Control de la calidad y ensayos de rendimiento de CromoCen® SALM según la norma ISO 11133:2014/Amd. 1: 2018 / Quality control and performance testing of CromoCen® SALM according to ISO 11133: 2014 / Amd.1: 2018

Introducción: En Cuba, se desarrolló un medio de cultivo cromogénico y fluorogénico, para la detección, aislamiento y diferenciación de Salmonella de otras bacterias Gram negativas. El método que emplea el medio fue validado y su uso se adoptó en una norma cubana. El aseguramiento de la calidad y el control del rendimiento de los medios garantizan la confiabilidad de los resultados analíticos. La norma ISO 11133 establece criterios mínimos y métodos para evaluarlos. Objetivo: Evaluar los criterios de control de la calidad y de rendimiento de CromoCen® SALM, establecidos en la ISO 11133:2014/Amd.1:2018, para demostrar su fiabilidad para el análisis microbiológico de los alimentos de consumo humano. Métodos: Se evaluaron los indicadores de calidad físico-químicos de tres lotes y se definió un conjunto de ellos que caracteriza la calidad del medio antes y después de terminado, así como la consistencia entre lotes. Para el ensayo de rendimiento se seleccionaron 10 cepas de diferentes géneros. Se determinó la relación de productividad, el factor de selectividad y la electividad de CromoCen® SALM, según la ISO 11133. Resultados: La evaluación físico-química mostró una consistencia entre lotes en color, homogeneidad, apariencia del polvo y del medio preparado. Los valores de contenido de humedad y pH se encontraron dentro de los valores establecidos para este producto. La relación de productividad de CromoCen® SALM con respecto al agar triptona soya, fue superior al 50 por ciento, mientras que el factor de selectividad resultó de 4. Se demostró que en el medio de cultivo se puede diferenciar un grupo representativo de géneros microbianos de Salmonella. Conclusiones: CromoCen® SALM cumple con los requisitos de calidad establecidos para este tipo de productos, según la ISO 11133 vigente. La correcta formulación de los lotes, así como el cumplimiento de los requisitos de calidad aseguran el funcionamiento adecuado para lo que fue diseñado(AU)

Introduction: In Cuba, a new chromogenic and fluorogenic culture medium was developed for the detection, isolation and differentiation of Salmonella from other Gram negative bacteria. The method and medium were validated and their use was adopted as a Cuban standard. Quality assurance and control of media is essential and mandatory to ensure the reliability of the results of the analysis in which they are used. ISO 11133 establishes minimum criteria and methods to evaluate them. Objective: To evaluate the quality and performance criteria of CromoCen® SALM, as recommended in ISO 11133:2014/Amd.1:2018 to demonstrate its reliability for the microbiological analysis of food for human consumption. Methods: The physical-chemical quality indicators of three batches were evaluated and a group of them was defined to characterize its quality before and after finishing, as well to evaluate the consistency between batches. For the performance test, 12 strains of different genera were selected. The productivity ratio, the selectivity factor and the electivity of CromoCen® SALM were determined. Results: The physico-chemical evaluation showed a consistency between batches in color, homogeneity, appearance of the powder and of the prepared medium. The moisture content and pH values ranged within the established values for this product. The productivity ratio of CromoCen® SALM with respect to tryptone soy agar was greater than 50 percent, while the selectivity factor was 4. It was shown that in the culture medium a representative group of Salmonella microbial genera can be differentiated. Conclusions: CromoCen® SALM meets the quality requirements established for this type of products, according to the current ISO 11133 standard. The correct formulation of the batches, as well as the fulfillment of the quality requirements ensure the proper functionality and match the design purpose(AU)

Validação de metodologia analítica para determinação de endotoxina bacteriana em água para diálise por meio de método cromogênico cinético / Analytical methodology validation for determining the bacterial endotoxin in the dialysis water by means of kinetic chromogenic technique

O nível de endotoxina presente na água tratada para hemodiálise é um importante indicador de qualidade, uma vez que altas concentrações de endotoxina atuam como a principal fonte de inflamação crônica em pacientes submetidos à diálise. Este estudo visa validar o método analítico para determinar quantitativamente a endotoxina bacteriana em amostras de água de hemodiálise pelo método cromogênico cinético e de comparar com o método de coagulação em gel. Os ensaios pelo método de coagulação em gel foram realizados de acordo com a Farmacopeia Brasileira em três amostras de água de hemodiálise. A validação do método cromogênico cinético foi realizada utilizando-se as mesmas amostras por meio de sistema de teste portátil. As médias geométricas das concentrações dos pontos finais obtidos nos testes de confirmação de sensibilidade LAL e de interferência por método de coagulação em gel apresentaram resultado de 0,125 UE/mL. Os resultados obtidos pelo método cromogênico para a recuperação do controle positivo do produto variaram de 89 a 186% e o coeficiente de variação de 2,5 a 18,2%, demonstrando que as amostras não apresentaram interferência. Foram obtidos resultados equivalentes em ambos os métodos, o que permite a implementação do método em laboratórios de saúde pública.

