RESUMEN
Pertussis toxin (PT) in its detoxified form is one of the major protective antigens in vaccines against Bordetella pertussis (whooping cough). Reference preparations of native PT are required for the quality control of pertussis vaccines. Stocks of the first WHO International Standard (IS) for PT (JNIH-5) were low and a replacement was required. One candidate material was donated by a vaccine manufacturer to NIBSC. It was formulated, lyophilised into sealed glass ampoules and coded 15/126. An international collaborative study assessed the suitability of this material to replace JNIH-5. Fourteen laboratories from 12 countries took part in the study. Eleven laboratories performed lethal murine histamine sensitisation assay (HIST), 14 performed Chinese Hamster Ovary (CHO) cell clustering assay. International Units (IU) were assigned to the material using these assays as they were used to assign units to JNIH-5. It was found that, unlike JNIH-5, the activities of 15/126 in HIST and CHO cell assays did not agree and therefore different unitage for each assay was assigned. The preparation 15/126 was established as the Second WHO IS for PT for HIST and CHO cell assays. It was assigned a unitage of 1,881 IU/ampoule in HIST and 680 IU/ampoule in the CHO cell clustering assay.
Asunto(s)
Bordetella pertussis , Toxina del Pertussis , Vacuna contra la Tos Ferina , Animales , Células CHO , Calibración , Cricetulus , Liofilización , Histamina , Toxina del Pertussis/análisis , Toxina del Pertussis/química , Toxina del Pertussis/normas , Vacuna contra la Tos Ferina/análisis , Vacuna contra la Tos Ferina/química , Vacuna contra la Tos Ferina/normasRESUMEN
Whooping cough is caused by the bacterium Bordetella pertussis. There are currently two types of vaccines that can prevent the disease; whole cell vaccines (WCV) and acellular vaccines (ACV). The main virulence factor produced by the organism is pertussis toxin (PTx). This toxin is responsible for many physiological effects on the host, but it is also immunogenic and in its detoxified form is the main component of all ACVs. In producing toxoid for vaccines, it is vital to achieve a balance between sufficiently detoxifying PTx to render it safe while maintaining enough molecular structure that it retains its protective immunogenicity. To ensure that the first part of this balancing act has been successfully achieved, assays are required to accurately measure residual PTx activity in ACV products accurately. Quality control assays are also required to ensure that the detoxification procedures are robust and stable. This manuscript reviews the methods that have been used to achieve this aim, or may have the potential to replace them, and highlights their continuing requirement as vaccines that induce a longer lasting immunity are developed to prevent the re-occurrence of outbreaks that have been observed recently.
Asunto(s)
Toxina del Pertussis/análisis , Vacuna contra la Tos Ferina/análisis , Animales , Bioensayo , Humanos , Toxina del Pertussis/toxicidadRESUMEN
Introduced for mass immunization in the 1920s, vaccines against diphtheria are among the oldest and safest vaccines known. The basic principle of their production is the inactivation of purified diphtheria toxin by formaldehyde cross-linking, which converts the potentially fatal toxin in a completely harmless protein aggregate, which is still immunogenic. Since in addition to diphtheria toxin also other proteins may be secreted by Corynebacterium diphtheriae during cultivation, we assumed that diphtheria toxoid might not be the only component present in the vaccine. To address this question, we established a protocol to reverse formaldehyde cross-linking and carried out mass spectrometric analyses. Different secreted, membrane-associated and cytoplasmic proteins of C. diphtheriae were detected in several vaccine preparations from across the world. Based on these results, bioinformatics and Western blot analyses were applied to characterize if these proteins are immunogenic and may therefore support protection against C. diphtheriae. In frame of this study, we could show that the C. diphtheriae toxoid vaccines induce antibodies against different C. diphtheriae proteins and against diphtheria toxin secreted by Corynebacterium ulcerans, an emerging pathogen which is outnumbering C. diphtheriae as cause of diphtheria-like illness in Western Europe.
