Variability of Bacillus thuringiensis strains by ERIC-PCR and biofilm formation.

Bacillus thuringiensis (Bt) is a soil-dwelling bacterium of great interest for agronomical research because of its use as biological pesticide. There are some limitations regarding the subspecies classification. Phenotyping and genotyping studies are important to ascertain its variability. The diversity of 40 environmental strains, isolated from different regions in Mexico, was analyzed by ERIC-PCR and the ability of biofilm formation. Thirty-nine different fingerprinting patterns revealed enough data to discriminate among the 40 strains. A total of 24 polymorphic fragments with sizes between 139 and 1,468 bp were amplified. Almost all (95 %) strains showed biofilm formation after 96 h of incubation. At 96 h of incubation the biofilm-forming strains from the CINVESTAV collection showed a more heterogeneous ability as biofilms producers. Results showed a large intra-species genomic variability in Bt. However, some strains could be correlated as they were found within clusters depending on the location of isolation.

Absence of high molecular weight proteins 1 and/or 2 is associated with decreased adherence among non-typeable Haemophilus influenzae clinical isolates.

High molecular weight (Hmw) proteins 1 and 2, type IV pilin protein (PilA), outer-membrane protein P5 (OmpP5), Haemophilus protein D (Hpd) and Haemophilus adhesive protein (Hap) are surface proteins involved in the adherence of non-typeable Haemophilus influenzae. One hundred clinical isolates were evaluated for the presence of the genes encoding these proteins by PCR and for their adherence capacity (AC) to Detroit 562 nasopharyngeal cells (D562). The majority of isolates were from blood (77/100); other sites were also represented. Confluent D562 monolayers (1.2×10(5) cells per well) were inoculated with standardized minimal infective doses (m.o.i.) of 10(2), 10(3) or 10(4) c.f.u. per well. The AC was categorized as low (<10 %) or high (≥10 %) depending on the percentage of c.f.u. adhering per well. All the isolates evaluated showed adherence: 69/100 (69 %) demonstrated high adherence, while 31/100 (31 %) showed low adherence. Of all the genes evaluated, hmw1A and/or hmw2A were detected in 69/100 (69 %) of isolates. The presence of hmw1A and/or hmw2A was associated with increased adherence to D562 cells (P≤0.001). Dot immunoblots were performed to detect protein expression using mAbs 3D6, AD6 and 10C5. Among the high-adherence isolates (n = 69), 72 % reacted with 3D6 and 21 % with 10C5. Our data indicate that the absence of Hmw1 and/or Hmw2 was associated with decreased adherence to D562 cells.

Measurement of Haemophilus influenzae type a capsular polysaccharide antibodies in cord blood sera.

We measured anti-Haemophilus influenzae type a capsular polysaccharide serum immunoglobulin G antibodies in cord blood sera from Mexican (n = 68) and Chilean mothers (n = 72) by enzyme-linked immunosorbent assay. Measurable antibodies were found in 79.3% of samples. Immunoglobulin G antibodies correlated with serum bactericidal activity (r = 0.66). This enzyme-linked immunosorbent assay can be used for the evaluation of adaptive immune responses to Haemophilus influenzae type a and serosurveillance studies in populations at risk.

Evaluation of serum bactericidal antibody assays for Haemophilus influenzae serotype a.

Haemophilus influenzae type a (Hia) is an important pathogen for some American Indian, Alaskan native, and Northern Canada aboriginal populations. Assays to measure serum bactericidal activity (SBA) to Hia have not been developed or validated. Here, we describe two methods for the measurement of SBA: SBA with a viability endpoint (CFU counts) and SBA with a fluorometric endpoint using alamarBlue as the metabolic indicator. Both SBA assays measure Hia-specific functional antibody and correlate with anti-Hia IgG enzyme-linked immunosorbent assay (ELISA) concentration of naturally acquired antibodies.

Immunoglobulin G avidities in infants in Mexico after primary immunization with three doses of polyribosylribitol phosphate-tetanus toxoid Haemophilus influenzae type b vaccine.

Serum immunoglobulin G concentrations and avidities specific to Haemophilus influenzae type b (Hib) were measured in 208 children living in Guadalajara and Mexico City. Protective concentrations were found in 98.9% and 100.0% of participants, respectively. Geometric mean concentrations differed between both populations and/or among age groups. Mean avidities differed only among the 7- to 12-month-old children. Diphtheria-tetanus-whole-cell pertussis-hepatitis B-Hib primary vaccination seems to induce protection in Mexican children.

Avidity determinations for Haemophilus influenzae Type b anti-polyribosylribitol phosphate antibodies.

Determination of antibody avidity measurements can be difficult in human serum depending on the population evaluated. We evaluated three approaches for the determination of antibody avidity for immunoglobulin G (IgG). These approaches were (i) elution of bound antibody with increasing concentrations of a chaotropic agent using a single serum dilution, (ii) binding interference of multiple serum dilutions by a single concentration of a chaotrope, and (iii) elution of multiple serum dilutions by a single concentration of a chaotrope. Parameters that affect the determination of avidity measurements and their limitations were evaluated with pre- and post-Haemophilus influenzae type b conjugate vaccination sera (n=89). We determined that elution of low-avidity antibodies present in multiple dilutions of the serum sample by a single concentration of a chaotrope (0.15 M sodium thiocyanate [NaSCN]) was optimal for the determination of avidity measurements throughout a wide range of IgG concentrations (0.94 to 304.6 microg/ml). The percent reduction in concentration as determined by the elution assay with 0.15 M NaSCN correlated highly (r=0.84) with weighted averages obtained by an elution assay with multiple solutions of NaSCN. The correlation (r=0.57) between elution and binding interference, when a single concentration of a chaotrope was used, was lower than the correlation between the two elution methods (r=0.84). We found that the serum dilution, the heterogeneity of the antibody population, and the concentration of the chaotrope were the primary variables affecting avidity determinations. In this study, we present multiple analysis methods depending on the methodology used. We also present the factors that affect the analysis of avidity determinations given the polyclonal nature of human sera. This experimental approach should benefit the evaluation of similar antibodies induced by other bacterial polysaccharide vaccines.

