Estandarización de un ELISA para la cuantificación de anticuerpos IgG humanos contra células enteras de Bordetella pertussis / Standardization of an ELISA for the quantification of human IgG antibodies against whole cells of Bordetella pertussis

Bordetella pertussis es un patógeno exclusivo de humanos que causa la tos ferina, enfermedad respiratoria aguda que afecta principalmente a la población pediátrica. Existen dos tipos de vacunas comercializadas contra este patógeno: celulares y acelulares. Las vacunas celulares han sido extensamente utilizadas y siguen teniendo gran relevancia. El presente trabajo tuvo como objetivo la estandarización de un ELISA para la cuantificación de anticuerpos IgG contra células enteras de Bordetella pertussis. Para ello se determinó la concentración de recubrimiento, el rango lineal de la curva, los parámetros de precisión intra e interensayo, la especificidad, el valor de corte y el límite de detección. Se determinó como concentración de recubrimiento 0,5 UO/mL de células enteras. La curva estándar utilizando un suero de referencia internacional presentó un buen ajuste a una función polinómica en un intervalo entre las diluciones 1/100 y 1/24.300 con un coeficiente de correlación R2≥0,98. Los coeficientes de variación en los ensayos de precisión intra e interensayo estuvieron en los intervalos establecidos para cada uno (≤10 por ciento, ≤20 por ciento respectivamente). Los resultados obtenidos avalan el empleo de este ELISA cuantitativo para la evaluación de la respuesta a células enteras de Bordetella pertussis en ensayos clínicos(AU)

Bordetella pertussis is a pathogen exclusive to humans that causes pertussis, an acute respiratory disease that mainly affects the pediatric population. There are two types of vaccines commercially available against this pathogen: cellular and acellular. Cellular vaccines have been widely used and continue to be of great relevance. The aim of the present work was to standardize an ELISA for the quantification of IgG antibodies against whole cells of Bordetella pertussis. For this purpose, the coating concentration, the linear range of the curve, the intra- and inter-assay precision parameters, the specificity, the cut-off value and the detection limit were determined. The coating concentration was determined as 0.5 UO/mL of whole cells. The standard curve using an international reference serum presented a good fit to a polynomial function in a range between dilutions 1/100 and 1/24,300 with a correlation coefficient R2≥0.98. The coefficients of variation in the intra- and inter-assay precision tests were in the intervals established for each (≤10percent, ≤20percent respectively). The results obtained support the use of this quantitative ELISA for the evaluation of whole-cell response to Bordetella pertussis in clinical trials(AU)

Severe immune thrombocytopenia following diphtheria, tetanus, pertussis and polio vaccination in a 36-year-old Caucasian woman: a case report.

BACKGROUND: Immune thrombocytopenia (ITP) is a rare autoimmune disorder characterized by low platelet counts and increased bleeding risk. The disease may be induced by other disorders, including malignancies, autoimmune diseases, infectious agents or drugs. However, ITP has also been described following vaccinations, such as the measles-mumps-rubella vaccination. In rare cases, ITP may occur in children who received a DTaP-IP (diphtheria, tetanus, acellular pertussis vaccine and inactivated poliovirus) vaccine. Hereinafter, we report the first well-documented cases of ITP in an adult patient in the temporal context of a DTaP-IP vaccination. CASE PRESENTATION: This case report attempts to capture the life-threatening picture of a 36-year-old otherwise healthy Caucasian woman with newly diagnosed severe immune thrombocytopenia in the temporal context of a DTaP-IP vaccination. Four days after receiving the vaccine, the women presented to her primary care physician with malaise, fever and recurrent epistaxis. Clinical examination revealed oral petechiae, ecchymoses, and non-palpable petechiae on both legs. The patient was immediately referred to a local hematology unit where she developed hematuria and an intestinal bleeding (WHO Bleeding Grade III) requiring multiple transfusions. After receiving oral corticosteroids and intravenous immunoglobulins, her platelets gradually recovered. Common causes of secondary ITP were ruled out by laboratory investigations, bone marrow and peripheral blood examinations. This raises the possibility of a (secondary) vaccination-associated thrombocytopenia. To the best of our knowledge, this is the first well-documented case of a DTaP-IP vaccination-related ITP in an adult patient in the English literature. CONCLUSION: Although a causal connection between both entities may not be established, we would like to raise awareness in clinicians that ITP following DTaP-IP vaccinations is potentially not limited to children, but may also occur in adults. Users of DTaP-IP booster vaccines should be alert of the possibility of such adverse reactions.