Asunto(s)
Proteínas Bacterianas/inmunología , Toxoide Diftérico/inmunología , Difteria/prevención & control , Vacuna contra la Tos Ferina/inmunología , Anticuerpos Antibacterianos/inmunología , Corynebacterium , Corynebacterium diphtheriae , Reactivos de Enlaces Cruzados , Citoplasma/química , Europa (Continente) , Formaldehído/química , Humanos , Espectrometría de Masas , Vacuna contra la Tos Ferina/análisis , Proteómica , VacunaciónRESUMEN
Detoxified pertussis toxin (pertussis toxoid) is a major antigen in acellular pertussis vaccines. Testing these vaccines on the presence of residual pertussis toxin (PTx) and reversion to toxicity is performed by the regulatory required in vivo Histamine Sensitization test (HIST). Lack of mechanistic understanding of the HIST, technical handicaps and animal welfare concerns, have promoted the development of alternative methods. As the majority of the cellular effects of PTx depend on its ability to activate intracellular pathways involving cAMP, the in vitro cAMP-PTx assay was developed. Although this assay could be used to detect PTx activity, it lacked sensitivity and robustness for use in a quality control setting. In the present study, novel reporter cell lines (CHO-CRE and A10-CRE) were generated that stably express a reporter construct responsive to changes in intracellular cAMP levels. These reporter cell lines were able to detect PTx in a concentration-dependent manner when combined with fixed amounts of forskolin. The CHO-CRE cell line enabled detection of PTx in the context of a multivalent vaccine containing aP, with a sensitivity equal to the HIST. However, the sensitivity of the A10-CRE cells was insufficient for this purpose. The experiments also suggest that the CHO-CRE reporter cell line might be suitable for assessment of cellular effects of PTd reverted to PTx. The CHO-CRE reporter cell line provides a platform that meets the criteria for specificity and sensitivity and is a promising in vitro model with potential to replace the HIST.
Asunto(s)
Bioensayo , Efecto Fundador , Toxina del Pertussis/análisis , Vacuna contra la Tos Ferina/química , Elementos de Respuesta , Animales , Células CHO , Línea Celular , Colforsina/farmacología , Cricetulus , AMP Cíclico/metabolismo , Genes Reporteros , Histamina/metabolismo , Histamina/farmacología , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Vacuna contra la Tos Ferina/análisis , Ratas , Sensibilidad y Especificidad , Vacunas AcelularesRESUMEN
BACKGROUND: Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. RESULTS: We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. CONCLUSIONS: We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.
Asunto(s)
Adhesinas Bacterianas/análisis , Bordetella pertussis/efectos de los fármacos , Células Epiteliales/microbiología , Ensayos Analíticos de Alto Rendimiento/métodos , Vacuna contra la Tos Ferina/análisis , Sistema Respiratorio/microbiología , Células A549/efectos de los fármacos , Células A549/microbiología , Anticuerpos Antibacterianos/efectos de los fármacos , Bordetella pertussis/patogenicidad , Técnicas de Cultivo de Célula , Línea Celular/efectos de los fármacos , Línea Celular/microbiología , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Modelos Biológicos , Vacuna contra la Tos Ferina/uso terapéutico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Vacunación , Vacunas Acelulares/análisis , Vacunas Acelulares/uso terapéutico , Tos Ferina/tratamiento farmacológico , Tos Ferina/microbiologíaRESUMEN
Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.