The occurrence of endotoxin in the treated water for hemodialysis is an important indicator of quality, since high concentrations of endotoxin constitute the main source for causing chronic inflammation in patients undergoing dialysis. This study aims at validating the analytical method for determining quantitatively the bacterial endotoxin in hemodialysis water samples. The data from the kinetic chromogenic method were compared with the results obtained from the gel coagulation technique. The gel coagulation assays were performed in three samples of hemodialysis water, according to the Brazilian Pharmacopoeia. The validation of the kinetic chromogenic method was performed using the same samples through the portable test system. The geometric means of the concentrations of the endpoints obtained from the tests for confirming the LAL sensitivity and the interference by gel coagulation method showed a result of 0.125 EU/mL. The results obtained by the chromogenic method for recovering the product positive control varied from 89 to 186% and the coefficient of variation from 2.5 to 18.2%, demonstrating that the samples did not show interference. Equivalent results were obtained in both methods, therefore being viable the implementation of this methodology in the public health laboratories.

Kappa and lambda determination by single colored immunohistochemistry and chromogenic in situ hybridization techniques in B-cell malignancies

Background: kappa and lambda light chains detection in bone marrow trephine sections help in the determination of B-cell clonality through evaluation of light chain restriction

Aim of the Work: was to compare the efficacy of single color detection-based immunohistochemistry [IHC] and chromogenic in situ hybridization [CISH] in evaluating kappa/lambda expression in tissues harboring B-lymphoid lesions

Patients and Methods: Forty patients were enrolled in this study. They were divided into three groups chronic lymphocytic leukemia [CLL/SLL] group I [n=13], non-Hodgkin lymphoma [NHL] group II [n=24] and hairy cell leukemia [HCL] group III [n=3]. The 24 NHL cases comprised of [11 diffuse large B-cell lymphoma, 6 mantle cell lymphomas, 3 marginal zone lymphoma, 2 lymphoplasmacytic lymphoma, 1 follicular lymphomas and 1 Burkitt's lymphoma]. Kappa and lambda light chains were detected in their bone marrow trephine sections using single colored immunohistochemistry, chromogenic in situ hybridization and the results were compared to the flowcytometry as reference method

Results: Light chain restriction [LCR] was detected by FCM in 100% of the cases followed by CISH [52.1%; 12/23] of the cases and finally IHC [43%; 18/40]

Conclusion: Both conventional CISH and IHC are effective in determining monoclonality in cases of mature B- cell neoplasm that has plasmacytic differentiation and with high amount of cytoplasmic Ig light chains such as MZL and LP. However, they are not effective in determining monoclonality in cases with low amount of Ig light chain such as cases of pregerminal and germinal center lymphoma. Yet, CISH is more informative than IHC due to the lack of background staining which allowed for greater discrimination between absence and presence of monoclonality

Recuperación y diferenciación de aeromonas con CromoCen AE y CromoCen AGN / Recovery and differentiation of Aeromonas with CromoCen AE and CromoCen AGN

INTRODUCCIÓN: en la actualidad las especies del género Aeromonas han emergido como un problema de salud pública, son ellas los agentes etiológicos de las enfermedades diarreicas con el aumento de la atención médica por años. Los procedimientos convencionales para su diagnóstico son muy engorrosos, laboriosos y duraderos. Una nueva metodología que emplea medios de cultivo cromogénicos ha permitido la simplificación y aceleración de su diagnóstico, que ofrece resultados altamente específicos. OBJETIVO: estudiar el efecto de la combinación de diferentes agentes selectivos de los microorganismos grampositivos sobre el aumento de la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas. MÉTODOS: se estudió el efecto inhibidor de la combinación de agentes selectivos (desoxicolato de sodio (0,05-0,2 g·L-1), sales biliares (0,65 g·L-1), verde brillante (0,025-0,03 g·L-1), cristal violeta (0,001-0,01 g·L-1) y sulfito de sodio (0,8 g·L-1) sobre los microorganismos grampositivos, así como la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas. Como base se utilizó la formulación de CromoCen AGN, sin el desoxicolato de sodio. RESULTADOS: los valores de las productividades de los medios CromoCen AE y CromoCen AGN a partir del inóculo 1,5 × 102 UFC·mL-1 resultaron, para: A. hydrophila 116,8 % y 23,9 %, A. caviae 100,8 % y 3,95 %, A. bestiarium 93,6 % y 28,8 %, A. culicicola 85,1 % y 66,12 %, A. veronii 116,7 % y 59,2 %, A. popoffi 86,56 % y 13,2 %, A. trota 94,8 % y 11,25 % y para A. eucrinophila 103,9 % y 2,80 %. La nueva composición cromogénica logró la diferenciación de los microorganismos por sus características culturales: color, forma, superficie, bordes en las colonias y proteólisis del medio circundante. CONCLUSIONES: la combinación de diferentes agentes selectivos para la inhibición de los microorganismos grampositivos coadyuvo el aumento de la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas.

