[Validation of an ELISA assay for the quantification of antibodies against Haemophilus influenzae type b capsular polysaccharide]. / Validación de un ensayo tipo ELISA para la cuantificación de anticuerpos contra el polisacárido capsular de Haemophilus influenzae tipo b.

The validation study of an ELISA assay -internationally accepted for this purpose- was conducted aimed at having a method for the quantification of antibodies against Haemophilus influenzae type b (Hib) capsular polysaccharide in the clinical trials used to prove the immunogenicity of a new conjugated vaccine composed of a synthetic antigen. The validation was carried out in the National Haemophilus Reference Laboratory, in "Pedro Kouri" Institute of Tropical Medicine, Havana, Cuba. A protocol was designed, in which the determination of accuracy, exactness, linearity and limit of the detection of the assay were considered. The Hib Batch 1983 international reference serum was used in the preparation of the standard pattern curve. Dispersion indexes lower than 10 and 20% were observed for the repeatability and reproducibility of the system, respectively. The limit of detection was 3.6 ng/m and the recovery and linearity trials showed the high accuracy of the method. It was concluded that during the clinical evaluation of the candidate vaccine obtained by chemical synthesis, the quantification of antibodies against Hib capsular polysaccharide may be faced with an appropriate precision and exactness by using the proposed method.

[Methicillin-resistant Staphylococcus aureus. Detection of carriers among hospitalized children and healthy children from the community]. / Staphylococcus aureus resistente a la meticilina: detección de portadores entre niños hospitalizados y niños sanos de la comunidad.

A descriptive punctual study was conducted to search for nasal carriers of Staphylococcus aureus strains resistant to methycilin (SARM) among sound children attended in nurseries and hospitalized children exposed to one or more risk factors predisposing to the colonization by this type of strains from October, 2000, to February, 2001, in Marianao municipality. For the primary isolation of the SARM strains, it was used the oxacillin-manitol-salt-agar (OMSA) and the sensitivity to antimicrobial drugs was determined in all strains identified as S. aureus by using the Kirby-Bauer method. In order to identify the resistance to oxacillin in the strains recovered from OMSA, the oxacillin salt-agar screening-plate method and the determination of inhibitory minimum concentration (IMC) in agar and broth were applied. The presence of the mecA gene was nvestigated by polymerase chain reaction in those strains with an IMC > or = 4 mg/mL of oxacillin. Of the sound children. 0.35 % and of the hospitalized, 2 %, were nasal carriers of SARM strains. The strains isolated in both study groups showed high indexes of resistance to penicillin, tetracycline and erythromycin by the Kirby-Bauer method. The mecA gene was identified in a SARM strain isolated from a hospitalized nasal carrier and it was defined that its behaviour agreed with the one described for a strain homoresistant to oxacillin.

Naturally acquired immunity to Haemophilus influenzae type B in healthy Cuban children.

We have evaluated the prevalence of antibody to immunogenicity of Haemophilus influenzae type b (Hib) in a group of 4 to 5 years old healthy children, who were too old to be included in the first vaccinated cohort when Hib vaccination begun in Cuba in 1999. Serum capsular polysaccharide specific IgG antibody concentrations were measured in 974 healthy children, between February and May 2002. The prevalence of Hib nasopharyngeal carriage was also estimated. The majority of children (99.7%) had more than 1 microg/ml of antibody. The preliminary report of the nasopharyngeal cultures was positive for H. influenzae in 16 children, but in only one was confirmed as Hib after serotyping (0.1% Hib nasopharyngeal carrier). These results provide evidence that in Cuba the natural active immunity to Hib can be acquired at an early age.

Validación de un ensayo tipo ELISA para la cuantificación de anticuerpos contra el polisacárido capsular de Haemophilus influenzae tipo b / Validation of an ELISA assay for the quantification of antibodies against Haemophilus influenzae type b capsular polysaccharide