Asunto(s)
Bioensayo/métodos , Contaminación de Medicamentos , Agonistas de los Receptores Histamínicos/farmacología , Histamina/farmacología , Toxina del Pertussis , Vacuna contra la Tos Ferina , Temperatura Cutánea/efectos de los fármacos , Animales , Humanos , Ratones , Toxina del Pertussis/análisis , Toxina del Pertussis/farmacología , Vacuna contra la Tos Ferina/análisis , Vacuna contra la Tos Ferina/farmacología , Sensibilidad y EspecificidadRESUMEN
An important step in vaccine production and quality control is the analysis of identity of different lots. For that purpose chicken was immunized with acellular Pertussis components (Pertussis toxoid, Filamenteous haemagglutinin, Pertactin, Fimbriae 2/3 antigen). The resulting antibodies (IgY) were non-invasive extracted from egg yolk and used for rocket immunoelectrophoresis (RIE). We demonstrated that the Ab reacted with characteristic peaks ("rockets") with the corresponding antigen. The shape of the peaks varied depending on the manufacturer and the nature of antigen (adsorbed or non-adsorbed). The coefficients of variation was about 20% during a year period. In summary, our data illustrate that an IgY-based RIE is not only a cost-effective method but also proficient for monitoring Pertussis vaccines.
Asunto(s)
Bordetella pertussis/inmunología , Pollos/inmunología , Inmunoelectroforesis/métodos , Inmunoglobulinas/inmunología , Vacuna contra la Tos Ferina/análisis , Animales , Reacciones Cruzadas , Vacuna contra la Tos Ferina/inmunologíaRESUMEN
Safety requirements stipulate the performance of the in vivo Histamine Sensitization (HS) test for quality control of acellular pertussis (aP) vaccines. For reasons of reproducibility and animal welfare concern, an in vitro assay was developed. The assay reflects the mechanism of histamine sensitization and is based on cAMP production in A10 cells to residual pertussis toxin (PT). We showed that PT induces cAMP levels in a dose-dependent manner while the sensitivity of the assay equals the sensitivity of the HS test. Neither the individual components nor the combination vaccine DTaP-IP did affect the assay. The cAMP assay meets the criteria for specificity and sensitivity and therefore might be a promising candidate to replace the HS test.
Asunto(s)
AMP Cíclico/análisis , Toxina del Pertussis/análisis , Vacuna contra la Tos Ferina/análisis , Animales , Relación Dosis-Respuesta a Droga , Histamina/análisis , Humanos , Toxina del Pertussis/farmacología , Vacuna contra la Tos Ferina/farmacología , Control de Calidad , Ratas , Sensibilidad y EspecificidadRESUMEN
All current acellular pertussis vaccines (ACVs) contain detoxified pertussis toxin (PT) as a major component. An essential part of the safety evaluation of these vaccines, required by regulatory authorities, is to monitor their active PT content and to check for reversion to toxicity of the detoxified PT. Although various in vitro tests are under investigation, the only practicable means for detecting active PT at present is the histamine sensitization test. The methods given in the European Pharmacopoeia and in the US Pharmacopoeia are based on recording a binary response to histamine challenge (using a lethal end point). A more sensitive method based on measurement of rectal temperature is given in the Japanese Minimum Requirements for Biological Products. More recently, a refinement of this method based on dermal temperature measurement has been developed for ACVs in combination with diphtheria and tetanus vaccines (DTaP). We show that this method also can be used for more complex combination vaccines and is readily transferable. Furthermore use of dermal temperature provides a more precise quantitative estimate of toxin activity than the binary response, leading to an increase in information from a specified number of animals, or allowing a reduction in the number of animals required. We suggest that, pending the development of an alternative in vitro replacement method, the temperature based method may serve as an intermediate solution to the estimation of PT activity giving a precise estimate with reduction in animal numbers.