Identfication of viral and bacterial etiologic agents of the pertussis-like syndrome in children under 5 years old hospitalized.

BACKGROUND: Acute respiratory infections (ARIs) represent an important cause of morbidity and mortality in children, remaining a major public health concern, especially affecting children under 5 years old from low-income countries. Unfortunately, information regarding their epidemiology is still limited in Peru. METHODS: A secondary data analysis was performed from a previous cross-sectional study conducted in children with a probable diagnosis of Pertussis from January 2010 to July 2012. All samples were analyzed via Polymerase Chain Reaction (PCR) for the following etiologies: Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Mycoplasma pneumoniae and Chlamydia pneumoniae. RESULTS: A total of 288 patients were included. The most common pathogen isolated was Adenovirus (49%), followed by Bordetella pertussis (41%) from our previous investigation, the most prevelant microorganisms were Mycoplasma pneumonia (26%) and Influenza-B (19.8%). Coinfections were reported in 58% of samples and the most common association was found between B. pertussis and Adenovirus (12.2%). CONCLUSIONS: There was a high prevalence of Adenovirus, Mycoplasma pneumoniae and other etiologies in patients with a probable diagnosis of pertussis. Despite the presence of persistent cough lasting at least two weeks and other clinical characteristics highly suspicious of pertussis, secondary etiologies should be considered in children under 5 years-old in order to give a proper treatment.

Identifying Children at Risk of Malignant Bordetella pertussis Infection.

OBJECTIVE: To identify factors associated with malignant pertussis. DESIGN: A retrospective case notes review from January 2003 to August 2013. Area under the receiver-operator characteristic curve was used to determine how well vital sign and white cell characteristics within 48 hours of hospital presentation identified children with malignant pertussis. SETTING: The national children's hospital in Auckland, New Zealand. PATIENTS: One hundred fifty-two children with pertussis. MEASUREMENTS AND MAIN RESULTS: There were 152 children with confirmed pertussis identified, including 11 children with malignant pertussis. The area under the receiver-operator characteristic curve was 0.88 (95% CI, 0.78-0.97) for maximum heart rate. The optimal cut-point was 180 beats/min, which predicted malignant pertussis with a sensitivity of 73% and a specificity of 91%. The area under the receiver-operator characteristic curve was 0.92 (95% CI, 0.81-1.0) for absolute neutrophil count, 0.85 (95% CI, 0.71-0.99) for total WBC count, 0.80 (95% CI, 0.63-0.96) for neutrophil-to-lymphocyte ratio, and 0.77 (95% CI, 0.58-0.92) for absolute lymphocyte count. All children with malignant pertussis had one or more of heart rate greater than 180 beats/min, total WBC count greater than 25 × 10/L, and neutrophil-to-lymphocyte ratio greater than 1.0 with an area under the receiver-operator characteristic curve of 0.96 (95% CI, 0.91-1.0) for a multivariate model that included these three variables. CONCLUSIONS: Clinical predictors of malignant pertussis are identifiable within 48 hours of hospital presentation. Early recognition of children at risk of malignant pertussis may facilitate early referral to a PICU for advanced life support and selection for trials of investigational therapies.

Many Inflammatory Bowel Disease Patients Are Not Immune to Measles or Pertussis.

BACKGROUND: Current guidelines emphasize vaccination for influenza and pneumococcus for IBD patients and the avoidance of live virus vaccines for those who are on immunosuppressive (ISS) therapy. Given the recent resurgence of measles and pertussis infections, we assessed the immune status of our IBD population in order to advise about these risks. METHODS: We prospectively collected measles and pertussis titers in our IBD patients from February 1-May 1, 2015. Immune status based on standard threshold values was determined: measles antibodies ≤0.8 antibody index (AI) = negative immunity, 0.9-1.1 AI = equivocal immunity and titers ≥1.2 AI = positive immunity. For pertussis immunity, anti-pertussis antibodies ≤5 IU/mL were considered negative immunity. Univariate analysis was performed to examine predictive factors including age, disease duration, and current medical therapies. RESULTS: A total of 122 patients' titers were assessed (77 Crohn's disease, 1 indeterminate colitis, and 45 ulcerative colitis). Sixteen (13.1 %) patients lacked detectable immunity to measles, and four (3 %) had equivocal immunity. Twelve (75 %) of the measles non-immune patients were on ISS therapy versus 65 (64 %) of 102 immune patients (OR 1.7, 95 % CI 0.5-5.9, p = 0.34). Out of 96 patients, 58 (60 %) were not immune to pertussis. Disease duration ≥10 years and age ≥50 were associated with significant lower measles titers. CONCLUSIONS: A significant number of our IBD patients lack immunity to measles, and a majority of our IBD patients do not have detectable immunity to pertussis. Importantly, the majority of the measles non-immune patients are on ISS therapy and therefore unable to receive a booster.