Se llevó a cabo el estudio de validación de un ensayo tipo ELISA —aceptado internacionalmente para este propósito—, con el objetivo de disponer de un método para la cuantificación de anticuerpos contra el polisacárido capsular de Haemophilus influenzae tipo b (Hib), en los ensayos clínicos previstos para demostrar la inmunogenicidad de una nueva vacuna conjugada, compuesta por un antígeno sintético. La validación fue conducida en el Laboratorio Nacional de Referencia para Haemophilus, en el Instituto de Medicina Tropical “Pedro Kourí”, La Habana, Cuba, y se diseñó para ello un protocolo en el cual se consideró la determinación de la precisión, exactitud, linealidad y límite de detección del ensayo. En la preparación de la curva patrón estándar se empleó el suero de referencia internacional Hib Lote 1983. Para la repetibilidad y reproducibilidad del sistema se observaron índices de dispersión inferiores a 10 y 20 por ciento, respectivamente. El límite de detección fue de 3,6 ng/m, y los ensayos de recuperación y linealidad revelaron la alta exactitud del método. Se concluyó que durante la evaluación clínica del candidato a vacuna, obtenido por síntesis química, la cuantificación de anticuerpos contra el polisacárido capsular de Hib podía ser afrontada con apropiada precisión y exactitud mediante el método propuesto

Staphylococcus aureus resistente a la meticilina: detección de portadores entre niños hospitalizados y niños sanos de la comunidad

Se realizó un estudio descriptivo puntual para la búsqueda de portadores nasales de cepas de Staphylococcus aureus resistentes a la meticilina (SARM), entre niños sanos atendidos en círculos infantiles y entre niños hospitalizados expuestos a uno de los factores de riesgo o más que predisponen para la colonización por este tipo de cepas; durante el período comprendido entre octubre de 2000 a febrero de 2001, en el municipio Marianao. Para el aislamiento primario de las cepas SARM se utilizó el medio oxacilina-manitol-sal-agar (OMSA) y a todas las cepas identificadas como S. aureus se les determinó la sensibilidad a drogas antimicrobianas por el método de Kirby-Bauer. Para identificar la resistencia a la oxacilina en las cepas recuperadas en OMSA se aplicaron los métodos: oxacillin salt-agar screening-plate y la determinación de la concentración mínima inhibitoria (CMI) en agar y en caldo. Se investigó la presencia del gen mecA mediante la reacción en cadena de la polimerasa en aquellas cepas que presentaron una CMI ³ 4 µg/mL de oxacilina. De los niños sanos, 0,35 % y de los hospitalizados, 2 % constituyeron portadores nasales de cepas SARM. Las cepas aisladas en ambos grupos de estudio mostraron índices altos de resistencia para la penicilina, tetraciclina y eritromicina por el método de Kirby-Bauer. Se identificó el gen mecA en una cepa SARM aislada de un portador nasal hospitalizado y se definió que su comportamiento se correspondía con el descrito para una cepa homorresistente para la oxacilina.

A descriptive punctual study was conducted to search for nasal carriers of Staphylococcus aureus strains resistant to methycilin (SARM) among sound children attended in nurseries and hospitalized children exposed to one or more risk factors predisposing to the colonization by this type of strains from October, 2000, to February, 2001, in Marianao municipality. For the primary isolation of the SARM strains, it was used the oxacillin-manitol-salt-agar (OMSA) and the sensitivity to antimicrobial drugs was determined in all strains identified as S. Aereus by using the Kirby-Bauer method. In order to identify the resistance to oxacillin in the strains recovered from OMSA, the oxacillin salt-agar screening -plate method and the determination of inhibitory minimum concentration (IMC) in agar and broth were applied. The presence of the mecA gene was nvestigated by polymerase chain reaction in those strains with an IMC ³ 4 mg/mL of oxacillin. Of the sound children, 0.35 % and of the hospitalized, 2 %, were nasal carriers of SARM strains. The strains isolated in both study groups showed high indexes of resistance to penicillin, tetracycline and erythromycin by the Kirby-Bauer method. The mecA gene was identified in a SARM strain isolated from a hospitalized nasal carrier and it was defined that its behaviour agreed with the one described for a strain homoresistant to oxacillin.

Naturally acquired immunity to Haemophilus influenzae type B in healthy Cuban children

We have evaluated the prevalence of antibody to immunogenicity of Haemophilus influenzae type b (Hib) in a group of 4 to 5 years old healthy children, who were too old to be included in the first vaccinated cohort when Hib vaccination begun in Cuba in 1999. Serum capsular polysaccharide specific IgG antibody concentrations were measured in 974 healthy children, between February and May 2002. The prevalence of Hib nasopharyngeal carriage was also estimated. The majority of children (99.7 percent) had more than 1 µg/ml of antibody. The preliminary report of the nasopharyngeal cultures was positive for H. influenzae in 16 children, but in only one was confirmed as Hib after serotyping (0.1 percent Hib nasopharyngeal carrier). These results provide evidence that in Cuba the natural active immunity to Hib can be acquired at an early age.