% confidence interval [CI] 96.0-99.5%) of GNB with known class A (KPC and Sme) and class B (NDM, IMP, VIM, and SIM) carbapenemases within 60 min, but failed to detect GES-5 carbapenemase. The DCT also detected all two Escherichia coli isolates with OXA-48, but failed to detect GNB with OXA-232, and other OXA carbapenemases. The DCT showed 100% specificity (95% CI, 99.2-100%) in the test of 448 imipenem-nonsusceptible, but carbapenemase genes not tested, clinical isolates of GNB. CONCLUSIONS: The DCT is simple and can be easily performed, even in small laboratories, for the rapid detection of GNB with KPC, NDM and the majority of IMP, VIM, and SIM carbapenemases.

Evaluación del Agar Azul Bromotimol Lactosa introducido en BioCen con cepas de referencia / Assessment of Agar Blue Bromotimol Lactose introduced in BioCen using reference strans

Exponer los resultados obtenidos en las evaluaciones realizadas a los medios de cultivo Agar azul bromotimol lactosa producido en BioCen, Cuba, y el comercializado por la Merck, Alemania, destinado para la diferenciación de microorganismos especialmente las enterobacterias, por su capacidad de fermentar la lactosa. Se ensayaron un total de 13 cepas certificadas y una aislada de muestra de agua, pertenecientes al Departamento de Investigaciones de Medios de Cultivo (BioCen). Las cepas evaluadas en ambos productos mostraron similitud en cuanto a su respuesta y a las características morfológicas de las colonias. Los valores del índice relativo de crecimiento (IRC) para 10 cepas superaron el 90 por ciento, resultando el 71,5 por ciento del total, mientras que solo 4 cepas reflejaron valores inferiores, resultando el 28,5 por ciento del total. Todas las cepas sobrepasaron el valor recomendado (> 70 por ciento), además las características culturales desarrolladas respondían a las reportadas. Los resultados alcanzados en la determinación del IRC demuestran la buena calidad del medio producido en BioCen frente a cepas de referencia, tomando como criterio el hecho de que todos los microorganismos ensayados mostraron valores del IRC superiores al valor recomendado.

To show results obtained in evaluations performed in agar blue bromotimol lactose culture media produced in BioCen, Cuba, and that marketed by Merck, Germany, created for microorganism differentiation, specially the Enterobacter ones, due to its ability for lactose fermentation. We assayed a total of 13 certified strains and another isolated from water sample, from Research Department of Culture Media (BeioCen). Values or Growing Relative Index for 10 strains were above 90 percent, with 71,5 percent of total, while that only 4 strains shoed lower values to a 28,5 percent of total. All strains exceeded the recommended value (>70 percent). Strains evaluated in both products showed similarity as regard its response and to morphologic features of colonies.

Stability-indicating spectrophotometric method for determination of Moexipril-HCL in bulk and pharmaceutical formulation

Three simple, sensitive and accurate spectrophotometric procedures have been established for the assay of Moexipril-HCI in bulk form, in pharmaceutical formulations, and in the presence of its degradation products. The procedures are based on the reaction between the examined drug and bromocresol purple [BCP], bromophenol blue [BPB], and bromothymol blue [BTB] in aqueous acidic medium producing an ion-pair complexes extracted in chloroform and measured at the optimum wavelengths. Reaction conditions were studied and optimized to obtain the maximum color intensity. The reactions were extremely rapid at room temperature and the absorbance values remains unchanged for 48 h. Beer's law was obeyed in the concentration ranges 4-32, 4-24, and 4-40 microg ml[-1] with molar absorptivities of 1.7x10[4], 2.1x10[4], and 1.5x10[4] mol[-1] cm[-1] and detection limit of 0.064, 0.065, and 0.077 microg ml[-1] for BCP, BPB, and BTB methods, respectively. The proposed methods have been applied successfully for the analysis of the drug in pure form and in its dosage forms with percentage recoveries range from 99.29 - 100.11. Statistical comparison of the results with those obtained by second derivatives spectrophotometric method shows excellent agreement and indicates no significant difference in accuracy and precision

Utilidad de la D-prolina en la diferenciación de las variedades de Cryptococcus neoformans / Usefulness of D-proline in the differentiation of varieties of Cryptococcus neoformans