Asunto(s)
Toxina del Pertussis/toxicidad , Vacuna contra la Tos Ferina/análisis , Temperatura Cutánea , Pruebas de Toxicidad/métodos , Animales , Femenino , Humanos , Japón , Ratones , Control de CalidadAsunto(s)
Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/química , Vacuna contra la Tos Ferina/química , Factores de Virulencia de Bordetella/análisis , Tos Ferina/prevención & control , Proteínas Bacterianas/análisis , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/análisis , Humanos , Lactante , Japón , Vacuna contra la Tos Ferina/análisis , Vacunas Acelulares/químicaRESUMEN
Pertussis toxin (PTx) in its detoxified form is an important component of both whole cell and acellular pertussis vaccines (ACVs). For safety reasons, it is imperative to ensure that the quantity of residual PTx in vaccines does not exceed permissible levels. The majority of the toxic effects of PTx have been attributed to the consequences of PTx-catalyzed ribosylation of the alpha-subunits of signal-transducing guanine-nucleotide-binding proteins. In this report PTx ribosylation activity was determined by an improved enzymatic-high performance liquid chromatography coupled assay using a fluorescein labeled Galpha(i3)C20 peptide. The effect of aluminum salts and other vaccine components on the assay system were also studied. The enzymatic assay system was shown to be a convenient, sensitive method and correlate well with the toxicity observed in vivo by the histamine sensitization assay. This method forms the basis of a new assay which could replace the unsatisfactory animal test currently used in pertussis vaccines control.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Toxina del Pertussis/análisis , Vacuna contra la Tos Ferina/química , Animales , Bioensayo/métodos , Células CHO , Cricetinae , Colorantes Fluorescentes , Proteínas de Unión al GTP Heterotriméricas , Vacuna contra la Tos Ferina/análisis , Vacuna contra la Tos Ferina/normas , Sensibilidad y Especificidad , Vacunas Acelulares/análisis , Vacunas Acelulares/química , Vacunas Acelulares/normasRESUMEN
The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Toxina del Pertussis , Vacuna contra la Tos Ferina/farmacología , Factores de Virulencia de Bordetella/farmacología , Secuencia de Aminoácidos , Animales , Bioensayo , Cromatografía Líquida de Alta Presión , Colorantes Fluorescentes , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Técnicas In Vitro , Espectrometría de Masas , Datos de Secuencia Molecular , NAD/metabolismo , Péptidos/química , Vacuna contra la Tos Ferina/análisis , Vacuna contra la Tos Ferina/normas , Vacunas Acelulares/análisis , Vacunas Acelulares/farmacología , Vacunas Acelulares/normas , Factores de Virulencia de Bordetella/análisisRESUMEN
The first British Standard (BS) (Code 66/303) for pertussis whole cell vaccine was prepared from the same suspension of Bordetella pertussis as the now exhausted Second International Standard for Pertussis Vaccine (2ndIS). The BS and the 2ndIS were compared and calibrated in a previous international study. This report describes a small international study, which included the BS, the 1stIS and the 2ndIS so that the present relationship of these preparations to one another could be assessed. The results of this study show that the relationship of the BS to the 1stIS is consistent with its established unitage of 46 IU per ampoule. The results further show that the potency of the BS is broadly consistent with that of the 2ndIS and that the BS has not lost activity relative to the 2ndIS (from which it was previously found to be indistinguishable). Based on its original calibration and supported by the results of this present study, the BS has been established as the Third International Standard for Pertussis Vaccine, with an assigned unitage of 46 IU per ampoule.
Asunto(s)
Vacuna contra la Tos Ferina/normas , Humanos , Vacuna contra la Tos Ferina/análisis , Estándares de Referencia , Reino Unido , Organización Mundial de la SaludRESUMEN
According to WHO and the European Pharmacopoeia, the current potency test for acellular pertussis vaccines is a mouse immunogenicity assay assessing consistency of production from batch to batch. The assay compares the batch under control with a reference vaccine of documented clinical efficacy. This study describes and illustrates critical aspects of the assay, based on our experience on a tricomponent vaccine: validation of immunoassay to quantify mouse antibody response, choice of vaccine immunising doses in the three-doses model, treatment of non-responder mice for calculations, establishment of assay validity criteria.