A Ten-Year Case-Control Study of Passive Smoke Exposure as a Risk Factor for Pertussis in Children.

The authors conducted a matched case-control study of laboratory-confirmed pertussis cases, occurring from 1/1/1996 to 12/31/2005, in children up to 12 years of age who were members of a large managed care organization. Sixty-five laboratoryconfirmed cases of pertussis were identified. Using multivariable conditional logistic regression analysis, the authors did not detect a statistically significant association between pertussis and household passive exposure to cigarette smoking.

[The return of pertussis]. / Le retour de la coqueluche.

Pertussis (whooping cough) is a contagious respiratory tract bacterial infection due to Bordetella pertussis who makes global comeback despite routine vaccination. Bordetella pertussis produces a series of virulence factors that are involved in one of several steps of the pathogenesis of whooping cough (adhesins, toxins). Unusual presentations are now most common. Clinical diagnosis of whooping cough is often difficult, and microbiologic evaluation is helpful. Treatment is based on antibiotherapy and sometimes intensive care management. Complications are most common in children younger than 6 months (death, malignant pertussis, respiratory and neurological complications). Pertussis can be controlled by treatment of exposed people and by vaccination.

Coqueluche en adultos y adolescentes / Pertussis in adults and adolescents

La coqueluche en adultos y adolescentes ha sido invocada como uno de los factores que condicionan la persistencia de la enfermedad en comunidades con altas coberturas de vacunación. Mejores recursos de laboratorio, incluyendo serología y reacción de polimerasa en cadena, han confirmado que un número significativo de las infecciones se presentan en estos grupos de edad. Sus síntomas pueden ser tan típicos como en niños o atípicos, siendo un hecho constante la tos persistente sobre 7 a 14 días. Se revisan los criterios de laboratorio para certificar el diagnóstico, las recomendaciones actuales de tratamiento y quimioprofilaxis en contactos y se discute la factibilidad de aplicar vacuna acelular en adolescentes y adultos, su potencial impacto e indicaciones.

Manual de vigilância epidemiológica; coqueluche: normas e instruções 2000 / Epidemiologic surveillance manual; whooping cough: standards and instructions 2000

A coqueluche, também conhecida pela designação expressiva de "tosse comprida" é uma doença infecciosa aguda e transmissível que compromete predominantemente o aparelho respiratório, caracterizando-se por típicos acessos paroxísticos de tosse. Sendo um agravo de notificação nacional, a principal dificuldade na vigilância dessa doença com todos os seus pressupostos, está na confirmação etiológica. Outras doenças respiratórias agudas, virais ou bacterianas, podem provocar a "síndrome pertussis" ou "doenças coqueluchóides" (ítem 7). Aparecem, com maior frequência, nos mesmos grupos populacionais onde ocorre a coqueluche, e também apresentam maior gravidade nos lactentes e crianças menores de dois anos. Esses agravos podem, então, serem confundidos e classificados como coqueluche, clinicamente. Dessa forma, um sistema de notificação passivo para a coqueluche tem baixo valor preditivo positivo, ou seja, confirma casos (clinicamente e/ou por métodos laboratoriais não específicos) que não o são, podendo induzir à investigação e adoção de medidas de controle de epidemias que de fato não tenham ocorrido, provocando custos desnecessários ao sistema de vigilância. O que se propõe no presente documento é a implantação de um sistema sentinela de vigilância para a coqueluche no Estado de São Paulo...