Se realizó un estudio comparativo entre la asimilación de la D-prolina y el crecimiento en medio canavanina-glicina-azul de bromotimol (CGB) utilizados para la clasificación de las variedades de Cryptococcus neoformans. En las 86 cepas estudiadas, 100 por ciento de coincidencia entre ambos métodos, permitió afirmar que 95,34 por ciento pertenecían a la var. neoformans y el resto (4,65 por ciento) a la var. gattii. Los resultados obtenidos corroboraronn que todos los aislamientos clínicos autóctonos, hasta el presente, corresponden a la var. neoformans y permitieron sugerir el uso de la D-prolina para la evaluación inicial de las cepas, como un método alternativo y sencillo que presentó, en estas condiciones, alta coincidencia con el método de referencia (crecimiento en CGB)

Identificación de cryptococcus neoformans var. gattii mediante el uso del medio canavaninaglicina-azul de bromotinol (CGB) / Identification of cryptococcus neoformans var. gattii by canavanine glycine bromothymol blue medium (CGB)

Se conocen 2 variedades de cryptococcus neoformans y 5 serotipos: cryptococcus neoformans var. neoformans (serotipos A,D y AD), usualmente aislados a partir de muestras clínicas, encontrándose cryptococcus neoformans var. neoformans serotipo A, con mayor incidencia en pacientes con SIDA; cryptococcus neoformans var. gattii (serotipos B y C), los cuales son aislados menos frecuentemente principalmente en pacientes VIH seronegativos y excepcionalmente han sido asociados con SIDA. Se estudiaron 43 cepas de cryptococcus neoformans. El medio de canavanina-glicina-azul de bromotinol (CGB) permite diferenciar en crytococcus la variedad gattii de la variedad neoformans. Nuestro estudio refleja que las 2 cepas (4,65 por ciento) de cryptococcus neoformans var. gattii, obtenidas a partir del aislamiento de muestras de líquido cefalorraquídeo, procedían de una paciente con inmunodeficiencia celular y humoral catalogada como ideopática, y de un paciente con SIDA, asociación rara vez reportada. Son escasos los estudios epidemiológicos de esta micosis en Venezuela, y pocos hacen identificación más allá de la especie, por lo cual es dificil comparar nuestros resultados

New Selective Medium for Rapid Identification of Vibrio vulnificus from Patients with V. vulnificus Sepsis / 대한피부과학회지

BACKGROUND: Vibrio(V.) vulnificus is a halophilic, gram-negative bacillus that causes a fatal sepsis in patients with underlying chronic disease such as liver cirrhosis and alcoholic abuse. Because V. vulnificus infection has a fulminant course and high mortality rate, early recognition and rapid diagnosis with prompt therapy are necessary to improve survival rate. OBJECTIVE: The purpose of this study was to develop a new selective medium for rapid identification of V. vulnificus through color change of medium according to pH from patients suspected of having V. vulnificus sepsis. METHODS: Rapid isolation and identification of V. vulnificus can be possible by modifying the component of PNC(5% peptone, 1% NaCl, and 0.08% cellobiose [pH 8.0]) broth medium. From this PNC broth, a basal broth(5% peptone+1% NaCl+cellobiose) was prepared and used to evaluate additional medium supplements(cellobiose concentration [0.08, 0.2, 0.1%], pH [6.8, 7.5, 8.0] and pH indicator dye [bromthymol blue, thymol blue, phenol red, bromcresol purple, crystal violet, cresol red, and neutral red]). To examine the rapid identification and selectivity of this basal medium according to various conditions, V. vulnificus was tested by using saline and normal human blood containing these bacteria(1, 000 bacteria/ml), respectively at 37degrees C. A positive reaction(V. vulnificus growth) appeared as color change. The selectivity and identification capacity of this new broth was tested by using other 6 Vibrio species and 14 strains of other bacteria. RESULTS: Color change appeared only in the medium including bromthymol blue and thymol blue as a pH indicator dye. It was called the basal medium containing blue dyes as PNCB(peptone, NaCl, cellobiose and blue dye) medium. It took an average time of 4.8hr for becoming aware of yellow color change in PNCB broth after cultivating with saline mixed with V. vulnificus and 6hr in PNCB broth after cultivating with blood mixed with V. vulnificus. One Vibrio species and another 3 bacteria produced color change. So we confirmed that the final composition and pH of PNCB broth medium was 5% peptone, 1% NaCl, 0.2% cellobiose, 0.0004% bromthymol blue and 0.0004% thymol blue [pH 7.5] CONCLUSIONS: PNCB broth could be used as a selective and differential medium for rapid isolation and identification of V. vulnificus in patients with V. vulnificus sepsis.