Asunto(s)
Inmunoensayo/métodos , Vacuna contra la Tos Ferina/análisis , Vacuna contra la Tos Ferina/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Bordetella pertussis/inmunología , Femenino , Inmunoensayo/normas , Ratones , Vacuna contra la Tos Ferina/normas , Control de Calidad , Estándares de Referencia , Vacunas Acelulares/análisis , Vacunas Acelulares/inmunología , Vacunas Acelulares/normasRESUMEN
The current potency test for whole cell pertussis vaccines, the intracerebral mouse protection test, is still the only assay which has shown a correlation with protection in children. However, it has considerable disadvantages as it uses a severe challenge procedure and the results tend to show significant intra- and inter-laboratory variation. An alternative assay based on non-lethal aerosol challenge of mice has been investigated as a replacement for the current intracerebral mouse protection test. Evaluation of this indicated that the aerosol system allowed consistent inoculation of bacteria into mice and gave good reproducibility. The protective capacity of different vaccine preparations was distinguished by this assay. Furthermore, the viable counts of Bordetella pertussis in the lungs of challenged mice were immunisation dose-dependent, which allowed the relative potency of vaccines to be calculated. Comparison of potency of five batches of vaccine from different manufacturers assayed by both the intracerebral and the aerosol challenge methods ranked the vaccines in identical order. The results suggest that this method has potential for use as a potency test for whole cell pertussis vaccine which would result in a great reduction in the number of animals used. It would also replace the lethal challenge by a non-lethal procedure and thereby avoid the use of the severe intracerebral challenge procedure.
Asunto(s)
Bioensayo/métodos , Vacuna contra la Tos Ferina/análisis , Aerosoles , Animales , Bordetella pertussis/inmunología , Bordetella pertussis/aislamiento & purificación , Bordetella pertussis/patogenicidad , Encéfalo/microbiología , Niño , Recuento de Colonia Microbiana , Femenino , Humanos , Inmunización , Pulmón/inmunología , Pulmón/microbiología , Ratones , Modelos Animales , Vacuna contra la Tos Ferina/administración & dosificación , Reproducibilidad de los Resultados , Tos Ferina/inmunología , Tos Ferina/prevención & controlAsunto(s)
Alternativas a las Pruebas en Animales , Bienestar del Animal , Animales de Laboratorio , Bioensayo/métodos , Ciencia de los Animales de Laboratorio/métodos , Vacunas/análisis , Animales , Reacciones Falso Negativas , Reacciones Falso Positivas , Humanos , Vacuna contra la Tos Ferina/análisis , Reproducibilidad de los ResultadosRESUMEN
The intracerebral mouse protection test (Kendrick test) for the potency assay of pertussis vaccines is a complex and time consuming in vivo test which has a significant intra- and interlaboratory variation. Thus, there is a pressing need to develop a replacement for the Kendrick test. There is now convincing evidence to suggest that Bordetella pertussis can be taken up and survive within macrophages in the lungs and that cell-mediated immunity plays a role in protection. It was hypothesised that murine macrophages could be activated by immunisation with whole cell pertussis vaccines and therefore induce NO production. An alternative in vitro assay based on the determination of reactive nitrogen intermediates produced as a result of macrophage activation has been examined as a possible replacement for the current intracerebral (i.c.) mouse protection test. NO induction was studied in the peritoneal macrophages of female NIH mice immunised with normal and denatured whole cell B. pertussis vaccines respectively. Compared with controls receiving diluent only, macrophages and spleen cells from mice immunised with whole cell pertussis vaccine responded in vitro to selected pertussis antigens by NO synthesis. The production of NO in response to in vitro culture with bacterial antigen was immunisation dose dependent and was correlated with protective immunity in vivo as determined by i.c. challenge. The results suggest that NO production may serve as a marker of macrophage activation in mice immunised with whole cell vaccine, and could form the basis of a potential replacement potency assay.