INTRODUCTION: Species of the genus Aeromonas are a current public health problem, for they are the etiological agents responsible for the growing incidence of diarrheal diseases requiring medical care. Conventional procedures for their diagnosis are very complicated, laborious and time-consuming. A new methodology based on the use of chromogenic culture media allows diagnostic simplification and acceleration, yielding highly specific results. OBJECTIVE: Study the effect of combining several selective agents for gram-positive microorganisms upon an increased capacity for recovery, quantification and differentiation of Aeromonas species. METHODS: Assessment was conducted of the inhibiting effect of combined selective agents (sodium deoxycholate (0.05-0.2 g·L-1), bile salts (0.65 g·L-1), brilliant green (0.025-0.03 g·L-1), crystal violet (0.001-0.01 g·L-1) and sodium sulfite (0.8 g·L-1)) on gram-positive microorganisms, as well as their capacity for recovery, quantification and differentiation of Aeromonas species. The base used was the CromoGen AGN formulation without sodium deoxycholate. RESULTS: Productivity values for the media CromoCen AE and CromoCen AGN based on inoculation of 1.5 × 102 CFU·mL-1 were 116.8 % and 23.9 % for A. hydrophila, 100.8 % and 3.95 % for A. caviae, 93.6 % and 28.8 % for A. bestiarium, 85.1 % and 66.12 % for A. culicicola, 116.7 % and 59.2 % for A. veronii, 86.56 % and 13.2 % for A. popoffi, 94.8 % and 11.25 % for A. trota, and 103.9 % and 2.8 0% for A. eucrinophila. The new chromogenic composition enabled differentiation of microorganisms based on their cultural characteristics: color, shape, surface, colony borders and environmental proteolysis. CONCLUSIONS: Combination of various selective agents for the inhibition of grampositive microorganisms led to an increased capacity for recovery, quantification and differentiation of Aeromonas species.

Efecto de una combinación nutritiva y selectiva con sustratos cromogénicos para diagnóstico de cocos grampositivos / Effect of a selective nutrient combination of chromogenic substrates for diagnosis of Grampositive cocci

INTRODUCCIÓN: la reemergencia de infecciones por bacterias grampositivas y el aumento de su patogenicidad, requiere de un diagnóstico microbiológico rápido y certero. En BioCen se desarrolló una composición cromogénica para el aislamiento, cultivo y diferenciación rápida y presuntiva de microorganismos grampositivos por medio de reacciones cromogénicas específicas, donde las bacterias gramnegativas se encuentran inhibidas de manera parcial o total. OBJETIVO: evaluar el efecto de la combinación de bases nutritivas, inhibidores selectivos y sustratos cromogénicos para aumentar la selectivad y capacidad diferencial para especies de los géneros Enterococcus, Streptococcus y Staphylococcus de importancia clínica. MÉTODOS: se evaluaron 21 cepas microbianas de la American Type Culture Collection y 24 aislamientos clínicos de Streptococcus, Enterococcus y Staphylococcus y otros microorganismos gramnegativos. Se evaluaron diferentes combinaciones de bases nutritivas, acetato de talio, ácido nalidíxico y sustratos cromogénicos para la promoción del crecimiento y diferenciación de las bacterias grampositivas. Se evaluó la funcionalidad microbiológica y se le determinaron los parámetros de calidad diagnóstica. RESULTADOS: la combinación de bases nutritivas permitió el desarrollo de los microorganismos grampositivos, en 24 h y su diferenciación por reacciones cromogénicas específicas. El crecimiento de los microorganismos gramnegativos fue inhibido por la acción del acetato de talio (0,014 g·L-1) y ácido nalidíxico (0,008 g·L-1), excepto Proteus mirabilis y Pseudomonas aeruginosa, cuyas características morfológicas no interfieren en la diferenciación de los microorganismos diana. La sensibilidad, especificidad y exactitud diagnósticas fueron del 100 %. CONCLUSIÓN: la combinación de las bases nutritivas, los inhibidores selectivos y los sustratos cromogénicos permitió el desarrollo y diferenciación de especies de los microorganismos evaluados. La inoculación en el medio cromogénico de microorganismos diana y no diana y la diferenciación de aquellas cepas donde se detectó color similar de las colonias por medio de pruebas complementarias rápidas, le confirió al medio elevadas sensibilidad, especificidad y exactitud diagnóstica.