Measurement of serum bactericidal activity specific for Haemophilus influenzae type b by using a chromogenic and fluorescent metabolic indicator.

We evaluated alamarBlue as a metabolic indicator in a standardized assay for the measurement of serum bactericidal activity (SBA) to Haemophilus influenzae type b (Hib) using sera containing natural and vaccine-induced anticapsular (polyribosylribitol phosphate) antibodies. SBA assays with a colorimetric and a fluorometric end point in the presence of alamarBlue were developed and compared to a standard SBA assay, where colony counts are performed to determine the titer (12). A colorimetric end point required a spectrophotometer, whereas a fluorometric end point required a fluorometer. Prevaccination sera (n = 27) and postvaccination sera (n = 13) were tested by all three methodologies, and the SBA titers obtained in the presence of alamarBlue were compared to those from the standard method. Both the colorimetric and the fluorometric SBA titers were significantly correlated (r = 0.87 and r = 0.95, respectively) with those of the standard assay (>/= 50% killing as the SBA titer end point), and titers were not significantly different when compared to those of the standard assay (P > 0.68). However, the fluorometric end point had superior performance and ease of titer determination compared to the colorimetric end point (95 versus 87% of SBA titers were within 2 dilutions of the standard titer). Hib SBA assays with alamarBlue are reproducible, faster (same-day assay), and easier to perform than the standardized assay, which requires manual or automated colony counts. These semiautomated methodologies result in increased sample throughput and collection of data in digital formats that can be exported to data analysis programs for determination of SBA titers.

Haemophilus influenzae b: una revisión sobre los determinantes de patogenicidad y de la respuesta inmune a la infección / Haemophilus influenzae b: a review of the determinants of the pathogenicity and immune response

Hemophilus influenzae continuá siendo en nuestro país un patógeno importante en gran variedad de procesos infecciosos que se presentan principalmente en la edad pediátrica. Los datos epidemiológicos señalan al sistema nervioso central (SNC) y a las vías respiratorias altas y bajas como los sitios más afectados. Para que se manifieste la enfermedad infecciosa por este organismo deben participar diversos factores, tanto el hospedero como del propio parásito. Dentro de estos factores, se encuentran los determinantes de patogenicidad del parásito y los de resistencia del hospedero. La interación de determinantes superficiales, subcelulares y/o productos extracelulares de este organismo patógeno y los mecanismos de resistencia del hospedero, conducen o no al establecimiento de enfermedad. El conocimiento y análisis de los determinantes de patogenicidad del parásito y de los factores de respuesta inmune del hospedero, permitirán identificar componentes útiles para el diagnóstico de la enfermedad, para señalar componentes bacterianos como candidatos importantes para la prevensión del padecimiento y para implementar estrategias adecuadas de tratamiento antimicrobiano apropiado

Haemophilus influenzae is still one of the main causes of diverse invasive diseases in children in México. Epidemiologic data indicate that these processes affect primarily the central nervous system and the respiratory tract. Several factors are involved in the expression of infectious disease by this organism, among them the pathogenic determinants of the parasite and those related with resistance in the host. Ocurrence of disease is usually the result of the interaction between these determinants. Knowledge of these pathogenic determinants of the parasite and of factors involved in the immune response of the host have allowed an understanding of the infectious process and have directed research in a least three areas: 1) identification of bacterial membrane fractions related with diagnosis of the disease, 2) screening for immunogenic components in the bacterias as vaccine candidates to be used in the prevention of the disease and, 3) the planning of appropriate alternatives for specific antimicrobial therapy.

Diseño e implementación del sistema de garantía de calidad en el hospital Occidente de Kennedy / Design and implementation of the quality assurance system at Hospital Occidente de Kennedy

El objetivo general de este trabajo es caracterizar e implementar un esquema de coordinación del sistema de garantía de la calidad en el hospital Occidente de Kennedy que integre las acciones y recursos destinados al control interno, al control de la calidad asistencial, la atención al usuario y la recepción y trámite de quejas. Los objetivos específicos consistieron en: caracterizar los mecanismos y procedimientos de garantía de la calidad en el hospital Occidente de Kennedy considerando el control interno, la garantía de la calidad y la atención a los usuarios; investigación aplicada para el diseño del sistema de garantía de la calidad que integre control interno, auditoría médica, quejas y reclamos, y atención al usuario; definir la oficina de garantía de la calidad y los ajustes a la estructura organizacional vigente; definir por consenso las funciones y procesos del sistema de garantía de la calidad (subsistema de control interno, subsistema de auditoría médica y subsistema de interacción con los usuarios); capacitar a los funcionarios para la puesta en operación; acompañar para la implementación del sistema de garantía de la calidad.

The general objective of this work is to characterize and implement a coordination scheme for the quality assurance system at the Hospital Occidente de Kennedy that integrates the actions and resources destined to internal control, quality control of care, user care and the reception and processing of complaints. The specific objectives consisted of: characterize the mechanisms and procedures for quality assurance in the hospital Occidente de Kennedy considering internal control, quality assurance and attention to users; applied research for the design of the quality assurance system that integrates internal control, medical audit, complaints and claims, and attention to the user; define the quality assurance office and adjustments to the current organizational structure; define by consensus the functions and processes of the quality assurance system (internal control subsystem, medical audit subsystem and subsystem of interaction with users); train staff to put it into operation; accompany the implementation of the quality assurance system.