Manual de vigilância epidemiológica; coqueluche: normas e instruções 2000

A coqueluche, também conhecida pela designação expressiva de "tosse comprida" é uma doença infecciosa aguda e transmissível que compromete predominantemente o aparelho respiratório, caracterizando-se por típicos acessos paroxísticos de tosse. Sendo um agravo de notificação nacional, a principal dificuldade na vigilância dessa doença com todos os seus pressupostos, está na confirmação etiológica. Outras doenças respiratórias agudas, virais ou bacterianas, podem provocar a "síndrome pertussis" ou "doenças coqueluchóides" (ítem 7). Aparecem, com maior frequência, nos mesmos grupos populacionais onde ocorre a coqueluche, e também apresentam maior gravidade nos lactentes e crianças menores de dois anos. Esses agravos podem, então, serem confundidos e classificados como coqueluche, clinicamente. Dessa forma, um sistema de notificação passivo para a coqueluche tem baixo valor preditivo positivo, ou seja, confirma casos (clinicamente e/ou por métodos laboratoriais não específicos) que não o são, podendo induzir à investigação e adoção de medidas de controle de epidemias que de fato não tenham ocorrido, provocando custos desnecessários ao sistema de vigilância. O que se propõe no presente documento é a implantação de um sistema sentinela de vigilância para a coqueluche no Estado de São Paulo

En coqueluche: ¿es posible? / In pertussis: is it possible?

Existen esquemas alternativos a los recomendados por la Academia Americana de Pediatría para el tratamiento antimicrobiano de la coqueluche. Estudios controlados apoyan la posibilidad de reducir el plazo de tratamiento a 7 días empleando eritromicina. Desde cuidarse en especial la dosficación de eritromicina (para etilsuccinato debe ser de 50 a 60 mg/kg/ día y administrarse siempre inmediatamente después de las comidas). El empleo de nuevos macrólidos requiere mayor base experimental para establecer dosis diaria, duración y eficacia bacteriológica. Es conveniente implementar el cultivo en centros centros centinelas para vigilar la susceptibilidad in vitro de bordetella pertussis y ratificar la eficacia bacteriológica de los esquemas abreviados de terapia específica. Los pasos abreviados de tratamiento de la infección establecida por bordetella pertussis parecen ser extrapolables a los esquemas recomendados para la profilaxis en los contactos

Fatal Bordetella pertussis infection: report of two cases with novel pathologic findings.

We report two infants less than 2 months of age who died of Bordetella pertussis infection: one of primary B. pertussis infection and the other of secondary bronchopneumonia. We describe histopathologic findings in the lung, including transmission and immunoelectron microscopy, studies showing close association between B. pertussis organisms and ciliated cells. A novel finding in both cases was striking dilatation and inspissation of proteinaceous material in pancreatic ducts, reminiscent of changes described in cystic fibrosis. The possible mechanism for these changes may be related to cellular and molecular actions of pertussis toxin-a powerful inhibitor of G proteins and adenyl cyclase important in cellular signal transduction.

Bordetella pertussis adenylate cyclase toxin: intoxication of host cells by bacterial invasion.

Bordetella pertussis produces extracytoplasmic adenylate cyclase toxin (AC toxin) which penetrates target cells and, upon activation by host calmodulin, generates high levels of intracellular cyclic AMP (cAMP). As a result, bactericidal functions of immune effector cells are impaired. Since a considerable amount of AC toxin is associated with the bacterium, it was proposed that the toxin may be delivered by direct interaction of the organism with the target cells (E. L. Hewlett, M. C. Gray, and R. D. Pearson, Clin. Res. 35:477A, 1987). Incubation of CHO cells with intact B. pertussis led to formation of intracellular cAMP at levels comparable to those produced in CHO cells by equivalent activities of isolated AC toxin. cAMP accumulation induced by the whole bacteria appeared after a lag of 40 to 60 min and reached high levels within 2 to 3 h, whereas adherence of the bacteria proceeded rapidly and reached a maximal level within 80 min. Sera of pertussis patients completely blocked cAMP accumulation induced by the whole bacteria without having a major effect on either bacterial adherence or cAMP production by the AC toxin. Cytochalasins B and D, inhibitors of bacterial invasion, abrogated the cAMP response to the whole bacteria but not the response to the AC toxin. These agents did not affect bacterial adherence. Transmission electron micrographs revealed that B. pertussis, within the time course of cAMP induction, invaded CHO cells. We suggest that cAMP induction by B. pertussis is caused by the entry of the whole bacteria into CHO cells rather than by delivery of AC toxin during bacterial adherence. This route of cell intoxication may be relevant to the pathogenesis of whooping cough.