INTRODUCTION: reemergence of Grampositive bacteria infections and the rise of their pathogenicity require a quick and accurate microbiological diagnosis. BioCen has developed a chromogenic composition for isolation, culturing and rapid and presumptive differentiation of gram-positive microorganisms through specific chromogenic reactions in which the inhibition of gramnegative bacteria is partial or total. OBJECTIVE: to evaluate the effect of a combination of nutrient bases, selective inhibitors and chromogenic substrates to increase the selectivity and differential capacity to detect Enterococcus, Streptococcus and Staphylococcus species of clinical importance. METHODS: twenty one microbial strains from the American Type Culture Collection and 24 clinical isolates of Enterococcus, Streptococcus and Staphylococcus and of other gramnegative microorganisms were evaluated. Various combinations of nutrient bases, thallium acetate, nalidixic acid and chromogenic substrates were also assessed for the promotion, growth and differentiation of grampositive bacteria. The microbiological functionality was evaluated whereas the diagnostic quality parameters were determined. RESULTS: the combination of nutrient bases allowed the development of grampositive microorganisms in 24 hours and their differentiation through specific chromogenic reactions. The growth of gramnegative microorganisms was inhibited by the thallium acetate (0.014 g·L-1) and nalidixic acid (0,008 g·L-1) except for Proteus mirabilis and Pseudomonas aeruginosa whose morphological characteristics do not interfere with differentiation of target microorganisms. Sensitivity, specificity and accuracy for diagnosis were 100 %. CONCLUSIONS: the combination of nutrient bases, selective inhibitors and chromogenic substrates allowed the development and differentiation of the evaluated microorganism species. The inoculation of target and non-target microorganisms in the chromogenic medium and the differentiation of those strains where a similar color of the colonies was detected by means of supplementary rapid tests provided the medium with high diagnostic sensitivity, specificity and accuracy.

Identificación de Candida glabrata y otras especies comunes del género Candida mediante el uso secuencial del medio de cultivo cromógeno y la prueba del tubo germinal / Identification of Candida glabrata and other common Candida species using chromogenic agar and germ tube test / Identificação de Candida glabrata e outras espécies comuns do gênero Candida mediante o uso sequencial do meio de cultivo cromogênico e a prova do tubo germinal

Introdución: se han usado agar cromógeno (AC) y la prueba del tubo germinal (PTG) para identificar C. albicans. Sin embargo, ninguna de estas dos pruebas por separado permite la identificación de C. glabrata. Objetivo: analizar la eficacia diagnóstica del uso secuencial del AC y la PTG para identificar las especies más comunes de Candida. Métodos: se identificaron 436 aislamientos usando el AC y luego la PTG; se utilizaron las pruebas bioquímicas como estándar de oro, y se determinaron la sensibilidad y especificidad del esquema secuencial con sus intervalos de confianza. Resultados: el uso en serie del AC y la PTG tuvo sensibilidad del 97,9 % (IC95 %: 96,0-99,9) para identificar C. albicans/dubliniensis y del 90,9 % (IC95 %: 84,0-97,8) para identificar C. tropicalis, con especificidad del 100 % para ambas especies. El mismo esquema permitió identificar C. glabrata con sensibilidad del 92,5 % (IC95 %: 86,2- 98,8) y especificidad del 96,6 % (IC95 %: 95,0-98,6), y el complejo C. parapsilosis con especificidad del 96,3 % (IC95 %: 94,5-98,1). Conclusiones: el esquema propuesto permite la identificación de C. albicans/dubliniensis, C. tropicalis y C. glabrata con sensibilidad y especificidad adecuadas, y podría ser útil en entornos clínicos de bajos recursos.

Introduction: Chromogenic agar and germ tube test have been used for decades to identify C. albicans. However, neither of these tests separately allows identification of C. glabrata. Objective: To test the efficacy of chromogenic agar and germ tube test used serially to identify the most common Candida species found in clinical practice, with emphasis on C. glabrata. Methods: 436 isolates were identified using the chromogenic medium followed by the germ tube test. Biochemical tests were used as gold standard. Sensitivity, specificity and confidence intervals (95 %) of the serial identification system were determined. Results: Sensitivity was 97.9 % (IC95 %: 96.0-99.9) to identify C. albicans/dubliniensis, and 90.9 % (IC95 %: 84.0-97.8) for C. tropicalis; specificity was 100 % for both species. Sensitivity was 92.5 % (IC95 %: 86.2-98.8) for identification of C. glabrata with 96.6 % (IC95 %: 95.0-98.6) specificity. Concerning identification of the C. parapsilosis complex, specificity was 96.3 % (IC95 %: 94.5-98.1). Conclusion: The proposed serial scheme has adequate sensitivity and specificity for identification of C. albicans/dubliniensis, C. tropicalis and C. glabrata. It could be useful in low-resources clinical settings.

