In order to study the genetic variability of Mycobacterium tuberculosis circulating in the Carabobo State at Venezuela, 317 strains of M. tuberculosis isolated from patients living in different health districts whose acid fast smears were positive, were included. Each strain was characterized by spoligotyping and the pattern compared with the national and worldwide SpolDB4.0 databases; 220 isolates (69.4%) were grouped in 24 clusters, being LAM the most common family (34%). In contrast to other Venezuelan regions, the most common Carabobo spoligotype was number 605, with 46 isolates (14.5%). In addition, there were 97 (30.6%) orphan spoligotypes, 19 of which are found in SpolDB4.0, and 78 non described. Co-infection with human immunodeficiency virus was detected in 11 patients (3.5%). These results show high genotypic variability of M. tuberculosis in the region, contributing with new information for a better understanding of tuberculosis transmission in Venezuela.

Detección del gen bla VIM-2 en cepas de Pseudomonas aeruginosa productoras de metalo β-lactamasa aisladas en una unidad de cuidados intensivos en Ciudad Bolívar, VenezuelaArmando Guevara, Jacobus de Waard y María Araque bla VIM-2 gene detection in metallo-β-lactamase-producing Pseudomonas aeruginosa strains isolated in an Intensive Care Unit in Ciudad Bolívar, VenezuelaTen Pseudomonas aeruginosa strains with resistance to broad-spectrum cephalosporin and carbapenems were studied to determine the presence of genes that mediate the production of metallo-β-lactamases. These strains were isolated from patients with nosocomial infection at the Intensive Care Unit of the Complejo Hospitalario "Ruiz y Paéz" of Ciudad Bolívar, Bolívar State, Venezuela, from 2003 to 2006. In all isolates a metallo-enzyme activity was detected by using the double disk synergism test. PCR amplification of genes encoding the families IMP, VIM and SPM metallo-β-lactamases showed the presence of a bla VIM gene in all strains studied. DNA sequencing revealed that all isolates showed the presence of bla VIM-2 . These results suggest that it is necessary to keep these strains under epidemiologic surveillance, establish laboratory strategies for opportune detection and the implementation of new policies to ensure the appropriate use of antibiotics in this institution.

RESUMENEl diagnóstico de la infección por Mycobacterium avium subsp. paratuberculosis (MAP) al utilizar un sistema de cultivo líquido resulta en una mayor sensibilidad, rapidez y automatización. Sin embargo, tiene como desventajas una mayor tasa de contaminación en relación con los sistemas convencionales y también es menos específico. El presente estudio identificó algunas bacterias contaminantes del sistema de cultivo BACTEC-MGIT 960 al procesar muestras clínicas de ganado bovino del sur de Chile. No se detectaron micobacterias en las muestras falsas positivas a MAP mediante la técnica Reacción en Cadena de la Polimerasa-Análisis con Enzimas de Restricción (PRA)-hsp65. Por otra parte, el Análisis de los Espaciadores Intergénicos Ribosomales (RISA) seguido de un análisis de secuenciación, reveló la presencia de Paenibacillus sp., Enterobacterias y Pseudomonas aeruginosa como contaminantes comunes. Los protocolos de eliminación de contaminantes deberían considerar esta información para mejorar los resultados diagnósticos de los sistemas de cultivo líquido. Además se requieren estudios adicionales que consideran un mayor número de muestras para establecer conclusiones más representativas de la población bovina.Key words: Mycobacterium avium subsp. paratuberculosis, liquid culture, contaminant bacteria, RISA, PRA-hps65. SUMMARYDiagnosis of Mycobacterium avium subsp. paratuberculosis infection by liquid culture is sensitive, faster than conventional solid culture and automated. However, a disadvantage of these culture systems is the potential for high frequency of culture contamination. Contaminant bacteria were identified as a step toward better contaminant control. No mycobacteria were detected by mycobacterial Polymerase Chain Reaction-Restriction Enzyme Analysis (PRA)-hsp65. Ribosomal Intergenic Spacer Analysis (RISA) followed by sequence analysis identified Paenibacillus sp., Enterobacteriaceae and Pseudomonas aeruginosa as common contaminants. The present study aimed to identify a representative sample of contaminants encountered when culturing clinical faecal samples from Chilean cattle. Further studies involving a larger and more representative sample of animals are required to extrapolate the results to a broader population.Palabras clave: Mycobacterium avium subsp. paratuberculosis, cultivo líquido, RISA, PRA-hps65.

Background: Meliodosis in an infectious disease affecting multi-organ system of man and animals caused by a bacterium, Burkholderia pseudomallei. The disease is potentially fatal and early detection is crucial for institution of life saving antimicrobial therapy. Culture isolation remains as a mainstay for definitive laboratory diagnosis of melioidosis. But it is less sensitive, laborious and timeconsuming task. In this report, an improved dot EIA test (patent pending) with user friendly assay protocol for serodiagnosis of melioidosis is described.Methods: Two optimised concentrations of antigens were dotted onto a nitro-cellulose membrane (Microfiltration System, Ca, USA, 0.45uM) divided into 9 mm × 4 mm. The membranes were blocked for 30 minutes in tris-buffered saline (TBS) wiht 5% nonfat skim milk. The membranes were rinsed and cut according to the divided size into test strips and placed in 48 well flat tissue culture plates (Costar 3548, UK.) The test strips were then incubated with patient serum at 1:100 in TBS for 1 hour at room temperature. After incubation, the test strips were washed with TBS for three times and each washing was done for 5 minutes. Subsequently, the diluted alkaline phosphatase conjugated goat anti-human immunoglobulin IgM, IgG and IgA isotypes were added into the respective wells and incubated for 1 hour at room temperature. After incubation the strips were washed three times in TBS as described above. Chromogenic substrate was used in the colour development reaction. Aftwer after 15 minutes the strips washing with distilled water to stop coloue development. The positive and negative results were determined by visual comparison of the dot intensity for each test with the positive cut off control.Results: The sensitivity and the specificity of the assay was 95% and 92%, respectively for the detection of IgG, IgM and IgA antibody isotypes. However, detection of IgG alone showed sensitivity value of 89.2%. The sensitivity value for the detection of IgM alone and IgA alone were 47.7% and 60% respectively.

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