Evaluación de una prueba de ureasa rápida para la detección de Helicobacter pylori / Evaluation of a fast urease test for the detection of Helicobacter pylori

La mucosa gástrica coloniza el Helicobacter pylori siendo la adaptación al medio relacionada con su alta actividad de la ureasa. Este enzima hidroliza la urea gástrica neutralizando el medio ácido que rodea la bacteria. Sobre la base de esta reacción se desarrollaron numerosos test diagnósticos usando una solución de urea (habitualmente 6 por ciento) con un indicador de pH (Rojo Fenol 0.05 por ciento), pero el contenido de color es tan leve que a veces no se detecta. Por esta razón, se propone una modificación usando una mezcla de indicadores de pH (Rojo Fenol 0.05 por ciento y 0.002 de azul de Bromotimol) que induce un control de calor del verde suave al púrpura fuerte. También se usa solamente azul de bromotimol. El uso de este indicador muestra los valores más altos de sensibilidad y especificidad (69 y 56 por ciento respectivamente). La mezcla de ambos 58.8 y 66.6 por ciento y el azul de bromotimol 46 y 71 por ciento. La eficacia usando Rojo Fenol o la mezcla con bromotimol es de 64.2 y 62.2 por ciento respectivamente; sin embargo, la mezcla de estos indicadores induce un cambio de calor más facilmente detectable.

Quimiotipagem do Cryptococcus neoformans. Revisão da literatura. Novos dados epidemiológicos sobre a criptococose. Nossa experiência com o emprego do meio de C.G.B. no estudo daquela levedura / Cryptococcus neoformans chemotyping. Review of the literature. New epidemiologic data on cryptococcosis. Our experience with the use of C.G.B. media in the study of this yeast

The purpose of this work was to collect the main information from the literature about the biotyping of Cryptococcus neoformans. The more up-to date research concerning the epidemiology of cryptococcosis comprising quite a few articles, mainly after the advent of AIDS, was also reviewed. The Cryptococcus neoformans varieties neoformans and gattii are well defined biochemically nowadays chiefly through the C.G.B. medium, according to Kwon-Chung et al. (1982). The isolation of C. neoformans var. gattii from flowers and leaves of Eucalyptus camaldulensis and Eucalyptus tereticornis, specially in Australia, through the works of Ellis & Pfeiffer (1990) and Pfeiffer & Ellis (1992) permitted very interesting epidemiological investigations on C. neoformans, a capsulated yeast by which Sanfelice, in Italy (1894; 1895) attracted attention of medical class. Busse, in 1894, described the first human case of cryptococcosis under the presentation of a bone lesion simulating sarcoma. In this paper, the Brazilian researchers focused on this subject were pointed out, followed by the Author's experience with the C.G.B. medium (L-canavanine, glycine and bromothymol blue) proposed by Kwon-Chung et al. (1982) with very good results. It was possible with such medium the study of 50 C.N.S. liquor samples, being 39 from AIDS patients (78) and 11 from non-AIDS ones (22). Thirty-seven out of the 39 HIV-positive patients (74) were identified as C. neoformans var. gattii. From the negative HIV, 8 (16) were classified as C. neoformans var. neoformans and 3 (6) as C. neoformans var. gattii. We could not perform the serotyping of the above referred samples. It is evident anyway that in Brazil there exist both varieties gattii and neoformans, agents of neurocryptococcosis, including AIDS patients. The importance of neurocryptococcosis, mainly among AIDS patients, is stressed here, showing once more the value of C.G.B. medium in the typing of C. neoformans in its two varieties. Also, it is of relevant importance the demonstration that some species of eucalyptus may act as "host-trees" of C. neoformans var. gattii.