Se usaram agar cromogênico (AC) e a prova do tubo germinal (PTG) para identificar C. albicans. No entanto, nenhuma destas duas provas por separado permite a identificação de C. glabrata. Objetivo: analisar a eficácia diagnóstica do uso sequencial do AC e a PTG para identificar as espécies mais comuns de Candida. Métodos: identificaram-se 436 isolamentos usando o AC e depois a PTG; utilizaram-se as provas bioquímicas como padrão de ouro, e se determinaram a sensibilidade e especificidade do esquema sequencial com seus intervalos de confiança. Resultados: o uso em série do AC e a PTG teve sensibilidade de 97,9 % (IC95 %: 96,0-99,9) para identificar C. albicans/dubliniensis e de 90,9 % (IC95 %: 84,0-97,8) para identificar C. tropicalis, com especificidade de 100 % para ambas espécies. O mesmo esquema permitiu identificar C. glabrata com sensibilidade de 92,5 % (IC95 %: 86,2- 98,8) e especificidade de 96,6 % (IC95 %: 95,0-98,6), e o complexo C. parapsilose com especificidade de 96,3 % (IC95 %: 94,5-98,1). Conclusões: o esquema proposto permite a identificação de C. albicans/dubliniensis, C. tropicalis e C. glabrata com sensibilidade e especificidade adequadas, e poderia ser útil em meios clínicos de baixos recursos.

Evaluation of the chromogenic medium [CPS] in the isolation and identification of urinary tract pathogens

Urine cultures constitute the majority of the workload for a microbiology laboratory with only 20%-30% of urine sample resulting in significant growth. Chromogenic media [CM] are available for urine specimens to enable rapid identification of common pathogens and also has been reported to increase mixed culture detection, reducing unnecessary workup. Chromogenic media offers the potential to lower costs by providing decreased work time, storage space and identification costs. The present study focused on evaluation of the chromogenic medium [CPS] for the diagnosis of UTI in comparison with CLED as a conventional medium. Over the period of January to July 2014, fifty urine samples with >/=100 pus cells /HPF were examined. CPS and CLED media were used for direct inoculation in addition to conventional biochemical reactions and/or API as needed. In comparison with CLED, CPS showed a sensitivity of 93.5%, specificity of 100%, positive predictive value of 100%, negative predictive value of 57.1% and total agreement of 94%. The sensitivity of CPS for E. coli was 95%, for KESC was 75%, for Proteeae was 100% and for Enterococcus was 100%. The specificity of CPS for detection of E. coli was 100%, for KESC was 100%, for Proteeae was 97.9% and for Enterococcus was 100%. CPS proved to be a rapid, cost-effective diagnostic method for urinary tract infections. Therefore, CPS can replace the standard primary plating media used in routine diagnosis of urinary tract infection

Chromogenic Tube Test for Presumptive Identification and Susceptibility Test of Candida Spp.

Chromagar Candida is a new, modified, simple, rapid and cost effective method for the presumptive identification of Candida spp. after preliminary growth. 54 randomly selected clinical isolates of Candida were evaluated including, C.albicans (24), C.tropicalis (13), C.parapsilosis (6), C.krusei (5) & C.glabrata (4). The sensitivity and specificity appeared to be equal to that of conventional identification system except 4 C.glabrata strains which could only be identified by conventional method. Terbinafine, amphotericin B and nystatin were found to be highly sensitive drugs and clotrimazole and fluconazole showed the worst sensitivity results.

Determinación cuantitativa de enterococos en aguas utilizando un método cromogénico alternativo / Quantitative determination of enterococci in water using an alternative chromogenic method

INTRODUCCIÓN: el control de la calidad de las aguas es vital para la protección de la salud humana y la biodiversidad en el ambiente acuático. Varios microorganismos son utilizados como indicadores de contaminación fecal para este propósito, dentro de ellos los enterococos son considerados buenos indicadores, porque sobreviven más tiempo ante condiciones adversas en la naturaleza. OBJETIVOS: evaluar la capacidad de un nuevo método cromogénico alternativo para la detección y enumeración de enterococos en aguas por la técnica de filtración por membrana, en comparación con el método estándar ISO 7899:2. MÉTODOS: se recolectaron muestras de aguas de tres orígenes diferentes para determinar su calidad. Las muestras se ensayaron en paralelo por las dos metodologías (alternativa y referencia), empleando la técnica de filtración por membrana. Para evaluar el desempeño de los métodos se determinaron varios parámetros: sensibilidad, especificidad, exactitud, porcentaje de falsos positivos, porcentaje de falsos negativos, índice kappa. Los tres primeros parámetros se calcularon para ambos métodos antes y después de la confirmación por pruebas bioquímicas. Los resultados se analizaron desde el punto de vista estadístico, teniendo en cuenta los principales criterios definidos en las normas ISO 16140 e ISO 177994. RESULTADOS: con el método alternativo los resultados se obtuvieron a las 24 h, estos mostraron valores de sensibilidad (99 %) y exactitud (98 %) más elevados que con el de referencia (97 % y 97 %, respectivamente), el cual demoró más de 48 h. La eficiencia y exactitud de ambos métodos fue similar y se obtuvo una concordancia entre ellos casi perfecta (kappa = 0,96) con el total de las muestras de aguas ensayadas. CONCLUSIONES: los resultados alcanzados indican que el método alternativo es eficiente para el recuento de enterococos provenientes de diferentes tipos de muestras de agua. Resulta además un método simple, más rápido y económicamente factible.