Asunto(s)
Alternativas a las Pruebas en Animales , Macrófagos Peritoneales/efectos de los fármacos , Óxido Nítrico/biosíntesis , Vacuna contra la Tos Ferina/farmacología , Vacuna contra la Tos Ferina/normas , Alternativas a las Pruebas en Animales/métodos , Alternativas a las Pruebas en Animales/normas , Animales , Bordetella pertussis/inmunología , Células Cultivadas , Femenino , Linfocitos/inmunología , Linfocitos/microbiología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Ratones , Ratones Endogámicos , Vacuna contra la Tos Ferina/análisis , Bazo/inmunologíaAsunto(s)
Humanos , Bordetella pertussis/aislamiento & purificación , Tos Ferina/diagnóstico , Vacuna contra la Tos Ferina/análisis , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Técnica del Anticuerpo Fluorescente Directa , Pruebas Serológicas , Tos Ferina/prevención & controlRESUMEN
El síndrome coqueluchoídeo es frecuente en el lactante menor siendo causa importante de morbilidad. Con el objeto de estudiar la importancia de bordetella pertussis como causa de este síndrome, las características clínicas de coqueluche y la sensibilidad y especificidad de las distintas técnicas para el diagnóstico de B. pertussis, se realizó un estudio con 66 niños hospitalizados con el diagnóstico de síndrome coqueluchoídeo. B. pertussis fue el principal agente causal (56 por ciento) seguido de varios virus respiratorios (14 por ciento) como adenovirus, virus respiratorio sincicial y parainfluenza. Las características clínicas que permitieron diferenciar a la coqueluche de las otras causas del síndrome coqueluchoídeo fueron la asociación de tos paroxística, emetizante y estridor inspiratorio. La linfocitosis absoluta sobre 10.000/mm elevado a 3, especialmente si se asoció a tos característica, tuvo buena especificidad con fines diagnósticos. La sensibilidad del cultivo, IFD y RPC para el diagnóstico de coqueluche fue de 32, 36 y 93 por ciento respectivamente. La especificidad de la IFD y la RPC fue de 87 y 98 por ciento respectivamente. El uso previo de antibióticos o la obtención de la muestra en estapas avanzadas de la enfermedad no modificó la sensibilidad de la técnica de RPC, no sucedió así con el cultivo e IFD. Por todo esto la RPC sería el método de elección para el diagnóstico de coqueluche
Asunto(s)
Humanos , Masculino , Femenino , Lactante , Bordetella pertussis/aislamiento & purificación , Tos Ferina/diagnóstico , Técnica del Anticuerpo Fluorescente Directa , Vacuna contra la Tos Ferina/análisis , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
The development of acellular pertussis vaccines has raised a number of issues relevant to the control of these products. Of particular importance is the need for robust and accurate in vitro assays for the antigen content of the vaccines which might contain up to five different antigen components, each of which needs to be independently assayed. This paper describes a simple method for the quantification of three component antigens. Because relatively high doses of purified antigens are used in those preparations, the elimination of residual toxicity is a major concern. This is achieved by genetic modification of chemical treatment. The latter results in modification of the immunological reactivity of the antigens making direct assay by such methods as ELISA ineffective. A single radial diffusion technique using polyclonal antisera for the assay of pertussis toxoid (PTxd), chemically treated filamentous haemagglutinin (FHA) and pertactin (69 kDa) has been developed. The method uses low concentrations of antisera, allowing accurate and reproducible quantification of antigen content as low as 25 microg/ml of protein for pertussis toxoid and filamentous haemagglutinin and 5 microg/ml for pertactin. Since by the addition of detergent, diffusible subunits are produced irrespective of the original physical state of the antigens, the assay is suitable for assay of these antigens after detoxification/or stabilization by chemical treatment and is able to determine the differences between preparations which have the same protein concentration but different antigenic contents. This provides a means for assuring the consistency of the antigens after detoxification/or chemical stabilization which could be used as an in-process control method for acellular pertussis vaccines.