INTRODUCTION: water quality control is essential for the protection of human health and biodiversity in the aquatic environment. For this purpose, several microorganisms are used as indicators of fecal contamination. Among them are enterococci, which are considered to be good indicators, since they survive for a longer time in adverse natural conditions. OBJECTIVES: evaluate the suitability of a new alternative chromogenic method for the detection and enumeration of enterococci in water by membrane filtration technique, in comparison with the ISO 7899:2 standard method. METHODS: water samples were collected from three different sources to determine their quality. The samples were assayed in parallel with the two methodologies (alternative and reference) using the membrane filtration technique. The following parameters were determined to evaluate the performance of the methods: sensitivity, specificity, accuracy, percentage of false positives, percentage of false negatives, kappa index. The first three parameters were estimated for both methods before and after confirmation by biochemical testing. Results were analyzed statistically based on the main criteria defined by ISO standards 16140 and 177994. RESULTS: with the alternative method, results were obtained at 24 h, whereas the reference method required more than 48 h. Sensitivity and accuracy were higher with the alternative method (99 % and 98 %, respectively) than with the reference method (97% and 97%, respectively). Both methods had similar efficiency and accuracy, with almost perfect concordance between them (kappa = 0.96) in all the water samples tested. CONCLUSIONS: results show that the alternative method is efficient for the enumeration of enterococci from different types of water samples. It is also a simple, faster and economically feasible method.

Performance of chromID Clostridium difficile Agar Compared with BBL C. difficile Selective Agar for Detection of C. difficile in Stool Specimens

We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMerieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80degrees C, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.

Evaluation of a Chromogenic Culture Medium for the Detection of Clostridium difficile

PURPOSE: Clostridium difficile (C. difficile) is an important cause of nosocomial diarrhea. Diagnostic methods for detection of C. difficile infection (CDI) are shifting to molecular techniques, which are faster and more sensitive than conventional methods. Although recent advances in these methods have been made in terms of their cost-benefit, ease of use, and turnaround time, anaerobic culture remains an important method for detection of CDI. MATERIALS AND METHODS: In efforts to evaluate a novel chromogenic medium for the detection of C. difficile (chromID CD agar), 289 fecal specimens were analyzed using two other culture media of blood agar and cycloserine-cefoxitin-fructose-egg yolk agar while enzyme immunosorbent assay and polymerase chain reaction-based assay were used for toxin detection. RESULTS: ChromID showed the highest detection rate among the three culture media. Both positive rate and sensitivity were higher from chromID than other culture media. ChromID was better at detecting toxin producing C. difficile at 24 h and showed the highest detection rate at both 24 h and 48 h. CONCLUSION: Simultaneous use of toxin assay and anaerobic culture has been considered as the most accurate and sensitive diagnostic approach of CDI. Utilization of a more rapid and sensitive chromogenic medium will aid in the dianogsis of CDI.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

Prevalence and correlation of human papilloma virus and its types with prognostic markers in patients with invasive ductal carcinoma of the breast in Kuwait

This study aimed to document the association of human papilloma virus [HPV] and its types in breast carcinoma tissues in Kuwaiti women, and correlate this with known prognostic markers. The clinicopathological data of archived tissue from 144 cases of invasive ductal breast carcinoma were studied [age, histological grade, size of tumour, lymph node metastases, oestrogen/progesterone receptors and human epidermal growth factor receptor 2 status]. HPV frequency was documented using immunohistochemistry [IHC] and chromogenic in-situ hybridisation [CISH]. HPV types were documented by CISH using HPV probes. CISH and IHC techniques were compared and HPV correlated with prognostic parameters. The HPV prevalence as determined by CISH and IHC was 51 [35.4%] and 24 [16.7%] cases, respectively. The sensitivity of HPV by IHC was 37.3% and specificity was 94.6%. The sensitivity and specificity of HPV-CISH compared to HPVIHC was statistically significant [P <0.001]. HPV-CISH was seen in 51 cases. A combination of HPV 6 and 11, and 16 and 18 was seen in 2 [3.9%] cases, and a combination of HPV 6, 11, 31 and 33 was seen in 7 [13.7%] cases. All three HPV probes: 6 and 11, 16 and 18, as well as 31 and 33 were present in 2 [3.9%] cases. The prevalence of HPVCISH in the Kuwaiti and non-Kuwaiti populations was 27 [52.9%] and 19 [37.2%], respectively. No correlation was observed with the prognostic parameters. The frequency of HPV in breast carcinoma cases in Kuwait was 35.4% [CISH]. Of those, 52.9% were Kuwaitis in whom both low- and high-risk HPV types were detected

Accurate assessment of HER2 gene status for invasive component of breast cancer by combination of immunohistochemistry and chromogenic In Situ hybridization / 华中科技大学学报（医学）（英德文版）

The specimens of ductal carcinoma in situ (DCIS) with early invasion, and specimens collected by core needle biopsy (CNB) tend to contain limited amount of invasive component, so it is imperative to explore a new technique which can assess HER2 gene status accurately for the limited invasive cancer component in these specimens. Dual staining technique of combining immunohistochemistry (IHC) for myoepithelial cells and single or dual probe chromogenic in situ hybridization (CISH) for HER2 gene was performed on routinely processed paraffin sections from 20 cases diagnosed as having DCIS with invasive cancer. Among them, 10 had fluorescence in situ hybridization (FISH)-confirmed amplification of HER2 and 10 had FISH-confirmed non-amplification of HER2. We successfully detected HER2 genetic signals and myoepithelial IHC markers (SMM-HC or CK5/6) simultaneously on a single section in all 20 specimens. Myoepithelial markers and HER2 signals detected by dual staining assay were consistent with those by individual technique performed alone. HER2 gene amplification results determined by dual staining assay were 100% consistent with those of FISH. Dual staining technique which allows simultaneous detection of myoepithelial marker protein and cancerous HER2 gene is feasible, and it has potential to be used in clinical practice for effective determination of HER2 amplification in limited invasive component.

Comparative Evaluation of Three Chromogenic Media Combined with Broth Enrichment and the Real-Time PCR-Based Xpert MRSA Assay for Screening of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs

BACKGROUND: We evaluated the performance of three chromogenic media (Brilliance agar I [Oxoid, UK], Brilliance agar II [Oxoid], and ChromID MRSA [Biomerieux, France]) combined with broth enrichment and the Xpert MRSA assay for screening of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: We obtained 401 pairs of duplicate nasal swabs from 321 patients. One swab was suspended overnight in tryptic soy broth; 50-microL aliquots of suspension were inoculated on the three chromogenic media. Brilliance agar I and II were examined after 24 hr, and ChromID MRSA, after 24 and 48 hr. The paired swab was processed directly using real-time PCR-based Xpert MRSA assay. RESULTS: True positives, designated as MRSA growth in any of the culture media, were detected with the prevalence of 17% in our institution. We report the sensitivity, specificity, positive predictive value, and negative predictive value of MRSA growth as follows: 92.3%, 94.0%, 75.9%, and 98.4% in Brilliance agar I (24 hr); 92.7%, 97.9%, 90.0%, and 98.5% in Brilliance agar II (24 hr); 95.6%, 95.8%, 82.3%, and 99.1% in ChromID MRSA (24 hr); 100%, 92.5%, 73.1%, and 100% in ChromID MRSA (48 hr); 92.6%, 96.7%, 85.1%, and 98.5% in Xpert MRSA assay. The agreement between the enriched culture and Xpert MRSA assay was 96.0%. CONCLUSIONS: Three chromogenic culture media combined with enrichment and Xpert MRSA assay demonstrated similar capabilities in MRSA detection. The Xpert MRSA assay yielded results comparable to those of culture methods, saving 48-72 hr, thus facilitating earlier detection of MRSA in healthcare settings.

Comparative study of Candida by conventional and CHROMagar method in non-denture and denture wearers by oral rinse technique.

Introduction: Candidal species colonizes the oral cavities of healthy individuals without dentures and also of denture wearers. Soft liners and tissue conditioning materials have been found to support the growth of Candida albicans which may predispose to lesions. The most important and common candidal species are C. albicans, C. tropicalis, and C. glabrata. C albicans is usually isolated from both the fitting surface of the denture and the denture-bearing mucosa of the affected patients. The aim of this study was to isolate, quantify, and speciate candidal species in non-denture wearers (controls) and denture wearers (study group) by the oral rinse technique. Isolation was done using Sabouraud dextrose agar (SDA). Speciation was done using conventional methods like the germ tube test, carbohydrate fermentation test, urease test, as well as the CHROMagar method. Aims and Objective: 1) To assess the prevalence of Candida in non-denture wearers and in denture wearers by oral rinse technique, with isolation on SDA; 2) to speciate and quantify Candida in non-denture wearers and denture wearers by using conventional methods (germ tube test, carbohydrate fermentation test, urease test) and the CHROMagar method; 3) to assess the influence of smoking and diabetes on candidal species among the denture wearers; and 4) to assess the sensitivity and specificity of SDA and CHRO Magar Materials and Methods: Salivary samples for Candida evaluation were collected from the subjects in sterile sample containers, using the oral rinse technique. Results: C glabrata was the most commonly found species among denture wearers and non-denture wearers both by conventional and CHROMagar methods. In males, C. albicans was the predominant species, whereas C. glabrata was the predominant species in females. Candidal colonization was higher in denture wearers compared to non-denture wearers, especially among females. The CHROMagar method was more rapid compared to conventional methods. In the present study, CHROMagar Candida showed 100% specificity and 100% sensitivity when compared to SDA and conventional methods.

CHROMagar KPC. Comparación con el método propuesto por los Centros para el Control y Prevención de Enfermedades (CDC, EE.UU.) para el estudio de portación rectal y evaluación de falsos positivos / CHROMagar KPC. Comparison with the method proposed by the Centers for Disease Control and Prevention (CDC, USA) for rectal screening and evaluation of false positive results

Se cultivaron 81 hisopados rectales en el medio CHROMagar KPC y por el método del CDC. Fueron positivos para Klebsiella pneumoniae KPC en CHROMagar KPC, 9/81 y 6/81 con el método del CDC. El medio CHROMagar KPC tuvo dos falsos positivos: 1 K. pneumoniae y 1 Acinetobacter sp. Los falsos positivos del método CDC fueron: 25 Acinetobacter spp., 2 Escherichia coli y4K. pneumoniae. El empleo del medio CHROMagar KPC resultó ser un método con mayor recuperación de aislamientos productores de KPC y menos falsos positivos que el método del CDC. Para evaluar los falsos positivos en el medio CHROMagar KPC se cultivaron 1247 hisopados rectales. Se obtuvieron 1021 negativos, 171 K. pneumoniae KPC y 55 (4,4 %) falsos positivos. Debido al desarrollo de falsos positivos en el medio CHROMagar KPC, se debe confirmar por caracterización fenotípica la presencia de KPC en las bacterias aisladas.

Eighty one rectal swabs (RS) were cultured on CHROMagar KPC and the CDC method. Of the 81 samples, 9 were positive for KPC-producing Klebsiella pneumoniae on CHROMagar KPC, and 6 for the CDC method. CHROMagar KPC had two false positive (FP) results: 1 K. pneumoniae and 1 Acinetobacter sp. FP results on the CDC method were: 25 Acinetobacter spp., 2 Escherichia coli and 4 K. pneumoniae. CHROMagar KPC yielded a better recovery of KPC-producing bacteria and less FP results than CDC method. In order to evaluate FP results on CHROMagar KPC, 1247 RS were cultured and yielded 1021 negatives, 171 KPC-producing K. pneumoniae and 55 FP (4.4 %). Because of the FP results growing on CHROMagar KPC, KPC must be phenotypically confirmed in the bacteria isolated.

Comparative evaluation of expression of proliferating cell nuclear antigen in variants of ameloblastoma and ameloblastic carcinoma.

Aim: To evaluate the use of proliferating cell nuclear antigen index in the different histopathological variants of ameloblastoma, such as the follicular, plexiform, and unicystic types, and in ameloblastic carcinoma by immunohistochemical staining. The proliferating cell nuclear antigen index values of the variants of ameloblastomas and ameloblastic carcinomas are compared in order to determine the biological behavior of these tumors. Materials and Methods: For the present study, archival tissues that had been diagnosed as ameloblastoma and ameloblastic carcinoma were collected from the department of oral pathology. Specimens were embedded in paraffin wax and were sectioned at a thickness of 5 μm and stained with hematoxylin-eosin for reconfirming the histologic pattern. It was also stained immunohistochemically for anti-proliferating cell nuclear antigen antibody. Results: Positive proliferating cell nuclear antigen expression is seen as a light brown, granular stain. The proliferating cell nuclear antigen values of ameloblastic carcinoma were almost five times the value of ameloblastoma. Analysis of variance test, Fischer's exact test/variance ratio test, and Student's t-test were performed and the probability values were determined. Summary and Conclusion: This study showed that ameloblastic carcinoma had the maximum proliferative capacity. Among the variants of ameloblastoma, the plexiform variety had the maximum proliferative capacity, followed by the follicular and unicystic varieties. Altogether, these data indicate that proliferating cell nuclear antigen is related to the biological behavior and proliferation of tumor cells in the variants of ameloblastoma and ameloblastic carcinoma.

Biological activity analysis of native and recombinant streptokinase using clot lysis and chromogenic substrate assay

Determination of streptokinase activity is usually accomplished through two assay methods: a] Clot lysis, b] Chromogenic substrate assay. In this study the biological activity of two streptokinase products, namely Streptase[registered], which is a native product and Heberkinasa[registered], which is a recombinant product, was determined against the third international reference standard using the two forementioned assay methods. The results indicated that whilst the activity of Streptase[registered] was found to be 101 +/- 4% and 97 +/- 5% of the label claim with Clot lysis and Chromogenic substrate assay respectively, for Heberkinasa[registered] the potency values obtained were 42 +/- 5% and 92.5 +/- 2% of the label claim respectively. To shed some light on the reason for this finding, the n-terminal sequence of the streptokinase molecules present in the two products was determined. The results showed slight differences in the amino acid sequence of the recombinant product in comparison to the native one at the amino terminus. This finding supports those of other workers who found that n-terminal sequence of the streptokinase molecule can have significant effect on the activity of this protein